Posts Tagged ‘Endothelium’

Action of Hormones on the Circulation

Writer and Curator: Larry H. Bernstein, MD, FCAP 




This is perhaps the most difficult piece to write, unexpectedly. I have done a careful search for related material using different search phrases.  It is perhaps because of the great complexity of the topic, which is inextricably linked to sepsis, the Systemic Inflammatory Response Syndrome SIRS), and is poised differently than the neural innervation of the hormonal response and circulation, as in the previous piece.  In the SIRS mechanism, we find a very large factor in glucocorticoids, the cytokine shower (IL-1, IL-6, TNF-α), and gluconeogenesis, with circulatory changes.  In this sequence, it appears that we are focused on the arteriolar and bronchial smooth muscle architecture, the adrenal medulla, vasoconstriction and vasodilation, and another set of peptide interactions.  This may be concurrent with the other effects described.

Related articles in Pharmaceutical Intelligence Journal:

Endothelial Function and Cardiovascular Disease

Pathologist and Author: Larry H Bernstein, MD, FCAP


Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination?

Curator: Aviva Lev-Ari, PhD, RN


Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Author and Curator of an Investigator Initiated Study: Aviva Lev-Ari, PhD, RN


Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

Curator of an Investigator Initiated Study: Aviva Lev-Ari, PhD, RN


Cardiovascular Disease (CVD) and the Role of Agent Alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

Curator and Investigator Initiated Study: Aviva Lev-Ari, PhD, RN


Innervation of Heart and Heart Rate

Writer and Curator: Larry H Bernstein, MD, FCAP


αllbβ3 Antagonists As An Example of Translational Medicine Therapeutics

Larry H Bernstein, MD, FCAP, Reporter and curator


Cardiac Contractility & Myocardium Performance: Therapeutic Implications for Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC


The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN


Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Larry H Bernstein, MD, FCAP
Aviva Lev-Ari, PhD, RN


Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


Advanced Topics in Sepsis and the Cardiovascular System at its End Stage

Larry H Bernstein, MD, FCAP


For most comprehensive Bibliography on the Ryanodine receptor calcium release channel complex and for FIGURES illustrating the phenomenon, see

Pharmacol Ther. 2009 August; 123(2): 151–177.


PMCID: PMC2704947

Ryanodine receptor-mediated arrhythmias and sudden cardiac death

Lynda M. Blayney[low asterisk] and F. Anthony Lai


Oxidized Calcium Calmodulin Kinase and Atrial Fibrillation

Author: Larry H. Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN


Contributions to cardiomyocyte interactions and signaling

Author and Curator: Larry H Bernstein, MD, FCAP  and Curator: Aviva Lev-Ari, PhD, RN


Cardiac Contractility & Myocardium Performance: Therapeutic Implications for Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Editor: Justin Pearlman, MD, PhD, FACC, Author and Curator: Larry H Bernstein, MD, FCAP, and Article Curator: Aviva Lev-Ari, PhD, RN


The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN


 Action of hormones on the circulation

Limbic system mechanisms of stress regulation: Hypothalamo-pituitary-adrenocortical axis

James P. Herman, Michelle M. Ostrander, Nancy K. Muelle, Helmer Figueiredo
Prog in Neuro-Psychopharmacol & Biol Psychiatry 29 (2005) 1201 – 1213

Limbic dysfunction and hypothalamo-pituitary-adrenocortical (HPA) axis dysregulation are key features of affective disorders. The following review summarizes our current understanding of the relationship between limbic structures and control of ACTH and glucocorticoid release, focusing on the hippocampus, medial prefrontal cortex and amygdala. In general, the hippocampus and anterior cingulate/prelimbic cortex inhibit stress-induced HPA activation, whereas the amygdala and perhaps the infralimbic cortex may enhance glucocorticoid secretion. Several characteristics of limbic–HPA interaction are notable: first, in all cases, the role of given limbic structures is both region- and stimulus-specific. Second, limbic sites have minimal direct projections to HPA effector neurons of the paraventricular nucleus (PVN); hippocampal, cortical and amygdalar efferents apparently relay with neurons in the bed nucleus of the stria terminalis, hypothalamus and brainstem to access corticotropin releasing hormone neurons. Third, hippocampal, cortical and amygdalar projection pathways show extensive overlap in regions such as the bed nucleus of the stria terminalis, hypothalamus and perhaps brainstem, implying that limbic information may be integrated at subcortical relay sites prior to accessing the PVN. Fourth, these limbic sites also show divergent projections, with the various structures having distinct subcortical targets. Finally, all regions express both glucocorticoid and mineralocorticoid receptors, allowing for glucocorticoid modulation of limbic signaling patterns. Overall, the influence of the limbic system on the HPA axis is likely the end result of the overall patterning of responses to given stimuli and glucocorticoids, with the magnitude of the secretory response determined with respect to the relative contributions of the various structures.

representations of the HPA axis

representations of the HPA axis

Diagrammatic representations of the HPA axis of the rat. HPA responses are initiated by neurosecretory neurons of medial parvocellular paraventricular nucleus (mpPVN), which secretes ACTH secretagogues such as corticotropin releasing hormone (CRH) and arginine vasopressin (AVP) in the hypophysial portal circulation at the level of the median eminence. These secretagogues promote release of ACTH into the systemic circulation, whereby it promotes synthesis and release of glucocorticoids at the adrenal cortex.

When exposed to chronic stress, the HPA axis can show both response Fhabituation_ and response Ffacilitation_. FHabituation_ occurs when the same (homotypic) stressor is delivered repeatedly, and is characterized by progressive diminution of glucocorticoid responses to the stimulus. Systemic administration of a mineralocorticoid receptor antagonist is sufficient to block habituation, implying a role for MR signaling in this process. It should be noted that HPA axis habituation is highly dependent on both the intensity and predictability of the stressful stimulus. FFacilitation_ is observed when animals repeatedly exposed to one stimulus are presented with a novel (heterotypic). In chronically stressed animals, exposure to a novel stimulus results in rise in glucocorticoids that is as large as or greater than that seen in a chronic stress naıve animal. Importantly, facilitation can occur in the context of chronic stress-induced elevations in resting glucocorticoids levels, suggesting that this process involves a bypass or override of negative feedback signals.

Hippocampal regulation of the HPA axis appears to be both region- and stressor-specific. Using a sequential lesion approach, our group has noted that the inhibitory effects of the hippocampus on stress-induced corticosterone release and CRH/AVP mRNA expression are likely subserved by neurons resident in the ventral subiculum-caudotemporal CA1. In addition to spatial specificity, hippocampal regulation of the HPA axis also appears to be specific to certain stress modalities; our studies indicate that ventral subiculum lesions cause elevated glucocorticoid secretion following restraint, open field or elevated plus maze exposure, but not to ether inhalation or hypoxia.

The research posits an intricate topographical organization of prefrontal cortex output to HPA regulatory circuits. The anatomy of medial prefrontal cortex efferents may illuminate this issue. The infralimbic cortex projects extensively to the anterior bed nucleus of the stria terminalis, medial and central amygdala and the nucleus of the solitary tract, all of which are implicated in stress excitation. In contrast, the prelimbic cortex has minimal input to these structures, but projects to the ventrolateral preoptic area, dorsomedial hypothalamus and peri-PVN region, areas implicated in stress inhibition. Thus, the infralimbic and prelimbic/anterior cingulate components of the prefrontal cortex may play very different roles in HPA axis regulation. Like other limbic regions, the influence of the amygdala on the HPA axis is stressor- and region-specific. The medial amygdala shows intense c-fos induction following stressors such as restraint, swimming, predator exposure and social interaction.

Despite the prominent involvement of the hippocampus, medial prefrontal cortex and amygdala in HPA axis regulation, there is limited evidence of direct innervation of the PVN by these structures. Rather, these regions appear to project to a number of basal forebrain, hypothalamic and brainstem cell populations that in turn innervate the medial parvocellular PVN. Thus, in order to access principle stress effector neurons, information from the limbic system requires an intermediary synapse. In the bed nucleus of the stria terminalis and hypothalamus, the majority of these intermediary neurons are GABAergic. For example, the bed nucleus of the stria terminalis, ventrolateral preoptic area, dorsomedial hypothalamic nucleus and peri-PVN region all contain rich populations of neurons expressing the GABA marker glutamatic acid decarboxylase (GAD) 65/67.

The organization of the peri-PVN cell groups is particularly interesting. In the case of the ventral subiculum and to a lesser extent, the medial prefrontal cortex, terminal fields can be observed in the immediate surround of the PVN, corresponding to areas containing substantial numbers of GABA neurons. Importantly, dendrites of PVN neurons are largely confined within the nucleus proper, indicating that limbic afferents are unlikely to interact directly with the PVN neurons themselves. The peri-PVN GABA neurons are activated by glutamate, and likely express glutamate receptor subunits. These neurons also up-regulate GAD65 mRNA following chronic stress, commensurate with involvement in long-term HPA regulation. Injections of a general ionotroptic glutamate receptor antagonist into the PVN surround potentiates glucocorticoid responses to restraint, consistent with blockade of glutamate excitation of these GABA neurons. The data are consistent with an interaction between the excitatory limbic structures and inhibitory PVN-regulatory cells at the level of the PVN surround.

Brainstem stress-modulatory pathways likely relay excitatory information to the PVN. For example, the nucleus of the solitary tract provides both catecholaminergic (norepinephrine) and non-catecholaminergic (e.g., glucagon-like peptide-1 (GLP-1) input to the medial parvocellular. Norepinephrine is released into the PVN following stress and is believed to activate CRH neurons via alpha-1 adrenergic receptors. The role of this pathway is thought to be associated with systemic stressors, as selective destruction of PVN norepinephrine input using anti-dopamine beta hydroxylase-saporin conjugate blocks responses to 2-deoxy-glucose but not restraint.  In contrast, blockade of central GLP-1 receptors using exendin 9–36 markedly inhibits responsiveness to both lithium chloride and novelty, suggesting that this non-catecholaminergic cell population may play a more general role in stress integration.

The existence of these putative two-neuron circuits lends important insight into the nature of stress information processing. Anatomical data support the hypothesis that the vast majority of medial prefrontal cortex and ventral subicular inputs to subcortical stress relays are glutamate-containing. As can be appreciated, pyramidal cells of the medial prefrontal cortex and subiculum richly express mRNA encoding vesicular glutamate transporter-1 (VGlut1), a specific marker of glutamate neurons. Combined retrograde tracing/in situ hybridization studies performed in our lab indicate that the vast majority of cortical and hippocampal afferents to PVN-projecting regions (e.g., bed nucleus of the stria terminalis, dorsomedial hypothalamus, ventrolateral medial preoptic area) indeed contain VGlut1, verifying a glutamatergic input to these areas. In contrast, the majority of amygdalar areas implicated in stress regulation express glutamic acid decarboxylase (GAD) 65 or 67 mRNA, suggesting a GABAergic phenotype; indeed, the vast majority of medial and central amygdaloid projections to PVN relays are GABAergic.

representations of limbic stress-integrative pathways from the prefrontal cortex, amygdala and hippocampus

representations of limbic stress-integrative pathways from the prefrontal cortex, amygdala and hippocampus

Diagrammatic representations of limbic stress-integrative pathways from the prefrontal cortex, amygdala and hippocampus. The medial prefrontal cortex (mPFC) subsumes neurons of the prelimbic (pl), anterior cingulate (ac) and infralimbic cortices (il), which appear to have different actions on the HPA axis stress response. The pl/ac send excitatory projections (designated as dark circles, filled line with arrows) to regions such as the peri-PVN zone and bed nucleus of the stria terminalis (BST), both of which send direct GABAergic projections to the medial parvocellular PVN (delineated as open circles, dotted lines ending in squares). This two-neuron chain is likely to be inhibitory in nature. In contrast, the infralimbic cortex projects to regions such as the nucleus of the solitary tract (NTS), which sends excitatory projections to the PVN, implying a means of PVN excitation from this cortical region. The ventral subiculum (vSUB) sends excitatory projections to numerous subcortical regions, including the posterior BST, peri-PVN region, ventrolateral region of the medial preoptic area (vlPOA) and ventrolateral region of the dorsomedial hypothalamic nucleus (vlDMH), all of which send GABAergic projections to the PVN and are likely to communicate transsynaptic inhibition. The medial amygdaloid nucleus (MeA) sends inhibitory projections to GABAergic PVN-projecting populations, such as the BST, vlPOA and peri-PVN, eliciting a transsynaptic disinhibition. A similar arrangement likely exists for the central amygdaloid nucleus (CeA), which sends GABAergic outflow to the ventrolateral BST and to a lesser extent, the vlDMH. The CeA also projects to GABAergic neurons in the NTS, which may disinhibit ascending projections to the PVN.

Inotropes and vasopressors: more than haemodynamics!

Hendrik Bracht, E Calzia, M Georgieff,  J Singer, P Radermacher and JA Russell
British Journal of Pharmacology (2012) 165 2009–2011

Circulatory shock is characterized by arterial hypotension requiring fluid resuscitation combined with inotropes and/or vasopressors to correct the otherwise life-threatening impairment of oxygen supply to peripheral tissues. Catecholamines represent the current therapeutic choice, but this standard is only based on empirical clinical experience. Although there is evidence that some catecholamines may be better than others, it is a matter of debate which one may be the most effective and/or the safest for the different situations. In their review in this issue of the British Journal of Pharmacology, Bangash et al. provide an overview of the pharmacology as well as the available clinical data on the therapeutic use of endogenous catecholamines, their synthetic derivatives and a range of other agents (vasopressin and its analogues, PDE inhibitors and levosimendan). The authors point out that, despite well-established receptor pharmacology, the clinical effects of these treatments are poorly understood. Hence, further investigations are essential to determine which catecholamine, or, in a broader sense, which alternative vasopressor and/or inotrope is the most appropriate for a particular clinical condition.

LINKED ARTICLES   This article is a commentary on Bangash et al., pp. 2015–2033 of this issue and is commented on by De Backer and Scolletta, pp. 2012–2014 of this issue. To view Bangash et al. visit http://dx.doi.org/10.1111/j.1476-5381.2011.01588.x   and to view De Backer and Scolletta visit http://dx.doi.org/10.1111/j.1476-5381.2011.01746.x

In the present issue of the British Journal of Pharmacology, Bangash et al. (2012) review the pharmacology as well as the available clinical data on the therapeutic use of various inotropes and vasopressor agents used for the hemodynamic management of (septic) shock. By definition, circulatory shock is characterized by arterial hypotension that necessitates immediate intervention to maintain the balance of tissue oxygen supply and demand. In practice, the longer and the more frequent periods of hypotension are present in a patient, the less likely is survival, and early aggressive resuscitation is associated with improved outcome. Besides fluid administration to increase the circulating blood volume, in most cases, vasoactive drugs are required to restore an adequate perfusion pressure, and up to now, catecholamines represent the current therapeutic choice. According to their pharmacological profile, catecholamines are traditionally used for their predominant inotropic, vasodilating or constrictor effects.

Clinicians should not forget two fundamental aspects of catecholamine action. First, because of the ubiquitous presence of adrenoceptors, endogenous catecholamines. as well as their synthetic derivatives, have pronounced effects on virtually all tissues (many of which were described several years ago), in particular on the immune system (van der Poll et al., 1996; Flierl et al., 2008), on energy metabolism (Cori and Cori, 1928; Bearn et al., 1951) and on gastrointestinal motility (McDougal and West, 1954). Second, the adrenoceptor density and responsiveness to catecholamines are markedly altered by both the underlying disease and the ongoing catecholamine. Bangash et al. (2012) have to be commended that they not only describe the various endogenous catecholamines and their synthetic derivatives but also thoroughly discuss possible alternatives, such as vasopressin and its analogues, PDE inhibitors and levosimendan.

Inhibitory effects of cortisone and hydrocortisone on human Kv1.5 channel currents

Jing Yu, Mi-Hyeong Park, Su-Hyun Jo
Eur J Pharmacol 746 (2015) 158–166  http://dx.doi.org/10.1016/j.ejphar.2014.11.007

Glucocorticoids are the primary hormones that respond to stress and protect organisms from dangerous situations. The glucocorticoids hydrocortisone and its dormant form, cortisone, affect the cardiovascular system with changes such as increased blood pressure and cardioprotection. Kv1.5 channels play a critical role in the maintenance of cellular membrane potential and are widely expressed in pancreatic β-cells, neurons, myocytes, and smooth muscle cells of the pulmonary vasculature. We examined the electrophysiological effects of both cortisone and hydrocortisone on human Kv1.5 channels expressed in Xenopus oocytes using a two-microelectrode voltage clamp technique. Both cortisone and hydrocortisone rapidly and irreversibly suppressed the amplitude of Kv1.5 channel current with IC50 values of 50.2 + 74.2 μM and 33.4 + 73.2 μM, respectively, while sustained the current trace shape of Kv1.5 current. The inhibitory effect of cortisone on Kv1.5 decreased progressively from – 10mV to +30 mV, while hydrocortisone’s inhibition of the channel did not change across the same voltage range. Both cortisone and hydrocortisone blocked Kv1.5 channel currents in a non-use-dependent manner and neither altered the channel’s steady-state activation or inactivation curves. These results show that cortisone and hydrocortisone inhibited Kv1.5 channel currents differently. Kv1.5 channels were more sensitive to hydrocortisone than to cortisone.

In conclusion, cortisone and hydrocortisones rapidly and irreversibly blocked human Kv1.5 channels expressed in Xenopus oocytes in a closed state without altering activation and inactivation gating. These data provide a possible mechanism for GC effects on the cardiovascular system. The detailed mechanism of the interaction between GCs and human Kv1.5 channels merits further exploration.

Inflammasome and cytokine blocking strategies in autoinflammatory disorders

Monika Moll, Jasmin B. Kuemmerle-Deschner
Clinical Immunology (2013) 147, 242–275 http://dx.doi.org/10.1016/j.clim.2013.04.008

Autoinflammatory disorders are characterized by usually unprovoked recurrent episodes of features of inflammation caused by activation of the innate immune system. Many autoinflammatory disorders – the monogenetic defects in particular – are associated with alterations of inflammasomes. Inflammasomes are complex multimolecular structures, which respond to “danger” signals by activation of cytokines. Among these, IL-1 is the key player of the innate immune response and inflammation. Consequently, IL-1 blocking strategies are specific pathway targeting therapies in autoinflammatory diseases and applied in CAPS, colchicine-resistant FMF, TRAPS, HIDS and DIRA. A number of rare genetic disorders involve inflammasome malfunction resulting in enhanced inflammatory response. IL-1 inhibition to date is the most successful specific therapy in autoinflammatory disorders. Here, current treatment strategies in autoinflammatory disorders are reviewed with a focus on inflammasome and cytokine inhibition.

Autoinflammatory disorders have been defined as “clinical disorders marked by abnormally increased inflammation, mediated predominantly by the cells and molecules of the innate immune system.”  This means that in autoinflammatory disorders autoantibodies or antigen related T-cells are usually absent. These are features of the adaptive immune system and found in autoimmune diseases.
In general, autoinflammatory disorders are characterized by a large spectrum of rather non-specific systemic and organ-specific signs and symptoms of inflammation. In some diseases specific symptoms are observed like hearing loss in Muckle–Wells syndrome or CNS-disease in NOMID/CINCA. Most autoinflammatory disorders are associated with high levels of serum amyloid A (SAA) during inflammatory attacks and high risk of life-threatening amyloidosis. In most cases the disease will start in infancy and childhood. Only rarely primary manifestations in adulthood are reported.
Because recurrent fevers have been the most prominent feature of this group of diseases, historically they have been summarized under the term “hereditary periodic fever syndromes”.  With the deeper understanding of the underlying pathophysiologic mechanisms on the genetic and cellular level, the more comprehensive term “autoinflammatory syndromes”.
Along with the detection of the genetic origin of the autoinflammatory disorders, the cellular pathomechanism leading to the resulting inflammation has been described. A number of genes, which are affected by mutations in autoinflammatory disorders, encode proteins forming intracellular complexes called inflammasomes. External and endogenous “dangers” are recognized by these “danger sensors” and are able to induce an inflammatory reaction. Microbial components from infectious agents such as LPS, flagellin, lipoteichoic acid from bacteria, peptidoglycan or double-stranded DNA from viruses, or inorganic crystalline structures such as uric acid crystals, display pathogen-associated molecular patterns (PAMPs). These and endogenous damage-associated molecular patterns (DAMPs) like heat-shock proteins, the chromatin-associated protein high-mobility group box 1 (HMGB1), hyaluronan fragments, ATP, uric acid, and DNA which are released with cellular waste and injury stimulate the inflammasome. Also, the myeloid related proteins MRP8 and 14 (also known as S100A8 and S100A9) which are used as biomarkers, belong to the group of DAMPs. In addition to PAMPs and DAMPs, the inflammasome may interact with and be stimulated by proteins such as pyrin, proline–serine–threonine phosphatase interacting protein 1 (PSTPIP1), mevalonate kinase (MK) and NLRP7. All of these may also be altered in structure and function by monogenetic mutations.
As a consequence of inflammasome activation, a large variety of cytokines are produced and released by cells of the innate immune system (monocytes, macrophages, dendritic cells). They include the IL-1 family (IL-1, IL-18, IL-33), the TNF family (TNF-α, LT-α), the IL-6 family (IL-6, IL-11), the IL-17 family (IL-17A, IL-25), and type 1 IFNs (IFN-α, IFN-β). These cytokines play redundant roles depending on the cause and pathway of inflammation in the respective disease. Therefore, therapeutic strategies targeting only one cytokine should be expected to be inadequate to treat inflammatory disorders. However, improvement observed in diabetes mellitus Type 2 after blockade of IL-1 indicates that targeting one cytokine, even in a polygenic, complex inflammatory disorder, may cause beneficial effects. Regarding the inflammatory pathogenesis involved in the disease, Goldbach–Mansky and co-workers have classified the monogenetic autoinflammatory disorders as IL-1 mediated (CAPS and DIRA), partially IL-1 mediated (FMF, HIDS, PAPA) and mediated by other pathways (TRAPS, Blau-syndrome, Majeed’s syndrome, cherubism and IL-10 receptor deficiency).

Intracellular signaling pathways and therapeutic targets in autoinflammatory diseases. In autoinflammatory diseases, complex intracellular pathways lead to activation of the inflammatory response, particularly IL-1β activation and release, but also induction of NFκB and TNFα. Several mechanisms may activate the inflammasome, one crucial step in the IL-1 pathway. These include DAMPs (1), K+-efflux (2), activation of ROS (3) by ATP, anorganic crystals, membrane perturbation and proteases which are released from lysosomes damaged by β-amyloid, and heat shock proteins (4). NFκB may be induced by PAMPs via toll like receptors (5), IL-1β-signaling (6) or UPR (7). Activated NFκB eventually leads to the release of pro-inflammatory cytokines like IL-1, IL-6 and TNFα (8). Most of these steps to activation have been identified as targets for anti-inflammatory therapies, which are either already used in clinical practice or still experimental. IL-1- (a), TNF- (b), and IL-6 (c) inhibition are established safe and effective treatment strategies in many autoinflammatory diseases. Thalidomide (d) probably inhibits activation of IκB and is also part of routine treatment. Still experimental strategies include inhibition of PAMPs (e), DAMPs (f), potassium efflux (g), ROS by antioxidants (h), heat shock proteins (i), or caspase-1 (k). Caspase-inhibitors have entered clinical trials.

Colchicine has been used for the treatment of inflammatory disorders for centuries. Colchicine is effective in gout, but also in Behcet’s disease and FMF, where it is able to prevent amyloidosis. The drug affects many cell types and accumulates preferentially in neutrophils. Although its mode of action is still unclear it has microtubule destabilizing properties which may be part of its effects. Additional effects such as alteration of adhesion molecule expression, chemotaxis, and ROS generation also impact inflammation. Colchicine is generally tolerated well. However gastrointestinal, hematologic, and neuromuscular side-effects occur, when the administered dose is too high.

Inflammasome activation by heat shock proteins may be prevented by direct inhibition of HSP. HSP90 inhibition was effective in reducing gout-like arthritis in an animal model. Targeting caspase-1 (caspase-1-inhibitors) may be a strategy which has even greater potential in the treatment of autoimmune diseases and autoinflammatory disorders. IL-1 converting enzyme/caspase inhibitor VX-765 was able to inhibit IL-β-secretion in LPS-stimulated cells from FCAS and control subjects. A new IL-1 inhibitor, gevokizumab or Xoma 052 has entered clinical pilot trials. Therapeutic targets particularly for the protein-misfolding autoinflammatory diseases could be chemical chaperones and drugs that stimulate autophagy. Also inhibiting the signaling molecules that mediate the UPR activation which causes activation of the innate immune system and exacerbate inflammation could be a target.

To date IL-1 blockade is the most effective therapy in most monogenetic autoinflammatory diseases — in intrinsic and in extrinsic inflammasom-opathies. The most favorable effects are seen in the treatment of cryopyrin associated periodic syndromes like FACS, MWS and CINCA. But IL-1-blockade is also effective in other diseases like DIRA, TRAPS, PFAPA, colchicine-resistant FMF etc. IL-1 inhibition also has a role in multifactorial and common autoinflammatory diseases like diabetes, gout and artherosclerosis.

Endothelin—Biology and disease

Al-karim Khimji, Don C. Rockey
Cellular Signalling 22 (2010) 1615–1625

Endothelins are important mediators of physiological and pathophysiologic processes including cardiovascular disorders, pulmonary disease, renal diseases and many others. Additionally, endothelins are involved in many other important processes such as development, cancer biology, wound healing, and even neurotransmission. Here, we review the cell and molecular biology as well as the prominent pathophysiological aspects of the endothelin system.

Endothelin-1 (ET-1) was originally isolated from porcine aortic endothelial cells  and is a 21 amino acid cyclic peptide, with two disulphide bridges joining the cysteine amino acids (positions 1–15 and 3–11) at the N-terminal end and hydrophobic amino acids at the c-terminal end of the peptide (Fig. 1). The C-terminal end contains the amino acids that bind to the receptor, the N-terminal end determines the peptide’s binding affinity to the receptor (see Fig. 1). There appear to be at least 2 other endothelin isoforms including endothelin-2 (ET-2) and endothelin-3 (ET-3), which differ from ET-1 in two and six amino acid residues, respectively.

Endothelin (ET) structure

Endothelin (ET) structure

Endothelin (ET) structure. Endothelin is a 21 amino acid cyclic peptide, with two disulphide bridges joining the cysteine residues at positions 1–15 and 3–11. The C-terminal end containsamino acids that appear tomediate receptor binding,while the N-terminal residues determine the peptide’s binding affinity to the receptor. The amino acids highlighted in black in panels (b) and (c) show differences in ET-2 and ET-3 compared to ET-1. As can be seen, the remainder of the primary sequence of the different family members is identical.

Endothelin-1 biosynthetic pathway

Endothelin-1 biosynthetic pathway

Endothelin-1 biosynthetic pathway. Preproendothelin mRNA is synthesized via transcriptional activation of the preproendothelin gene. The translational product is a 203-amino acid peptide known as preproendothelin, which is cleaved at dibasic sites by furin-like endopeptidases to form big endothelins. These biologically inactive, 37- to 41-amino acid intermediates, are cleaved at Trp21–Val 22 by a family of endothelin-converting enzymes (ECE) to produce mature ET-1. The pathway for endothelin-2 and -3 is presumed to be similar.

The endothelin peptides are produced through a set of complex molecular processes. Preproendothelins are synthesized via transcriptional activation of the preproendothelin gene, which is regulated by c-fos and c-jun, nuclear factor-1, AP-1 and GATA-2. The translational product is a 203-amino acid peptide known as preproendothelin which is cleaved at dibasic sites by furin-like endopeptidases to form big endothelins. These biologically inactive 37- to 41-amino acid intermediates are cleaved at Trp21–Val 22 by a family of endothelin-converting enzymes (ECE) to produce mature ET-1.

Three isoforms of ECE have been reported, namely ECE-1, ECE-2 and ECE-3; ECE-1 and ECE-2 are most prominent. (Endothelin receptors are widely distributed in many different tissues and cells, there is a marked difference in cell and tissue distribution patterns between the two receptor subtypes i.e. ETA and ETB. [ET Receptors: Endothelial cells -ETB Vascular tone, clearance of circulating ET-1]).  ECEs belong to the M13 group of proteins—which is a family that includes neutral endopeptidases, kell blood group antigens (Kell), a peptide from phosphate regulating gene (PEX), X-converting enzyme (XCE), “secreted” endopeptidases, and the ECEs. M13 family members contain type II integral membrane proteins with zinc metalloprotease activity, and their function is inhibited by phosphoramidon. Four variants of ECE-1 have been reported in humans, namely ECE-1a, ECE-1b, ECE-1c and ECE-1d which are a result of alternate splicing of ECE-1mRNA. ECE-1 appears to be localized in the plasma cell membrane and its optimal activity is atpH7; it processes big ETs both intracellularly and on the cell surface. It is distributed predominantly in smooth muscle cells. ECE-1 can also hydrolyze other proteins including bradykinin, substance P, and insulin. ECE-2 is localized to the trans-Golgi network and is expressed abundantly in neural tissues and endothelial cells. Its optimal activity is at pH5; the acidic activity marks ECE-2 as an intracellular enzyme. Substrate selectivity experiments indicate that both ECE-1 and ECE-2 show preference for big ET-1 over big ET-2 or big ET-3.

Although there has been controversy about the precise repertoire of endothelin receptors, it appears that the endothelins exert their actions through two major receptor subtypes known as ETA and ETB receptors. ETA and ETB receptors belong to the superfamily of G-protein coupled receptors and contain seven transmembrane domains of 22–26 hydrophobic amino acids among approximately 400 total amino acids. The ETA receptor is found predominantly in smooth muscle cells and cardiac muscles, whereas the ETB receptor is abundantly expressed in endothelial cells.

ET-1 signaling is extremely complicated and ET receptor activation leads to diverse cellular responses through interaction in a chain of pathways that includes the G-protein-activated cell surface receptor, coupling G-proteins and phospholipase (PLC) pathway and other G protein-activated effectors. In one of the canonical signaling pathways, ETA induced activation of phospholipase C leads to the formation of inositol triphosphate and diacylglcerol from phosphatidylinositol. Inositol 1,4,5 triphosphate (IP3) then diffuses to specific receptors on the endoplasmic reticulum and releases stored Ca2+ into the cytosol. This causes a rapid elevation in intracellular Ca2+, which in turn causes cellular contraction and then vasoconstriction; the vasoconstrictive effects of ET persist despite dissociation of ET-1 from the receptor, perhaps because the levels of intracellular calcium remain elevated or because endothelin signaling pathways remain activated for prolonged time periods.

Endothelin signaling – smooth muscle cells

Endothelin signaling – smooth muscle cells

Endothelin signaling – smooth muscle cells. ET receptor stimulation leads to diverse cellular responses in a chain of pathways that include the G protein bg activation. This is followed by activation of a variety of different downstream cascades. For example, shown on the left, ETA induced activation of phosphatidyl inositol specific phospholipase C (PI-PLC) leads to the formation of inositol triphosphate (IP3) and diacylglcerol (DAG) from phosphoinositol 4,5 bisphosphate (PIP2). Inositol 1, 4, 5 triphosphate (IP3) then diffuses to specific receptors on the endoplasmic reticulum and releases stored Ca2+ into the cytosol. This causes a rapid elevation in intracellular Ca2+, which in turn causes cellular contraction

Endothelin signaling – endothelial cells.

Endothelin signaling – endothelial cells.

Endothelin signaling – endothelial cells. ET-1 stimulates NO production in endothelial cells by activation of endothelial cell NO synthase (eNOS). This occurs via ET-1’s activation of the ET-B receptor and the PI3-K/Akt pathway, which in turn stimulates phosphorylation of eNOS, with subequent conversion of L-arginine to L-citrulline and at the same time, generating NO. In addition shear stress, G-protein coupled receptors (GPCR), transient receptor potential channel (TRPC) and receptor tyrosine kinase (RTK) are also activators of eNOS. As a result, NO diffuses to stellate cell, where it directly activates the heme moiety of soluble guanylate cyclase, leading to the production of cyclic GMP. Intracellular cyclic GMP leads to activation of protein kinase G (PKG) resulting in relaxation of stellate cells – offsetting ET’s contractile effect on stellate cells.

The plasma levels of endothelin do not correlate with either the presence of essential hypertension or its severity, presumably, due to the fact that endothelin appears to be biologically active in a paracrine or autocrine fashion (i.e., rather than in an endocrine fashion. Systemic administration of ET-1 in low doses produces a modest increase in blood pressure which is normalized by selective ETA receptor blockade. In experimental models, long-term infusion with ET-1 leads to stroke and renal injury, which can be prevented with long-term administration of selective ETA receptor antagonists. Apart from its direct vasoconstrictor effects, mediated by smooth muscle cell contraction in the arterial system, ET-1 also indirectly enhances the vasoconstrictor effects of other neurohumoral and endocrine factors and may potentiate essential hypertension via this mechanism. For example, ET-1 induces conversion of angiotensin I to angiotensin II in in vitro models and stimulates adrenal synthesis of epinephrine and aldosterone. Thus there is cross-talk between the endothelin and renin–angiotensin–aldosterone systems—to synergistically act to facilitate vasoconstriction. In aggregate, the data suggest that dysregulation of the endothelin system contributes to multisystem complications of hypertension such as progressive renal disease, cerebrovascular diseases, atherosclerosis, and cardiac disease.

ET-1 in the renal system is synthesized in vascular endothelial cells and epithelial cells of the collecting ducts. Both ET receptors are present in renal vasculature and epithelial cells where ETB is the predominant receptor type. Renal vasculature is relatively more sensitive to the vasoconstrictive effects of ET-1 than any other vasculature and it causes constriction of both afferent and efferent renal arterioles.

ET-1 administration in humans significantly reduces renal blood flow, glomerular filtration rate and urine volume. In addition to its hemodynamic effects, ET-1 system is also involved in salt and water reabsorption, acid-base balance, promotion of mesangial cell growth and activation of inflammatory cells. ET-1 has been implicated in the pathophysiology of acute renal injury, chronic renal failure as well as renal remodeling. Transgenic mice overexpressing ET-1 develop glomerulosclerosis, interstitial fibrosis and reduced renal function. Increased ET-1 and ET receptor upregulation has been described in various animal models of acute renal injury and also in patients with chronic renal failure. Additionally, plasma ET-1 levels have been shown to correlate with the severity of chronic renal failure.

ET-1 is produced and released by airway epithelial cells, macrophages, and pulmonary vascular endothelial cells. Endothelin receptors are similarly widely distributed in airway smooth muscle cells, the pulmonary vasculature, and in the autonomic neuronal network lining tracheal muscles. ET-1 has a potent bronchoconstrictor effect.  In animal models, intravenous ET-1 injection led to a dose-dependent increase in airway resistance. The increase in airway resistance is in part due to enhanced production of thromboxanes with subsequent activation of thromboxane receptors and smooth muscle cell proliferation. The ET system has been emphasized in a number of pulmonary disorders, including asthma, cryptogenic fibrosing alveolitis, and pulmonary hypertension. Increased lung vasculature ET-1 immunoreactivity has been reported in both animals and patients with pulmonary hypertension and increases in ET-1 immunoreactivity correlate with the degree of pulmonary vascular resistance, disorders such as pulmonary hypertension, myocardial infarction, heart failure, neoplasia, vascular disorders, wound healing, and many others.

Endothelin and endothelin antagonism: Roles in cardiovascular health and disease

Praveen Tamirisa, William H. Frishman, and Anil Kumar
Am Heart J 1995;130:601-10

Endothelin is a naturally occurring polypeptide substance with potent vasoconstrictive actions. It was originally described as endotensin or endothelial contracting factor in 1985 by Hickey et al., who reported on the finding of a potent stable vasoconstricting substance produced by cultured endothelial cells. Subsequently, Yanagisawa et al. isolated and purified the substance from the supernatant of cultured porcine aortic and endothelial
cells and then went on to prepare its complementary deoxyribonucleic acid (cDNA). This substance was renamed endothelin.

Endothelin is the most potent vasoconstrictor known to date. Its chemical structure is closely related to certain neurotoxins (sarafotoxins) produced by scorpions and the burrowing asp (Atractaspis engaddensis).  Endothelins have now been isolated in various cell lines from several organisms. They are now considered to be autocoids or cytokines 4 because of their wide distribution, their expression during ontogeny and adult life, their primary role as intracellular factors, and the complexity of their biologic effects.

The superfamily of endothelins and sarafotoxins have two main branches with four members each. Endothelin is a polypeptide consisting of 21 amino acids. There are three closely related isoforms endothelin-1, endothelin-2, and endothelin-3 (ET1, ET2, and ET3, respectively), which differ in a few of the amino acid constituents. The fourth member, called ET4 or vasoactive intestinal constrictor, is considered to be the murine form ofET2. The endothelin molecules have several conserved amino acids, including the last six carboxyl (C)-terminal amino acids and four cysteine residues, which form two intrachain disulfide bonds between residues 1 and 15 and 3 and 11. These residues may have biologic implications particularly in relation to three dimensional structure and function. The main differences in the endothelin isopeptides reside in their amino (N)-terminal segments. There is a very high degree of sequence similarity between the two branches (approximately 60%) and within the constituent members of a branch (71% to 95%).

Endothelin has been demonstrated to be produced from endothelial and nonendothelial cells. The synthesis of endothelins parallels that of the various peptide hormones in that a precursor polypeptide is sequentially cleaved to generate the active form. Recently, endothelin-converting enzyme (ECE) was cloned. ECE acts at an essential step in the production of active forms of endothelins. The fully formed molecule is then broken down into inactive peptides by as yet uncharacterized proteases. Some candidates are the lysosomal protective protein (deamidase) and enkephalinase (neutral endopeptidase EC 24.11). The regulation of endothelin production occurs predominantly at the levels of transcription and translation. No storage
vesicles containing endothelin have been identified. The genes for the various endothelin isoforms have been sequenced and are found to be scattered in different chromosomes. Current evidence suggests that they arose from a common ancestor by exon duplication.

Factors known to release endothelinThrombinTransforming growth factor-~Arginine vasopressinHypoxia

Phorbol ester


Angiotensin II


Insulinlike growth factor



Low-density lipeprotein cholesterol


Changes in shear stress on vascular wall

Receptor affinities
Receptor Affinity
ETA ET1 > ET2 > ET3
ETB ET1 = ET2 = ET3
Intracellular signal transduction pathways activated by endothelins (ETs)

Intracellular signal transduction pathways activated by endothelins (ETs)

Intracellular signal transduction pathways activated by endothelins (ETs). Activated ET receptor stimulates phospholipase C (PLC) and phospholipase A2 (PLA2). Activated ET receptor also stimulates voltage-dependent calcium channels (VDC) and probably receptor-operated calcium channel (ROC). Inositol triphosphate (IP3) elicits release of calcium ion from caffeine-sensitive calcium store. Protein kinase C (PKC) activated by diacylglycerol (DG) sensitizes contractile apparatus. Increased concentration of intracellular free calcium ion ([Ca2+]i induces contraction. Cyclooxygenase products (prostacyclin [PGI2], prostaglandin E2 [PGE2], and thromboxane A2 [TXA2]) modify contraction. G, G protein; IP2, inositol biphosphate; IP3, inositol triphosphate; PIP2, phosphatidyl inositol biphosphate. (From Masaki T et al. Circulation 1991;84: 1460.)

Systemic hypertension. Endothelin is the most potent vasoconstrictor known to date and has an exceptionally long duration of physiologic action. The influence of endothelin in maintaining normal blood pressure and its role in the cause of systemic hypertension remain unclear. Intravenous injections of endothelin in animals cause a transient decrease in systolic blood pressure (ETB) followed by a prolonged pressor response (ETA). The vasoconstrictor action is mediated by ETA receptors in the vascular smooth muscle, whereas the predominant vasodilation effect is mediated by the ETB receptors on the endothelial cells that cause release of prostacyclin and nitric oxide. Therefore the overall predominant hemodynamic effect of endothelin in a given organ depends on the receptor type being stimulated, its location, and its relative abundance.

Angiotensin II has been found to increase endothelin concentrations in vitro from endo thelial cells, suggesting one mechanism by which angiotensin-converting-enzyme (ACE) inhibition could function in vivo. ACE inhibitors also can indirectly interfere with endothelin: increased concentrations of bradykinin decrease endothelin release (by acting through bradykinin 2 receptors, stimulation of which cause increased nitric oxide release). ACE inhibitors can cause regression of intimal hyperplasia, whereas other antihypertensive drugs are ineffective in this regard.

Myocardial ischemia. Myocardial ischemia can enhance the release of endothelin by cardiomyocytes and increase its vasoactive effects. Infusion of the ET1 isoform directly into the coronary circulation of animals results in the development of myocardial infarction, with impaired ventricular functioning and the development of arrhythmias. Endothelin has been shown to lower the threshold for ventricular fibrillation in dogs. An increase in ET1 has been observed in cardiac tissue after experimental myocardial infarction in rats, and pretreatment with an antiendothelin ϒ-globulin in this model can reduce infarct size by as much as 40%. Infusion of ETA receptor antagonist drugs before an ischemic insult can also reduce infarct size in animals.

Plasma endothelin concentrations can predict hemodynamic complications in patients with myocardial infarction. Patients with the highest plasma endothelin concentrations after myocardial infarction have the highest creatine phosphokinase (CPK) and CPK MB-isoenzyme concentrations and the lowest angiographically determined ejection fractions.

Left ventricular function and congestive heart failure. Endothelin exhibits potent inotropic activity in isolated hearts, cardiac muscle strips, isolated cells, and instrumented intact animals. High-affinity receptors for endothelin have been demonstrated in the atria and the ventricles. Intravenous administration of the ET1 isoform produces delayed prolonged augmentation of left ventricular performance in addition to its biphasic vasoactive effects of transient vasodilation followed by sustained vasocontraction.

Endothelin is a potent secretogogue of atrial natriuretic factor, which is a naturally occurring antagonist of endothelin. The ETA receptor appears to mediate endothelin’s actions of vasoconstriction and the stimulation of atrial natriuretic factor secretion, and the ETB receptor mediates endothelin-induced vasodilation and activation of the renin-angiotensin-aldosterone system. Urinary water excretion is mediated through both receptors, but sodium excretion is mediated through the ETA receptor.

Increased concentrations of endothelin described in patients with congestive heart failure are predictive of increased mortality risk. It also has been suggested that increased concentrations of endothelin may play an important role in the increased systemic vascular resistance observed in congestive heart failure.

There is early clinical evidence that treatment with ETA receptor antagonists and ECE inhibitors can influence favorably the course of human heart failure.  ACE inhibitors may also benefit patients with heart failure because of their antiendothelin actions.

Pulmonary hypertension. Expression of ET1 in the lung has been studied by immunocytochemistry and hybridization in situ in specimens from patients with pulmonary hypertension of primary or secondary causes. In contrast to normal lung, specimens from patients with pulmonary hypertension exhibit abundant ET2 immunostaining, particularly over endothelium of markedly hypertrophied muscular pulmonary arteries and plexogenic lesions. Endothelin has been suggested as a potent vasoconstrictor and growth-promoting factor in the pathophysiologic pathophysiologic mechanisms of pulmonary hypertension.

Ventricular and vascular hypertrophy. Endothelin increases DNA synthesis in vascular smooth-muscle ceils, cardiomyocytes, fibroblasts, glial cells, mesangial cells, and other cells; causes expression of protooncogenes; causes cell proliferation; and causes hypertrophy. It acts in synergy with various factors such as transforming growth factor, epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor and insulin to potentiate cellular transformation and replication. This synergy suggests that all of these factors act through common pathways involving PKC and cyclic adenosine monophosphate. Endothelin per se may not be a direct mediator of angiogenesis but may function as a comitogenic factor.

Neointima formation after vascular wall trauma. The efficacy of coronary angioplasty is limited by the high incidence of restenosis. ET1 induces cultured vascular smooth-muscle cell proliferation by activation of the ETA-receptor subtype, a response that normally is attenuated by an intact, functional endothelium. In addition, ET1 also induces the expression and release of several protooncogenes and growth factors that modulate smooth-muscle cell migration, proliferation, and matrix formulation. In addition to inhibiting smooth-muscle cell proliferation in vitro, endothelin-receptor antagonism with SB 209670 ameliorates the degree of neointima formation observed after rat carotid artery angioplasty. The observations raise the possibility that ET1 antagonists will serve as novel therapeutic agents in the control of restenosis.

Nonspecific endothelin antagonists
ECE inhibitorsAngiotensin-converting-enzyme inhibitorsAngiotensin II receptor blocking agentsCalcium-entry blocking agentsPotassium-channel opening agentsAdenosineNitroglycerin






Endothelin is the most potent mammalian vasoconstrictor yet discovered. Its three isoforms play leading roles in regulating vascular tone and causing mitogenesis. The isoforms bind to two major receptor subtypes (ETA and ETB), which mediate a wide variety of physiologic actions in several organ systems. Endothelin may also be a disease marker or an etiologic factor in ischemic heart disease, atherosclerosis, congestive heart failure, renal failure, myocardial and vascular wall hypertrophy, systemic hypertension, pulmonary hypertension, and subarachnoid hemorrhage. Specific and nonspecific receptor antagonists and ECE inhibitors that have been developed interfere with endothelin’s function. Many available cardiovascular therapeutic agents, such as angiotensin-converting-enzyme inhibitors, calcium-entry blocking drugs, and nitroglycerin, also may interfere with endothelin release or may modify its activity. The endothelin antagonists have great potential as agents for use in the treatment of a wide spectrum of disease entities and as biologic probes for understanding the actions of endothelin in human beings.

Endothelin receptor antagonists

Sophie Motte, Kathleen McEntee, Robert Naeije
Pharmacology & Therapeutics 110 (2006) 386 – 414

Endothelin receptor antagonists (ERAs) have been developed to block the effects of endothelin-1 (ET-1) in a variety of cardiovascular conditions. ET-1 is a powerful vasoconstrictor with mitogenic or co-mitogenic properties, which acts through the stimulation of 2 subtypes of receptors [endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) receptors]. Endogenous ET-1 is involved in a variety of conditions including systemic and pulmonary hypertension (PH), congestive heart failure (CHF), vascular remodeling (restenosis, atherosclerosis), renal failure, cancer, and cerebrovascular disease. The first dual ETA/ETB receptor blocker, bosentan, has already been approved by the Food and Drug Administration for the treatment of pulmonary arterial hypertension (PAH). Trials of endothelin receptor antagonists in heart failure have been completed with mixed results so far. Studies are ongoing on the effects of selective ETA antagonists or dual ETA/ETB antagonists in lung fibrosis, cancer, and subarachnoid hemorrhage. While non-peptidic ET-1 receptor antagonists suitable for oral intake with excellent bioavailability have become available, proven efficacy is limited to pulmonary hypertension, but it is possible that these agents might find a place in the treatment of several cardiovascular and non-cardiovascular diseases in the coming future.

Proposed mechanism by which ET-1 triggers vasoconstriction and vascular remodeling. Activation of G-protein-coupled endothelin receptors leads to stimulation of phospholipase C (PLC) which hydrolyses phosphatidyl inositol  biphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG). DAG opens receptor-operated Ca++ channels (ROC) while IP3 induces Ca++ mobilization from the sarcoplasmic reticulum (SR) and opens store-operated Ca++ channels (SOC) directly or indirectly by store depletion to further increase cytosolic Ca++. This Ca++ increase may also trigger Ca++ release from the SR through ryanodine receptors. Depolarization induced by the opening of non-selective cationic channels (NSCC) via ET-1 and Ca++-activated Cl[1] channels as well as by the inhibition of voltage-gated K+ channels (Kv), opens voltage-dependent Ca++ channels (VDCC) to further increase the Ca++ entry across the plasma membrane. The cytosolic Ca++ increase may also activate Na/H exchangers resulting in alkalinization of the cells and promoting Ca++ influx by activating the Na/Ca exchanger. In addition, the elevated cytosolic Ca++ concentrations and DAG activate the protein kinase C and thus promote cell cycle progression by the Ca++/calmodulin complex (Ca++/CaM) and induction of proto-oncogenes. The intracellular signaling cascade induced by activation of ETB receptor is similar to the ETA receptor one, in stimulating the activation of PLC, generating IP3 and DAG and mobilizing of calcium. However, the PLA2 is also activated via ETB receptors to release prostaglandins (PG) and thromboxane A2 (TXA2).

Endothelin-1 increases isoprenaline-enhanced cyclic AMP levels in cerebral cortex

Marıa J. Perez-Alvareza, MC Calcerrada, F Hernandez, RE Catalan, AM Martınez
Regulatory Peptides 88 (2000) 41–46  PII: S0167-0115(99)00118-4

We examined the effect of ET-1 on cyclic AMP levels in rat cerebral cortex. The peptide caused a concentration-dependent increase of [3 H] cyclic AMP accumulation after 10 min of treatment. This effect was due to adenosine accumulation since it was inhibited by the treatment with adenosine deaminase. ET-1, apart from being able to increase cyclic AMP, also potentiated the cyclic AMP generated by isoprenaline in the presence of adenosine deaminase. Experiments performed in the presence of BQ-123 or BQ-788, specific ETA or ETB receptor antagonists respectively indicated that ET was the receptor involved. This effect was dependent on extracellular and B intracellular calcium concentration. These findings suggest that ET-1 plays a modulatory role in cyclic AMP generation systems in cerebral cortex.

