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Protein kinase C-[beta]II (PKC[beta]II) is an important modulator of cellular stress responses. To test the hypothesis that PKC[beta]II modulates the response to myocardial ischemia-reperfusion (I/R) injury, we subjected mice to occlusion and reperfusion of the left anterior descending coronary artery. Homozygous PKC[beta]-null ([PKC[beta].sup.-/-]) and wildtype mice fed the PKC[beta] inhibitor ruboxistaurin displayed significantly decreased infarct size and enhanced recovery of left ventricular (LV) function and reduced markers of cellular necrosis and serum creatine phosphokinase and lactate dehydrogenase levels compared with wild-type or vehicle-treated animals after 30 rain of ischemia followed by 48 h of reperfusion. Our studies revealed that membrane translocation of PKC[beta]II in LV tissue was sustained after I/R and that gene deletion or pharmacological blockade of PKC[beta] protected ischemic myocardium. Homozygous deletion of PKC[beta] significantly diminished phosphorylation of c-Jun N[H.sub.2]-terminal mitogen-activated protein kinase and expression of activated caspase-3 in LV tissue of mice subjected to I/R. These data implicate PKC[beta] in I/R-mediated myocardial injury, at least in part via phosphorylation of JNK, and suggest that blockade of PKC[beta] may represent a potent strategy to protect the vulnerable myocardium. myocardial ischemia; JNK; caspase-3

The receptor for advanced glycation end-products (RAGE) has been implicated in the pathogenesis of ischemia-reperfusion (I/R) injury in the isolated perfused heart. To test the hypothesis that RAGE-dependent mechanisms modulated responses to I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD), we subjected male homozygous [RAGE.sup.-/-] mice and their wild-type age-matched littermates to 30 min of occlusion of the LAD followed by reperfusion. At 48 h of reperfusion, hematoxylin and eosin staining revealed significantly larger infarct size in wild-type versus [RAGE.sup.-/-] mice. Contractile function, as evaluated by echocardiography 48 h after reperfusion, revealed that fractional shortening was significantly higher in [RAGE.sup.-/-] versus wild-type mice. Plasma levels of creatine kinase were markedly decreased in [RAGE.sup.-/-] versus wild-type animals. Integral to the impact of RAGE deletion on diminished myocardial damage after infarction was significantly decreased apoptosis in the heart, as assessed by TUNEL staining, release of cytochrome c, and caspase-3 activity. Experiments investigating the impact of RAGE on early signaling pathways influencing myocardial ischemic injury revealed attenuation of JNK and STAT5 phosphorylation in [RAGE.sup.-/-] mouse hearts versus robust activation observed in wild-type mice upon ischemia and reperfusion. Solidifying the link to RAGE, these experiments revealed that infarction stimulated the rapid production of advanced glycation endproducts in the heart. Thus, we tested the effect of ligand decoy soluble RAGE (sRAGE). Administration of sRAGE protected the myocardium from ischemic damage, similar to the effects observed in [RAGE.sup.-/-] mouse hearts. Taken together, these data implicate RAGE and its ligands in the pathogenesis of I/R injury and identify JNK and STAT signal transduction as central downstream effector pathways of the ligand-RAGE axis in the heart subjected to I/R injury. receptor for advanced glycation end-products; glycation end-products; myocardial infarction; left anterior descending coronary artery

Endothelial dysfunction is a key triggering event in atherosclerosis. Following the entry of lipoproteins into the vessel wall, their rapid modification results in the generation of advanced glycation endproduct epitopes and subsequent infiltration of inflammatory cells. These inflammatory cells release receptor for advanced glycation endproduct (RAGE) ligands, specifically S100/calgranulins and high-mobility group box 1, which sustain vascular injury. Here, we demonstrate critical roles for RAGE and its ligands in vascular inflammation, endothelial dysfunction, and atherosclerotic plaque development in a mouse model of atherosclerosis, apoE-/- mice. Experiments in primary aortic endothelial cells isolated from mice and in cultured human aortic endothelial cells revealed the central role of JNK signaling in transducing the impact of RAGE ligands on inflammation. These data highlight unifying mechanisms whereby endothelial RAGE and its ligands mediate vascular and inflammatory stresses that culminate in atherosclerosis in the vulnerable vessel wall., Introduction The metabolic and biochemical milieu generated upon entry of lipid into the vessel wall drives the generation of ligands of the receptor for advanced glycation endproduct (RAGE), including advanced [...]

