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Molecular Pathogenesis of Progressive Lung Diseases

Author: Larry H. Bernstein, MD, FCAP

 

Abstract

The lung and its airways are constantly exposed to the air we breath, its contaminants, microparticulates (asbestose), and incidental microorganisms, such as viruses. These are sources of acute and chronic pulmonary diseases. Just as the lung remodels in normal growth and development, the lung remodels following acute injuries, but in the case of chronic conditions, the remodeling capacity is stressed. The lung is potentially stressed even without exposure to external contaminants or viruses. This stress is related to its normal function of gas exchange between oxygen and carbon dioxide across the alveolar wall. This involves a mechanism for tissue repair initiated by signaling pathways that are triggered in response to oxidative stress that result in a process called the unfolded protein response (UPR). The UPR does not necessarily lead to tissue damage. Damage only occurs when there is sustained stress that exceeds the ability of the tissue to repair the cellular framework. Here, we shall visit the underlying repair process that may be undermined in different lung diseases, all of which involve the inflammatory response, but not necessarily under the same course and conditions.

 

Introduction

The lung develops as an outpouching of the foregut and consists of the trachea and bronchi, and the alveoli. Air exchange occurs in the alveoli. In utero, the lungs are filled with fluid, and breathing occurs at the time of birth. When birth is premature, the surfactant produced by the alveolar lining cells that is necessary for passage of air into and expand the lungs may be insufficient, leading to alveolar collapse. Another problem may be neonatal hypertension. The discussion that follows will only deal with a common metabolic condition that underlies the conditions that underlie the development of chronic pulmonary diseases in the neonate and the adult.

The main feature of the alveoli is that they consist of a single layer of epithelium lining the airspaces beneath which lies a capillary, ideally suited for the exchange of O2 and CO2. There is a basement membrane between the epithelial cells and the capillaries. Two types of alveolar epithelial cells cover 90% of the airway surface. The alveolar type I epithelial cells (ATI), whose main function is gas interchange, are the larger flattened phenotype. Alveolar type II epithelial cells (ATII) are the most abundant epithelial cell type functioning to maintain the alveolar space by secretion of several types of surfactant proteins and other ECM components. There is also a basement membrane beneath the epithelium to be considered. Secretory Clara and goblet cells, ciliated, basal and neuroendocrine cells are also found in the tracheo-bronchial pseudostratified epithelium. Ciliated and secretory cells are involved in clearing the airway passages from microorganisms, air pollutants and other inhaled pathogens. Mucous and goblet cells secrete mucous into the apical surface of the epithelium, which traps foreign particles. These are then cleared out by the action of ciliated cells.

In cellular senescence there are secretory phenotypes that produce pro-inflammatory and pro-fibrotic factors. In the case of subepithelial fibrosis immune cells, like macrophages and neutrophils as well as activated myofibroblasts populate the subcellular matrix and release of pro-fibrotic transforming growth factor beta and continuous deposition of ECM stiffens the basement membrane. This is accompanied by interstitial fibrosis (1).

The remainder of this review will consider how the lung reacts to stresses that may be functionally inherent, genetic mediated, environmental, or virus. This requires an understanding of the UPR, a common mechanism for cellular repair in response to oxidative and nitrosative stress, which is the common mechanism for protecting the alveolar cell, but becomes pathogenic when the stress exceeds the clearance mechanism.

 

The unfolded protein response (UPR)

The mitochondria (mi) and the endoplasmic reticulum (ER) play key roles in the response to stress, and the mitochondria are also involved by way of signaling mechanisms. We shall begin by considering the ER role (ERUPR). The ER are tubular structures that have smooth and rough portions. The rough ER are essential for translation of the genetic code into an amino acid sequence. The smooth ER is involved in lipid synthesis, and other processes. Just as tRNAs are important building blocks for protein, microRNAs come into the picture as well. The microRNAs have a regulatory role in that they are noncoding, but they repress gene expression and thereby, protein homeostasis (protostasis) under the influence of ERUPR signaling. They have their expression under the influence of UPR signaling when there is oxidative/nitrosative stress (2).

The ER-induced ERUPR is mediated by three major ER-resident transmembrane sensors named PKR-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6a and isoforms), and inositol requiring enzyme 1 (IRE1a and isoforms)(2-4). BiP is an abundant ER chaperone that dissociates from these three sensors, leading to their activation of the ER stress response.

The first step is activation of IRE1a, which dimerizes, forms oligomers, and autophosphorylates. This results in a conformational change that activates RNAse. IRE1a RNAse excises a 26-nucleotide intron of the mRNA encoding the transcription factor X-box binding protein-1 (XBP1). This in turn is ligated resulting in a coding reading phase frame shift in the mRNA and leads to the expression of a more stable and active transcription factor, termed XBP1 spliced (XBP1s). XBP1s trans-activates target genes, which depends on the context of tissue and the stress stimuli. The targets of XBP1s are genes involved in protein folding, endoplasmic reticulum-associated degradation (ERAD), protein translocation to the ER, and protein secretion. IRE1a also signals through the assembly of many adapter proteins and regulators, referred to as the UPRosome, as well as control gene expression through ER stress-dependent XBP1 mRNA splicing (2-5)).

The ER is the site where protein is synthesis and maturation occurs. It is also where the transportation and release of correctly folded proteins together with the Golgi apparatus. ER dysfunction has been viewed in the context of adaptation to protein processing and folding in the ER lumen (3).

Activation of the UPR results in accumulation of reactive oxygen species (ROS) in cells devoid of PERK. ATF4 and PERK knockout cells require amino acid and cysteine supplementation. This is thought to be to replenish amino acids lost during secretion and to increase glutathione levels. ATF4 is essential for regulating amino acid metabolism and oxidative stress response. (In addition, PERK knockout cells cannot activate eIF2a dependent translational up-regulation of ATF4, and ATF4-/- cells lack ATF4 protein. ATF4 induces the transcription of genes involved in amino acid import, glutathione biosynthesis and resistance to oxidative stress (3).

NFD-kB is released from its inhibitor IkB as a result of PERK-mediated attenuation of translation. A variety of different genes involved in inflammatory pathways are expressed, such as those encoding the cytokines IL-1 and TNF-a, as NF-kB moves to the nucleus and switches on. Activated IRE1a recruits tumor necrosis factor-a (TNF-a)-receptor-associated factor 2 (TRAF2) in the second branch of the UPR. TRAF2 then activates JNK and IkB kinase (IKK). These are inflammatory kinases that phosphorylate and activate downstream mediators of inflammation. The third branch of the UPR, the ATF6 pathway, also activates NF-kB. The crosstalk between the three branches is evidenced by the spliced X-box binding protein 1 (XBP1s) and ATF4 both inducing production of the cytokines IL-8, IL-6, and monocyte chemoattractant protein 1 (MCP1) by endothelial cells. XBP1s and IFN-b are both initiated in IFN-b production when ER stress is combined with activation of Toll-like receptor (TLR) signaling and in IFN-a production by dendritic cells. ER calcium stores are mediated in calcium-dependent inflammatory responses that produce IL-8. The XBP1s expand the capacity of the ER for protein folding and results in the assembly of the metainflammasome. This protein complex integrates pathogen and nutrient sensing with ER stress, inflammatory kinases, insulin action, and metabolic homeostasis. The eIF2a kinase PKR (double-stranded RNA-activated protein kinase) is a core component of the metaflammasome which interacts directly with several inflammatory kinases such as IKK and JNK, insulin receptor signaling components such as IRS1, and the translational machinery via eIF2a (4).

The conformational alteration of IRE1 via phosphorylation which exposes the RNAse that removes an intron from XBP1 mRNA generates of a protein that is a transcriptional regulator of genes involved in protein folding and degradation, both necessary mechanisms needed to restore ER homeostasis. GRP78 dissociation activates PERK, which in turn phosphorylates eIF2a, an inhibitor of new protein translation and activator of the transcription factor ATF4. The phosphorylation of PERK observed in primary aveolar epithelial cells comes with significant increase in the expression of ATF4 (5).

ER stress-dependent activation of UPR-mediated ER Ca2+ store expansion (via XBP-1 mRNA splicing) is induced by inflammation. This response is coupled to amplification of Ca2+-dependent inflammation and may be beneficial or adverse for the airways. This depends on whether airways are competent to clear or are obstructed. In normal airways (competent to clear), the airway epithelial ER Ca2+ store expansion provides a beneficial response, and reverses the expanded ER Ca2+ stores back to normal levels. However, the airway epithelial ER Ca2+ store expansion-mediated amplification of airway inflammation may be maladaptive for CF and COPD airways. This results in persistent airway epithelial ER Ca2+ store expansion that leads to chronic airway inflammation (3).

XBP1s launches a transcriptional program to produce chaperones (such as Grp78) and proteins involved in ER biogenesis, phospholipid synthesis, ER-associated protein degradation (ERAD), and secretion alone or in conjunction with ATF6a, which are key regulators of the transcriptional response programs.

There are five proteins that have sequence similarity with ATF6a and are anchored to the ER and in response to activation by specific stimuli. They undergo regulated intramembrane proteolysis in the Golgi and subsequent translocation to the nucleus. They all have been implicated in the ER stress response due to their ability to respond to traditional ER stressors. They activate known UPR targets, or show activity at UPR response elements.

Activation of the third arm of the UPR through PERK results in phosphorylation of eukaryotic translational initiation factor 2a (eIF2a) converting eIF2a to a competitor of eIF2b, which then results in reduced global protein synthesis. PERK is one of four protein kinases that can mediate eIF2a phosphorylation; the other three kinases are double stranded RNA-activated protein kinase (PKR), GCN2 general control non-derepressible kinase 2 (GCN2), and heme-regulated inhibitor kinase (HRI)(4).

 

Oxidative stress

Several oxidants give rise to reactive oxygen species (ROS) by inflammatory and epithelial cells within the lung as part of an inflammatory-immune response. These interfere with protein folding in the ER and the compensatory response is the ‘‘unfolded protein response’’ (UPR). Superoxide radicals (O2 •−) can either react with nitric oxide (NO) to form highly reactive peroxynitrite molecules (ONOO) or are rapidly converted into hydrogen peroxide (H2O2) under the influence of superoxide dismutase (SOD) by activation of NADPH oxidase 2 (Nox2) on macrophages, neutrophils and epithelium. The non-enzymatic production of damaging hydroxyl radical (OH) from H2O2 occurs in the presence of Fe2+. Glutathione peroxidases (Gpxs) and catalase catalyze H2O2 to formH2O and O2. The ROS O2•−, ONOO, H2O2 andOH trigger extensive inflammation, DNA damage, protein denaturation and lipid peroxidation. Lipid peroxidation products are 8-isoprostane, 4-hydroxy-2-nonenal (4-OH-2-nonenal) and malondialdehyde (MDA)], LTB4, carbon monoxide and myeloperoxidase (MPO)(6).

 

Mitochondrial phase of UPR (mtUPR)

Mitochondria have a role in regulating alveolar epithelial cell (AEC) programmed cell death (apoptosis), and they are impaired by the generation of ROS, previously discussed. mtDNA encodes for 13 proteins, and that includes several essential for oxidative phosphorylation. The role of hemoglobin in O2/CO2 exchange given the redox state of iron, and the significant concentration of mitochondria in the AEC provides a suitable environment for the generation of ROS, which trigger an AEC mtDNA damage response and apoptosis (7). AEC mtDNA damage repair depends on 8-oxoguanine DNA glycosylase (OGG1) and mitochondrial aconitase (ACO-2), as they actively maintain mtDNA integrity. Reactive oxygen species (ROS)-driven mitochondrial metabolism is modulated by SIRTs. Indeed, SIRT3 is a mitochondrial deactylase linked to mitochondrial metabolism and mtDNA integrity. Moreover, it is known that there is crosstalk between mitochondrial ROS production, mtDNA damage, p53 activation, OGG1, and ACO-2 acting as a mitochondrial redox-sensor involved in mtDNA maintenance (7). Oxidative stress-induced mtROS induces mtDNA damage. It decreases the concentration of SIRT3, ACo-2 and mtOGG1 in AEC, and thereby causes a defective electron transport (ETC) that results in mitochondrial dysfunction, AEC apoptosis, and pulmonary fibrosis.

MtDNA encodes only 3% of mitochondrial proteins, and the rest are nuclear DNA proteins that are transported into the mitochondrion by transfer from the cytosol into the inner membrane. However, OGG1, ACO-2, mitochondrial transcription factor A (Tfam) are among those proteins encoded by nDNA essential for maintaining mtDNA integrity, as are those proteins involved in mtDNA repair. Nevertheless, mtDNA is ~50-fold more sensitive to oxidative damage because of proximity to the ETC, and are without histone protection, and repair mechanisms are limited. Consequently, stress-induced mtDNA damage has a mutation rate that is 10-fold greater than nDNA mtDNA damage. mtDNA mutations can then lead to mitochondrial dysfunction, including the collapse in the mitochondrial membrane potential (ΔΨm) and release of pro-apoptogenic agents (7).

The mitochondrial ETC generates hydroxyl radicals (HO), superoxide anions (O2•−), and hydrogen peroxide (H2O2) generated from redox-active ferrous (Fe2+) iron or contact with asbestos fibers that impair ETC function by decreasing SIRT3, ACo-2 and mtOGG1 in AEC, causing mtDNA damage creating energy imbalance leading to apoptosis. (Not shown. from Seok-Jo Kim, P Cheresh, RP Jablonski, DB Williams and DW Kamp. Int. J. Mol. Sci. 2015; 16: 21486-21519. http://dx.doi.org:/10.3390/ijms160921486)

 

AEC apoptosis and pulmonary fibrosis

AEC apoptosis is followed by pulmonary fibrosis (PF) because of mutation –related damage to AEC Type 2 (AT2) cells (i.e., surfactant C and A2 genes, MUC5b). Oxidative stress occurs in the majority of AT2 cells, many of them having shortened telomeres, and PF occurs in the underlying matrix (7). This is evidenced with activation by various fibrotic stimuli that stimulate pro-apoptotic Bcl-2 family members action (i.e., ROS, DNA damage, asbestos, etc.). The intrinsic apoptotic death pathway acting in mitochondria results in increased permeability of the outer mitochondrial membrane, reduced ΔΨm. This is accompanied by the release of apoptotic proteins, such as cytochrome c, that activate pro-apoptotic caspase-9 and caspase-3.

Pulmonary fibrosis is driven by PINK1 expression and AEC apoptosis. Pro-apoptotic Bim activation is associated with mitochondria-regulated apoptosis and fibrosis. In addition, mitochondrial quality control pathway disruptions lead to accumulation of mtDNA mutations. These mtDNA mutations may compromise ETC function, and they also drive AEC to aerobic glycolysis, associated with the lung cancer phenotype (7).

AEC mtDNA damage is modulated by p53 in the pro-fibrotic lung response (8). In this process, plasminogen activator inhibitor (PAI-1) promotes AEC apoptosis, and at the same time reduces fibroblast proliferation and collagen production. At the same time there is crosstalk between the p53-uPA fibrinolytic system in AT2 cells. A change in phenotype in lung fibroblasts and tissue injury includes lung fibrosis. This is brought on by mtDNA damage and a DNA damage-associated molecular pattern (DAMP) that activates innate immun responses, especially toll like receptor (TLR)-9 signaling (8).
Concurrently, ACO-2 can be relocated from the TCA cycle to the nucleosome to stabilize the mtDNA with subsequent removal of oxidized Aco-2 by Lon protease (9).

Consider the role that UPR plays a role in lung diseases caused by the expression of genetically mutated, misfolded proteins. In cystic fibrosis, The UPR in airway epithelial cells is activated by mutant cystic fibrosis transmembrane conductance regulator (CFTR) delta F508, which interferes with CFTR expression and activates the innate immune response. The UPR in AT2 induces AT2 apoptosis concomitant with epithelial–mesenchymal transformation and extracellular matrix production in mutant surfactant protein C–induced interstitial pulmonary fibrosis (IPF)(10). It also is assumed to play a role in the pathogenesis of COPD. Potential mechanisms that activate the UPR in AT2 cells include direct oxidation of client proteins or chaperones, impaired function of the proteasome or autophagosomes, and decreased expression of miRNAs.

 

Disease specific UPR involvement in pulmonary fibrosis

  • Idiopathic Pulmonary Fibrosis (IPF)

Idiopathic pulmonary fibrosis (IPF) is characterized by repeated injury to the alveolar epithelium with loss of lung epithelial cells and abnormal tissue repair, which results in accumulation of fibroblasts and myofibroblasts, deposit of extracellular matrix components and distorted lung architecture (11). The expression of heme oxygenase-1, a critical defender against oxidative stress, is decreased in macrophages of idiopathic pulmonary fibrosis patients, suggesting an oxidant–antioxidant imbalance in the pathogenesis of idiopathic pulmonary fibrosis (12).

Epithelial apoptosis leads to the release of growth factors and chemokines, which recruit fibroblasts to the site of injury (fibroblastic foci). Thus, myofibroblasts proliferate and extracellular matrix is deposited continues unabated in IPF. The transformation of epithelial cells into mesenchymal cells is a process known as epithelial mesenchymal transition. It allows direct communication between cells, and may explain the buildup of myofibroblasts in interstitial pulmonary fibrosis (IPF). When the distal epithelium in the lung becomes injured the basement membrane loses its integrity. It has to re-epithelialize the surface. Growth factors locally produced can potentially recruit fibroblasts or myofibroblasts (11). TGFb
-/- mice are devoid of avb6 integrin. Hence, they are unable to activate latent TGF-b1 and are protected from bleomycin-induced pulmonary fibrosis. Primary AECs were found to produce ET-1 at physiologically active levels and increased synthesis of TGF-b1 and the induction of EMT in AECs (11). The fibrosis of IPF occurs only in the lung, is the major source of surfactant proteins (SPs), such as SP-C. This protein appears vulnerable to mutations that disrupt folding and secretion. Recent studies found that IPF patients carry increased number of apoptotic cells in alveolar and bronchial epithelia. The bleomycin mouse model supports an hypothesis that inhibition of epithelial cell apoptosis prevents the development of the fibrosis (1).

  • Interstitial pulmonary fibrosis (IPF)

IPF is the most common variety of lung fibrosis and carries a sobering mortality approaching 50% at 3–4 years (7). Increased oxidative DNA damage is seen in IPF, silicosis, and asbestosis patients, as well in experimental animal models. Ras-related C3 botulinum toxin substrate 1 (Rac1), is a protein encoded by the RAC1 gene found in human cells, which has a variety of alternatively spliced versions of the Rac1 protein (13). The UPR is activated in AT2 cells and induces epithelial–mesenchymal transformation, extracellular matrix production, and type II cell apoptosis In mutant surfactant protein C–induced interstitial pulmonary fibrosis (IPF) (10). The fibrotic phenotype of activated myofibroblasts show inhibition of the ER stress-induced IRE1a signaling pathway by using the inhibitor 4l8C that blocks TGFb-induced activation of myofibroblasts in vitro (13). IRE1a cleaves miR-150 releasing the suppressive effect that miR-150 exerts on aSMA expression through c-Myb. It also blocks ER expansion through an XBP-1-dependent pathway. In addition, prominent expression of UPR markers in AECs has been shown in the lungs of patients with surfactant protein C (SFTPC) mutation-associated fibrosis (14). Patients without SFTPC mutations with familial interstitial pneumonia and patients with sporadic IPF had selective UPR activation of AECs lining areas where there was fibrotic remodeling.
Activation of the UPR pathways may result from altered surfactant protein processing or chronic herpesvirus infection.

Fibroblasts in fibroblastic foci of IPF showed immunoreactivity for GRP78. In addition, TGF-b1 increased expression of GRP78, XBP-1, and ATF6a, which was accompanied by increases in a-SMA and collagen type I expression in mouse and human fibroblasts (15). TGF-b1–induced UPR and a-SMA and collagen type I induction were suppressed by the 4-PBA chaperone. Therefore, UPR is involved in myofibroblastic differentiation during fibrosis.

Initial observations linking ER stress and IPF were made in cases of familial interstitial pneumonia (FIP), the familial form of IPF, in a family with a mutation in surfactant protein C (SFTPC). ER stress markers are highly expressed in the alveolar epithelium in IPF and FIP (15). ER stress is induced in the alveolar epithelium predisposed to enhanced lung fibrosis after treatment with bleomycin, which is mediated at least in part by increased alveolar epithelial cell (AEC) apoptosis. In another study, aged mice developed greater ER stress in the AEC population linked to MHV68 infection as a result of increased BiP expression and increased XBP1 splicing, as well as increased AEC apoptosis, compared with young mice (16).