Endothelins And Asthma

Roy G. Goldie and Peter J. Henry
Life Sciences I999; 65(1), pp. I-15, PI1 SOO24-3205(98)00614-6

In the decade since endothelin-1 (ET-l) and related endogenous peptides were first identified as vascular endothelium-derived spasmogens, with potential pathophysiological roles in vascular diseases, there has been a significant accumulation of evidence pointing to mediator roles in obstructive respiratory diseases such as asthma. Critical pieces of evidence for this concept include the fact that ET-l is an extremely potent spasmogen in human and animal airway smooth muscle and that it is synthesised in and released from the bronchial epithelium. Importantly, symptomatic asthma involves a marked enhancement of these processes, whereas asthmatics treated with anti-inflammatory glucocorticoids exhibit reductions in these previously elevated indices. Despite this profile, a causal link between ET-l and asthma has not been definitively established. This review attempts to bring together some of the evidence suggesting the potential mediator roles for ET-l in this disease.

Endothelial Cell Peroxisome Proliferator–Activated Receptor ϒ Reduces Endotoxemic Pulmonary Inflammation and Injury

Aravind T. Reddy, SP Lakshmi, JM Kleinhenz, RL Sutliff, CM Hart, and R. Reddy
J Immunol 2012; 189:5411-5420

Bacterial endotoxin (LPS)-mediated sepsis involves severe, dysregulated inflammation that injures the lungs and other organs Bacterial endotoxin (LPS)-mediated sepsis involves severe, dysregulated inflammation that injures the lungs and other organs, often fatally. Vascular endothelial cells are both key mediators and targets of LPS-induced inflammatory responses. The nuclear hormone receptor peroxisome proliferator–activated receptor ϒ (PPARϒ) exerts anti-inflammatory actions in various cells, but it is unknown whether it modulates inflammation through actions within endothelial cells. To determine whether PPARϒ acts within endothelial cells to diminish endotoxemic lung inflammation and injury, we measured inflammatory responses and mediators in mice with endothelial-targeted deletion of PPARϒ. Endothelial cell PPARϒ (ePPARϒ) knockout exacerbated LPS-induced pulmonary inflammation and injury as shown by several measures, including infiltration of inflammatory cells, edema, and production of reactive oxygen species and proinflammatory cytokines, along with upregulation of the LPS receptor TLR4 in lung tissue and increased activation of its downstream signaling pathways. In isolated LPS-stimulated endothelial cells in vitro, absence of PPARϒ enhanced the production of numerous inflammatory markers. We hypothesized that the observed in vivo activity of the ligand-activated ePPARϒ may arise, in part, from nitrated fatty acids (NFAs), a novel class of endogenous PPARϒ ligands.
Supporting this idea, we found that treating isolated endothelial cells with physiologically relevant concentrations of the endogenous NFA 10-nitro-oleate reduced LPS-induced expression of a wide range of inflammatory markers in the presence of PPARϒ, but not in its absence, and also inhibited neutrophil mobility in a PPARϒ-dependent manner. Our results demonstrate a key protective role of ePPARϒ against endotoxemic injury and a potential ePPARϒ-mediated anti-inflammatory role for NFAs.

Endothelins in health and disease

Rahman Shah
European Journal of Internal Medicine 18 (2007) 272–282

Endothelins are powerful vasoconstrictor peptides that also play numerous other roles. The endothelin (ET) family consists of three peptides produced by a variety of tissues. Endothelin-1 (ET-1) is the principal isoform produced by the endothelium in the human cardiovascular system, and it exerts its actions through binding to specific receptors, the so-called type A (ETA) and type B (ETB) receptors. ET-1 is primarily a locally acting paracrine substance that appears to contribute to the maintenance of basal vascular tone. It is also activated in several diseases, including congestive heart failure, arterial hypertension, atherosclerosis, endothelial dysfunction, coronary artery diseases, renal failure, cerebrovascular disease, pulmonary arterial hypertension, and sepsis. Thus, ET-1 antagonists are promising new agents. They have been shown to be effective in the management of primary pulmonary hypertension, but disappointing in heart failure. Clinical trials are needed to determine whether manipulation of the ET system will be beneficial in other diseases.

The production of ET receptors is affected by several factors. Hypoxia, cyclosporine, epidermal growth factor, basic fibroblast growth factor, cyclic AMP, and estrogen upregulate ETA receptors in some tissues, and C-type natriuretic hormone, angiotensin II, and perhaps basic fibroblast growth factor up-regulate ETB receptors. In contrast, the endothelins, angiotensin II, platelet-derived growth factor, and transforming growth factor down-regulate ETA receptors, whereas cyclic AMP and catecholamines down-regulate ETB receptors.

The ETA receptor contains 427 amino acids and binds with the following affinity: ET-1N>T-2>ET-3. It is predominantly expressed in vascular smooth muscle cells and cardiac myocytes. Its interaction with ET-1 results in vasoconstriction and cell proliferation. In contrast, the ETB receptor contains 442 amino acids and binds all endothelins with equal affinity. It is predominantly expressed on vascular endothelial cells and is linked to an inhibitory G protein. Activation of ETB receptors stimulates the release of NO and prostacyclin, prevents apoptosis, and inhibits ECE-1 expression in endothelial cells. ETB receptors also mediate the pulmonary clearance of circulating ET-1 and the re-uptake of ET-1 by endothelial cells.

All three endothelins cause transient endothelium dependent vasodilatation before the development of constriction, though this is most apparent for ET-1. Endothelins induce vasodilatation via the endothelial cell ETB receptors through generation of endothelium-derived dilator substances (Fig. 3), including nitric oxide (NO), which perhaps acts by physiologically antagonizing ETA receptor mediated vasoconstriction. The transient early vasodilator actions of the endothelins are attenuated by NO synthase inhibitors.  Additionally, ET-1 increases generation of prostacyclin by cultured endothelial cells, whereas cyclo-oxygenase inhibitors potentiate ET-1-induced constriction, suggesting that vasodilator prostaglandins play a similar modulatory role.

It has been proposed that ET-1 can affect vascular tone indirectly through its effect on the sympathetic nervous system, and it has been shown that that ET-1 may increase peripheral sympathetic activity through postsynaptic potentiation of the effects of norepinephrine. While in vitro low concentrations of ET-1 potentiate the effects of other vasoconstrictor hormones, including norepinephrine and serotonin, these findings have not been confirmed in vivo in the forearm resistance bed of healthy subjects.  In addition to its action on vascular vasomotion, ET-1 is thought to be a mediator in the vascular remodeling process. It seems that ET-1 interactions with the renin–angiotensin–aldosterone system play a significant role in this remodeling process.

Vascular actions of endothelin-1

Vascular actions of endothelin-1

Vascular actions of endothelin-1. Modified from – Galie N, Manes A, Branzi A; The endothelin system in pulmonary arterial hypertension. Cardiovasc Res 2004;61:227–37.

ET-1 appears to have a diverse role as a modulator of vascular tone and growth and as a mediator in many cardiovascular and non-cardiovascular diseases. To date, no disease entity, however, has been attributed solely to an abnormality in ET-1. Yet, ET-1 receptor antagonists have been studied in clinical trials involving a wide spectrum of cardiovascular diseases, though the only proven efficacy has been in patients with PAH.

Learning points

  • Endothelins are powerful vasoconstrictors and major regulators of vascular tone.
  • The endothelin (ET) family consists of three peptides (ET-1 ∼60%, ET-2 ∼30%, and ET-3 ∼10%) produced by a variety of tissues.
  • ET-1 is the principal isoform produced by the endothelium in the human cardiovascular system and appears to be foremost a locally acting paracrine substance rather than a circulating endocrine hormone.
  • Several human studies suggest that circulating ET-1 levels, which are elevated in heart failure and pulmonary hypertension, correlate with the prognosis of the disease.
  • ET-1 antagonists have been shown to be effective in the management of primary pulmonary hypertension, but disappointing in heart failure.
  • Clinical trials are needed to investigate the role of ET-1 receptor antagonists for other conditions, as ET-1 levels have been shown to be elevated in arterial hypertension, atherosclerosis, endothelial dysfunction, coronary artery disease, renal failure, cerebrovascular disease, and sepsis.

In Vitro Stability and Intestinal Absorption Characteristics of Hexapeptide Endothelin Receptor Antagonists

Hyo-kyung Han, BH Stewart, AM Doherty, WL Cody and GL Amidon
Life Sciences. I998; 63(18), pp. 1599-1609. PI1 SOO24-3205(98)00429-9

Endothelins are potent vasoconstrictor peptides which have a wide range of tissue distribution and three receptor subtypes (ETA ETB and ETC). Among the linear hexapeptide ETA / ETB receptor antagonists, PD 145065 (Ac-D-Bhg-L-Leu-L-Asp-L-Ile-L-Ile-L-Trp,  Bhg = (10,ll -dihydro-5H-dibenzo[a,d]cyclohepten-5-yl)-Gly) and PD 156252 (Ac-o-Bhg-L-Leu-L-Asp-L-Ile-(N-methyl)-L-Ile-L-Trp) were selected to evaluate the metabolic stability and intestinal absorption in the absence and/or in the presence of protease inhibitors. In vitro stability of both compounds was investigated in fresh plasma, lumenal perfusate, intestinal and liver homogenates. PD 156252 was more stable than PD 145065 in intestinal tissue homogenate (63.4% vs. 20.5% remaining) and liver homogenate (74.4% vs. 35.5 % remaining), while both compounds showed relatively good stability in the fresh plasma (94.5% vs. 86.7% remaining) and lumenal perfusate (85.8% vs. 72.3% remaining). The effect of protease inhibitors on the degradation of PD 145065 and PD 156252 was also investigated. Amastatin, thiorphan, chymostatin and the mixture of these three inhibitors were effective in reducing the degradation of both compounds. The pharmacokinetic parameters of PD 156252, calculated by using a non-compartmental model, were 6.95 min (terminal half-life), 191 mL (Vss), and 25.5 mL/min (Cltot) after intravenous administration in rats. The intestinal absorption of PD 156252 in rats was evaluated in the absence and/or in the presence of protease inhibitors. The results indicate that the major elimination pathway of PD 156252 appears to be the biliary excretion and protease inhibitors increase the intestinal absorption of PD 156252 through increasing metabolic stability.

Inhibitory and facilitatory presynaptic effects of endothelin on sympathetic cotransmission in the rat isolated tail artery

Violeta N. Mutafova-Yambolieva & David P. Westfall
British Journal of Pharmacology (1998) 123, 136 – 142

1 The present study was undertaken to determine the modulatory effects of the endothelin peptides on the neurogenically-induced release of endogenous noradrenaline (NA) and the cotransmitter adenosine 5′-triphosphate (ATP) from the sympathetic nerves of endothelium-free segments of the rat isolated tail artery. The electrical field stimulation (EFS, 8 Hz, 0.5 ms, 3 min) evoked over¯ow of NA and ATP, in the absence of endothelins, was 0.035+0.002 pmol mg71 tissue and 0.026+0.002 pmol mg71 tissue, respectively.

2 Endothelin-1 (ET-1; 1 ± 30 nM) significantly reduced the EFS evoked overflow of both NA and ATP.  The maximum inhibitory effect was produced by a peptide concentration of 10 nM, the amount of NA overflow being 0.020+0.002 pmol mg71 and that of ATP overflow 0.015+0.001 pmol mg71. Higher peptide concentrations (100 and 300 nM) reversed the EFS-evoked overflow of NA to control levels and that of ATP to above control levels. The inhibitory effect of ET-1 (10 nM) was resistant to the selective ETA receptor antagonist cyclo-D-Trp-D-Asp(ONa)-Pro-D-Val-Leu (BQ-123) but was prevented by ETB receptor desensitization with sarafotoxin S6c (StxS6c) or by ETB receptor blockade with N, cis-2,6-dimethyl-piperidinocarbonyl-L-gmethylleucyl-D-1-methoxycarbonyl-tryptophanyl-D-norleucine (BQ-788).

3 StxS6c, upon acute application, exerted a dual effect on transmitter release. At concentrations of 0.001 ± 0.3 nM the peptide significantly reduced the EFS-evoked NA overflow, whereas at concentrations of 1 ± 10 nM it caused a significant increase in the evoked overflow of both ATP and NA. Both the maximum inhibitory effect of StxS6c at a concentration of 0.003 nM approximately 85% reduction of NA overflow and 40% of ATP overflow) and the maximum facilitatory effect of the peptide at a concentration of 3 nM (approximately 400% increase of ATP overflow and 200% of NA overflow) were completely antagonized by either BQ-788 or by StxS6c-induced ETB receptor desensitization.

4 ET-3 (10 ± 100 nM) did not a€ect the EFS evoked overflow of either ATP or NA, but at a concentration of 300 nM significantly potentiated the release of both transmitters (0.118+ 0.02 pmol mg71 tissue ATP overflow and .077+0.004 pmol mg71 NA overflow). This effect was prevented either by BQ-123 or by BQ-788.

5 In summary, the endothelin peptides exerted both facilitatory and inhibitory effects on the neurogenically-induced release of the sympathetic cotransmitters ATP and NA in the rat tail artery. Both transmitters were modulated in parallel indicating that the endothelins do not differentially modulate the release of NA and ATP in this tissue.

Involvement of the central adrenomedullin peptides in the baroreflex

Meghan M. Taylo, Cynthia A. Keown, Willis K. Samson
Regulatory Peptides 112 (2003) 87– 93

The peptides derived from post-translational processing of preproadreno-medullin are produced in and act on areas of the autonomic nervous system important for blood pressure regulation. We examined the role of endogenous, brain-derived adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) in the central nervous system arm of the baroreflex by using passive immunoneutralization to block the actions of the endogenous peptides. Our results indicate that the preproadrenomedullin-derived peptides do not play a role in sensing changes in blood pressure (baroreflex sensitivity), but the adrenomedullin peptides do regulate the speed with which an animal returns to a normal, stable blood pressure. These findings suggest that endogenous, brain-derived AM and PAMP participate in the regulation of autonomic activity in response to baroreceptor activation and inactivation.

Pharmacological characterization of cardiovascular responses induced by endothelin-1 in the perfused rat heart

Keiji Kusumoto, A Fujiwara, S Ikeda, T Watanabe, M Fujino
Eur J Pharmacology 296 (1996) 65-74 SSDI 0014-2999(95)00680-X

The effects of the endothelin receptor antagonist TAK-044 (cyclo[D-α-aspartyl-3-[(4-phenylpiperazin-l-yl)carbonyl]-L-alanyl-L-α-aspartyl-D-2-(2-thienyl)-glycyl-L-leucyl-D-tryptophyl] disodium salt) and BQ-123 (cyclo[D-Asp-Pro-D-VaI-Leu-D-Trp]) were studied in the rat heart to characterize the receptor subtypes responsible for the cardiovascular actions of endothelin-1. Endothelin-1 induced a transient decrease and subsequent increase in perfusion pressure in perfused rat hearts, and increased left ventricular developed pressure. TAK-044 diminished these endothelin-l-induced responses (100 pmol/heart) with IC50 values of 140, 57 and 1.3 nM, respectively. BQ-123 (1-30/µM) partially inhibited the endothelin-l-induced hypertension (30-40%) in the rat heart, and failed to inhibit the hypotension. The positive inotropic effect of endothelin-1 was abolished by BQ-123. Neither indomethacin (10/µM) nor N’°-nitro-L-arginine methyl ester (100/pM) attenuated the  endothelin-l-induced hypotension. TAK-044 and BQ-123 attenuated the positive inotropic effect of endothelin-1 in rat papillary muscles. In rat cardiac membrane fractions, TAK-044 and BQ-123 inhibited [125I]endothelin-1 binding to endothelin ET A receptors with IC50 values of 0.39 + 0.6 and 36 + 9 nM, respectively, whereas only TAK-044 potently blocked the endothelin ET B receptor subtype (IC50 value: 370 + 180 nM). These results suggest that endothelin-1 modulates cardiovascular functions in the rat heart by activating both endothelin ET A and endothelin ET B receptors, all of which are sensitive to TAK-044.

Molecular Pharmacology and Pathophysiological Significance of Endothelin

Katsutoshi Goto, Hiroshi Hama and Yoshitoshi Kasuya
Jp J Pharmacol 1996; 72: 261-290

Since the discovery of the most potent vasoconstrictor peptide, endothelin, in 1988, explosive investigations have rapidly clarified much of the basic pharmacological, biochemical and molecular biological features of endothelin, including the presence and structure of isopeptides and their genes (endothelin- 1, -2 and -3), regulation of gene expression, intracellular processing, specific endothelia converting enzyme (ECE), receptor subtypes (ETA and ETB), intracellular signal transduction following receptor activation, etc. ECE was recently cloned, and its structure was shown to be a single transmembrane protein with a short intracellular N-terminal and a long extracellular C-terminal that contains the catalytic domain and numerous N-glycosylation sites. In addition to acute contractile or secretory actions, endothelin has been shown to exert long-term proliferative actions on many cell types. In this case, intracellular signal transduction appears to converge to activation of mitogen-activated protein kinase. As a recent dramatic advance, a number of non-peptide and orally active receptor antagonists have been developed. They, as well as current peptide antagonists, markedly accelerated the pace of investigations into the true pathophysiological roles of endogenous endothelin-1 in mature animals.

The discovery of endothelin in 1988 soon triggered explosive investigations of a worldwide scale, presumably due to its unusual characteristics; i.e., marked potency and long-lasting pressor actions. As a result, most of the basic problems concerned with the science of endothelin have rapidly been solved; e.g., features and regulations of the expression of endothelin genes,  biosynthetic pathways including characterization and cloning of endothelin converting enzyme, pharmacological, biochemical and molecular-biological identification of endothelin receptor subtypes, intracellular signal transduction following receptor activation, and discovery of various receptor agonists and antagonists. In addition to its potent cardiovascular actions, endothelin-1 shows a wide variety of biological effects, including contraction of nonvascular smooth muscle (intestinal, tracheal, broncheal, mesangial, bladder, uterine and prostatic smooth muscle), stimulation of neuropeptides, pituitary hormone and atrial natriuretic peptide release and aldosterone biosynthesis, modulation of neurotransmitter release, and increase of bone resorption. Furthermore, endothelin-1 has mitogenic properties and causes proliferation and hypertrophy of a number of cell types, including vascular smooth muscle cells, cardiac myocytes, mesangial cells, bronchial smooth muscle cells and fibroblasts. Endothelin-1 also induces the expression of several protooncogenes (c fos, C -Jun, c-myc, etc.).

These actions, whereby endothelin- 1 might influence the development of cellular hypertrophy/hyperplasia, are of potential significance in pathophysiological conditions associated with long-term changes in cardiovascular tissues, e.g., hypertension, myocardial infarction, chronic heart failure, vascular restenosis following balloon angioplasty, and atherosclerosis. These pathophysiological conditions are usually associated with increased plasma levels of endothelin-1, although the correlation is relatively poor. Nevertheless, a considerable increase in the tissue content of endothelin-1 has been gradually uncovered in many cases of these conditions. Even if the concentration of endothelin-1 at the cell surface is not high enough to induce contraction, it is well known that subthreshold concentrations of endothelin will enhance or potentiate the contraction produced by other vasoconstrictors (e.g., norepinephrine, serotonin, angiotensin II), indicating the existence of cross-talk among various vasoactive substances. Another important cross-talk among these substances may be mutual enhancement or inhibition of their expression in various tissues. In addition to these interactions, the true physiological and/or pathophysiological roles of each of the endothelin family peptide and receptor subtypes remain to be investigated.

Hydrogen Sulfide and Endothelium-Dependent Vasorelaxation

Jerzy Bełtowski, and Anna Jamroz-Wiśniewska
Molecules 2014, 19, 21183-21199; http://dx.doi.org:/10.3390/molecules191221183

In addition to nitric oxide and carbon monoxide, hydrogen sulfide (H2S), synthesized enzymatically from L-cysteine or L-homocysteine, is the third gasotransmitter in mammals. Endogenous H2S is involved in the regulation of many physiological processes, including vascular tone. Although initially it was suggested that in the vascular wall H2S is synthesized only by smooth muscle cells and relaxes them by activating ATP-sensitive potassium channels, more recent studies indicate that H2S is synthesized in endothelial cells as well. Endothelial H2S production is stimulated by many factors, including acetylcholine, shear stress, adipose tissue hormone leptin, estrogens and plant flavonoids. In some vascular preparations H2S plays a role of endothelium-derived hyperpolarizing factor by activating small and intermediate-conductance calcium-activated potassium channels. Endothelial H2S signaling is up-regulated in some pathologies, such as obesity and cerebral ischemia-reperfusion. In addition, H2S activates endothelial NO synthase and inhibits cGMP degradation by phosphodiesterase thus potentiating the effect of NO-cGMP pathway. Moreover, H2S-derived polysulfides directly activate protein kinase G. Finally, H2S interacts with NO to form nitroxyl (HNO)—a potent vasorelaxant. H2S appears to play an important and multidimensional role in endothelium-dependent vasorelaxation.

GPCR modulation by RAMPs

Debbie L. Hay, David R. Poyner, Patrick M. Sexton
Pharmacology & Therapeutics 109 (2006) 173 – 197

Our conceptual understanding of the molecular architecture of G-protein coupled receptors (GPCRs) has transformed over the last decade. Once considered as largely independent functional units (aside from their interaction with the G-protein itself), it is now clear that a single GPCR is but part of a multifaceted signaling complex, each component providing an additional layer of sophistication. Receptor activity modifying proteins (RAMPs) provide a notable example of proteins that interact with GPCRs to modify their function. They act as pharmacological switches, modifying GPCR pharmacology for a particular subset of receptors. However, there is accumulating evidence that these ubiquitous proteins have a broader role, regulating signaling and receptor trafficking. This article aims to provide the reader with a comprehensive appraisal of RAMP literature and perhaps some insight into
the impact that their discovery has had on those who study GPCRs.

RAMPs were first identified during attempts to expression clone a receptor for the neuropeptide calcitonin gene related peptide (CGRP; McLatchie et al., 1998). Historical evidence had suggested that CGRP acted through a GPCR, as its binding had proven sensitive to GTP analogues and stimulation of various tissues and cells led to the accumulation of cAMP, suggesting activation of a Gs-coupled GPCR. However, attempts to clone such a receptor proved difficult. A putative canine CGRP receptor, RDC-1, was identified in 1995, but the original findings have not been replicated and current IUPHAR guidelines do not consider this receptor a genuine CGRP receptor (Kapas & Clark, 1995; Poyner et al., 2002). Shortly afterward, a further orphan receptor (CL, a close homologue of the calcitonin receptor) was shown to be activated by CGRP when transfected into HEK293 cells (Aiyar et al., 1996). This finding posed something of a conundrum since earlier attempts to examine the function of this receptor (or its rat homologue) in Cos 7 cells had not given positive results with CGRP.
Given the apparent functionality of the human CL receptor in HEK293 cells, the rat homologue was also transfected into this cell type and now responded to CGRP (Han et al., 1997). The authors speculated that there was a factor present in HEK293 cells that conferred high affinity for CGRP on the receptor.

In 1998, McLatchie and colleagues confirmed this speculation and provided new insights into the way that GPCRs and their pharmacology can be regulated (McLatchie et al., 1998). It was discovered that a novel family of single transmembrane domain proteins, termed RAMPs, was required for functional expression of CL at the cell surface, explaining why it had been so difficult to observe CGRP binding or function when CL was transfected into cells lacking RAMP expression (Fluhmann et al., 1995; Han et al., 1997; McLatchie et al., 1998). RAMPs were first identified from a library derived from SK-N-MC cells, cells known to express CGRP receptors. An expression-cloning strategy was utilized, whereby an SK-N-MC cDNA library was transcribed and the corresponding cRNA was used for injection into Xenopus oocytes. Cystic
fibrosis transmembrane regulator chloride conductance, a reporter for cAMP formation, was strongly potentiated by a single cRNA pool (in the presence of CGRP). Subsequently, a single cDNA encoding a 148-amino-acid protein comprising RAMP1 was isolated. The structure of the protein was unexpected, as it was not a GPCR and it did not respond to CGRP in mammalian cells. Thus, it was postulated that RAMP1 might potentiate CGRP receptors. A CL/RAMP1 co-transfection experiment supported this hypothesis.

CGRP/AM on the outside of the cell and did not simply act as anchoring/chaperone proteins for CL. RAMPs therefore provide a novel mechanism for modulating receptor–ligand specificity. The unique pharmacological profiles supported by RAMPs are discussed in later sections.

Fig. (not shown).  CGRP1 receptor-specific small molecule antagonists. The small molecule antagonist BIBN4096 BS (brown) is a specific antagonist of the CGRP1 receptor, acting at the interface between RAMP1 and the CL receptor to inhibit CGRP action. At least part of the binding affinity for BIBN4096 BS arises from interaction with Trp74 (red) of RAMP1. In contrast, antagonists that bind principally to the CL component of the complex will not discriminate between different CL/RAMP complexes.

The classic function attributed to RAMPs is their ability to switch the pharmacology of CL, thus providing a novel mechanism for modulating receptor specificity. Thus, the CL/RAMP1 complex is a high affinity CGRP receptor, but in the presence of RAMP2, CL specificity is radically altered, the related peptide AM being recognized with the highest affinity and the affinity for CGRP being reduced ¨100-fold. While AM is the highest affinity peptide, CGRP is recognized with moderate, rather than low affinity. Indeed, depending on the species and the form of CGRP (h vs. a), the separation between the 2 peptides can be as little as 10-fold (Hay et al., 2003a). This may particularly be true if receptor components of mixed species are used. The detailed pharmacology of the CGRP and AM receptors formed by RAMP interaction with CL has recently been reviewed (Born et al., 2002; Poyner et al., 2002; Hay et al., 2004; Kuwasako et al., 2004).

Fig. (not shown). The broadening spectrum of RAMP–receptor interactions. RAMPs can interact with multiple receptor partners. All RAMPs interact with the calcitonin receptor-like receptor (CL-R), the calcitonin receptor (CTR), and the VPAC1 receptor, while the glucagon and PTH1 receptors interact with RAMP2, the PTH2 receptor with RAMP3, and the calcium sensing receptor (CalS-R) with RAMP1 or RAMP3. The consequence of RAMP interaction varies. For the CL and CalS receptors, RAMPs play a chaperone role, allowing cell surface expression. For the CL and calcitonin receptors, RAMP interaction leads to novel receptor binding phenotypes . There is also evidence that RAMP interaction will modify signaling, and this has been seen for the VPAC1–RAMP2 heterodimer and for calcitonin receptor/RAMP complexes. In many instances, however, the consequence of RAMP interaction has yet to be defined.

Overall, the distribution data presented so far are supportive of the hypothesis that RAMP and CL or calcitonin receptor combinations are able to account for the observed CGRP, AM, and AMY pharmacology. A salient point for CGRP receptors relates to the cerebellum, where the lack of CL mRNA in some studies despite abundant CGRP binding has prompted speculation of alternative CGRP receptors (Oliver et al., 2001; Chauhan et al., 2003). Nevertheless, this apparent lack is study dependent and CL has been identified in cerebellum in other studies.

Some consideration has been given to the potential role that RAMPs may have in modifying receptor behaviors other than ligand binding pharmacology. An additional functional consequence might be that of alteration of receptor signaling characteristics.

While there is currently little evidence for signaling modifications of CL-based receptors in association with RAMPs, a completely different paradigm is evident for the VPAC1 receptor. This receptor has strong interactions with all 3 RAMPs, but its pharmacology, in terms of agonist binding, does not appear to be modified by their presence. On the other hand, there was a clear functional consequence of RAMP2 overexpression with the VPAC1 receptor where PI hydrolysis was specifically augmented relative to cAMP, which did not change. The potency of the response (EC50 of vasoactive intestinal peptide) was not altered, but the maximal PI hydrolysis response was elevated in the presence of RAMP2 . It has been suggested that this may reflect a change in compartmentalization of the receptor signaling complex. Such augmentation was not evident for the interaction of the VPAC1 receptor with RAMP1 or RAMP3; in these cases, the outcome of heterodimerization may be more subtle or involve the modification of different receptor parameters such as trafficking.

RAMPs transformed our understanding of how receptor pharmacology can be modulated and provided a novel mechanism for generating receptor subtypes within a subset of family B GPCRs. Their role has now broadened and they have been shown to interact with several other family B GPCRs, in 1 case modifying signaling parameters. There is now evidence to suggest that their interactions also reach into family C, and possibly family A, GPCRs, indicating that their function may not be restricted to modulation of a highly specific subset of receptors. Indeed, many aspects of RAMP function remain poorly understood, and the full extent of their action remains to be explored.

Receptor activity modifying proteins

Patrick M. Sexton, Anthony Albiston, Maria Morfis, Nanda Tilakaratne
Cellular Signalling 13 (2001) 73-83  PII: S0898-6568(00)00143-1

Our understanding of G protein-coupled receptor (GPCR) function has recently expanded to encompass novel protein interactions that underlie both cell-surface receptor expression and the exhibited phenotype. The most notable examples are those involving receptor activity modifying proteins (RAMPs). RAMP association with the calcitonin (CT) receptor-like receptor (CRLR) traffics this receptor to the cell surface where individual RAMPs dictate the expression of unique phenotypes. A similar function has been ascribed to RAMP interaction with the CT receptor (CTR) gene product. This review examines
our current state of knowledge of the mechanisms underlying RAMP function.

It is now evident that RAMPs can interact with receptors other than CRLR. Expression of amylin receptor phenotypes requires the coexpression of
RAMPs with the CTR gene product. However, as seen in CRLR, the phenotype engendered by individual RAMPs was distinct. In COS-7 or rabbit aortic endothelial cells (RAECs), RAMP1 and RAMP3 induced amylin receptors that differ in their affinity for CGRP, while RAMP2 was relatively ineffective in inducing amylin receptor phenotype. RAMP2 can also induce an amylin receptor phenotype, which is distinct from either the RAMP1- or RAMP3-induced receptors. However, the efficacy of RAMP2 was highly dependent upon the cellular background and the isoform of CTR used in the study.

In humans, the major CTR variants differ by the presence or absence of a 16 amino acid insert in the first intracellular domain, with the insert negative isoform (hCTRI1ÿ) being the most commonly expressed form and the variant used for initial studies with RAMPs. Unlike hCTRI1ÿ, cotransfection of the hCTRI1+ variant with any of the RAMPs into COS-7 cells caused strong induction of amylin receptor phenotype. The hCTR isoforms differ in their ability to activate signaling pathways (presumably due to an effect on G protein coupling) and to internalize in response to agonist treatment, which may suggest a role for G proteins in the ability of RAMPs to alter receptor phenotype.

There are at least three potential consequences of RAMP interaction with its associating receptors. The first is trafficking of receptor protein from an intracellular compartment to the cell surface. The second is an alteration in
the terminal glycosylation of the receptor, and the third is alteration of receptor phenotype, presumably through a direct or indirect effect on the ligand-binding site.

potential actions of RAMPs

potential actions of RAMPs

Schematic diagram illustrating potential actions of RAMPs. (A) RAMPs facilitate the trafficking of CRLR from an intracellular compartment to the cell surface. (B) RAMP1 (but not RAMP2 or RAMP3) modifies the terminal glycosylation
of CRLR. (C) The cell surface RAMP1±CRLR complex is a Type 1 CGRP receptor, which displays a 1:1 stoichiometry. (D,E) Cell surface RAMP2±CRLR and  RAMP3±CRLR complexes are adrenomedullin receptors. (F,G) For at least RAMP1 and RAMP3, RAMPs form stable homodimers, although the function
of these complexes is unknown. (H) Unlike CRLR, the CTR gene product is trafficked to the cell surface in the absence of RAMPs, where it displays classical CTR phenotype. (I,J) RAMP1± and RAMP3±CTR complexes form distinct amylin receptors. RAMP2 can also generate a separate amylin receptor phenotype (not illustrated). (C ±E,I,J) RAMPs are trafficked with either receptor to the plasma membrane. (K) For all three RAMP±CRLR complexes, agonist treatment causes clathrin-mediated internalization of both CRLR and RAMP.
(L) The majority of the internalized complex is targeted to the lysosomal-degradation pathway.

The data from Zumpe et al. suggest that RAMP2 interacts more weakly with the hCTRI1ÿ than RAMP1, and that the affinity of this interaction derives principally from the transmembrane domain/C-terminus (Ct) of the RAMPs. As RAMP3 induces an amylin receptor phenotype in COS-7 cells where RAMP2 is relatively weak, it is inferred that RAMP3 interaction with the hCTRI1ÿ is probably greater than that of RAMP2. Nonetheless, this has not been examined empirically. Given the recent data suggesting a potential role for G protein coupling in expression of RAMP-induced phenotype, it is also possible that the strength of RAMP interaction is, at least partially, dictated by receptor-G protein or RAMP-G protein interaction.

The discovery of RAMPs has led to a greater understanding of the nature of receptor diversity. However, although much progress has been made into elucidating the molecular mechanism of RAMP action, emerging data continue to open up new areas for investigation. These include identification of other RAMP-interacting receptors, understanding of the role of specific G proteins in RAMP-receptor function and the potential importance of RAMP regulation in disease progression. It also seems likely that the RAMP-receptor interface can provide a useful target for future drug development.

Cardiovascular endothelins: Essential regulators of cardiovascular homeostasis

Friedrich Brunner, C Bras-Silva, AS Cerdeira, AF Leite-Moreira
Pharmacology & Therapeutics 111 (2006) 508 – 531

The endothelin (ET) system consists of 3 ET isopeptides, several isoforms of activating peptidases, and 2 G-protein-coupled receptors, ETA and ETB, that are linked to multiple signaling pathways. In the cardiovascular system, the components of the ET family are expressed in several tissues, notably the vascular endothelium, smooth muscle cells, and cardiomyocytes. There is general agreement that ETs play important physiological roles in the regulation of normal cardiovascular function, and excessive generation of ET isopeptides has been linked to major cardiovascular pathologies, including hypertension and heart failure. However, several recent clinical trials with ET receptor antagonists were disappointing.

In the present review, the authors take the stance that ETs are mainly and foremost essential regulators of cardiovascular function, hence that antagonizing normal ET actions, even in patients, will potentially do more harm than good. To support this notion, we describe the predominant roles of ETs in blood vessels, which are (indirect) vasodilatation and ET clearance from plasma and interstitial spaces, against the background of the subcellular mechanisms mediating these effects. Furthermore, important roles of ETs in regulating and adapting heart functions to different needs are addressed, including recent progress in understanding the effects of ETs on diastolic function, adaptations to changes in preload, and the interactions between endocardial-derived ET-1 and myocardial pump function. Finally, the potential dangers (and gains) resulting from the suppression of excessive generation or activity of ETs occurring in some cardiovascular pathological states, such as hypertension, myocardial ischemia, and heart failure, are discussed.

Figure (not shown):  Synthesis of ET and its regulation. The release of active ET-1 is controlled via regulation of gene transcription and/or endothelin converting enzyme activity. ET-1 synthesis is stimulated by several factors, of which hypoxia seems to be the most potent in humans (see text). ET-1 formation is down-regulated by activators of the NO/cGMP pathway and other factors.

Figure (not shown): Vascular actions of ET. In healthy blood vessels, the main action of ET-1 is indirect vasodilatation mediated by ETB receptors located on endothelial cells. Their activation generates a Ca2+ signal via PLC that turns on the generation of NO, prostacyclin, adrenomedullin, and other mediators that are powerful relaxants of smooth muscle. On the other hand, binding of ET-1 to ETA receptors located on smooth muscle cells will lead to vascular contraction (physiological effect) and/or wall thickening, inflammation, and tissue remodeling (pathological effects). These latter effects may partly be mediated by vascular ETB2 receptors in certain disease states. Smooth muscle cell signaling involves DAG formation, PKC activation, and extracellular Ca2+ recruited via different cation channels. The specificity of the cellular response resides at the level of G proteins, that is, G-as or G-aq in the case of ETA, G-ai or G-aq for ETB.

signal transduction mechanisms involved in ET-1-mediated positive (left) and negative (right) inotropic effects

signal transduction mechanisms involved in ET-1-mediated positive (left) and negative (right) inotropic effects

Summary of proposed signal transduction mechanisms involved in ET-1-mediated positive (left) and negative (right) inotropic effects. Left: Stimulation of ETA receptors causes Gq protein-directed activation of PLC, formation of IP3 and DAG, and activation of NHE-1. Increased contractile force is the result of (i) Ca2+ release from the sarco(endo)plasmic reticulum, (ii) sensitization of cardiac myofilaments to Ca2+ due to cellular alkalosis, and (iii) increased Ca2+ influx through the NCX operating in reverse mode. The contribution of voltage-gated L-type Ca2+ channels to the systolic Ca2+ transient is unknown, as is the role of myocyte ETB2 receptors. Right: The ET receptor subtypes mediating negative inotropic effects are poorly known. Two main signaling mechanisms involve (i) inhibition of adenylyl cyclase (AC), guided by a G protein, of unknown binding preference, which results in decreased levels of cAMP; (ii) cGMP-mediated activation of phosphatases that dephosphorylate putative targets resulting from cAMP/protein kinase A (PKA) activation. Other kinases like PKC and PKG have also been implicated in accentuated force antagonism.

Adrenomedullin (11–26): a novel endogenous hypertensive peptide isolated from bovine adrenal medulla

Kazuo Kitamuraa,*, Eizaburo Matsuia, Jhoji Katoa, Fumi Katoha
Peptides 22 (2001) 1713–1718 PII: S0196-9781(01)00529-0

Adrenomedullin (AM) is a potent hypotensive peptide originally isolated from pheochromocytoma tissue. Both the ring structure and the C-terminal amide structure of AM are essential for its hypotensive activity. We have developed an RIA which recognizes the ring structure of human AM. Using this RIA, we have characterized the molecular form of AM in bovine adrenal medulla. Gel filtration chromatography revealed that three major peaks of immunoreactive AM existed in the adrenal medulla. The peptide corresponding to Mr 1500 Da was further purified to homogeneity. The peptide was determined to be AM (11–26) which has one intramolecular disulfide bond. Amino acid sequences of bovine AM and its precursor were deduced from the analyses of cDNA encoding bovine AM precursor. The synthetic AM (11–26) produced dose-dependent strong pressor responses in unanesthetized rats in vivo. The hypertensive activity lasted about one minute, and a dose dependent increase in heart rate was also observed. The present data indicate that AM (11–26) is a major component of immunoreactive AM in bovine adrenal medulla and shows pressor activity.

The pressor effect of AM(11–26) was examined by methods similar to those reported for Neuropeptide Y.

We have established a sensitive RIA system using a monoclonal antibody which recognizes the ring structure of human AM. Human AM antiserum recognized the peptide with high affinity at a final dilution of 1:2,800,000. The half maximal inhibition of radioiodinated ligand binding by human AM was observed at 10 fmol/tube. From 1 to 128 fmol/tube of AM was measurable by this RIA system. The intra- and inter-assay coefficients of variance were less than 6% and 9%, respectively. This RIA had 100% cross-reactivity with human AM(13–31), (1–25), (1–52)Gly and AM(1–52)CONH2, but less than 1% cross-reactivity with rat AM.

Sephadex G-50 gel-filtration of strongly basic peptide extract (SP-III) in bovine adrenal medulla identified three major peaks of immunoreactive AM. One emerged at the identical position of authentic AM, the other two unknown peaks were eluted later at molecular weights estimated to be 3000 and 1500 Da, respectively. The peptide corresponding to Mr 1500 Da was further purified.

The purified peptide (20 pmol) was subjected to a gas phase sequencer, and the amino acid sequence was determined up to the 16th residue, which was found to be C terminus . It was found that the purified peptide was AM (11–26). The structure of AM (11–26) was confirmed by chromatographic comparison with native AM (11–26) as well as a synthetic AM (11–26), which has one intramolecular disulfide bond.

3 clones were isolated, and the clone designated pBAM-2, which harbored the longest insert of 1,438 base, was used for sequencing. The bovine AM cDNA contained a single open reading frame encoding a putative 188 amino acid polypeptide. The first 21-residue peptide is thought to be a signal peptide. The bovine AM propeptide contains three signals of dibasic amino acid sequences, Lys-Arg or Arg-Arg. The first Lys-Arg followed proadrenomedullin N-terminal 20 peptide (PAMP) sequences. AM is located between the second signal of Lys-Arg and the third signal of Arg-Arg. Gly residues, which are donors of C-terminal amide structure of PAMP and AM, are found before the first and third signal of Lys-Arg and Arg-Arg. Bovine AM consists of 52 amino acids and is identical to human AM with exception of four amino acids. Bovine PAMP consists of 20 amino acids and is identical to human PAMP with exception of one amino acid. The present cDNA sequence encoding bovine AM precursor is almost identical to those of the reported AM cDNA sequences from bovine aortic endothelial cells. However, a difference in one amino acid was found in the sequences of signal peptide. In addition, three different residues of nucleotides were found in the noncoding region of cDNA encoding bovine preproadreno-medullin.

AM(11–26) elicited a potent hypertensive effects in unanesthetized rats.
When AM(11–26) at 20 nmol/kg was injected i.v., the maximum increase of mean blood pressure was 50  7.1 mmHg. Similarly, the synthetic AM(11–26) produced dose-dependent strong pressor responses in unanesthetized rats in vivo. (Blood pressure increase; F(3, 20 = 13.845, P < 0.0001). Injection of saline did not affects blood pressure and heart rate. The hypertensive activity lasted about 70 s, and a dose dependent increase of heart rate was also observed (Heart rate increase; F(3, 20) = 6.151, P = 0.0039).

We have isolated and characterized bovine AM(11–26) from bovine adrenal medulla as an endogenous peptide. The hallmark biological effects of AM are vasodilation and hypotensive effects in the vascular systems of most species. The mature form of AM has one ring structure formed by an intramolecular disulfide bond and a C terminal amide structure, both of which are essential for the hypotensive and other biological activities of AM. Watanabe et al. reported that the synthetic N-terminal fragment of human AM, AM (1–25)COOH and other related peptides, show vasopressor activity in anesthetized rats. The present purification and characterization of AM(11–26) indicate that the ring structure of AM may function as a biologically active endogenous peptide. The peptide corresponding to Mr 1,500 Da was further purified to homogeneity.

The purified peptide was found to be AM(11–26) which has one intramolecular disulfide bond. The structure of AM(11–26) was confirmed by chromatographic comparison with native AM(11–26) as well as a synthetic specimen which was prepared according to the determined sequence. The structure of bovine AM and related peptides were determined by cDNA analysis encoding bovine AM. Bovine AM consists of 52 amino acids whose sequence is identical to the human sequences with the exception of four amino acids. Furthermore, according to the cDNA analysis and chromatographic comparison of the synthetic AM(11–26) and purified AM, is now determined to be cystine. It should be noted that the structure of bovine AM(11–26) is identical to human AM(11–26).

It is well known that many peptide hormones and neuropeptides are processed from larger, biologically inactive precursors by the specific processing enzyme. It usually recognizes pairs of basic amino acids, processing signals, such as primarily Lys-Arg and Arg-Arg. AM (11–26) is not flanked by such a processing signal, but it was reproducibly observed in bovine adrenal medulla peptide extract. The molar ratio of AM(11–26)/AM was estimated to be 40%. The ratio varied from 5% to 50% according to the individual specimen, but the minor peak corresponding to 1,500 Da was reproducibly observed, suggesting that AM(11–26) is an endogenous peptide. It is likely that AM(11–26) is biosynthesized from AM or AM precursor by a specific enzyme.

In contrast to AM, synthetic bovine AM(11–26) caused potent hypertensive effects in unanesthetized rats. The hypertensive activity of AM(11–26) seems to be comparable to that of AM(1–25) as reported by Watanabe et al.  It was unexpected that AM(11–26) would cause a dose dependent increase of heart rate in unanesthetized rats because vasopressor activity normally causes bradycardia through baroreceptor activation. The hypertensive mechanism is not fully understood, but it has been reported that the vasopressor effect of AM(1–25) might be caused by the release of endogenous catecholamine. We speculate that the released catecholamine counters the baroreceptor function resulting in an increased heart rate and blood pressure. It is possible that AM(11–26) participates in blood pressure control as an endogenous peptide.

A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and vasodilating peptides.

Tanenao Eto
Peptides 22 (2001) 1693–1711 PII: S0196-9781(01)00513-7

Adrenomedullin (AM), identified from pheochromocytoma and having 52 amino acids, elicits a long-lasting vasodilatation and diuresis. AM is mainly mediated by the intracellular adenylate cyclase coupled with cyclic adenosine monophosphate (cAMP) and nitric oxide (NO) -cyclic guanosine monophosphate (cGMP) pathway through its specific receptor. The calcitonin receptor-like receptor (CLCR) and receptor-activity modifying protein (RAMP) 2 or RAMP3 models have been proposed as the candidate receptor. AM is produced mainly in cardiovascular tissues in response to stimuli such as shear stress and stretch, hormonal factors and cytokines. Recently established AM knockout mice lines revealed that AM is essential for development of vitelline vessels of embryo. Plasma AM levels elevate in cardiovascular diseases such as heart failure, hypertension and septic shock, where AM may play protective roles through its characteristic biological activities. Human AM gene delivery improves hypertension, renal function, cardiac hypertrophy and nephrosclerosis in the hypertensive rats. AM decreases cardiac preload and afterload and improves cardiac contractility and diuresis in patients with heart failure and hypertension. Advances in gene engineering and receptor studies may contribute to further understandings of biological implication and therapeutic availability of AM.

AM acts as a circulating hormone as well as elicits multiple biological activities in a paracrine or autocrine manner. Among them the most characteristic biological activity of AM is a very powerful hypotensive activity caused by dilatation of resistance vessels. A sensitive and specific radioimmunoassay demonstrated that AM circulates in blood and occurs in a variety of tissues. Plasma AM levels elevate in various diseases including cardiovascular and renal disorders or septic shock. Thus, AM may be involved in pathophysiological processes in these diseases, especially in disorders controlling circulation and body fluid. In this short review, the history of AM and proadrenomedullin N-terminal 20 peptide (PAMP) will be reviewed with special references to biological properties and function, receptors, gene engineering and clinical viewpoints. This review includes oral presentations from the aforementioned symposium; some of which have not yet been published. These unpublished oral presentations are quoted in this paper from the abstracts of this symposium.

Preproadrenomedullin, which consists of 185 amino acids and contains a 21-amino acid signal peptide, is processed to synthesize proadrenomedullin and finally AM. In the proadrenomedullin, a unique twenty amino acid sequence followed by a typical amidation signal known as Gly-Lys-Arg, is included in the N-terminal region. This novel 20 residues peptide with carboxyl terminus of Arg-CONH2 is also present in vivo and is termed “proadrenomedullin N-terminal 20 peptide (PAMP).” PAMP elicits a potent hypotensive activity in anesthetized rats.

Although widely distributed in the adenophypophysis and the neural lobe of pituitary glands, AM and PAMP occur in cell-specific, but not overlapping, patterns in the anterior pituitary. This cell-specific expression of each peptide may be explained by differences in posttranslational processing of AM gene. As such, potential pituitary specific transcription factor binding sites, gonadotropic-specific element (GSE) and a binding site for steroidogenic factor-l (SF-1) are found in the 5flanking region of human and mouse AM gene.  SF-1 is a member of the steroid receptor superfamily that has been shown necessary for gonadotrope differentiation within the pituitary. In addition, one putative binding sequence of Pit-1 has been reported in mouse AM gene promoter position.

A specific AM binding protein (AMBP-1) in human plasma was isolated and the purified protein was identified as human complement factor H. AM and factor H interaction may interfere with the radioimmunoassay quantification of circulating AM. Factor H enhances AM-mediated induction of cAMP in fibroblast; augments the AM-mediated growth of a cancer cell line; and suppresses the bactericidal capability of AM on Escherichia coli. Conversely, AM influences the complement regulatory function of factor H by enhancing the cleavage of C3b via factor I. The augmentation of AM actions indicates that AMBP may facilitate the binding of AM to its receptor. In addition, the existence of AMBP suggests that large amounts of AM may circulate bound to this plasma protein.

In rat vascular smooth muscle cells, the CGRP, CGRP1 receptor antagonist, competitively inhibits the intracellular accumulation of cAMP induced by AM. Vasodilation of the rat mesenteric vascular bed elicited by AM and CGRP is also blocked by CGRP. Similar effects of CGRP are observed in the isolated rat heart and its microvasculature. Thus, CGRP1 receptor can mediate some effects of AM, but AM has a low affinity at CGRP2 receptor. Two distinct AM labeled bands with a molecular weight of 120 and 70 kDa was reported in the cultured rat vascular smooth muscle cell membrane. Therefore, the binding specificity and characteristics of the AM receptor may differ regionally by organ or tissue.

