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Posts Tagged ‘Endothelium’


Action of Hormones on the Circulation

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

 

 

Introduction

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

https://pharmaceuticalintelligence.com/2012/10/25/endothelial-function-and-cardiovascular-disease/

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

https://pharmaceuticalintelligence.com/2012/10/19/clinical-trials-results-for-endothelin-system-pathophysiological-role-in-chronic-heart-failure-acute-coronary-syndromes-and-mi-marker-of-disease-severity-or-genetic-determination/

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

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

https://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

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

https://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

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

https://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Innervation of Heart and Heart Rate

Writer and Curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2015/02/15/innervation-of-heart-and-heart-rate/

αllbβ3 Antagonists As An Example of Translational Medicine Therapeutics

Larry H Bernstein, MD, FCAP, Reporter and curator

https://pharmaceuticalintelligence.com/2013/10/12/%CE%B1llb%CE%B23-antagonists-as-an-example-of-translational-medicine-therapeutics/

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

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/

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

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-differences/

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

Larry H Bernstein, MD, FCAP
and
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-exocytosis/

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

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/

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

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

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

https://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-and-calcium-release-related-contractile-dysfunction-ryanopathy/

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

https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

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

Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/08/18/advanced-topics-in-sepsis-and-the-cardiovascular-system-at-its-end-stage/

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.

http://dx.doi.org:/10.1016/j.pharmthera.2009.03.006

PMCID: PMC2704947

Ryanodine receptor-mediated arrhythmias and sudden cardiac death

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

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2704947/

Oxidized Calcium Calmodulin Kinase and Atrial Fibrillation

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

https://pharmaceuticalintelligence.com/2013/10/26/oxidized-calcium-calmodulin-kinase-and-atrial-fibrillation/

Contributions to cardiomyocyte interactions and signaling

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

https://pharmaceuticalintelligence.com/2013/10/21/contributions-to-cardiomyocyte-interactions-and-signaling/

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

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/

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

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/

 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
http://dx.doi.org:/10.1016/j.pnpbp.2005.08.006

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
http://dx.doi.org:/10.1111/j.1476-5381.2011.01776.x

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
http://dx.doi.org:/10.1016/j.cellsig.2010.05.002

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

Glucose

Angiotensin II

Cyclosporin

Insulinlike growth factor

Bombesin

Cortisol

Low-density lipeprotein cholesterol

Hypercholesterolemia

Changes in shear stress on vascular wall

Receptor affinities
Receptor Affinity
ETA ET1 > ET2 > ET3
ETB ET1 = ET2 = ET3
ETC ET3 > ET1
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

 

 

 

 

SUMMARY

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
http://dx.doi.org:/10.1016/j.pharmthera.2005.08.012

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
http://www.jimmunol.org/content/189/11/5411

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
http://dx.doi.org:/10.1016/j.ejim.2007.04.002

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
http://dx.doi.org:/10.1016/S0167-0115(03)00026-0

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
http://dx.doi.org:/10.1016/j.pharmthera.2005.06.015

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
http://dx.doi.org:/10.1016/j.pharmthera.2005.11.001

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
CGRPR CGRP>AM>>amylin=CT
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
http://dx.doi.org:/10.1161/CIRCRESAHA.114.303045

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
https://pharmaceuticalintelligence.com/2013/11/10/biomarkers-diagnosis-and-management/

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
https://pharmaceuticalintelligence.com/2013/03/15/accurate-identification-and-treatment-of-emergent-cardiac-events/

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.
http://dx.doi.org:/10.4137/BMI.S12703

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
http://dx.doi.org/10.1055/s-0031-1272552

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 .
http://dx.doi.org/10.1016/j.jchf.2013.02.007

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.
http://dx.doi.org:/10.1136/bmjopen-2013-004387

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
http://circ.ahajournals.org/content/102/6/636
http://dx.doi.org:/10.1161/01.CIR.102.6.636

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.
http://www.ncbi.nlm.nih.gov/pubmed/7917682

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.
http://www.ncbi.nlm.nih.gov/pubmed/22905340

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.
http://dx.doi.org:/10.1136/bmjopen-2013-004399

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.
http://dx.doi.org:/10.1136/bmjopen-2013-003208

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
http://www.ncbi.nlm.nih.gov/pubmed/22951621

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

Proteomics

  1. The Human Proteome Map Completed

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

https://pharmaceuticalintelligence.com/2014/08/28/the-human-proteome-map-completed/

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

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/06/24/proteomics-the-pathway-to-
understanding-and-decision-making-in-medicine/