 

Chronic Obstructive Lung Disease (COPD)

Inflammatory and infectious factors are present in diseased airways that interact with G-protein coupled receptors (GPCRs), such as purinergic receptors and bradykinin (BK) receptors, to stimulate phospholipase C [PLC]. This is followed by the activation of inositol 1,4,5-trisphosphate (IP3)-dependent activation of IP3 channel receptors in the ER, which results in channel opening and release of stored Ca2+ into the cytoplasm. When ER Ca2+ stores are depleted a pathway for Ca2+ influx across the plasma membrane is activated. This has been referred to as “capacitative Ca2+ entry”, and “store-operated calcium entry” (3). In the next step PLC mediated Ca2+ i is mobilized as a result of GPCR activation by inflammatory mediators, which triggers cytokine production by Ca2+ i-dependent activation of the transcription factor nuclear factor kB (NF-kB) in airway epithelia. Ca2+ binding proteins including calmodulin, protein kinases C (PKCs) and the phosphatidylinositol 3-kinase (PI3K) can link Ca2+ i mobilization to NF-kB activation. Ca2+ i from ER Ca2+ release and/or a Ca2+ influx through the plasma membrane can be sensed by Ca2+ binding proteins (3). Chronically infected/inflamed native human bronchial epithelia exhibit UPR activation-dependent XBP-1 mRNA splicing and ER Ca2+ store expansion.

Protein secretion can constitute an irreversible loss of amino acids into the extracellular environment and produce net loss of equivalents from the cell. The greater the secretory burden, the greater the loss of amino acids and reducing equivalents from the cell. Activation of the UPR results in accumulation of ROS in PERK knockout cells. ATF4(-/-) and PERK (-/-) knockout cells require amino acid and cysteine supplementation to replenish amino acids lost during secretion. ATF4 would be required to induce the transcription of genes involved in amino acid import, glutathione biosynthesis and resistance to oxidative stress (3). Oxidative stress is a hallmark of CF airways disease and ATF4-induced amino acid transport is necessary for a protective role in inflamed CF airway epithelia.

Airway epithelial infection/inflammation induces ER stress-dependent activation of UPR-mediated ER Ca2+ store expansion (via XBP-1 mRNA splicing). The airway epithelial ER Ca2+ store is beneficial to the clearing of infection in normal airways. Epithelial ER Ca2+ store expansion-mediated amplification of airway inflammation may not be adequate for cystic fibrosis (CF) and COPD airways (3).

Changes in phosphor-eIF2a and CHOP expression correlate directly with the severity of airflow obstruction in COPD (10). An increase in CHOP in COPD was associated with increases in caspase 3 and 7, suggesting that the PERK pathway was contributing to heightened apoptosis in COPD. Mucous hypersecretion contributes to symptomatology and morbidity in COPD. IRE1b expression in airway epithelial cells promotes mucus cell development and mucin production.

 

Cigarette Smoke and COPD

Cigarette smoking is the major cause of COPD and accounts for more than 95% of cases in industrialized countries. It is the third largest cause of death in the world. It is now well established that cardiovascular -related comorbidities such as stroke contribute to morbidity and mortality in COPD (18). COPD involves chronic obstructive bronchiolitis with fibrosis, obstruction of small airways, emphysema with enlargement of airspaces, destruction of lung parenchyma, and loss of lung elasticity and closure of small airways. Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation and loss of lung function (18). Chronic obstructive bronchiolitis, emphysema and mucus plugging are all characteristic features.

Proteomes of lung samples were taken from chronic cigarette smokers. There were 26 differentially expressed proteins (20 were up-regulated, 5 were down-regulated, and 1 was detected only in the smoking group) compared with nonsmokers. Several UPR proteins were up-regulated in smokers compared with nonsmokers and ex-smokers, including the chaperones, glucose-regulated protein 78 (GRP78) and calreticulin; a foldase, protein disulfide isomerase (PDI), and enzymes involved in antioxidant defense (18). Indeed, a UPR response in the human lung occurs in cigarette smoking that is rapid in onset, concentration dependent, and may be partially reversible with smoking cessation.
Of the proteins reported in chronic smokers, four are involved in translation and ribosome formation (60S acidic ribosomal protein P2, heat shock protein 27, and elongation factors-1b and -1d). Heat shock protein 27 inhibits formation of the large and small ribosomal complex, and 60S acidic ribosomal protein P2 associates with elongation factor-2 to form the large and small ribosomal complex. Glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, and ATP synthase subunit beta were up-regulated, and the inflammatory protein S100-A9/calgranulin C, an EF hand calcium-binding protein, was down-regulated (19).

Conclusion

The current status of a consolidated view of chronic pulmonary fibrotic diseases could not have been envisioned in a 19th century scientific framework. There was no scientific guideline for constructing such a perspective. I have written this perspective on lung diseases keeping in memory the contributions of my mentor, Averill A. Liebow. I have not included pulmonary carcinoma in this discussion, although it too has a place. It was in 1927 that Otto Warburg conducted his historic work with rediscovery of the observation of Louis Pasteur more than a half century earlier in his observation of aerobic glycolysis in cancer cells. The mitochondrion was not known then, which he referred to as “grana”. There was no clear mechanism for such a phenomenon. This discussion based on a growing body of work brings greater clarity to the relationship between lung development, the aging of pulmonary tissue, and the process of tissue remodeling, with a more unified view of pulmonary degeneration that even applies to pulmonary hypertension.

 

References

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Vaccine for Heart Disease

Writer and Curator: Larry, MD, FCAP 

 

 

Introduction

Research investigators at Wayne State University in collaboration with La Jolla Institute for Allergy and Immunology (LJAI) are developing a T-cell peptide-based vaccine for cardiovascular disease, specifically, to reduce immune-based inflammatory plaques in arteries.  The scientists published their findings in the December 2013 issue of Frontiers in Immunology, titled “Atheroprotective vaccination with MCH-II restricted peptides from Apo B-100.”  These experiments show proof of concept for the development of an autoantigen-specific vaccine for reducing the amount of atherosclerotic plaques in mice.
The published work was done in the laboratory of Klaus Ley, M.D., a prominent vascular biolist of LIAI based on the discovery by Harley Tse, Ph.D., Professor of immunology and microbiology at Wayne Stae University School of Medicine, and Wayne State’s Cardiovascular Research Institute with Michael Shae, Ph.D., adjunct assistant professor of immunology and microbiology.Shaw and Tse are the first to demonstrate that two T-cell epitopes of the autoantigen apoB100 are deeply involved in the development of the disease. The discovery is reported in J Immunol Clin Res Apr-Jun, 2014; 2: “Identification of two immunogenic T cell epitopes of ApoB100 and their Autoimmune Implications.”

 

Atheroprotective Vaccination with MHC-II Restricted Peptides from ApoB-100.

Tse K, Gonen A, Sidney J, Ouyang H, Witztum JL, Sette A, Tse H, Ley K
Front Immunol. 2013 Dec 27; 4:493.
http://dx.doi.org:/10.3389/fimmu.2013.00493 eCollection 2013.

BACKGROUND:  Subsets of CD4(+) T-cells have been proposed to serve differential roles in the development of atherosclerosis. Some T-cell types are atherogenic (T-helper type 1), while others are thought to be protective (regulatory T-cells). Lineage commitment toward one type of helper T-cell versus another is strongly influenced by the inflammatory context in which antigens are recognized. Immunization of atherosclerosis-prone mice with low-density lipoprotein (LDL) or its oxidized derivative (ox-LDL) is known to be atheroprotective. However, the antigen specificity of the T-cells induced by vaccination and the mechanism of protection are not known.

METHODS: Identification of two peptide fragments (ApoB3501-3516 and ApoB978-993) from murine ApoB-100 was facilitated using I-Ab prediction models, and their binding to I-Ab determined. Utilizing a vaccination scheme based on complete and incomplete Freund’s adjuvant (CFA and IFA) [1 × CFA + 4 × IFA], we immunized Apoe(-/-)mice with ApoB3501-3516 or ApoB978-993 emulsified in CFA once and subsequently boosted in IFA four times over 15 weeks. Spleens, lymph nodes, and aortas were harvested and evaluated by flow cytometry and real time RT-PCR. Total atherosclerotic plaque burden was determined by aortic pinning and by aortic root histology.

RESULTS:  Mice immunized with ApoB3501-3516 or ApoB978-993 demonstrated 40% reduction in overall plaque burden when compared to adjuvant-only control mice. Aortic root frozen sections from ApoB3501-3516 immunized mice showed a >60% reduction in aortic sinus plaque development. Aortas from both ApoB3501-3516 and ApoB978-993 immunized mice contained significantly more mRNA for IL-10. Both antigen-specific IgG1 and IgG2c titers were elevated in ApoB3501-3516 or ApoB978-993 immunized mice, suggesting helper T-cell immune activity after immunization.

CONCLUSION: Our data show that MHC Class II restricted ApoB-100 peptides can be atheroprotective, potentially through a mechanism involving elevated IL-10.

Atherosclerosis is decreased in ApoB3501–3516 and ApoB978–993

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873602/bin/fimmu-04-00493-g001.jpg

Atherosclerosis is decreased in ApoB3501–3516 and ApoB978–993-treated mice compared to controls. (A) Vaccination schedule: 8-week-old female Apoe−/− mice were immunized once with either PBS or peptide in CFA, then boosted four more times with PBS or peptide in IFA. WD was maintained for 13 weeks. Mice were sacrificed and organs harvested at 23 weeks of age. (B,C) Results of aortic pinning analysis after Sudan IV staining are shown with representative photographs. N = 12–15 in each group, *p < 0.05 when compared to 1× CFA + 4× IFA group. (D) Representative aortic root staining sections after ORO staining, counter-stained with hematoxylin. (E) Plaque area from aortic roots stained from each group. Lesion sizes from 30 to 40 μm distal to start of the aortic valve were averaged per group. N = 5 in each group, *p < 0.05 when compared to 1× CFA + 1× IFA control group.

 

Inhibition of T cell response to native low density lipoprotein reduces atherosclerosis

Andreas Hermansson, DFJ Ketelhuth, D Strodthoff, M Wurm, E. Hansson, et al.
J. Exp. Med. Mar 2015; 207(5): 1081-1093
http://www.jem.org/cgi/doi/10.1084/jem.20092243

Atherosclerosis is a chronic inflammatory disease in which lipoproteins accumulate, eliciting an inflammatory response in the arterial wall. Adaptive immune responses that engage clonally expanded T cell populations contribute to this process, as do innate immune responses that are mounted by macrophages and other cells. Several studies have suggested that components of low-density lipoprotein (LDL) particles trigger vascular inflammation (Tabas et al., 2007; Hartvigsen et al., 2009).

As a consequence of oxidation, the double bonds of fatty acid residues in phospholipids, cholesteryl esters, and triglycerides are cleaved, thus generating reactive aldehydes and truncated lipids (Esterbauer et al., 1990). Among the latter, modified phospholipids, such as lysophosphatidylcholine and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (ox-PAPC), induce endothelial cells, macrophages, and B1-type B cells to initiate innate immune responses, effecting adhesion molecule expression, chemokine production, and secretion of natural antibodies containing germline IgM sequences (Leitinger et al., 1997; Binder et al., 2004; Gharavi et al., 2007).

Immune responses to oxidized low-density lipoprotein (oxLDL) are proposed to be important in atherosclerosis. To identify the mechanisms of recognition that govern T cell responses to LDL particles, we generated T cell hybridomas from human ApoB100 transgenic (huB100tg) mice that were immunized with human oxLDL. Surprisingly, none of the hybridomas responded to oxidized LDL, only to native LDL and the purified LDL apolipoprotein ApoB100.

However, sera from immunized mice contained IgG antibodies to oxLDL, suggesting that T cell responses to native ApoB100 help B cells making antibodies to oxLDL. ApoB100 responding CD4+ T cell hybridomas were MHC class II–restricted and expressed a single T cell receptor (TCR) variable (V)  chain, TRBV31, with different V chains. Immunization of huB100tgxLdlr/ mice with a TRBV31-derived peptide induced anti-TRBV31 antibodies that blocked T cell recognition of ApoB100. This treatment significantly reduced atherosclerosis by 65%, with a concomitant reduction of macrophage infiltration and MHC class II expression in lesions. In conclusion, CD4+ T cells recognize epitopes on native ApoB100 protein, this response is associated with a limited set of clonotypic TCRs, and blocking TCR-dependent antigen recognition by these T cells protects against atherosclerosis.

 

Impact of multiple antigenic epitopes from ApoB100, hHSP60 and Chlamydophila pneumoniae on atherosclerotic lesion development in Apobtm2SgyLdlrtm1HerJ mice

Xinjie Lu, Min Xia, V Endresz, I Faludi, A Szabo, et al.
Atherosclerosis Nov 2012; 225(1): 56–68
http://www.sciencedirect.com.scopeesprx.elsevier.com/science/article/pii/S0021915012004935
http://dx.doi.org:/10.1016/j.atherosclerosis.2012.07.021

Highlights

► We produced 5 constructs using dendroaspin as a scaffold for immunization study. ► All constructs have the effect on lesion reduction. ► Modulation in atherosclerosis-related autoimmunity appears by Tregs.

Atherosclerosis is increasingly recognized as a complex chronic inflammatory disease of the arterial walls [1], [2] and [3], as evidenced by the presence of inflammatory cells, activated immune cells and cytokines in lesions, all of which indicate involvement of the immune system. Atherosclerotic plaques are known to contain macrophage-derived foam cells in which macrophages interact with T-cells to produce a wide array of cytokines that can exert both pro- and anti-inflammatory effects.

 

Antibodies against aldehyde-modified ApoB100, a major constituent of low-density lipoprotein, reduce atherosclerosis in mice expressing human ApoB100, suggesting an immunogenic role of ApoB100. Antibodies against epitopes of the human heat shock protein 60 (hHSP60) molecule (hHSP60153–163: AELKKQSKPVT and hHSP60303-312: PGFGDNRKNQ) are present in atherosclerotic patients and share considerable homology with human cytomegalovirus (HCMV)-derived protein (immediate early protein UL122) and Porphyromonas gingivalis microbial HSP60. Sequence homology between microbial HSP60 and hHSP60 has been suggested to result in immunological cross-reactivity, which may play a role in atherogenesis. Titers of Cpn antibodies are not always positively associated with the Cpn organism in atheroma; however, these antibodies might exert cross-reactivity to non-Cpn antigens.

Immunization of mice with a single construct containing multiple epitopes derived from ApoB100, hHSP60 and Cpn was more effective in reducing early atherosclerotic lesions through the induction of a specific Treg-cell response than was the construct containing either mono- or bi-epitopes. This approach offers attractive opportunities for the design of protein-based, multivalent vaccines against atherosclerosis.

 

Immunization with a combination of ApoB and HSP60 epitopes significantly reduces early atherosclerotic lesion in Apobtm2SgyLdlrtm1Her/J mice

Xinjie Lu, Daxin Chen, Valeria Endreszb, Min Xia, Ildiko Faludi, et. al.
Atherosclerosis 212 (2010) 472–480
http://dx.doi.org:/10.1016/j.atherosclerosis.2010.06.007

Objective: HSP60 is emerging as an immune-dominant target of autoantibodies in atherosclerosis and recent studies have revealed oxLDL as a key antigen in the development of atherosclerosis. In this study, we assay whether immunizing Apobtm2SgyLdlrtm1Her/J mice with a combination of ApoB and human HSP60 peptides has an additive effect on athero-protection compared to ApoB or HSP60 peptides applied alone by following atherosclerotic lesion development. Methods and results: In this study, 2 weeks after the first immunization, Apobtm2SgyLdlrtm1Her/J mice were placed on a high-fat diet for 8 weeks followed by 2 weeks on a normal diet allowing the mice to adapt to the environment before sacrifice. High levels of ApoB and HSP60 antibodies were detectable in week 2 and week 12 following the first immunization with KLH-conjugated ApoB and HSP60 peptides either individually or in combination. Histological analyses demonstrated that mice immunized with both, ApoB and HSP60 peptides, showed the most significant reduction in atherosclerotic lesions (41.3%; p < 0.001) compared to a reduction of 14.7% (p < 0.05) and 21.1% (p < 0.01) in mice immunized with ApoB or HSP60 peptides, respectively; control mice were immunized with either PBS or adjuvant alone. These results

were further supported by significant differences in the cellular and humoral immune responses between test animals. Conclusions: Immunization with a combination of ApoB and HSP60 peptide antigens significantly reduced early atherosclerotic lesions in the Apobtm2SgyLdlrtm1Her/J mouse model of atherosclerosis. This approach offers promise as a novel strategy for developing anti-atherosclerotic agents.

 

Chlamydophila (Chlamydia) pneumoniae infection promotes vascular smooth muscle cell adhesion and migration through IQ domain GTPase-activating protein 1

Lijun Zhang, Xiankui Li, Lijun Zhang, Beibei Wang, Tengteng Zhang, Jing Ye
Microb Pathogen 2012; 53(5–6): 207–213
http://dx.doi.org:/10.1016/j.micpath.2012.07.005

Highlights

► C. pneumoniae infection increases the adhesion of vascular smooth muscle cells. ► C. pneumoniae infection promotes the migration of vascular smooth muscle cells. ► IQGAP1 expression was increased in the infected vascular smooth muscle cells. ► Depletion of IQGAP1 inhibits the infection-induced cell adhesion and migration.

The mechanisms for Chlamydophila (Chlamydia) pneumoniae (C. pneumoniae) infection-induced atherosclerosis are still unclear. Cell adhesion has important roles in vascular smooth muscle cell (VSMC) migration required in the development of atherosclerosis. However, it is still unknown whether IQ domain GTPase-activating protein 1 (IQGAP1) plays pivotal roles in C. pneumoniae infection-induced the adhesion and migration of rat primary VSMCs. Accordingly, in this study, we demonstrated that rat primary VSMC adhesion (P < 0.001) and migration (P < 0.01) measured by cell adhesion assay and Transwell assay, respectively, were significantly enhanced after C. pneumoniae infection. Reverse transcription-polymerase chain reaction analysis revealed that the mRNA expression levels of IQGAP1 in the infected rat primary VSMCs were found to increase gradually to reach a peak and then decrease gradually to a level similar to the control. We further showed that the increases in rat primary VSMC adhesion to Matrigel (P < 0.001) and migration (P < 0.01) caused by C. pneumoniae infection were markedly inhibited after IQGAP1 knockdown by a pool of four short hairpin RNAs. Taken together, our results suggest that C. pneumoniae infection may promote the adhesion and migration of VSMCs possibly by upregulating the IQGAP1 expression.

 

Rosiglitazone negatively regulates c-Jun N-terminal kinase and toll-like receptor 4 proinflammatory signalling during initiation of experimental aortic aneurysms

Grisha Pirianov, Evelyn Torsney, Franklyn Howe, Gillian W. Cockerill
Atherosclerosis 2012; 225(1): 69–75
http://dx.doi.org:/10.1016/j.atherosclerosis.2012.07.034

Highlights

► Rosiglitazone has a marked effect on both aneurysm rupture and development. ► Rosiglitazone modulates inflammation by blocking TLR4/JNK signalling. ► Specific antagonists of JNK and TLR4 may be therapeutic for aneurysms.

Development and rupture of aortic aneurysms (AA) is a complex process involving inflammation, cell death, tissue and matrix remodelling. The thiazolidinediones (TZDs) including Rosiglitazone (RGZ) are a family of drugs which act as agonists of the nuclear peroxisome proliferator-activated receptors and have a broad spectrum of effects on a number of biological processes in the cardiovascular system. In our previous study we have demonstrated that RGZ has a marked effect on both aneurysm rupture and development, however, the precise mechanism of this is unknown.

Methods and results  In the present study, we examined possible targets of RGZ action in the early stages of Angiotensin II-induced AA in apolipoprotein E-deficient mice. For this purpose we employed immunoblotting, ELISA and antibody array approaches. We found that RGZ significantly inhibited c-Jun N-terminal kinase (JNK) phosphorylation and down-regulated toll-like receptor 4 (TLR4) expression at the site of lesion formation in response to Angiotensin II infusion in the initiation stage (6–72 h) of experimental AA development. Importantly, this effect was also associated with a decrease of CD4 antigen and reduction in production of TLR4/JNK-dependant proinflammatory chemokines MCP-1 and MIP-1α.  Conclusion These data suggest that RGZ can modulate inflammatory processes by blocking TLR4/JNK signalling in initiation stages of AA development.