Two more RAMP proteins, RAMP2 and RAMP3, were discovered from database searches. These proteins share approximately 30% homology with RAMP1. Co-expression of RAMP2 or RAMP3 with CRLR appears to constitute AM receptor. RAMP2 and RAMP3 are indistinguishable in terms of AM binding. The RAMPs are required to transport CRLR to the plasma membrane. RAMP1 presents CRLR as a mature glycoprotein at the cell surface to form a CGRP receptor. However, receptors transported by RAMP2 or RAMP3 are core glycosylated and then become AM receptors. Three putative N-glycosylation sites Asn 60, Asn 112 and Asn 117 are present in the amino-terminal extracellular domain of the human CRLR. When the glycosylation of a myc-tagged CRLR was inhibited, specific 125I-CGRP and -AM binding were blocked in parallel. Substitution of the Asn 117 by threonine abolished CGRP and AM binding in the face of intact N-glycosylation and cell surface expression. RAMPs are accessory proteins of CTR and CRLR at the cell surface where they define AM, amylin, calcitonin and CGRP specificity.

The receptor component protein (RCP) was cloned on the basis of its ability to potentiate the endogenous Xenopus oocyte CGRP receptor. RCP is a cytosolic protein with no similarity to RAMPs, consists of a hydrophobic 146 amino acids and is obtained from the Corti organ of guinea pig. RCF plays an essential role for signal-transduction of CGRP and AM, and interacts with CRLR directly within the cells. Thus, a functional AM or CGRP receptor seems to consist of at least three proteins: CRLR, RAMP and RCP, coupling the receptor to the intracellular signal-transduction pathway.

By using a chimera of the CRLR and green fluorescent protein (GFP), the study demonstrated that CRLR-GFP failed to generate responses to CGRP or AM without RAMP2 or RAMP3 in HEK 293 cells. When coexpressed with RAMP2 or RAMP3, CRLR-GFP appeared on the cell membrane and activated an intracellular cAMP production and calcium mobilization. Agonist-mediated internalization of CRLR-GFP was observed in RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM, which occurred with similar kinetics, indicating the existence of ligand-specific regulation of CRLR internalization by RAMPs.

The discovery of RAMPs has promoted our understandingthat some of the biological activities of AM are blocked by CGRP receptor antagonist, whereas other biological activities are blocked only by AM receptor antagonist, which indicates the possible existence of AM receptor in dual nature. RAMP association with CRLR traffics this receptor to the cell surface where individual RAMPs dictate the expression of unique phenotypes such as CGRP receptor or AM receptors. Apart from receptor trafficking and glycosylation, the RAMPs may interact directly with the receptors in the cell surface modifying their affinities for the ligands.

Since AM was discovered by monitoring the elevating activity of cAMP in rat platelets, cAMP appears to be its major second messenger. Dose-dependent intracellular production of cAMP induced by AM has been confirmed in various tissues and cells. Moreover, information on the role of NO in alternative signal-transduction pathways for AM is available.

The vasodilating effect of AM is reduced by the blockade of NO synthetase activity with NG-nitro-L-arginine methylester (L-NAME), indicating that NO may at least partly contribute to the AM-induced vasodilation. However, the degree of NO contribution to vasodilation varies depending upon the organ or tissue and the species. NO synthetase inhibitor in the pulmonary vascular beds of rat significantly attenuates the AM-induced vasodilation, but it does not occur in cats. Thus, NO seems to be an important AM mediator despite regional and interspecies variation.

In bovine aortic endothelial cells, AM increases intracellular ionic calcium (Ca2+) and causes the accumulation of cAMP. This increase in intracellular Ca2+ may be involved in the activation of phospholipase C, thereby producing inducible NO synthetase and subsequently NO. NO transferred to medial smooth muscle cells may activate cGMP-mediating smooth muscle cells vasodilatation. In contrast, AM lowers both cytosolic Ca2+ and Ca2+ sensitivity in smooth muscle cells of pig coronary arteries and intracellular Ca2+ in rat renal arterial smooth muscle cells.

Among the multi-functional properties of AM, the most characteristic one is an intensive, long-lasting hypotension that is dose-dependent in humans, rats, rabbits, dogs, cats and sheep. AM dilates resistance vessels in the kidneys, brain, lung, hindlimbs in animals as well as in the mesentery. Moreover, AM elicits relaxation of ring preparations of the aorta and cerebral arteries. An i.v. injection of human AM to conscious sheep causes a dose dependent fall of blood pressure, an increase in heart rate and cardiac output with a small reduction in stroke volume, as well as a marked decrease in total peripheral resistance. Coronary blood flow increases in parallel with the increase in coronary conductance. These cardiovascular responses return to the control level by 40 min after the injection.

The low-dose infusion of AM administered to conscious sheep on a low-salt diet antagonizes the vasopressor actions of administered angiotensin II while stimulating cardiac output and heart rate. AM may control cardiovascular homeostasis in part through antagonism of the vasopressor action of angiotensin II. AM inhibits the secretion of endothelin-1 from the vascular endothelial cells and proliferation of vascular smooth muscle cells. In the cultured cardiomyocytes as well as cardiac fibroblasts, AM inhibits protein synthesis in these cells in an autocrine or a paracrine manner, which may result in modulating the cardiac growth. AM inhibits bronchial constriction induced by acetylcholine or histamine in a dose-dependent  manner, indicating the important role of AM on airway function and its usefulness for the management of bronchial asthma. AM inhibits secretion of aldosterone from the adrenal cortex. When infused directly into the adrenal arterial supply of conscious sheep, AM directly inhibits the acute stimulation of aldosterone by angiotensin II,  KCl and ACTH while not affecting basal or chronic aldosterone secretion or cortisol secretion stimulated by ACTH. AM co-exists in insulin-producing cells and it inhibits insulin secretion dose-dependently in isolated rat islets.

The N-terminal region of preproadrenomedullin, the precursor of AM, contains a unique 20-residue sequence followed by Gly-Lys-Arg, a typical amidation signal, which was termed as proadrenomedullin N-terminal 20 peptide (PAMP). PAMP was purified from porcine adrenal medulla and human pheochromo-cytoma by using radioimmunoassay for the peptide and its complete amino acid sequence was determined. In addition to the original form of PAMP [1–20], PAMP [9–20] has recently been purified from the bovine adrenal medulla. The amino acid sequences of both forms of PAMP are identical to amino acid sequences deduced by cDNA analysis and their carboxyl terminus of Arg is amidated. The distribution of PAMP is similar to that of human AM, due to the fact that PAMP as well as human AM is biosynthesized from an AM precursor.

AM is processed from its precursor, proadrenomedullin, as the intermediate or immature form, AM-glycine (AM[1–52]-COOH, immature AM). Subsequently, immature AM is converted to the biologically active mature form, AM [1–52]-CONH2 (mature AM) by enzymatic amidation. The AM circulating in the human blood stream (total AM), thus, consists of both mature AM and immature AM. In earlier studies, plasma AM levels were measured by using radioimmunoassay recognizing the entire AM molecule (AM [1–52]), which reflects plasma total AM levels, as previously described.

In healthy volunteers severe exercise elevates the plasma AM levels with an increase in plasma norepinephrine and exaggerated sympathetic nerve activity. In heart transplant recipients, maximal exercise induces an increase in plasma AM that is inversely related to mean blood pressure. AM, therefore, may participate in blood pressure regulation during exercise even after heart transplantation.

When compared with healthy controls, the plasma AM levels are increased in patients with a variety of diseases: congestive heart failure, myocardial infarction, renal diseases, hypertensive diseases, diabetes mellitus, acute phase of stroke, and septic shock.

Adrenomedullin and central cardiovascular regulation

Meghan M. Taylor, Willis K. Samson
Peptides 22 (2001) 1803–1807 PII: S0196-9781(01)00522-8

Adrenomedullin gene products have been localized to neurons in brain that innervate sites known to be important in the regulation of cardiovascular function. Those sites also have been demonstrated to possess receptors for the peptide and central administrations of adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) elevate blood pressure and heart rate in both conscious and anesthetized animals. The accumulated evidence points to a role of the sympathetic nervous system in these cardiovascular effects. These sympathostimulatory actions of AM and PAMP have been hypothesized to be cardioprotective in nature and to reflect the central nervous system (CNS) equivalent of the direct cardiostimulatory effects of the peptides in the periphery. This review summarizes the most recent data on the CNS actions of the adrenomedullin gene-derived peptides and suggests future strategies for the elucidation of the physiologic relevance of the already demonstrated, pharmacologic actions of these peptides.

Adrenomedullin and related peptides: receptors and accessory proteins

Roman Muff, Walter Born, Jan A. Fischer
Peptides 22 (2001) 1765–1772  PII: S0196-9781(01)00515-0
Adrenomedullin (AM), α- and β-calcitonin gene-related peptide (CGRP), amylin and calcitonin (CT) are structurally and functionally related peptides. The structure of a receptor for CT (CTR) was elucidated in 1991 through molecular cloning, but the structures of the receptors for the other three peptides had yet to be elucidated. The discovery of receptor-activity-modifying proteins (RAMP) 1 and -2 and their co-expression with an orphan receptor, calcitonin receptor-like receptor (CRLR) has led to the elucidation of functional CGRP and AM receptors, respectively. RAMP1 and -3 which are co-expressed with CTR revealed two amylin receptor isotypes. Molecular interactions between CRLR and RAMPs are involved in their transport to the cell surface. Heterodimeric complexes between CRLR or CTR and RAMPs are required for ligand recognition.

Pharmacological profiles of receptors of the adrenomedullin peptidefamily
AMR AM>CGRP>>amylin=CT
CTR CT>amylin>>CGRP=AM
AmylinR AmylinsCT­CGRP>>hCT>AM

Specific AM binding sites have been identified in many tissues including the heart, blood vessels, lung and spleen. Based on pharmacological evidence two receptor isotypes have been distinguished, for instance in rat astrocytes and NG108–15 cells. One AM receptor isotype recognizes CGRP and CGRP(8–37). The other receptor isotype specific for the AM ligand and antagonized by AM(22–52) does not recognize CGRP to any great extent. Both isotypes of the receptors have been shown to interact poorly with amylin and CT (Table). Biological actions of AM include vaso- and bronchodilation, and CNS transmitted inhibition of water intake.

CGRP receptors are widely distributed in the nervous and cardiovascular systems. To date, two isotypes have been described. On pharmacological evidence, CGRP1 receptors, such as those identified in human SK-N-MC neuroblastoma cells, recognize intact CGRP and CGRP(8–37) with similar potency, unlike a linear analog lacking the disulfide bridge. CGRP2 receptors,
on the other hand, interact with the linear analog but not with CGRP(8–37). These CGRP receptor isotypes cross-react with AM to some extent, but only minimally with amylin and CT. CGRP shares potent vasodilatory actions with AM, and has chronotropic and inotropic actions in the heart. The ionotropic actions are indirectly brought about via activation of the sympathetic nervous system. There is evidence to suggest the existence of α- or β-CGRP preferring receptor isotypes in both the central nervous system and peripheral tissues.

RAMP1, -2 and -3 are widely expressed, suggesting that RAMPs may have
important functions beyond those of the adrenomedullin family of receptors. To this end, RAMP1 and -3 are thought to reduce cell surface expression of angiotensin (AT) AT1 and AT2 receptors.

RAMP2 and CRLR are expressed in vascular smooth muscle cells, and RAMP1 expression was increased by dexamethasone. Moreover, increased levels of RAMP2 and CRLR were observed in the kidney and heart of rats with obstructive nephropathy and congestive heart failure, respectively. RAMP2
and CRLR levels were reduced, and RAMP3 levels were increased during lipopolysaccharide induced sepsis in rats.

The GABAB receptor 1 is retained as an immature glycoprotein in the cytosol unless co-expressed with GABAB receptor 2 isotype. Heterodimers of fully functional opioid receptors δ and κ result in a novel receptor displaying binding and functional properties distinct from those of the δ or κ receptors alone. Heterodimerization therefore facilitates receptor expression and defines ligand specificity also in G protein-coupled receptor families A and C. Moreover, heterodimers of metabotropic glutamate 1receptor (family C) and adenosine A1 receptors (family A) have been observed. As yet there is no evidence for homo or heterodimerization of family B receptors. Cysteines conserved in the extracellular N-terminal domain in all the receptors of family B and RAMPs suggest that RAMPs are truncated forms of receptors that interact as heterodimers with CRLR and CTR.

The discovery of RAMPs in combination with CRLR and CTR has led to the molecular identification of CGRP1, CGRP/amylin, AM and amylin receptor complexes. The physiological advantage of heterodimers between seven transmembrane domain receptors and the RAMPs required for the functional expression of the adrenomedullin, CGRP and amylin receptors remains to be demonstrated.

Angiotensin II, From Vasoconstrictor to Growth Factor: A Paradigm Shift

Sasa Vukelic, Kathy K. Griendling
Circ Res. 2014;114:754-757

Angiotensin II (Ang II) is today considered as one of the essential factors in the pathophysiology of cardiovascular disease, producing acute hemodynamic and chronic pleiotropic effects. Although now it is widely accepted that these chronic effects are important, Ang II was initially considered only a short-acting, vasoactive hormone. This view was modified a quarter of a century ago when Dr Owens and his group published an article in Circulation Research with initial evidence that Ang II can act as a growth factor that regulates cell hypertrophy. They showed in a series of elegant experiments that Ang II promotes hypertrophy and hyperploidy of cultured rat aortic smooth muscle cells. However, Ang II had no effect on hyperplasia. These findings led to a paradigm shift in our understanding of the roles of growth factors and vasoactive substances in cardiovascular pathology and helped to redirect basic and clinical renin–angiotensin system research during the next 25 years. Ang II is now known to be a pleiotropic hormone that uses multiple signaling pathways to influence most processes that contribute to the development and progression of cardiovascular diseases, ranging from hypertrophy, endothelial dysfunction, cardiac remodeling, fibrosis, and inflammation to oxidative stress.

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Biomarkers and risk factors for cardiovascular events, endothelial dysfunction, and thromboembolic complications

Curator: Larry H Bernstein, MD, FCAP



Acute Coronary Syndrome

Predictive Cardiovascular and Circulation Biomarkers

Biomarkers are chemistry analytes measured in plasma, serum or whole blood that potentially identify injury or risk for injury.  They may be measured in the laboratory or at the bedside (point of care technology).  They may be measured as an enzyme (CK isoenzyme MB), a protein (troponins I & T), or as a micro RNA (miRNA).  In the last decade the discovery and use of cardiac biomarkers has moved toward very small quantities, even 100 times below the picogram range using Quanterix Simoa, compared with an enzyme immunoassay.

The time of sampling was based on time to appearance from time of damage, and the release of the biomarker is a stochastic process. The earliest studies of CK-MB appearance, peak height, and disappearance was by Burton Sobel and associates related to measuring the extent of damage, and determined that reperfusion had an effect.

There has been a nonlinear introduction of new biomarkers in that period, with an explosion of methods discovery and large studies to validate them in concert with clinical trials. The improvement of interventional methods, imaging methods, and the unraveling of patient characteristics associated with emerging cardiovascular disease is both cause for alarm (technology costs) and for raised expectations for both prevention, risk reduction, and treatment. What is strikingly missing is the kind of data analyses on the population database that could alleviate the burden of physician overload. It is an urgent requirement for the EHR, and it needs to be put in place to facilitate patient care.


Biomarkers: Diagnosis and Management, Present and Future

Curator: Larry H Bernstein, MD, FCAP
Biomarkers of Cardiovascular Disease : Molecular Basis and Practical Considerations.
RS Vasan .
Circulation. 2006;113:2335-2362. http://dx.doi.org/10.1161/CIRCULATIONAHA.104.482570

sCD40L indicates soluble CD40 ligand; Fbg, fibrinogen; FFA, free fatty acid; ICAM, intercellular adhesion molecule; IL, interleukin; IMA, ischemia modified albumin; MMP, matrix metalloproteinases; MPO, myeloperoxidase; Myg, myoglobin; NT-proBNP, N-terminal proBNP; Ox-LDL, oxidized low-density lipoprotein; PAI-1, plasminogen activator inhibitor; PAPP-A, pregnancy-associated plasma protein-A; PlGF, placental growth factor; TF, tissue factor; TNF, tumor necrosis factor; TNI, troponin I; TNT, troponin T; VCAM, vascular cell adhesion molecule; and VWF, von Willebrand factor.


Accurate Identification and Treatment of Emergent Cardiac Events  

Author: Larry H Bernstein, MD, FCAP

The main issue that we have a consensus agreement that PLAQUE RUPTURE is not the only basis for a cardiac ischemic event. The introduction of  high sensitivity troponin tests has made it no less difficult after throwing out the receiver-operator characteristic curve (ROC) and assuming that any amount of cardiac troponin released from the heart is pathognomonic of an acute ischemic event.  This has resulted in a consensus agreement that

  • ctn measurement at a coefficient of variant (CV) measurement in excess of 2 Std dev of the upper limit of normal is a “red flag” signaling AMI? or other cardiomyopathic disorder

This is the catch.  The ROC curve established AMI in ctn(s) that were accurate for NSTEMI – (and probably not needed with STEMI or new Q-wave, not previously seen) –

  1. ST-depression
  2. T-wave inversion
  3. in the presence of other findings
  • suspicious for AMI

Wouldn’t it be nice if it was like seeing a robin on your lawn after a harsh winter?  Life isn’t like that.  When acute illness hits the patient may well present with ambiguous findings.   We are accustomed to relying on

  • clinical history
  • family history
  • co-morbidities, eg., diabetes, obesity, limited activity?, diet?
  • stroke and/or peripheral vascular disease
  • hypertension and/or renal vascular disease
  • aortic atherosclerosis or valvular heart disease

these are evidence, and they make up syndromic classes

  • Electrocardiogram – 12 lead EKG (as above)
  • Laboratory tests
  • isoenzyme MB of creatine kinase (CK)… which declines after 12-18 hours
  • isoenzyme-1 of LD if the time of appearance is > day-1 after initial symptoms (no longer used)
  1. cardiac troponin cTnI or cTnT
  • genome testing
  • advanced analysis of EKG

This may result in more consults for cardiologists, but it lays the ground for better evaluation of the patient, in the long run.

Perspectives on the Value of Biomarkers in Acute Cardiac Care and Implications for Strategic Management
Antoine Kossaify, … STAR-P Consortium
Biomarker Insights 2013:8 115–126.

In addition to the conventional use of natriuretic peptides, cardiac troponin, and C-reactive protein, other biomarkers are outlined in variable critical conditions that may be related to acute cardiac illness. These include ST2 and chromogranin A in acute dyspnea and acute heart failure, matrix metalloproteinase in acute chest pain, heart-type fatty acid binding protein in acute coronary syndrome, CD40 ligand and interleukin-6 in acute myocardial infarction, blood ammonia and lactate in cardiac arrest, as well as tumor necrosis factor-alpha in atrial fibrillation. Endothelial dysfunction, oxidative stress and inflammation are involved in the physiopathology of most cardiac diseases, whether acute or chronic. In summary, natriuretic peptides, cardiac troponin, C-reactive protein are currently the most relevant biomarkers in acute cardiac care.

 Inverse Association between Cardiac Troponin-I and Soluble Receptor for Advanced Glycation End Products in Patients with Non-ST-Segment Elevation Myocardial Infarction

ED. McNair, CR. Wells, A.M. Qureshi, C Pearce, G Caspar-Bell, and K Prasad
Int J Angiol 2011;20:49–54

Interaction of advanced glycation end products (AGEs) with the receptor for advanced AGEs (RAGE) results in activation of nuclear factor kappa-B, release of cytokines, expression of adhesion molecules, and induction of oxidative stress. Oxygen radicals are involved in plaque rupture contributing to thromboembolism, resulting in acute coronary syndrome (ACS). Thromboembolism and the direct effect of oxygen radicals on myocardial cells cause cardiac damage that results in the release of cardiac troponin-I (cTnI) and other biochemical markers. The soluble RAGE (sRAGE) compete with RAGE for binding with AGE, thus functioning as a decoy and exerting a cytoprotective effect. Low levels of serum sRAGE would allow unopposed serum AGE availability for binding with RAGE, resulting in the generation of oxygen radicals and proinflammatory molecules that have deleterious consequences and promote myocardial damage. sRAGE may stabilize atherosclerotic plaques. It is hypothesized that low levels of sRAGE are associated with high levels of serum cTnI in patients with ACS.
The levels of cTnI were higher in NSTEMI patients (2.180.33 mg/mL) as compared with control subjects (0.0120.001 mg/mL). Serum sRAGE levels were negatively correlated with the levels of cTnI. In conclusion, the data suggest that low levels of serum sRAGE are associated with high serum levels of cTnI and that there is a negative correlation between sRAGE and cTnI.

Correlation of soluble receptor for advanced glycation end products (sRAGE) with cardiac troponin-I

Correlation of soluble receptor for advanced glycation end products (sRAGE) with cardiac troponin-I


Figure 1 Serum levels of soluble receptor for advanced glycation end products (sRAGE) in control subjects and in patients with non-ST-elevation myocardial infarction (NSTEMI). Results are expressed as meanstandard error. *p<0.05, control versus NSTEMI.


Serum levels of soluble receptor for advanced glycation end products

Serum levels of soluble receptor for advanced glycation end products

Figure 3 Correlation of soluble receptor for advanced glycation end products (sRAGE) with cardiac troponin-I (cTnI) in patients with non-ST-segment elevation myocardial infarction.


Heart Failure Complicating Non–ST-Segment Elevation Acute Coronary Syndrome

MC Bahit, RD. Lopes, RM. Clare, et al.
JACC: HtFail 2013; 1(3):223–9 .

This study sought to describe the occurrence and timing of heart failure (HF), associated clinical factors, and 30-day outcomes in patients with non–ST-segment elevation acute coronary syndromes (NSTE-ACS). Of 46,519 NSTE-ACS patients, 4,910 (10.6%) had HF at presentation. Of the 41,609 with no HF at presentation, 1,194 (2.9%) developed HF during hospitalization. A total of 40,415 (86.9%) had no HF at any time. Patients presenting with or developing HF during hospitalization were older, more often female, and had a higher risk of death at 30 days than patients without HF (adjusted odds ratio [OR]: 1.74; 95% confidence interval: 1.35 to 2.26). Older age, higher presenting heart rate, diabetes, prior myocardial infarction (MI), and enrolling MI were significantly associated with HF during hospitalization.

Other risk factors

Additive influence of genetic predisposition and conventional risk factors in the incidence of coronary heart disease: a population-based study in Greece
N Yiannakouris, M Katsoulis, A Trichopoulou, JM Ordovas, DTrichopoulos
BMJ Open 2014;4:e004387.

Genetic predisposition to CHD, operationalised through a multilocus GRS, and ConvRFs have essentially additive effects on CHD risk.

PTX3, A Prototypical Long Pentraxin, Is an Early Indicator of Acute Myocardial Infarction

G Peri, M Introna, D Corradi, G Iacuitti, S Signorini, et al.
Circulation. 2000;102:636-641

PTX3 is a long pentraxin whose expression is induced by cytokines in endothelial cells, mononuclear phagocytes, and myocardium. PTX3 is present in the intact myocardium, increases in the blood of patients with AMI, and disappears from damaged myocytes. We suggest that PTX3 is an early indicator of myocyte irreversible injury in ischemic cardiomyopathy.

Early release of glycogen phosphorylase inpatients with unstable angina and transient ST-T alterations

J Mair, B Puschendorf, J Smidt, P Lechleitner, F Dienstl, et al.
BrHeartJ 1994;72:125-127.

Glycogen phosphorylase BB (molecular weight 96000 kDa as a monomer) is the predominant isotype in human myocardium where it occurs alongside the MM subtype. The release of glycogen phosphorylase from injured myocardium may reflect the burst in glycogenolysis initiated during acute myocardial ischaemia. This is supported by a rapid increase in serum concentrations of glycogen phosphorylase BB in patients with acute myocardial infarction before concentrations of creatine kinase, creatine kinase MB, myoglobin, and cardiac troponin T increase. Unstable angina, however, ranges from no myocardial cell damage to non-Q wave myocardial infarction.
All variables except for creatine kinase and creatine kinase MB activities were significantly higher on admission in patients with unstable angina and transient ST-T alterations than in patients without. However, glycogen phosphorylase BB concentration was the only marker that was significantly (p = 0-0001) increased above its discriminator value in most patients.

Endothelium and Vascular

Endothelial Dysfunction: An Early Cardiovascular Risk Marker in Asymptomatic Obese Individuals with Prediabetes
AK. Gupta, E Ravussin, DL. Johannsen, AJ. Stull, WT. Cefalu and WD. Johnson
Br J Med Med Res 2012; 2(3): 413-423.

Adults with desirable weight [n=12] and overweight [n=8] state, had normal fasting plasma glucose [Mean(SD)]: FPG [91.1(4.5), 94.8(5.8) mg/dL], insulin [INS, 2.3(4.4), 3.1(4.8) μU/ml], insulin sensitivity by homeostasis model assessment [HOMA-IR, 0.62(1.2), 0.80(1.2)] and desirable resting clinic blood pressure [SBP/DBP, 118(12)/74(5), 118(13)/76(8) mmHg]. Obese adults [n=22] had prediabetes [FPG, 106.5(3.5) mg/dL], hyperinsulinemia [INS 18.0(5.2) μU/ml], insulin resistance [HOMA-IR 4.59(2.3)], prehypertension [PreHTN; SBP/DBP 127(13)/81(7) mmHg] and endothelial dysfunction [ED; reduced RHI 1.7(0.3) vs. 2.4(0.3); all p<0.05]. Age-adjusted RHI correlated with BMI [r=-0.53; p<0.001]; however, BMI-adjusted RHI was not correlated with age [r=-0.01; p=0.89].

Association of digital vascular function with cardiovascular risk factors: a population study.
T Kuznetsova, E Van Vlierberghe, J Knez, G Szczesny, L Thijs, et al.
BMJ Open 2014; 4:e004399.

Our study is the first to implement the new photoplethysmography (PPG) technique to measure digital pulse amplitude hyperemic in a sample of a general population. The correlates of hyperaemic response were as expected and constitute an internal validation of the PPG technique in assessment of digital vascular function.

Thrombotic/Embolic Events

Risk marker associations with venous thrombotic events: a cross-sectional analysis 
BA Golomb, VT Chan, JO Denenberg, S Koperski,  & MH Criqui.
BMJ Open 2014;4:e003208.

To examine the interrelations among, and risk marker associations for, superficial and deep venous events—superficial venous thrombosis (SVT), deep venous thrombosis (DVT) and pulmonary embolism (PE). Significant correlates on multivariable analysis were, for SVT: female sex, ethnicity (African-American=protective), lower educational attainment, immobility and family history of varicose veins. For DVT and DVE, significant correlates included: heavy smoking, immobility and family history of DVEs (borderline for DVE). For PE, significant predictors included immobility and, in contrast to DVT, blood pressure (BP, systolic or diastolic). In women, estrogen use duration for hormone replacement therapy, in all and among estrogen users, predicted PE and DVE, respectively.

Endothelium and hemorheology
T Gori, S Dragoni, G Di Stolfo and S Forconi
Ann Ist Super Sanità 2007 | Vol. 43, No. 2: 124-129

The mechanisms underlying the regulation of its function are extremely complex, and are principally determined by physical forces imposed on the endothelium by the flowing blood. In the present paper, we describe the interactions between the rheological properties of blood and the vascular endothelium.The role of shear stress, viscosity, cell-cell interactions, as well as the molecular mechanisms that are important for the transduction of these signals are discussed both in physiology and in pathology, with a particular attention to the role of reactive oxygen species. In the final conclusions, we propose an hypothesis regarding the implications of changes in blood viscosity, and particularly on the significance of secondary hyperviscosity syndromes..

Fig. 1 | Endothelial “function” (i.e.,the production of protective autacoids by the vascular endothelium) and “dysfunction” (i.e., the involvement of the endothelium in vascular pathology). EDHF: En d o t h e l i um-De r i v e d Hyperpolarizing Factor; LDL:Low-Density Lipoprotein

Fig. 2 | Endothelial production of nitric oxide (NO) is stimulated by oscillatory shear stress, transmitted by the endothelial surface layer to the endothelial cells. NO: Nitric Oxide; NOS: Nitrous Oxide Systems; ESL: Endothelial Surface Layer





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Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation: a Compilation of Articles in the Journal http://pharmaceuticalintelligence.com

Compilation of References by Leaders in Pharmaceutical Business Intelligence in the Journal http://pharmaceuticalintelligence.com about
Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation

Curator: Larry H Bernstein, MD, FCAP


  1. The Human Proteome Map Completed

Reporter and Curator: Larry H. Bernstein, MD, FCAP


  1. Proteomics – The Pathway to Understanding and Decision-making in Medicine

Author and Curator, Larry H Bernstein, MD, FCAP


3. Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/10/22/advances-in-separations-technology-for-the-omics-and-clarification-         of-therapeutic-targets/

  1. Expanding the Genetic Alphabet and Linking the Genome to the Metabolome

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/09/24/expanding-the-genetic-alphabet-and-linking-the-genome-to-the-                metabolome/

5. Genomics, Proteomics and standards

Larry H Bernstein, MD, FCAP, Author and Curator


6. Proteins and cellular adaptation to stress

Larry H Bernstein, MD, FCAP, Author and Curator




  1. Extracellular evaluation of intracellular flux in yeast cells

Larry H. Bernstein, MD, FCAP, Reviewer and Curator


  1. Metabolomic analysis of two leukemia cell lines. I.

Larry H. Bernstein, MD, FCAP, Reviewer and Curator


  1. Metabolomic analysis of two leukemia cell lines. II.

Larry H. Bernstein, MD, FCAP, Reviewer and Curator


  1. Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics

Reviewer and Curator, Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/22/metabolomics-metabonomics-and-functional-nutrition-the-next-step-          in-nutritional-metabolism-and-biotherapeutics/

  1. Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation

Larry H. Bernstein, MD, FCAP, Reviewer and curator

https://pharmaceuticalintelligence.com/2014/08/27/buffering-of-genetic-modules-involved-in-tricarboxylic-acid-cycle-              metabolism-provides-homeomeostatic-regulation/

Metabolic Pathways

  1. Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief

Reviewer and Curator: Larry H. Bernstein, MD, FCAP


  1. Mitochondria: More than just the “powerhouse of the cell”

Ritu Saxena, PhD


  1. Mitochondrial fission and fusion: potential therapeutic targets?

Ritu saxena


4.  Mitochondrial mutation analysis might be “1-step” away

Ritu Saxena


  1. Selected References to Signaling and Metabolic Pathways in PharmaceuticalIntelligence.com

Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/14/selected-references-to-signaling-and-metabolic-pathways-in-                     leaders-in-pharmaceutical-intelligence/

  1. Metabolic drivers in aggressive brain tumors

Prabodh Kandal, PhD


  1. Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes

Writer and Curator, Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/10/22/metabolite-identification-combining-genetic-and-metabolic-                        information-genetic-association-links-unknown-metabolites-to-functionally-related-genes/

  1. Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation

Larry H Bernstein, MD, FCAP, author and curator

https://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-            glycolysis-metabolic-adaptation/

  1. Therapeutic Targets for Diabetes and Related Metabolic Disorders

Reporter, Aviva Lev-Ari, PhD, RD


10.  Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation

Larry H. Bernstein, MD, FCAP, Reviewer and curator

https://pharmaceuticalintelligence.com/2014/08/27/buffering-of-genetic-modules-involved-in-tricarboxylic-acid-cycle-              metabolism-provides-homeomeostatic-regulation/

11. The multi-step transfer of phosphate bond and hydrogen exchange energy

Larry H. Bernstein, MD, FCAP, Curator:

https://pharmaceuticalintelligence.com/2014/08/19/the-multi-step-transfer-of-phosphate-bond-and-hydrogen-                          exchange-energy/

12. Studies of Respiration Lead to Acetyl CoA


13. Lipid Metabolism

Author and Curator: Larry H. Bernstein, MD, FCAP


14. Carbohydrate Metabolism

Author and Curator: Larry H. Bernstein, MD, FCAP


15. Update on mitochondrial function, respiration, and associated disorders

Larry H. Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2014/07/08/update-on-mitochondrial-function-respiration-and-associated-                   disorders/

16. Prologue to Cancer – e-book Volume One – Where are we in this journey?

Author and Curator: Larry H. Bernstein, MD, FCAP


17. Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/04/04/introduction-the-evolution-of-cancer-therapy-and-cancer-research-          how-we-got-here/

18. Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K

Author and Curator: Larry H. Bernstein, MD, FCAP


19. The Binding of Oligonucleotides in DNA and 3-D Lattice Structures

Author and Curator: Larry H. Bernstein, MD, FCAP


20. Mitochondrial Metabolism and Cardiac Function

Author and Curator: Larry H. Bernstein, MD, FCAP


21. How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia

Curator: Larry H. Bernstein, MD, FCAP


22. AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo

Author and Curator: Stephen J. Williams, PhD

https://pharmaceuticalintelligence.com/2013/03/12/ampk-is-a-negative-regulator-of-the-warburg-effect-and-suppresses-         tumor-growth-in-vivo/

23. A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/12/03/a-second-look-at-the-transthyretin-nutrition-inflammatory-                         conundrum/

24. Mitochondrial Damage and Repair under Oxidative Stress

Author and Curator: Larry H. Bernstein, MD, FCAP


25. Nitric Oxide and Immune Responses: Part 2

Author and Curator: Aviral Vatsa, PhD, MBBS


26. Overview of Posttranslational Modification (PTM)

Writer and Curator: Larry H. Bernstein, MD, FCAP


27. Malnutrition in India, high newborn death rate and stunting of children age under five years

Writer and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/15/malnutrition-in-india-high-newborn-death-rate-and-stunting-of-                   children-age-under-five-years/

28. Update on mitochondrial function, respiration, and associated disorders

Writer and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/08/update-on-mitochondrial-function-respiration-and-associated-                  disorders/

29. Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease

Larry H. Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2014/07/06/omega-3-fatty-acids-depleting-the-source-and-protein-insufficiency-         in-renal-disease/

30. Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine

Larry H. Bernstein, MD, FCAP, writer, and Aviva Lev- Ari, PhD, RN

https://pharmaceuticalintelligence.com/2014/04/27/larryhbernintroduction_to_cardiovascular_diseases-                                  translational_medicine-part_2/

31. Epilogue: Envisioning New Insights in Cancer Translational Biology
Series C: e-Books on Cancer & Oncology

Author & Curator: Larry H. Bernstein, MD, FCAP, Series C Content Consultant


32. Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone                         and Neurotransmitter

Writer and Curator: Larry H Bernstein, MD, FCAP and
Curator and Content Editor: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-                    hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocy

33. Cardiac Contractility & Myocardial Performance: Therapeutic Implications of Ryanopathy (Calcium Release-                           related Contractile Dysfunction) and Catecholamine Responses

Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
Author and Curator: Larry H Bernstein, MD, FCAP
and Article Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-      and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-                    contractile/

34. Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

Author and Curator: Larry H Bernstein, MD, FCAP Author: Stephen Williams, PhD, and Curator: Aviva Lev-Ari, PhD, RN


35. Identification of Biomarkers that are Related to the Actin Cytoskeleton

Larry H Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-                           cytoskeleton/

36. Advanced Topics in Sepsis and the Cardiovascular System at its End Stage

Author: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/08/18/advanced-topics-in-Sepsis-and-the-Cardiovascular-System-at-its-              End-Stage/

37. The Delicate Connection: IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology

Demet Sag, PhD, Author and Curator

https://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-               immunology/

38. IDO for Commitment of a Life Time: The Origins and Mechanisms of IDO, indolamine 2, 3-dioxygenase

Demet Sag, PhD, Author and Curator

https://pharmaceuticalintelligence.com/2013/08/04/ido-for-commitment-of-a-life-time-the-origins-and-mechanisms-of-             ido-indolamine-2-3-dioxygenase/

39. Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Homeostasis of Immune Responses for Good and Bad

Curator: Demet Sag, PhD, CRA, GCP

https://pharmaceuticalintelligence.com/2013/07/31/confined-indolamine-2-3-dehydrogenase-controls-the-hemostasis-           of-immune-responses-for-good-and-bad/

40. Signaling Pathway that Makes Young Neurons Connect was discovered @ Scripps Research Institute

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/06/26/signaling-pathway-that-makes-young-neurons-connect-was-                     discovered-scripps-research-institute/

41. Naked Mole Rats Cancer-Free

Writer and Curator: Larry H. Bernstein, MD, FCAP


42. Late Onset of Alzheimer’s Disease and One-carbon Metabolism

Reporter and Curator: Dr. Sudipta Saha, Ph.D.


43. Problems of vegetarianism

Reporter and Curator: Dr. Sudipta Saha, Ph.D.


44.  Amyloidosis with Cardiomyopathy

Writer and Curator: Larry H. Bernstein, MD, FCAP


45. Liver endoplasmic reticulum stress and hepatosteatosis

Larry H Bernstein, MD, FACP


46. The Molecular Biology of Renal Disorders: Nitric Oxide – Part III

Curator and Author: Larry H Bernstein, MD, FACP


47. Nitric Oxide Function in Coagulation – Part II

Curator and Author: Larry H. Bernstein, MD, FCAP


48. Nitric Oxide, Platelets, Endothelium and Hemostasis

Curator and Author: Larry H Bernstein, MD, FACP


49. Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Curator and Author: Larry H Bernstein, MD, FACP


50. Nitric Oxide and Immune Responses: Part 1

Curator and Author:  Aviral Vatsa PhD, MBBS


51. Nitric Oxide and Immune Responses: Part 2

Curator and Author:  Aviral Vatsa PhD, MBBS


52. Mitochondrial Damage and Repair under Oxidative Stress

Curator and Author: Larry H Bernstein, MD, FACP


53. Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/10/17/is-the-warburg-effect-the-cause-or-the-effect-of-cancer-a-21st-                 century-view/

54. Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-                  proteolysis-and-cell-apoptosis/

55. Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-                   proteolysis-and-cell-apoptosis-reconsidered/

56. Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II

Curator and Author: Larry H Bernstein, MD, FACP


57. New Insights on Nitric Oxide donors – Part IV

Curator and Author: Larry H Bernstein, MD, FACP


58. Crucial role of Nitric Oxide in Cancer

Curator and Author: Ritu Saxena, Ph.D.


59. Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-         a-concomitant-influence-on-mitochondrial-function/

60. Targeting Mitochondrial-bound Hexokinase for Cancer Therapy

Curator and Author: Ziv Raviv, PhD, RN 04/06/2013


61. Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I

Curator and Author: Larry H Bernstein, MD, FACP


Genomics, Transcriptomics, and Epigenetics

  1. What is the meaning of so many RNAs?

Writer and Curator: Larry H. Bernstein, MD, FCAP


  1. RNA and the transcription the genetic code

Larry H. Bernstein, MD, FCAP, Writer and Curator


  1. A Primer on DNA and DNA Replication

Writer and Curator: Larry H. Bernstein, MD, FCAP


4. Synthesizing Synthetic Biology: PLOS Collections

Reporter: Aviva Lev-Ari


5. Pathology Emergence in the 21st Century

Author and Curator: Larry Bernstein, MD, FCAP


6. RNA and the transcription the genetic code

Writer and Curator, Larry H. Bernstein, MD, FCAP


7. A Great University engaged in Drug Discovery: University of Pittsburgh

Larry H. Bernstein, MD, FCAP, Reporter and Curator


8. microRNA called miRNA-142 involved in the process by which the immature cells in the bone  marrow give                              rise to all the types of blood cells, including immune cells and the oxygen-bearing red blood cells

Aviva Lev-Ari, PhD, RN, Author and Curator

https://pharmaceuticalintelligence.com/2014/07/24/microrna-called-mir-142-involved-in-the-process-by-which-the-                   immature-cells-in-the-bone-marrow-give-rise-to-all-the-types-of-blood-cells-including-immune-cells-and-the-oxygen-             bearing-red-blood-cells/

9. Genes, proteomes, and their interaction

Larry H. Bernstein, MD, FCAP, Writer and Curator


10. Regulation of somatic stem cell Function

Larry H. Bernstein, MD, FCAP, Writer and Curator    Aviva Lev-Ari, PhD, RN, Curator


11. Scientists discover that pluripotency factor NANOG is also active in adult organisms

Larry H. Bernstein, MD, FCAP, Reporter

https://pharmaceuticalintelligence.com/2014/07/10/scientists-discover-that-pluripotency-factor-nanog-is-also-active-in-           adult-organisms/

12. Bzzz! Are fruitflies like us?

Larry H Bernstein, MD, FCAP, Author and Curator


13. Long Non-coding RNAs Can Encode Proteins After All

Larry H Bernstein, MD, FCAP, Reporter


14. Michael Snyder @Stanford University sequenced the lymphoblastoid transcriptomes and developed an
allele-specific full-length transcriptome

Aviva Lev-Ari, PhD, RN, Author and Curator

https://pharmaceuticalintelligence.com/014/06/23/michael-snyder-stanford-university-sequenced-the-lymphoblastoid-            transcriptomes-and-developed-an-allele-specific-full-length-transcriptome/

15. Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: Views by Larry H                                     Bernstein, MD, FCAP

Author: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/16/commentary-on-biomarkers-for-genetics-and-genomics-of-                        cardiovascular-disease-views-by-larry-h-bernstein-md-fcap/

16. Observations on Finding the Genetic Links in Common Disease: Whole Genomic Sequencing Studies

Author an curator: Larry H Bernstein, MD, FCAP


17. Silencing Cancers with Synthetic siRNAs

Larry H. Bernstein, MD, FCAP, Reviewer and Curator


18. Cardiometabolic Syndrome and the Genetics of Hypertension: The Neuroendocrine Transcriptome Control Points

Reporter: Aviva Lev-Ari, PhD, RN


19. Developments in the Genomics and Proteomics of Type 2 Diabetes Mellitus and Treatment Targets

Larry H. Bernstein, MD, FCAP, Reviewer and Curator

https://pharmaceuticalintelligence.com/2013/12/08/developments-in-the-genomics-and-proteomics-of-type-2-diabetes-           mellitus-and-treatment-targets/

20. Loss of normal growth regulation

Larry H Bernstein, MD, FCAP, Curator


21. CT Angiography & TrueVision™ Metabolomics (Genomic Phenotyping) for new Therapeutic Targets to Atherosclerosis

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/11/15/ct-angiography-truevision-metabolomics-genomic-phenotyping-for-           new-therapeutic-targets-to-atherosclerosis/

22.  CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics

Genomics Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/30/cracking-the-code-of-human-life-the-birth-of-bioinformatics-                      computational-genomics/

23. Big Data in Genomic Medicine

Author and Curator, Larry H Bernstein, MD, FCAP


24. From Genomics of Microorganisms to Translational Medicine

Author and Curator: Demet Sag, PhD

https://pharmaceuticalintelligence.com/2014/03/20/without-the-past-no-future-but-learn-and-move-genomics-of-                      microorganisms-to-translational-medicine/

25. Summary of Genomics and Medicine: Role in Cardiovascular Diseases

Author and Curator, Larry H Bernstein, MD, FCAP


 26. Genomic Promise for Neurodegenerative Diseases, Dementias, Autism Spectrum, Schizophrenia, and Serious                      Depression

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/02/19/genomic-promise-for-neurodegenerative-diseases-dementias-autism-        spectrum-schizophrenia-and-serious-depression/

 27.  BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair

Sudipta Saha, PhD

https://pharmaceuticalintelligence.com/2012/12/04/brca1-a-tumour-suppressor-in-breast-and-ovarian-cancer-functions-         in-transcription-ubiquitination-and-dna-repair/

28. Personalized medicine gearing up to tackle cancer

Ritu Saxena, PhD


29. Differentiation Therapy – Epigenetics Tackles Solid Tumors

Stephen J Williams, PhD


30. Mechanism involved in Breast Cancer Cell Growth: Function in Early Detection & Treatment

     Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/17/mechanism-involved-in-breast-cancer-cell-growth-function-in-early-          detection-treatment/

31. The Molecular pathology of Breast Cancer Progression

Tilde Barliya, PhD


32. Gastric Cancer: Whole-genome reconstruction and mutational signatures

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/12/24/gastric-cancer-whole-genome-reconstruction-and-mutational-                   signatures-2/

33. Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine –                                                       Part 1 (pharmaceuticalintelligence.com)

Aviva  Lev-Ari, PhD, RN


34. LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer                                         Personalized Treatment: Part 2

A Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/13/leaders-in-genome-sequencing-of-genetic-mutations-for-therapeutic-       drug-selection-in-cancer-personalized-treatment-part-2/

35. Personalized Medicine: An Institute Profile – Coriell Institute for Medical Research: Part 3

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/13/personalized-medicine-an-institute-profile-coriell-institute-for-medical-        research-part-3/

36. Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of                           Cancer Scientific Leaders @http://pharmaceuticalintelligence.com

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/13/7000/Harnessing_Personalized_Medicine_for_ Cancer_Management-      Prospects_of_Prevention_and_Cure/

37.  GSK for Personalized Medicine using Cancer Drugs needs Alacris systems biology model to determine the in silico
effect of the inhibitor in its “virtual clinical trial”

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/11/14/gsk-for-personalized-medicine-using-cancer-drugs-needs-alacris-             systems-biology-model-to-determine-the-in-silico-effect-of-the-inhibitor-in-its-virtual-clinical-trial/

38. Personalized medicine-based cure for cancer might not be far away

Ritu Saxena, PhD


39. Human Variome Project: encyclopedic catalog of sequence variants indexed to the human genome sequence

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/11/24/human-variome-project-encyclopedic-catalog-of-sequence-variants-         indexed-to-the-human-genome-sequence/

40. Inspiration From Dr. Maureen Cronin’s Achievements in Applying Genomic Sequencing to Cancer Diagnostics

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/10/inspiration-from-dr-maureen-cronins-achievements-in-applying-                genomic-sequencing-to-cancer-diagnostics/

41. The “Cancer establishments” examined by James Watson, co-discoverer of DNA w/Crick, 4/1953

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/09/the-cancer-establishments-examined-by-james-watson-co-discover-         of-dna-wcrick-41953/

42. What can we expect of tumor therapeutic response?

Author and curator: Larry H Bernstein, MD, FACP


43. Directions for genomics in personalized medicine

Author and Curator: Larry H. Bernstein, MD, FCAP


44. How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis.

Stephen J Williams, PhD

https://pharmaceuticalintelligence.com/2012/10/31/how-mobile-elements-in-junk-dna-prote-cancer-part1-transposon-            mediated-tumorigenesis/

45. mRNA interference with cancer expression

Author and Curator, Larry H. Bernstein, MD, FCAP


46. Expanding the Genetic Alphabet and linking the genome to the metabolome

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/09/24/expanding-the-genetic-alphabet-and-linking-the-genome-to-the-               metabolome/

47. Breast Cancer, drug resistance, and biopharmaceutical targets

Author and Curator: Larry H Bernstein, MD, FCAP


48.  Breast Cancer: Genomic profiling to predict Survival: Combination of Histopathology and Gene Expression                            Analysis

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/12/24/breast-cancer-genomic-profiling-to-predict-survival-combination-of-           histopathology-and-gene-expression-analysis

49. Gastric Cancer: Whole-genome reconstruction and mutational signatures

Aviva  Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/12/24/gastric-cancer-whole-genome-reconstruction-and-mutational-                   signatures-2/

50. Genomic Analysis: FLUIDIGM Technology in the Life Science and Agricultural Biotechnology

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/08/22/genomic-analysis-fluidigm-technology-in-the-life-science-and-                   agricultural-biotechnology/

51. 2013 Genomics: The Era Beyond the Sequencing Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

Aviva Lev-Ari, PhD, RD


52. Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/Paradigm Shift in Human Genomics_/

Signaling Pathways

  1. Proteins and cellular adaptation to stress

Larry H Bernstein, MD, FCAP, Curator


  1. A Synthesis of the Beauty and Complexity of How We View Cancer:
    Cancer Volume One – Summary

Author and Curator: Larry H. Bernstein, MD, FCAP


  1. Recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes in
    serous endometrial tumors

Sudipta Saha, PhD

https://pharmaceuticalintelligence.com/2012/11/19/recurrent-somatic-mutations-in-chromatin-remodeling-ad-ubiquitin-           ligase-complex-genes-in-serous-endometrial-tumors/

4.  Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition

Stephen J Williams, PhD

https://pharmaceuticalintelligence.com/2012/11/30/histone-deacetylase-inhibitors-induce-epithelial-to-mesenchymal-              transition-in-prostate-cancer-cells/

5. Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Author and Curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-                   proteolysis-and-cell-apoptosis/

6. Signaling and Signaling Pathways

Larry H. Bernstein, MD, FCAP, Reporter and Curator


7.  Leptin signaling in mediating the cardiac hypertrophy associated with obesity

Larry H. Bernstein, MD, FCAP, Reporter and Curator

https://pharmaceuticalintelligence.com/2013/11/03/leptin-signaling-in-mediating-the-cardiac-hypertrophy-associated-            with-obesity/

  1. Sensors and Signaling in Oxidative Stress

Larry H. Bernstein, MD, FCAP, Reporter and Curator


  1. The Final Considerations of the Role of Platelets and Platelet Endothelial Reactions in Atherosclerosis and Novel

Larry H. Bernstein, MD, FCAP, Reporter and Curator

https://pharmaceuticalintelligence.com/2013/10/15/the-final-considerations-of-the-role-of-platelets-and-platelet-                      endothelial-reactions-in-atherosclerosis-and-novel-treatments

10.   Platelets in Translational Research – Part 1

Larry H. Bernstein, MD, FCAP, Reporter and Curator


11.  Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and
Cardiovascular Calcium Signaling Mechanism

Author and Curator: Larry H Bernstein, MD, FCAP, Author, and Content Consultant to e-SERIES A:
Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-             smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

12. The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and
Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia,
Similarities and Differences, and Pharmaceutical Targets

     Author and Curator: Larry H Bernstein, MD, FCAP, Author, and Content Consultant to
e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and
Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-       kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-           differen/

13.  Nitric Oxide Signalling Pathways

Aviral Vatsa, PhD, MBBS


14. Immune activation, immunity, antibacterial activity

Larry H. Bernstein, MD, FCAP, Curator


15.  Regulation of somatic stem cell Function

Larry H. Bernstein, MD, FCAP, Writer and Curator    Aviva Lev-Ari, PhD, RN, Curator


16. Scientists discover that pluripotency factor NANOG is also active in adult organisms

Larry H. Bernstein, MD, FCAP, Reporter


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Selected References to Signaling and Metabolic Pathways in PharmaceuticalIntelligence.com

Curator: Larry H. Bernstein, MD, FCAP


This is an added selection of articles in Leaders in Pharmaceutical Intelligence after the third portion of the discussion in a series of articles that began with signaling and signaling pathways. There are fine features on the functioning of enzymes and proteins, on sequential changes in a chain reaction, and on conformational changes that we shall return to.  These are critical to developing a more complete understanding of life processes.  I have indicated that many of the protein-protein interactions or protein-membrane interactions and associated regulatory features have been referred to previously, but the focus of the discussion or points made were different.