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

https://pharmaceuticalintelligence.com/2014/07/06/genomics-proteomics-and-standards/

6. Proteins and cellular adaptation to stress

Larry H Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2014/07/08/proteins-and-cellular-adaptation-to-stress/

 

Metabolomics

  1. Extracellular evaluation of intracellular flux in yeast cells

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

https://pharmaceuticalintelligence.com/2014/08/25/extracellular-evaluation-of-intracellular-flux-in-yeast-cells/

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

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

https://pharmaceuticalintelligence.com/2014/08/23/metabolomic-analysis-of-two-leukemia-cell-lines-_i/

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

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

https://pharmaceuticalintelligence.com/2014/08/24/metabolomic-analysis-of-two-leukemia-cell-lines-ii/

  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

https://pharmaceuticalintelligence.com/2014/08/21/pentose-shunt-electron-transfer-galactose-more-lipids-in-brief/

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

Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

  1. Mitochondrial fission and fusion: potential therapeutic targets?

Ritu saxena

https://pharmaceuticalintelligence.com/2012/10/31/mitochondrial-fission-and-fusion-potential-therapeutic-target/

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

Ritu Saxena

https://pharmaceuticalintelligence.com/2012/08/14/mitochondrial-mutation-analysis-might-be-1-step-away/

  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

https://pharmaceuticalintelligence.com/2012/11/11/metabolic-drivers-in-aggressive-brain-tumors/

  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

https://pharmaceuticalintelligence.com/2012/08/20/therapeutic-targets-for-diabetes-and-related-metabolic-disorders/

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

https://pharmaceuticalintelligence.com/2014/08/18/studies-of-respiration-lead-to-acetyl-coa/

13. Lipid Metabolism

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

https://pharmaceuticalintelligence.com/2014/08/15/lipid-metabolism/

14. Carbohydrate Metabolism

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

https://pharmaceuticalintelligence.com/2014/08/13/carbohydrate-metabolism/

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

https://pharmaceuticalintelligence.com/2014/04/13/prologue-to-cancer-ebook-4-where-are-we-in-this-journey/

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

https://pharmaceuticalintelligence.com/2013/11/01/inhibition-of-the-cardiomyocyte-specific-kinase-tnni3k/

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

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

https://pharmaceuticalintelligence.com/2013/05/15/the-binding-of-oligonucleotides-in-dna-and-3-d-lattice-structures/

20. Mitochondrial Metabolism and Cardiac Function

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

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

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

Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/04/04/sulfur-deficiency-leads_to_hyperhomocysteinemia/

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

https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

25. Nitric Oxide and Immune Responses: Part 2

Author and Curator: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/10/28/nitric-oxide-and-immune-responses-part-2/

26. Overview of Posttranslational Modification (PTM)

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

https://pharmaceuticalintelligence.com/2014/07/29/overview-of-posttranslational-modification-ptm/

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

https://pharmaceuticalintelligence.com/2014/03/29/epilogue-envisioning-new-insights/

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

https://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/

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

https://pharmaceuticalintelligence.com/2013/06/20/naked-mole-rats-cancer-free/

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

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

https://pharmaceuticalintelligence.com/2013/05/06/alzheimers-disease-and-one-carbon-metabolism/

43. Problems of vegetarianism

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

https://pharmaceuticalintelligence.com/2013/04/22/problems-of-vegetarianism/

44.  Amyloidosis with Cardiomyopathy

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

https://pharmaceuticalintelligence.com/2013/03/31/amyloidosis-with-cardiomyopathy/

45. Liver endoplasmic reticulum stress and hepatosteatosis

Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/03/10/liver-endoplasmic-reticulum-stress-and-hepatosteatosis/

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

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/26/the-molecular-biology-of-renal-disorders/

47. Nitric Oxide Function in Coagulation – Part II

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

https://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-function-in-coagulation/

48. Nitric Oxide, Platelets, Endothelium and Hemostasis

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/08/nitric-oxide-platelets-endothelium-and-hemostasis/

49. Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/09/14/interaction-of-nitric-oxide-and-prostacyclin-in-vascular-endothelium/

50. Nitric Oxide and Immune Responses: Part 1

Curator and Author:  Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/10/18/nitric-oxide-and-immune-responses-part-1/

51. Nitric Oxide and Immune Responses: Part 2

Curator and Author:  Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/10/28/nitric-oxide-and-immune-responses-part-2/

52. Mitochondrial Damage and Repair under Oxidative Stress

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

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

https://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-and-inos-have-key-roles-in-kidney-diseases/

57. New Insights on Nitric Oxide donors – Part IV

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/26/new-insights-on-no-donors/