 

Atheroprotective immunization with malondialdehyde-modified LDL is hapten specific and dependent on advanced MDA adducts: implications for development of an atheroprotective vaccine.

Gonen A, Hansen LF, Turner WW, Montano EN, Que X,…, Hartvigsen K.
J Lipid Res. 2014 Oct;55(10):2137-55.
http://dx.doi.org:/10.1194/jlr.M053256.  Epub 2014 Aug 20.

Immunization with homologous malondialdehyde (MDA)-modified LDL (MDA-LDL) leads to atheroprotection in experimental models supporting the concept that a vaccine to oxidation-specific epitopes (OSEs) of oxidized LDL could limit atherogenesis. However, modification of human LDL with OSE to use as an immunogen would be impractical for generalized use. Furthermore, when MDA is used to modify LDL, a wide variety of related MDA adducts are formed, both simple and more complex. To define the relevant epitopes that would reproduce the atheroprotective effects of immunization with MDA-LDL, we sought to determine the responsible immunodominant and atheroprotective adducts. We now demonstrate that fluorescent adducts of MDA involving the condensation of two or more MDA molecules with lysine to form malondialdehyde-acetaldehyde (MAA)-type adducts generate immunodominant epitopes that lead to atheroprotective responses. We further demonstrate that a T helper (Th) 2-biased hapten-specific humoral and cellular response is sufficient, and thus, MAA-modified homologous albumin is an equally effective immunogen. We further show that such Th2-biased humoral responses per se are not atheroprotective if they do not target relevant antigens. These data demonstrate the feasibility of development of a small-molecule immunogen that could stimulate MAA-specific immune responses, which could be used to develop a vaccine approach to retard or prevent atherogenesis.

 

Low density lipoprotein oxidation and atherogenesis: from experimental models to clinical studies.

Napoli C
G Ital Cardiol. 1997 Dec; 27(12):1302-14.

Oxidative modifications of low-density lipoproteins (LDL) (“oxidation hypothesis”) appears to be the pathophysiologic mechanism implicated in early atherogenesis. Oxidized LDL (ox-LDL) may also induce several pro-atherogenic mechanisms, such as the regulation of vascular tone, by interfering with nitric oxide, the stimulation of cytokines and chemotactic factors (MCP-1, M-CSF, VCAM-1, etc.) and transcription factors (AP1 and NFk beta). These phenomena complicate the spectrum of direct and indirect actions of ox-LDL. The immunogenicity of ox-LDL was used to generate monoclonal antibodies against many epitopes of ox-LDL, such as malondialdehyde-lysine (MDA-2) or 4-hydroxynonenal-lysine (NA59). These antibodies showed the occurrence of ox-LDL in vivo. Another issue is the role of the humoral and cellular immune system in atherogenesis, in particular whether the immune response to ox-LDL enhances or reduces early atherogenesis. Moreover, the induction of autoantibodies against ox-LDL and the recognition by “natural” antibodies, and the use of the antigens to screen human sera may serve as a marker of atherosclerosis. In this review, we have stressed the importance of methodologic approach in the assessment of LDL-oxidation and the fact that lipoprotein (a) may also undergo oxidative modifications. Several clinical conditions are associated with increased rate of LDL-oxidation. Recently, we have observed the presence of LDL oxidation-specific epitopes in human fetal aortas. Antioxidants studies in primary prevention of atherosclerosis have produced contradictory results. This may be explained in part by the selection of patients who had advanced lesions and were often smokers. New trails suggest that antioxidants be administered early in children. Lastly, antioxidant studies in the secondary prevention of coronary heart disease (CHAOS, WACS, and HOPE) show clear evidence of the benefits of antioxidants in reducing new cardiovascular events.

 

Summary:

Atheroprotective Vaccine

Tech ID: 19640 / UC Case 2006-250-0
http://www.ucop.edu//ncd/12343.html

Atherosclerosis is a chronic inflammatory disease and immunological mechanisms are of central importance. It is known that oxidized LDL and its oxidized moieties were a major class of immunodominant epitopes within the atherosclerotic plaque. Oxidation of LDL leads to the generation of a variety of oxidized lipids and oxidized lipid-apo-B adducts.

Technology Description

UC San Deigo researchers proposed that an immunization strategy could be used to inhibit the progression of atherosclerosis by showing that immunization of rabbits and/or mice (and ultimately humans) with MDA-LDL could inhibit atherosclerosis. To develop a safe vaccine for human use would require the identification of the specific immunogenic oxidation-specific epitope(s) that provides the atheroprotective immunity. Until now, the mechanism of the protection, that is, the immunodominant epitope(s) has not yet been determined.

UC San Diego researchers have been able to identify a small group of MDA-derived adducts which are immunodominant and atheroprotective in mice following immunization. The invention described here has the potential to provide an antigen to formulate a wholly synthetic vaccine to inhibit  the development of atherosclerosis in man. Furthermore, in vivo levels of the adducts, and the autoantibodies recognizing them, may be used as diagnostic tools in patients with cardiovascular and other inflammatory diseases.

State Of Development

Mice have been immunized with the adducts resulting in atheroprotection. Techniques are currently being developed for a totally synthetic immunogen suitable for human clinical studies. Assays are also being developed.

Intellectual Property Info

A patent application has been filed on this technology.

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The Vibrant Philly Biotech Scene: Focus on KannaLife Sciences and the Discipline and Potential of Pharmacognosy

Curator and Interviewer: Stephen J. Williams, Ph.D.

 

philly2nightThis post is the third in a series of posts highlighting interviews with Philadelphia area biotech startup CEO’s and show how a vibrant biotech startup scene is evolving in the city as well as the Delaware Valley area. Philadelphia has been home to some of the nation’s oldest biotechs including Cephalon, Centocor, hundreds of spinouts from a multitude of universities as well as home of the first cloned animal (a frog), the first transgenic mouse, and Nobel laureates in the field of molecular biology and genetics. Although some recent disheartening news about the fall in rankings of Philadelphia as a biotech hub and recent remarks by CEO’s of former area companies has dominated the news, biotech incubators like the University City Science Center and Bucks County Biotechnology Center as well as a reinvigorated investment community (like PCCI and MABA) are bringing Philadelphia back. And although much work is needed to bring the Philadelphia area back to its former glory days (including political will at the state level) there are many bright spots such as the innovative young companies as outlined in these posts.

In today’s post, I had the opportunity to talk with both Dr. William Kinney, Chief Scientific Officer and Thoma Kikis, Founder/CMO of KannaLife Sciences based in the Pennsylvania Biotech Center of Bucks County.   KannaLifeSciences, although highlighted in national media reports and Headline news (HLN TV)for their work on cannabis-derived compounds, is a phyto-medical company focused on the discipline surrounding pharmacognosy, the branch of pharmacology dealing with natural drugs and their constituents.

Below is the interview with Dr. Kinney and Mr. Kikis of KannaLife Sciences and Leaders in Pharmaceutical Business Intelligence (LPBI)

 

PA Biotech Questions answered by Dr. William Kinney, Chief Scientific Officer of KannaLife Sciences

 

 

LPBI: Your parent company   is based in New York. Why did you choose the Bucks County Pennsylvania Biotechnology Center?

 

Dr. Kinney: The Bucks County Pennsylvania Biotechnology Center has several aspects that were attractive to us.  They have a rich talent pool of pharmaceutically trained medicinal chemists, an NIH trained CNS pharmacologist,  a scientific focus on liver disease, and a premier natural product collection.

 

LBPI: The Blumberg Institute and Natural Products Discovery Institute has acquired a massive phytochemical library. How does this resource benefit the present and future plans for KannaLife?

 

Dr. Kinney: KannaLife is actively mining this collection for new sources of neuroprotective agents and is in the process of characterizing the active components of a specific biologically active plant extract.  Jason Clement of the NPDI has taken a lead on these scientific studies and is on our Advisory Board. 

 

LPBI: Was the state of Pennsylvania and local industry groups support KannaLife’s move into the Doylestown incubator?

 

Dr. Kinney: The move was not State influenced by state or industry groups. 

 

LPBI: Has the partnership with Ben Franklin Partners and the Center provided you with investment opportunities?

 

Dr. Kinney: Ben Franklin Partners has not yet been consulted as a source of capital.

 

LPBI: The discipline of pharmacognosy, although over a century old, has relied on individual investigators and mainly academic laboratories to make initial discoveries on medicinal uses of natural products. Although there have been many great successes (taxol, many antibiotics, glycosides, etc.) many big pharmaceutical companies have abandoned this strategy considering it a slow, innefective process. Given the access you have to the chemical library there at Buck County Technology Center, the potential you had identified with cannabanoids in diseases related to oxidative stress, how can KannaLife enhance the efficiency of finding therapeutic and potential preventive uses for natural products?

 

Dr. Kinney: KannaLife has the opportunity to improve upon natural molecules that have shown medically uses, but have limitations related to safety and bioavailability. By applying industry standard medicinal chemistry optimization and assay methods, progress is being made in improving upon nature.  In addition KannaLife has access to one of the most commercially successful natural products scientists and collections in the industry.

 

LPBI: How does the clinical & regulatory experience in the Philadelphia area help a company like Kannalife?

 

Dr. Kinney: Within the region, KannaLife has access to professionals in all areas of drug development either by hiring displaced professionals or partnering with regional contract research organizations.

 

LPBI  You are focusing on an interesting mechanism of action (oxidative stress) and find your direction appealing (find compounds to reverse this, determine relevant disease states {like HCE} then screen these compounds in those disease models {in hippocampal slices}).  As oxidative stress is related to many diseases are you trying to develop your natural products as preventative strategies, even though those type of clinical trials usually require massive numbers of trial participants or are you looking to partner with a larger company to do this?

 

Dr. Kinney: Our strategy is to initially pursue Hepatic Encephalophy (HE) as the lead orphan disease indication and then partner with other organizations to broaden into other areas that would benefit from a neuroprotective agent.  It is expected the HE will be responsive to an acute treatment regimen.   We are pursuing both natural products and new chemical entities for this development path.

 

 

General Questions answered by Thoma Kikis, Founder/CMO of KannaLife Sciences

 

LPBI: How did KannaLife get the patent from the National Institutes of Health?

 

My name is Thoma Kikis I’m the co-founder of KannaLife Sciences. In 2010, my partner Dean Petkanas and I founded KannaLife and we set course applying for the exclusive license of the ‘507 patent held by the US Government Health and Human Services and National Institutes of Health (NIH). We spent close to 2 years working on acquiring an exclusive license from NIH to commercially develop Patent 6,630,507 “Cannabinoids as Antioxidants and Neuroprotectants.” In 2012, we were granted exclusivity from NIH to develop a treatment for a disease called Hepatic Encephalopathy (HE), a brain liver disease that stems from cirrhosis.

 

Cannabinoids are the chemicals that compose the Cannabis plant. There are over 85 known isolated Cannabinoids in Cannabis. The cannabis plant is a repository for chemicals, there are over 400 chemicals in the entire plant. We are currently working on non-psychoactive cannabinoids, cannabidiol being at the forefront.

 

As we started our work on HE and saw promising results in the area of neuroprotection we sought out another license from the NIH on the same patent to treat CTE (Chronic Traumatic Encephalopathy), in August of 2014 we were granted the additional license. CTE is a concussion related traumatic brain disease with long term effects mostly suffered by contact sports players including football, hockey, soccer, lacrosse, boxing and active military soldiers.

 

To date we are the only license holders of the US Government held patent on cannabinoids.

 

 

LPBI: How long has this project been going on?

 

We have been working on the overall project since 2010. We first started work on early research for CTE in early-2013.

 

 

LPBI: Tell me about the project. What are the goals?

 

Our focus has always been on treating diseases that effect the Brain. Currently we are looking for solutions in therapeutic agents designed to reduce oxidative stress, and act as immuno-modulators and neuroprotectants.

 

KannaLife has an overall commitment to discover and understand new phytochemicals. This diversification of scientific and commercial interests strongly indicates a balanced and thoughtful approach to our goals of providing standardized, safer and more effective medicines in a socially responsible way.

 

Currently our research has focused on the non-psychoactive cannabidiol (CBD). Exploring the appropriate uses and limitations and improving its safety and Metered Dosing. CBD has a limited therapeutic window and poor bioavailability upon oral dosing, making delivery of a consistent therapeutic dose challenging. We are also developing new CBD-like molecules to overcome these limitations and evaluating new phytochemicals from non-regulated plants.

 

KannaLife’s research is led by experienced pharmaceutically trained professionals; Our Scientific team out of the Pennsylvania Biotechnology Center is led by Dr. William Kinney and Dr. Douglas Brenneman both with decades of experience in pharmaceutical R&D.

 

 

LPBI: How do cannabinoids help neurological damage? -What sort of neurological damage do they help?

 

Cannabinoids and specifically cannabidiol work to relieve oxidative stress, and act as immuno-modulators and neuroprotectants.

 

So far our pre-clinical results show that cannabidiol is a good candidate as a neuroprotectant as the patent attests to. Our current studies have been to protect neuronal cells from toxicity. For HE we have been looking specifically at ammonia and ethanol toxicity.

 

 

– How did it go from treating general neurological damage to treating CTE? Is there any proof yet that cannabinoids can help prevent CTE? What proof?

 

We started examining toxicity first with ammonia and ethanol in HE and then posed the question; If CBD is a neuroprotectant against toxicity then we need to examine what it can do for other toxins. We looked at CTE and the toxin that causes it, tau. We just acquired the license in August from the NIH for CTE and are beginning our pre-clinical work in the area of CTE now with Dr. Ron Tuma and Dr. Sara Jane Ward at Temple University in Philadelphia.

 

 

LPBI: How long until a treatment could be ready? What’s the timeline?

 

We will have research findings in the coming year. We plan on filing an IND (Investigational New Drug application) with the FDA for CBD and our molecules in 2015 for HE and file for CTE once our studies are done.

 

 

LPBI: What other groups are you working with regarding CTE?

 

We are getting good support from former NFL players who want solutions to the problem of concussions and CTE. This is a very frightening topic for many players, especially with the controversy and lawsuits surrounding it. I have personally spoken to several former NFL players, some who have CTE and many are frightened at what the future holds.

 

We enrolled a former player, Marvin Washington. Marvin was an 11 year NFL vet with NY Jets, SF 49ers and won a SuperBowl on the 1998 Denver Broncos. He has been leading the charge on KannaLife’s behalf to raise awareness to the potential solution for CTE.

 

We tried approaching the NFL in 2013 but they didn’t want to meet. I can understand that they don’t want to take a position. But ultimately, they’re going to have to make a decision and look into different research to treat concussions. They have already given the NIH $30 Million for research into football related injuries and we hold a license with the NIH, so we wanted to have a discussion. But currently cannabinoids are part of their substance abuse policy connected to marijuana. Our message to the NFL is that they need to lead the science, not follow it.

 

Can you imagine the NFL’s stance on marijuana treating concussions and CTE? These are topics they don’t want to touch but will have to at some point.

 

LPBI: Thank you both Dr. Kinney and Mr. Kikis.

 

Please look for future posts in this series on the Philly Biotech Scene on this site

Also, if you would like your Philadelphia biotech startup to be highlighted in this series please contact me or

http://pharmaceuticalintelligence.com at:

sjwilliamspa@comcast.net or @StephenJWillia2  or @pharma_BI.

Our site is read by ~ thousand international readers DAILY and thousands of Twitter followers including venture capital.

 

Other posts on this site in this VIBRANT PHILLY BIOTECH SCENE SERIES OR referring to PHILADELPHIA BIOTECH include:

The Vibrant Philly Biotech Scene: Focus on Computer-Aided Drug Design and Gfree Bio, LLC

RAbD Biotech Presents at 1st Pitch Life Sciences-Philadelphia

The Vibrant Philly Biotech Scene: Focus on Vaccines and Philimmune, LLC

What VCs Think about Your Pitch? Panel Summary of 1st Pitch Life Science Philly

1st Pitch Life Science- Philadelphia- What VCs Really Think of your Pitch

LytPhage Presents at 1st Pitch Life Sciences-Philadelphia

Hastke Inc. Presents at 1st Pitch Life Sciences-Philadelphia

PCCI’s 7th Annual Roundtable “Crowdfunding for Life Sciences: A Bridge Over Troubled Waters?” May 12 2014 Embassy Suites Hotel, Chesterbrook PA 6:00-9:30 PM

Pfizer Cambridge Collaborative Innovation Events: ‘The Role of Innovation Districts in Metropolitan Areas to Drive the Global an | Basecamp Business

Mapping the Universe of Pharmaceutical Business Intelligence: The Model developed by LPBI and the Model of Best Practices LLC

 

 

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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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A Future for Plasma Metabolomics in Cardiovascular Disease Assessment

Curator: Larry H Bernstein, MD, FCAP

 

 

Plasma metabolomics reveals a potential panel of biomarkers for early diagnosis
in acute coronary syndrome  

CM. Laborde, L Mourino-Alvarez, M Posada-Ayala,
G Alvarez-Llamas, MG Serranillos-Reus, et al.
Metabolomics – manuscript draft

In this study, analyses of peripheral plasma from Non-ST Segment Elevation
Acute Coronary Syndrome patients and healthy controls by gas chromatography-
mass spectrometry permitted the identification of 15 metabolites with statistical
differences (p<0.05) between experimental groups.
In our study, 6 amino acids were found decreased in NSTEACS patients when
compared with healthy control group suggesting either a decrease in anabolic
activity of these metabolites or an increase in the catabolic pathways. Of both
possibilities, the increased catabolism of the amino acids can be explained
considering simultaneously the capacity of glycogenic and ketogenic amino
acids along with the gradual hypoxic condition to which cardiac muscle cells
have been exposed.

Additionally, validation by gas chromatography-mass spectrometry and liquid
chromatography-mass spectrometry permitted us to identify a potential panel
of biomarkers formed by 5-OH tryptophan, 2-OH-butyric acid and 3-OH-butyric
acid. Oxidative stress conditions dramatically increase the rate of hepatic
synthesis of glutathione. It is synthesized from the amino acids cysteine, glutamic
acid and glycine. Under these conditions of metabolic stress, the supply of cysteine
for glutathione synthesis become limiting and homocysteine is used to form
cystathionine, which is cleaved to cysteine and 2-OH-butyric acid. Thus elevated
plasma levels of 2-OH-butyric acid can be a good biomarker of cellular oxidative
stress for the early diagnosis of ACS.  Another altered metabolite of similar
structure was 3-OH-butyric acid, a ketone body together with the acetoacetate,
and acetone. Elevated levels of ketone bodies in blood and urine mainly occur
in diabetic ketoacidosis. Type 1 diabetes mellitus (DMI) patients have decreased
levels of insulin in the blood that prevent glucose enter cells so these cells use
the catabolism of fats as energy source that produce ketones as final products.
This panel of biomarkers reflects the oxidative stress and the hypoxic state that
disrupts the myocardial cells and consequently constitutes a metabolomic
signature that could be used for early diagnosis of acute coronary syndrome.
We hypothesize that the hypoxia situation comes to “mimic” the physiological
situation that occurs in DMI. In this case, the low energy yield of glucose
metabolism “forces” these cells to use fat as energy source (through catabolism
independent of aerobic/anaerobic conditions) occurring ketones as final
products. In our experiment, the 3-OH-butyric acid was strongly elevated in
NSTEACS patients.