  1. Signaling and signaling pathways
  2. Signaling transduction tutorial.
  3. Carbohydrate metabolism3.1  Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence
  4. Lipid metabolism
  5. Protein synthesis and degradation
  6. Subcellular structure
  7. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

Selected References to Signaling and Metabolic Pathwayspublished in this Open Access Online Scientific Journal, include the following:

Update on mitochondrial function, respiration, and associated disorders

Curator and writer: Larry H. Benstein, MD, FCAP


A Synthesis of the Beauty and Complexity of How We View Cancer

Cancer Volume One – Summary

A Synthesis of the Beauty and Complexity of How We View Cancer

Author: Larry H. Bernstein, MD, FCAP


Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Author and Curator: Larry H Bernstein, MD, FCAP


 The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Author and Curator: Larry H Bernstein, MD, FCAP, 
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
And Curator: Aviva Lev-Ari, PhD, RN


Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease

Author and Curator: Larry H. Bernstein, MD, FCAP
Curator:  Stephen J. Williams, PhD
and Curator: Aviva Lev-Ari, PhD, RN


Mitochondrial Metabolism and Cardiac Function

Curator: Larry H Bernstein, MD, FACP


Mitochondrial Dysfunction and Cardiac Disorders

Curator: Larry H Bernstein, MD, FACP


Reversal of Cardiac mitochondrial dysfunction

Curator: Larry H Bernstein, MD, FACP


Advanced Topics in Sepsis and the Cardiovascular System  at its End Stage

Author: Larry H Bernstein, MD, FCAP


Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Curator: Larry H Bernstein, MD, FACP


Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III

Curator: Larry H Bernstein, MD, FCAP



Nitric Oxide, Platelets, Endothelium and Hemostasis (Coagulation Part II)

Curator: Larry H. Bernstein, MD, FCAP 


Mitochondrial Damage and Repair under Oxidative Stress

Curator: Larry H Bernstein, MD, FCAP


Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation

Reporter and Curator: Larry H Bernstein, MD, FACP



Nitric Oxide has a Ubiquitous Role in the Regulation of Glycolysis – with a Concomitant Influence on Mitochondrial Function

Reporter, Editor, and Topic Co-Leader: Larry H. Bernstein, MD, FCAP


Mitochondria and Cancer: An overview of mechanisms

Author and Curator: Ritu Saxena, Ph.D.


Mitochondria: More than just the “powerhouse of the cell”

Author and Curator: Ritu Saxena, Ph.D.


Overview of Posttranslational Modification (PTM)

Curator: Larry H. Bernstein, MD, FCAP


Ubiquitin Pathway Involved in Neurodegenerative Diseases

Author and curator: Larry H Bernstein, MD,  FCAP


Is the Warburg Effect the Cause or the Effect of Cancer: A 21st Century View?

Author: Larry H. Bernstein, MD, FCAP 


New Insights on Nitric Oxide donors – Part IV

Curator and Author: Larry H. Bernstein, MD, FCAP


Perspectives on Nitric Oxide in Disease Mechanisms [Kindle Edition]

Margaret Baker PhD (Author), Tilda Barliya PhD (Author), Anamika Sarkar PhD (Author), Ritu Saxena PhD (Author), Stephen J. Williams PhD (Author), Larry Bernstein MD FCAP (Editor), Aviva Lev-Ari PhD RN (Editor), Aviral Vatsa PhD (Editor)




Nitric oxide and its role in vascular biology

Signal transmission by a gas that is produced by one cell, penetrates through membranes and regulates the function of another cell represents an entirely new principle for signaling in biological systems.   All compounds that inhibit endothelium-derived relaxation-factor (EDRF) have one property in common, redox activity, which accounts for their inhibitory action on EDRF. One exception is hemoglobin, which inactivates EDRF by binding to it. Furchgott, Ignarro and Murad received the Nobel Prize in Physiology and Medicine for discovery of EDRF in 1998 and demonstrating that it might be nitric oxide (NO) based on a study of the transient relaxations of endothelium-denuded rings of rabbit aorta.  These investigators working independently demonstrated that NO is indeed produced by mammalian cells and that NO has specific biological roles in the human body. These studies highlighted the role of NO in cardiovascular, nervous and immune systems. In cardiovascular system NO was shown to cause relaxation of vascular smooth muscle cells causing vasodilatation, in nervous system NO acts as a signaling molecule and in immune system it is used against pathogens by the phagocytosis cells. These pioneering studies opened the path of investigation of role of NO in biology.

NO modulates vascular tone, fibrinolysis, blood pressure and proliferation of vascular smooth muscles. In cardiovascular system disruption of NO pathways or alterations in NO production can result in preponderance to hypertension, hypercholesterolemia, diabetes mellitus, atherosclerosis and thrombosis. The three enzyme isoforms of NO synthase family are responsible for generating NO in different tissues under various circumstances.

Reduction in NO production is implicated as one of the initial factors in initiating endothelial dysfunction. This reduction could be due to

  • reduction in eNOS production
  • reduction in eNOS enzymatic activity
  • reduced bioavailability of NO

Nitric oxide is one of the smallest molecules involved in physiological functions in the body. It is seeks formation of chemical bonds with its targets.  Nitric oxide can exert its effects principally by two ways:

  • Direct
  • Indirect

Direct actions, as the name suggests, result from direct chemical interaction of NO with its targets e.g. with metal complexes, radical species. These actions occur at relatively low NO concentrations (<200 nM)

Indirect actions result from the effects of reactive nitrogen species (RNS) such as NO2 and N2O3. These reactive species are formed by the interaction of NO with superoxide or molecular oxygen. RNS are generally formed at relatively high NO concentrations (>400 nM)

Although it can be tempting for scientists to believe that RNS will always have deleterious effects and NO will have anabolic effects, this is not entirely true as certain RNS mediated actions mediate important signalling steps e.g. thiol oxidation and nitrosation of proteins mediate cell proliferation and survival, and apoptosis respectively.

  • Cells subjected to NO concentration between 10-30 nM were associated with cGMP dependent phosphorylation of ERK
  • Cells subjected to NO concentration between 30-60 nM were associated with Akt phosphorylation
  • Concentration nearing 100 nM resulted in stabilisation of hypoxia inducible factor-1
  • At nearly 400 nM NO, p53 can be modulated
  • >1μM NO, it nhibits mitochondrial respiration


Nitric oxide signaling, oxidative stress,  mitochondria, cell damage

Recent data suggests that other NO containing compounds such as S- or N-nitrosoproteins and iron-nitrosyl complexes can be reduced back to produce NO. These NO containing compounds can serve as storage and can reach distant tissues via blood circulation, remote from their place of origin. Hence NO can have both paracrine and ‘endocrine’ effects.

Intracellularly the oxidants present in the cytosol determine the amount of bioacitivity that NO performs. NO can travel roughly 100 microns from NOS enzymes where it is produced.

NO itself in low concentrations have protective action on mitochondrial signaling of cell death.

The aerobic cell was an advance in evolutionary development, but despite the energetic advantage of using oxygen, the associated toxicity of oxygen abundance required adaptive changes.

Oxidation-reduction reactions that are necessary for catabolic and synthetic reactions, can cumulatively damage the organism associated with cancer, cardiovascular disease, neurodegerative disease, and inflammatory overload.  The normal balance between production of pro-oxidant species and destruction by the antioxidant defenses is upset in favor of overproduction of the toxic species, which leads to oxidative stress and disease.

We reviewed the complex interactions and underlying regulatory balances/imbalances between the mechanism of vasorelaxation and vasoconstriction of vascular endothelium by way of nitric oxide (NO), prostacyclin, in response to oxidative stress and intimal injury.

Nitric oxide has a ubiquitous role in the regulation of glycolysis with a concomitant influence on mitochondrial function. The influence on mitochondrial function that is active in endothelium, platelets, vascular smooth muscle and neural cells and the resulting balance has a role in chronic inflammation, asthma, hypertension, sepsis and cancer.

Potential cytotoxic mediators of endothelial cell (EC) apoptosis include increased formation of reactive oxygen and nitrogen species (ROSRNS) during the atherosclerotic process. Nitric oxide (NO) has a biphasic action on oxidative cell killing with low concentrations protecting against cell death, whereas higher concentrations are cytotoxic.

ROS induces mitochondrial DNA damage in ECs, and this damage is accompanied by a decrease in mitochondrial RNA (mtRNA) transcripts, mitochondrial protein synthesis, and cellular ATP levels.

NO and circulatory diseases

Blood vessels arise from endothelial precursors that are thin, flat cells lining the inside of blood vessels forming a monolayer throughout the circulatory system. ECs are defined by specific cell surface markers that characterize their phenotype.

Scientists at the University of Helsinki, Finland, wanted to find out if there exists a rare vascular endothelial stem cell (VESC) population that is capable of producing very high numbers of endothelial daughter cells, and can lead to neovascular growth in adults.

VESCs discovered that reside at the blood vessel wall endothelium are a small population of CD117+ ECs capable of self-renewal.  These cells are capable of undergoing clonal expansion unlike the surrounding ECs that bear limited proliferating potential. A single VESC cell isolated from the endothelial population was able to generate functional blood vessels.

Among many important roles of Nitric oxide (NO), one of the key actions is to act as a vasodilator and maintain cardiovascular health. Induction of NO is regulated by signals in tissue as well as endothelium.

Chen et. al. (Med. Biol. Eng. Comp., 2011) developed a 3-D model consisting of two branched arterioles and nine capillaries surrounding the vessels. Their model not only takes into account of the 3-D volume, but also branching effects on blood flow.

The model indicates that wall shear stress changes depending upon the distribution of RBC in the microcirculations of blood vessels, lead to differential production of NO along the vascular network.

Endothelial dysfunction, the hallmark of which is reduced activity of endothelial cell derived nitric oxide (NO), is a key factor in developing atherosclerosis and cardiovascular disease. Vascular endothelial cells play a pivotal role in modulation of leukocyte and platelet adherence, thrombogenicity, anticoagulation, and vessel wall contraction and relaxation, so that endothelial dysfunction has become almost a synonym for vascular disease. A single layer of endothelial cells is the only constituent of capillaries, which differ from other vessels, which contain smooth muscle cells and adventitia. Capillaries directly mediate nutritional supply as well as gas exchange within all organs. The failure of the microcirculation leads to tissue apoptosis/necrosis.

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Contributions to Cardiomyocyte Interactions and Signaling

Author and Curator: Larry H Bernstein, MD, FCAP


Curator: Aviva Lev-Ari, PhD, RN


This is Part II of the ongoing research in the Lee Laboratory, concerned with Richard T Lee’s dissection of the underlying problems that will lead to a successful resolution of myocardiocyte regeneration unhampered by toxicity, and having a suffuciently sustained effect for an evaluation and introduction to the clinic.  This would be a milestone in the treatment of heart failure, and an alternative to transplantation surgery.  This second presentation focuses on the basic science work underpinning the therapeutic investigations.  It is work that, if it was unsupported and did not occur because of insufficient funding, the Part I story could not be told.

Cardiomyocyte hypertrophy and degradation of connexin43 through spatially restricted autocrine/paracrine heparin-binding EGF

J Yoshioka, RN Prince, H Huang, SB Perkins, FU Cruz, C MacGillivray, DA Lauffenburger, and RT Lee *
Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; and Biological Engineering Division, MIT, Cambridge, MA
PNAS 2005; 302(30):10622-10627.  http://pnas.org/cgi/doi/10.1073/pnas.0501198102

Growth factor signaling can affect tissue remodeling through autocrine/paracrine mechanisms. Recent evidence indicates that EGF receptor transactivation by heparin-binding EGF (HB-EGF) contributes to hypertrophic signaling in cardiomyocytes. Here, we show that HB-EGF operates in a spatially restricted circuit in the extracellular space within the myocardium, revealing the critical nature of the local microenvironment in intercellular signaling. This highly localized microenvironment of HB-EGF signaling was demonstrated with 3D morphology, consistent with predictions from a computational model of EGF signaling. HB-EGF secretion by a given cardiomyocyte in mouse left ventricles led to cellular hypertrophy and reduced expression of connexin43 in the overexpressing cell and in immediately adjacent cells but not in cells farther away. Thus, HB-EGF acts as an autocrine and local paracrine cardiac growth factor that leads to loss of gap junction proteins within a spatially confined microenvironment. These findings demonstrate how cells can coordinate remodeling with their immediate neighboring cells with highly localized extracellular EGF signaling. Within 3D tissues, cells must coordinate remodeling in response to stress or growth signals, and this communication may occur by direct contact or by secreted signaling molecules. Cardiac hypertrophy is a physiological response that enables the heart to adapt to an initial stress; however, hypertrophy can ultimately lead to the deterioration in cardiac function and an increase in cardiac arrhythmias. Although considerable progress has been made in elucidating the molecular pathogenesis of cardiac hypertrophy, the precise mechanisms guiding the hypertrophic process remain unknown. Recent evidence suggests that myocardial heparin-binding (HB)-epidermal growth factor participates in the hypertrophic response. In cardiomyocytes, hypertrophic stimuli markedly increase expression of the HB-EGF gene, suggesting that HB-EGF can act as an autocrine trophic factor that contributes to cellular growth. HB-EGF is first synthesized as a membrane-anchored form (proHB-EGF), and subsequent ectodo-main shedding at the cell surface releases the soluble form of HB-EGF. Soluble HB-EGF is a diffusible factor that can be captured by the receptors to activate the intracellular EGF receptor signaling cascade. Indeed, EGF receptor (EGFR) transactivation, triggered by shedding of HB-EGF from the cell surface, plays an important role in cardiac hypertrophy resulting from pressure overload in the aortic-banding model. EGFR activation can occur through autocrine and paracrine signaling. In autocrine signaling, a cell produces and responds to the same signaling molecules. Paracrine signaling molecules can target groups of distant cells or act as localized mediators affecting only cells in the immediate environment of the signaling cell. Thus, although locally produced HB-EGF may travel through the extra-cellular space, it may also be recaptured by the EGFR close to the point where it was released from the cell surface. The impact of spatially localized microenvironments of signaling could be extensive heterogeneous tissue remodeling, which can be particularly important in an electrically coupled tissue like myocardium. Interestingly, recent data suggest that EGF can regulate protea-some-dependent degradation of connexin43 (Cx43), a major trans-membrane gap junction protein, in liver epithelial cells, along with a rapid inhibition of cell–cell communication through gap junctions. One of the critical potential myocardial effects of HB-EGF could therefore be to increase degradation of Cx43 and reduce electrical stability of the heart. Reduced content of Cx43 is commonly observed in chronic heart diseases such as hypertrophy, myocardial infarction, and failure. Thus, we hypothesized that HB-EGF signals may operate in a spatially restricted local circuit in the extracellular space. We also hypothesized that HB-EGF secretion by a given cardiomyocyte could create a local remodeling microenvironment of decreased Cx43 within the myocardium. To explore whether HB-EGF signaling is highly spatially constrained, we took advantage of the nonuniform gene transfer to cardiac myocytes in vivo, normally considered a pitfall of gene therapy. We also performed computational modeling to predict HB-EGF dynamics and developed a 3D approach to measure cardiomyocyte hypertrophy.


Autocrine HB-EGF and Cardiomyocyte Growth.

To assess the effects of gene transfer of HB-EGF on cardiomyocyte hypertrophy, cells were infected with adenoviral vectors expressing GFP alone (Ad-GFP) or HB-EGF and GFP (Ad-HB-EGF). At this level of infection, 99% of cardiomyocytes were transduced. The incidence of apoptotic cell death (sub-G1 fraction) was not different between Ad-GFP cells, suggesting that expression of GFP by the adenoviral vector was not cardiotoxic in these conditions. Western analysis by using an anti-HB-EGF antibody confirmed successful gene transfer of HB-EGF in cardiomyocytes (18 ± 5-fold, n = 4, P < 0.01); HB-EGF appeared electrophoretically as several bands from 15 to 30 kDa (Fig. 1A). The strongest band corresponds to the soluble 20-kDa form of HB-EGF. To confirm that Ad-HB-EGF results in cellular hypertrophy, cell size and protein synthesis were measured. Ad-HB-EGF enlarged cardiomyocytes compared with Ad-GFP-infected cells by phase-contrast microscopy (24 ± 10% increase in cell surface area, n = 27, P < 0.05) and with flow cytometry analysis (26 ± 10% increase of Ad-GFP infected cells, P < 0.01, Fig. 1B). Overexpression of HB-EGF increased total protein synthesis in cardiomyocytes as measured by [3H]leucine uptake (34 ± 6% of Ad-GFP, n = 6, P < 0.01, Fig. 1C). Uninfected cells within the same dish (and thus sharing the same culture media) did not develop hypertrophy. Additionally, medium from cultures previously infected with Ad-HB-EGF for 48 h was collected and applied to adenovirus-free cultures. Conditioned medium from Ad-HB-EGF-infected cardiomyocytes failed to stimulate hypertrophy in naive cardiomyocytes (Fig. 1C), and there were no significant differences in cell size between noninfected cells from Ad-GFP and Ad-HB-EGF dishes. These results suggest that HB-EGF acts primarily as an autocrine growth factor in cardiomyocytes in vitro.
Because the dilution factor in culture media is important for autocrine/paracrine signaling, we determined the concentration of soluble HB-EGF in the conditioned medium and the effective concentration to stimulate cardiomyocyte growth. HB-EGF levels in the conditioned medium from Ad-HB-EGF dishes were 258 ± 73 pg/ml (n = 4), whereas HB-EGF levels from Ad-GFP dishes (n = 8) were below the limit of detection (6.7 pg/ml). The addition of 300 pg/ml of exogenous recombinant HB-EGF into fresh media failed to stimulate hypertrophy in cardiomyocytes as measured by [3H]leucine uptake (-12 ± 5.0% compared with control, n = 5,P = not significant), but 2,000 pg/ml of recombinant HB-EGF did result in a significant effect (+24 ± 5.5% compared with control, n = 6, P < 0.05). This comparison implies that the local concentration of autocrine ligand is substantially greater than that indicated by a bulk measurement of conditioned media, consistent with previous experimental and theoretical studies.

Fig. 1. Effects of gene transfer of HB-EGF on rat neonatal cardiomyocyte growth.

(A) Cells were infected with adenoviral vectors expressing GFP (Ad-GFP), or HB-EGF and GFP (Ad-HB-EGF). Western analysis showed the successful gene transfer of HB-EGF. (8) FACS analysis of 5,000 cardiomyocytes demonstrated that overexpression of HB-EGF produced a 26 ± 10% increase in cell size that was significantly greater than the overex-pression of GFP. Bar graphs with errors represent mean ± SEM from three independent experiments. **, P < 0.01 vs. Ad-HB-EGF-nonin-fected cells and Ad-GFP nonin-fected cells. , P < 0.05 vs. Ad-GFP infected cells. (C) Overexpression of HB-EGF resulted in a 34 ± 6% increase in [3H]leucine uptake compared with Ad-GFP (n = 6), whereas conditioned medium from Ad-HB-EGF cells caused an only insignificant increase. **, P < 0.05 vs. Ad-GFP control and conditioned medium Ad-GFP.

 Effects of HB-EGF on Cx43 Content in Cultured Cardiomyocytes

Because EGF can induce degradation of the gap junction protein Cx43 in other cells, we then determined whether Cx43 is regulated by HB-EGF in cardiomyocytes. Fig. 2A shows a representative immunoblot from three separate experiments in which Cx43 migrated as three major bands at 46, 43, and 41 kDa, as reported in ref. 16. Overexpression of HB-EGF decreased total Cx43 content (27 ± 11% compared with Ad-GFP, n = 4, P < 0.05) without affecting the intercellular adhesion protein, N-cadherin. The phosphorylation of ERK1/2, an intracellular signaling kinase downstream of EGFR transactivation, was augmented by HB-EGF (3.2 ± 1.0-fold compared with Ad-GFP, n = 4, P < 0.05). Northern analysis showed that HB-EGF did not reduce Cx43 gene expression, suggesting that HB-EGF decreases Cx43 by posttranslational modification (Fig. 2B). AG 1478 (10 iLM), a specific inhibitor of EGFR tyrosine kinase, abolished the effect of HB-EGF on Cx43 (Fig. 2C), indicating that the decrease in Cx43 content depends on EGFR transactivation by HB-EGF. The conditioned medium from Ad-HB-EGF-infected cells did not change expression of Cx43 in naive cells, even though ERK1/2 was slightly activated by the conditioned medium (Fig. 1D). These data are consistent with the hypertrophy data presented above, demonstrating that HB-EGF can act as a predominantly autocrine factor both in hypertrophy and in the reduction of Cx43 content in cardiomyocytes.

Computational Analysis Predicts HB-EGF Autocrine/Paracrine Signaling in Vivo.

Although these in vitro experiments showed HB-EGF as a predominantly autocrine cardiac growth factor, HB-EGF signaling in vivo takes place in a very different environment. Therefore, we sought to determine the extent that soluble HB-EGF may travel in the interstitial space of the myocardium with a simple 2D model of HB-EGF diffusion (Fig. 3A). An approximate geometric representation of myocytes in cross-section is a square (15 x 15 iLm), with each of the corners occupied by a capillary (diameter 5 iLm). The cell shape was chosen so that the extracellular matrix width (0.5 iLm), in which soluble HB-EGF is free to diffuse, was constant around all tissue features. This model geometry is based on a square array of capillaries; although a hexagonal pattern of capillary distribution is commonly accepted, the results are not expected to be substantially different with this simpler construction, because both have four capillaries surrounding each myocyte. The model represents a single central cell that is releasing HB-EGF at a constant rate, Rgen, (approximated from the HB-EGF concentration measurement in conditioned medium) into the extracellular space. HB-EGF then can diffuse throughout this space, or enter a capillary and leave the system. This system is governed by
  • the diffusion equation at steady state (DV2C = 0),
  • the boundary condition for the ligand producing cell (—DVC = Rgen),
  • the boundary condition for all other cells (—DVC = 0), and
  • the capillary boundary condition (DVC = h(C — Cblood)).

C denotes HB-EGF concentration, D is the diffusivity constant, h is the mass transfer coefficient, and Cblood is the concentration of HB-EGF in the blood, approximated to be zero.  The numerical solution in Fig. 3B illustrates that HB-EGF remained localized around the cell which produced it and did not diffuse farther because of the sink-like effect of the capillaries. The maximum concentration of soluble HB-EGF achieved is 0.27 nM, which is near the threshold level of HB-EGF measured to stimulate cardiomyocyte growth (2,000 pg/ml). Therefore, the central HB-EGF-producing cell only signals to its four adjacent neighbors where the HB-EGF concentration reaches this threshold. However, if the model geometry is altered to reflect a 50% and 150% increase in cross-sectional area in all cells because of hypertrophy, estimated from 1 and 4 weeks of transverse aortic constriction, the maximum concentration achieved increases slightly to 0.29 nM and 0.37 nM, respectively. As the cell width increases, HB-EGF must diffuse farther to reach a capillary, exposing adjacent cells to a higher concentration during hypertrophy. However, no additional cells are exposed to HB-EGF. 

Fig. 3. Computational modeling of HB-EGF diffusion in the myocardium.

Red areas represent capillaries, green represents the HB-EGF ligand producing cell, pink represents adjacent cells, and white is an extracellular matrix where HB-EGF is free to diffuse. (A) The model geometry where HB-EGF is generated by the ligand-producing cell at a constant rate, Rgen, and diffuses throughout the extracellular space or enters a capillary and leaves the system with a mass transfer coefficient, h. (B) Numerical solution of the steady-state HB-EGF concentration profile with Rgen = 10 cell-1s-1, D = 0.7 µm2/s, and h = 0.02 µm/s, where concentration is shown by the color scale and height depicted. The maximum concentration achieved with the stated parameters was 0.27 nM from a capillary. Myocyte length was assumed to be 100 µm.

The driving force that determined the extent to which HB-EGF traveled was the rate of HB-EGF transfer into the capillaries and the diffusivity of HB-EGF. The exact mechanism of macromolecule transport into capillaries is unknown; however, it is most likely through diffusion, transcytosis, or a combination of the two. In the case of diffusion, the mass transfer coefficient governing the flux of HB-EGF through the capillary wall is coupled to the diffusivity of HB-EGF, whereas the terms are uncoupled for the case of transcytosis. Therefore, this model assessed transcytosis as a conservative scenario for HB-EGF localization. Parameter perturbation with uncoupled diffusion and capillary mass transfer showed that HB-EGF remained localized around the origin of production and diffused only to immediate neighbors for mass transfer coefficients >0.002 µm/s. For values <0.002 µm/s, HB-EGF diffused distances more than two cells away from the origin. Although the actual mass transfer coefficient of ligands in the size range of HB-EGF is unknown, values for O2 (0.02 µm/s, 0.032 kDa) (19) and LDL (1.7 x 10-5 µm/s, 2,000–3,000 kDa) (20) have been reported, and we assumed HB-EGF is in the upper end of that range due to its small size. HB-EGF also binds to EGFRs, the extracellular matrix, and cell surface heparan sulfate proteoglycans. EGFR binding and internalization could serve to further localize HB-EGF. The number of extracellular binding sites does not affect the steady-state HB-EGF concentration profile if this binding is reversible. However, these binding sites could serve to localize HB-EGF as the cell begins to produce the ligand by slowing the travel of HB-EGF to the capillaries in the approach to the steady state, or as a source of HB-EGF as the cell slows or stops HB-EGF production. At a diffusivity of 0.7 µm2/s (21), HB-EGF traveled only one cell away, but traveled approximately five cells away at 51.8 µm2/s (22), with a peak concentration below the estimated threshold for stimulating.

Overexpression of HB-EGF Causes Hypertrophy on the Infected Cell and Its Immediate Neighbor in Vivo.

To explore whether HB-EGF signals operate in a spatially restricted local circuit in the in vivo myocardial extracellular space as predicted by computational modeling, adenoviral vectors were injected directly into the left ventricular free wall in 26 male mice (Ad-GFP, n = 12; Ad-HB-EGF, n = 14). Of the 26 mice, 5 (4 Ad-GFP and 1 Ad-HB-EGF) mice died after the surgery. Gene expression was confirmed as positive cellular fluorescence in the presence of GFP, allowing determination of which cells were infected at 7 days (Fig. 4A). Immunohis-tochemical staining revealed that HB-EGF was localized on the Ad-HB-EGF-infected cell membrane or in the extracellular space around the overexpressing cell (Fig. 4A). For comparison, remote cells were defined as noninfected cells far (15–20 cell dimensions) from the adenovirus-infected area and in the same field as infected cells. Conventional 2D cross-sectional analysis blinded to treatment group (Fig. 4B) showed that Ad-GFP-infected cells (n = 102) resulted in no cellular hypertrophy compared with noninfected, adjacent (n = 92), or remote (n = 97) cells (2D myocyte cross-sectional area, 250 ± 7 versus 251 ± 7 or 255 ± 6 µm2, respectively). These data suggest that expression of GFP in these conditions does not cause cellular hypertrophy. However, overexpression of HB-EGF caused hypertrophy in both Ad-HB-EGF-infected cells (a 41 ± 5% increase of Ad-GFP-infected cells, n = 119, P < 0.01) and noninfected adjacent cells (a 33 ± 5% increase of Ad-GFP-adjacent cells, n = 97, P < 0.01) compared with remote cells (n = 109). Because 2D analysis of cardiomyocyte hypertrophy can be influenced by the plane of sectioning, we then developed a 3D histology approach that allowed reconstruction of cardiomyocytes in situ (Fig. 4C). We performed an independent 3D histology analysis of cardiomyocytes to determine cell volumes, blinded to treatment group (Fig. 4B). The volumes of both HB-EGF-infected cells (n = 19, 42,700 ± 4,000 µm3) and their adjacent cells (n = 11, 33,500 ± 3,300 µm3) were significantly greater than volumes of remote cells (n = 13, 18,600 ± 1,700 µm3, P < 0.01 vs. HB-EGF-infected cells and P < 0.05 vs. HB-EGF-adjacent cells, Fig. 4D). In contrast, cells treated with Ad-GFP (n = 12) showed no hypertrophy in the Ad-GFP-adjacent (n = 10) or remote cells (n = 9). These data demonstrate that HB-EGF acts as both an autocrine and local paracrine growth factor within myocardium, as predicted by computational modeling.

Degradation of Cx43 Through Local Autocrine/Paracrine HB-EGF

To determine whether the spatially confined effect of HB-EGF reduces local myocardial Cx43 in vivo, Cx43 was assessed with immunohistochemistry and confocal fluorescence imaging. Cells infected with Ad-HB-EGF had significant decreases in Cx43 immunoreactive signal compared with Ad-GFP cells, consistent with the results of in vitro immunoblotting (Fig. 5A). Quantitative digital image analyses of Cx43 in a total of 22 fields in 6 Ad-HB-EGF hearts and 19 fields in 4 Ad-GFP hearts were analyzed (Fig. 5B). Although Ad-GFP-infected cells showed immunoreactive Cx43 at the appositional membrane, overexpression of HB-EGF increased Cx43 in intracellular vesicle-like components (Fig. 5C), with reduced gap junction plaques (percent Cx43 area per cell area, 52 ± 8% of Ad-GFP control, P < 0.01). These data suggest that reduced expression of Cx43 can be attributed to an increased rate of internalization and degradation in gap junction plaques in cardiomyocytes. Interestingly, HB-EGF secretion by a given cardiomyocyte caused a 37 ± 13% reduction of Cx43 content in its adjacent cells compared with GFP controls (P < 0.05). As degradation of Cx43 may accompany structural changes with marked rearrangement of intercellular connections.  In contrast to Cx43, there was no significant difference in total area occupied by N-cadherin immunoreactive signal in between Ad-GFP (n = 19) and Ad-HB-EGF hearts (1.8 ± 0.5-fold compared with Ad-GFP, n = 17, P = not significant), indicating that HB-EGF has a selective effect on Cx43. Taken together, these data show that HB-EGF leads to cardiomyocyte hypertrophy and degradation of Cx43 in the infected cell and its immediately adjacent neighbors because of autocrine/ paracrine signaling. It should be noted, however, that quantifying the Cx43 from immunostaining could be limited by a nonlinear relation between the amount of Cx43 present and the area of staining.

Fig. 4. Effects of gene transfer of HB-EGF on cardiomyocyte hypertrophy in vivo.

(A) Adenoviral vectors (Ad-GFP or Ad-HB-EGF) were injected into the left ventricular free wall in mice. Myocytes were grouped as infected or noninfected on the basis of GFP fluorescence. Overex-pression of HB-EGF was confirmed by im-munohistochemistry. The presented image was pseudocolored with blue from that stained with Alexa Fluor 555 for the presence of HB-EGF. (Scale bars: 20 sm.) (B) 2D cross-sectional area of cardiomyo-cytes was measured in infected and non-infected cells in the same region of the same animal. Overexpression of HB-EGF caused cellular hypertrophy in both infected and adjacent cells. **, P < 0.01 vs. Ad-GFP infected; , P < 0.01 vs. Ad-HB-EGF remote; and §, P < 0.01 vs. Ad-GFP adjacent cells. GFP (infected 102 cells, adjacent 92 cells, and remote 97 cells from 5 mice), and HB-EGF (infected 119 cells, adjacent 97 cells, and remote 109 cells from 7 mice). The 3D histology also revealed cellular hypertrophy in both Ad-HB-EGF-infected cell and its adjacent cell. **, P < 0.01 vs. Ad-GFP infected; , P < 0.01; and *, P < 0.05 vs. Ad-HB-EGF remote cells. GFP (infected 12 cells, adjacent 10 cells, and remote 9 cells), and HB-EGF (infected 19 cells, adjacent 11 cells, and remote 13 cells). Statistical analysis was performed with one-way ANOVA. (C) Sample image of extracted myocytes in three dimensions.


We have demonstrated in this study that HB-EGF secreted by cardiomyocytes leads to cellular growth and reduced expression of the principal ventricular gap junction protein Cx43 in a local autocrine/paracrine manner. Although proHB-EGF is biologically active as a juxtacrine growth factor that can signal to immediately neighboring cells in a nondiffusible mannerseveral studies have revealed the crucial role of metalloproteases in the enzymatic conversion of proHB-EGF to soluble HB-EGF, which binds to and activates the EGFR. Hypertrophic stimuli such as mechanical strain and G protein-coupled receptors agonists mediate cardiac hypertrophy through the shedding of membrane-bound proHB-EGF. Thus, an autocrine/paracrine loop, which requires the diffusible, soluble form of HB-EGF, is necessary for subsequent transactivation of the EGFR to produce the hypertrophic response.

To our knowledge, there have been no previous reports concerning the spatial extent of autocrine/paracrine ligand distribution and signaling in myocardial tissue. A theoretical analysis by Shvartsman et al. predicted, from computational modeling in an idealized cell culture environment, that autocrine ligands may remain highly localized, even within subcellular distances; this prediction has support from experimental data in the EGFR system. In contrast, a theoretical estimate by Francis and Palsson has suggested that cytokines might effectively communicate larger distances, approximated to be 200–300 m from the point of release. However, these studies have all focused on idealized cell culture systems, so our combined experimental and computational investigation here aimed at understanding both in vitro and in vivo situations offers insight.
Our computational model of diffusion in the extracellular space predicts that HB-EGF acts as both an autocrine and spatially restricted paracrine growth factor for neighboring cells. We studied the responses of the signaling cell and its immediate neighbors compared with more distant cells. For a paracrine signal to be delivered to its proper target, the secreted signaling molecules cannot diffuse too far; in vitro experiments, in fact, indicated that HB-EGF acts as a predominantly autocrine signal in cell culture, where diffusion into the medium is relatively unconstrained.
In contrast, in the extracellular space of the myocardium, HB-EGF is localized around the source of production because of tissue geometry, thereby acting in a local paracrine or autocrine manner only. Indeed, our results from in vivo gene transfer demonstrated that both the cell releasing soluble HB-EGF and its surrounding cells undergo hypertrophy. This localized conversation between neighboring cells may allow remodeling to be fine-tuned on a highly spatially restricted level within the myocardium and in other tissues.

Common genetic variation at the IL1RL1locus regulates IL-33/ST2 signaling

JE Ho, Wei-Yu Chen, Ming-Huei Chen, MG Larson, ElL McCabe, S Cheng, A Ghorbani, E Coglianese, V Emilsson, AD Johnson,….. CARDIoGRAM Consortium, CHARGE Inflammation Working Group, A Dehghan, C Lu, D Levy, C Newton-Cheh, CHARGE Heart Failure Working Group, …. JL Januzzi, RT Lee, and TJ Wang J Clin Invest Oct 2013; 123(10):4208-4218.  http://dx.doi.org/10.1172/JCI67119

Abstract and Introduction

The suppression of tumorigenicity 2/IL-33 (ST2/IL-33) pathway has been implicated in several immune and inflammatory diseases. ST2 is produced as 2 isoforms. The membrane-bound isoform (ST2L) induces an immune response when bound to its ligand, IL-33. The other isoform is a soluble protein (sST2) that is thought to be a decoy receptor for IL-33 signaling. Elevated sST2 levels in serum are associated with an increased risk for cardiovascular disease. We investigated the determinants of sST2 plasma concentrations in 2,991 Framing­ham Offspring Cohort participants. While clinical and environmental factors explained some variation in sST2 levels, much of the variation in sST2 production was driven by genetic factors. In a genome-wide associ­ation study (GWAS), multiple SNPs within IL1RL1 (the gene encoding ST2) demonstrated associations with sST2 concentrations. Five missense variants of IL1RL1 correlated with higher sST2 levels in the GWAS and mapped to the intracellular domain of ST2, which is absent in sST2. In a cell culture model, IL1RL1 missense variants increased sST2 expression by inducing IL-33 expression and enhancing IL-33 responsiveness (via ST2L). Our data suggest that genetic variation in IL1RL1 can result in increased levels of sST2 and alter immune and inflammatory signaling through the ST2/IL-33 pathway. Suppression of tumorigenicity 2 (ST2) is a member of the IL-1 receptor (IL-1R) family that plays a major role in immune and inflammatory responses. Alternative promoter activation and splicing produces both a membrane-bound protein (ST2L) and a soluble form (sST2). The transmembrane form of ST2 is selectively expressed on Th2- but not Th1-type T cells, and bind­ing of its ligand, IL-33, induces Th2 immune responses.  In contrast, the soluble form of ST2 acts as a decoy receptor by sequestering IL-33. The IL-33/ST2 pathway has important immunomodulatory effects. Clinically, the ST2/IL-33 signaling pathway participates in the pathophysiology of a number of inflammatory and immune diseases related to Th2 activation, including asthma, ulcera­tive colitis, and inflammatory arthritis. ST2 expression is also upregulated in cardiomyocytes in response to stress and appears to have cardioprotective effects in experimental studies. As a biomarker, circulating sST2 concentrations have been linked to worse prognosis in patients with heart failure, acute dyspnea, and acute coronary syndrome, and also predict mortality and incident cardiovascular events in individuals without existing cardiovascular disease. Both sST2 and its transmembrane form are encoded by IL-1R– like 1 (IL1RL1). Genetic variation in this pathway has been linked to a number of immune and inflammatory diseases. The contribution of IL1RL1 locus variants to interindividual variation in sST2 has not been investigated. The emergence of sST2 as an important predictor of cardiovascular risk and the important role outside of the ST2/IL-33 pathway in inflammatory diseases highlight the value of understanding genetic determinants of sST2. The fam­ily-based FHS cohort provides a unique opportunity to examine the heritability of sST2 and to identify specific variants involved using a genome-wide association study (GWAS). Thus, we per­formed a population-based study to examine genetic determinants of sST2 concentrations, coupled with experimental studies to elu­cidate the underlying molecular mechanisms.


Clinical characteristics of the 2,991 FHS participants are presented in Supplemental Table 1 (supplemental material available online with this article; doi:10.1172/JCI67119DS1). The mean age of participants was 59 years, and 56% of participants were women. Soluble ST2 concentrations were higher in men compared with those in women (P < 0.001). Soluble ST2 concentrations were positively associated with age, systolic blood pressure, body-mass index, antihypertensive medication use, and diabetes mellitus (P < 0.05 for all). Together, these variables accounted for 14% of the variation in sST2 concentrations. The duration of hypertension or diabetes did not materially influence variation in sST2 concentra­tions. After additionally accounting for inflammatory conditions, clinical variables accounted for 14.8% of sST2 variation.

Heritability of sS72.

The age- and sex-adjusted heritability (h2) of sST2 was 0.45 (P = 5.3 x 10–16), suggesting that up to 45% of the vari­ation in sST2 not explained by clinical variables was attributable to genetic factors. Multivariable adjustment for clinical variables pre­viously shown to be associated with sST2 concentrations (21) did not attenuate the heritability estimate (adjusted h2 = 0.45, P = 8.2 x 10–16). To investigate the influence of shared environmental fac­tors, we examined the correlation of sST2 concentrations among 603 spousal pairs and found no significant correlation (r = 0.05, P = 0.25).

Genetic correlates of sS72.

We conducted a GWAS of circulating sST2 concentrations. Quantile-quantile, Manhattan, and regional linkage disequilibrium plots are shown in Supplemental Figures 1–3.  All genome-wide significant SNPs were located in a 400-kb linkage disequilibrium block that included IL1RL1 (the gene encoding ST2), IL1R1, IL1RL2, IL18R1, IL18RAP, and SLC9A4 (Figure 1). Results for 11 genome-wide significant “indepen­dent” SNPs, defined as pairwise r2 < 0.2, are shown in Table 1. In aggregate, these 11 “independent” genome-wide significant SNPs across the IL1RL1 locus accounted for 36% of heritability of sST2. In conditional analyses, 4 out of the 11 SNPs remained genome-wide significant, independent of each other (rs950880, rs13029918, rs1420103, and rs17639215), all within the IL1RL1 locus. The most significant SNP (rs950880, P = 7.1 x 10–94) accounted for 12% of the residual interindividual variability in circulating sST2 concentrations. Estimated mean sST2 concen­trations were 43% higher in major homozygotes (CC) compared with minor homozygotes (AA). Tree loci outside of the IL1RL1 locus had suggestive associations with sST2 (P < 1 x 10–6) and are displayed in Supplemental Table 3.

In silico association with expression SNPs.

The top 10 sST2 SNPs (among 11 listed in Table 1) were explored in collected gene expression databases. There were 5 genome-wide significant sST2 SNPs associated with gene expression across a variety of tissue types (Table 2). Specifically, rs13001325 was associated with IL1RL1 gene expression (the gene encoding both soluble and transmembrane ST2) in several subtypes of brain tissue (prefrontal cortex, P = 1.95 x 10–12; cerebellum, P = 1.54 x 10–5; visual cortex, P = 1.85 x 10–7). The CC genotype of rs13001325 was associated with a higher IL1RL1 gene expression level as well as a higher circulating sST2 concentration when compared with the TT genotype (Supplemental Figure 4). Other ST2 variants were significantly associated with IL18RAP (P = 8.50 x 10–41, blood) and IL18R1 gene expression (P = 2.99 x 10–12, prefrontal cortex).

In silico association with clinical phenotypes in published data

The G allele of rs1558648 was associated with lower sST2 concentra­tions in the FHS (0.88-fold change per G allele, P = 3.94 x 10–16) and higher all-cause mortality (hazard ratio [HR] 1.10 per G allele, 95% CI 1.03–1.16, P = 0.003) in the CHARGE consortium, which observed 8,444 deaths in 25,007 participants during an average fol­low-up of 10.6 years (22). The T allele of rs13019803 was associated with lower sST2 concentrations in the FHS (0.87-fold change per G allele, P = 5.95 x 10–20), higher mortality in the CHARGE consor­tium (HR 1.06 per C allele, 95% CI 1.01–1.12, P = 0.03), and higher risk of coronary artery disease (odds ratio 1.06, 95% CI 1.00–1.11, P = 0.035) in the CARDIoGRAM consortium, which included 22,233 individuals with coronary artery disease and 64,762 controls (23). In relating sST2 SNPs to other clinical phenotypes (including blood pressure, body-mass index, lipids, fasting glucose, natriuretic peptides, C-reactive protein, and echocardiographic traits) in pre­viously published studies, we found nominal associations with C-reactive protein for 2 SNPs (Supplemental Table 4).

Putative functional variants.

Using GeneCruiser, we examined nonsynonymous SNPs (nSNPs) (missense variants) that had at least suggestive association with sST2 (P < 1 x 10–4), includ­ing SNPs that served as proxies (r2 = 1.0) for nSNPs within the 1000 Genomes Pilot 1 data set (ref. 24 and Table 3). There were 6 missense variants located within the IL1RL1 gene, 5 of which had genome-wide significant associations with sST2 concen­trations, including rs6749114 (proxy for rs10192036, Q501K), rs4988956 (A433T), rs10204137 (Q501R), rs10192157 (T549I), rs10206753 (L551S), and rs1041973 (A78AE). Base substitutions and corresponding amino acid changes for these coding muta­tions are listed in Table 3. In combination, these 6 missense muta­tions accounted for 5% of estimated heritability, with an effect estimate of 0.23 (standard error [s.e.] 0.02, P = 2.4 x 10–20). When comparing major homozygotes with minor homozygotes, the esti­mated sST2 concentrations for these missense variants differed by 11% to 15% according to genotype (Supplemental Table 5). In conditional analyses, intracellular and extracellular variants appeared to be independently associated with sST2. For instance, in a model containing rs4988956 (A433T) and rs1041973 (A78E), both SNPs remained significantly associated with sST2 (P = 2.61 x 10–24 and P = 7.67 x 10–15, respectively). In total, missence variants added little to the proportion of sST2 variance explained by the 11 genome-wide significant nonmissense variants listed in Table 1. In relating these 6 missense variants to other clinical phenotypes in large consortia, we found an association with asthma for 4 out of the 6 variants (lowest P = 4.8 x 10–12 for rs10204137) (25).

Homology map of IL1RL1 missense variants and ST2 structure.

Of the 6 missense variants mapping to IL1RL1, 5 were within the cytoplas­mic Toll/IL-1R (TIR) domain of the transmembrane ST2 receptor (Figure 2A), and these intracellular variants are thus not part of the circulating sST2 protein. Of these cytoplasmic domain variants, A433T was located within the “box 2” region of sequence conserva­tion, described in the IL-1R1 TIR domain as important for IL-1 sig­naling . Q501R/K was within a conserved motif called “box 3,” but mutants of IL-1R1 in box 3 did not significantly affect IL-1 signaling in previous experiments (26). Both T549I and L551S were near the C terminus of the transmembrane ST2 receptor and were not predicted to alter signaling function based on previous exper­iments with the IL-1R . The A78E SNP was located within the extracellular domain of ST2 and is thus present in both the sST2 isoform and the transmembrane ST2 receptor. In models of the ST2/IL-33/IL-1RAcP complex derived from a crystal structure of the IL-1RII/IL-1β/IL-1RAcP complex (protein data bank ID 1T3G and 3O4O), A78E was predicted to be located on a surface loop within the first immunoglobulin-like domain (Figure 2B), distant from the putative IL-33 binding site or the site of interaction with IL-1RAcP. There were 2 rare extracellular variants that were not cap­tured in our GWAS due to low minor allele frequencies (A80E, MAF 0.008; A176T, MAF 0.002). Both were distant from the IL-33 bind­ing site on homology mapping and unlikely to affect IL-33 binding.

Functional effects of IL1RL1 missense variants on sST2 expression and promoter activity.

Since 5 of the IL1RL1 missense variants asso­ciated with sST2 levels mapped to the intracellular domain of ST2L and hence are not present on sST2 itself, we hypothesized that these missense variants exert effects via intracellular mecha­nisms downstream of ST2 transmembrane receptor signaling to regulate sST2 levels. To investigate the effect of IL1RL1 missense variants (identified by GWAS) on sST2 expression, stable cell lines expressing WT ST2L, IL1RL1 variants (A78E, A433T, T549I, Q501K, Q501R, and L551S), and a construct containing the 5 IL1RL1 intracellular domain variants (5-mut) were generated. Expression of ST2L mRNA and protein (detected in membrane fractions) was confirmed (Supplemental Figures 5 and 6). Eight different stable clones in each group were analyzed to reduce bias from clonal selection. Intracellular domain variants (A433T, T549I, Q501K, Q501R, L551S, and 5-mut), but not the extracellular domain variant (A78E), were associated with increased basal sST2 expression when compared with WT expression (P < 0.05 for all, Figure 3A). sST2 expression was highest in the 5-mut construct, suggesting that intracellular ST2L variants cooperatively regulate sST2 levels. This same pattern was consistent across different cell types (U937, Jurkat T, and A549 cells; Supplemental Figure 7). These findings suggest that intracellular domain variants of the transmembrane ST2 receptor may functionally regulate downstream signaling.   IL1RL1 transcription may occur via two alternative promoters (proximal vs. distal), which leads to differential expression of the soluble versus membrane-bound ST2 proteins. Similar to the sST2 protein expression results above, the intracellular domain variants, but not the extracellular domain variant, were associated with higher basal proximal promoter activity. Dis­tal promoter activity was also increased for most intracellular domain variants (Supplemental Figure 8).

IL1RL1 intracellular missense variants resulted in higher IL-33 pro­tein levels.

In addition to upregulation of sST2 protein levels, IL1RL1 intracellular missense variants caused increased basal IL-33 protein expression (Figure 3B), suggesting a possible autoregulatory loop whereby IL-33 signaling positively induces sST2 expression. IL-33 induced sST2 protein expression in cells expressing both WT and IL1RL1 missense variants. Interest­ingly, this effect was particularly pronounced in the A433T and Q501R variants (Supplemental Figure 9A).

Enhanced IL-33 responsiveness is mediated by IL-113 in A433T and Q501R variants.

Interaction among IL-33, sST2, and IL-113.