Multiple sclerosis (MS) is a devastating neuroinflammatory disorder of the central nervous system (CNS) in which T cells that are reactive with major components of myelin sheaths have a central role. The receptor for advanced glycation end products (RAGE) is present on T cells, mononuclear phagocytes and endothelium. Its pro-inflammatory ligands, S100-calgranulins, are upregulated in MS and in the related rodent model, experimental autoimmune encephalomyelitis (EAE). Blockade of RAGE suppressed EAE when disease was induced by myelin basic protein (MBP) peptide or encephalitogenic T cells, or when EAE occurred spontaneously in T-cell receptor (TCR)-transgenic mice devoid of endogenous TCR-[alpha] and TCR-[beta] chains. Inhibition of RAGE markedly decreased infiltration of the CNS by immune and inflammatory cells. Transgenic mice with targeted overexpression of dominant-negative RAGE in CD4[sup.+] T cells were resistant to MBP-induced EAE. These data reinforce the importance of RAGE-ligand interactions in modulating properties of CD4[sup.+] T cells that infiltrate the CNS., Author(s): Shirley ShiDu Yan (corresponding author) [1]; Zhi-Ying Wu [1, 2]; Hui Ping Zhang [1]; Glaucia Furtado [3]; Xi Chen [4]; Shi Fang Yan [1]; Ann Marie Schmidt [1]; Chris [...]

Carbon monoxide (CO) can arrest cellular respiration, but paradoxically, it is synthesized endogenously by heme oxygenase type 1 (Ho-1) in response to ischemic stress. Ho-1-deficient (Hmox1[sup.-/-]) mice exhibited lethal ischemic lung injury, but were rescued from death by inhaled CO. CO drove ischemic protection by activating soluble guanylate cyclase and thereby suppressed hypoxic induction of the gene encoding plasminogen activator inhibitor-1 (PAI-1) in mononuclear phagocytes, which reduced accrual of microvascular fibrin. CO-mediated ischemic protection observed in wild-type mice was lost in mice null for the gene encoding PAI-1 (Serpine1). These data establish a fundamental link between CO and prevention of ischemic injury based on the ability of CO to derepress the fibrinolytic axis. These data also point to a potential therapeutic use for inhaled CO., Author(s): Tomoyuki Fujita [1]; Koichi Toda [1]; Ann Karimova [1]; Shi-Fang Yan [1]; Yoshifumi Naka [1]; Shaw-Fang Yet [2]; David J. Pinsky (corresponding author) [1] Carbon monoxide (CO), once thought [...]

Activation of the zinc-finger transcription factor early growth response (Egr)-1, initially linked to developmental processes, is shown here to function as a master switch activated by ischemia to trigger expression of pivotal regulators of inflammation, coagulation and vascular hyperpermeability. Chemokine, adhesion receptor, procoagulant and permeability-related genes are coordinately upregulated by rapid ischemia-mediated activation of Egr-1. Deletion of the gene encoding Egr-1 strikingly diminished expression of these mediators of vascular injury in a murine model of lung ischemia/reperfusion, and enhanced animal survival and organ function. Rapid activation of Egr-1 in response to oxygen deprivation primes the vasculature for dysfunction manifest during reperfusion. These studies define a central and unifying role for Egr-1 activation in the pathogenesis of ischemic tissue damage., Author(s): Shi-Fang Yan (corresponding author) [1]; Tomoyuki Fujita [1]; Jiesheng Lu [1]; Kenji Okada [1]; Yu Shan Zou [1]; Nigel Mackman [4]; David J. Pinsky [3]; David M. Stern [1, [...]