 

Current Methods Used in the Protein Carbonyl Assay
Nicoleta Carmen Purdel, Denisa Margina and Mihaela Ilie.
Ann Res & Rev in Biol 2014; 4(12): 2015-2026.
http://www.sciencedomain.org/download.php?f=Purdel4122013ARRB8763-1

The attack of reactive oxygen species on proteins and theformation of
protein carbonyls were investigated only in the recent years. Taking into
account that protein carbonyls may play an important role in the early
diagnosis of pathologies associated with reactive oxygen species
overproduction, a robust and reliable method to quantify the protein
carbonyls in complex biological samples is also required. Oxidative
stress represents the aggression produced at the molecular level by
the imbalance between pro-oxidant and antioxidant agents, in favor of
pro-oxidants, with severe functional consequences in all organs and
tissues. An overproduction of ROS results in oxidative damages
especially to proteins (the main target of ROS), as well as in lipids,or
DNA. Glycation and oxidative stress are closely linked, and both
phenomena are referred to as ‘‘glycoxidation’’. All steps of glycoxidation
generate oxygen-free radical production, some of them being common
with lipidic peroxidation pathways.
The initial glycation reaction is followed by a cascade of chemical
reactions resulting in the formation of intermediate products (Schiff base,
Amadori and Maillard products) and finally to a variety of derivatives
named advanced glycation end products (AGEs). In hyperglycemic
environments and in natural aging, AGEs are generated in increased
concentrations; their levels can be evaluated in plasma due to the fact
that they are fluorescent compounds. Specific biomarkers of oxidative
stress are currently investigated in order to evaluate the oxidative status
of a biological system and/or its regenerative power. Generaly, malondi-
aldehyde, 4-hydroxy-nonenal (known together as thiobarbituric acid
reactive substances – TBARS), 2-propenal and F2-isoprostanes are
investigated as markers of lipid peroxidation, while the measurement
of protein thiols, as well as S-glutathionylated protein are assessed
as markers of oxidative damage of proteins. In most cases, the
oxidative damage of the DNA has 8-hydroxy-2l-deoxyguanosine
(8-OHdG) as a marker.  The oxidative degradation of proteins plays an
important role in the early diagnosis of pathologies associated with
ROS overproduction. Oxidative modification of the protein structure
may take a variety of forms, including the nitration of tyrosine residues,
carbonylation, oxidation of methionine, or thiol groups, etc.

The carbonylation of protein represents the introduction of carbonyl
groups (aldehyde or ketone) in the protein structure, through several
mechanisms: by direct oxidation of the residues of lysine, arginine,
proline and threonine residues from the protein chain, by interaction
with lipid peroxidation products with aldehyde groups (such as 4-
hydroxy-2-nonenal, malondialdehyde, 2-propenal), or by the
interaction with the compounds with the carbonyl groups resulting
from the degradation of the lipid or glycoxidation. All of these
molecular changes occur under oxidative stress conditions.
There is a pattern of carbonylation, meaning that only certain
proteins can undergo this process and protein structure determines
the preferential sites of carbonylation. The most investigated
carbonyl derivates are represented by gamma-glutamic
semialdehyde (GGS) generated from the degradation of arginine
residue and α-aminoadipic semialdehyde (AAS) derived from lysine.

A number of studies have shown that the generation of protein
carbonyl groups is associated with normal cellular phenomena like
apoptosis, and cell differentiation and is dependent on age, species
and habits (eg. smoking) or severe conditions’ exposure (as
starvation or stress). The formation and accumulation of protein
carbonyls is increased in various human diseases, including –
diabetes and cardiovascular disease.

Recently, Nystrom [7] suggested that the carbonylation process
is associated with the physiological and not to the chronological
age of the organism and the carbonylation may be one of the causes
of aging and cell senescence; therefore it can be used as the marker
of these processes. Jha and Rizvi, [15] proposed the quantification of
protein carbonyls in the erythrocyte membrane as a biomarker of aging

PanelomiX: A threshold-based algorithm to create panels of
biomarkers

X Robin, N Turck, A Hainard, N Tiberti, F Lisacek. 
T r a n s l a t i o n a l  P r o t e o m i c s   2 0 1 3; 1: 57–64.
http://dx.doi.org/10.1016/j.trprot.2013.04.003

The computational toolbox we present here – PanelomiX – uses
the iterative combination of biomarkers and thresholds (ICBT) method.
This method combines biomarkers andclinical scores by selecting
thresholds that provide optimal classification performance. Tospeed
up the calculation for a large number of biomarkers, PanelomiX selects
a subset ofthresholds and parameters based on the random forest method.
The panels’ robustness and performance are analysed by cross-validation
(CV) and receiver operating characteristic(ROC) analysis.

Using 8 biomarkers, we compared this method against classic
combination procedures inthe determination of outcome for 113 patients
with an aneurysmal subarachnoid hemorrhage. The panel classified the
patients better than the best single biomarker (< 0.005) and compared
favourably with other off-the-shelf classification methods.

In conclusion, the PanelomiX toolbox combines biomarkers and evaluates
the performance of panels to classify patients better than single markers
or other classifiers. The ICBT algorithm proved to be an efficient classifier,
the results of which can easily be interpreted. 

Multiparametric diagnostics of cardiomyopathies by microRNA
signatures.
CS. Siegismund, M Rohde, U Kühl,  D  Lassner.
Microchim Acta 2014 Mar.
http://dx.doi.org:/10.1007/s00604-014-1249-y

MicroRNAs (miRNAs) represent a new group of stable biomarkers
that are detectable both in tissue and body fluids. Such miRNAs
may serve as cardiological biomarkers to characterize inflammatory
processes and to differentiate various forms of infection. The predictive
power of single miRNAs for diagnosis of complex diseases may be further
increased if several distinctly deregulated candidates are combined to
form a specific miRNA signature. Diagnostic systems that generate
disease related miRNA profiles are based on microarrays, bead-based
oligo sorbent assays, or on assays based on real-time polymerase
chain reactions and placed on microfluidic cards or nanowell plates.
Multiparametric diagnostic systems that can measure differentially
expressed miRNAs may become the diagnostic tool of the future due
to their predictive value with respect to clinical course, therapeutic
decisions, and therapy monitoring.

Nutritional lipidomics: Molecular metabolism, analytics, and
diagnostics
JT. Smilowitz, AM. Zivkovic, Yu-Jui Y Wan, SM. Watkins, et al.
Mol. Nutr. Food Res2013, 00, 1–17.
http://dx.doi.org:/10.1002/mnfr.201200808

The term lipidomics is quite new, first appearing in 2001. Its definition
is still being debated, from “the comprehensive analysis of all lipid
components in a biological sample” to “the full characterization of
lipid molecular species and their biological roles with respect to the
genes that encode proteins that regulate lipid metabolism”. In principle,
lipidomics is a field taking advantage of the innovations in the separation
sciences and MS together with bioinformatics to characterize the lipid
compositions of biological samples (biofluids, cells, tissues, organisms)
compositionally and quantitatively.

Biochemical pathways of lipid metabolism remain incomplete and the
tools to map lipid compositional data to pathways are still being assembled.
Biology itself is dauntingly complex and simply separating biological
structures remains a key challenge to lipidomics. Nonetheless, the
strategy of combining tandem analytical methods to perform the sensitive,
high-throughput, quantitative, and comprehensive analysis of lipid
metabolites of very large numbers of molecules is poised to drive
the field forward rapidly. Among the next steps for nutrition to understand
the changes in structures, compositions, and function of lipid biomolecules
in response to diet is to describe their distribution within discrete functional
compartments lipoproteins. Additionally, lipidomics must tackle the task
of assigning the functions of lipids as signaling molecules, nutrient sensors,
and intermediates of metabolic pathways.

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Larry H Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2014/06/22/Proteomics – The Pathway to Understanding and Decision-making in Medicine

This dialogue is a series of discussions introducing several perspective on proteomics discovery, an emerging scientific enterprise in the -OMICS- family of disciplines that aim to clarify many of the challenges toward the understanding of disease and aiding in the diagnosis as well as guiding treatment decisions. Beyond that focus, it will contribute to personalized medical treatment in facilitating the identification of treatment targets for the pharmaceutical industry. Despite enormous advances in genomics research over the last two decades, there is a still a problem in reaching anticipated goals for introducing new targeted treatments that has seen repeated failures in stage III of clinical trials, and even when success has been achieved, it is temporal.  The other problem has been toxicity of agents widely used in chemotherapy.  Even though the genomic approach brings relieve to the issues of toxicity found in organic chemistry derivative blocking reactions, the specificity for the target cell without an effect on normal cells has been elusive.

This is not confined to cancer chemotherapy, but can also be seen in pain medication, and has been a growing problem in antimicrobial therapy.  The stumbling block has been inability to manage a multiplicity of reactions that also have to be modulated in a changing environment based on 3-dimension structure of proteins, pH changes, ionic balance, micro- and macrovascular circulation, and protein-protein and protein- membrane interactions. There is reason to consider that the present problems can be overcome through a much better modification of target cellular metabolism as we peel away the confounding and blinding factors with a multivariable control of these imbalances, like removing the skin of an onion.

This is the first of a series of articles, and for convenience we shall here  only emphasize the progress of application of proteomics to cardiovascular disease.

growth in funding proteomics 1990-2010

growth in funding proteomics 1990-2010

Part I.

Panomics: Decoding Biological Networks  (Clinical OMICs 2014; 5)

Technological advances such as high-throughput sequencing are transforming medicine from symptom-based diagnosis and treatment to personalized medicine as scientists employ novel rapid genomic methodologies to gain a broader comprehension of disease and disease progression. As next-generation sequencing becomes more rapid, researchers are turning toward large-scale pan-omics, the collective use of all omics such as genomics, epigenomics, transcriptomics, proteomics, metabolomics, lipidomics and lipoprotein proteomics, to better understand, identify, and treat complex disease.

Genomics has been a cornerstone in understanding disease, and the sequencing of the human genome has led to the identification of numerous disease biomarkers through genome-wide association studies (GWAS). It was the goal of these studies that these biomarkers would serve to predict individual disease risk, enable early detection of disease, help make treatment decisions, and identify new therapeutic targets. In reality, however, only a few have gone on to become established in clinical practice. For example in human GWAS studies for heart failure at least 35 biomarkers have been identified but only natriuretic peptides have moved into clinical practice, where they are limited primarily for use as a diagnostic tool.

Proteomics Advances Will Rival the Genetics Advances of the Last Ten Years

Seventy percent of the decisions made by physicians today are influenced by results of diagnostic tests, according to N. Leigh Anderson, founder of the Plasma Proteome Institute and CEO of SISCAPA Assay Technologies. Imagine the changes that will come about when future diagnostics tests are more accurate, more useful, more economical, and more accessible to healthcare practitioners. For Dr. Anderson, that’s the promise of proteomics, the study of the structure and function of proteins, the principal constituents of the protoplasm of all cells.

In explaining why proteomics is likely to have such a major impact, Dr. Anderson starts with a major difference between the genetic testing common today, and the proteomic testing that is fast coming on the scene. “Most genetic tests are aimed at measuring something that’s constant in a person over his or her entire lifetime. These tests provide information on the probability of something happening, and they can help us understand the basis of various diseases and their potential risks. What’s missing is, a genetic test is not going to tell you what’s happening to you right now.”

Mass Spec-Based Multiplexed Protein Biomarkers

Clinical proteomics applications rely on the translation of targeted protein quantitation technologies and methods to develop robust assays that can guide diagnostic, prognostic, and therapeutic decision-making. The development of a clinical proteomics-based test begins with the discovery of disease-relevant biomarkers, followed by validation of those biomarkers.

“In common practice, the discovery stage is performed on a MS-based platform for global unbiased sampling of the proteome, while biomarker qualification and clinical implementation generally involve the development of an antibody-based protocol, such as the commonly used enzyme linked ELISA assays,” state López et al. in Proteome Science (2012; 10: 35–45). “Although this process is potentially capable of delivering clinically important biomarkers, it is not the most efficient process as the latter is low-throughput, very costly, and time-consuming.”

Part II.  Proteomics for Clinical and Research Use: Combining Protein Chips, 2D Gels and Mass Spectrometry in 

The next Step: Exploring the Proteome: Translation and Beyond

N. Leigh Anderson, Ph.D., Chief Scientific Officer, Large Scale Proteomics Corporation

Three streams of technology will play major roles in quantitative (expression) proteomics over the coming decade. Two-dimensional electrophoresis and mass spectrometry represent well-established methods for, respectively, resolving and characterizing proteins, and both have now been automated to enable the high-throughput generation of data from large numbers of samples.

These methods can be powerfully applied to discover proteins of interest as diagnostics, small molecule therapeutic targets, and protein therapeutics. However, neither offers a simple, rapid, routine way to measure many proteins in common samples like blood or tissue homogenates.

Protein chips do offer this possibility, and thus complete the triumvirate of technologies that will deliver the benefits of proteomics to both research and clinical users. Integration of efforts in all three approaches are discussed, highlighting the application of the Human Protein Index® database as a source of protein leads.

leighAnderson

leighAnderson

N. Leigh Anderson, Ph D. is Chief Scientific Officer of the Proteomics subsidiary of Large Scale Biology Corporation (LSBC).
Dr. Anderson obtained his B.A. in Physics with honors from Yale and a Ph.D. in Molecular Biology from Cambridge University
(England) where he worked with M. F. Perutz as a Churchill Fellow at the MRC Laboratory of Molecular Biology. Subsequently
he co-founded the Molecular Anatomy Program at the Argonne National Laboratory (Chicago) where his work in the development
of 2D electrophoresis and molecular database technology earned him, among other distinctions, the American Association for
Clinical Chemistry’s Young Investigator Award for 1982, the 1983 Pittsburgh Analytical Chemistry Award, 2008 AACC Outstanding
Research Award, and 2013 National Science Medal..

In 1985 Dr. Anderson co-founded LSBC in order to pursue commercial development and large scale applications of 2-D electro-
phoretic protein mapping technology. This effort has resulted in a large-scale proteomics analytical facility supporting research
work for LSBC and its pharmaceutical industry partners. Dr. Anderson’s current primary interests are in the automation of proteomics
technologies, and the expansion of LSBC’s proteomics databases describing drug effects and disease processes in vivo and in vitro.
Large Scale Biology went public in August 2000.

Part II. Plasma Proteomics: Lessons in Biomarkers and Diagnostics

Exposome Workshop
N Leigh Anderson
Washington 8 Dec 2011

QUESTIONS AND LESSONS:

CLINICAL DIAGNOSTICS AS A MODEL FOR EXPOSOME INDICATORS
TECHNOLOGY OPTIONS FOR MEASURING PROTEIN RESPONSES TO EXPOSURES
SCALE OF THE PROBLEM: EXPOSURE SIGNALS VS POPULATION NOISE

The Clinical Plasma Proteome
• Plasma and serum are the dominant non-invasive clinical sample types
– standard materials for in vitro diagnostics (IVD)
• Proteins measured in clinically-available tests in the US
– 109 proteins via FDA-cleared or approved tests
• Clinical test costs range from $9 (albumin) to $122 (Her2)
• 90% of those ever approved are still in use
– 96 additional proteins via laboratory-developed tests (not FDA
cleared or approved)
– Total 205 proteins (≅ products of 211genes, excluding Ig’s)
• Clinically applied proteins thus account for
– About 1% of the baseline human proteome (1 gene :1 protein)
– About 10% of the 2,000+ proteins observed in deep discovery
plasma proteome datasets

“New” Protein Diagnostics Are FDA-Cleared at a Rate of ~1.5/yr:
Insufficient to Meet Dx or Rx Development Needs

FDA clearance of protein diagnostics

FDA clearance of protein diagnostics

A  Major Technology Gulf Exists Between Discovery

Proteomics and Routine Diagnostic Platforms

Two Streams of Proteomics
A.  Problem Technology
Basic biology: maximum proteome coverage (including PTM’s, splices) to
provide unbiased discovery of mechanistic information
• Critical: Depth and breadth
• Not critical: Cost, throughput, quant precision

B.  Discovery proteomics
Specialized proteomics field,
large groups,
complex workflows and informatics

Part III.  Addressing the Clinical Proteome with Mass Spectrometric Assays

N. Leigh Anderson, PhD, SISCAPA Assay Technologies, Inc.

protein changes in biological mechanisms

protein changes in biological mechanisms

No Increase in FDA Cleared Protein Tests in 20 yr

“New” Protein Tests in Plasma Are FDA-Cleared at a Rate of ~1.5/yr:
Insufficient to Meet Dx or Rx Development Needs

See figure above

An Explanation: the Biomarker Pipeline is Blocked at the Verification Step

Immunoassay Weaknesses Impact Biomarker Verification

1) Specificity: what actually forms the immunoassay sandwich – or prevents its
formation – is not directly visualized

2) Cost: an assay developed to FDA approvable quality costs $2-5M per
protein

Major_Plasma_Proteins

Major_Plasma_Proteins

Immunoassay vs Hybrid MS-based assays

Immunoassay vs Hybrid MS-based assays

MASS SPECTROMETRY: MRM’s provide what is missing in..IMMUNOASSAYS:

– SPECIFICITY
– INTERNAL STANDARDIZATION
– MULTIPLEXING
– RAPID CONFIGURATION PROVIDED A PROTEIN CAN ACT LIKE A SMALL
MOLECULE

MRM of Proteotypic Tryptic Peptides Provides Highly Specific Assays for Proteins > 1ug/ml in Plasma

Peptide-Level MS Provides High Structural Specificity
Multiple Reaction Monitoring (MRM) Quantitation

ADDRESSING MRM LIMITATIONS VIA SPECIFIC ENRICHMENT OF ANALYTE  PEPTIDES: SISCAPA

– SENSITIVITY
– THROUGHPUT (LC-MS/MS CYCLE TIME)

SISCAPA combines best features of immuno and MS

SISCAPA combines best features of immuno and MS

SISCAPA Process Schematic Diagram
Stable Isotope-labeled Standards with Capture on Anti-Peptide Antibodies

An automated process for SISCAPA targeted protein quantitation utilizes high affinity capture antibodies that are immobilized on magnetic beads

An automated process for SISCAPA targeted protein quantitation utilizes high affinity capture antibodies that are immobilized on magnetic beads

Antibodies sequence specific peptide binding

Antibodies sequence specific peptide binding

SISCAP target enrichmant

SISCAP target enrichmant

Multiple reaction monitoring (MRM) quantitation

Multiple reaction monitoring (MRM) quantitation

protein-quantitation-via-signature-peptides.png

protein-quantitation-via-signature-peptides.png

First SISCAP Assay - thyroglobulin

First SISCAP Assay – thyroglobulin

personalized reference range within population range

Glycemic control in DM

Glycemic control in DM

Part IV. National Heart, Lung, and Blood Institute Clinical

Proteomics Working Group Report
Christopher B. Granger, MD; Jennifer E. Van Eyk, PhD; Stephen C. Mockrin, PhD;
N. Leigh Anderson, PhD; on behalf of the Working Group Members*
Circulation. 2004;109:1697-1703 doi: 10.1161/01.CIR.0000121563.47232.2A
http://circ.ahajournals.org/content/109/14/1697

Abstract—The National Heart, Lung, and Blood Institute (NHLBI) Clinical Proteomics Working Group
was charged with identifying opportunities and challenges in clinical proteomics and using these as a
basis for recommendations aimed at directly improving patient care. The group included representatives
of clinical and translational research, proteomic technologies, laboratory medicine, bioinformatics, and
2 of the NHLBI Proteomics Centers, which form part of a program focused on innovative technology development.

This report represents the results from a one-and-a-half-day meeting on May 8 and 9, 2003. For the purposes
of this report, clinical proteomics is defined as the systematic, comprehensive, large-scale identification of
protein patterns (“fingerprints”) of disease and the application of this knowledge to improve patient care
and public health through better assessment of disease susceptibility, prevention of disease, selection of
therapy for the individual, and monitoring of treatment response. (Circulation. 2004;109:1697-1703.)
Key Words: proteins diagnosis prognosis genetics plasma

Part V.  Overview: The Maturing of Proteomics in Cardiovascular Research

Jennifer E. Van Eyk
Circ Res. 2011;108:490-498  doi: 10.1161/CIRCRESAHA.110.226894
http://circres.ahajournals.org/content/108/4/490

Abstract: Proteomic technologies are used to study the complexity of proteins, their roles, and biological functions.
It is based on the premise that the diversity of proteins, comprising their isoforms, and posttranslational modifications
(PTMs) underlies biology.

Based on an annotated human cardiac protein database, 62% have at least one PTM (phosphorylation currently dominating),
whereas 25% have more than one type of modification.

The field of proteomics strives to observe and quantify this protein diversity. It represents a broad group of technologies
and methods arising from analytic protein biochemistry, analytic separation, mass spectrometry, and bioinformatics.
Since the 1990s, the application of proteomic analysis has been increasingly used in cardiovascular research.

prevalence-of-cardiovascular-diseases-in-adults-by-age-and-sex-u-s-2007-2010.

prevalence-of-cardiovascular-diseases-in-adults-by-age-and-sex-u-s-2007-2010.