Inhibition of IL-113 by anti–IL-113 mAb reduced basal expression of sST2 (Supplemental Figure 11A). Blocking of IL-33 by sST2 did not reduce the induction of IL-113 levels by the IL1RL1 variants (Supplemental Figure 11B). Furthermore, inhibition of IL-113 by anti–IL-113 reduced the basal IL-33 levels. IL-33 itself upregulated sST2 levels, which in turn reduced IL-33 levels (Supplemental Figure 11C). Our results revealed that both IL-33 and IL-113 drive sST2 expression and that IL-113 acts as an upstream inducer of IL-33 and maintains IL-33 expression by intracellular IL1RL1 vari­ants (Supplemental Figure 11D). This suggests that IL1RL1 vari­ants upregulated sST2 mainly through IL-33 autoregulation and that the enhanced IL-33 responsiveness by A433T and Q501R was mediated by IL-113 upregulation.

IL1RL1 missense variants modulate ST2 signaling pathways

The effect of IL1RL1 missense variants on known ST2 downstream regulatory pathways, including NF-KB, AP-1/c-Jun, AKT, and STAT3 , was examined in the presence and absence of IL-33 (Figure 4 and Supplemental Figure 12). The IL1RL1 intracellular missense vari­ants (A433T, T549I, Q501K, Q501R, and L551S) were associated with higher basal phospho–NF-KB p65 and phospho–c-Jun levels (Figure 4, A and B). Consistent with enhanced IL-33 responsive­ness in A433T and Q501R cells, levels of IL-33–induced NF-KB and c-Jun phosphorylation were enhanced in these 2 variants (Figure 4, B and D). In contrast, A433T and Q501R variants showed lower basal phospho-AKT levels (Figure 4E). ……….. The majority of sST2 gene variants in our study were located within or near IL1RL1, the gene coding for both transmembrane ST2 and sST2. IL1RL1 resides within a linkage disequilibrium block of 400 kb on chromosome 2q12, a region that includes a number of other cytokines, including IL-18 receptor 1 (IL18R1) and IL-18 receptor accessory protein (IL18RAP). Polymorphisms in this gene cluster have been associated previously with a num­ber of immune and inflammatory conditions, including asthma, celiac disease, and type 1 diabetes mellitus . Many of these variants were associated with sST2 concentrations in our analysis (Supplemental Table 6). The immune effects of ST2 are corroborated by experimental evidence: membrane-bound ST2 is selectively expressed on Th2- but not Th1-type T helper cells, and activation of the ST2/IL-33 axis elaborates Th2 responses. In general, the allergic phenotypes above are thought to be Th2-mediated processes, in contrast to atherosclerosis, which appears to be a Th1-driven process.

Fig 2  Models of ST2 illustrate IL1RL1 missense variant locations.

Figure 2 Models of ST2 illustrate IL1RL1 missense variant locations.

Models of the (A) intracellular TIR domain (ST2-TIR) and the (B) extracellular domain (ST2-ECD) of ST2 (protein data bank codes 3O4O and 1T3G, respectively). Domains of ST2 are shown in yellow, with identified mis-sense SNP positions represented as red spheres and labels. Note that positions 549 and 551 are near the C terminus of ST2, which is not defined in the crystal structure (protein data bank ID 1T3G, shown as dashed black line in A). Arrows point toward the transmembrane domain, which is also not observed in crystal structures.

Fig 3 IL1RL1 intracellular missense variants resulted in higher sST2 and IL-33.

Figure 3   IL1RL1 intracellular missense variants resulted in higher sST2 and IL-33.

Media from KU812 cells expressing WT and IL1RL1 missense variants were collected for ELISA analysis of (A) sST2, (B) IL-33, and (C) IL-113 levels. Horizontal bars indicate mean values, and symbols represent indi­vidual variants. *P < 0.05, **P < 0.01 vs. WT. (D) Effect of anti–IL-113 mAb on IL-33–induced sST2 expression. Dashed line indicates PBS-treated cells as referent group. Error bars represent mean ± SEM from 2 independent experiments. *P < 0.05 vs. IL-33.

Fig 4  IL1RL1 missense variants modulated ST2 signaling pathways

Figure 4  IL1RL1 missense variants modulated ST2 signaling pathways. 

KU812 cells expressing WT or IL1RL1 variants were treated with PBS or IL-33. Levels of the following phosphorylated proteins were detected in cell lysates using ELISA: (A and B) phospho-NF-KB p65; (C and D) phospho-c-Jun activity; and (E and F) phospho-AKT. (A, C, and E) White bars represent basal levels, and (B, D, and F) gray bars represent relative fold increase (compared with PBS-treated group) after IL-33 treatment. *P < 0.05 vs. WT; **P < 0.01 vs. PBS-treated group. Dashed line in B, D, and F represents PBS-treated cells as referent group. Error bars represent mean ± SEM from 2 independent experiments. Fig 5   IL-33–induced sST2 expression is enhanced with mTOR inhibition and occurs via ST2L-dependent signaling.

Figure 5  IL-33–induced sST2 expression is enhanced with mTOR inhibition and occurs via ST2L-dependent signaling.

(A) sST2 mRNA expression in KU812 cells after treatment with DMSO, IL-33, or IL-33 plus signal inhibitors (wortmannin, LY294002, rapamycin, PD98059, SP60125, BAY11-7082, or SR11302). (B) ST2L mRNA and (C) sST2 mRNA expression in KU812 cells treated with PBS (white columns), rapamycin (rapa), anti-ST2 mAb, IL-33, IL-33 plus anti-ST2, IL-33 plus rapamycin, IL-33 plus rapamycin plus anti-ST2 mAb, or rapamycin plus anti-ST2. (D) IL33 mRNA expression in KU812 cells after treatment with DMSO, signal inhibitors, IL-33 plus signal inhibitors, and IL-1n plus signal inhibitors. *P < 0.05 vs. PBS-treated group; #P < 0.05 vs. IL-33–treated group; &P < 0.05 vs. IL-1n–treated group. Error bars represent mean ± SEM from 2 independent experiments. (E) A schematic model illustrating the regulation of sST2 expression by IL1RL1 missense variants through enhanced induction of IL-33 via enhanced NF-KB and AP-1 signaling and enhanced IL-33 responsiveness via increasing ST2L expression.

Quantitating subcellular metabolism with multi-isotope imaging mass spectrometry

ML Steinhauser, A Bailey, SE Senyo, C Guillermier, TS Perlstein, AP Gould, RT Lee, and CP Lechene
Department of Medicine, Divisions of Cardiovascular Medicine & Genetics, Brigham and Women’s Hospital, Harvard Medical School & Harvard Stem Cell Institute Division of Physiology and Metabolism, Medical Research Council National Institute for Medical Research, Mill Hill, London, UK National Resource for Imaging Mass Spectroscopy
Nature 2012;481(7382): 516–519.   http://dx. do.org/10.1038/nature10734

Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter, but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with sub-micron resolution. Here we apply MIMS to diverse organisms, including Drosophila, mice, and humans. We test the “immortal strand hypothesis,” which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labeling mice with 15N-thymidine from gestation through post-natal week 8, we find no 15N label retention by dividing small intestinal crypt cells after 4wk chase. In adult mice administered 15N-thymidine pulse-chase, we find that proliferating crypt cells dilute label consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human hematopoietic system. These studies show that MIMS provides high-resolution quantitation of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research. MIMS combines ion microscopy with secondary ion mass spectrometry (SIMS), stable isotope reporters, and intensive computation (Supplemental Fig 1). MIMS allows imaging and measuring stable isotope labels in cell domains smaller than one micron cubed. We tested the potential of MIMS to quantitatively track DNA labeling with 15N-thymidine in vitro. In proliferating fibroblasts, we detected label incorporation within the nucleus by an increase in the 15N/14N ratio above natural ratio (Fig 1a). The labeling pattern resembled chromatin with either stable isotope-tagged thymidine or thymidine analogs (Fig 1b). We measured dose-dependent incorporation of 15N-thymidine over three orders of magnitude (Fig 1d, Supplemental Fig 2). We also tracked fibroblast division after a 24-hour label-free chase (Fig 1d,e, Supplemental Fig 3). Cells segregated into two populations, one indistinguishable from control cells suggesting no division, the other with halving of label, consistent with one division during chase. We found similar results by tracking cell division in vivo in the small intestine (Fig 1f,g, Supplemental Figs 4–6). We measured dose-dependent 15N-thymidine incorporation within nuclei of actively dividing crypt cells (Fig 1g, Supplemental Fig 4), down to a dose of 0.1µg/ g (Supplemental Fig 2). The cytoplasm was slightly above natural ratio, likely due to low level soluble 15N-thymidine or mitochondrial incorporation (Supplemental Fig 2). We measured halving of label with each division during label-free chase (Supplemental Fig 6). We then tested the “immortal strand hypothesis,” a concept that emerged from autoradiographic studies and that predicted long-term label retaining cells in the small intestinal crypt. It proposes that asymmetrically dividing stem cells also asymmetrically segregate DNA, such that older template strands are retained by daughter cells that will remain stem cells and newer strands are passed to daughters committed to differentiation (Supplemental Fig 7)5,6. Modern studies continue to argue both for or against the hypothesis, leading to the suggestion that definitive resolution of the debate will require a new experimental approach. Although prior evidence suggests a concentration of label-retaining cells in the +4 anatomic position, we searched for DNA label retention irrespective of anatomic position or molecular identity. We labeled mice with 15N-thymidine for the first 8 wks of life when intestinal stem cells are proposed to form. After a 4-wk chase, mice received bromodeoxyuridine (BrdU) for 24h prior to sacrifice to identify proliferating cells(Fig 2a, Supplemental Fig 8: Exp 1), specifically crypt base columnar (CBC) cells and transit amplifying cells (TA) (Supplemental Fig 9), which cycle at a rate of one and two times per 24h, respectively (Supplemental Fig 10). All crypt cell nuclei were highly labeled upon completion of 15N-thymidine; after a four-week chase, however, we found no label retention by non-Paneth crypt cells (Fig 2b–f; n=3 mice, 136 crypts analysed). 15N-labeling in BrdU/15N+ Paneth and mesenchymal cells was equivalent to that measured at pulse completion (Fig2b,c) suggesting quiescence during the chase (values above 15N/14N natural ratio: Paneth pulse=107.8 +/− 5.0% s.e.m. n=51 vs Paneth pulse-chase=96.3+/−2.8% s.e.m. n=218; mesenchymal pulse=92.0+/−5.0% s.e.m. n=89 vs mesenchymal pulse-chase=90.5+/ −2.2% s.e.m. n=543). The number of randomly selected crypt sections was sufficient to detect a frequency as low as one label-retaining stem cell per crypt irrespective of anatomic location within the crypt. Because each anatomic level contains approximately 16 circumferentially arrayed cells, a 2-dimensional analysis captures approximately 1/8th of the cells at each anatomic position (one on each side of the crypt; Supplemental Fig 9a). Therefore, assuming only 1 label-retaining stem cell per crypt we should have found 17 label-retaining cells in the 136 sampled crypts (1/8th of 136); we found 0 (binomial test p<0.0001). The significance of this result held after lowering the expected frequency of label-retaining cells by 25% to account for the development of new crypts, a process thought to continue into adulthood. In three additional experiments, using shorter labeling periods and including in utero development, we also found no label-retaining cells in the crypt other than Paneth cells (Supplemental Fig 8, Exps 2–4).

Fig 1 post-natal human DNA synthesis in the heart

In recent years, several protocols have been developed experimentally in an attempt to identify novel therapeutic interventions aiming at the reduction of infarct size and prevention of short and long term negative ventricular remodeling following ischemic myocardial injury. Three main strategies have been employed and a significant amount of work is being conducted to determine the most effective form of action for acute ischemic heart failure. The delivery of bone marrow progenitor cells (BMCs) has been highly controversial, but recent clinical data have shown improvement in ventricular performance and clinical outcome. These observations have not changed the nature of the debate concerning the efficacy of this cell category for the human disease and the mechanisms involved in the impact of BMCs on cardiac structure and function. Whether BMCs transdifferentiate and acquire the cardiomyocyte lineage has faced strong opposition and data in favor and against this possibility have been reported. However, this is the only cell class which has been introduced in the treatment of heart failure in patients and large clinical trials are in progress.
Human embryonic stem cells (ESCs) have repeatedly been utilized in animal models to restore the acutely infarcted myocardium, but limited cell engraftment, modest ability to generate vascular structures, teratoma formation and the apparent transient beneficial effects on cardiac hemodynamics have questioned the current feasibility of this approach clinically. Tremendous efforts are being performed to reduce the malignant tumorigenic potential of ESCs and promote their differentiation into cardiomyocytes with the expectation that these extremely powerful cells may be applied to human beings in the future. Additionally, the study of ESCs may provide unique understanding of the mechanisms of embryonic development that may lead to therapeutic interventions in utero and the correction of congenital malformations.
The recognition that a pool of primitive cells with the characteristics of stem cells resides in the myocardium and that these cells form myocytes, ECs and SMCs has provided a different perspective of the biology of the heart and mechanisms of cardiac homeostasis and tissue repair. Regeneration implies that dead cells are replaced by newly formed cells restoring the original structure of the organ. In adulthood, this process occurs during physiological cell turnover, in the absence of injury. However, myocardial damage interferes with recapitulation of cell turnover and restitutio ad integrum of the organ. Because of the inability of the adult heart to regenerate itself after infarction, previous studies have promoted tissue repair by injecting exogenously expanded CPCs in proximity of the necrotic myocardium or by activating resident CPCs through the delivery of growth factors known to induce cell migration and differentiation. These strategies have attenuated ventricular dilation and the impairment in cardiac function and in some cases have decreased animal mortality.

Although various subsets of CPCs have been used to reconstitute the infarcted myocardium and different degrees of muscle mass regeneration have been obtained, in all cases the newly formed cardiomyocytes possessed fetal-neonatal characteristics and failed to acquire the adult cell phenotype. In the current study, to enhance myocyte growth and differentiation, we have introduced cell therapy together with the delivery of self-assembly peptide nanofibers to provide a specific and prolonged local myocardial release of IGF-1. IGF-1 increases CPC growth and survival in vitro and in vivo and this effect resulted here in a major increase in the formation of cardiomyocytes and coronary vessels, decreasing infarct size and restoring partly cardiac performance. This therapeutic approach was superior to the administration of CPCs or NF-IGF-1 only. Combination therapy appeared to be additive; it promoted myocardial regeneration through the activation and differentiation of resident and exogenously delivered CPCs. Additionally, the strategy implemented here may be superior to the utilization of BMCs for cardiac repair. CPCs are destined to form myocytes, and vascular SMCs and ECs and, in contrast to BMCs, do not have to transdifferentiate to acquire cardiac cell lineages. Transdifferentiation involves chromatin reorganization with activation and silencing of transcription factors and epigenetic modifications.

Selected References

  1. Hsieh PC, Davis ME, Gannon J, MacGillivray C, Lee RT. Controlled delivery of PDGF-BB for myocardial protection using injectable self-assembling peptide nanofibers. J Clin Invest 2006;116:237–248. [PubMed: 16357943]
  2. Davis ME, Hsieh PC, Takahashi T, Song Q, Zhang S, Kamm RD, Grodzinsky AJ, Anversa P, Lee RT. Local myocardial insulin-like growth factor 1 (IGF-1) delivery with biotinylated peptide nanofibers improves cell therapy for myocardial infarction. Proc Natl Acad Sci USA 2006;103:8155–8160. [PubMed: 16698918]
  3. Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K, Leri A, Kajstura J, Nadal-Ginard B, Anversa P. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114:763–776. [PubMed: 14505575]
  4. Rota M, Padin-Iruegas ME, Misao Y, De Angelis A, Maestroni S, Ferreira-Martins J, Fiumana E, Rastaldo R, Arcarese ML, Mitchell TS, Boni A, Bolli R, Urbanek K, Hosoda T, Anversa P, Leri A, Kajstura J. Local activation or implantation of cardiac progenitor cells rescues scarred infarcted myocardium improving cardiac function. Circ Res 2008;103:107–116. [PubMed: 18556576]

Cardiac anatomy.

Figure 2.  Cardiac anatomy.

(A and B) Cardiac weights and infarct size. R and L correspond, respectively, to the number of myocytes remaining and lost after infarction. (C–G) LV dimensions. Sham-operated: SO. *Indicates P<0.05 vs SO; **vs untreated infarcts (UN); †vs infarcts treated with CPCs; ‡vs infarcts treated with NF-IGF-1.

Ventricular function

Figure 3.  Ventricular function.

Combination therapy (CPC-NF-IGF-1) attenuated the most the negative impact of myocardial infarction on cardiac performance. See Figure 2 for symbols.

Endothelial Cells Promote Cardiac Myocyte Survival and Spatial Reorganization: Implications for Cardiac Regeneration

Daria A. Narmoneva, Rada Vukmirovic, Michael E. Davis, Roger D. Kamm,  and Richard T. Lee
Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, and the Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA
Circulation. 2004 August 24; 110(8): 962–968.        http://dx.doi.org/10.1161/01.CIR.0000140667.37070.07


Endothelial-cardiac myocyte (CM) interactions play a key role in regulating cardiac function, but the role of these interactions in CM survival is unknown. This study tested the hypothesis that endothelial cells (ECs) promote CM survival and enhance spatial organization in a 3-dimensional configuration.

Methods and Results

Microvascular ECs and neonatal CMs were seeded on peptide hydrogels in 1 of 3 experimental configurations:

  1. CMs alone,
  2. CMs mixed with ECs (coculture), or
  3. CMs seeded on preformed EC networks (prevascularized).

Capillary-like networks formed by ECs promoted marked CM reorganization along the EC structures, in contrast to limited organization of CMs cultured alone. The presence of ECs markedly inhibited CM apoptosis and necrosis at all time points. In addition, CMs on preformed EC networks resulted in significantly less CM apoptosis and necrosis compared with simultaneous EC-CM seeding (P<0.01, ANOVA). Furthermore, ECs promoted synchronized contraction of CMs as well as connexin 43 expression.


These results provide direct evidence for a novel role of endothelium in survival and organization of nearby CMs. Successful strategies for cardiac regeneration may therefore depend on establishing functional CM-endothelium interactions.

Keywords:  endothelium; cardiomyopathy; heart failure; tissue


Recent studies suggest that the mammalian heart possesses some ability to regenerate itself through several potential mechanisms, including generation of new cardiomyocytes (CMs) from extracardiac progenitors, CM proliferation, or fusion with stem cells with subsequent hybrid cell division. These mechanisms are insufficient to regenerate adequate heart tissue in humans, although some vertebrates can regenerate large volumes of injured myocardium.
Several approaches in cell transplantation and cardiac tissue engineering have been investigated as potential treatments to enhance cardiac function after myocardial injury. Implantation of skeletal muscle cells, bone marrow cells, embryonic stem cell-derived CMs, and myoblasts can enhance cardiac function. Cell-seeded grafts have been used instead of isolated cells for in vitro cardiac tissue growth or in vivo transplantation. These grafts can develop a high degree of myocyte spatial organization, differentiation, and spontaneous and coordinated contractions. On implantation in vivo, cardiac grafts can integrate into the host tissue and neovascularization can develop. However, the presence of scar tissue and the death of cells in the graft can limit the amount of new myocardium formed, most likely due to ischemia. Therefore, creating a favorable environment to promote survival of transplanted cells and differentiation of progenitor cells remains one of the most important steps in regeneration of heart tissue.
One of the key factors for myocardial regeneration is revascularization of damaged tissue. In the normal heart, there is a capillary next to almost every CM, and endothelial cells (ECs) outnumber cardiomyocytes by ≈3:1. Developmental biology experiments reveal that myocardial cell maturation and function depend on the presence of endocardial endothelium at an early stage. Experiments with inactivation or overexpression of vascular endothelial growth factor (VEGF) demonstrated that at later stages, either an excess or a deficit in blood vessel formation results in lethality due to cardiac dysfunction. Both endocardium and myocardial capillaries have been shown to modulate cardiac performance, rhythmicity, and growth. In addition, a recent study showed the critical importance of CM-derived VEGF in paracrine regulation of cardiac morphogenesis. These findings and others highlight the significance of interactions between CMs and endothelium for normal cardiac function. However, little is known about the specific mechanisms for these interactions, as well as the role of a complex, 3-dimensional organization of myocytes, ECs, and fibroblasts in the maintenance of healthy cardiac muscle.
The critical relation of CMs and the microvasculature suggests that successful cardiac regeneration will require a strategy that promotes survival of both ECs and CMs. The present study explored the hypothesis that ECs (both as preexisting capillary-like structures and mixed with myocytes at the time of seeding) promote myocyte survival and enhance spatial reorganization in a 3-dimensional configuration. The results demonstrate that CM interactions with ECs markedly decrease myocyte death and show that endothelium may be important not only for the delivery of blood and oxygen but also for the formation and maintenance of myocardial structure.


  • Three-Dimensional Culture
  • Immunohistochemistry and Cell Death Assays
  • Evaluation of Contractile Areas


  • EC-CM Interactions Affect Myocyte Reorganization
  • ECs Improve Survival of CMs
  • Preformed Endothelial Networks Promote Coordinated, Spontaneous Contractions
  • ECs Promote Cx43 Expression

EC-CM Interactions Affect Myocyte Reorganization

To explore interactions between CMs and ECs in 3-dimensional culture, we used peptide hydrogels, a tissue engineering scaffold. Cells seeded on the surface of the hydrogel attach and then migrate into the hydrogel. When CMs alone were used, cells attached on day 1 and then formed small clusters of cells at days 3 and 7 (Figure 1). In contrast, when CMs were seeded together with ECs, cells formed interconnected linear networks, as commonly seen with ECs in 3-dimensional culture environments, with increasing spatial organization from day 1 to day 7 (Figure 1).

Figure 1.  ECs promote CM reorganization. 

When CMs were cultured alone (left column), they aggregated into sparse clusters. When CMs were cultured with ECs (center), cells organized into capillary-like networks. There was no difference in morphological appearance between coculture or prevascularized cultures (not shown) and ECs alone (right column). Bar=100 μm. Abbreviations are as defined in text.

To establish whether preformed endothelial networks enhanced the organization of myocytes, we also seeded ECs 1 day before myocytes were added. These ECs formed similar interconnected networks in the absence of myocytes; preforming the vascular network did not lead to significant differences in morphology (data not shown). Furthermore, to exclude the possibility that the increasing cell density of added ECs caused the spatial organization, we also performed control experiments with varying numbers and combinations of cells; there was no effect of doubling or halving cell numbers, indicating that the spatial organization effect was specifically due to ECs. To establish that both myocytes and ECs were forming networks together, we performed immunofluorescence studies with specific antibodies, as well as analysis of cross sections of CM-EC cocultures, whereby cells were labeled with CellTracker dyes before seeding. Immunofluorescent staining demonstrated that >95% of CMs were present within these networks, suggesting that CMs preferentially migrate to or survive better near ECs (Figure 2).

Figure 2.  CMs appear on outside of endothelial networks.

CMs appear on outside of endothelial networks. High-magnification, double-immunofluorescence image of structures formed in EC-CM coculture at day 7 demonstrating CMs (sarcomeric actinin, red) spread on top of ECs (von Willebrand factor, green) with no myocytes present outside structure. Bar=100 μm. Abbreviations are as defined in text.

The analysis of cross sections demonstrated the presence of what appeared to be EC-derived, tubelike structures (Figure 3), with myocytes spread on the outer part of the capillary wall. Along with the capillary-like structures, clusters of intermingled cells (both myocytes and ECs) not containing the lumen were also observed (not shown). However, when the lumen was present, ECs were always on the inner side and myocytes on the outer side of the structure.

Figure 3.  ECs form tubelike structures with myocytes spreading on outer wall.

Cross section of paraffin-embedded sample of 3-day coculture of myocytes (red) and ECs (green) incubated in CellTracker dye before seeding on hydrogel. Bar=50 μm. Abbreviations are as defined in text.

In CM-fibroblast cocultures, cells rapidly (within 24 hours) formed large clusters consisting of cells of both types (not shown). At later time points, fibroblast proliferation resulted in their migration outside the clusters and spreading on the hydrogel without any pattern. However, in contrast to EC-CM cocultures, CMs remained in the clusters and demonstrated only limited spreading. Immunofluorescent staining revealed that there was no orientation of myocytes relative to the fibroblasts in the clusters. In cultures with EC-conditioned medium, myocyte morphology and spatial organization remained similar to those of myocyte controls.

ECs Improve Survival of CMs

To test the hypothesis that ECs promote CM survival, we assessed apoptosis and necrosis in the 3-dimensional cultures. Quantitative analyses of CMs positive for TUNEL and necrosis staining demonstrated significantly decreased myocyte apoptosis and necrosis when cultured with ECs, compared with CM-only cultures (Figure 4, P<0.01). This effect was observed at all 3 time points, although the decreased necrosis was most pronounced at day 1. In addition, CMs seeded on the preformed EC networks had a lower rate of apoptosis at day 1 relative to same-time seeding cultures (P<0.05, post hoc test), suggesting that early EC-CM interactions provided by the presence of well-attached and prearranged ECs may further promote CM survival. In contrast to the ECs, cardiac fibroblasts did not affect myocyte survival (P>0.05, Figure 4), with ratios for myocyte apoptosis and necrosis in the myocyte-fibroblast cocultures being similar to those for myocyte-only controls. However, addition of EC-conditioned medium resulted in a significant decrease in apoptosis and necrosis ratios of myocytes (P<0.01). Interestingly, the effect of conditioned medium on myocyte necrosis was similar in magnitude to the effect of ECs, whereas myocyte apoptosis ratios in the conditioned-medium group were only partially decreased compared with those in the presence of ECs. These results suggest that the prosurvival effect of ECs on CMs may not only be merely due to the local interactions between myocytes and ECs during myocyte attachment but may also involve direct signaling between myocytes and ECs.

Figure 4.  ECs prolong survival of CMs

Top, dual immunostaining of CMs and EC-myocyte prevascularized groups at day 3 in culture, with TUNEL-positive cells in red; green indicates sarcomeric actinin; blue, DAPI. Bottom, presence of ECs decreased CM apoptosis and necrosis, both in coculture conditions and when cultures were prevascularized by seeding with ECs 1 day before CMs (mean±SD, P<0.01). EC-conditioned medium decreased myocyte apoptosis and necrosis (P<0.01), whereas fibroblasts did not have any effect (P>0.05). *Different from myocytes alone; **different from EC-myocyte coculture and pre-vascularized. Bar=100 μm. Abbreviations are as defined in text.

Preformed Endothelial Networks Promote Coordinated, Spontaneous Contractions

In the prevascularized group with preformed vascular structures, synchronized, spontaneous contractions of large areas (Figure 5, top panels) were detected as early as days 2 to 3after seeding, in contrast to the coculture group, wherein such contractions were observed on days 6 to 7. In CM-only cultures, beating of separate cells and small cell clusters was also detected at days 2 to 3, similar to that in the prevascularized group. However, the average area of synchronized beating at day 3 in the myocyte-only group (3.5±0.5×102 μm2) was nearly 3 orders of magnitude smaller than the synchronously contracting area in the prevascularized group (4.3±2.5×105 μm2, mean±SD, n=5). These data suggest that ECs promote synchronized CM contraction, particularly when vascular networks are already formed.

Figure 5.  ECs promote large-scale, synchronized contraction of CMs.

Left, phase-contrast video of beating areas in CM-only and prevascularized groups (day 3). Right, motion analysis of video showing regions of synchronized contractions (connected areas in purple are contracting synchronously) and nonmoving areas in blue. Bars=100 μm. Abbreviations are as defined in text.

ECs Promote Cx43 Expression

Staining for Cx43 showed striking differences in the distribution pattern of this gap junction protein between EC-CM cocultures and CMs cultured alone. In myocyte-only cultures, Cx43 expression was barely detectable at day 1 (not shown); at days 3 and 7, Cx43 expression was sparse throughout the cell clusters (Figure 6). In the presence of ECs (in both coculture and prevascularized groups), Cx43 staining was evident at day 1, both between ECs and distributed among CMs. As early as day 3 in culture, patches of localized junction-like Cx43, in addition to diffuse staining, were observed for myocytes in the coculture group (Figure 6). In the prevascularized group at day 3, wherein spontaneous contractions were already observed, more junction-like patches of Cx43 were observed compared with the coculture group, indicating electrical connections between myocytes (Figure 6). In addition to junctions between myocytes, there was also evidence of Cx43 localized at the interface between ECs and myocytes (Figure 6) detected in both the coculture group (at day 7) and the preculture group (as early as day 3). When myocytes and myocyte-EC coculture groups were cultured for 3 days with or without addition of 100 ng/mL of neutralizing anti-mouse VEGF antibody (R&D Systems), we observed no differences in either apoptosis or Cx43 staining between VEGF antibody-containing cultures and controls.

Figure 6. ECs promote Cx43 expression

Cultures at 3 days immunostained for Cx43 (red) and anti-sarcomeric actinin (green); nuclei are stained with DAPI (blue). For CMs alone (left), Cx43 staining is diffuse and sparse, with no evidence of gap junctions; for coculture (center), both diffuse (yellow arrow) and patchlike (thin, white arrow) Cx43 staining is observed; for prevascularized (right), increased patchlike staining indicates presence of gap junctions. Thick arrow-heads indicate junctions between myocytes and ECs. Bar=50 μm. Abbreviations are as defined in text.

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Endothelial-Cardiomyocyte Interactions in Cardiac Development and Repair: Implications for Cardiac Regeneration

Patrick C.H. Hsieh, Michael E. Davis, Laura K. Lisowski, and Richard T. Lee

Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
Annu Rev Physiol.    PMC 2009 September 30

The ongoing molecular conversation between endothelial cells and cardiomyocytes is highly relevant to the recent excitement in promoting cardiac regeneration. The ultimate goal of myocardial regeneration is to rebuild a functional tissue that closely resembles mature myocardium, not just to improve systolic function transiently. Thus, regenerating myocardium will require rebuilding the vascular network along with the cardiomyocyte architecture. Here we review evidence demonstrating crucial molecular interactions between endothelial cells and cardiomyocytes. We first discuss endothelial-cardiomyocyte interactions during embryonic cardiogenesis, followed with morphological and functional characteristics of endothelial-cardiomyocyte interactions in mature myocardium. Finally, we consider strategies exploiting endothelial-cardiomyocyte interplay for cardiac regeneration.

Signaling from Cardiomyocytes to Endothelial Cells

The examples of neuregulin-1, NF1, and PDGF-B demonstrate that signals from endothelial cells regulate the formation of primary myocardium. Similarly, signaling from myocardial cells to endothelial cells is also required for cardiac development. Two examples of myocardial-to-endothelial signaling are vascular endothelial growth factor (VEGF)-A and angiopoietin-1.


VEGF-A is a key regulator of angiogenesis during embryogenesis. In mice, a mutation in VEGF-A causes endocardial detachment from an underdeveloped myocardium. A mutation in VEGF receptor-2 (or Flk-1) also results in failure of the endocardium and myocardium to develop (18). Furthermore, cardiomyocyte-specific deletion of VEGF-A results in defects in vasculogenesis/angiogenesis and a thinned ventricular wall, further confirming reciprocal signaling from the myocardial cell to the endothelial cell during cardiac development. Interestingly, this cardiomyocyte-selective VEGF-A-deletion mouse has underdeveloped myocardial microvasculature but preserved coronary artery structure, implying a different signaling mechanism for vasculogenesis/angiogenesis in the myocardium and in the epicardial coronary arteries.
Cardiomyocyte-derived VEGF-A also inhibits cardiac endocardial-to-mesenchymal transformation. This process is essential in the formation of the cardiac cushions and requires delicate control of VEGF-A concentration. A minimal amount of VEGF initiates endocardial-to-mesenchymal transformation, whereas higher doses of VEGF-A terminate this transformation. Interestingly, this cardiomyocyte-derived VEGF-A signaling for endocardial-to-mesenchymal transformation may be controlled by an endothelial-derived feedback mechanism through the calcineurin/NFAT pathway (24), demonstrating the importance of endothelial-cardiomyocyte interactions for cardiac morphogenesis.


Another mechanism of cardiomyocyte control of endothelial cells during cardiac development is the angiopoietin-Tie-2 system. Both angiopoietin-1 and angiopoietin-2 may bind to Tie-2 receptors in a competitive manner, but with opposite effects: Angiopoietin-1 activates the Tie-2 receptor and prevents vascular edema, whereas angiopoietin-2 blocks Tie-2 phosphorylation and increases vascular permeability. During angiogenesis/vasculogenesis, angiopoietin-1 is produced primarily by pericytes, and Tie-2 receptors are expressed on endothelial cells. Angiopoietin-1 regulates the stabilization and maturation of neovasculature; genetic deletion of angiopoietin-1 or Tie-2 causes a defect in early vasculogenesis/angiogenesis and is lethal.
Cardiac endocardium is one of the earliest vascular components (along with the dorsal aorta and yolk sac vessels) and the adult heart can be regarded as a fully vascularized organ, angiopoietin-Tie-2 signaling may also be required for early cardiac development. Indeed, mice with mutations in Tie-2 have underdeveloped endocardium and myocardium. These Tie-2 knockout mice display defects in the endocardium but have normal vascular morphology at E10.5, suggesting that the endocardial defect is the fundamental cause of death. In addition, a recent study showed that overexpression, and not deletion, of angiopoietin-1 from cardiomyocytes caused embryonic death between E12.5-15.5 due to cardiac hemorrhage. The mice had defects in the endocardium and myocardium and lack of coronary arteries, suggesting that, as with VEGF-A, a delicate control of angiopoietin-1 concentration is critical for early heart development.


Cardiac Endothelial Cells Regulate Cardiomyocyte Contraction

The vascular endothelium senses the shear stress of flowing blood and regulates vascular smooth muscle contraction. It is therefore not surprising that cardiac endothelial cells—the endocardial endothelial cells as well as the endothelial cells of intramyocardial capillaries— regulate the contractile state of cardiomyocytes. Autocrine and paracrine signaling molecules released or activated by cardiac endothelial cells are responsible for this contractile response (Figure 2).


Three different nitric oxide synthase isoenzymes synthesize nitric oxide (NO) from L-arginine. The neuronal and endothelial NO synthases (nNOS and eNOS, respectively) are expressed in normal physiological conditions, whereas the inducible NO synthase is induced by stress or cytokines. Like NO in the vessel, which causes relaxation of vascular smooth muscle, NO in the heart affects the onset of ventricular relaxation, which allows for a precise optimization of pump function beat by beat. Although NO is principally a paracrine effector secreted by cardiac endothelial cells, cardiomyocytes also express both nNOS and eNOS. Endothelial expression of eNOS exceeds that in cardiomyocytes by greater than 4:1. Cardiomyocyte autocrine eNOS signaling can regulate β-adrenergic and muscarinic control of contractile state.
Barouch et al. demonstrated that cardiomyocyte nNOS and eNOS may have opposing effects on cardiac structure and function. Using mice with nNOS or eNOS deficiency, they found that nNOS and eNOS have not only different localization in cardiomyocytes but also opposite effects on cardiomyocyte contractility; eNOS localizes to caveolae and inhibits L-type Ca2+ channels, leading to negative inotropy, whereas nNOS is targeted to the sarcoplasmic reticulum and facilitates Ca2+ release and thus positive inotropy (31). These results demonstrate that spatial confinement of different NO synthase isoforms contribute independently to the maintenance of cardiomyocyte structure and phenotype.
As indicated above, mutation of neuregulin or either of two of its cognate receptors, erbB2 and erbB4, causes embryonic death during mid-embryogenesis due to aborted development of myocardial trabeculation . Neuregulin also appears to play a role in fully developed myocardium. In adult mice, cardiomyocyte-specific deletion of erbB2 leads to dilated cardiomyopathy. Neuregulin from endothelial cells may induce a negative inotropic effect in isolated rabbit papillary muscles. This suggests that, along with NO, the neuregulin signaling pathway acts as an endothelial-derived regulator of cardiac inotropism.  In fact, the negative inotropic effect of neuregulin may require NO synthase because L-NMMA, an inhibitor of NO synthase, significantly attenuates the negative inotropy of neuregulin.

Studies to date indicate that cardiac regeneration in mammals may be feasible, but the response is inadequate to preserve myocardial function after a substantial injury. Thus, understanding how normal myocardial structure can be regenerated in adult hearts is essential. It is clear that endothelial cells play a role in cardiac morphogenesis and most likely also in survival and function of mature cardiomyocytes. Initial attempts to promote angiogenesis in myocardium were based on the premise that persistent ischemia could be alleviated. However, it is also possible that endothelial-cardiomyocyte interactions are essential in normal cardiomyocyte function and for protection from injury. Understanding the molecular and cellular mechanisms controlling these cell-cell interactions will not only enhance our understanding of the establishment of vascular network in the heart but also allow the development of new targeted therapies for cardiac regeneration by improving cardiomyocyte survival and maturation.

Endothelial-cardiomyocyte assembly

Figure 1.  Endothelial-cardiomyocyte assembly in adult mouse myocardium.
Normal adult mouse myocardium is stained with intravital perfusion techniques to demonstrate cardiomyocyte (outlined in red) and capillary (green; stained with isolectin-fluorescein) assembly. Nuclei are blue (Hoechst). Original magnification: 600X

Endothelial dysfunction

Intramyocardial Fibroblast – Myocyte Communication

Rahul Kakkar, M.D. and Richard T. Lee, M.D.
From the Cardiology Division, Massachusetts General Hospital and the Cardiovascular Division, Brigham and Women’s Hospital, Department of Medicine, Harvard Medical School, Boston, MA
Circ Res. 2010 January 8; 106(1): 47–57.    http://dx.doi.org/10.1161/CIRCRESAHA.109.207456

Cardiac fibroblasts have received relatively little attention compared to their more famous neighbors, the cardiomyocytes. Cardiac fibroblasts are often regarded as the “spotters”, nonchalantly watching the cardiomyocytes do the real weight-lifting, and waiting for a catastrophe that requires their actions. However, emerging data now reveal the fibroblast as not only a critical player in the response to injury, but also as an active participant in normal cardiac function.
Interest in cardiac fibroblasts has grown with the recognition that cardiac fibrosis is a prominent contributor to diverse forms of myocardial disease. In the early 1990’s, identification of angiotensin receptors on the surface of cardiac fibroblasts linked the renin-angiotensin-aldosterone system directly with pathologic myocardial and matrix extracellular remodeling.  Fibroblasts were also revealed as a major source of not only extracellular matrix, but the proteases that regulate and organize matrix. New research has uncovered paracrine and well as direct cell-to-cell interactions between fibroblasts and their cardiomyocyte neighbors, and cardiac fibroblasts appear to be dynamic participants in ventricular physiology and pathophysiology.
This review will focus on several aspects of fibroblast-myocyte communication, including mechanisms of paracrine communication.  Ongoing efforts at regeneration of cardiac tissue focus primarily on increasing the number of cardiomyocytes in damaged myocardium. Although getting cardiomyocytes into myocardium is an important goal, understanding intercellular paracrine communication between different cell types, including endothelial cells but also fibroblasts, may prove crucial to regenerating stable myocardium that responds to physiological conditions appropriately.

An area of active research in cardiovascular therapeutics is the attempt to engineer, ex vivo, functional myocardial tissue that may be engrafted onto areas of injured ventricle. Recent data suggests that the inclusion of cardiac fibroblasts in three-dimensional cultures greatly enhances the stability and growth of the nascent myocardium. Cardiac fibroblasts when included in polymer scaffolds seeded with myocytes and endothelial cells have the ability to promote and stabilize vascular structures. Naito and colleagues constructed three dimensional cultures of neonatal rat cell isolates on collagen type I and Matrigel (a basement membrane protein mixture), and isolates of a mixed cell population versus a myocyte-enriched population were compared. The mixed population cultures, which contained a higher fraction of cardiac fibroblasts than the myocyte-enriched cultures, displayed improved contractile force generation and greater inotropic response despite an equivalent overall cell number. Greater vascularity was also seen in the mixed-pool cultures.(160) Building on this, Nichol and colleagues demonstrated that in a self-assembling nanopeptide scaffold, embedded rat neonatal cardiomyocytes exhibit greater cellular alignment and reduced apoptosis when cardiac fibroblasts were included in the initial culture. A similar result was noted when polymer scaffolds were pre-treated with cardiac fibroblasts before myocyte seeding, suggesting a persistent paracrine effect. These data reinforce the concept that engineering functional myocardium, either in situ or ex vivo will require attention to the nature of cell-cell interactions, including fibroblasts.

To date, a broad initial sketch of cardiac fibroblast-myocyte interactions has been drawn. Future studies in this field will better describe these interactions. How do multiple paracrine factors interact to produce a cohesive and coordinated communication scheme? What are the changes in coordinated bidirectional signaling that during development promotes myocyte progenitor proliferation but have different roles in the adult? Might fibroblasts actually be required for improved cardiac repair and regeneration?
Recent studies have begun to apply genetic and cellular fate-mapping techniques to document the origins of cardiac fibroblasts, the dynamic nature of their population, and how that population may be in flux during time of injury or pressure overload. It is crucial to define on a more specific molecular basis the origins and fates of cardiac fibroblasts. Do fibroblasts that have been resident within the ventricle since development fundamentally differ from those that arise from endothelial transition or that infiltrate from the bone marrow during adulthood? Do fibroblasts with these different origins behave differently or take on different roles in the face of ventricular strain or injury?

Our understanding of the nature of the cardiac fibroblast is evolving from the concept of the fibroblast as a bystander that causes unwanted fibrosis to the picture of a more complex role of fibroblasts in the healthy as well as diseased heart. The pathways used by cardiac fibroblasts to communicate with their neighboring myocytes are only partially described, but the data to date indicate that these pathways will be important for cardiac repair and regeneration.

. Paracrine bidirectional cardiac fibroblast-myocyte crosstalk

Figure 2. Paracrine bidirectional cardiac fibroblast-myocyte crosstalk

Under biomechanical overload, cardiac fibroblasts and myocytes respond to an altered environment via multiple mechanisms including integrin-extracellular matrix interactions and renin-angiotensin-aldosterone axis activation. Cardiac fibroblasts increase synthesis of matrix proteins and secrete a variety of paracrine factors that can stimulate myocyte hypertrophy. Cardiac myocytes similarly respond by secreting a conglomerate of factors. Hormones such as TGFβ1, FGF-2, and the IL-6 family members LIF and CT-1 have all been implicated in this bidirectional fibroblast-myocyte hormonal crosstalk.

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Platelets in Translational Research – Part 2

Subtitle: Discovery of Potential Anti-platelet Targets

Reviewer and Curator: Larry H. Bernstein, MD, FCAP 


This presentation is the the second of a series on Platelets in Translational Medicine: Part I:  Platelet structure, interactions between platelets and endothelium, and intracellular transcription

Part II: Discovery of Potential Anti-platelet Targets

Endothelium-dependent vasodilator effects of platelet activating factor on rat resistance vessels

1Katsuo Kamata, Tatsuya Mori, *Koki Shigenobu & Yutaka Kasuya Department of Pharmacology, School of Pharmacy, Hoshi University, Tokyo and *Department of Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashi, Chiba, Jp Br. J. Pharmacol. (1989), 98, 1360-1364 To elucidate the mechanisms of the powerful and long-lasting hypotension produced by platelet activating factor (PAF), its effects on perfusion pressure in the perfused mesenteric arterial bed of the rat were examined. 2 Infusion of PAF (10-11 to 3 x 10-10M; EC50 = 4.0 x 10′ m; 95%CL = 1.6 x 10-11 — 9.4 x 10-11 M) and acetylcholine (ACh) (10′ to 10-6m; EC50 = 3.0 ± 0.1 x 10-9m) produced marked concentration-dependent vasodilatations which were significantly inhibited by treatment with detergents (0.1% Triton X-100 for 30 s or 0.3% CHAPS for 90 s). 3 Pretreatment with CV-6209, a PAF antagonist, inhibited PAF- but not ACh-induced vasodila­tation. 4 Treatment with indomethacin (10-6m) had no effect on PAF- or ACh-induced vasodilatation. 5


These results demonstrate that extremely low concentrations of PAF produce vasodilatation of resistance vessels through the release of endothelium-derived relaxing factor (EDRF). This may account for the strong hypotension produced by PAF in vivo. Platelet activating factor (PAF, acetyl glyceryl ether phosphorylcholine) has been shown to produce strong and long-lasting hypotension in various animal species, e.g. normotensive and spontaneously hypertensive rats, rabbits, guinea-pigs, and dogs (Tanaka et al., 1983). This action of PAF is thought to be endothelium-dependent (Kamitani et al., 1984; Kasuya et al., 1984a,b; Shigenobu et al., 1985; 1987). In a previous study (Shigenobu et al., 1987), we found that relatively low concentrations of PAF (10-9-10-7m) produced endothelium-dependent relaxation of the rat aorta in the presence of bovine serum albumin. This vasodilator action of PAF at low concentrations might be the cause of its hypo­tensive action in vivo. While the aorta will offer a resistance to flow, it is obvious that the contribution of vessels of smaller diameter to peripheral vascular resistance is much greater. In this regard, the mesen­teric circulation of the rat receives approximately one-fifth of the cardiac output (Nichols et al., 1985) and, thus, regulation of this bed may make a signifi­cant contribution towards systemic blood pressure and circulating blood volume.  Therefore, we examined the effect of PAF on the resistance vessels of the rat mesenteric vascular bed and found that extremely low concentrations (10 -11 to 3 x 10-16 m) can produce endothelium-dependent vasodilatation. Figure 1 Effects of PAF on the perfusion pressure of the methoxamine (10-3N)-constricted mesenteric vascu­lar bed. (a) Upper panel: relaxation induced by PAF (3 x 10-10 M). Lower panel: effects of the PAF-antagonist, CV-6209 (3 x 10-914), on the relaxation induced by PAF (3 x 10“N). (b) Concentration-response curve for the relaxation produced by PAF (10-11 to 3 x 10-10N) in the methoxamine (10-51)-constricted mesenteric vascular bed. Each point is the mean and vertical bars represent the s.e.mean from 5 experiments. Figure 2 Effects of detergents on acetylcholine (ACh)-induced relaxation of the methoxamine (10-5M)-con­stricted mesenteric vascular bed. Concentration-response curves are shown for ACh-induced vasodilatation before (0) and after treatment with 0.3% CHAPS (❑) or 0.1% Triton X-100 (0). Each point is the mean and vertical bars represent the s.e.mean from 5 experiments. Infusions of extremely low concentrations of PAF (10-11 to 3.1 x 10-1° m) produced a marked and long-lasting vasodilatation which was significantly suppressed by treatment with detergents ar bed. Concentration-response curves are shown for ACh-induced vasodilatation before (0) and after treatment with 0.3% CHAPS (❑) or 0.1% Triton X-100 (0). Each point is the mean and vertical bars represent the s.e.mean from 5 experiments. Since Furchgott & Zawadzki (1980) demonstrated the obligatory role of endothelium in vascular relax­ation by ACh, many studies have suggested that endothelium-derived relaxing factor (EDRF) is re­leased from endothelial cells in response to a large number of agonists (Furchgott, 1984). In the present study with perfused resistance vessels, ACh produced vasodilatation in a concentration-dependent manner and the vasorelaxant responses were significantly suppressed by perfusion with detergents such as CHAPS or Triton X-100.  These data strongly suggest the pos­sible involvement of the endothelium in the relax­ation induced by PAF. CV-6209, a PAF antagonist, inhibited PAF-induced but not ACh-induced vasodilatation in a concentration-dependent manner. Specific antago­nism by CV-6209 has already been obtained with respect to PAF-induced hypotension or platelet aggregation (Terashita et al., 1987). An accumulating body of evidence suggests that hypotension resulting from endotoxin challenge is due to the endogenous release of PAF from endothelial cells (Camussi et al., 1983), leukocytes (Demopoules et al., 1979), macro­phages (Mencia-Huerta & Benveniste, 1979; Camussi et al., 1983) and platelets (Chingard et al., 1979). Indeed, PAF antagonists can reverse estab­lished endotoxin-induced hypotension (Terashita et al., 1985; Handley et al., 1985a,b). From the above data and the results of the present study, one pos­sible explanation for endotoxin-induced hypotension may be that the release of PAF occurs, which then binds to its receptors located on the endothelial cells, stimulating production of EDRF. In conclusion, we demonstrated that extremely low concentrations of PAF produce long-lasting vasodilatation in a resistance vessel of the mesenteric vasculature. Moreover, we showed that this PAF-induced vasodilatation is mediated by a vasodilator substance released from endothelial cells (EDRF) which is not a prostaglandin. Since the PAF-induced endothelium-dependent relaxation observed in the present study was elicited at low concentrations and was long-lasting, it may be the main mechanism by which PAF induces hypotension in vivo.

Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study

Andreas C Eriksson*1, Lena Jonasson2, Tomas L Lindahl3, Bo Hedbäck2 and Per A Whiss1 1Divisions of Drug Research/Pharmacology and 2Cardiology, Department of Medical and Health Sciences, Linköping University, Linköpin, Sw, and 3Department of Clinical Chemistry, University Hospital, Linköping, Sw Journal of Translational Medicine 2009, 7:42     http:/dx.doi.org/10.1186/1479-5876-7-42   http://www.translational-medicine.com/content/7/1/42


Background: Despite the use of anti-platelet agents such as acetylsalicylic acid (ASA) and clopidogrel in coronary heart disease, some patients continue to suffer from atherothrombosis. This has stimulated development of platelet function assays to monitor treatment effects. However, it is still not recommended to change treatment based on results from platelet function assays. This study aimed to evaluate the capacity of a static platelet adhesion assay to detect platelet inhibiting effects of ASA and clopidogrel. The adhesion assay measures several aspects of platelet adhesion simultaneously, which increases the probability of finding conditions sensitive for anti-platelet treatment.