Local hypoxemia and stasis trigger thrombosis. We have demonstrated previously that in a murine model of normobaric hypoxia pulmonary fibrin deposition is a result of expression of tissue factor, especially in oxygen-deprived mononuclear phagocytes (MPs). We now show that transcription factor early-growth-response gene product (Egr-1) is rapidly activated in hypoxia, both in vitro and in vivo, and is responsible for transcription and expression of tissue factor in hypoxic lung. MPs and HeLa cells subjected to hypoxia (p[O.sub.2] [approximately equal to]13 torr) had increased levels of tissue factor transcripts ([approximately equal to]18-fold) and an increased rate of transcription ([approximately equal to] 15-fold), based on nuclear run-on analysis. Gel-shift analysis of nuclear extracts from hypoxic MPs and HeLa cells demonstrated increased DNA-binding activity at the serum response region (SRR; -111/+14 bp) of the tissue factor promoter at Egr-1 motifs. Using 32P-labeled Egr consensus oligonucleotide, we observed induction of DNA-binding activity in nuclear extracts from hypoxic lung and HeLa cells because of activation of Egr-1, by means of supershift analysis. Transient transfection of HeLa cells with chimeric plasmids containing wild-type or mutant SRR from the tissue factor promoter showed that intact Spl sites are necessary for basal promoter activity, whereas the integrity of Egr-1 sites was required for hypoxia-enhanced expression. A central role for Egr-1 in hypoxia-mediated tissue factor expression was confirmed by experiments with homozygous Egr-1 null mice; wild-type mice subjected to oxygen deprivation expressed tissue factor and showed fibrin deposition, but hypoxic homozygous Egr-1 null mice displayed neither tissue factor nor fibrin. These data delineate a novel biology for hypoxia-induced fibrin deposition, in which oxygen deprivation-induced activation of Egr-1, resulting in expression of tissue factor, has an unexpected and central role.

In Alzheimer disease (AD), neurons are thought to be subjected to the deleterious cytotoxic effects of activated microglia. We demonstrate that binding of amyloid-beta peptide (A[Beta]) to neuronal Receptor for Advanced Glycation Endproduct (RAGE), a cell surface receptor for A[Beta], induces macrophage-colony stimulating factor (M-CSF) by an oxidant sensitive, nuclear factor [Kappa]B-dependent pathway. AD brain shows increased neuronal expression of M-CSF in proximity to A[Beta] deposits, and in cerebrospinal fluid from AD patients there was [approximately equal to]5-fold increased M-CSF antigen (P < 0.01), compared with age-matched controls. M-CSF released by A[Beta]-stimulated neurons interacts with its cognate receptor, c-fms, on microglia, thereby triggering chemotaxis, cell proliferation, increased expression of the macrophage scavenger receptor and apolipoprotein E, and enhanced survival of microglia exposed to A[Beta], consistent with pathologic findings in AD. These data delineate an inflammatory pathway triggered by engagement of A[Beta] on neuronal RAGE. We suggest that M-CSF, thus generated, contributes to the pathogenesis of AD, and that M-CSF in cerebrospinal fluid might provide a means for monitoring neuronal perturbation at an early stage in AD.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.01.002 Byline: Ravichandran Ramasamy (1), Shi Fang Yan (1), Ann Marie Schmidt (1) Abstract: The posttranslational modification of proteins by methylglyoxal, a highly reactive compound derived from glycolysis, may contribute to aging, diabetes, and other disorders. In this issue of Cell, Brownlee and colleagues () demonstrate a specific mechanism by which methylglyoxal modifies a transcriptional corepressor to enhance gene expression. Author Affiliation: (1) Department of Surgery, Columbia University Medical Center, New York, NY 10032, USA