Technology development and adaptation have been at the heart of this progress. Technology undergoes a maturation,

becoming routine and ultimately obsolete, being replaced by newer methods. Because of extensive methodological
improvements, many proteomic studies today observe 1000 to 5000 proteins.

Only 5 years ago, this was not feasible. Even so, there are still road blocks. Nowadays, there is a focus on obtaining
better characterization of protein isoforms and specific PTMs. Consequentl, new techniques for identification and
quantification of modified amino acid residues are required, as is the assessment of single-nucleotide polymorphisms
in addition to determination of the structural and functional consequences.

In this series, 4 articles provide concrete examples of how proteomics can be incorporated into cardiovascular
research and address specific biological questions. They also illustrate how novel discoveries can be made and
how proteomic technology has continued to evolve. (Circ Res. 2011;108:490-498.)
Key Words: proteomics technology protein isoform posttranslational modification polymorphism

Part VI.   The -omics era: Proteomics and lipidomics in vascular research

Athanasios Didangelos, Christin Stegemann, Manuel Mayr∗

King’s British Heart Foundation Centre, King’s College London, UK

Atherosclerosis 2012; 221: 12– 17     http://dx.doi.org/10.1016/j.atherosclerosis.2011.09.043

a b s t r a c t

A main limitation of the current approaches to atherosclerosis research is the focus on the investigation of individual
factors, which are presumed to be involved in the pathophysiology and whose biological functions are, at least in part, understood.

These molecules are investigated extensively while others are not studied at all. In comparison to our detailed
knowledge about the role of inflammation in atherosclerosis, little is known about extracellular matrix remodelling
and the retention of individual lipid species rather than lipid classes in early and advanced atherosclerotic lesions.

The recent development of mass spectrometry-based methods and advanced analytical tools are transforming
our ability to profile extracellular proteins and lipid species in animal models and clinical specimen with the goal
of illuminating pathological processes and discovering new biomarkers.

Fig. 1. ECM in atherosclerosis

Fig. 1. ECM in atherosclerosis. The bulk of the vascular ECM is synthesised by smooth muscle cells and composed primarily of collagens, proteoglycans and glycoproteins.During the early stages of atherosclerosis, LDL binds to the proteoglycans of the vessel wall, becomes modified, i.e. by oxidation (ox-LDL), and sustains a proinflammatory cascade that is proatherogenic

Lipidomics of atherosclerotic plaques

Lipidomics of atherosclerotic plaques

Fig. 2. Lipidomics of atherosclerotic plaques. Lipids were separated by ultra performance reverse phase
liquid chromatography on a Waters® ACQUITY UPLC® (HSS T3 Column, 100 mm × 2.1 mm i.d., 1.8 _m
particle size, 55 ◦C, flow rate 400 _L/min, Waters, Milford MA, USA) and analyzed on a quadrupole time-of-flight
mass spectrometer (Waters® SYNAPTTM HDMSTM system) in both positive (A) and negative ion mode (C).
In positive MS mode, lysophosphatidyl-cholines (lPCs) and lysophosphatidylethanolamines (lPEs) eluted first;
followed by phosphatidylcholines (PCs), sphingomyelin (SMs), phosphatidylethanol-amines (PEs) and cholesteryl
esters (CEs); diacylglycerols (DAGs) and triacylglycerols (TAGs) had the longest retention times. In negative MS mode,
fatty acids (FA) were followed by phosphatidyl-glycerols (PGs), phosphatidyl-inositols (PIs), phosphatidylserines (PS)
and PEs. The chromatographic peaks corresponding to the different classes were detected as retention time-mass to
charge ratio (m/z) pairs and their areas were recorded. Principal component analyses on 629 variables from triplicate
analysis (C1, 2, 3 = control 1, 2, 3; P1, 2, 3 = endarterectomy patient 1, 2, 3) demonstrated a clear separation of
atherosclerotic plaques and control radial arteries in positive (B) and negative (D) ion mode. The clustering of the
technical replicates and the central projection of the pooled sample within the scores plot confirm the reproducibility
of the analyses, and the Goodness of Fit test returned a chi-squared of 0.4 and a R-squared value of 0.6.

Challenges in mass spectrometry

Mass spectrometry is an evolving technology and the technological advances facilitate the detection and quantification
of scarce proteins. Nonetheless, the enrichment of specific subproteomes using differential solubilityor isolation of cellular
organelleswill remain important to increase coverage and, at least partially, overcome the inhomogeneity of diseased tissue,
one of the major factors affecting sample-to-sample variation.

Proteomics is also the method of choice for the identification of post-translational modifications, which play an essential
role in protein function, i.e. enzymatic activation, binding ability and formation of ECM structures. Again, efficient enrichment
is essential to increase the likelihood of identifying modified peptides in complex mixtures. Lipidomics faces similar challenges.
While the extraction of lipids is more selective, new enrichment methods are needed for scarce lipids as well as labile lipid
metabolites, that may have important bioactivity. Another pressing issue in lipidomics is data analysis, in particular the lack
of automated search engines that can analyze mass spectra obtained from instruments of different vendors. Efforts to
overcome this issue are currently underway.

Conclusions

Proteomics and lipidomics offer an unbiased platform for the investigation of ECM and lipids within atherosclerosis. In
combination, these innovative technologies will reveal key differences in proteolytic processes responsible for plaque rupture
and advance our understanding of ECM – lipoprotein interactions in atherosclerosis.

references

Virtualization in Proteomics: ‘Sakshat’ in India, at IIT Bombay(tginnovations.wordpress.com)

Proteome Portraits (the-scientist.com)

A Protease for ‘Middle-down’ Proteomics(pharmaceuticalintelligence.com)

Intrinsic Disorder in the Human Spliceosomal Proteome(ploscompbiol.org)

proteome

proteome

active site of eNOS (PDB_1P6L) and nNOS (PDB_1P6H).

active site of eNOS (PDB_1P6L) and nNOS (PDB_1P6H).

Table - metabolic  targets

Table – metabolic targets

HK-II Phosphorylation

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A Tribute to Johannes Everse

Author: Larry H Bernstein, MD, FCAP

 

Johannes Everse was a retired Tenured Professor at Texas Tech University Health Sciences Center in Lubbock, Texas, who dies on June 10, 2013.  He survived the Nazi invasion of Netherlands during World War II, and worked in the pharmaceutical industry after finishing a unique technical education the surpassed any that existed in United States that included an extensive knowledge of analytical instruments and expertise in organic chemical syntheses.  Given a unique opportunity, he applied for and obtained a position as a technician in the Laboratory of Nathan O Kaplan’s Laboratory at the time of Kaplan’s move from John’s Hopkins University to Brandeis University, where Kaplan with Sidney Colowick established the prestigious Methods in Enzymology series, and in a few short years built a worldclass Graduate Department of Biochemistry.  Kaplan was very sharp in selecting graduate students, postdoctoral students, and at administration, but his ability to recognioze potential talent was seen in his recruitment of Francis Stolzenbach and Johannes Everse.  He also gave considerable support to those who he had confidence in.  Consequently, Everse was able to take exams completing a BS degree, and eventually, the PhD degree at the University of California, San Diego, in the 1970s. When Prof. Kaplan was recruited to the UCSD campus by Martin Kamens, he was also installed in the National Academy of Sciences.

I worked with Jo Everse for several years as postdoctoral biochemist and resident-USPHS Fellow in  Pathology on the mechanism of the malate dehydrogenase (MDH) reaction and the regulatory function of the mitochondrial and cytoplasmic MDHs.   These were important formative years in my scientific training, and it was by no accident that I was sent to work in that laboratory by my previous mentor, a pathologist and biochemist who had worked on adenylate kinases, as I had been attracted to that problem as a medical student working on the ontogeny of the lactic dehydrogenases in the embryonic lens.  Jo Everse was responsible for synthesizing the pyridine nucleotide adducts that proved to be critical to understanding the pyridine nucleotide related dehydrogenase reactions.  Jo was undoubtedly a driving force in that laboratory.

It was at that time that my first daughter was born, and she had the opportunity to play with the Everse children, who as adults are both PhD biochemists.  I have been fortunate to live through a dynamic period in the history of scientific discovery, and most amazingly, at a time of decline in funding for science that has not been deterred since the Vietnam War.  You may consider it the cost of hegemony after the treaty that ended WWII and brought us the cold war.

Jo went on to a tenured faculty position at TTUHSS, and his retirement came shortly before his death at 80. While he stayed longer than his superiors wanted, his welcome was not so warm after he criticized the administration of the graduate program.  Unfortunately, he did not have the kind of backing that a colleague at Berkeley, Howard Schachman, Professor of the Graduate School Division of Biochemistry, Biophysics and Structural Biology, enjoyed.  It should not be a surprise how good health, power and money makes a difference in how it plays out.

Schachman was asked to retire in 2002 having a busy, well-funded study, that involved allostery and precisely – in the structure, function, assembly and interactions of biological macromolecules, with particular emphasis on the regulatory enzyme, aspartate transcarbamylase (ATCase).  The studies challenged earlier studies that designated the complex of ATCase with a bisubstrate ligand as the R state of the enzyme. but changes in the conformation were reinterpreted to be the result of the actual binding event rather than the allosteric transition whereby the enzyme is converted from an inactive, taut (T) state to the activated R conformation and they developed methods for understanding the formation of domains and the effect of deletions of helical regions on stability and the folding and assembly pathways.

Jo Everse came out of the depression in Europe (1931 birth), lived through WWII, and he managed to get a unique technical education that took him to Boston.  He became an excellent teacher.  He had a good marriage and father of two children.  He collected Packard automobiles and rebuilt them.  He also played the organ, and he made and maintained an organ for his home.  He lived a good life.

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


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

Author and Curator: Larry H Bernstein, MD, FCAP

The evolution of progress we have achieved in cancer research, diagnosis, and therapeutics has  originated from an emergence of scientific disciplines and the focus on cancer has been recent. We can imagine this from a historical perspective with respect to two observations. The first is that the oldest concepts of medicine lie with the anatomic dissection of animals and the repeated recurrence of war, pestilence, and plague throughout the middle ages, and including the renaissance.  In the awakening, architecture, arts, music, math, architecture and science that accompanied the invention of printing blossomed, a unique collaboration of individuals working in disparate disciplines occurred, and those who were privileged received an education, which led to exploration, and with it, colonialism.  This also led to the need to increasingly, if not without reprisal, questioning long-held church doctrines.

It was in Vienna that Rokitansky developed the discipline of pathology, and his student Semelweis identified an association between then unknown infection and childbirth fever. The extraordinary accomplishments of John Hunter in anatomy and surgery came during the twelve years war, and his student, Edward Jenner, observed the association between cowpox and smallpox resistance. The development of a nursing profession is associated with the work of Florence Nightengale during the Crimean War (at the same time as Leo Tolstoy). These events preceded the work of Pasteur, Metchnikoff, and Koch in developing a germ theory, although Semelweis had committed suicide by infecting himself with syphilis. The first decade of the Nobel Prize was dominated by discoveries in infectious disease and public health (Ronald Ross, Walter Reed) and we know that the Civil War in America saw an epidemic of Yellow Fever, and the Armed Services Medical Museum was endowed with a large repository of osteomyelitis specimens. We also recall that the Russian physician and playwriter, Anton Checkov, wrote about the conditions in prison camps.

But the pharmacopeia was about to open with the discoveries of insulin, antibiotics, vitamins, thyroid action (Mayo brothers pioneered thyroid surgery in the thyroid iodine-deficient midwest), and pitutitary and sex hormones (isolatation, crystal structure, and synthesis years later), and Karl Landsteiner’s discovery of red cell antigenic groups (but he also pioneered in discoveries in meningitis and poliomyelitis, and conceived of the term hapten) with the introduction of transfusion therapy that would lead to transplantation medicine.  The next phase would be heralded by the discovery of cancer, which was highlighted by the identification of leukemia by Rudolph Virchow, who cautioned about the limitations of microscopy. This period is highlighted by the classic work – “Microbe Hunters”.

John Hunter

John Hunter

Walter Reed

Walter Reed

Robert Koch

Robert Koch

goldberger 1916 Pellagra

goldberger 1916 Pellagra

Louis Pasteur

Louis Pasteur

A multidisciplinary approach has led us to a unique multidisciplinary or systems view of cancer, with different fields of study offering their unique expertise, contributions, and viewpoints on the etiology of cancer.  Diverse fields in immunology, biology, biochemistry, toxicology, molecular biology, virology, mathematics, social activism and policy, and engineering have made such important contributions to our understanding of cancer, that without cooperation among these diverse fields our knowledge of cancer would never had evolved as it has. In a series of posts “Heroes in Medical Research:” the work of researchers are highlighted as examples of how disparate scientific disciplines converged to produce seminal discoveries which propelled the cancer field, although, at the time, they seemed like serendipitous findings.  In the post Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin (Translating Basic Research to the Clinic) discusses the seminal yet serendipitous discoveries by bacteriologist Dr. Barnett Rosenberg, which eventually led to the development of cisplatin, a staple of many chemotherapeutic regimens. Molecular biologist Dr. Robert Ting, working with soon-to-be Nobel Laureate virologist Dr. James Gallo on AIDS research and the associated Karposi’s sarcoma identified one of the first retroviral oncogenes, revolutionizing previous held misconceptions of the origins of cancer (described in Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer).   Located here will be a MONTAGE of PHOTOS of PEOPLE who made seminal discoveries and contributions in every field to cancer   Each of these paths of discovery in cancer research have led to the unique strategies of cancer therapeutics and detection for the purpose of reducing the burden of human cancer.  However, we must recall that this work has come at great cost, while it is indeed cause for celebration. The current failure rate of clinical trials at over 70 percent, has been a cause for disappointment, and has led to serious reconsideration of how we can proceed with greater success. The result of the evolution of the cancer field is evident in the many parts and chapters of this ebook.  Volume 4 contains chapters that are in a predetermined order:

  1. The concepts of neoplasm, malignancy, carcinogenesis,  and metastatic potential, which encompass:

(a)     How cancer cells bathed in an oxygen rich environment rely on anaerobic glycolysis for energy, and the secondary consequences of cachexia and sarcopenia associated with progression

invasion

invasion

ARTS protein and cancer

ARTS protein and cancer

Glycolysis

Glycolysis

Krebs cycle

Krebs cycle

Metabolic control analysis of respiration in human cancer tissue

Metabolic control analysis of respiration in human cancer tissue

akip1-expression-modulates-mitochondrial-function

akip1-expression-modulates-mitochondrial-function

(b)     How advances in genetic analysis, molecular and cellular biology, metabolomics have expanded our basic knowledge of the mechanisms which are involved in cellular transformation to the cancerous state.

nucleotides

nucleotides

Methylation of adenine

Methylation of adenine

ampk-and-ampk-related-kinase-ark-family-

ampk-and-ampk-related-kinase-ark-family-

ubiquitylation

ubiquitylation

(c)  How molecular techniques continue to advance our understanding  of how genetics, epigenetics, and alterations in cellular metabolism contribute to cancer and afford new pathways for therapeutic intervention.

 genomic effects

genomic effects

LKB1AMPK pathway

LKB1AMPK pathway

mutation-frequencies-across-12-cancer-types

mutation-frequencies-across-12-cancer-types

AMPK-activating drugs metformin or phenformin might provide protection against cancer

AMPK-activating drugs metformin or phenformin might provide protection against cancer

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

2. The distinct features of cancers of specific tissue sites of origin

3.  The diagnosis of cancer by

(a)     Clinical presentation

(b)     Age of onset and stage of life

(c)     Biomarker features

hairy cell leukemia

hairy cell leukemia

lymphoma leukemia

lymphoma leukemia

(d)     Radiological and ultrasound imaging

  1. Treatments
  2. Prognostic differences within and between cancer types

We have introduced the emergence of a disease of great complexity that has been clouded in more questions than answers until the emergence of molecular biology in the mid 20th century, and then had to await further discoveries going into the 21st century.  What gave the research impetus was the revelation of

1     the mechanism of transcription of the DNA into amino acid sequences

Proteins in Disease

Proteins in Disease

2     the identification of stresses imposed on cellular function

NO beneficial effects

NO beneficial effects

3     the elucidation of the substructure of the cell – cell membrane, mitochondria, ribosomes, lysosomes – and their functions, respectively

pone.0080815.g006  AKIP1 Expression Modulates Mitochondrial Function

AKIP1 Expression Modulates Mitochondrial Function

4     the elucidation of oligonucleotide sequences

nucleotides

nucleotides

dna-replication-unwinding

dna-replication-unwinding

dna-replication-ligation

dna-replication-ligation

dna-replication-primer-removal

dna-replication-primer-removal

dna-replication-leading-strand

dna-replication-leading-strand

dna-replication-lagging-strand

dna-replication-lagging-strand

dna-replication-primer-synthesis

dna-replication-primer-synthesis

dna-replication-termination

dna-replication-termination

5     the further elucidation of functionally relevant noncoding lncDNA

lncRNA-s   A summary of the various functions described for lncRNA

6     the technology to synthesis mRNA and siRNA sequences

RNAi_Q4 Primary research objectives

Figure. RNAi and gene silencing

7     the repeated discovery of isoforms of critical enzymes and their pleiotropic properties

8.     the regulatory pathways involved in signaling

signaling pathjways map

Figure. Signaling Pathways Map

This is a brief outline of the modern progression of advances in our understanding of cancer.  Let us go back to the beginning and check out a sequence of  Nobel Prizes awarded and related discoveries that have a historical relationship to what we know.  The first discovery was the finding by Louis Pasteur that fungi that grew in an oxygen poor environment did not put down filaments.  They did not utilize oxygen and they produced used energy by fermentation.  This was the basis for Otto Warburg sixty years later to make the comparison to cancer cells that grew in the presence of oxygen, but relied on anaerobic glycolysis. He used a manometer to measure respiration in tissue one cell layer thick to measure CO2 production in an adiabatic system.

video width=”1280″ height=”720″ caption=”1741-7007-11-65-s1 Macromolecular juggling by ubiquitylation enzymes.” mp4=”https://pharmaceuticalintelligence.files.wordpress.com/2014/04/1741-7007-11-65-s1-macromolecular-juggling-by-ubiquitylation-enzymes.mp4“][/video]

An Introduction to the Warburg Apparatus

http://www.youtube.com/watch?v=M-HYbZwN43o

Lavoisier Antoine-Laurent and Laplace Pierre-Simon (1783) Memoir on heat. Mémoirs de l’Académie des sciences. Translated by Guerlac H, Neale Watson Academic Publications, New York, 1982.

Instrumental background 200 years later:   Gnaiger E (1983) The twin-flow microrespirometer and simultaneous calorimetry. In Gnaiger E, Forstner H, eds. Polarographic Oxygen Sensors. Springer, Heidelberg, Berlin, New York: 134-166.

otto_heinrich_warburg

otto_heinrich_warburg

Warburg apparatus

The Warburg apparatus is a manometric respirometer which was used for decades in biochemistry for measuring oxygen consumption of tissue homogenates or tissue slices.

The Warburg apparatus has its name from the German biochemist Otto Heinrich Warburg (1883-1970) who was awarded the Nobel Prize in physiology or medicine in 1931 for his “discovery of the nature and mode of action of the respiratory enzyme” [1].

The aqueous phase is vigorously shaken to equilibrate with a gas phase, from which oxygen is consumed while the evolved carbon dioxide is trapped, such that the pressure in the constant-volume gas phase drops proportional to oxygen consumption. The Warburg apparatus was introduced to study cell respiration, i.e. the uptake of molecular oxygen and the production of carbon dioxide by cells or tissues. Its applications were extended to the study of fermentation, when gas exchange takes place in the absence of oxygen. Thus the Warburg apparatus became established as an instrument for both aerobic and anaerobic biochemical studies [2, 3].

The respiration chamber was a detachable glass flask (F) equipped with one or more sidearms (S) for additions of chemicals and an open connection to a manometer (M; pressure gauge). A constant temperature was provided by immersion of the Warburg chamber in a constant temperature water bath. At thermal mass transfer equilibrium, an initial reading is obtained on the manometer, and the volume of gas produced or absorbed is determined at specific time intervals. A limited number of ‘titrations’ can be performed by adding the liquid contained in a side arm into the main reaction chamber. A Warburg apparatus may be equipped with more than 10 respiration chambers shaking in a common water bath.   Since temperature has to be controlled very precisely in a manometric approach, the early studies on mammalian tissue respiration were generally carried out at a physiological temperature of 37 °C.