Methods: With a randomised cross-over design we evaluated the anti-platelet effects of ASA combined with clopidogrel as well as monotherapy with either drug alone in 29 patients with a recent acute coronary syndrome. Also, 29 matched healthy controls were included to evaluate intra-individual variability over time. Platelet function was measured by flow cytometry, serum thromboxane B2 (TXB2)-levels and by static platelet adhesion to different protein surfaces. The results were subjected to Principal Component Analysis followed by ANOVA, t-tests and linear regression analysis.

Results: The majority of platelet adhesion measures were reproducible in controls over time denoting that the assay can monitor platelet activity. Adenosine 5′-diphosphate (ADP)-induced platelet adhesion decreased significantly upon treatment with clopidogrel compared to ASA. Flow cytometric measurements showed the same pattern (r2 = 0.49). In opposite, TXB2-levels decreased with ASA compared to clopidogrel. Serum TXB2 and ADP-induced platelet activation could both be regarded as direct measures of the pharmacodynamic effects of ASA and clopidogrel respectively. Indirect pharmacodynamic measures such as adhesion to albumin induced by various soluble activators as well as SFLLRN-induced activation measured by flow cytometry were lower for clopidogrel compared to ASA. Furthermore, adhesion to collagen was lower for ASA and clopidogrel combined compared with either drug alone. Conclusion: The indirect pharmacodynamic measures of the effects of ASA and clopidogrel might be used together with ADP-induced activation and serum TXB2 for evaluation of anti-platelet treatment. This should be further evaluated in future clinical studies where screening opportunities with the adhesion assay will be optimised towards increased sensitivity to anti-platelet treatment. The benefits of ASA have been clearly demonstrated by the Anti-platelet Trialists’ Collaboration. They found that ASA therapy reduces the risk by 25% of myocardial infarction, stroke or vascular death in “high-risk” patients. When using the same outcomes as the Anti-platelet Trialists’ Collaboration on a comparable set of “high-risk” patients, the CAPRIE-study showed a slight benefit of clopidogrel over ASA. Furthermore, the combination of clopidogrel and ASA has been shown to be more effective than ASA alone for preventing vascu­lar events in patients with unstable angina and myo­cardial infarction as well as in patients undergoing percutaneous coronary intervention (PCI). Despite the obvious benefits from anti-platelet therapy in coro­nary disease, low response to clopidogrel has been described by several investigators. A lot of attention has also been drawn towards low response to ASA, often called “ASA resistance”. The concept of ASA resistance is complicated for several reasons. First of all, different stud­ies have defined ASA resistance in different ways. In its broadest sense, ASA resistance can be defined either as the inability of ASA to inhibit platelets in one or more platelet function tests (laboratory resistance) or as the inability of ASA to prevent recurrent thrombosis (i.e. treatment fail­ure, here denoted clinical resistance). The lack of a general definition of ASA resistance results in difficulties when trying to measure the prevalence of this phenome­non. Estimates of laboratory resistance range from approximately 5 to 60% depending on the assay used, the patients studied and the way of defining ASA resistance. Likewise, lack of a standardized definition of low response to clopidogrel makes it difficult to estimate the prevalence of this phenomenon as well. The principles of existing platelet assays, as well as their advantages and disadvantages, have been described elsewhere. In short, assays potentially useful for monitoring treatment effects include those commonly used in research such as platelet aggregometry and flow cytometry as well as immunoassays for measuring metabolites of thromboxane A2 (TXA2). Also, the PFA-100TM, MultiplateTM and the VerifyNowTM are examples of instruments commercially developed for evaluation of anti-platelet therapy. How­ever, no studies have investigated the usefulness of alter­ing treatment based on laboratory findings of ASA resistance. Regarding clopidogrel, there are recent studies showing that adjustment of clopidogrel loading doses according to vasodilator-stimulated phosphoprotein phosphorylation index measured utilising flow cytometry decrease major adverse cardiovascular events in patients with clopidogrel resistance. Static adhesion is an aspect of platelet function that has not been investigated in earlier studies of the effects of platelet inhibiting drugs. Consequently, static platelet adhesion is not measured by any of the current candidate assays for clinical evaluation of platelet function. The static platelet adhesion assay offers an opportunity for simultaneous measurements of the combined effects of several different platelet activators on platelet function. In this study, platelet adhesion to albumin, collagen and fibrinogen was investigated in the presence of soluble platelet activators including adenosine 5′-diphosphate (ADP), adrenaline, lysophosphatidic acid (LPA) and ris-tocetin. Collagen, fibrinogen, ADP and adrenaline are physiological agents that are well-known for their interac­tions with platelets. Ristocetin is a compound derived from bacteria that facilitates the interaction between von Willebrand factor (vWf) and glycoprotein (GP)-Ib-IX-V on platelets, which otherwise occurs only at flow condi­tions. The static nature of the assay therefore prompted us to include ristocetin in order to get a rough estimate on GPIb-IX-V dependent events. LPA is a phospholipid that is produced and released by activated platelets and that also can be generated through mild oxi­dation of LDL. It was included in the present study since it is present in atherosclerotic vessels and suggested to be important for platelet activation after plaque rup­ture. Finally, albumin was included as a surface since the platelet activating effect of LPA can be detected when measuring adhesion to such a surface. Thus, by the use of different platelet activators, several measures of platelet adhesion were obtained simultaneously This means that the possibilities to screen for conditions potentially important for detecting effects of platelet-inhibiting drugs far exceeds the screening abilities of other platelet function tests. Consequently, the static platelet adhesion assay is very well suited for development into a clinically useful device for monitoring platelet inhibiting treatment. Also, it has earlier been proposed that investi­gating the combined effects of two activators on platelet activity might be necessary in order to detect effects of ASA and other antiplatelet agents [26]. This is a criterion that can easily be met by the static platelet adhesion assay. Through the screening procedure we found different con­ditions where the static adhesion was influenced by the drug given.

The inclusion of patients and controls. Patients and controls were included consecutively. Blood samples from controls were drawn at two different occasions separated by 2–5.5 months. All patients entering the study received ASA combined with clopidogrel and blood sampling was performed 1.5–6.5 months after initiating the treatment. This was followed by a randomised cross-over enabling all patients to receive monotherapy with both ASA and clopidogrel. The patients received monotherapy for at least 3 weeks and for a maximum of 4.5 months before performing blood sampling. A total of 33 patients and 30 controls entered the study. In the end, 29 patients and 29 controls completed the study. Blood was drawn from patients at three different occa­sions (Figure 1). The first sample was drawn after all patients had received combined treatment with ASA (75 mg/day) and clopidogrel (75 mg/day) for 1.5–6.5 months after the index event. The study then used a randomised cross-over design meaning that half of the patients received ASA as monotherapy while half received only clopidogrel (75 mg/day for both monotherapies). The monotherapy was then switched for every patient so that all patients in total received all three therapies. Samples for evaluation of the monotherapies were drawn after therapy for at least 3 weeks and at the most for 4.5 months. Most of the differences in treatment length can be ascribed to the fact that the national recommendations for treatment in this patient group were changed during the course of the study. The allocation to monotherapy was blinded for the laboratory personnel. In general, the use of three different treatments for intra-individual com­parisons in a cross-over design is different from previous studies on ASA and clopidogrel, which have mainly been concerned with only two treatment alternatives.

Intra-individual variation in healthy controls

Measurements of platelet adhesion and serum TXB2-levels were performed on healthy controls on two separate occa­sions (2–5.5 months interval) in order to investigate the presence of intraindividual variation in platelet reactivity and clotting-induced TXB2-production. The standardised Z-scores from the simplified factors were used for analysis by Repeated Measures ANOVA of the data from the healthy controls. We found significantly decreased plate­let adhesion at the second compared to the first visit for ADP-induced adhesion (Factor 1, p = 0.012) and for adhe­sion to fibrinogen (Factor 5, p = 0.012). This intra-indi-vidual variability over time makes it difficult to draw any conclusions regarding effects of anti-platelet treatment. We therefore further analysed the individual variables constituting Factors 1 and 5 with Repeated Measures ANOVA in order to distinguish the variables that varied significantly over time. Variables being significantly dif­ferent between visit 1 and visit 2 were then excluded and a new Repeated Measures ANOVA was performed on the new factors. After this modification, none of the factors corresponding to adhesion showed variation over time and these factors were then used for analysis on patients. Serum levels of TXB2, which constituted a separate factor, varied significantly in healthy controls at two separate occasions (Figure 2). flow chart of patients and controls_Image_1 Effect of platelet inhibiting treatment on serum TXB2-levels (Factor 13). Serum TXB2-levels (Factor 13) for patients (n = 29) and healthy controls (n = 29) are presented as mean + SEM. ASA alone or in combination with clopidogrel was signif­icantly different from clopidogrel alone and compared to the mean of the controls (p < 0.001). Also, the difference between controls at visit 1 and visit 2 was significant. ***p < 0.001, ns = not significant. When investigating possible effects of platelet-inhibiting treatment with Repeated Measures ANOVA, significant effects were seen for four of the factors corresponding to platelet adhesion. The factors that were not able to detect significant treatment effects were adrenaline-induced adhesion (Factor 3), ristocetin-induced adhesion (Factor 4) and adhesion to fibrinogen (Factor 5). Regarding adhe­sion factors detecting treatment effects, ADP-induced adhesion (Factor 1, Figure 3A inset) was significantly decreased by clopidogrel alone or by clopidogrel plus ASA compared with ASA alone. Surprisingly, platelet adhesion induced by ADP was lower for the monotherapy with clopidogrel compared to dual therapy. ADP-induced adhesion to albumin is shown as a representative example of the variables of Factor 1 (Figure 3A). Ristocetin-induced adhesion to albumin (Factor 6, Figure 3B inset) was signif­icantly decreased by clopidogrel alone compared with ASA alone. This difference was also seen for ristocetin combined with LPA, which is shown as an example of a variable belonging to Factor 6 (Figure 3B). In Factor 7 (Figure 3C inset), corresponding to LPA-induced adhe­sion to albumin, we found clopidogrel to decrease adhe­sion compared with ASA and compared with ASA plus clopidogrel. These differences were reflected by the com­bined activation through LPA and adrenaline, which was a variable included in Factor 7 (Figure 3C). Finally, adhe­sion to collagen (Factor 8, Figure 3D) was significantly decreased by dual therapy compared with ASA alone or clopidogrel alone. As can be seen from the above descrip­tion, monotherapy with clopidogrel resulted in signifi­cantly decreased adhesion compared to clopidogrel combined with ASA for Factors 1 and 7. This was also observed for the variable shown as a representative exam­ple of Factor 6 (Figure 3B). The two factors corresponding to flow cytometric measurements (Factors 14 and 15, Fig­ure 4) both showed that ASA-treated platelets were more active than platelets treated with clopidogrel alone or clopidogrel plus ASA. Furthermore, serum TXB2-levels (Figure 2) was significantly decreased by ASA alone or by ASA plus clopidogrel compared with clopidogrel alone. Regarding the other measurements not directly measuring platelet function, significant differences were found for Factor 10 including HDL and for platelet count (Factor 12) but neither for the factor corresponding to inflamma­tion (Factor 9) nor for Factor 11 including LDL. Factor 10 including HDL was found to be elevated by both ASA and clopidogrel monotherapies compared with dual therapy (p = 0.003 for ASA, p = 0.019 for clopidogrel, data not shown). Platelet count were found to be increased after dual therapy compared with both monotherapies (p < 0.001, data not shown). flow chart of patients and controls_Image_2 The influence of ASA and clopidogrel on platelet adhesion. The main figures are representative examples of the varia­bles constituting the respective factors. The insets show the Z-scores for each factor. Also shown in the insets are the compar­isons between the control means of visit 1 and 2 and treatment with ASA (A), clopidogrel (C) and the combination of ASA and clopidogrel (A+C). The respective figures show the effect of platelet inhibiting treatment on ADP-induced adhesion (Factor 1, Fig A), ristocetin-induced adhesion to albumin (Factor 6, Fig B), LPA-induced adhesion to albumin (Factor 7, Fig C) and adhe­sion to collagen (Factor 8, Fig D) for patients (n = 29) and healthy controls (n = 29). All values are presented as mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. flow chart of patients and controls_Image_4 The influence of ASA and clopidogrel on platelet activity measured by flow cytometry. The effects of platelet inhibiting treatment on platelet activation detected by flow cytometry induced by ADP (Factor 14, Fig A) and SFLLRN (Factor 15, Fig B) on patients (n = 29). The main figures are representative examples of the variables constituting the respective fac­tors. The insets show the Z-scores for each factor. All values are presented as mean + SEM. ***p < 0.001, ns = not significant. Platelets from patients (n = 29) were activated in vitro with adenosine 5′-diphosphate (ADP; 0.1 and 0.6 μmol/L) or SFLLRN (5.3 μmol/L) followed by flow cytometric measurements of fibrinogen-binding or expression of P-selectin. Presented results are the mean-% of fibrinogen-binding and P-selectin expression ± SEM. Reference values (obtained earlier during routine analysis at the accredited Dept. of Clinical Chemistry at the University hospital in Linköping) are shown as mean with reference interval within parenthesis. Stars indicate significant differences for patients compared to reference values. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.  (Table not shown)


With the aim of finding variables sensitive to clopidogrel and ASA-treatment, this study used a screening approach and measured several different variables simultaneously. To reduce the complexity of the material we performed PCA in order to find correlating variables that measured the same property. In this way the 54 measurements of platelet adhesion were reduced to 8 factors. Visual inspec­tion revealed that each factor represented a separate entity of platelet adhesion and the factors could therefore be renamed according to the aspect they measured. We thus conclude that future studies must not involve all 54 adhe­sion variables, but instead, one variable from each factor should be enough to cover 8 different aspects of platelet adhesion. In addition to the adhesion data, the remaining 15 variables also formed distinct factors that were possible to rename according to measured property. It is notable that serum TXB2 formed a distinct group not correlated to any of the other measurements.

It is important that laboratory assays used for clinical pur­poses are reproducible and that they measure parameters that are not confounded by other variables. Some of the measurements performed in this study (clinical chemistry variables and platelet function measured by flow cytome-try) are used for clinical analysis at accredited laboratories at the University hospital in Linköping. However, the reproducibility of the platelet adhesion assay was mostly unknown before this study. Our initial results suggested that the factors corresponding to ADP-induced adhesion and adhesion to fibrinogen were not reproduci­ble. We therefore excluded the most varied variables con­stituting these factors, which resulted in no intra-individual effects for healthy controls in the platelet adhe­sion assay. From this we conclude that many, but not all, measures of platelet adhesion are reproducible. Moreover, the static condition might limit the possibilities for trans­lating the results from the adhesion assay into in vivo platelet adhesion occurring during flow conditions. How­ever, platelet adhesion to collagen and fibrinogen is dependent on α2131– and αIIb133-receptors respectively in the current assay. This suggests that the static platelet adhesion assay can measure important aspects of platelet function despite its simplicity. Furthermore, vWf depend­ent adhesion is not directly covered in the present assay although ristocetin-induced adhesion appears to be dependent on GPIb-IX-V and vWf . From this discussion it is evident that the adhesion assay as well as flow cytometry can measure effects of clopidog-rel when using ADP as activating stimuli. It is also evident that serum-TXB2 levels measure the effects of ASA. How­ever, these measures focus on the primary interaction between the drugs and the platelets, which could be prob­lematic when trying to evaluate the complex in vivo treat­ment effect. It has previously been found that only 12 of 682 ASA-treated patients (≈ 2%) had residual TXB2 serum levels higher than 2 standard deviations from the popula­tion mean. Measurements of the effect of arachidonic acid on platelet aggregometry have also led to the conclu­sion that ASA resistance is a very rare phenomenon. Thus, our study supports these previous findings that assays measuring the pharmacodynamic activity of ASA (to inhibit the COX-enzyme) seldom recognizes patients as ASA-resistant. This suggests that the cause of ASA-resistance is not due to an inability of ASA to act as a COX-inhibitor.

We suggest that direct measurements of ADP and TXA2-effects (in our case ADP-induced activation measured by adhesion or flow cytometry and serum TXB2-levels) must be combined with measures that are only partly dependent on ADP and TXA2 respectively. For instance, an adhesion variable partly dependent on TXA2 might be able to detect ASA resistance caused by increased signalling through other activating pathways. Such a scenario would be character­ized by serum TXB2 values showing normal COX-inhibi­tion while platelet adhesion is increased. This study employed a screening procedure in order to find such indirect measures of the effects of ASA and clopidogrel. Our results show inhibiting effects of clopidogrel com­pared to ASA on adhesion to albumin in the presence of LPA or ristocetin. This was also observed for our flow cytometric measurements with SFLLRN as activator, which confirms that SFLLRN is able to induce release of granule contents in platelets. SFLLRN- and ADP-induced platelet activation, as measured by flow cytometry, was moderately correlated to each other and adhesion induced by LPA as well as ristocetin showed weak correla­tions with ADP-induced adhesion. These results further confirm that these measures of platelet activity are partly dependent on ADP. We have earlier shown that adhesion to albumin induced by simultaneous stimulation by LPA and adrenaline (a variable belonging to the LPA-factor in the present study) can be inhibited by inhibition of ADP-signalling in vitro. This strengthens our conclusion that the effect on LPA-induced adhesion observed for clopidogrel is caused by inhibition of ADP-signalling. Also, the presence of LPA in atherosclerotic plaques and its possible role in thrombus formation after plaque rup­ture makes it especially interesting for the in vivo set­ting of myocardial infarction. Assays of static platelet adhesion that have been used in previous studies aimed at investigating treatment effects of platelet inhibiting drugs. Importantly, this study shows that the static platelet adhesion assay is reproducible over time. We also showed that the static platelet adhesion assay as well as flow cytometry detected the ability of clopidogrel to inhibit platelet activation induced by ADP. Our results further suggest that other measures of platelet adhesion and platelet activation measured by flow cytometry are indirectly dependent on secreted ADP or TXA2. One such measure is adhesion to a collagen surface, which should be more thoroughly investigated for its ability to detect effects of clopidogrel and ASA. Likewise, due to its connection to atherosclerosis and myocardial infarction, the LPA-induced effect should be further evaluated for its ability to detect effects of clopidogrel. In conclusion, the screening procedure undertaken in this study has revealed suggestions on which measures of platelet activity to com­bine in order to evaluate platelet function.

Effect of protein kinase C and phospholipase A2 inhibitors on the impaired ability of human platelets to cause vasodilation

*,1Helgi J. Oskarsson, 1Timothy G. Hofmeyer, 1Lawrence Coppey & 1Mark A. Yorek 1Department of Internal Medicine, University of Iowa and VA Medical Center, Iowa City, IA British Journal of Pharmacology (1999) 127, 903-908   http://www.stockton-press.co.uk/bjp

1   The aim of this study was to examine the mechanism of impaired platelet-mediated endothelium-dependent vasodilation in diabetes. Exposure of human platelets to high glucose in vivo or in vitro impairs their ability to cause endothelium-dependent vasodilation. While previous data suggest that the mechanism for this involves increased activity of the cyclo-oxygenase pathway, the signal transduction pathway mediating this effect is unknown. 2 Platelets from diabetic patients as well as normal platelets and normal platelets exposed to high glucose concentrations were used to determine the role of the polyol pathway, diacylglycerol (DAG) production, protein kinase C (PKC) activity and phospholipase A2 (PLA2) activity on vasodilation in rabbit carotid arteries. 3 We found that two aldose-reductase inhibitors, tolrestat and sorbinil, caused only a modest improvement in the impairment of vasodilation by glucose exposed platelets. However, sorbitol and fructose could not be detected in the platelets, at either normal or hyperglycaemic conditions. We found that incubation in 17 mM glucose caused a significant increase in DAG levels in platelets. Furthermore, the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused significant impairment of platelet-mediated vasodilation. The PKC inhibitors calphostin C and H7 as well as inhibitors of PLA2 activity normalized the ability of platelets from diabetic patients to cause vasodilation and prevented glucose-induced impairment of platelet-mediated vasodilation in vitro. 4 These results suggest that the impairment of platelet-mediated vasodilation caused by high glucose concentrations is mediated by increased DAG levels and stimulation of PKC and PLA2 activity. Keywords: Glucose; signal-transduction; platelet; vasodilation; diabetes Abbreviations: ADP, adenosine diphosphate; DAG, diacyglycerol; DEDA, dimethyleicosadienoic acid; EDNO, endothelium-derived nitric oxide; OAG, 1-oleoyl-2-acetyl-sn-glycerol; PKC, protein kinase C; PLA2, phospholipase A2; PMA, phorbol 12-myristate 13-acetate


Activated normal platelets produce vasodilation via release of platelet-derived adenosine diphosphate (ADP), which in turn stimulates the release of endothelium-derived nitric oxide (EDNO) . EDNO causes vascular smooth muscle relaxation and inhibits platelet aggregation and excessive thrombus formation. Recent reports suggest that platelets from patients with diabetes mellitus lack the ability to produce EDNO-dependent vasodilation. This platelet defect can be reproduced in vitro by exposure of normal human platelets to high glucose concentrations, in a time and concentration dependent manner. This glucose-induced platelet defect appears to involve activation of the cyclo-oxygenase pathway, including thromboxane synthase. However, it remains unknown how exposure of platelets to high concentrations of glucose in vivo or in vitro, leads to increased activity of these enzymes. Previous studies indicate that high glucose concentrations mediate some of their adverse biologic effects via the polyol pathway high glucose increases intracellular diacylglycer-ol (DAG) levels, upregulates protein kinase C (PKC) activity and can lead to increased arachidonic acid release via PKC-mediated increase in phospholipase A2 activity, which in turn increases activity of cyclo-oxygenase. In this study we explore the possible role of these metabolic pathways in mediating the inability of diabetic and hyperglycaemia-induced platelets to produce vasodilation. In this study we show that in vitro incubation of normal human platelets in high glucose causes a significant increase in platelet DAG levels, which is evident after 30 min.

The role of protein kinase-C (PKC)

DAG and OAG are known activators of PKC. Data in Figure 2 show that normal human platelets incubated with the DAG analogue, (OAG), in order to mimic the effect of increased intracellular DAG, lost their ability to cause vasodilation.  Next we tested whether enhanced PKC activity plays a role in the signalling pathway leading to impaired ability of diabetic platelets to cause vasodilation. We found that platelets from patients with diabetes mellitus that were treated with the PKC-inhibitor calphostin-C produced normal vasodilation, while untreated platelets from the same patients lacked the ability to cause vasorelaxation (Figure 3A). Similarly, while normal platelets incubated in high glucose lost their ability to cause vasorelaxation, co-incubation with calphostin-C prevented the glucose-mediated impairment of platelet-mediated vasodila-tion (Figure 3B). Calphostin-C did not affect the ability of normal platelets to mediate vasodilation: 35±3 vs 37±4% increase in vessel diameter, with or without the inhibitor (n=5), respectively. Similar results were obtained with the PKC-inhibitor H7 (50 ILM) (results not shown).  In addition, normal platelets  `primed’ by a 20 min incubation in Tyrode’s buffer containing PMA (80 nM) completely lost their ability to produce vasorelaxation (Figure 4). Figure 3 (A) Platelets were isolated from patients with diabetes mellitus (n=6). Platelets were incubated in Tyrode’s buffer for 2 h with or without calphostin-C (50 nM). Subsequently the platelets were thrombin (0.1 U ml1) activated and perfused through a phenylephrine (10 jIM) preconstricted normal rabbit carotid artery, and the change in vessel diameter measured. *P<0.01. (B) Platelets isolated from healthy donors (n=6) were incubated in Tyrode’s buffer containing either 6.6 mM (118 mg dl1) [NL Plts] or 17 mM (300 mg dl1) [Glucose Plts] glucose for 4 h. For the last 2 h the PKC-inhibitor calphostin-C (50 nM) was added to some of the high glucose treated platelets. Subsequently the three groups of platelets were thrombin (0.1 U ml1) activated and perfused through a phenylephrine (10 jIM) preconstricted normal rabbit carotid artery, and the change in vessel diameter measured. *P<0.01 vs NL-Plts and Gluc-Plts+Calp-C. (noy shown) Figure 4 Platelets from healthy donors (n=8) were isolated separated into two groups and treated with or without phorbol 12-myristate 13-acetate (PMA) (80 nM) for 20 min. After a washout period, treated and untreated platelets were thrombin (0.1 U ml1) activated and perfused through a phenylephrine (10 jIM) precon-stricted rabbit carotid artery, and the change in vessel diameter measured. *P<0.01 for PMA-Plts vs NL-Plts. (not shown)


In summary, the results of this study along with recently published data (Oskarsson & Hofmeyer 1997; Oskarsson et al., 1997) suggest that high glucose levels cause an increase in platelet DAG that upregulates the activity of PKC, which in turn increases the activity of phospholipase A2 that causes release of arachidonic acid which leads to increased activity of cyclo-oxygenase and thromboxane synthase in platelets (Oskarsson et al., 1997). From a clinical perspective this pathway is of considerable interest since it lends itself to therapeutic interventions with inhibitors both at the level of cyclo-oxygenase and the thromboxane-synthase.


OSKARSSON, H.J. & HOFMEYER, T.G. (1996). Platelet-mediated endothelium-dependent vasodilation is impaired by platelets from patients with diabetes mellitus. J. Am. Coll. Cardiol., 27, 1464 – 1470. OSKARSSON, H.J. & HOFMEYER, T.G. (1997). Diabetic human platelets release a substance which inhibits platelet-mediated vasodilation. Am. J. Phys., 273, H371 – H379. OSKARSSON, H.J., HOFMEYER, T.G. & KNAPP, H.R. (1997). Malondialdehyde inhibits platelet-mediated vasodilation by interfering with platelet-derived ADP. JACC, 29 (Suppl A): 304A.

G-Protein−Coupled Receptors as Signaling Targets for Antiplatele t Therapy

Susan S. Smyth, Donna S. Woulfe, Jeffrey I. Weitz, Christian Gachet, Pamela B. Conley, et al. Participants in the 2008 Platelet Colloquium Arterioscler Thromb Vasc Biol. 2009;29:449-457.     http://dx.doi.org/10.1161/ATVBAHA.108.176388    Online ISSN: 1524-4636    http://atvb.ahajournals.org/content/29/4/449


Platelet G protein–coupled receptors (GPCRs) initiate and reinforce platelet activation and thrombus formation. The clinical utility of antagonists of the P2Y12 receptor for ADP suggests that other GPCRs and their intracellular signaling pathways may represent viable targets for novel antiplatelet agents. For example, thrombin stimulation of platelets is mediated by 2 protease-activated receptors (PARs), PAR-1 and PAR-4. Signaling downstream of PAR-1 or PAR-4 activates phospholipase C and protein kinase C and causes autoamplification by production of thromboxane A2, release of ADP, and generation of more thrombin. In addition to ADP receptors, thrombin and thromboxane A2 receptors and their downstream effectors—including phosphoinositol-3 kinase, Rap1b, talin, and kindlin—are promising targets for new antiplatelet agents. The mechanistic rationale and available clinical data for drugs targeting disruption of these signaling pathways are discussed. The identification and development of new agents directed against specific platelet signaling pathways may offer an advantage in preventing thrombotic events while minimizing bleeding risk. (Arterioscler Thromb Vasc Biol. 2009;29:449-457.) Key Words: platelets . signaling . G proteins . receptors . thrombosis


Since the first observations of agonist-induced platelet aggregation in 1962, remarkable progress has been made in identifying cell surface receptors and intracellular signaling pathways that regulate platelet function. These discoveries have translated into estab­lished, new, and emerging therapeutics to treat and prevent acute ischemic events by targeting platelet signal transduction.  Indeed, antiplatelet therapy is a mainstay of initial management of patients with ACS and those undergoing percutaneous coronary intervention (PCI). Evidence-based refinements in anticoagulant and antiplatelet therapies have played an important role in the progressive decline in the death rate from coronary disease observed from 1994 to 2004. Despite these therapeutic advances, however, ACS patients receiving “optimal” antithrombotic therapy still suf­fer cardiovascular events. Platelet Signaling Pathways

Vascular injury—whether caused by spontaneous rupture of atherosclerotic plaque, plaque erosion, or PCI-related or other trauma—exposes adhesive proteins, tissue factor, and lipids promoting platelet tethering, adhesion, and activation. Once bound and activated, platelets release soluble mediators such as ADP, thromboxane A2, and serotonin and facilitate throm­bin generation. These mediators, in turn, stimulate GPCRs on the platelet surface that are critical to initiation of various intracellular signaling pathways, including activa­tion of phospholipase C (PLC), protein kinase C (PKC), and phosphoinositide (PI)-3 kinase. Both calcium and PKC con­tribute to activation of the small G protein,  Recently, members of the kindlin family of focal adhesion proteins have been identified as integrin activators, perhaps functioning to facilitate talin–integrin interactions. Platelet signaling pathways Figure. Role of G protein–coupled receptors in the thrombotic process. In humans, protease-activated receptors (PAR)-1 and PAR-4 are coupled to intracellular signaling pathways through molecular switches from the Gq, G12, and Gi protein families. When thrombin (scissors) cleaves the amino-terminal of PAR-l and PAR-4, several signaling pathways are activated, one result of which is ADP secretion. By binding to its receptor, P2Y12, ADP activates additional Gi-mediated pathways. In the absence of wounding, platelet activation is counteracted by signaling from PG I2 (PGI2). Adapted from references 26–28 with permission. Ca2 indicates calcium; CalDAG-GEF1, calcium and diacylglcerol-regulated guanine-nucleotide exchange factor 1; GP, glycoprotein; IP, prostacyclin; PKC, pro­tein kinase C; PLC, phospholipase C; RIAM, Rap1-GTP–interacting adapter molecule.

Future Directions: P2Y1 and P2X Inhibition

Given the clinical success of the P2Y12 antagonists, it is worthwhile to investigate other purinergic signaling pathways in platelets. Although platelets have 2 P2Y receptors acting synergistically through different signaling pathways, the overall platelet response to ADP is relatively modest. For example, ADP alone elicits only reversible responses and does not promote platelet secretion. The low number of ADP receptors on the platelet surface also may limit signal­ing.

Thrombin Signaling in Platelets

Thrombin, the most potent platelet agonist, has diverse effects on various vascular cells. For example, thrombin promotes chemotaxis, adhesion, and inflammation through its effects on neutrophils and monocytes. Thrombin also influ­ences vascular permeability through its effects on endothelial cells and triggers smooth muscle vasoconstriction and mitogenesis.54 Thrombin interacts with 2 protease-activated receptors (PARs) on the surface of human platelets—PAR-1 and PAR-4. Signaling through the PARs is triggered by thrombin-mediated cleavage of the extracellular domain of the receptor and exposure of a “tethered ligand” at the new end of the receptor (Figure 1). Signaling through either PAR can activate PLC and PKC and cause autoamplification through the production of thromboxane A2, the release of ADP, and generation of more thrombin on the platelet surface.

PAR-1 Antagonists as Antithrombotic Therapy

The expression profiles of PARs on platelets differ between humans and nonprimates. Mouse platelets lack PAR-1 and largely signal through PAR-4 in response to thrombin, with PAR-3 serving a cofactor function. Platelets from cynomol-gus monkeys contain primarily PAR-1 and PAR-4, and a peptide-mimetic PAR-1 antagonist extends the time to throm­bosis after carotid artery injury. The nonpeptide antagonist SCH 530348 (described below) inhibits thrombin- and PAR-1 agonist peptide (TRAP)-induced platelet aggregation (inhibitory concentrations of 47 nmol/L and 25 nmol/L, respectively), but it has no effect on ADP, collagen, U46619, or PAR-4 agonist peptide stimulation of platelets. SCH 530348 has excellent bioavailability in rodents and monkeys (82%; 1 mg/kg) and completely inhibits ex vivo platelet aggregation in response to TRAP within 1 hour of oral administration in monkeys with no effect on prothrombin or activated partial thromboplastin times. Of the PAR-1 antagonists, SCH 530348 and E5555 are the compounds farthest along in development and clinical testing. SCH 530348 is an oral reversible PAR-1 antagonist de­rived from himbacine, a compound found in the bark of the Australian magnolia tree. In clinical trials, 68% of patients showed ~80% inhibition of platelet aggregation in response to thrombin receptor activating peptide (TRAP; 15 mol/L) 60 minutes after receiving a 40-mg loading dose of SCH 530348. By 120 minutes, the proportion had risen to 96%. In a Phase 2 trial of SCH 530348, 1031 patients scheduled for angiography and possible stenting were randomized to re­ceive SCH 530348 or placebo plus aspirin, clopidogrel, and antithrombin therapy (heparin or bivalirudin). Major and minor bleeding did not differ substantially between the placebo and individual or combined SCH 530348 groups.

Future Directions: PAR-4 Inhibition

Activation and signaling of PAR-1 and PAR-4 provoke a biphasic “spike and prolonged” response, with PAR-1 acti­vated at thrombin concentrations 50% lower than those required to activate PAR-4. A 4-amino acid segment, YEPF, on the extracellular domain of PAR-1 appears to account for the receptor’s high-affinity interactions with thrombin. The YEPF sequence has homology to the COOH-terminal of hirudin and its synthetic GEPF analog, bivaliru-din, which can interact with exosite-1 on thrombin. Thus, thrombin may interact in tandem with PAR-1 and PAR-4, with the initial interactions involving exosite-1 and PAR-1, and subsequent docking at PAR-4 via the thrombin active site.56 PAR-1 and PAR-4 may form a stable heterodimer that enables thrombin to act as a bivalent functional agonist, rendering the PAR-1–PAR-4 heterodimer complex a unique target for novel antithrombotic therapies. Pepducins, or cell-permeable peptides derived from the third intracellular loop of either PAR-1 or PAR-4, disrupt signaling between the receptors and G proteins and inhibit thrombin-induced platelet aggregation. In mice, a PAR-4 pepducin has been shown to prolong bleeding times and attenuate platelet activation. Combining bivalirudin with a PAR-4 pepducin (P4pal-i1) inhibited aggregation of human platelets from 15 healthy volunteers, even in response to high concentrations of thrombin. In addition, although bivaliru-din and P4pal-i1 each delayed the time to carotid artery occlusion after ferric chloride-induced injury in guinea pigs, their combination prolonged the time to occlusion more than did bivalirudin alone. Additional blockade of the PAR-4 receptor may confer a benefit beyond that achieved by inhibition of thrombin activity.

Targeting Thromboxane Signaling

Thromboxane A2 acts on the thromboxane A2/prostaglandin (PG) H2 (TP) receptor, causing PLC signaling and platelet activation. Several drugs have been tested and developed that prevent thromboxane synthesis—most notably, aspirin. Be­yond the documented success of aspirin, however, results have been uniformly disappointing with a wide variety of thromboxane synthase inhibitors.  Likewise, a multitude of TP receptor antagonists have been developed, but few have progressed beyond Phase 2 trials because of safety concerns. More recently, the thromboxane A2 receptor antagonist terutroban (S18886) showed rapid, potent inhibition of platelet aggregation in a porcine model of in-stent thrombosis that was comparable to the combination of aspirin and clopidogrel but with a more favorable bleeding profile. Ramatroban, another TP inhibitor approved in Japan for treatment of allergic rhinitis, has shown antiaggre-gatory effects in vitro comparable to those of aspirin and cilostazol.

Novel Downstream Signaling Targets

Signaling pathways stimulated by GPCR activation are es­sential for thrombus formation and may represent potential targets for drug development. One pathway involved in platelet activation is signaling through lipid kinases. PI-3 kinases transduce signals by generating lipid second­ary messengers, which then recruit signaling proteins to the plasma membrane. A principal target for PI-3K signaling is the protein kinase Akt (Figure 1). Platelets contain both the Akt1 and Akt2 isoforms.28 In mice, both Akt1 and Akt2 are required for thrombus formation. Mice lacking Akt2 have aggregation defects in response to low concentrations of thrombin or thromboxane A2 and corresponding defects in dense and a-granule secretion. The Akt isoforms have multiple substrates in platelets. Glycogen synthase kinase (GSK)-3(3 is phosphorylated by Akt in platelets and sup­presses platelet function and thrombosis in mice. Akt-mediated phosphorylation of GSK-3(3 inhibits the kinase activity of the enzyme, and with it, its suppression of platelet function. Akt activation also stimulates nitric oxide produc­tion in platelets, which results in protein kinase G–dependent degranulation. Finally, Akt has been implicated in activa­tion of cAMP-dependent phosphodiesterase (PDE3A), which plays a role in reducing platelet cAMP levels after thrombin stimulation.67 Each of these Akt-mediated events is expected to contribute to platelet activation. Rap1 members of the Ras family of small G proteins have been implicated in GPCR signaling and integrin activation. Rap1b, the most abundant Ras GTPase in platelets, is activated rapidly after GPCR stimulation and plays a key role in the activation of integrin aIIb(3) Stimulation of Gq-linked receptors, such as PAR-4 or PAR-1, activates PLC and, with consequent increases in intracellular calcium, PKC. These signals in turn activate calcium and diacylglcerol-regulated guanine-nucleotide exchange factor 1 (CalDAG-GEF1), which has been implicated in activation of Rap1 in plate-lets. Experiments in CalDAG-GEF1-deficient platelets indicate that PKC- and CalDAG-GEF1–dependent events represent independent synergistic pathways leading to Rap1-mediated integrin aIIb(33 activation. Consistent with this concept, ADP can stimulate Rap1b activation in a P2Y12– and PI-3K-dependent, but calcium-independent, manner. A final common step in integrin activation involves bind­ing of the cytoskeletal protein talin to the integrin-(33-subunit cytoplasmic tail. Rap1 appears to be required to form an activation complex with talin and the Rap effector RIAM, which redistributes to the plasma membrane and unmasks the talin binding site, resulting in integrin activation. Mice that lack Rap1b or platelet talin have a bleeding disorder with impaired platelet aggregation because of the lack of integrin aIIb( (3activation. In contrast, mice with a integrin-(33 subunit mutation that prevents talin binding have impaired agonist-induced platelet aggregation and are protected from throm­bosis, but do not display pathological bleeding, suggest­ing that this interaction may be an attractive therapeutic target. Recently, members of the kindlin family of focal adhesion proteins, kindlin-2 and kindlin-3, have been identi­fied as coactivators of integrins, required for talin activation of integrins. Kindlin-2 binds and synergistically en­hances talin activation of aIIb. Of note, deficiency in kindlin-3, the predominant kindlin family member found in hematopoietic cells, results in severe bleeding and protection from thrombosis in mice.


Antiplatelet therapy targeting thromboxane production, ADP effects, and fibrinogen binding to integrin aIIb(33 have proven benefit in preventing or treating acute arterial thrombosis. New agents that provide greater inhibition of ADP signaling and agents that impede thrombin’s actions on platelets are currently in clinical trials. Emerging strategies to inhibit platelet function include blocking alternative platelet GPCRs and their intracellular signaling pathways. The challenge remains to determine how to best combine the various current and pending antiplatelet therapies to maximize benefit and minimize harm. It is well documented that aspirin therapy increases bleeding compared with pla­cebo; that when clopidogrel is added to aspirin therapy, bleeding increases relative to the use of aspirin therapy alone; and that when even greater P2Y12 inhibition with prasugrel is added to aspirin therapy, bleeding is further increased com­pared with the use of clopidogrel and aspirin combination therapy. Does this mean that improved antiplatelet efficacy is mandated to come at the price of increased bleeding? Not necessarily, but it will require a far better understanding of platelet signaling pathways and what aspects of platelet function must be blocked to minimize arterial thrombosis. One of the best clinical examples of the disconnect between antiplatelet-related bleeding and antithrombotic ef­ficacy is the case of the oral platelet glycoprotein (GP) IIb/IIIa antagonists. The use of these agents uniformly led to significantly greater bleeding compared with aspirin but no greater efficacy; in fact, mortality was increased among patients receiving the oral glycoprotein IIb/IIIa inhibitors.77 Through an improved understanding of platelet signaling pathways, antiplatelet therapies likely can be developed not based on their ability to inhibit platelets from aggregating, as current therapies are, but rather based on their ability to prevent the clinically meaningful consequences of platelet activation. What exactly these are remains the greatest obstacle.

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(no name assigned is Larry H Bernstein, MD)

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αllbβ3 Antagonists As An Example of Translational Medicine Therapeutics   http://phrmaceuticalintelligence.com/2013-10-12/larryhbern_BS-Coller/αllbβ3_Antagonists_As_An_Example_of_Translational_Medicine_Therapeutics

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Introducing Dr. Tim Wu – Interventional Cardiologist, Inventor and Entrepreneur

Author: Ed Kislauskis, PhD

Welcome readers to the first in a series of interviews with future scientific leaders in biotechnology and medicine.  In this post I interview a close colleague and clinical scientist who appears to be on a fast-track to achieving his vision for the future of interventional cardiology – at the very vanguard of applied nanotechnology.

Tim (Tiangen) Wu, M.D has graciously accepted my invitation to answer a few questions about how his career path and primary goal to develop and commercialize his first product, a fully-biodegradable drug-eluting stent he calls the PowerStent® Absorb (see insert).  This technology combines three especially innovations:  a unique balloon-expandable stent design (PowerStent®), a bioabsorbable nanoparticle composition (BioDe®), and a formulation of two commercially-available anti-restenosis drugs (Combo®).


About the Subject

Dr. Wu received his clinical education in China and research training in the USA. In 1988, he graduated with an MD from the prestigious Linyli Medical School and completed a fellowship in clinical cardiology at the Tonji Medical University.  In 1993, presented with an opportunity to travel to the US, he uprooted to accept a position as visiting scholar, and ultimately post-doctoral fellow,in Jeffrey Isner’s lab at St. Elizabeth Hospital (Tufts University) and the Beth Israel Medical Center (Harvard Medical).  There he investigated the biology of stenosis, and directed sponsored research projects to evaluate the safety and efficacy of the latest commercially-developed drug-coated stents (DES) in animals.

After  a decade in academia, Dr. Wu made the successful transition to industry and joined Nitromed Inc. as a Research Scientist.  His next stop was as a Research Director at Biomedical Research Models, Inc (2000-2006) where we met and collaborated on developing and characterizing macrovascular disease in an inbred, type 2 diabetic rat model.  After a 20 year career, and upon gaining additional qualification in Mechanical Engineering (Wentworth Institute), Business Administration (MIT), Clinical Research Affairs (Mass. Biotech Council), and Medical Device Regulatory Affairs (North Eastern Univ.), he was ready to take the entrepreneurial leap.  His first company, VasoTech would aim to re-engineer the clinical standards of stent design and drug delivery.

In 2007, Dr. Wu founded VasoTech, Inc. from inside his home garage. Less than a year later, VasoTech received a $1.5M SBIR fast-track grant award from the NIH.  With funding, VasoTech joined the newly announced M2D2 facility on the University of Massachusetts Lowell campus, and expanded operations in China.  With the support of one of his closest advisors, Dr. Stephen McCarthy and other research faculty, Dr. Wu was appointed as an adjunct faculty in the Dept. of BioMedical Engineering at the UMass/Lowell where he mentored a number of talented graduate students.  Dr. Wu is recognized as a senior reviewer on the NIH Bioengineering, Surgical Science and Technology Study Section, and Biomaterials, Delivery Systems and Nanotechnology Special Emphasis Panels servicing the  Small Business Innovation Research (SBIR) grant program.

Dr. Wu’s work at Vasotech is devoted to developing a 3rd generation of fully biodegradable DES coronary stents to solve two major complications associated with stenting, restenosis and late-stage thrombosis. Thusfar, his ideas have attracted well over $1.5 Million (USD) in Small Business Innovation Research (SBIR) grant awards from the National Institute of Diabetes and Digestive and Kidney Diseases, and $1million (USD) from China Innovative Talent Leadership Program.  Through his efforts VasoTech is well positioned to attract the strategic partnerships and venture capital investments necessary to translate his research through clinical stages of development both in China and the US.

The Interview

Kislauskis:  Please help our readers understand the current clinical approach to CAD.

Wu:  Most patients with advanced atherosclerosis diseases are at risk for occlusive coronary arterial disease and stroke. Consequently, it is recommended they undergo a percutaneous intervention (PCI); essentially, balloon angioplasty followed by instillation of one or more expandable metal stents. A properly expanded stent will dilate the vessel and increase blood flow to cardiac muscle tissue. Current 2nd generation drug-eluting-stents (DES) release drugs to inhibit the process of vascular remodeling leading to restenosis. Because the DES approach is remarkably successful and lowers the rate of restenosis to < 10%, DESs is now performed in 85% of the 2 million percutaneous coronary interventions (PCI) procedures annually in the U.S.

Kislauskis:  What is your impression of the recent 5 yr update of the FREEDOM trial comparing effectiveness of coronary artery bypass grafting (CABG) to PCI among diabetics? 1

Wu:  It makes perfect sense. There are other reports evaluating PCI in patients within high risk categories, including those with small diameter vessels, diabetes, and extensive, systemic vascular disease, showing unacceptably high rates of restenosis with bare metal stents (30%-60%) and DESs (6%-18%) 2-4.  We also know first-hand using an inbred rat strain that develops macrovascular disease 4 months after onset of spontaneous diabetes.  In our experiment model, just 4weeks following balloon-induced injury to the coratid artery (PTCA),  we observed 2x greater restenosis in female obese rats, and 4x greater stenosis in obese, diabetic rats  littermates (syndrome X) relative to the non-obese, non-diabetic littermates.  These results predicted that obesity (dyslipidemia) and diabetes (severe hyperglycemia) were major risk factors promoting the complication of restenosis (Wu and Kislauskis, unpublished).

Kislauskis: Can you tell our readers a bit more about the significance of restenosis and thrombosis and the concept behind your approach.

Wu: Two significant drawbacks to conventional PCI are the need for costly, long-term anti-platelet therapy; and having a metal artifact within the coronary vessel. In fact, once installed, the purpose of DES is to maintain patency and provide a scaffold until remodeling is complete, maybe 6 months.  The period of drug elution is typically shorter in duration.  In the event of restenosis, a second DES procedure is recommended and performed with satisfactory results.  However, leaving another metal artifact is problematic.

Most concerning to PCI patients, however, should be an increased risk of sudden death from heart attack from a clot (thrombosis) and tissue ischemia (myocardial infarction).  No available DES technology (eg. Cypher®or Taxus® DES) demonstrates any advantage over bare metal stents in this regard 5-7.  So the thinking is a metal artifact create an irregular vessel surface and micro-eddys in blood flow which ultimately result in late-stage thrombosis, particularly in patients who go off anti-their platelet therapy too soon 8.  Therefore and conceptually, by combining potent DES technology with a fully-biodegradable scaffold, designed to be absorbed fully into the tissue, likely will reduce the rate in-stent stenosis and prevents late-stage thrombosis.

Kislauskis: How did you come up with your unique polymer formulation?

Wu: It turns out that through a process of trial and error in the lab I was able to identify a biodegradable formulation which reduces the local inflammatory response common to all DES formulations while improving the stent’s radial strength.  With a stable drug delivery platform (BioDe®), the process of remodeling will contribute far less to restenosis.  Furthermore, and unlike all prior art, my BioDe® formulation can neutralize acidic intermediates generated during stent degradation that induce inflammation.  The combination of anti-restenosis drugs (Combo®) also is effective at inhibiting signaling pathways that contribute to restenosis.

Kislauskis:  How did you come to design the PowerStent®?

Wu: Again, a long process of trial and error, initially using computer applied design (CAD) principals I learned while earning attending a mechanical engineering certificate program at Wentworth Institute of Technology in Boston. Elements behind my concept for BioDe® came to me while I was involved in a home renovation project, working with grout.  Although the formulation is simple and may be duplicated, the process of manufacturing is complicated.

Kislauskis: So it’s your trade secret.

Wu: Absolutely.

Kislauskis: Can you summary its other advantages and your plans to commercialize the PowerStent®?

Wu: Preclinical, short duration (30 day) studies in porcine models with the PowerStent® Absorb deployed indicate that it will be non-inferior to the current metal DES and competing biodegradable stent technologies. Important functional attributes of the BioDe® polymer include better biocompatibility (less inflammatory), excellent radial strength, potent anti-restenosis activity, and a unique microporous surface that promotes integration into neointimal layer of stented vessel.  Ongoing and much longer duration studies may also support our contention that this design can reduce risks of late-stage in-stent thrombosis.

Kislauskis: What path and difficulties to you foresee in obtaining a regulatory approval to conduct clinical trials with the PowerStent® Absorb?

Wu:  FDA Guidance to commercialize conventional DES technology is available. Unfortunately, no guidance is published for a fully-biodegradable stent.  Therefore, I anticipate seeking advice from the regulatory bodies prior to petitioning for approval to perform clinical trials.  It will no doubt be a complicated process as this technology involves a novel drug combination (albeit FDA-approved drugs), and a novel formulation (albeit FDA-approved components), and a novel indwelling and bioabsorbable medical device (stent).  We are presently completing several required engineering studies for the final phase of pre-clinical safety and efficacy testing, in China. The goals are to obtain FDA pre-market and NDA approvals, and to receive a CE mark from major international markets including Europe and the BRICK nations.

Kislauskis: How will you commercialize this 3rd generation, fully-biodegradable stent?