58. Crucial role of Nitric Oxide in Cancer

Curator and Author: Ritu Saxena, Ph.D.

https://pharmaceuticalintelligence.com/2012/10/16/crucial-role-of-nitric-oxide-in-cancer/

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

https://pharmaceuticalintelligence.com/2013/04/06/targeting-mitochondrial-bound-hexokinase-for-cancer-therapy/

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

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/26/biochemistry-of-the-coagulation-cascade-and-platelet-aggregation/

Genomics, Transcriptomics, and Epigenetics

  1. What is the meaning of so many RNAs?

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

https://pharmaceuticalintelligence.com/2014/08/06/what-is-the-meaning-of-so-many-rnas/

  1. RNA and the transcription the genetic code

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

https://pharmaceuticalintelligence.com/2014/08/02/rna-and-the-transcription-of-the-genetic-code/

  1. A Primer on DNA and DNA Replication

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

https://pharmaceuticalintelligence.com/2014/07/29/a_primer_on_dna_and_dna_replication/

4. Synthesizing Synthetic Biology: PLOS Collections

Reporter: Aviva Lev-Ari

https://pharmaceuticalintelligence.com/2012/08/17/synthesizing-synthetic-biology-plos-collections/

5. Pathology Emergence in the 21st Century

Author and Curator: Larry Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/03/pathology-emergence-in-the-21st-century/

6. RNA and the transcription the genetic code

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

https://pharmaceuticalintelligence.com/2014/08/02/rna-and-the-transcription-of-the-genetic-code/

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

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

https://pharmaceuticalintelligence.com/2014/07/15/a-great-university-engaged-in-drug-discovery/

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

https://pharmaceuticalintelligence.com/2014/07/28/genes-proteomes-and-their-interaction/

10. Regulation of somatic stem cell Function

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

https://pharmaceuticalintelligence.com/2014/07/29/regulation-of-somatic-stem-cell-function/

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

https://pharmaceuticalintelligence.com/2014/07/07/bzzz-are-fruitflies-like-us/

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

Larry H Bernstein, MD, FCAP, Reporter

https://pharmaceuticalintelligence.com/2014/06/29/long-non-coding-rnas-can-encode-proteins-after-all/

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

https://pharmaceuticalintelligence.com/2013/05/18/observations-on-finding-the-genetic-links/

17. Silencing Cancers with Synthetic siRNAs

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

https://pharmaceuticalintelligence.com/2013/12/09/silencing-cancers-with-synthetic-sirnas/

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

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/12/cardiometabolic-syndrome-and-the-genetics-of-hypertension-the-neuroendocrine-transcriptome-control-points/

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

https://pharmaceuticalintelligence.com/2014/07/06/loss-of-normal-growth-regulation/

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

https://pharmaceuticalintelligence.com/2012/12/17/big-data-in-genomic-medicine/

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

https://pharmaceuticalintelligence.com/2014/01/06/summary-of-genomics-and-medicine-role-in-cardiovascular-diseases/

 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

https://pharmaceuticalintelligence.com/2013/01/07/personalized-medicine-gearing-up-to-tackle-cancer/

29. Differentiation Therapy – Epigenetics Tackles Solid Tumors

Stephen J Williams, PhD

      https://pharmaceuticalintelligence.com/2013/01/03/differentiation-therapy-epigenetics-tackles-solid-tumors/

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

https://pharmaceuticalintelligence.com/2013/01/10/the-molecular-pathology-of-breast-cancer-progression

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

http://pharmaceuticalntelligence.com/2013/01/13/paradigm-shift-in-human-genomics-predictive-biomarkers-and-personalized-medicine-part-1/

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

  https://pharmaceuticalintelligence.com/2012/11/20/personalized-medicine-based-cure-for-cancer-might-not-be-far-away/

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

https://pharmaceuticalintelligence.com/2012/12/05/what-can-we-expect-of-tumor-therapeutic-response/

43. Directions for genomics in personalized medicine

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

https://pharmaceuticalintelligence.com/2013/01/27/directions-for-genomics-in-personalized-medicine/

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

 https://pharmaceuticalintelligence.com/2012/10/26/mrna-interference-with-cancer-expression/

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

https://pharmaceuticalintelligence.com/2012/09/18/breast-cancer-drug-resistance-and-biopharmaceutical-targets/

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

https://pharmaceuticalintelligence.com/2013_Genomics

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

https://pharmaceuticalintelligence.com/2014/07/08/proteins-and-cellular-adaptation-to-stress/