The Warburg apparatus has been replaced by polarographic instruments introduced by Britton Chance in the 1950s. Since Chance and Williams (1955) measured respiration of isolated mitochondria simultaneously with the spectrophotometric determination of cytochrome redox states, a water chacket could not be used, and measurements were carried out at room temperature (or 25 °C). Thus most later studies on isolated mitochondria were shifted to the artifical temperature of 25 °C.

Today, the importance of investigating mitochondrial performance at in vivo temperatures is recognized again in mitochondrial physiology.  Incubation times of 1 hour were typical in experiments with the Warburg apparatus, but were reduced to a few or up to 20 min, following Chance and Williams, due to rapid oxygen depletion in closed, aqueous phase oxygraphs with high sample concentrations.  Today, incubation times of 1 hour are typical again in high-resolution respirometry, with low sample concentrations and the option of reoxygenations.

“The Nobel Prize in Physiology or Medicine 1931”. Nobelprize.org. 27 Dec 2011 www.nobelprize.org/nobel_prizes/medicine/laureates/1931/

  1. Oesper P (1964) The history of the Warburg apparatus: Some reminiscences on its use. J Chem Educ 41: 294.
  2. Koppenol WH, Bounds PL, Dang CV (2011) Otto Warburg’s contributions to current concepts of cancer metabolism. Nature Reviews Cancer 11: 325-337.
  3. Gnaiger E, Kemp RB (1990) Anaerobic metabolism in aerobic mammalian cells: information from the ratio of calorimetric heat flux and respirometric oxygen flux. Biochim Biophys Acta 1016: 328-332. – “At high fructose concen­trations, respiration is inhibited while glycolytic end products accumulate, a phenomenon known as the Crabtree effect. It is commonly believed that this effect is restric­ted to microbial and tumour cells with uniquely high glycolytic capaci­ties (Sussman et al, 1980). How­ever, inhibition of respiration and increase of lactate production are observed under aerobic condi­tions in beating rat heart cell cultures (Frelin et al, 1974) and in isolated rat lung cells (Ayuso-Parrilla et al, 1978). Thus, the same general mechanisms respon­sible for the integra­tion of respiration and glycolysis in tumour cells (Sussman et al, 1980) appear to be operating to some extent in several isolated mammalian cells.”

Mitochondria are sometimes described as “cellular power plants” because they generate most of the cell’s supply of adenosine triphosphate (ATP), used as a source of chemical energy.[2] In addition to supplying cellular energy, mitochondria are involved in other tasks such as signalingcellular differentiationcell death, as well as the control of the cell cycle and cell growth.[3]   The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria,[9   Leonor Michaelis discovered that Janus green can be used as a supravital stain for mitochondria in 1900.  Benjamin F. Kingsbury, in 1912, first related them with cell respiration, but almost exclusively based on morphological observations.[13] In 1913 particles from extracts of guinea-pig liver were linked to respiration by Otto Heinrich Warburg, which he called “grana”. Warburg and Heinrich Otto Wieland, who had also postulated a similar particle mechanism, disagreed on the chemical nature of the respiration. It was not until 1925 when David Keilin discovered cytochromes that the respiratory chain was described.[13]    

The Clark Oxygen Sensor

Dr. Leland Clark – inventor of the “Clark Oxygen Sensor” (1954); the Clark type polarographic oxygen sensor remains the gold standard for measuring dissolved oxygen in biomedical, environmental and industrial applications .   ‘The convenience and simplicity of the polarographic ‘oxygen electrode’ technique for measuring rapid changes in the rate of oxygen utilization by cellular and subcellular systems is now leading to its more general application in many laboratories. The types and design of oxygen electrodes vary, depending on the investigator’s ingenuity and specific requirements of the system under investigation.’Estabrook R (1967) Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol. 10: 41-47.   “one approach that is underutilized in whole-cell bioenergetics, and that is accessible as long as cells can be obtained in suspension, is the oxygen electrode, which can obtain more precise information on the bioenergetic status of the in situ mitochondria than more ‘high-tech’ approaches such as fluorescent monitoring of Δψm.” Nicholls DG, Ferguson S (2002) Bioenergetics 3. Academic Press, London.

Great Figures in Cancer

Dr. Elizabeth Blackburn,

Dr. Elizabeth Blackburn,

j_michael_bishop onogene

j_michael_bishop onogene

Harold Varmus

Harold Varmus

Potts and Habener (PTH mRNA, Harvard MIT)  JCI

Potts and Habener (PTH mRNA, Harvard MIT) JCI

JCI Fuller Albright and hPTH AA sequence

JCI Fuller Albright and hPTH AA sequence

Dr. E. Donnall Thomas  Bone Marrow Transplants

Dr. E. Donnall Thomas Bone Marrow Transplants

Dr Haraldzur Hausen  EBV HPV

Dr Haraldzur Hausen EBV HPV

Dr. Craig Mello

Dr. Craig Mello

Dorothy Hodgkin  protein crystallography

Lee Hartwell - Hutchinson Cancer Res Center

Lee Hartwell – Hutchinson Cancer Res Center

Judah Folkman, MD

Judah Folkman, MD

Gertrude B. Elien (1918-1999)

Gertrude B. Elien (1918-1999)

The Nobel Prize in Physiology or Medicine 1922   

Archibald V. Hill, Otto Meyerhof

AV Hill –

“the production of heat in the muscle” Hill started his research work in 1909. It was due to J.N. Langley, Head of the Department of Physiology at that time that Hill took up the study on the nature of muscular contraction. Langley drew his attention to the important (later to become classic) work carried out by Fletcher and Hopkins on the problem of lactic acid in muscle, particularly in relation to the effect of oxygen upon its removal in recovery. In 1919 he took up again his study of the physiology of muscle, and came into close contact with Meyerhof of Kiel who, approaching the problem differently, arrived at results closely analogous to his study. In 1919 Hill’s friend W. Hartree, mathematician and engineer, joined in the myothermic investigations – a cooperation which had rewarding results.

Otto Meyerhof

otto-fritz-meyerhof

otto-fritz-meyerhof

lactic acid production in muscle contraction Under the influence of Otto Warburg, then at Heidelberg, Meyerhof became more and more interested in cell physiology.  In 1923 he was offered a Professorship of Biochemistry in the United States, but Germany was unwilling to lose him.  In 1929 he was he was placed in charge of the newly founded Kaiser Wilhelm Institute for Medical Research at Heidelberg.  From 1938 to 1940 he was Director of Research at the Institut de Biologie physico-chimique at Paris, but in 1940 he moved to the United States, where the post of Research Professor of Physiological Chemistry had been created for him by the University of Pennsylvania and the Rockefeller Foundation.  Meyerhof’s own account states that he was occupied chiefly with oxidation mechanisms in cells and with extending methods of gas analysis through the calorimetric measurement of heat production, and especially the respiratory processes of nitrifying bacteria. The physico-chemical analogy between oxygen respiration and alcoholic fermentation caused him to study both these processes in the same subject, namely, yeast extract. By this work he discovered a co-enzyme of respiration, which could be found in all the cells and tissues up till then investigated. At the same time he also found a co-enzyme of alcoholic fermentation. He also discovered the capacity of the SH-group to transfer oxygen; after Hopkins had isolated from cells the SH bodies concerned, Meyerhof showed that the unsaturated fatty acids in the cell are oxidized with the help of the sulfhydryl group. After studying closer the respiration of muscle, Meyerhof investigated the energy changes in muscle. Considerable progress had been achieved by the English scientists Fletcher and Hopkins by their recognition of the fact that lactic acid formation in the muscle is closely connected with the contraction process. These investigations were the first to throw light upon the highly paradoxical fact, already established by the physiologist Hermann, that the muscle can perform a considerable part of its external function in the complete absence of oxygen.

But it was indisputable that in the last resort the energy for muscle activity comes from oxidation, so the connection between activity and combustion must be an indirect one, and observed that in the absence of oxygen in the muscle, lactic acid appears, slowly in the relaxed state and rapidly in the active state, disappearing in the presence of oxygen. Obviously, then, oxygen is involved when muscle is in the relaxed state. http://upload.wikimedia.org/wikipedia/commons/e/e1/Glycolysis.jpg

The Nobel Prize committee had been receiving nominations intermittently for the previous 14 years (for Eijkman, Funk, Goldberger, Grijns, Hopkins and Suzuki but, strangely, not for McCollum in this period). Tthe Committee for the 1929 awards apparently agreed that it was high time to honor the discoverer(s) of vitamins; but who were they? There was a clear case for Grijns, but he had not been re-nominated for that particular year, and it could be said that he was just taking the relatively obvious next steps along the new trail that had been laid down by Eijkman, who was also now an old man in poor health, but there was no doubt that he had taken the first steps in the use of an animal model to investigate the nutritional basis of a clinical disorder affecting millions. Goldberger had been another important contributor, but his recent death put him out of consideration. The clearest evidence for lack of an unknown “something” in a mammalian diet was presented by Gowland Hopkins in 1912. This Cambridge biochemist was already well known for having isolated the amino acid tryptophan from a protein and demonstrated its essential nature. He fed young rats on an experimental diet, half of them receiving a daily milk supplement, and only those receiving milk grew well, Hopkins suggested that this was analogous to human diseases related to diet, as he had suggested already in a lecture published in 1906. Hopkins, the leader of the “dynamic biochemistry” school in Britain and an influential advocate for the importance of vitamins, was awarded the prize jointly with Eijkman. A door was opened. Recognition of work on the fat-soluble vitamins begun by McCollum. The next award related to vitamins was given in 1934 to George WhippleGeorge Minot and William Murphy “for their discoveries concerning liver therapy in cases of [then incurable pernicious] anemia,” The essential liver factor (cobalamin, or vitamin B12) was isolated in 1948, and Vitamin B12  was absent from plant foods. But William Castle in 1928 had demonstrated that the stomachs of pernicious anemia patients were abnormal in failing to secrete an “intrinsic factor”.

1937   Albert von Szent-Györgyi Nagyrápolt

” the biological combustion processes, with special reference to vitamin C and the catalysis of fumaric acid”

http://www.biocheminfo.org/klotho/html/fumarate.html

structure of fumarate

Szent-Györgyi was a Hungarian biochemist who had worked with Otto Warburg and had a special interest in oxidation-reduction mechanisms. He was invited to Cambridge in England in 1927 after detecting an antioxidant compound in the adrenal cortex, and there, he isolated a compound that he named hexuronic acid. Charles Glen King of the University of Pittsburgh reported success In isolating the anti-scorbutic factor in 1932, and added that his crystals had all the properties reported by Szent-Györgyi for hexuronic acid. But his work on oxidation reactions was also important. Fumarate is an intermediate in the citric acid cycle used by cells to produce energy in the form of adenosine triphosphate (ATP) from food. It is formed by the oxidation of succinate by the enzyme succinate dehydrogenase. Fumarate is then converted by the enzyme fumarase to malate. An enzyme adds water to the fumarate molecule to form malate. The malate is created by adding one hydrogen atom to a carbon atom and then adding a hydroxyl group to a carbon next to a terminal carbonyl group.

In the same year, Norman Haworth from the University of Birmingham in England received a Nobel prize from the Chemistry Committee for having advanced carbohydrate chemistry and, specifically, for having worked out the structure of Szent-Györgyi’s crystals, and then been able to synthesize the vitamin. This was a considerable achievement. The Nobel Prize in Chemistry was shared with the Swiss organic chemist Paul Karrer, cited for his work on the structures of riboflavin and vitamins A and E as well as other biologically interesting compounds. This was followed in 1938 by a further Chemistry award to the German biochemist Richard Kuhn, who had also worked on carotenoids and B-vitamins, including riboflavin and pyridoxine. But Karrer was not permitted to leave Germany at that time by the Nazi regime. However, the American work with radioisotopes at Lawrence Livermore Laboratory, UC Berkeley, was already ushering in a new era of biochemistry that would enrich our studies of metabolic pathways. The importance of work involving vitamins was acknowledged in at least ten awards in the 20th century.

1.   Carpenter, K.J., Beriberi, White Rice and Vitamin B, University of California Press, Berkeley (2000).

2.  Weatherall, M.W. and Kamminga, H., The making of a biochemist: the construction of Frederick Gowland Hopkins’ reputation. Medical History vol.40, pp. 415-436 (1996).

3.  Becker, S.L., Will milk make them grow? An episode in the discovery of the vitamins. In Chemistry and Modern Society (J. Parascandela, editor) pp. 61-83, American Chemical Society,

Washington, D.C. (1983).

4.  Carpenter, K.J., The History of Scurvy and Vitamin C, Cambridge University Press, New York (1986).

Transport and metabolism of exogenous fumarate and 3-phosphoglycerate in vascular smooth muscle.

D R FinderC D Hardin

Molecular and Cellular Biochemistry (Impact Factor: 2.33). 05/1999; 195(1-2):113-21.  http://dx.doi.org/10.1023/A:1006976432578

The keto (linear) form of exogenous fructose 1,6-bisphosphate, a highly charged glycolytic intermediate, may utilize a dicarboxylate transporter to cross the cell membrane, support glycolysis, and produce ATP anaerobically. We tested the hypothesis that fumarate, a dicarboxylate, and 3-phosphoglycerate (3-PG), an intermediate structurally similar to a dicarboxylate, can support contraction in vascular smooth muscle during hypoxia. 3-PG improved maintenance of force (p < 0.05) during the 30-80 min period of hypoxia. Fumarate decreased peak isometric force development by 9.5% (p = 0.008) but modestly improved maintenance of force (p < 0.05) throughout the first 80 min of hypoxia. 13C-NMR on tissue extracts and superfusates revealed 1,2,3,4-(13)C-fumarate (5 mM) metabolism to 1,2,3,4-(13)C-malate under oxygenated and hypoxic conditions suggesting uptake and metabolism of fumarate. In conclusion, exogenous fumarate and 3-PG readily enter vascular smooth muscle cells, presumably by a dicarboxylate transporter, and support energetically important pathways.

Comparison of endogenous and exogenous sources of ATP in fueling Ca2+ uptake in smooth muscle plasma membrane vesicles.

C D HardinL RaeymaekersR J Paul

The Journal of General Physiology (Impact Factor: 4.73). 12/1991; 99(1):21-40.   http://dx.doi.org:/10.1085/jgp.99.1.21

A smooth muscle plasma membrane vesicular fraction (PMV) purified for the (Ca2+/Mg2+)-ATPase has endogenous glycolytic enzyme activity. In the presence of glycolytic substrate (fructose 1,6-diphosphate) and cofactors, PMV produced ATP and lactate and supported calcium uptake. The endogenous glycolytic cascade supports calcium uptake independent of bath [ATP]. A 10-fold dilution of PMV, with the resultant 10-fold dilution of glycolytically produced bath [ATP] did not change glycolytically fueled calcium uptake (nanomoles per milligram protein). Furthermore, the calcium uptake fueled by the endogenous glycolytic cascade persisted in the presence of a hexokinase-based ATP trap which eliminated calcium uptake fueled by exogenously added ATP. Thus, it appears that the endogenous glycolytic cascade fuels calcium uptake in PMV via a membrane-associated pool of ATP and not via an exchange of ATP with the bulk solution. To determine whether ATP produced endogenously was utilized preferentially by the calcium pump, the ATP production rates of the endogenous creatine kinase and pyruvate kinase were matched to that of glycolysis and the calcium uptake fueled by the endogenous sources was compared with that fueled by exogenous ATP added at the same rate. The rate of calcium uptake fueled by endogenous sources of ATP was approximately twice that supported by exogenously added ATP, indicating that the calcium pump preferentially utilizes ATP produced by membrane-bound enzymes.

Evidence for succinate production by reduction of fumarate during hypoxia in isolated adult rat heart cells.

C HohlR OestreichP RösenR WiesnerM Grieshaber

Archives of Biochemistry and Biophysics (Impact Factor: 3.37). 01/1988; 259(2):527-35. http://dx.doi.org:/10.1016/0003-9861(87)90519-4   It has been demonstrated that perfusion of myocardium with glutamic acid or tricarboxylic acid cycle intermediates during hypoxia or ischemia, improves cardiac function, increases ATP levels, and stimulates succinate production. In this study isolated adult rat heart cells were used to investigate the mechanism of anaerobic succinate formation and examine beneficial effects attributed to ATP generated by this pathway. Myocytes incubated for 60 min under hypoxic conditions showed a slight loss of ATP from an initial value of 21 +/- 1 nmol/mg protein, a decline of CP from 42 to 17 nmol/mg protein and a fourfold increase in lactic acid production to 1.8 +/- 0.2 mumol/mg protein/h. These metabolite contents were not altered by the addition of malate and 2-oxoglutarate to the incubation medium nor were differences in cell viability observed; however, succinate release was substantially accelerated to 241 +/- 53 nmol/mg protein. Incubation of cells with [U-14C]malate or [2-U-14C]oxoglutarate indicates that succinate is formed directly from malate but not from 2-oxoglutarate. Moreover, anaerobic succinate formation was rotenone sensitive.

We conclude that malate reduction to succinate occurs via the reverse action of succinate dehydrogenase in a coupled reaction where NADH is oxidized (and FAD reduced) and ADP is phosphorylated. Furthermore, by transaminating with aspartate to produce oxaloacetate, 2-oxoglutarate stimulates cytosolic malic dehydrogenase activity, whereby malate is formed and NADH is oxidized.

In the form of malate, reducing equivalents and substrate are transported into the mitochondria where they are utilized for succinate synthesis.

1953 Hans Adolf Krebs –

 ” discovery of the citric acid cycle” and In the course of the 1920’s and 1930’s great progress was made in the study of the intermediary reactions by which sugar is anaerobically fermented to lactic acid or to ethanol and carbon dioxide. The success was mainly due to the joint efforts of the schools of Meyerhof, Embden, Parnas, von Euler, Warburg and the Coris, who built on the pioneer work of Harden and of Neuberg. This work brought to light the main intermediary steps of anaerobic fermentations.

In contrast, very little was known in the earlier 1930’s about the intermediary stages through which sugar is oxidized in living cells. When, in 1930, I left the laboratory of Otto Warburg (under whose guidance I had worked since 1926 and from whom I have learnt more than from any other single teacher), I was confronted with the question of selecting a major field of study and I felt greatly attracted by the problem of the intermediary pathway of oxidations.

These reactions represent the main energy source in higher organisms, and in view of the importance of energy production to living organisms (whose activities all depend on a continuous supply of energy) the problem seemed well worthwhile studying.   http://www.johnkyrk.com/krebs.html

Interactive Krebs cycle

There are different points where metabolites enter the Krebs’ cycle. Most of the products of protein, carbohydrates and fat metabolism are reduced to the molecule acetyl coenzyme A that enters the Krebs’ cycle. Glucose, the primary fuel in the body, is first metabolized into pyruvic acid and then into acetyl coenzyme A. The breakdown of the glucose molecule forms two molecules of ATP for energy in the Embden Meyerhof pathway process of glycolysis.

On the other hand, amino acids and some chained fatty acids can be metabolized into Krebs intermediates and enter the cycle at several points. When oxygen is unavailable or the Krebs’ cycle is inhibited, the body shifts its energy production from the Krebs’ cycle to the Embden Meyerhof pathway of glycolysis, a very inefficient way of making energy.  

Fritz Albert Lipmann –

 “discovery of co-enzyme A and its importance for intermediary metabolism”.

In my development, the recognition of facts and the rationalization of these facts into a unified picture, have interplayed continuously. After my apprenticeship with Otto Meyerhof, a first interest on my own became the phenomenon we call the Pasteur effect, this peculiar depression of the wasteful fermentation in the respiring cell. By looking for a chemical explanation of this economy measure on the cellular level, I was prompted into a study of the mechanism of pyruvic acid oxidation, since it is at the pyruvic stage where respiration branches off from fermentation.

For this study I chose as a promising system a relatively simple looking pyruvic acid oxidation enzyme in a certain strain of Lactobacillus delbrueckii1.   In 1939, experiments using minced muscle cells demonstrated that one oxygen atom can form two adenosine triphosphate molecules, and, in 1941, the concept of phosphate bonds being a form of energy in cellular metabolism was developed by Fritz Albert Lipmann.