Wu: There are likely 3 scenarios to complete development and commercialization.  One involves securing bridge funding from the NIH SBIR program, supplemented with angel financing to complete preclinical program. I project that a minimum of $6 Million (USD) will be required to complete regulatory approval and pivotal clinical trials.  Therefore, it is conceivable that a Series A round of equity financing from venture capitalists, in either US or China, will be required. A third scenario is to partner or sell the technology to a major player in this space to complete clinical testing and commercialization. Potential partners include Boston Scientific Company, J&J, etc. Any of these partners could facilitate the processes of regulatory approval, manufacturing, global distribution and marketing.  Discussions are underway with one such prospective partner and with several VC groups.

Kislauskis: What is its likely impact of this product on patient care and the field of interventional cardiology?

Wu: According to US statistics, approximately 14 million Americans suffer from CAD, and 500,000 people die from acute myocardial infarction. One million more survive but with a 1.5 to 15 times greater risk of mortality or morbidity than the rest of the population each year.  In the U.S., the annual health care costs of CAD are estimated to be in excess of $112 billion, and the estimated annual total direct cost associated with PCI with stents is over $2 billion.  I anticipate that our PowerStent® Absorb stent will be competitive in a marketplace estimated to be over $5 billion in 2010. Although CAD patients are the primary market, other related applications for our PowerStent Absorb technology include peripheral arteries, intracerebral vascular and small vessels which are also significant.

Kislauskis:  Thank you for your contribution to this site.  For more information about MMG, LLC and Dr. Wu’s technology please refer to his publications 9-13 or contact him directly at tiangenwu@yahoo.com.


1.   Mark A. Hlatky, M.D. Compelling Evidence for Coronary-Bypass Surgery in Patients with Diabetes.   N Engl J Med 2012; 367:2437-2438.

2.  Stamler, J. (1989) Epidemiology.  Established major risk factors, and the primary prevention of coronary heart disease. In: Chatterjee K, Karliner J, Rapaport E, Cheitlin MD, Parmlee WW, Sheinman, M eds. Cardiology, Philadelphia Penn: JB Lippincott, 1991, 7.2-7.35. (volume 2).

3. Tanabe, K, Regar, E et al.  Sirolimus-eluting stent for treatment of in-stentrestenosis: One-year angiographic and intravascular ultrasound follow-up. J. Am Col.Cardi.   (2003) 41: 12A.

4. Grube, Eberhard;  Silber, Sigmund.  Six- and twelve-month results from a randomized, double-blind trial on a slow-release paclitaxel-eluting stent for de novo coronary lesions. Circulation 2003: 107, 38-42.

5.  Iakovou I, Schmidt T, Bonizzoni E, et al. Incidence, predictors, and outcome of thrombosis after successful implantation of drug-eluting stents. JAMA 2005;293:2126–2130.

6.  Ong AT, McFadden EP, Regar E, et al. Late angiographic stent thrombosis (LAST) events with drug-eluting stents. J Am Coll Cardiol 2005;45:2088–2092.

7. Wang F, Stouffer GA, Waxman S, et al. Late coronary stent thrombosis: Early vs late stent thrombosis in the stent era. Catheter Cardiovasc Interven 2002;55:142–147.

8. McFadden EP, Stabile E, Regar E, et al. Late thrombosis in drug-eluting coronary stents after discontinuation of antiplatelet therapy. Lancet 2004;364:1519–1521.

9. Ma X, Oyamada S, Wu T, Robich MP, Wu H, Wang X, Buchholz B, McCarthy S, Bianchi CF, Sellke FW, Laham R. In vitro and in vivo degradation of poly(D, L-lactide-co-glycolide)/amorphous calcium phosphate copolymer coated on metal stents. J Biomed Mater Res A. 2011 Mar 15;96(4):632-8. doi: 10.1002/jbm.a.33016. Epub 2011 Jan 25.

10. Oyamada S, Ma X, Wu T, Robich MP, Wu H, Wang X, Buchholz B, McCarthy S, Bianchi CF, Sellke FW, Laham R. Trans-iliac rat aorta stenting: a novel high throughput preclinical stent model for restenosis and thrombosis. J Surg Res. 2011 Mar;166(1):e91-5. Erratum in: J Surg Res. 2012 May 1;174(1):184.

11. Ma X, Oyamada S, Gao F, Wu T, Robich MP, Wu H, Wang X, Buchholz B, McCarthy S, Gu Z, Bianchi CF, Sellke FW, Laham R Paclitaxel/sirolimus combination coated drug-eluting stent: in vitro and in vivo drug release studies. J Pharm Biomed Anal. 2011 Mar 25;54(4):807-11. Erratum in: J Pharm Biomed Anal. 2012 Feb 5;59:217.

12. Ma X, Wu T, Robich MP, Wang X, Wu H, Buchholz B, McCarthy S. Drug-eluting stents. Int J Clin Exp Med. 2010 Jul 15;3(3):192-201.

Other articles related to this subject were published in this Open Access OnlIne Scientific Journal:

Lev-Ari, A. (2012aa). Renal Sympathetic Denervation: Updates on the State of Medicine



Lev-Ari, A. (2012U). Imbalance of Autonomic Tone: The Promise of Intravascular Stimulation of Autonomics


Lev-Ari, A. (2012R). Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents https://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/


Lev-Ari, A. (2012K). Percutaneous Endocardial Ablation of Scar-Related Ventricular Tachycardia



Lev-Ari, A. (2012C). Treatment of Refractory Hypertension via Percutaneous Renal Denervation


Lev-Ari, A. (2012D). Competition in the Ecosystem of Medical Devices in Cardiac and Vascular Repair: Heart Valves, Stents, Catheterization Tools and Kits for Open Heart and Minimally Invasive Surgery (MIS)


Lev-Ari, A. (2012E). Executive Compensation and Comparator Group Definition in the Cardiac and Vascular Medical Devices Sector: A Bright Future for Edwards Lifesciences Corporation in the Transcatheter Heart Valve Replacement Market



Lev-Ari, A. (2012F). Global Supplier Strategy for Market Penetration & Partnership Options (Niche Suppliers vs. National Leaders) in the Massachusetts Cardiology & Vascular Surgery Tools and Devices Market for Cardiac Operating Rooms and Angioplasty Suites



Lev-Ari, A. (2012G).  Heart Remodeling by Design: Implantable Synchronized Cardiac Assist Device: Abiomed’s Symphony



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Telling NO to Cardiac Risk

DDAH Says NO to ADMA(1); The DDAH/ADMA/NOS Pathway(2)

Author-Writer-Reporter:  Stephen J. Williams, PhD

Endothelium-derived nitric oxide (NO) has been shown to be vasoprotective.  Nitric oxide enhances endothelial cell survival, inhibits excessive proliferation of vascular smooth muscle cells, regulates vascular smooth muscle tone, and prevents platelets from sticking to the endothelial wall.  Together with evidence from preclinical and human studies, it is clear that impairment of the NOS pathway increases risk of cardiovascular disease (3-5).

This post contains two articles on the physiological regulation of nitric oxide (NO) by an endogenous NO synthase inhibitor asymmetrical dimethylarginine (ADMA) and ADMA metabolism by the enzyme DDAH(1,2).  Previous posts on nitric oxide, referenced at the bottom of the page, provides excellent background and further insight for this posting. In summary plasma ADMA levels are elevated in patients with cardiovascular disease and several large studies have shown that plasma ADMA is an independent biomarker for cardiovascular-related morbidity and mortality(6-8).



Figure 1 A. Cardiac risks of ADMA B. Effects of ADMA (Photo credit: Wikipedia)

ADMA Production and Metabolism

Nuclear proteins such as histones can be methylated on arginine residues by protein-arginine methyltransferases, enzymes which use S-adenosylmethionine as methyl groups.  This methylation event is thought to regulate protein function, much in the way of protein acetylation and phosphorylation (9).  And much like phosphorylation, these modifications are reversible through methylesterases.   The proteolysis of these arginine-methyl modifications lead to the liberation of free guanidine-methylated arginine residues such as L-NMMA, asymmetric dimethylarginine (ADMA) and symmetrical methylarginine (SDMA).

The first two, L-NMMA and ADMA, have been shown to inhibit the activity of the endothelial NOS.  This protein turnover is substantial: for instance the authors note that each day 40% of constitutive protein in adult liver is newly synthesized protein. And in several diseases, such as muscular dystrophy, ischemic heart disease, and diabetes, it has been known since the 1970’s that protein catabolism rates are very high, with corresponding increased urinary excretion of ADMA(10-13).  Methylarginines are excreted in the urine by cationic transport.  However, the majority of ADMA and L-NMMA are degraded within the cell by dimethylaminohydrolase (DDAH), first cloned and purified in rat(14).

endogenous NO inhibitors from pubchem

Figure 2.  Endogenous inhibitors of NO synthase.  Chemical structures generated from PubChem.


DDAH specifically hydrolyzes ADMA and L-NMMA to yield citruline and demethylamine and usually shows co-localization with NOS. Pharmacologic inhibition of DDAH activity causes accumulation of ADMA and can reverse the NO-mediated bradykinin-induced relaxation of human saphenous vein.

Two isoforms have been found in human:

  • DDAH1 (found in brain and kidney and associated with nNOS) and
  • DDAH2 (highly expressed in heart, placenta, and kidney and associated with eNOS).

DDAH2 can be upregulated by all-trans retinoic acid (atRA can increase NO production).  Increased reactive oxygen species and possibly homocysteine, a risk factor for cardiovascular disease, can decrease DDAH activity(15,16).

  • The importance of DDAH activity can also be seen in transgenic mice which overexpress DDAH, exhibiting increased NO production, increased insulin sensitivity, and reduced vascular resistance  (17).  Likewise,
  • Transgenic mice, null for the DDAH1, showed increase in blood pressure, decreased NO production, and significant increase in tissue and plasma ADMA and L-NMMA.


Figure 3.  The DDAH/ADMA/NOS cycle. Figure adapted from Cooke and Ghebremarian (1).

As mentioned in the article by Cooke and Ghebremariam, the authors state: the weight of the evidence indicates that DDAH is a worthy therapeutic target. Agents that increase DDAH expression are known, and 1 of these, a farnesoid X receptor agonist, is in clinical trials


An alternate approach is to

  • develop an allosteric activator of the enzyme.  Although
  • development of an allosteric activator is not a typical pharmaceutical approach, recent studies indicate that this may be achievable aim(18).


1.            Cooke, J. P., and Ghebremariam, Y. T. : DDAH says NO to ADMA.(2011) Arteriosclerosis, thrombosis, and vascular biology 31, 1462-1464

2.            Tran, C. T., Leiper, J. M., and Vallance, P. : The DDAH/ADMA/NOS pathway.(2003) Atherosclerosis. Supplements 4, 33-40

3.            Niebauer, J., Maxwell, A. J., Lin, P. S., Wang, D., Tsao, P. S., and Cooke, J. P.: NOS inhibition accelerates atherogenesis: reversal by exercise. (2003) American journal of physiology. Heart and circulatory physiology 285, H535-540

4.            Miyazaki, H., Matsuoka, H., Cooke, J. P., Usui, M., Ueda, S., Okuda, S., and Imaizumi, T. : Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis.(1999) Circulation 99, 1141-1146

5.            Wilson, A. M., Shin, D. S., Weatherby, C., Harada, R. K., Ng, M. K., Nair, N., Kielstein, J., and Cooke, J. P. (2010): Asymmetric dimethylarginine correlates with measures of disease severity, major adverse cardiovascular events and all-cause mortality in patients with peripheral arterial disease. Vasc Med 15, 267-274

6.            Kielstein, J. T., Impraim, B., Simmel, S., Bode-Boger, S. M., Tsikas, D., Frolich, J. C., Hoeper, M. M., Haller, H., and Fliser, D. : Cardiovascular effects of systemic nitric oxide synthase inhibition with asymmetrical dimethylarginine in humans.(2004) Circulation 109, 172-177

7.            Kielstein, J. T., Donnerstag, F., Gasper, S., Menne, J., Kielstein, A., Martens-Lobenhoffer, J., Scalera, F., Cooke, J. P., Fliser, D., and Bode-Boger, S. M. : ADMA increases arterial stiffness and decreases cerebral blood flow in humans.(2006) Stroke; a journal of cerebral circulation 37, 2024-2029

8.            Mittermayer, F., Krzyzanowska, K., Exner, M., Mlekusch, W., Amighi, J., Sabeti, S., Minar, E., Muller, M., Wolzt, M., and Schillinger, M. : Asymmetric dimethylarginine predicts major adverse cardiovascular events in patients with advanced peripheral artery disease.(2006) Arteriosclerosis, thrombosis, and vascular biology 26, 2536-2540

9.            Kakimoto, Y., and Akazawa, S.: Isolation and identification of N-G,N-G- and N-G,N’-G-dimethyl-arginine, N-epsilon-mono-, di-, and trimethyllysine, and glucosylgalactosyl- and galactosyl-delta-hydroxylysine from human urine. (1970) The Journal of biological chemistry 245, 5751-5758

10.          Inoue, R., Miyake, M., Kanazawa, A., Sato, M., and Kakimoto, Y.: Decrease of 3-methylhistidine and increase of NG,NG-dimethylarginine in the urine of patients with muscular dystrophy. (1979) Metabolism: clinical and experimental 28, 801-804

11.          Millward, D. J.: Protein turnover in skeletal muscle. II. The effect of starvation and a protein-free diet on the synthesis and catabolism of skeletal muscle proteins in comparison to liver. (1970) Clinical science 39, 591-603

12.          Goldberg, A. L., and St John, A. C.: Intracellular protein degradation in mammalian and bacterial cells: Part 2. (1976) Annual review of biochemistry 45, 747-803

13.          Dice, J. F., and Walker, C. D.: Protein degradation in metabolic and nutritional disorders. (1979) Ciba Foundation symposium, 331-350

14.          Ogawa, T., Kimoto, M., and Sasaoka, K.: Purification and properties of a new enzyme, NG,NG-dimethylarginine dimethylaminohydrolase, from rat kidney. (1989) The Journal of biological chemistry 264, 10205-10209

15.          Ito, A., Tsao, P. S., Adimoolam, S., Kimoto, M., Ogawa, T., and Cooke, J. P.: Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase. (1999) Circulation 99, 3092-3095

16.          Stuhlinger, M. C., Tsao, P. S., Her, J. H., Kimoto, M., Balint, R. F., and Cooke, J. P. : Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine.(2001) Circulation 104, 2569-2575

17.          Sydow, K., Mondon, C. E., Schrader, J., Konishi, H., and Cooke, J. P.: Dimethylarginine dimethylaminohydrolase overexpression enhances insulin sensitivity. (2008) Arteriosclerosis, thrombosis, and vascular biology 28, 692-697

18.          Zorn, J. A., and Wells, J. A.: Turning enzymes ON with small molecules. (2010) Nature chemical biology 6, 179-188

Other research papers on Nitric Oxide and Cardiac Risk  were published on this Scientific Web site as follows:

The Nitric Oxide and Renal is presented in FOUR parts:

Part I: The Amazing Structure and Adaptive Functioning of the Kidneys: Nitric Oxide

Part II: Nitric Oxide and iNOS have Key Roles in Kidney Diseases

Part III: The Molecular Biology of Renal Disorders: Nitric Oxide

Part IV: New Insights on Nitric Oxide donors

Cardiac Arrhythmias: A Risk for Extreme Performance Athletes

What is the role of plasma viscosity in hemostasis and vascular disease risk?

Cardiovascular Risk Inflammatory Marker: Risk Assessment for Coronary Heart Disease and Ischemic Stroke – Atherosclerosis.

Endothelial Dysfunction, Diminished Availability of cEPCs, Increasing CVD Risk for Macrovascular Disease – Therapeutic Potential of cEPCs

Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I

Nitric Oxide Function in Coagulation

Read Full Post »

Special Considerations in Blood Lipoproteins, Viscosity, Assessment and Treatment

Special Considerations in Blood Lipoproteins, Viscosity, Assessment and Treatment

Author: Larry H. Bernstein, MD, FCAP


Curator: Aviva Lev-Ari, PhD, RN

This is the second of a two part discussion of viscosity, hemostasis, and vascular risk

This is Part II of a series on blood flow and shear stress effects on hemostasis and vascular disease.

See Part I on viscosity, triglycerides and LDL, and thrombotic risk.


Hemostatic Factors in Thrombophilia

Objectives.—To review the state of the art relating to elevated hemostatic factor levels as a potential risk factor for thrombosis, as reflected by the medical literature and the consensus opinion of recognized experts in the field, and to make recommendations for the use of specific measurements of hemostatic factor levels in the assessment of thrombotic risk in individual patients.

Data Sources.—Review of the medical literature, primarily from the last 10 years.

Data Extraction and Synthesis.—After an initial assessment of the literature, key points were identified. Experts were assigned to do an in-depth review of the literature and to prepare a summary of their findings and recommendations.

A draft manuscript was prepared and circulated to every participant in the College of American Pathologists Conference XXXVI: Diagnostic Issues in Thrombophilia prior to the conference. Each of the key points and associated recommendations was then presented for discussion at the conference. Recommendations were accepted if a consensus of the 27 experts attending the conference was reached. The results of the discussion were used to revise the manuscript into its final form.

Consensus was reached on 8 recommendations concerning the use of hemostatic factor levels in the assessment of thrombotic risk in individual patients.

The underlying premise for measuring elevated coagulation factor levels is that if the average level of the factor is increased in the patient long-term, then the patient may be at increased risk of thrombosis long-term. Both risk of thrombosis and certain factors increase with age (eg, fibrinogen, factor VII, factor VIII, factor IX, and von Willebrand factor). Are these effects linked or do we need age specific ranges? Do acquired effects like other diseases or medications affect factor levels, and do the same risk thresholds apply in these instances? How do we assure that the level we are measuring is a true indication of the patient’s average baseline level and not a transient change? Fibrinogen, factor VIII, and von Willebrand factor are all strong acute-phase reactants.

Risk of bleeding associated with coagulation factor levels increases with roughly log decreases in factor levels. Compared to normal (100%), 60% to 90% decreases in a coagulation factor may be associated with excess bleeding with major trauma, 95% to 98% decreases with minor trauma, and .99% decrease with spontaneous hemorrhage. In contrast, the difference between low risk and high risk for thrombosis may be separated by as little as 15% above normal.

It may be possible to define relative cutoffs for specific factors, for example, 50% above the mean level determined locally in healthy subjects for a certain factor. Before coagulation factor levels can be routinely used to assess individual risk, work must be done to better standardize and calibrate the assays used.

Detailed discussion of the rationale for each of these recommendations is presented in the article. This is an evolving area of research. While routine use of factor level measurements is not recommended, improvements in assay methodology and further clinical studies may change these recommendations in the future.

Chandler WL, Rodgers GM, Sprouse JT, Thompson AR.  Elevated Hemostatic Factor Levels as Potential Risk Factors for Thrombosis.  Arch Pathol Lab Med. 2002;126:1405–1414

Model System for Hemostatic Behavior

This study explores the behavior of a model system in response to perturbations in

  • tissue factor
  • thrombomodulin surface densities
  • tissue factor site dimensions
  • wall shear rate.

The classic time course is characterized by

  • initiation and
  • amplification of thrombin generation
  • the existence of threshold-like responses

This author defines a new parameter, the „effective prothrombotic zone‟,  and its dependence on model parameters. It was found that prothrombotic effects may extend significantly beyond the dimensions of the spatially discrete site of tissue factor expression in both axial and radial directions. Furthermore, he takes advantage of the finite element modeling approach to explore the behavior of systems containing multiple spatially distinct sites of TF expression in a physiologic model. The computational model is applied to assess individualized thrombotic risk from clinical data of plasma coagulation factor levels. He proposes a systems-based parameter with deep venous thrombosis using computational methods in combination with biochemical panels to predict hypercoagulability for high risk populations.


The Vascular Surface

The ‘resting’ endothelium synthesizes and presents a number of antithrombogenic molecules including

  • heparan sulfate proteoglycans
  • ecto-adenosine diphosphatase
  • prostacyclin
  • nitric oxide
  • thrombomodulin.

In response to various stimuli

  • inflammatory mediators
  • hypoxia
  • oxidative stress
  • fluid shear stress

the cell surface becomes ‘activated’ and serves to organize membrane-associated enzyme complexes of coagulation.

Fluid Phase Models of Coagulation

Leipold et al. developed a model of the tissue factor pathway as a design aid for the development of exogenous serine protease inhibitors. In contrast, Guo et al. focused on the reactions of the contact, or intrinsic pathway, to study parameters relevant to material-induced thrombosis, including procoagulant surface area.

Alternative approaches to modeling the coagulation cascade have been pursued including the use of stochastic activity networks to represent the intrinsic, extrinsic, and common pathways through fibrin formation and a kinetic Monte Carlo simulation of TF-initiated thrombin generation. Generally, fluid phase models of the kinetics of coagulation are both computationally and experimentally less complex. As such, the computational models are able to incorporate a large number of species and their reactions, and empirical data is often available for regression analysis and model validation. The range of complexity and motivations for these models is wide, and the models have been used to describe various phenomena including the ‘all-or-none’ threshold behavior of thrombin generation. However, the role of blood flow in coagulation is well recognized in promoting the delivery of substrates to the vessel wall and in regulating the thrombin response by removing activated clotting factors.

Flow Based Models of Coagulation

In 1990, Basmadjian presented a mathematical analysis of the effect of flow and mass transport on a single reactive event at the vessel wall and consequently laid the foundation for the first flow-based models of coagulation. It was proposed that for vessels greater than 0.1 mm in diameter, reactive events at the vessel wall could be adequately described by the assumption of a concentration boundary layer very close to the reactive surface, within which the majority of concentration changes took place. The height of the boundary layer and the mass transfer coefficient that described transport to and from the vessel wall were shown to stabilize on a time scale much shorter than the time scale over which concentration changes were empirically observed. Thus, the vascular space could be divided into two compartments, a boundary volume and a bulk volume, and furthermore, changes within the bulk phase could be considered negligible, thereby reducing the previously intractable problem to a pseudo-one compartment model described by a system of ordinary differential equations.

Basmadjian et al. subsequently published a limited model of six reactions, including two positive feedback reactions and two inhibitory reactions, of the common pathway of coagulation triggered by exogenous factor IXa under flow. As a consequence of the definition of the mass transfer coefficient, the kinetic parameters were dependent on the boundary layer height. Furthermore, the model did not explicitly account for intrinsic tenase or prothrombinase formation, but rather derived a rate expression for reaction in the presence of a cofactor. The major finding of the study was the predicted effect of increased mass transport to enhance thrombin generation by decreasing the induction time up to a critical mass transfer rate, beyond which transport significantly decreased peak thrombin levels thereby reducing overall thrombin production.

Kuharsky and Fogelson formulated a more comprehensive, pseudo-one compartment model of tissue factor-initiated coagulation under flow, which included the description of 59 distinct fluid- and surface-bound species. In contrast to the Baldwin-Basmadjian model, which defined a mass transfer coefficient as a rate of transport to the vessel surface, the Kuharsky-Fogelson model defined the mass transfer coefficient as a rate of transport into the boundary volume, thus eliminating the dependence of kinetic parameters on transport parameters. The computational study focused on the threshold response of thrombin generation to the availability of membrane binding sites. Additionally, the model suggested that adhered platelets may play a role in blocking the activity of the TF/ VIIa complex. Fogelson and Tania later expanded the model to include the protein C and TFPI pathways.

Modeling surface-associated reactions under flow uses finite element method (FEM), which is a technique for solving partial differential equations by dividing the vascular space into a finite number of discrete elements. Hall et al. used FEM to simulate factor X activation over a surface presenting TF in a parallel plate flow reactor. The steady state model was defined by the convection-diffusion equation and Michaelis-Menten reaction kinetics at the surface. The computational results were compared to experimental data for the generation of factor Xa by cultured rat vascular smooth muscle cells expressing TF.

Based on discrepancies between numerical and experimental studies, the catalytic activity of the TF/ VIIa complex may be shear-dependent. Towards the overall objective of developing an antithrombogenic biomaterial, Tummala and Hall studied the kinetics of factor Xa inhibition by surface-immobilized recombinant TFPI under unsteady flow conditions. Similarly, Byun et al. investigated the association and dissociation kinetics of ATIII inactivation of thrombin accelerated by surface-immobilized heparin under steady flow conditions. To date, finite element models that detail surface-bound reactions under flow have been restricted to no more than a single reaction catalyzed by a single surface-immobilized species.


Models of Coagulation Incorporating Spatial Parameter

Major findings include the roles of these specific coagulation pathways in the

  • initiation
  • amplification
  • termination phases of coagulation.

Coagulation near the activating surface was determined by TF/VIIa catalyzed factor Xa production, which was rapidly inhibited close to the wall. In contrast, factor IXa diffused farther from the surface, and thus factor Xa generation and clot formation away from the reactive wall was dependent on intrinsic tenase (IXa/ VIIIa) activity. Additionally, the concentration wave of thrombin propagated away from the activation zone at a rate which was dependent on the efficiency of inhibitory mechanisms.

Experimental and ‘virtual’ addition of plasma-phase thrombomodulin resulted in dose-dependent termination of thrombin generation and provided evidence of spatial localization of clot formation by TM with final clot lengths of 0.2-2 mm under diffusive conditions.

These studies provide an interesting analysis of the roles of specific factors in relation to space due to diffusive effects, but neglect the essential role of blood flow in the transport analysis. Additionally, the spatial dynamics of clot localization by thrombomodulin would likely be affected by restricting the inhibitor to its physiologic site on the vessel surface.

Finite Element Modeling

Finite element method (FEM) is a numerical technique for solving partial differential equations. Originally proposed in the 1940s to approach structural analysis problems in civil engineering, FEM now finds application in a wide variety of disciplines. The computational method relies on mesh discretization of a continuous domain which subdivides the space into a finite number of ‘elements’. The physics of each element are defined by its own set of physical properties and boundary conditions, and the simultaneous solution of the equations describing the individual elements approximate the behavior of the overall domain.

Sumanas W. Jordan, PhD Thesis. A Mathematical Model of Tissue Factor-Induced Blood Coagulation: Discrete Sites of Initiation and Regulation under Conditions of Flow.

Doctor of Philosophy in Biomedical Engineering. Emory University, Georgia Institute of Technology. May 2010.  Under supervision of: Dr. Elliot L. Chaikof, Departments of Surgery and Biomedical Engineering.

Blood Coagulation (Thrombin) and Protein C Pat...

Blood Coagulation (Thrombin) and Protein C Pathways (Blood_Coagulation_and_Protein_C_Pathways.jpg) (Photo credit: Wikipedia)

Coagulation cascade

Coagulation cascade (Photo credit: Wikipedia)


Cardiovascular Physiology: Modeling, Estimation and Signal Processing

With cardiovascular diseases being among the main causes of death in the world, quantitative modeling, assessment and monitoring of cardiovascular dynamics, and functioning play a critical role in bringing important breakthroughs to cardiovascular care. Quantification of cardiovascular physiology and its control mechanisms from physiological recordings, by use of mathematical models and algorithms, has been proved to be of important value in understanding the causes of cardiovascular diseases and assisting the diagnostic and prognostic process. This E-Book is derived from the Frontiers in Computational Physiology and Medicine Research Topic entitled “Engineering Approaches to Study Cardiovascular Physiology: Modeling, Estimation and Signal Processing.”

There are two review articles. The first review article by Chen et al. (2012) presents a unified point process probabilistic framework to assess heart beat dynamics and autonomic cardiovascular control. Using clinical recordings of healthy subjects during Propofol anesthesia, the authors demonstrate the effectiveness of their approach by applying the proposed paradigm to estimate

  • instantaneous heart rate (HR),
  • heart rate variability (HRV),
  • respiratory sinus arrhythmia (RSA)
  • baroreflex sensitivity (BRS).

The second review article, contributed by Zhang et al. (2011), provides a comprehensive overview of tube-load model parameter estimation for monitoring arterial hemodynamics.

The remaining eight original research articles can be mainly classified into two categories. The two articles from the first category emphasize modeling and estimation methods. In particular, the paper “Modeling the autonomic and metabolic effects of obstructive sleep apnea: a simulation study” by Cheng and Khoo (2012), combines computational modeling and simulations to study the autonomic and metabolic effects of obstructive sleep apnea (OSA).

The second paper, “Estimation of cardiac output and peripheral resistance using square-wave-approximated aortic flow signal” by Fazeli and Hahn (2012), presents a model-based approach to estimate cardiac output (CO) and total peripheral resistance (TPR), and validates the proposed approach via in vivo experimental data from animal subjects.

The six articles in the second category focus on application of signal processing techniques and statistical tools to analyze cardiovascular or physiological signals in practical applications. the paper “Modulation of the sympatho-vagal balance during sleep: frequency domain study of heart rate variability and respiration” by Cabiddu et al. (2012), uses spectral and cross-spectral analysis of heartbeat and respiration signals to assess autonomic cardiac regulation and cardiopulmonary coupling variations during different sleep stages in healthy subjects.

The paper “increased non-gaussianity of heart rate variability predicts cardiac mortality after an acute myocardial infarction” by Hayano et al. (2011) uses a new non-gaussian index to assess the HRV of cardiac mortality using 670 post-acute myocardial infarction (AMI) patients. the paper “non-gaussianity of low frequency heart rate variability and sympathetic activation: lack of increases in multiple system atrophy and parkinson disease” by Kiyono et al. (2012), applies a non-gaussian index to assess HRV in patients with multiple system atrophy (MSA) and parkinson diseases and reports the relation between the non-gaussian intermittency of the heartbeat and increased sympathetic activity. The paper “Information domain approach to the investigation of cardio-vascular, cardio-pulmonary, and vasculo-pulmonary causal couplings” by Faes et al. (2011), proposes an information domain approach to evaluate nonlinear causality among heartbeat, arterial pressure, and respiration measures during tilt testing and paced breathing protocols. The paper “integrated central-autonomic multifractal complexity in the heart rate variability of healthy humans” by Lin and Sharif (2012), uses a relative multifractal complexity measure to assess HRV in healthy humans and discusses the related implications in central autonomic interactions. Lastly, the paper “Time scales of autonomic information flow in near-term fetal sheep” by Frasch et al. (2012), analyzes the autonomic information flow (AIF) with kullback–leibler entropy in fetal sheep as a function of vagal and sympathetic modulation of fetal HRV during atropine and propranolol blockade.

In summary, this Research Topic attempts to give a general panorama of the possible state-of-the-art modeling methodologies, practical tools in signal processing and estimation, as well as several important clinical applications, which can altogether help deepen our understanding about heart physiology and pathology and further lead to new scientific findings. We hope that the readership of Frontiers will appreciate this collected volume and enjoy reading the presented contributions. Finally, we are grateful to all contributed authors, reviewers, and editorial staffs who had all put tremendous effort to make this E-Book a reality.

Cabiddu, R., Cerutti, S., Viardot, G., Werner, S., and Bianchi, A. M. (2012). Modulation of the sympatho-vagal balance during sleep: frequency domain study of heart rate variability and respiration. Front. Physio. 3:45. doi: 10.3389/fphys.2012.00045

Chen, Z., Purdon, P. L., Brown, E. N., and Barbieri, R. (2012). A unified point process probabilistic framework to assess heartbeat dynamics and autonomic cardiovascular control. Front. Physio. 3:4. doi: 10.3389/fphys.2012.00004

Cheng, L., and Khoo, M. C. K. (2012). Modeling the autonomic and metabolic effects of obstructive sleep apnea: a simulation study. Front. Physio. 2:111. doi: 10.3389/fphys.2011.00111

Faes, L., Nollo, G., and Porta, A. (2011). Information domain approach to the investigation of cardio-vascular, cardio-pulmonary, and vasculo-pulmonary causal couplings. Front. Physio. 2:80. doi: 10.3389/fphys.2011.00080

Fazeli, N., and Hahn, J.-O. (2012). Estimation of cardiac output and peripheral resistance using square-wave-approximated aortic flow signal. Front. Physio. 3:298. doi: 10.3389/fphys.2012.00298

Frasch, M. G., Frank, B., Last, M., and Müller, T. (2012). Time scales of autonomic information flow in near-term fetal sheep. Front. Physio. 3:378. doi: 10.3389/fphys.2012.00378

Hayano, J., Kiyono, K., Struzik, Z. R., Yamamoto, Y., Watanabe, E., Stein, P. K., et al. (2011). Increased non-gaussianity of heart rate variability predicts cardiac mortality after an acute myocardial infarction. Front. Physio. 2:65. doi: 10.3389/fphys.2011.00065

Kiyono, K., Hayano, J., Kwak, S., Watanabe, E., and Yamamoto, Y. (2012). Non-Gaussianity of low frequency heart rate variability and sympathetic activation: lack of increases in multiple system atrophy and Parkinson disease. Front. Physio. 3:34. doi: 10.3389/fphys.2012.00034

Lin, D. C., and Sharif, A. (2012). Integrated central-autonomic multifractal complexity in the heart rate variability of healthy humans. Front. Physio. 2:123. doi: 10.3389/fphys.2011.00123

Zhang, G., Hahn, J., and Mukkamala, R. (2011). Tube-load model parameter estimation for monitoring arterial hemodynamics. Front. Physio. 2:72. doi: 10.3389/fphys.2011.00072

Citation: Chen Z and Barbieri R (2012) Editorial: engineering approaches to study cardiovascular physiology: modeling, estimation, and signal processing. Front. Physio. 3:425. doi: 10.3389/fphys.2012.00425

fluctuations of cerebral blood flow and metabolic demand following hypoxia in neonatal brain

Most of the research investigating the pathogenesis of perinatal brain injury following hypoxia-ischemia has focused on excitotoxicity, oxidative stress and an inflammatory response, with the response of the developing cerebrovasculature receiving less attention. This is surprising as the presentation of devastating and permanent injury such as germinal matrix-intraventricular haemorrhage (GM-IVH) and perinatal stroke are of vascular origin, and the origin of periventricular leukomalacia (PVL) may also arise from poor perfusion of the white matter. This highlights that cerebrovasculature injury following hypoxia could primarily be responsible for the injury seen in the brain of many infants diagnosed with hypoxic-ischemic encephalopathy (HIE).

The highly dynamic nature of the cerebral blood vessels in the fetus, and the fluctuations of cerebral blood flow and metabolic demand that occur following hypoxia suggest that the response of blood vessels could explain both regional protection and vulnerability in the developing brain.

This review discusses the current concepts on the pathogenesis of perinatal brain injury, the development of the fetal cerebrovasculature and the blood brain barrier (BBB), and key mediators involved with the response of cerebral blood vessels to hypoxia.

Baburamani AA, Ek CJ, Walker DW and Castillo-Melendez M. Vulnerability of the developing brain to hypoxic-ischemic damage: contribution of the cerebral vasculature to injury and repair? Front. Physio. 2012;  3:424. doi: 10.3389/fphys.2012.00424

remodeling of coronary and cerebral arteries and arterioles 

Effects of hypertension on arteries and arterioles often manifest first as a thickened wall, with associated changes in passive material properties (e.g., stiffness) or function (e.g., cellular phenotype, synthesis and removal rates, and vasomotor responsiveness). Less is known, however, regarding the relative evolution of such changes in vessels from different vascular beds.

We used an aortic coarctation model of hypertension in the mini-pig to elucidate spatiotemporal changes in geometry and wall composition (including layer-specific thicknesses as well as presence of collagen, elastin, smooth muscle, endothelial, macrophage, and hematopoietic cells) in three different arterial beds, specifically aortic, cerebral, and coronary, and vasodilator function in two different arteriolar beds, the cerebral and coronary.

Marked geometric and structural changes occurred in the thoracic aorta and left anterior descending coronary artery within 2 weeks of the establishment of hypertension and continued to increase over the 8-week study period. In contrast, no significant changes were observed in the middle cerebral arteries from the same animals. Consistent with these differential findings at the arterial level, we also found a diminished nitric oxide-mediated dilation to adenosine at 8 weeks of hypertension in coronary arterioles, but not cerebral arterioles.

These findings, coupled with the observation that temporal changes in wall constituents and the presence of macrophages differed significantly between the thoracic aorta and coronary arteries, confirm a strong differential progressive remodeling within different vascular beds.

These results suggest a spatiotemporal progression of vascular remodeling, beginning first in large elastic arteries and delayed in distal vessels.

Hayenga HN, Hu J-J, Meyer CA, Wilson E, Hein TW, Kuo L and Humphrey JD  Differential progressive remodeling of coronary and cerebral arteries and arterioles in an aortic coarctation model of hypertension. Front. Physio. 2012; 3:420. doi: 10.3389/fphys.2012.00420

C-reactive protein oxidant-mediated release of pro-thrombotic  factor

Inflammation and the generation of reactive oxygen species (ROS) have been implicated in the initiation and progression of atherosclerosis. Although C-reactive protein (CRP) has traditionally been considered to be a biomarker of inflammation, recent in vitro and in vivo studies have provided evidence that CRP, itself, exerts pro-thrombotic effects on vascular cells and may thus play a critical role in the development of atherothrombosis. Of particular importance is that CRP interacts with Fcγ receptors on cells of the vascular wall giving rise to the release of pro-thrombotic factors. The present review focuses on distinct sources of CRP-mediated ROS generation as well as the pivotal role of ROS in CRP-induced tissue factor expression. These studies provide considerable insight into the role of the oxidative mechanisms in CRP-mediated stimulation of pro-thrombotic factors and activation of platelets. Collectively, the available data provide strong support for ROS playing an important intermediary role in the relationship between CRP and atherothrombosis.

Zhang Z, Yang Y, Hill MA and Wu J.  Does C-reactive protein contribute to atherothrombosis via oxidant-mediated release of pro-thrombotic factors and activation of platelets? Front. Physio.  2012; 3:433. doi: 10.3389/fphys.2012.00433

CRP association with Peripheral Vascular Disease

To determine whether the increase in plasma levels of C-Reactive Protein (CRP), a non-specifi c reactant in the acute-phase of systemic infl ammation, is associated with clinical severity of peripheral arterial disease (PAD).

This is a cross-sectional study at a referral hospital center of institutional practice in Madrid, Spain.  These investigators took a stratifi ed random sampling of 3370 patients with symptomatic PAD from the outpatient vascular laboratory database in 2007 in the order of their clinical severity:

  • the fi rst group of patients with mild chronological clinical severity who did not require surgical revascularization,
  • the second group consisted of patients with moderate clinical severity who had only undergone only one surgical revascularization procedure and
  • the third group consisted of patients who were severely affected and had undergone two or more surgical revascularization procedures of the lower extremities in different areas or needed late re-interventions.

The Neyman affi xation was used to calculate the sample size with a fi xed relative error of 0.1.

A homogeneity analysis between groups and a unifactorial analysis of comparison of medians for CRP was done.

The groups were homogeneous for

  • age
  • smoking status
  • Arterial Hypertension
  • diabetes mellitus
  • dyslipemia
  • homocysteinemia and
  • specifi c markers of infl ammation.

In the unifactorial analysis of multiple comparisons of medians according to Scheffé, it was observed that

the median values of CRP plasma levels were increased in association with higher clinical severity of PAD

  • 3.81 mg/L [2.14–5.48] vs.
  • 8.33 [4.38–9.19] vs.
  • 12.83 [9.5–14.16]; p  0.05

as a unique factor of tested ones.

Plasma levels of CRP are associated with not only the presence of atherosclerosis but also with its chronological clinical severity.

De Haro J, Acin F, Medina FJ, Lopez-Quintana A, and  March JR.  Relationship Between the Plasma Concentration of C-Reactive Protein and Severity of Peripheral Arterial Disease.
Clinical Medicine: Cardiology 2009;3: 1–7

Hemostasis induced by hyperhomocysteinemia

Elevated concentration of homocysteine (Hcy) in human tissues, defined as hyperhomocysteinemia has been correlated with some diseases, such as

  • cardiovascular
  • neurodegenerative
  • kidney disorders

L-Homocysteine (Hcy) is an endogenous amino acid, containing a free thiol group, which in healthy cells is involved in methionine and cysteine synthesis/resynthesis. Indirectly, Hcy participates in methyl, folate, and cellular thiol metabolism. Approximately 80% of total plasma Hcy is protein-bound, and only a small amount exists as a free reduced Hcy (about 0.1 μM). The majority of the unbound fraction of Hcy is oxidized, and forms dimers (homocystine) or mixed disulphides consisting of cysteine and Hcy.

Two main pathways of Hcy biotoxicity are discussed:

  1. Hcy-dependent oxidative stress – generated during oxidation of the free thiol group of Hcy. Hcy binds via a disulphide bridge with

—     plasma proteins

—     or with other low-molecular plasma  thiols

—     or with a second Hcy molecule.

Accumulation of oxidized biomolecules alters the biological functions of many cellular pathways.

  1. Hcy-induced protein structure modifications, named homocysteinylation.

Two main types of homocysteinylation exist: S-homocysteinylation and N-homocysteinylation; both considered as posttranslational protein modifications.

a)      S-homocysteinylation occurs when Hcy reacts, by its free thiol group, with another free thiol derived from a cysteine residue in a protein molecule.

These changes can alter the thiol-dependent redox status of proteins.

b)      N-homocysteinylation takes place after acylation of the free ε-amino lysine groups of proteins by the most reactive form of Hcy — its cyclic thioester (Hcy thiolactone — HTL), representing up to 0.29% of total plasma Hcy.

Homocysteine occurs in human blood plasma in several forms, including the most reactive one, the homocysteine thiolactone (HTL) — a cyclic thioester, which represents up to 0.29% of total plasma Hcy. In human blood, N-homocysteinylated (N-Hcy-protein) and S-homocysteinylated proteins (S-Hcy-protein) such as NHcy-hemoglobin, N-(Hcy-S-S-Cys)-albumin, and S-Hcyalbumin are known. Other pathways of Hcy biotoxicity might be apoptosis and excitotoxicity mediated through glutamate receptors. The relationship between homocysteine and risk appears to hold for total plasma concentrations of homocysteine between 10 and 30 μM.

Different forms of homocysteine present in human blood.

*Total level of homocysteine — the term “total homocysteine” describes the pool of homocysteine released by reduction of all disulphide bonds in the sample (Perla-Kajan et al., 2007; Zimny, 2008; Manolescu et al., 2010, modified).

The form of Hcy The concentration in human blood
Homocysteine thiolactone (HTL) 0–35 nM
Protein N-linked homocysteine:
N-Hcy-hemoglobin, N-(Hcy-S-S-Cys)-albumin
about 15.5 μM: 12.7 μM, 2.8 μM
Protein S-linked homocysteine — S-Hcy-albumin about 7.3 μM*
Homocystine (Hcy-S-S-Hcy) and combined with cysteine to from mixed disulphides (Hcy-S-S-Cys) about 2 μM*
Free reduced Hcy about 0.1 μM*

As early as in the 1960s it was noted that the risk of atherosclerosis is markedly increased in patients with homocystinuria, an inherited disease resulting from homozygous CBS deficiency and characterized by episodes of

—     thromboembolism

—     mental retardation

—     lens dislocation

—     hepatic steatosis

—     osteoporosis.

—     very high concentrations of plasma homocysteine and methionine.

Patients with homocystinuria have very severe hyperhomocysteinemia, with plasma homocysteine concentration reaching even 400 μM, and represent a very small proportion of the population (approximately 1 in 200,000 individuals). Heterozygous lack of CBS, CBS mutations and polymorphism of the methylenetetrahydrofolate reductase gene are considered to be the most probable causes of hyperhomocysteinemia.

The effects of hyperhomocysteinemia include the complex process of hemostasis, which regulates the properties of blood flow. Interactions of homocysteine and its different derivatives, including homocysteine thiolactone, with the major components of hemostasis are:

  • endothelial cells
  • platelets
  • fibrinogen
  • plasminogen

Elevated plasma Hcy (>15 μM; Hcy) is associated with an increased risk of cardiovascular diseases

  • thrombosis
  • thrombosis related diseases
  • ischemic brain stroke (independent of other, conventional risk factors of this disease)

Every increase of 2.5 μM in plasma Hcy may be associated with an increase of stroke risk of about 20%.  Total plasma Hcy level above 20 μM are associated with a nine-fold increase of the myocardial infarction and stroke risk, in comparison to the concentrations below 9 μM. The increase of Hcy concentration has been also found in other human pathologies, including neurodegenerative diseases

Modifications of hemostatic proteins (N-homocysteinylation or S-homocysteinylation) induced by Hcy or its thiolactone seem to be the main cause of homocysteine biotoxicity in hemostatic abnormalities.

Hcy and HTL may act as oxidants, but various polyphenolic antioxidants are able to inhibit the oxidative damage induced by Hcy or HTL. Therefore, we have to consider the role of phenolic antioxidants in hyperhomocysteinemia –induced changes in hemostasis.

The synthesis of homocysteine thiolactone is associated with the activation of the amino acid by aminoacyl-tRNA synthetase (AARS). Hcy may also undergo erroneous activation, e.g. by methionyl-t-RNA synthetase (MetRS). In the first step of conversion of Hcy to HTL, MetRS misactivates Hcy giving rise to homocysteinyl-adenylate. In the next phase, the homocysteine side chain thiol group reacts with the activated carboxyl group and HTL is produced. The level of HTL synthesis in cultured cells depends on Hcy and Met levels.

Hyperhomocysteinemia and Changes in Fibrinolysis and Coagulation Process

The fibrinolytic activity of blood is regulated by specific inhibitors; the inhibition of fibrinolysis takes place at the level of plasminogen activation (by PA-inhibitors: plasminogen activator inhibitor type-1, -2; PAI-1 or PAI-2) or at the level of plasmin activity (mainly by α2-antiplasmin). Hyperhomocysteinemia disturbs hemostasis and shifts the hemostatic mechanisms in favor of thrombosis. The recent reports indicate that the prothrombotic state observed in hyperhomocysteinemia may arise not only due to endothelium dysfunction or blood platelet and coagulation activation, but also due to impaired fibrinolysis. Hcy-modified fibrinogen is more resistant to the fibrinolytic action. Oral methionine load increases total Hcy, but may diminish the fibrinolytic activity of the euglobulin plasma fraction. Homocysteine-lowering therapies may increase fibrinolytic activity, thereby, prevent atherothrombotic events in patients with cardiovascular diseases after the first myocardial infarction.

Homocysteine — Fibronectin Interaction and its Consequences

Fibronectin (Fn) plays key roles in

  • cell adhesion
  • migration
  • embryogenesis
  • differentiation
  • hemostasis
  • thrombosis
  • wound healing
  • tissue remodeling

Interaction of FN with fibrin, mediated by factor XIII transglutaminase, is thought to be important for cell adhesion or cell migration into fibrin clots. After tissue injury, a blood clot formation serves the dual role of restoring vascular integrity and serving as a temporary scaffold for the wound healing process. Fibrin and plasma FN, the major protein components of blood clots, are essential to perform these functions. In the blood clotting process, after fibrin deposition, plasma FN-fibrin matrix is covalently crosslinked, and it then promotes fibroblast adhesion, spreading, and migration into the clot.

Homocysteine binds to several human plasma proteins, including fibronectin. If homocysteine binds to fibronectin via a disulphide linkage, this binding results in a functional change, namely, the inhibition of fibrin binding by fibronectin. This inhibition may lead to a prolonged recovery from a thrombotic event and contribute to vascular occlusion.

Grape seeds are one of the richest plant sources of phenolic substances, and grape seed extract reduces the toxic effect of Hcys and HTL on fibrinolysis. The grape seed extract (12.5–50 μg/ml) supported plasminogen to plasmin conversion inhibited by Hcys or HTL. In vitro experiments showed in the presence of grape seed extract (at the highest tested concentration — 50 μg/ml) the increase of about 78% (for human plasminogen-treated with Hcys) and 56% (for human plasma-treated with Hcys). Thus, in the in vitro model system, that the grape seed extract (12.5–50 μg/ml) diminished the reduction of thiol groups and of lysine ε-amino groups in plasma proteins treated with Hcys (0.1 mM) or HTL (1 μM). In the presence of the grape seed extract at the concentration of 50 μg/ml, the level of reduction of thiol groups reached about 45% (for plasma treated with Hcys) and about 15% (for plasma treated with HTL).

In the presence of the grape seed extract at the concentration of 50 μg/ml, the level of reduction of thiol groups reached about 45% (for plasma treated with Hcys) and about 15% (for plasma treated with HTL).Very similar protective effects of the grape seed extract were observed in the measurements of lysine ε-amino groups in plasma proteins treated with Hcys or HTL. These results indicated that the extract from berries of Aronia melanocarpa (a rich source of phenolic substances) reduces the toxic effects of Hcy and HTL on the hemostatic properties of fibrinogen and plasma. These findings indicate a possible protective action of the A. melanocarpa extract in hyperhomocysteinemia-induced cardiovascular disorders. Moreover, the extract from berries of A. melanocarpa, due to its antioxidant action, significantly attenuated the oxidative stress (assessed by measuring of the total antioxidant status — TAS) in plasma in a model of hyperhomocysteinemia.

Proposed model for the protective role of phenolic antioxidants on selected elements of hemostasis during hyperhomocysteinemia.

various antioxidants (present in human diet), including phenolic compounds, may reduce the toxic effects of Hcy or its derivatives on hemostasis. These findings give hope for the develop development of dietary supplements, which will be capable of preventing thrombosis which occurs under pathological conditions, observed also in hyperhomocysteinemia, such as plasma procoagulant activity and oxidative stress.