  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

https://pharmaceuticalintelligence.com/2014/03/26/a-synthesis-of-the-beauty-and-complexity-of-how-we-view-cancer/

  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

https://pharmaceuticalintelligence.com/2014/08/12/signaling-and-signaling-pathways/

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

https://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

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

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

https://pharmaceuticalintelligence.com/2013/10/07/platelets-in-translational-research-1/

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

https://pharmaceuticalintelligence.com/2012/08/22/nitric-oxide-signalling-pathways/

14. Immune activation, immunity, antibacterial activity

Larry H. Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2014/07/06/immune-activation-immunity-antibacterial-activity/

15.  Regulation of somatic stem cell Function

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

https://pharmaceuticalintelligence.com/2014/07/29/regulation-of-somatic-stem-cell-function/

16. 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/

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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

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

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

https://pharmaceuticalintelligence.com/2014/03/26/a-synthesis-of-the-beauty-and-complexity-of-how-we-view-cancer/

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/

 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

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

https://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-exchange-mechanism-in-health-and-disease/

Mitochondrial Metabolism and Cardiac Function

Curator: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

Mitochondrial Dysfunction and Cardiac Disorders

Curator: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

Reversal of Cardiac mitochondrial dysfunction

Curator: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

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/

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

Curator: Larry H Bernstein, MD, FACP

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

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

Curator: Larry H Bernstein, MD, FCAP

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

 

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

Curator: Larry H. Bernstein, MD, FCAP 

https://pharmaceuticalintelligence.com/2012/11/08/nitric-oxide-platelets-endothelium-and-hemostasis/


Mitochondrial Damage and Repair under Oxidative Stress

Curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

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

Reporter and Curator: Larry H Bernstein, MD, FACP

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

 

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

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


Mitochondria and Cancer: An overview of mechanisms

Author and Curator: Ritu Saxena, Ph.D.

https://pharmaceuticalintelligence.com/2012/09/01/mitochondria-and-cancer-an-overview/

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

Author and Curator: Ritu Saxena, Ph.D.

https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

Overview of Posttranslational Modification (PTM)

Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/29/overview-of-posttranslational-modification-ptm/


Ubiquitin Pathway Involved in Neurodegenerative Diseases

Author and curator: Larry H Bernstein, MD,  FCAP

https://pharmaceuticalintelligence.com/2013/02/15/ubiquitin-pathway-involved-in-neurodegenerative-diseases/

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

Author: Larry H. Bernstein, MD, FCAP 

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

New Insights on Nitric Oxide donors – Part IV

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

https://pharmaceuticalintelligence.com/2012/11/26/new-insights-on-no-donors/

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)

https://pharmaceuticalintelligence.com/biomed-e-books/series-a-e-books-on-cardiovascular-diseases/perspectives-on-nitric-oxide-in-disease-mechanisms-v2/

 

Summary

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

and

Curator: Aviva Lev-Ari, PhD, RN

Introduction

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.

Results

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.

Discussion

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.

Results

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

Background

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.

Conclusions

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

Introduction

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.

Methods

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

Results

  • 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.

VASCULAR ENDOTHELIAL GROWTH FACTOR-A

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.

ANGIOPOIETIN-1

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.

ENDOTHELIAL-CARDIOMYOCYTE INTERACTIONS IN NORMAL CARDIAC FUNCTION

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).

NITRIC OXIDE

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

Abstract

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)

Discussion

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

Introduction

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)

Conclusions

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.

References

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

Abstract

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

Introduction

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.

Conclusions

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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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®).

Stent

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.

REFERENCES

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

https://pharmaceuticalintelligence.com/2012/12/31/renal-sympathetic-denervation-updates-on-the-state-of-medicine/

 

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

https://pharmaceuticalintelligence.com/2012/09/02/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

https://pharmaceuticalintelligence.com/2012/07/18/percutaneous-endocardial-ablation-of-scar-related-ventricular-tachycardia/

 

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

https://pharmaceuticalintelligence.com/2012/06/13/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)

https://pharmaceuticalintelligence.com/2012/06/22/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

https://pharmaceuticalintelligence.com/2012/06/19/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-replace/

 

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

https://pharmaceuticalintelligence.com/2012/06/22/global-supplier-strategy-for-market-penetration-partnership-options-niche-suppliers-vs-national-leaders-in-the-massachusetts-cardiology-vascular-surgery-tools-and-devices-market-for-car/

 

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

https://pharmaceuticalintelligence.com/2012/07/23/heart-remodeling-by-design-implantable-synchronized-cardiac-assist-device-abiomeds-symphony/

 

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