In the following years, the mechanism behind cellular respiration was further elaborated, although its link to the mitochondria was not known.[13]The introduction of tissue fractionation by Albert Claude allowed mitochondria to be isolated from other cell fractions and biochemical analysis to be conducted on them alone. In 1946, he concluded that cytochrome oxidase and other enzymes responsible for the respiratory chain were isolated to the mitchondria. Over time, the fractionation method was tweaked, improving the quality of the mitochondria isolated, and other elements of cell respiration were determined to occur in the mitochondria.[13]

The most important event during this whole period, I now feel, was the accidental observation that in the L. delbrueckii system, pyruvic acid oxidation was completely dependent on the presence of inorganic phosphate. This observation was made in the course of attempts to replace oxygen by methylene blue. To measure the methylene blue reduction manometrically, I had to switch to a bicarbonate buffer instead of the otherwise routinely used phosphate. In bicarbonate, pyruvate oxidation was very slow, but the addition of a little phosphate caused a remarkable increase in rate. The phosphate effect was removed by washing with a phosphate free acetate buffer. Then it appeared that the reaction was really fully dependent on phosphate.

A coupling of this pyruvate oxidation with adenylic acid phosphorylation was attempted. Addition of adenylic acid to the pyruvic oxidation system brought out a net disappearance of inorganic phosphate, accounted for as adenosine triphosphate.   The acetic acid subunit of acetyl CoA is combined with oxaloacetate to form a molecule of citrate. Acetyl coenzyme A acts only as a transporter of acetic acid from one enzyme to another. After Step 1, the coenzyme is released by hydrolysis to combine with another acetic acid molecule and begin the Krebs’ Cycle again.

Hugo Theorell

the nature and effects of oxidation enzymes”

From 1933 until 1935 Theorell held a Rockefeller Fellowship and worked with Otto Warburg at Berlin-Dahlem, and here he became interested in oxidation enzymes. At Berlin-Dahlem he produced, for the first time, the oxidation enzyme called «the yellow ferment» and he succeeded in splitting it reversibly into a coenzyme part, which was found to be flavin mononucleotide, and a colourless protein part. On return to Sweden, he was appointed Head of the newly established Biochemical Department of the Nobel Medical Institute, which was opened in 1937.

Succinate is oxidized by a molecule of FAD (Flavin Adenine Dinucleotide). The FAD removes two hydrogen atoms from the succinate and forms a double bond between the two carbon atoms to create fumarate.

1953

double-stranded-dna

double-stranded-dna

crick-watson-with-their-dna-model.

crick-watson-with-their-dna-model.

Watson & Crick double helix model 

A landmark in this journey

They followed the path that became clear from intense collaborative work in California by George Beadle, by Avery and McCarthy, Max Delbruck, TH Morgan, Max Delbruck and by Chargaff that indicated that genetics would be important.

1965

François Jacob, André Lwoff and Jacques Monod  –

” genetic control of enzyme and virus synthesis”.

In 1958 the remarkable analogy revealed by genetic analysis of lysogeny and that of the induced biosynthesis of ß-galactosidase led François Jacob, with Jacques Monod, to study the mechanisms responsible for the transfer of genetic information as well as the regulatory pathways which, in the bacterial cell, adjust the activity and synthesis of macromolecules. Following this analysis, Jacob and Monod proposed a series of new concepts, those of messenger RNA, regulator genes, operons and allosteric proteins.

Francois Jacob

Having determined the constants of growth in the presence of different carbohydrates, it occurred to me that it would be interesting to determine the same constants in paired mixtures of carbohydrates. From the first experiment on, I noticed that, whereas the growth was kinetically normal in the presence of certain mixtures (that is, it exhibited a single exponential phase), two complete growth cycles could be observed in other carbohydrate mixtures, these cycles consisting of two exponential phases separated by a-complete cessation of growth.

Lwoff, after considering this strange result for a moment, said to me, “That could have something to do with enzyme adaptation.”

“Enzyme adaptation? Never heard of it!” I said.

Lwoff’s only reply was to give me a copy of the then recent work of Marjorie Stephenson, in which a chapter summarized with great insight the still few studies concerning this phenomenon, which had been discovered by Duclaux at the end of the last century.  Studied by Dienert and by Went as early as 1901 and then by Euler and Josephson, it was more or less rediscovered by Karström, who should be credited with giving it a name and attracting attention to its existence.

Lwoff’s intuition was correct. The phenomenon of “diauxy” that I had discovered was indeed closely related to enzyme adaptation, as my experiments, included in the second part of my doctoral dissertation, soon convinced me. It was actually a case of the “glucose effect” discovered by Dienert as early as 1900.   That agents that uncouple oxidative phosphorylation, such as 2,4-dinitrophenol, completely inhibit adaptation to lactose or other carbohydrates suggested that “adaptation” implied an expenditure of chemical potential and therefore probably involved the true synthesis of an enzyme.

With Alice Audureau, I sought to discover the still quite obscure relations between this phenomenon and the one Massini, Lewis, and others had discovered: the appearance and selection of “spontaneous” mutants.   We showed that an apparently spontaneous mutation was allowing these originally “lactose-negative” bacteria to become “lactose-positive”. However, we proved that the original strain (Lac-) and the mutant strain (Lac+) did not differ from each other by the presence of a specific enzyme system, but rather by the ability to produce this system in the presence of lactose.  This mutation involved the selective control of an enzyme by a gene, and the conditions necessary for its expression seemed directly linked to the chemical activity of the system.

1974

Albert Claude, Christian de Duve and George E. Palade –

” the structural and functional organization of the cell”.

I returned to Louvain in March 1947 after a period of working with Theorell in Sweden, the Cori’s, and E Southerland in St. Louis, fortunate in the choice of my mentors, all sticklers for technical excellence and intellectual rigor, those prerequisites of good scientific work. Insulin, together with glucagon which I had helped rediscover, was still my main focus of interest, and our first investigations were accordingly directed on certain enzymatic aspects of carbohydrate metabolism in liver, which were expected to throw light on the broader problem of insulin action. But I became distracted by an accidental finding related to acid phosphatase, drawing most of my collaborators along with me. The studies led to the discovery of the lysosome, and later of the peroxisome.

In 1962, I was appointed a professor at the Rockefeller Institute in New York, now the Rockefeller University, the institution where Albert Claude had made his pioneering studies between 1929 and 1949, and where George Palade had been working since 1946.  In New York, I was able to develop a second flourishing group, which follows the same general lines of research as the Belgian group, but with a program of its own.

1968

Robert W. Holley, Har Gobind Khorana and Marshall W. Nirenberg –

“interpretation of the genetic code and its function in protein synthesis”.

1969

Max Delbrück, Alfred D. Hershey and Salvador E. Luria –

” the replication mechanism and the genetic structure of viruses”.

1975 David Baltimore, Renato Dulbecco and Howard Martin Temin –

” the interaction between tumor viruses and the genetic material of the cell”.

1976

Baruch S. Blumberg and D. Carleton Gajdusek –

” new mechanisms for the origin and dissemination of infectious diseases” The editors of the Nobelprize.org website of the Nobel Foundation have asked me to provide a supplement to the autobiography that I wrote in 1976 and to recount the events that happened after the award. Much of what I will have to say relates to the scientific developments since the last essay. These are described in greater detail in a scientific memoir first published in 2002 (Blumberg, B. S., Hepatitis B. The Hunt for a Killer Virus, Princeton University Press, 2002, 2004).

1980

Baruj Benacerraf, Jean Dausset and George D. Snell 

” genetically determined structures on the cell surface that regulate immunological reactions”.

1992

Edmond H. Fischer and Edwin G. Krebs 

“for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism”

1994

Alfred G. Gilman and Martin Rodbell –

“G-proteins and the role of these proteins in signal transduction in cells”

2011

Bruce A. Beutler and Jules A. Hoffmann –

the activation of innate immunity and the other half to Ralph M. Steinman – “the dendritic cell and its role in adaptive immunity”.

Renato L. Baserga, M.D.

Kimmel Cancer Center and Keck School of Medicine

Dr. Baserga’s research focuses on the multiple roles of the type 1 insulin-like growth factor receptor (IGF-IR) in the proliferation of mammalian cells. The IGF-IR activated by its ligands is mitogenic, is required for the establishment and the maintenance of the transformed phenotype, and protects tumor cells from apoptosis. It, therefore, serves as an excellent target for therapeutic interventions aimed at inhibiting abnormal growth. In basic investigations, this group is presently studying the effects that the number of IGF-IRs and specific mutations in the receptor itself have on its ability to protect cells from apoptosis.

This investigation is strictly correlated with IGF-IR signaling, and part of this work tries to elucidate the pathways originating from the IGF-IR that are important for its functional effects. Baserga’s group has recently discovered a new signaling pathway used by the IGF-IR to protect cells from apoptosis, a unique pathway that is not used by other growth factor receptors. This pathway depends on the integrity of serines 1280-1283 in the C-terminus of the receptor, which bind 14.3.3 and cause the mitochondrial translocation of Raf-1.

Another recent discovery of this group has been the identification of a mechanism by which the IGF-IR can actually induce differentiation in certain types of cells. When cells have IRS-1 (a major substrate of the IGF-IR), the IGF-IR sends a proliferative signal; in the absence of IRS-1, the receptor induces cell differentiation. The extinction of IRS-1 expression is usually achieved by DNA methylation.

Janardan Reddy, MD

Northwestern University

The central effort of our research has been on a detailed analysis at the cellular and molecular levels of the pleiotropic responses in liver induced by structurally diverse classes of chemicals that include fibrate class of hypolipidemic drugs, and phthalate ester plasticizers, which we designated hepatic peroxisome proliferators. Our work has been central to the establishment of several principles, namely that hepatic peroxisome proliferation is associated with increases in fatty acid oxidation systems in liver, and that peroxisome proliferators, as a class, are novel nongenotoxic hepatocarcinogens.

We introduced the concept that sustained generation of reactive oxygen species leads to oxidative stress and serves as the basis for peroxisome proliferator-induced liver cancer development. Furthermore, based on the tissue/cell specificity of pleiotropic responses and the coordinated transcriptional regulation of fatty acid oxidation system genes, we postulated that peroxisome proliferators exert their action by a receptor-mediated mechanism. This receptor concept laid the foundation for the discovery of

  • a three member peroxisome proliferator-activated receptor (PPARalpha-, ß-, and gamma) subfamily of nuclear receptors.
  •  PPARalpha is responsible for peroxisome proliferator-induced pleiotropic responses, including
    • hepatocarcinogenesis and energy combustion as it serves as a fatty acid sensor and regulates fatty acid oxidation.

Our current work focuses on the molecular mechanisms responsible for PPAR action and generation of fatty acid oxidation deficient mouse knockout models. Transcription of specific genes by nuclear receptors is a complex process involving the participation of multiprotein complexes composed of transcription coactivators.  

Jose Delgado de Salles Roselino, Ph.D.

Leloir Institute, Brazil

Warburg effect, in reality “Pasteur-effect” was the first example of metabolic regulation described. A decrease in the carbon flux originated at the sugar molecule towards the end metabolic products, ethanol and carbon dioxide that was observed when yeast cells were transferred from anaerobic environmental condition to an aerobic one. In Pasteur´s works, sugar metabolism was measured mainly by the decrease of sugar concentration in the yeast growth media observed after a measured period of time. The decrease of the sugar concentration in the media occurs at great speed in yeast grown in anaerobiosis condition and its speed was greatly reduced by the transfer of the yeast culture to an aerobic condition. This finding was very important for the wine industry of France in Pasteur time, since most of the undesirable outcomes in the industrial use of yeast were perceived when yeasts cells took very long time to create a rather selective anaerobic condition. This selective culture media was led by the carbon dioxide higher levels produced by fast growing yeast cells and by a great alcohol content in the yeast culture media. This finding was required to understand Lavoisier’s results indicating that chemical and biological oxidation of sugars produced the same calorimetric results. This observation requires a control mechanism (metabolic regulation) to avoid burning living cells by fast heat released by the sugar biological oxidative processes (metabolism). In addition, Lavoisier´s results were the first indications that both processes happened inside similar thermodynamics limits.

In much resumed form, these observations indicates the major reasons that led Warburg to test failure in control mechanisms in cancer cells in comparison with the ones observed in normal cells. Biology inside classical thermo dynamics poses some challenges to scientists. For instance, all classical thermodynamics must be measured in reversible thermodynamic conditions. In an isolated system, increase in P (pressure) leads to decrease in V (volume) all this in a condition in which infinitesimal changes in one affects in the same way the other, a continuum response. Not even a quantic amount of energy will stand beyond those parameters. In a reversible system, a decrease in V, under same condition, will led to an increase in P.

In biochemistry, reversible usually indicates a reaction that easily goes from A to B or B to A. This observation confirms the important contribution of E Schrodinger in his What´s Life: “This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject-matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

Hans Krebs describes the cyclic nature of the citrate metabolism. Two major research lines search to understand the mechanism of energy transfer that explains how ADP is converted into ATP. One followed the organic chemistry line of reasoning and therefore, searched how the breakdown of carbon-carbon link could have its energy transferred to ATP synthesis. A major leader of this research line was B. Chance who tried to account for two carbon atoms of acetyl released as carbon dioxide in the series of Krebs cycle reactions. The intermediary could store in a phosphorylated amino acid the energy of carbon-carbon bond breakdown. This activated amino acid could transfer its phosphate group to ADP producing ATP. Alternatively, under the possible influence of the excellent results of Hodgkin and Huxley a second line of research appears.

The work of Hodgkin & Huxley indicated the storage of electrical potential energy in transmembrane ionic asymmetries and presented the explanation for the change from resting to action potential in excitable cells. This second line of research, under the leadership of P Mitchell postulated a mechanism for the transfer of oxide/reductive power of organic molecules oxidation through electron transfer as the key for energetic transfer mechanism required for ATP synthesis. Paul Boyer could present how the energy was transduced by a molecular machine that changes in conformation in a series of 3 steps while rotating in one direction in order to produce ATP and in opposite direction in order to produce ADP plus Pi from ATP (reversibility). Nonetheless, a victorious Peter Mitchell obtained the correct result in the conceptual dispute, over the B. Chance point of view, after he used E. Coli mutants to show H gradients in membrane and its use as energy source.

However, this should not detract from the important work of Chance. B. Chance got the simple and rapid polarographic assay method of oxidative phosphorylation and the idea of control of energy metabolism that bring us back to Pasteur. This second result seems to have been neglected in searching for a single molecular mechanism required for the understanding of the buildup of chemical reserve in our body. In respiring mitochondria the rate of electron transport, and thus the rate of ATP production, is determined primarily by the relative concentrations of ADP, ATP and phosphate in the external media (cytosol) and not by the concentration of respiratory substrate as pyruvate. Therefore, when the yield of ATP is high as is in aerobiosis and the cellular use of ATP is not changed, the oxidation of pyruvate and therefore of glycolysis is quickly (without change in gene expression), throttled down to the resting state. The dependence of respiratory rate on ADP concentration is also seen in intact cells. A muscle at rest and using no ATP has very low respiratory rate.

I have had an ongoing discussion with Jose Eduardo de Salles Roselino, inBrazil. He has made important points that need to be noted.

  1. The constancy of composition which animals maintain in the environment surrounding their cells is one of the dominant features of their physiology. Although this phenomenon, homeostasis, has held the interest of biologists over a long period of time, the elucidation of the molecular basis for complex processes such as temperature control and the maintenance of various substances at constant levels in the blood has not yet been achieved. By comparison, metabolic regulation in microorganisms is much better understood, in part because the microbial physiologist has focused his attention on enzyme-catalyzed reactions and their control, as these are among the few activities of microorganisms amenable to quantitative study. Furthermore, bacteria are characterized by their ability to make rapid and efficient adjustments to extensive variations in most parameters of their environment; therefore, they exhibit a surprising lack of rigid requirements for their environment, and appears to influence it only as an incidental result of their metabolism. Animal cells on the other hand have only a limited capacity for adjustment and therefore require a constant milieu. Maintenance of such constancy appears to be a major goal in their physiology (Regulation of Biosynthetic Pathways H.S. Moyed and H EUmbarger Phys Rev,42 444 (1962)).
  2. A living cell consists in a large part of a concentrated mixture of hundreds of different enzymes, each a highly effective catalyst for one or more chemical reactions involving other components of the cell. The paradox of intense and highly diverse chemical activity on the one hand and strongly poised chemical stability (biological homeostasis) on the other is one of the most challenging problems of biology (Biological feedback Control at the molecular Level D.E. Atkinson Science vol. 150: 851, 1965). Almost nothing is known concerning the actual molecular basis for modulation of an enzyme`s kinetic behavior by interaction with a small molecule. (Biological feedback Control at the molecular Level D.E. Atkinson Science vol. 150: 851, 1965). In the same article, since the core of Atkinson´s thinking seems to be strongly linked with Adenylates as regulatory effectors, the previous phrases seems to indicate a first step towards the conversion of homeostasis to an intracellular phenomenon and therefore, one that contrary to Umbarger´s consideration could be also studied in microorganisms.
  3.  Most biochemical studies using bacteria, were made before the end of the third upper part of log growth phase. Therefore, they could be considered as time-independent as S Luria presented biochemistry in Life an Unfinished Experiment. The sole ingredient on the missing side of the events that led us into the molecular biology construction was to consider that proteins, a macromolecule, would never be affected by small molecules translational kinetic energy. This, despite the fact that in a catalytic environment and its biological implications S Grisolia incorporated A K Balls observation indicating that the word proteins could be related to Proteus an old sea god that changed its form whenever he was subjected to inquiry (Phys Rev v 4,657 (1964).
  1. In D.E. Atkinson´s work (Science vol 150 p 851, 1965), changes in protein synthesis acting together with factors that interfere with enzyme activity will lead to “fine-tuned” regulation better than enzymatic activity regulation alone. Comparison of glycemic regulation in granivorous and carnivorous birds indicate that when no important nutritional source of glucose is available, glycemic levels can be kept constant in fasted and fed birds. The same was found in rats and cats fed on high protein diets. Gluconeogenesis is controlled by pyruvate kinase inhibition. Therefore, the fact that it can discriminate between fasting alone and fasting plus exercise (carbachol) requirement of gluconeogenic activity (correspondent level of pyruvate kinase inhibition) the control of enzyme activity can be made fast and efficient without need for changes in genetic expression (20 minute after stimulus) ( Migliorini,R.H. et al Am J. Physiol.257 (Endocrinol. Met. 20): E486, 1989). Regrettably, this was not discussed in the quoted work. So, when the control is not affected by the absorption of nutritional glucose it can be very fast, less energy intensive and very sensitive mechanism of control despite its action being made in the extracellular medium (homeostasis).

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Sensors and Signaling in Oxidative Stress

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

This is article ELEVEN in the following series on Calcium Role in Cardiovascular Diseases

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-
that-are-related-to-the-actin-cytoskeleton/

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and 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/

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease
Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-
exchange-mechanism-in-health-and-disease/

Part IV: 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/

Part V: 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/

Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary
Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-in-cardiac-gene-therapy-inhalable-gene-therapy-
for-pulmonary-arterial-hypertension-and-percutaneous-intra-coronary-artery-infusion-for-heart-failure-contributions-by-roger-j-hajjar/

Part VII: 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/

Part VIII: 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/

Part IX: 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/

Part X: 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/

Part XI: Sensors and Signaling in Oxidative Stress
Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

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

https://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

This important article on oxidative stress was published in Free Radical Biol. and Med.

Nrf2:INrf2(Keap1) Signaling in Oxidative Stress

James W. Kaspar, Suresh K. Niture, and Anil K. Jaiswal*
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD

Free Radic Biol Med. 2009 Nov; 47(9): 1304–1309.           http://dx.doi.org/10.1016/j.freeradbiomed.2009.07.035

Nrf2:INrf2(Keap1) are cellular sensors of chemical and radiation induced oxidative and electrophilic stress. Nrf2 is a nuclear transcription factor that controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins. This is a mechanism of critical importance for cellular protection and cell survival. Nrf2 is retained in the cytoplasm by an inhibitor INrf2. INrf2 functions as an adapter for Cul3/Rbx1 mediated degradation of Nrf2. In response to oxidative/electrophilic stress, Nrf2 is switched on and then off by distinct early and delayed mechanisms. Oxidative/electrophilic modification of INrf2cysteine151 and/or PKC phosphorylation of Nrf2serine40 results in

  • the escape or release of Nrf2 from INrf2.