Malinowska J,  Kolodziejczyk J and Olas B. The disturbance of hemostasis induced by hyper-homocysteinemia; the role of antioxidants. Acta Biochimica Polonica 2012; 59(2): 185–194.

Lipoprotein (a)

Lipoprotein (a) (Lp(a)), for the first time described in 1963 by Berg belongs to the lipoproteins with the strongest atherogenic effect. Its importance for the development of various atherosclerotic vasculopathies (coronary heart disease, ischemic stroke, peripheral vasculopathy, abdominal aneurysm) was recognized considerably later.

Lipoprotein(a) (Lp(a)), an established risk marker of cardiovascular diseases, is independent from other risk markers. The main difference of Lp(a) compared to low density lipoprotein (LDL) is the apo(a) residue, covalently bound to apoB is covalently by a disulfide-bridge. Apo(a) synthesis is performed in the liver, probably followed by extracellular assembly to the apoB location of the LDL.


ApoB-100_______LDL¬¬___ S-S –    9

Apo(a) has been detected bound to triglyceride-rich lipoproteins (Very Low Density Lipoproteins; VLDL). Corresponding to the structural similarity to LDL, both particles are very similar to each other with regard to their composition. It is a glycoprotein which underlies a large genetic polymorphism caused by a variation of the kringle-IV-type-2 repeats of the protein, characterized by a structural homology to plasminogen. Apo(a)’s structural homology to plasminogen, shares the gene localization on chromosome 6. The kringle repeats present a particularly characteristic structure, which have a high similarity to kringle IV (K IV) of plasminogen. Apo(a) also has a kringle V structure of plasminogen and also a protease domain, which cannot be activated, as opposed to the one of plasminogen. At least 30 genetically determined apo(a) isoforms were identified in man.


  • Non covalent binding of kringle -4 types 7 and 8 of apo (a) to apo B
  • Disulfide bond at Cys4326 of ApoB (near its receptor binding domain ) and the only free cysteine group in K –IV type 9 (Cys4057) of apo(a )
  • Binding to fibrin and cell membranes
  • Enhancement by small isoforms ; high concentrations compared to plasminogen and homocysteine
  • Binding to different lysine rich components of the coagulation system (e. g. TFPI)
  • Intense homology to plasminogen but no protease activity
ApoB-100_______LDL¬¬___ S-S – 9

The synthesis of Lp(a), which thus occurs as part of an assembly, is a two-step process.

  • In a first step, which can be competitively inhibited by lysine analogues, the free sulfhydryl groups of apo(a) and apoB are brought close together.
  • The binding of apo(a) then occurs near the apoB domain which binds to the LDL receptor, resulting in a reduced affinity of Lp(a) to the LDL-receptor.

Particles that show a reduced affinity to the LDL receptor are not able to form stable compounds with apo(a). Thus the largest part of apo(a) is present as apo(a) bound to LDL. Only a small, quantitatively variable part of apo(a) remains as free apo(a) and probably plays an important role in the metabolism and physiological function of Lp(a).

The Lp(a) plasma concentration in the population is highly skewed and determined to more than 90 % by genetic factors. In healthy subjects the Lp(a)-concentration is correlated with its synthesis.

It is assumed that the kidney has a specific function in Lp(a) catabolizm, since nephrotic syndrome and terminal kidney failure are associated with an elevation of the Lp(a) plasma concentration. One consequence of the poor knowledge of the metabolic path of Lp(a) is the fact that so far pharmaceutical science has failed to develop drugs that are able to reduce elevated Lp(a) plasma concentrations to a desirable level.

Plasma concentrations of Lp(a) are affected by different diseases (e.g. diseases of liver and kidney), hormonal factors (e.g. sexual steroids, glucocorticoids, thyroid hormones), individual and environmental factors (e.g. age, cigarette smoking) as well as pharmaceuticals (e.g. derivatives of nicotinic acid) and therapeutic procedures (lipid apheresis). This review describes the physiological regulation of Lp(a) as well as factors influencing its plasma concentration.

Apart from its significance as an important agent in the development of atherosclerosis, Lp(a) has even more physiological functions, e.g. in

  • wound healing
  • angiogenesis
  • hemostasis

However, in the meaning of a pleiotropic mechanism the favorable action mechanisms are opposed by pathogenic mechanisms, whereby the importance of Lp(a) in atherogenesis is stressed.

Lp(a) in Atherosclerosis

In transgenic, hyperlipidemic and Lp(a) expressing Watanabe rabbits, Lp(a) leads to enhanced atherosclerosis. Under the influence of Lp(a), the binding of Lp(a) to glycoproteins, e.g. laminin, results – via its apo(a)-part – both in

  • an increased invasion of inflammatory cells and in
  • an activation of smooth vascular muscle cells

with subsequent calcifications in the vascular wall.

The inhibition of transforming growth factor-β1 (TGF-β1) activation is another mechanism via which Lp(a) contributes to the development of atherosclerotic vasculopathies. TGF-β1 is subject to proteolytic activation by plasmin and its active form leads to an inhibition of the proliferation and migration of smooth muscle cells, which play a central role in the formation and progression of atherosclerotic vascular diseases.

In man, Lp(a) is an important risk marker which is independent of other risk markers. Its importance, partly also under consideration of the molecular weight and other genetic polymorphisms, could be demonstrated by a high number of epidemiological and clinical studies investigating the formation and progression of atherosclerosis, myocardial infarction, and stroke.

Lp(a) in Hemostasis

Lp(a) is able to competitively inhibit the binding of plasminogen to fibrinogen and fibrin, and to inhibit the fibrin-dependent activation of plasminogen to plasmin via the tissue plasminogen activator, whereby apo(a) isoforms of low molecular weight have a higher affinity to fibrin than apo(a) isoforms of higher molecular weight. Like other compounds containing sulfhydryl groups, homocysteine enhances the binding of Lp(a) to fibrin.

Pleiotropic effect of Lp(a).

Prothrombotic :

  • Binding to fibrin
  • Competitive inhibition of plasminogen
  • Stimulation of plasminogen activator inhibitor I and II (PAI -I, PAI -II)
  • Inactivation of tissue factor pathway inhibitor (TFPI)

Antithrombotic :

  • Inhibition of platelet activating factor acetylhydrolase (PAF -AH)
  • Inhibition of platelet activating factor
  • Inhibition of collagen dependent platelet aggregation
  • Inhibition of secretion of serotonin und thromboxane

Lp(a) in Angiogenesis

Lp(a) is also important for the process of angiogenesis and the sprouting of new vessels.

  • angiogenesis starts with the remodelling of matrix proteins and
  • activation of matrix metalloproteinases (MMP).

The latter ones are usually synthesised as

  • inactive zymogens and
  • require activation by proteases,

Recall that Apo(a) is not activated by proteases. The angiogenesis is also accomplished by plasminogen. Lp(a) and apo(a) and its fragments has an antiangiogenetic and metastasis inhibiting effect related to the structural homology with plasminogen without the protease activity.

Siekmeier R, Scharnagl H, Kostner GM, T. Grammer T, Stojakovic T and März W.  Variation of Lp(a) Plasma Concentrations in Health and Disease.  The Open Clinical Chemistry Journal, 2010; 3: 72-89.


In 1985, Brown and Goldstein were awarded the Nobel Prize for medicine for their work on the regulation of cholesterol metabolism. On the basis of numerous studies, they were able to demonstrate that circulating low-density lipoprotein (LDL) is absorbed into the cell through receptor linked endocytosis. The absorption of LDL into the cell is specific and is mediated by a LDL receptor. In patients with familial hypercholesterolemia, this receptor is changed, and the LDL particles can no longer be recognized. Their absorption can thus no longer be mediated, leading to an accumulation of LDL in blood.

Furthermore, an excess supply of cholesterol also blocks the 3-hydrox-3 methylglutaryl-Co enzyme A (HMG CoA), reductase enzyme, which otherwise inhibits the cholesterol synthesis rate. Brown and Goldstein also determined the structure of the LDL receptor. They discovered structural defects in this receptor in many patients with familial hypercholesterolemia. Thus, familial hypercholesterolemia was the first metabolic disease that could be tracked back to the mutation of a receptor gene.

Dyslipoproteinemia in combination with diabetes mellitus causes a cumulative insult to the vasculature resulting in more severe disease which occurs at an earlier age in large and small vessels as well as capillaries. The most common clinical conditions resulting from this combination are myocardial infarction and lower extremity vascular disease. Ceriello et al. show an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial function, suggesting oxidative stress as common mediator of such effect. The combination produces greater morbidity and mortality than either alone.

As an antiatherogenic factor, HDL cholesterol correlates inversely to the extent of postprandial lipemia. A high concentration of HDL is a sign that triglyceride-rich particles are quickly decomposed in the postprandial phase of lipemia. Conversely, with a low HDL concentration this decomposition is delayed. Thus, excessively high triglyceride concentrations are accompanied by very low HDL counts. This combination has also been associated with an increased risk of pancreatitis.

The importance of lipoprotein (a) (Lp(a)) as an atherogenic substance has also been recognized in recent years. Lp(a) is very similar to LDL. But it also contains Apo(a), which is very similar to plasminogen, enabling Lp(a) to bind to fibrin clots. Binding of plasminogen is prevented and fibrinolysis obstructed. Thrombi are integrated into the walls of the arteries and become plaque components.

Another strong risk factor for accelerated atherogenesis, which must be mentioned here, are the widespread high homocysteine levels found in dialysis patients. This risk factor is independent of classic risk factors such as high cholesterol and LDL levels, smoking, hypertension, and obesity, and much more predictive of coronary events in dialysis patients than are these better-known factors. Homocysteine is a sulfur aminoacid produced in the metabolism of methionine. Under normal conditions, about 50 percent of homocysteine is remethylated to methionine and the remaining via the transsulfuration pathway.

Defining hyperhomocysteinemia as levels greater than the 90th percentile of controls and elevated Lp(a) level as greater than 30mg/dL, the frequency of the combination increased with declining renal function. Fifty-eight percent of patients with a GFR less than 10mL/min had both hyperhomocysteinemia and elevated Lp(a) levels, and even in patients with mild renal impairment, 20 percent of patients had both risk factors present.

The prognosis of patients suffering from severe hyperlipidemia, sometimes combined with elevated lipoprotein (a) levels, and coronary heart disease refractory to diet and lipid-lowering drugs is poor. For such patients, regular treatment with low-density lipoprotein (LDL) apheresis is the therapeutic option. Today, there are five different LDL-apheresis systems available: cascade filtration or lipid filtration, immunoadsorption, heparin-induced LDL precipitation, dextran sulfate LDL adsorption, and the LDL hemoperfusion. The requirement that the original level of cholesterol is to be reduced by at least 60 percent is fulfilled by all these systems.

There is a strong correlation between hyperlipidemia and atherosclerosis. Besides the elimination of other risk factors, in severe hyperlipidemia therapeutic strategies should focus on a drastic reduction of serum lipoproteins. Despite maximum conventional therapy with a combination of different kinds of lipid-lowering drugs, sometimes the goal of therapy cannot be reached. Hence, in such patients, treatment with LDL-apheresis is indicated. Technical and clinical aspects of these five different LDL-apheresis methods are depicted. There were no significant differences with respect to or concerning all cholesterols, or triglycerides observed.

High plasma levels of Lp(a) are associated with an increased risk for atherosclerotic coronary heart       disease
(CHD) by a mechanism yet to be determined. Because of its structural properties, Lp(a) can have both atherogenic and thrombogenic potentials. The means for correcting the high plasma levels of Lp(a) are still limited in effectiveness. All drug therapies tried thus far have failed. The most effective therapeutic methods in lowering Lp(a) are the LDL-apheresismethods. Since 1993, special immunoadsorption polyclonal antibody columns (Pocard, Moscow, Russia) containing sepharose bound anti-Lp(a) have been available for the treatment of patients with elevated Lp(a) serum concentrations.

With respect to elevated lipoprotein (a) levels, however, the immunoadsorption method seems to be most effective. The different published data clearly demonstrate that treatment with LDL-apheresis in patients suffering from severe hyperlipidemia refractory to maximum conservative therapy is effective and safe in long-term application.

LDL-apheresis decreases not only LDL mass but also improves the patient’s life expectancy. LDL-apheresis performed with different techniques decreases the susceptibility of LDL to oxidation. This decrease may be related to a temporary mass imbalance between freshly produced and older LDL particles. Furthermore, the baseline fatty acid pattern influences pretreatment and postreatment susceptibility to oxidation.

Bambauer R, Bambauer C, Lehmann B, Latza R, and Ralf Schiel R. LDL-Apheresis: Technical and Clinical Aspects. The Scientific World Journal 2012; Article ID 314283, pp 1-19. doi:10.1100/2012/314283

Summary:  This discussion is a two part sequence that first establishes the known strong relationship between blood flow viscosity, shear stress, and plasma triglycerides (VLDL) as risk factors for hemostatic disorders leading to thromboembolic disease, and the association with atherosclerotic disease affecting the heart, the brain (via carotid blood flow), peripheral circulation,the kidneys, and retinopathy as well.

The second part discusses the modeling of hemostasis and takes into account the effects of plasma proteins involved with red cell and endothelial interaction, which is related to part I.  The current laboratory assessment of thrombophilias is taken from a consensus document of the American Society for Clinical Pathology.  The problems encountered are sufficient for the most common problems of coagulation testing and monitoring, but don’t address the large number of patients who are at risk for complications of accelerated vasoconstrictive systemic disease that precede serious hemostatic problems.  Special attention is given to Lp(a) and to homocysteine.  Lp(a) is a protein that has both prothrombotic and antithrombotic characteristics, and is a homologue of plasminogen and is composed of an apo(a) bound to LDL.  Unlike plasminogen, it has no protease activity.   Homocysteine elevation is a known risk factor for downstream myocardial infarct.  Homocysteine is a mirror into sulfur metabolism, so an increase is an independent predictor of risk, not fully discussed here.  The modification of risk is discussed by diet modification.  In the most serious cases of lipoprotein disorders, often including Lp(a) the long term use of LDL-apheresis is described.

see Relevent article that appears in NEJM from American College of Cardiology

Apolipoprotein(a) Genetic Sequence Variants Associated With Systemic Atherosclerosis and Coronary Atherosclerotic Burden but Not With Venous Thromboembolism

Helgadottir A, Gretarsdottir S, Thorleifsson G, et al

J Am Coll Cardiol. 2012;60:722-729

Study Summary

The LPA gene codes for apolipoprotein(a), which, when linked with low-density lipoprotein particles, forms lipoprotein(a) [Lp(a)] — a well-studied molecule associated with coronary artery disease (CAD). The Lp(a) molecule has both atherogenic and thrombogenic effects in vitro , but the extent to which these translate to differences in how atherothrombotic disease presents is unknown.

LPA contains many single-nucleotide polymorphisms, and 2 have been identified by previous groups as being strongly associated with levels of Lp(a) and, as a consequence, strongly associated with CAD. However, because atherosclerosis is thought to be a systemic disease, it is unclear to what extent Lp(a) leads to atherosclerosis in other arterial beds (eg, carotid, abdominal aorta, and lower extremity), as well as to other thrombotic disorders (eg, ischemic/cardioembolic stroke and venous thromboembolism). Such distinctions are important, because therapies that might lower Lp(a) could potentially reduce forms of atherosclerosis beyond the coronary tree.

To answer this question, Helgadottir and colleagues compiled clinical and genetic data on the LPA gene from thousands of previous participants in genetic research studies from across the world. They did not have access to Lp(a) levels, but by knowing the genotypes for 2 LPA variants, they inferred the levels of Lp(a) on the basis of prior associations between these variants and Lp(a) levels. [1] Their studies included not only individuals of white European descent but also a significant proportion of black persons, in order to widen the generalizability of their results.

Their main findings are that LPA variants (and, by proxy, Lp(a) levels) are associated with CAD,  peripheral arterial disease, abdominal aortic aneurysm, number of CAD vessels, age at onset of CAD diagnosis, and large-artery atherosclerosis-type stroke. They did not find an association with cardioembolic or small-vessel disease-type stroke; intracranial aneurysm; venous thrombosis; carotid intima thickness; or, in a small subset of individuals, myocardial infarction.


The main conclusion to draw from this work is that Lp(a) is probably a strong causal factor in not only CAD, but also the development of atherosclerosis in other arterial trees. Although there is no evidence from this study that Lp(a) levels contribute to venous thrombosis, the investigators do not exclude a role for Lp(a) in arterial thrombosis.

Large-artery atherosclerosis stroke is thought to involve some element of arterial thrombosis or thromboembolism, [2] and genetic substudies of randomized trials of aspirin demonstrate that individuals with LPA variants predicted to have elevated levels of Lp(a) benefit the most from antiplatelet therapy. [3] Together, these data suggest that Lp(a) probably has clinically relevant effects on the development of atherosclerosis and arterial thrombosis.

Of  note, the investigators found no association between Lp(a) and carotid intima thickness, suggesting that either intima thickness is a poor surrogate for the clinical manifestations of atherosclerosis or that Lp(a) affects a distinct step in the atherosclerotic disease process that is not demonstrable in the carotid arteries.

Although Lp(a) testing is available, these studies do not provide any evidence that testing for Lp(a) is of clinical benefit, or that screening for atherosclerosis should go beyond well-described clinical risk factors, such as low-density lipoprotein cholesterol levels, high-density lipoprotein levels, hypertension, diabetes, smoking, and family history. Until evidence demonstrates that adding information on Lp(a) levels to routine clinical practice improves the ability of physicians to identify those at highest risk for atherosclerosis, Lp(a) testing should remain a research tool. Nevertheless, these findings do suggest that therapies to lower Lp(a) may have benefits that extend to forms of atherothrombosis beyond the coronary tree.

The finding of this study is interesting:

[1] It consistent with Dr. William LaFramboise..   examination specifically at APO B100, which is part of Lp(a) with some 14 candidate predictors for a more accurate exclusion of patients who don’t need intervention.          Apo B100 was not one of 5 top candidates.

William LaFramboise • Our study (http://www.ncbi.nlm.nih.gov/pubmed/23216991) comprised discovery research using targeted immunochemical screening of retrospective patient samples using both Luminex and Aushon platforms as opposed to shotgun proteomics. Hence the costs constrained sample numbers. Nevertheless, our ability to predict outcome substantially exceeded available methods:

The Framingham CHD scores were statistically different between groups (P <0.001, unpaired Student’s t test) but they classified only 16% of the subjects without significant CAD (10 of 63) at a 95% sensitivity for patients with CAD. In contrast, our algorithm incorporating serum values for OPN, RES, CRP, MMP7 and IFNγ identified 63% of the subjects without significant CAD (40 of 63) at 95% sensitivity for patients with CAD. Thus, our multiplex serum protein classifier correctly identified four times as many patients as the Framingham index.

This study is consistent with the concept of CAD, PVD, and atheromatous disease is a systemic vascular disease, but the point that is made is that it appears to have no relationship to venous thrombosis. The importance for predicting thrombotic events is considered serious.   The venous flow does not have the turbulence of large arteries, so the conclusion is no surprise.  The flow in capillary beds is a linear cell passage with minimal viscosity or turbulence.  The finding of no association with carotid artery disease  is interpreted to mean that the Lp(a) might be an earlier finding than carotid intimal thickness.  It is reassuring to find a recommendation for antiplatelet therapy for individuals with LPA variants based on randomized trials of aspirin substudies.

If that is the conclusion from the studies, and based on the strong association between the prothrombotic (pleiotropic) effect and the association with hyperhomocysteinemia, my own impression is that the recommendation is short-sighted.

[2]  Lp(a) is able to competitively inhibit the binding of plasminogen to fibrinogen and fibrin, and to inhibit the fibrin-dependent activation of plasminogen to plasmin via the tissue plasminogen activator, whereby apo(a) isoforms of low molecular weight have a higher affinity to fibrin than apo(a) isoforms of higher molecular weight. Like other compounds containing sulfhydryl groups, homocysteine enhances the binding of Lp(a) to fibrin.

Prothrombotic :

  • Binding to fibrin
  • Competitive inhibition of plasminogen
  • Stimulation of plasminogen activator inhibitor I and II (PAI -I, PAI -II)
  • Inactivation of tissue factor pathway inhibitor (TFPI)

Source for Lp(a)

Artherogenesis: Predictor of CVD – the Smaller and Denser LDL Particles


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Jax TW, Peters AJ, Plehn G, Schoebel FC. Hemostatic risk factors in patients with coronary artery disease and type 2 diabetes – a two year follow-up of 243 patients. Cardiovasc Diabetol 2009; 8:48.

Ernst E, Weihmayr T, et al. Cardiovascular risk factors and hemorheology. Physical fitness, stress and obesity. Atherosclerosis 1986; 59:263-9.

Hoieggen A, Fossum E, et al. Whole-blood viscosity and the insulin-resistance syndrome. J Hypertens 1998; 16:203-10.

Carroll S, Cooke CB, Butterly RJ. Plasma viscosity, fibrinogen and the metabolic syndrome: effect of obesity and cardiorespiratory fitness. Blood Coagul Fibrinolysis 2000; 11:71-8.

Ernst E, Koenig W, Matrai A, et al. Blood rheology in healthy cigarette smokers. Results from the MONICA project, Augsburg. Arteriosclerosis 1988; 8:385-8.

Ernst E. Haemorheological consequences of chronic cigarette smoking. J Cardiovasc Risk 1995; 2:435-9.

Lowe GD, Drummond MM, Forbes CD, Barbenel JC. The effects of age and cigarette-smoking on blood and plasma viscosity in men. Scott Med J 1980; 25:13-7.

Kameneva MV, Watach MJ, Borovetz HS. Gender difference in rheologic properties of blood and risk of cardiovascular diseases. Clin Hemorheol Microcirc 1999; 21:357-363.

Fowkes FG, Pell JP, Donnan PT, et al. Sex differences in susceptibility to etiologic factors for peripheral atherosclerosis. Importance of plasma fibrinogen and blood viscosity. Arterioscler Thromb 1994; 14:862-8.

Coppola L, Caserta F, De Lucia D, et al. Blood viscosity and aging. Arch Gerontol Geriatr 2000; 31:35-42.


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What is the Role of Plasma Viscosity in Hemostasis and Vascular Disease Risk?

Author: Larry H Bernstein, MD


Curator: Aviva Lev-Ari, PhD, RN

This is the first of a two part discussion of viscosity, hemostasis, and vascular risk

Part II:  Special Considerations in Blood Lipoproteins, Viscosity, Assessment and Treatment

Thesis Statement: The effects of low-density lipoprotein and high-density lipoprotein on blood viscosity correlate with their association with risk of atherosclerosis in humans.  (Seminal study)

G. D. Sloop, MD.
Department of Pathology, Louisiana State University School of Medicine,
New Orleans, LA 70112, U.S.A.

  •  Increased blood or plasma viscosity has been associated with increased atherogenesis, and that the effects of low-density lipoprotein and high-density lipoprotein on blood viscosity correlate with their association with atherosclerosis risk.
  • Low-density lipoprotein-cholesterol was more strongly correlated with blood viscosity than was total cholesterol (r = 0.4149, P = 0.0281, compared with r = 0.2790, P = 0.1505). High-density lipoprotein-cholesterol levels were inversely associated with blood viscosity (r = – 0.4018, P = 0.0341).
  • To confirm these effects, viscometry was performed on erythrocytes, suspended in saline, which had been incubated in plasma of various low-density lipoprotein/high-density lipoprotein ratios. Viscosity correlated directly with low-density lipoprotein/high-density lipoprotein ratio (n = 23, r = 0.8561, P < 0.01).
  • Low-density lipoprotein receptor occupancy data suggests that these effects on viscosity are mediated by erythrocyte aggregation.
  • These results demonstrate that the effects of low-density lipoprotein and high-density lipoprotein on blood viscosity in healthy subjects may play a role in atherogenesis by modulating the dwell or residence time of atherogenic particles in the vicinity of the endothelium.

This discussion is an additional perspective on the series on coagulation, and earlier posts that were on flow dynamics.

Stroke and Bleeding in Atrial Fibrillation with Chronic Kidney Disease

Atrial Fibrillation: The Latest Management Strategies

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB

Positioning a Therapeutic Concept for Endogenous Augmentation of cEPCs — Therapeutic Indications for Macrovascular Disease: Coronary, Cerebrovascular and Peripheral

New Definition of MI Unveiled, Fractional Flow Reserve (FFR)CT for Tagging Ischemia

Nitric Oxide Signalling Pathways            AviralvatsaEndothelial Dysfunction, Diminished Availability of cEPCs, Increasing CVD Risk for Macrovascular Disease – Therapeutic Potential of cEPCs

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Repair damaged blood vessels in heart disease, stroke, diabetes and trauma: Cellular Reprogramming amniotic fluid-derived cells into Endothelial Cells

Septic Shock: Drotrecogin Alfa (Activated) in Septic Shock

Statins’ Nonlipid Effects on Vascular Endothelium through eNOS Activation   LHB

Nitric Oxide Covalent Modifications: A Putative Therapeutic Target?  SJWilliamspa

Vascular Wall Shear Stress

Shear Stress

  1. The basic principles concerning mechanical stress applies to pathophysiological mechanisms in the vascular bed. In physics, stress is the internal distribution of forces within a body that balance and react to the external loads applied to it. Blood flow in the circulation leads to the development of superficial stresses near the vessel walls in either of two categories:

a) circumferential stress due to pulse pressure variation inside the vessel;
b) shear stress due to blood flow.

  1. The direction of the shear stress vector is determined the blood flow velocity vector adjacent to applied against the vessel wall.
  2. Friction is the opposing force applied by the wall.
  3. Shear stresses are disturbed by turbulent flow, regions of flow recirculation or flow separation.
  4. The notions of shear rate and fluid viscosity are crucial for the assessment of shear stress.

Fluid Flow and Shear Stress

  1. Shear rate is defined as the rate at which adjacent layers of fluid move with respect to each other, usually expressed as reciprocal seconds.
  2. The size of the shear rate gives an indication of the shape of the velocity profile for a given situation.
  3. The determination of shear stresses on a surface is based on the fundamental assumption of fluid mechanics, according to which the velocity of fluid upon the surface is zero (no-slip condition).
  4. Assuming that the blood is an ideal Newtonian fluid with constant viscosity, the flow is steady and laminar and the vessel is straight, cylindrical and inelastic, which is not the case. Under ideal conditions a parabolic velocity profile could be assumed.

The following assumptions have been made:

  1. The blood is considered as a Newtonian fluid.
  2. The vessel cross sectional area is cylindrical.
  3. The vessel is straight with inelastic walls.
  4. The blood flow is steady and laminar.

The Haagen-Poisseuille equation indicates that shear stress is directly proportional to blood flow rate and inversely proportional to vessel diameter.

  1. Viscosity is a property of a fluid that offers resistance to flow, and it is a measure of the combined effects of adhesion and cohesion.
  2. Viscosity increases as temperature decreases.
  3. Blood viscosity (non-Newtonian fluid) depends on shear rate, which is determined by blood platelets, red cells, etc.
  4. Blood viscosity is slightly affected by shear rate changes at low levels of hematocrit, but as hematocrit increases, the effect of shear rate changes becomes greater.
  5. the dependence of blood viscosity on hematocrit is more pronounced in the microcirculation than in larger vessels, due to hematocrit variations observed in small vessels (lumen diameter <100 Ìm).

The significant change of hematocrit in relation to vessel diameter is associated with the tendency of red blood cells to travel closer to the centre of the vessels. Thus, the greater the decrease in vessel lumen, the smaller the number of red blood cells that pass through, resulting in a decrease in blood viscosity.

Shear stress and vascular endothelium

  1. Endothelium responds to shear stress depending on the kind and the magnitude of shear stresses.
  2. the exposure of vascular endothelium to shear forces in the normal value range stimulates endothelial cells to release agents with direct or indirect antithrombotic properties, such as
  • prostacyclin,
  • nitric oxide (NO),
  • calcium,
  • thrombomodulin, etc.

Changes in shear stress magnitude activate cellular proliferation mechanisms as well as vascular remodeling processes.

  1. a high grade of shear stress increases wall thickness and expands the vessel’s diameter
  2. low shear stress induces a reduction in vessel diameter.
  3. Shear stresses are maintained at a mean of about 15 dynes/cm2.
  4. The presence of low shear stresses is frequently accompanied by unstable flow conditions
  • turbulence flow,
  • regions of blood recirculation,
  • “stagnant” blood areas.

(Papaioannou TG, Stefanadis C. Vascular Wall Shear Stress: Basic Principles and Methods. Hellenic J Cardiol 2005; 46: 9-15.)

Hemorheology and Microvascular Disorders

Blood flow in large arteries is dominated by inertial forces exhibited at high flow velocities, while viscosity is negligible. When the flow velocity is compromised by deceleration at a bifurcation, endothelial cell dysfunction can occur along the outer wall at the bifurcation.

In sharp contrast, the flow of blood in micro-vessels is dominated by viscous shear forces since the inertial forces are negligible due to low flow velocities. Shear stress is a critical parameter in micro-vascular flow, and a force-balance approach is proposed for determining micro-vascular shear stress. When the attractive forces between erythrocytes are greater than the shear force produced by micro-vascular flow, tissue perfusion itself cannot be sustained.

The yield stress parameter is presented as a diagnostic candidate for future clinical research, specifically, as a fluid dynamic biomarker for micro-vascular disorders. The relation between the yield stress and diastolic blood viscosity (DBV) is described using the Casson model for viscosity, from which one may be able determine thresholds of DBV where the risk of microvascular disorders is high.

Cho Y-Il, and Cho DJ. Hemorheology and Microvascular Disorders. Korean Circ J 2011; 41:287-295.
Print ISSN 1738-5520 / On-line ISSN 1738-5555

Blood Rheology in Genesis of Atherothrombosis

Elevated blood viscosity is an integral component of vascular shear stress that contributes to the

  • site specificity of atherogenesis,
  • rapid growth of atherosclerotic lesions, and
  • increases their propensity to rupture.

Ex vivo measurements of whole blood viscosity (WBV) is a predictor of cardiovascular events in apparently both healthy individuals and cardiovascular disease patients. The association of an elevated WBV and incident cardiovascular events remains significant in multivariate models that adjust for major cardiovascular risk factors.

These prospective data suggest that measurement of WBV may be valuable as part of routine cardiovascular profiling, thereby potentially useful data for risk stratification and therapeutic interventions.

The recent development of a high throughput blood viscometer, which is capable of rapidly performing blood viscosity measurements across 10,000 shear rates using a single blood sample, enables the assessment of blood flow characteristics in different regions of the circulatory system and opens new opportunities for detecting and monitoring cardiovascular diseases.

Cowan AQ, Cho DJ, & Rosenson RS. Importance of Blood Rheology in the Pathophysiology of Athero-thrombosis. Cardiovasc Drugs Ther 2012; 26:339–348. DOI 10.1007/s10557-012-6402-4


English: shear stress

English: shear stress (Photo credit: Wikipedia)

English: Shear rate dependency on fluid type a...

English: Shear rate dependency on fluid type and applied shear stress. (Photo credit: Wikipedia)

Inflammatory, haemostatic, and rheological markers

Markers of inflammation, hemostasis, and blood rheology have been ascertained to be risk factors for coronary heart disease and stroke. Their role in peripheral arterial disease (PAD) is not well established and some of them, including the pro-inflammatory cytokine interleukin-6 (IL-6), have not been examined before in prospective epidemiological studies.

In the Edinburgh Artery Study, we studied the development of PAD in the general population and evaluated 17 potential blood markers as predictors of incident PAD. At baseline (1987), 1519 men and women free of PAD aged 55–74 were recruited. After 17 years, 208 subjects had developed symptomatic PAD. In analysis adjusted for cardiovascular risk factors and baseline cardiovascular disease (CVD), only

  1. C-reactive protein 1.30 (1.08, 1.56)
  2. fibrinogen               1.16 (1.05, 1.17)
  3. lipoprotein (a)        1.22 (1.04, 1.44),
  4. hematocrit 1.22 (1.08, 1.38) [hazard ratio (95% CI) ]

-corresponding to an increase equal to the inter-tertile range-

were significantly (P , 0.01) associated with PAD.

These markers provided very little prognostic information for incident PAD to that obtained by cardiovascular risk factors and the ankle brachial index. Other markers included:

  • IL-6
  • intracellular adhesion molecule 1 (ICAM-1)
  • D-dimer
  • tissue plasminogen activator antigen
  • plasma and blood viscosities

having weak associations, were considerably attenuated when accounting for CVD risk factors.

Tzoulaki I, Murray GD, Lee AJ, Rumley A, et al. Inflammatory, haemostatic, and rheological markers for incident peripheral arterial disease: Edinburgh Artery Study. European Heart Journal (2007) 28, 354–362. doi:10.1093/eurheartj/ehl441


Leukocyte and platelet adhesion under flow

Leukocyte adhesion under flow in the microvasculature is mediated by

  • binding between cell surface receptors and
  • complementary ligands expressed on the surface of the endothelium.

Leukocytes adhere to endothelium in a two-step mechanism:

  1. rolling (primarily mediated by selectins) followed by
  2. firm adhesion (primarily mediated by integrins).

These investigators simulated the adhesion of a cell to a surface in flow, and elucidated the relationship between receptor–ligand functional properties and the dynamics of adhesion using a computational method called ‘‘Adhesive Dynamics.’’

Behaviors that are observed in simulations include

  • firm adhesion,
  • transient adhesion (rolling), and
  • no adhesion.

They varied the

  • dissociative properties,
  • association rate,
  • bond elasticity, and
  • shear rate

and found that the

  1. unstressed dissociation rate, kro,
  2. and the bond interaction length, γ,

are the most important molecular properties controlling the dynamics of adhesion.

(Chang KC, Tees DFJ andHammer DA. The state diagram for cell adhesion under flow: Leukocyte rolling and firm adhesion. PNAS 2000; 97(21):11262-11267.)

  • The effect of leukocyte adhesion on blood flow in small vessels is treated as a homogeneous Newtonian fluid is sufficient to explain resistance changes in venular microcirculation.
  • The Casson model represents the effect of red blood cell aggregation and requires the non-Newtonian fluid flow model of resistance changes in small venules.

In this model the blood vessel is considered as a circular cylinder and the leukocyte is considered as a truncated spherical protrusion in the inner side of the blood vessel.

Numerical simulations demonstrated that for a Casson fluid with hematocrit of 0.4 and flow rate Q = 0:072 nl/s, a single leukocyte increases flow resistance by 5% in a 32 m diameter and 100 m long vessel. For a smaller vessel of 18 m, the flow resistance increases by 15%.

(Das B, Johnson PC, and Popel AS. Computational fluid dynamic studies of leukocyte adhesion effects on non-Newtonian blood flow through microvessels. Biorheology  2000; 37:239–258.)

Adhesive interactions between leukocytes

The mechanics of how blood cells interact with one another and with biological or synthetic surfaces is quite complex: owing to

  • the deformability of cells,
  • the variation in vessel geometry, and
  • the large number of competing chemistries present

(Lipowski et al., 1991, 1996).

Adhesive interactions between white blood cells and the interior surface of the blood vessels they contact are important in

  • inflammation and in
  • the progression of heart disease.

Parallel-plate micro-channels have been used to characterize the strength of these interactions. Recent computational and experimental work by several laboratories are directed at bridging the gap between

  • behavior observed in flow chamber experiments, and
  • cell surface interactions observed in the micro-vessels

What follows is a computational simulation of specific adhesive interactions between cells and surfaces under flow. In the adhesive dynamics formulation, adhesion molecules are modeled as compliant springs. The Bell model is used to describe the kinetics of single biomolecular bond failure, which relates

  1. the rate of dissociation kr to
  2. the magnitude of the force on the bond F.

The rate of formation directly follows from the Boltzmann distribution for affinity. The expression for the binding rate must also incorporate the effect of the relative motion of the two surfaces. Unless firmly adhered to a surface, white blood cells can be effectively modeled as rigid spherical particles. This is consistent with good agreement between bead versus cell in vitro experiments (Chang and Hammer, 2000).

Various methods have been used to bring clarity to the complex range of transient interactions between

  • cells,
  • neighboring cells, and
  • bounding surfaces under flow.

Knowledge gained from these investigations of flow systems may prove useful in microfluidic applications where the transport of

  • blood cells and
  • solubilized, bioactive molecules is needed, or
  • in miniaturized diagnostic devices

where cell mechanics or binding affinities can be correlated with clinical pathologies.

(King MR. Cell-Surface Adhesive Interactions in Microchannels and Microvessels.   First International Conference on Microchannels and Minichannels. 2003, Rochester, NY. Pp 1-6. ICMM2003-1012.

Monitoring Blood Viscosity to Improve Cognitive Function

Blood viscosity, the metric for the thickness and stickiness of blood, is associated with all major risk factors for cardiovascular disease, complications of diabetes, and it is highly predictive of stroke and MI, as well as cognitive decline. While elevated blood viscosity has a role in the etiology of atherosclerosis,  there is strong evidence for a causal role in the development of dementia.  It follows that improving blood viscosity should lead to improvements in cognitive as well as cardiovascular function.

Factors Affecting Blood Viscosity

Five cardinal factors are:

  1. Hematocrit,
  2. erythrocyte deformability,
  3. plasma viscosity,
  4. erythrocyte aggregation, and
  5. temperature

First to consider is hematocrit. Erythrocyte deformability is the ability of red blood cells to elongate and fold themselves for better hemodynamic flow in large vessels as well as for more efficient passage through capillaries.  The more deformable the red blood cells, the less viscous the blood.  Young red blood cells are flexible and tend to stiffen over their 120 day life-span.  Erythrocyte deformability is, after hematocrit, the second most important determinant of blood viscosity.

The third factor is plasma viscosity.  An important determinant of plasma viscosity is hydration status, but it is also determined by the presence of high molecular-weight proteins, especially immune globulins and fibrinogen.

Erythrocyte aggregation, the tendency of red blood cells to be attracted to each other and stick together is not well understood, but erythrocyte deformability and plasma proteins play important roles.

Blood, like most other fluids, is less viscous at higher temperatures. It is estimated that a 1°C increase in temperature results in a 2% decrease in blood viscosity.

Viscous Blood is Abrasive Blood

Maintaining efficient blood flow through the vessels forms layers, or lamina, that slide easily over each other.

  • Faster flowing blood can be found in the central layers and
  • Slower moving blood in the outer layers near the vessel walls.
  • Hyper-viscous blood doesn’t slide as smoothly as less viscous blood.
  • The turbulence damages the delicate intima of the blood vessel.

One of the most common locations for the development of atherosclerotic plaques is at the bifurcation of the carotid arteries, and the positioning of these plaques can be mapped to the turbulent blood flow patterns of this region.

Blood viscosity is highly correlated with thickening of the carotid intima-media, a prelude to plaque formation.  As the carotid arteries become progressively more occluded, there is decreased blood supply to the brain.

Hyper-viscosity also impacts the brain at the level of micro-perfusion.  Stiffened red blood cells have a decreased ability to bend and fold as they pass through capillaries. This leads to endothelial abrasion.  The capillary walls thicken and diffusion of oxygen and nutrients into the tissues decreases. The effect is most pronounced in those tissues where perfusion is essential for unimpaired function, such as the brain.

Diabetes, Blood Viscosity, and Dementia

While diabetics have elevated blood viscosity, blood viscosity is a risk factor that predicts progression from metabolic syndrome to diabetes. Red blood cell flexibility is greatly reduced by fluctuations in the osmolality of the blood which is affected by blood glucose concentration.  Uncontrolled, this leads to  small vessel disease.

  • Blindness,
  • kidney insufficiency, and
  • leg ischemia

develop as these organs are the dependent on micro-perfusion.

The Rotterdam Study and other research point to decreased cognitive function and increased dementia among diabetics as being further manifestations of the decreased perfusion that accompanies elevated blood viscosity.


Blood Viscosity, Cognitive Decline, and Alzheimer’s

Multiple forms of cognitive decline, including dementia and Alzheimers’ are impacted by increased blood viscosity. The Edinburgh Artery Study (2010) showed that blood viscosity predicted cognitive decline over a four year period in 452 elderly subjects (p<0.05).  Blood viscosity, an important determinant of the circulatory flow, was significantly linked with cognitive function.  The associations between cardiovascular risk factors, vascular dementia, and Alzheimer’s disease were presented by de la Torre (2002) (nine points of evidence) in a compelling argument that Alzheimer’s is a vascular disorder characterized by impaired micro-perfusion to the brain.

Testing for Blood Viscosity

The most recent technology uses an automated scanning capillary tube viscometer capable of measuring viscosity over the complete range of physiologic values experienced in a cardiac cycle (10,000 shear rates) with a single continuous measurement. This test provides clinicians with measurements of blood viscosity at both systolic and diastolic pressures.

Blood viscosity testing is indicated for a wide range of patients, as good tissue perfusion is central to good health regardless of what system is being addressed.  Patients with signs of cognitive decline should be high on the list of those appropriate to test, and those patients with a history or family history of heart disease, stroke, hypertension, diabetes, metabolic syndrome, migraines, smoking, alcoholism or other risk factors associated with the development of Alzheimer’s disease.

Source: Larsen P, Monitoring Blood Viscosity to Improve Cognitive Function

  1. World Health Organization. Dementia: A Public Health Priority. April, 2012.
  2. Sloop GD. A unifying theory of atherogenesis. Med Hypotheses. 1996; 47:321-5.
  3. Kensey KR and Cho, Y. Physical Principles and Circulation: Hemodynamics. In: The Origin of Atherosclerosis: What Really Initiates the Inflammatory Process. 2nd Ed. Summersville, WV: SegMedica; 2007:33-50.
  4. Hofman A., Ott A, et. al. Atherosclerosis, apolipoprotein E, and prevalence of dementia and Alzheimer’s disease in the Rotterdam Study. Lancet, 1997, 349 (9046): 151-154


 Sleep Apnea and Blood Viscosity.

Obstructive sleep apnea (OSA) is an important public health concern, which affects around 2–4% of the population. Left untreated, it causes a decrease not only in quality of life, but also of life expectancy. Despite the fact that knowledge about the mechanisms of development of cardiovascular disease in patients with OSA is still incomplete, observations confirm a relationship between sleep disordered breathing and the rheological properties of blood.

Tażbirek M, Słowińska L, Kawalski M, Pierzchała W.   The rheological properties of blood and the risk of cardiovascular disease in patients with obstructive sleep apnea syndrome (OSAS) Folia Histochemica et Cytobiologica 2011; 49(2):206–210.

Hemostatic and Rheological Risk Factors and the Risk Stratification

Backgound: Thrombosis is regarded to be a key factor in the development of acute coronary syndromes in patients with coronary artery disease (CAD). We hypothesize, that hemostatic
and rheological risk factors may be of major relevance for the incidence and the risk stratification of these patients.

  • Methods: In 243 patients with coronary artery disease and stable angina pectoris parameters of metabolism, hemostasis, blood rheology and endogenous fibrinolysis were assessed.

Patients were prospectively followed for 2 years in respect to elective revascularizations and acute coronary syndromes.

Results: During follow-up 88 patients presented with cardiac events, 22 of those were admitted to the hospital because of acute events, 5 Patients were excluded due to non- cardiac death.

Patients with clinical events were found to be more frequently diabetic and presented with a more progressed coronary atherosclerosis. Even though patients with diabetes mellitus demonstrated a comparable level of multivessel disease (71% vs. 70%) the rate of elective revascularization was higher (41% vs. 28%, p < 0.05). The results were also unfavorable for
the incidence of acute cardiovascular events (18% vs. 8%, p < 0.01).

In comparison to non-diabetic patients diabetics demonstrated significantly elevated levels of

  • fibrinogen (352 ± 76 vs. 312 ± 64 mg/dl, p < 0.01),
  • plasma viscosity (1.38 ± 0.23 vs. 1.31 ± 0.16 mPas, p < 0.01),
  • red blood cell aggregation (13.2 ± 2.5 vs. 12.1 ± 3.1 E, p < 0.05) and

plasmin-activator-inhibitor (6.11 ± 3.4 vs. 4.7 ± 2.7 U/l, p < 0.05).

Conclusion: Pathological alterations of fibrinogen, blood rheology and plasminogen-activatorinhibtor as indicators of a procoagulant state are of major relevance for the
short-term incidence of cardiac events, especially in patients with diabetes mellitus type 2, and may be used to stratify patients to specific therapies.

parameters of metabolism, hemostasis, endogenous fibrinolysis and blood rheology for patients with and without diabetes mellitus.

diabetes mellitus non-diabetic patients p-value
glucose (mg/dl) 157 ± 67 88 ± 12 <0,0001
fibrinogen (mg/dl) 351 ± 76 312 ± 64 <0,01
plasma viscosity (mPa × s-1) 1,38 ± 0,23 1,31 ± 0,16 <0,01

Jax TW, Peters AJ, Plehn G, and  Schoebel FC. Hemostatic risk factors in patients with coronary artery disease and type 2 diabetes – a two year follow-up of 243 patients. Cardiovascular Diabetology 2009; 8:48-57.  doi:10.1186/1475-2840-8-48


Abnormal Viscosity in Pregnancy

Abnormal hemorheology has been shown to be in almost all conditions associated with accelerated atherosclerotic cardiovascular disorders. The aim of this study is to test the hypothesis that high concentration of plasma Triglyceride (TG) predicts altered hemorheological variables in normal pregnancy.

Sixty pregnant women attending antenatal clinic of the University of Ilorin Teaching Hospital at 14-36 weeks of gestation (aged 21-36 years) were recruited after giving informed consent to participate in the study. They consisted of 28 primigravidae and 32 multigravidae. Twenty-four healthy non-pregnant women of similar age and socioeconomical status were also recruited. The study showed that fasting plasma Triglyceride (TG) increased significantly in primigravidae and multigravidae.

There was a positive correlation between plasma TG level and blood viscosity (r = 0.36, p<0.01). TG also correlated positively with hematocrit (r = 0.48, p<0.001), hemoglobin concentration (r = 0.43, p<0.001) and white blood cell count (r = 0.38, p<0.01) in the pregnant group as a whole. In primigravidae, there was a strong correlation between TG and

o          blood viscosity (r = 0.63, p<0.001),

o          hematocrit (r = 0.88, p<0.001),

o          hemoglobin concentration (r = 0.85, p<0.001).

However, there was an insignificant correlation between TG and the hemorheological variables in multigravidae.

Plasma TG concentration in primigravidae is strongly associated with blood viscosity also with hematocrit and hemoglobin concentration, but the association is lost in multigravidae. Therefore, TG could be considered as an important potential indicator of altered blood rheology in primigravidae, but not in multigravidae.

Olatunji LA, Soladoye AO, Fawole AA, Jimoh RO and Olatunji VA. Association between Plasma Triglyceride and Hemorheological Variables in Nigerian Primigravidae and Multigravidae.

Research Journal of Medical Sciences 2008; 2(3):116-120. ISSN: 1815-9346.


Retinal Vein Occlusion

Retinal vein occlusion (RVO) is an important cause of permanent visual loss. Hyperviscosity, due to alterations of blood cells and plasma components, may play a role in the pathogenesis of RVO. Aim of this case-control study was to evaluate the possible association between hemorheology and RVO. In 180 RVO patients and in 180 healthy subjects comparable for age and gender we analysed the whole hemorheological profile: [whole blood viscosity (WBV), erythrocyte deformability index (DI), plasma viscosity (PLV), and fibrinogen]. WBV and PLV were measured using a rotational viscosimeter, whereas DI was measured by a microcomputer-assisted filtrometer. WBV at 0.512 sec-1 and 94.5 sec-1 shear rates as well as DI, but not PLV, were significantly different in patients as compared to healthy subjects.

At the logistic univariate analysis, a significant association between the

  • highest tertiles of WBV at 94.5 sec-1 shear rate (OR:4.91,95%CI 2.95–8.17;p<0.0001),
  • WBV at 0.512 sec-1 shear rate (OR: 2.31, 95%CI 1.42–3.77; p<0.0001), and
  • the lowest tertile of DI (OR: 0.18, 95%CI 0.10–0.32; p<0.0001) and RVO was found.

After adjustment for potential confounders,

  • the highest tertiles of WBV at 0.512 sec-1 shear rate (OR: 3.23, 95%CI 1.39–7.48; p=0.006),
  • WBV at 94.5 sec-1 shear rate (OR: 6.74, 95%CI 3.06–14.86; p<0.0001) and
  • the lowest tertile of DI (OR:0.20,95%CI 0.09–0.44,p<0.0001)

remained significantly associated with the disease. In conclusion, the data indicate that an alteration of hemorheological parameters may modulate the susceptibility to the RVO.

Sofi F, Mannini L, Marcucci R, Bolli P, Sodi A, et al.  Role of hemorheological factors in patients with retinal vein occlusion. In Blood Coagulation, Fibrinolysis and Cellular Haemostasis.  Thromb Haemost 2007; 98:1215–1219.

Summary:  This discussion is a two part sequence that first establishes the known strong relationship between blood flow viscosity, shear stress, and plasma triglycerides (VLDL) as risk factors for hemostatic disorders leading to thromboembolic disease, and the association with atherosclerotic disease affecting the heart, the brain (via carotid blood flow), peripheral circulation, the kidneys, and retinopathy as well.

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