Nrf2 is stabilized and

  • translocates to the nucleus,
  • forms heterodimers with unknown proteins, and
  • binds antioxidant response element (ARE) that
  • leads to coordinated activation of gene expression.
  • It takes less than fifteen minutes from the time of exposure to switch on nuclear import of Nrf2. This is followed by activation of a delayed mechanism that controls switching off of Nrf2 activation of gene expression. GSK3β phosphorylates Fyn at unknown threonine residue(s) leading to nuclear localization of Fyn. Fyn phosphorylates Nrf2tyrosine568
  • resulting in nuclear export of Nrf2, binding with INrf2 and
  • degradation of Nrf2.

The switching on and off of Nrf2 protects cells against free radical damage, prevents apoptosis and promotes cell survival.

Introduction

Oxidative stress is induced by a vast range of factors including xenobiotics, drugs, heavy metals and ionizing radiation. Oxidative stress leads to the generation of Reactive Oxygen Species (ROS) and electrophiles. ROS and electrophiles generated can have a profound impact on survival, growth development and evolution of all living organisms [1,2] ROS include

  • both free radicals, such as the superoxide anion and the hydroxyl radical, and
  • oxidants such as hydrogen peroxide [3].

ROS and electrophiles can cause diseases such as cancer, cardiovascular complications, acute and chronic inflammation, and neurodegenerative diseases [1]. Therefore, it is obvious that

  • cells must constantly labor to control levels of ROS, preventing them from accumulation.

Much of what we know about the mechanisms of protection against oxidative stress has come from the study of prokaryotic cells [4,5]. Prokaryotic cells utilize transcription factors OxyR and SoxRS to sense the redox state of the cell, and

  • during oxidative stress these factors induce the expression of nearly eighty defensive genes [5].

Eukaryotic cells have similar mechanisms to protect against oxidative stress [Fig. 1; ref. 3,6–9]. Initial effect of oxidative/electrophilic stress leads to activation of a battery of defensive gene expression that leads to detoxification of chemicals and ROS and prevention of free radical generation and cell survival [Fig. 1].

Fig 1.  Chemical and radiation exposure and coordinated induction of defensive genes.

Fig. 1. Chemical and radiation exposure and coordinated induction of defensive genes.

Of these genes, some are enzymes such as NAD(P)H:quinine oxidoreductase 1 (NQO1), NRH:quinone oxidoreductase 2 (NQO2), glutathione S-transferase Ya subunit (GST Ya Subunit), heme oxygenase 1 (HO-1), and γ-glutamylcysteine synthetase (γ-GCS), also known as glutamate cysteine ligase (GCL). Other genes have end products that regulate a wide variety of cellular activities including

  • signal transduction,
  • proliferation, and
  • immunologic defense reactions.

There is a wide variety of factors associated with the cellular response to oxidative stress. For example,

  • NF-E2 related factor 2 (Nrf2),
  • heat shock response activator protein 1, and
  • NF-kappaB promote cell survival,

whereas activation of c-jun, N-terminal kinases (JNK), p38 kinase and TP53 may lead to cell cycle arrest and apoptosis [10]. The Nrf2 pathway is regarded as the most important in the cell to protect against oxidative stress. [3,6–9]. It is noteworthy that accumulation of ROS and/or electrophiles leads to oxidative/electrophile stress,

  • membrane damage,
  • DNA adducts formation and
  • mutagenicity [Fig. 1].

These changes lead to degeneration of tissues and premature aging, apoptotic cell death, cellular transformation and cancer.

Antioxidant Response Element and Nrf2

Promoter analysis identified a cis-acting enhancer sequence designated as the antioxidant response element (ARE) that

  • controls the basal and inducible expression of antioxidant genes in response to xenobiotics, antioxidants, heavy metals and UV light [11].

The ARE sequence is responsive to a broad range of structurally diverse chemicals apart from β-nafthoflavone and phenolic antioxidants [12]. Mutational analysis revealed GTGACA***GC to be the core sequence of the ARE [11,13–14]. This core sequence is present in all Nrf2 downstream genes that respond to antioxidants and xenobiotics [3,6–9]. Nrf2 binds to the ARE and regulates ARE-mediated antioxidant enzyme genes expression and induction in response to a variety of stimuli including antioxidants, xenobiotics, metals, and UV irradiation [6,15–21].

Nrf2 is ubiquitously expressed in a wide range of tissue and cell types [22–24] and belongs to a subset of basic leucine zipper genes (bZIP) sharing a conserved structural domain designated as a cap’n’collar domain which is highly conserved in Drosphila transcription factor CNC (Fig. 2; ref. 25].

Fig. 2. Schematic Presentation of Various Domains of Nrf (Nrf1, Nrf2, Nrf3) and INrf2

Fig. 2. Schematic Presentation of Various Domains of Nrf (Nrf1, Nrf2, Nrf3) and INrf2

Nrf, NF-E2 Related Factor; INrf2, Inhibitor of Nrf2; NTR, N-Terminal Region; BTB, Broad complex, Tramtrack, Bric-a-brac; IVR, Intervening/linker Region; DGR, Kelch domain/ diglycine repeats; CTR, C-Terminal Region.

The basic region, just upstream of the leucine zipper region,

  • is responsible for DNA binding [3] and
  • the acidic region is required for transcriptional activation.

ARE-mediated transcriptional activation requires heterodimerization of Nrf2 with other bZIP proteins including Jun (c-Jun, Jun-D, and Jun-B) and small Maf (MafG, MafK, MafF) proteins [18– 20,26–27].

Initial evidence demonstrating the role of Nrf2 in antioxidant-induction of detoxifying enzymes came from studies on

  • the role of Nrf2 in ARE-mediated regulation of NQO1 gene expression [17].

Nrf2 was subsequently shown to be involved in

  • the transcriptional activation of other ARE-responsive genes such as
    • GST Ya, γ-GCS, HO-1, antioxidants, proteasomes, and drug transporters [3,6–9,28–33].

Overexpression of Nrf2 cDNA was shown to upregulate the expression and induction of the NQO1 gene in response to antioxidants and xenobiotics [17]. In addition, Nrf2-null mice exhibited a marked

  • decrease in the expression and induction of NQO1,
  • indicating that Nrf2 plays an essential role in the in vivo regulation of NQO1 in response to oxidative stress [26].

The importance of this transcription factor in upregulating ARE-mediated gene expression has been demonstrated by several in vivo and in vitro studies [reviewed in ref. 3]. The results indicate that Nrf2 is an important activator of phase II antioxidant genes [3,8].

Negative Regulation of Nrf2 mediated by INrf2

A cytosolic inhibitor (INrf2), also known as Keap1 (Kelch-like ECH-associating protein 1), of Nrf2 was identified and reported [Fig. 2; ref. 34–35]. INrf2, existing as a dimer [36], retains Nrf2 in the cytoplasm. Analysis of the INrf2 amino acid sequence and domain structure-function analyses have revealed that

  • INrf2 has a BTB (broad complex, tramtrack, bric-a-brac)/ POZ (poxvirus, zinc finger) domain and
  • a Kelch domain [34–35] also known as the DGR domain (Double glycine repeat) [37].

Keap1 has three additional domains/regions:

  1. the N-terminal region (NTR),
  2. the invervening region (IVR), and
  3. the C-terminal region (CTR) [8].

The BTB/POZ domain has been shown to be

  • a protein-protein interaction domain.

In the Drosophila Kelch protein, and in IPP,

  • the Kelch domain binds to actin [38–39]
  • allowing the scaffolding of INrf2 to the actin cytoskeleton
    • which plays an important role in Nrf2 retention in the cytosol [40].

The main function of INrf2 is to serve as

  • an adapter for the Cullin3/Ring Box 1 (Cul3/Rbx1) E3 ubiquitin ligase complex [41–43].

Cul3 serves as a scaffold protein that forms the E3 ligase complex with Rbx1 and recruits a cognate E2 enzyme [8].

INrf2

  1. via its N-terminal BTB/POZ domain binds to Cul3 [44] and
  2. via its C-terminal Kelch domain binds to the substrate Nrf2
  • leading to the ubiquitination and degradation of Nrf2 through the 26S proteasome [45–49].

Under normal cellular conditions, the cytosolic INrf2/Cul3-Rbx1 complex is constantly degrading Nrf2. When a cell is exposed to oxidative stress Nrf2 dissociates from the INrf2 complex, stabilizes and translocates into the nucleus leading to activation of ARE-mediated gene expression [3,6–9]. An alternative theory is that Nrf2 in response to oxidative stress escapes INrf2 degradation, stabilizes and translocates in the nucleus [49–50]. We suggested the theory of escape of Nrf2 from INrf2 [49] and similar suggestion was also made in another report [50]. However, the follow up studies in our laboratory could not support the escape theory. Escape theory is a possibility but has to be proven by experiments before it can be adapted. Therefore, we will use the release of Nrf2 from INrf2 in the rest of this review.

Numerous reports have suggested that

  • any mechanism that modifies INrf2 and/or Nrf2 disrupting the Nrf2:INrf2 interaction will result in the upregulation of ARE-mediated gene expression.

A model Nrf2:INrf2 signaling from antioxidant and xenobiotic to activation of ARE-mediated defensive gene expression is shown in Fig. 3.

Fig. 3. Nrf2 signaling in ARE-mediated coordinated activation of defensive genes

Fig. 3. Nrf2 signaling in ARE-mediated coordinated activation of defensive genes

Since the metabolism of antioxidants and xenobiotics results in the generation of ROS and electrophiles [51], it is thought that these molecules might act as second messengers, activating ARE-mediated gene expression. Several protein kinases including PKC, ERK, MAPK, p38, and PERK [49,52– 56] are known to modify Nrf2 and activate its release from INrf2. Among these mechanisms,

  1. oxidative/electrophilic stress mediated phosphorylation of Nrf2 at serine40 by PKC is necessary for Nrf2 release from INrf2, but
  2. is not required for Nrf2 accumulation in the nucleus [49,52–53].

In addition to post-translational modification in Nrf2, several crucial residues in INrf2 have also been proposed to be important for activation of Nrf2. Studies based on

  • the electrophile mediated modification,
  • location and
  • mutational analyses revealed
    • that three cysteine residues, Cys151, Cys273 and Cys288 are crucial for INrf2 activity [50].

INrf2 itself undergoes ubiquitination by the Cul3 complex, via a proteasomal independent pathway,

  • which was markedly increased in response to phase II inducers such as antioxidants [57].

It has been suggested that normally INrf2 targets Nrf2 for ubiquitin mediated degradation but

  • electrophiles may trigger a switch of Cul3 dependent ubiquitination from Nrf2 to INrf2 resulting in ARE gene induction.

The redox modulation of cysteines in INrf2

  • might be a mechanism redundant to the phosphorylation of Nrf2 by PKC, or that
  • the two mechanisms work in concert.

In addition to cysteine151 modification,

  • phosphorylation of Nrf2 has also been shown to play a role in INrf2 retention and release of Nrf2.

Serine104 of INrf2 is required for dimerization of INrf2, and

  • mutations of serine104 led to the disruption of the INrf2 dimer leading to the release of Nrf2 [36].

Recently, Eggler at al. demonstrated that modifying specific cysteines of the electrophile-sensing human INrf2 protein is insufficient to disrupt binding to the Nrf2 domain Neh2 (58). Upon introduction of electrophiles, modification of INrf2C151 leads to a change in the conformation of the BTB domain by means of perturbing the homodimerization site, disrupting Neh2 ubiquitination, and causing ubiquitination of INrf2. Modification of INrf2 cysteines by electrophiles does not lead to disruption of the INrf2–Nrf2 complex. Rather, the switch of ubiquitination from Nrf2 to INrf2 leads to Nrf2 nuclear accumulation.

More recently, our laboratory demonstrated that phosphorylation and de-phosphorylation of tyrosine141 in INrf2 regulates its stability and degradation, respectively [59]. The de-phosphorylation of tyrosine141 caused destabilization and degradation of INrf2 leading to the release of Nrf2. Furthermore, we showed that prothymosin-α mediates nuclear import of the INrf2/Cul3-Rbx1 complex [60]. The INrf2/Cul3-Rbx1 complex inside the nucleus exchanges prothymosin-α with Nrf2 resulting in degradation of Nrf2. These results led to the conclusion that prothymosin-α mediated nuclear import of INrf2/Cul3-Rbx1 complex leads to ubiquitination and degradation of nuclear Nrf2 presumably to regulate nuclear level of Nrf2 and rapidly switch off the activation of Nrf2 downstream gene expression. An auto-regulatory loop also exists within the Nrf2 pathway [61]. An ARE was identified in the INrf2 promoter that facilitates Nrf2 binding causing induction of the INrf2 gene. Nrf2 regulates INrf2 by controlling its transcription, and INrf2 controls Nrf2 by serving as an adaptor for degradation.

Other Regulatory Mediators of Nrf2

Bach1 (BTB and CNC homology 1, basic leucine zipper transcription factor 1) is a transcription repressor [62] that is ubiquitously expressed in tissues [63–64] and distantly related to Nrf2 [8]. In the absence of cellular stress, Bach1 heterodimers with small Maf proteins [65] that bind to the (ARE) [66] repressing gene expression. In the presence of oxidative stress, Bach1 releases from the ARE and is replaced by Nrf2. Bach1 competes with Nrf2 for binding to the ARE leading to suppression of Nrf2 downstream genes [66].

Nuclear import of Nrf2, from time of exposure to stabilization, takes roughly two hours [67]. This is followed by activation of a delayed mechanism involving Glycogen synthase kinase 3 beta (GSK3f3) that controls switching off of Nrf2 activation of gene expression (Fig. 3). GSK3f3 is a multifunctional serine/threonine kinase, which plays a major role in various signaling pathways [68]. GSK3f3 phosphorylates Fyn, a tyrosine kinase, at unknown threonine residue(s) leading to nuclear localization of Fyn [69]. Fyn phosphorylates Nrf2 tyrosine 568 resulting in nuclear export of Nrf2, binding with INrf2 and degradation of Nrf2 [70].

The negative regulation of Nrf2 by Bach1 and GSK3f3/Fyn are important in repressing Nrf2 downstream genes that were induced in response to oxidative/electrophilic stress. The tight control of Nrf2 is vital for the cells against free radical damage, prevention of apoptosis and cell survival [3,6–9,70].

Nrf2 in Cytoprotection, Cancer and Drug Resistance

Nrf2 is a major protective mechanism against xenobiotics capable of damaging DNA and initiating carcinogenesis [71]. Inducers of Nrf2 function as blocking agents that prevents carcinogens from reaching target sites, inhibits parent molecules undergoing metabolic activation, or subsequently preventing carcinogenic species from interacting with crucial cellular macromolecules, such as DNA, RNA, and proteins [72]. A plausible mechanism by which blocking agents impart their chemopreventive activity is the induction of detoxification and antioxidant enzymes [73]. Oltipraz, 3H-1,2,-dithiole-3-thione (D3T), Sulforaphane, and Curcumin can be considered potential chemopreventive agents because

  • these compounds have all been shown to induce Nrf2 [74–81].

Studies have shown a role of Nrf2 in protection against cadmium and manganese toxicity [82]. Nrf2 also plays an important role in reduction of methyl mercury toxicity [83]. Methylmercury activates Nrf2 and the activation of Nrf2 is essential for reduction of methylmercury by facilitating its excretion into extracellular space. In vitro and in vivo studies have shown a role of Nrf2 in neuroprotection and protection against Parkinson’s disease [84– 86]. Disruption of Nrf2 impairs the resolution of hyperoxia-induced acute lung injury and inflammation in mice [87]. Nrf2-knockout mice were more prone to

  • tumor growth when exposed to carcinogens such as benzo[a]pyrene, diesel exhaust, and N-nitrosobutyl (4-hydroxybutyl) amine [88–90].

INrf2/Nrf2 signaling is also shown to regulate oxidative stress tolerance and lifespan in Drosophila [91].

A role of Nrf2 in drug resistance is suggested based on its property to induce detoxifying and antioxidant enzymes (92–97). The loss of INrf2 (Keap1) function is shown to

  • lead to nuclear accumulation of Nrf2, activation of metabolizing enzymes and drug resistance (95).

Studies have reported mutations resulting in dysfunctional INrf2 in lung, breast and bladder cancers (96–100). A recent study reported that somatic mutations also occur in the coding region of Nrf2, especially in cancer patients with a history of smoking or suffering from squamous cell carcinoma (101). These mutations abrogate its interaction with INrf2 and nuclear accumulation of Nrf2. This gives advantage to

  • cancer cell survival and
  • undue protection from anti-cancer treatments.

However, the understanding of the mechanism of Nrf2 induced drug resistance remains in its infancy. In addition, the studies on Nrf2 regulated downstream pathways that contribute to drug resistance remain limited.

Future Perspectives

Nrf2 creates a new paradigm in cytoprotection, cancer prevention and drug resistance. Considerable progress has been made to better understand all mechanisms involved within the intracellular pathways regulating Nrf2 and its downstream genes. Preliminary studies demonstrate that

  • deactivation of Nrf2 is as important as activation of Nrf2.

Further studies are needed to better understand the negative regulation of Nrf2. Also better understanding of the negative regulation of Nrf2 could help design a new class of effective chemopreventive compounds not only targeting Nrf2 activation, but also

  • targeting the negative regulators of Nrf2.

Abbreviations: 

Nrf2    NF-E2 related factor 2;  INrf2   Inhibitor of Nrf2 also known as Keap1;   ROS    Reactive oxygen species.

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Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K

Author and Curator: Larry H Bernstein, MD, FCAP

 

 

This report from Science Translational Medicine is about the finding that a cardiomyocyte-specific kinase limits reperfusion injury in acute coronary syndrome, a phenomenon driven by oxidative stress, protecting cardiac cells from further damage.

Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K Limits Oxidative Stress, Injury, and Adverse Remodeling in the Ischemic Heart

Ronald J. Vagnozzi1,2,  Gregory J. Gatto Jr.3, Lara S. Kallander3, Nicholas E. Hoffman2, Karthik Mallilankaraman2, Victoria L. T. Ballard3, Brian G. Lawhorn3, …, and Thomas Force2,6,*
+ Author Affiliations
1Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA
2Center for Translational Medicine, and 6Cardiology Division, Temple University School of Medicine, Philadelphia, PA
3Heart Failure Discovery Performance Unit, Metabolic Pathways and Cardiovascular Therapeutic Area Unit, GlaxoSmithKline, King of Prussia, PA
4Platform Technology and Sciences, GlaxoSmithKline, King of Prussia, PA
5Cardiovascular Division, Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya 663-8131, Japan.

Sci Transl Med 16 Oct 2013; 
5(207), p. 207ra141     http://dx.doi.org/10.1126/scitranslmed.3006479
Percutaneous coronary intervention is first-line therapy for acute coronary syndromes (ACS) but can promote cardiomyocyte death and cardiac dysfunction via reperfusion injury, a phenomenon driven in large part by oxidative stress. Therapies to limit this progression have proven elusive, with no major classes of new agents since the development of anti-platelets/anti-thrombotics. We report that cardiac troponin I–interacting kinase (TNNI3K), a cardiomyocyte-specific kinase,
  1. promotes ischemia/reperfusion injury,
  2. oxidative stress,
  3. and myocyte death.
TNNI3K-mediated injury occurs
  • through increased mitochondrial superoxide production and
  • impaired mitochondrial function and is largely
  • dependent on p38 mitogen-activated protein kinase (MAPK) activation.

We developed a series of small-molecule TNNI3K inhibitors that

  1. reduce mitochondrial-derived superoxide generation,
  2. p38 activation, and
  3. infarct size
when delivered at reperfusion to mimic clinical intervention.
TNNI3K inhibition also preserves cardiac function and limits chronic adverse remodeling.
Our findings demonstrate that TNNI3K modulates reperfusion injury in the ischemic heart and is a tractable therapeutic target for ACS.  Pharmacologic TNNI3K inhibition would be cardiac-selective,
  • preventing potential adverse effects of systemic kinase inhibition.
Citation: R. J. Vagnozzi, G. J. Gatto, L. S. Kallander, N. E. Hoffman, K. Mallilankaraman, V. L. T. Ballard, B. G. Lawhorn, P. Stoy, J. Philp, A. P. Graves, Y. Naito, J. J. Lepore, E. Gao, M. Madesh, T. Force, Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K Limits Oxidative Stress, Injury, and Adverse Remodeling in the Ischemic Heart. Sci. Transl. Med. 5, 207ra141 (2013).

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