Faculty Bibliography

Myocardial infarction, stroke, and venous thromboembolism are characterized by oxygen deprivation. In hypoxia, biological responses are activated that evoke tissue damage. Rapid activation of early growth response-1 in hypoxia upregulates fundamental inflammatory and prothrombotic stress genes. We probed the mechanisms mediating regulation of early growth response-1 and demonstrate that hypoxia stimulates brisk generation of advanced glycation end products (AGEs) by endothelial cells. Via AGE interaction with their chief signaling receptor, RAGE, membrane translocation of protein kinase C-betaII occurs, provoking phosphorylation of c-Jun NH(2)-terminal kinase and increased transcription of early growth response-1 and its downstream target genes. These findings identify RAGE as a master regulator of tissue stress elicited by hypoxia and highlight this receptor as a central therapeutic target to suppress the tissue injury-provoking effects of oxygen deprivation

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(-/-) 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(-/-) mice. Contractile function, as evaluated by echocardiography 48 h after reperfusion, revealed that fractional shortening was significantly higher in RAGE(-/-) versus wild-type mice. Plasma levels of creatine kinase were markedly decreased in RAGE(-/-) 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(-/-) 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 end-products 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(-/-) 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

Protein kinase C-betaII (PKCbetaII) is an important modulator of cellular stress responses. To test the hypothesis that PKCbetaII 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 PKCbeta-null (PKCbeta(-/-)) and wild-type mice fed the PKCbeta 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 min of ischemia followed by 48 h of reperfusion. Our studies revealed that membrane translocation of PKCbetaII in LV tissue was sustained after I/R and that gene deletion or pharmacological blockade of PKCbeta protected ischemic myocardium. Homozygous deletion of PKCbeta significantly diminished phosphorylation of c-Jun NH(2)-terminal mitogen-activated protein kinase and expression of activated caspase-3 in LV tissue of mice subjected to I/R. These data implicate PKCbeta in I/R-mediated myocardial injury, at least in part via phosphorylation of JNK, and suggest that blockade of PKCbeta may represent a potent strategy to protect the vulnerable myocardium

The multiligand receptor for advanced glycation end products (RAGE) of the immunoglobulin superfamily is expressed on multiple cell types implicated in the immune-inflammatory response and in atherosclerosis. Multiple studies have elucidated that ligand-RAGE interaction on cells, such as monocytes, macrophages, and endothelial cells, mediates cellular migration and upregulation of proinflammatory and prothrombotic molecules. In addition, recent studies reveal definitive rules for RAGE in effective T lymphocyte priming in vivo. RAGE ligand AGEs may be formed in diverse settings; although AGEs are especially generated in hyperglycemia, their production in settings characterized by oxidative stress and inflammation suggests that these species, in part via RAGE, may contribute to the pathogenesis of atherosclerosis. In murine models of atherosclerosis, vascular inflammation is a key factor and one which is augmented, in parallel with even further increases in RAGE ligands, in diabetic macrovessels. The findings that antagonism and genetic disruption of RAGE in atherosclerosis-susceptible mice strikingly reduces vascular inflammation and atherosclerotic lesion area and complexity link RAGE intimately to these processes and suggest that RAGE is a logical target for therapeutic intervention in aberrant inflammatory mechanisms and in atherosclerosis

OBJECTIVE: Heightened expression of the receptor for advanced glycation end products (RAGE) contributes to development of systemic diabetic complications, but its contribution to diabetic neuropathy is uncertain. We studied experimental diabetic neuropathy and its relationship with RAGE expression using streptozotocin-induced diabetic mice including a RAGE(-/-) cohort exposed to long-term diabetes compared with littermates without diabetes. RESEARCH DESIGN AND METHODS: Structural indexes of neuropathy were addressed with serial (1, 3, 5, and 9 months of experimental diabetes) electrophysiological and quantitative morphometric analysis of dorsal root ganglia (DRG), peripheral nerve, and epidermal innervation. RAGE protein and mRNA levels in DRG, peripheral nerve, and epidermal terminals were assessed in WT and RAGE(-/-) mice, with and without diabetes. The correlation of RAGE activation with nuclear factor (NF)-kappaB and protein kinase C beta II (PKC beta II) protein and mRNA expression was also determined. RESULTS: Diabetic peripheral epidermal axons, sural axons, Schwann cells, and sensory neurons within ganglia developed dramatic and cumulative rises in RAGE mRNA and protein along with progressive electrophysiological and structural abnormalities. RAGE(-/-) mice had attenuated structural features of neuropathy after 5 months of diabetes. RAGE-mediated signaling pathway activation for NF-kappaB and PKC beta II pathways was most evident among Schwann cells in the DRG and peripheral nerve. CONCLUSIONS: In a long-term model of experimental diabetes resembling human diabetic peripheral neuropathy, RAGE expression in the peripheral nervous system rises cumulatively and relates to progressive pathological changes. Mice lacking RAGE have attenuated features of neuropathy and limited activation of potentially detrimental signaling pathways.

Soluble amyloid-beta (Abeta) peptide is likely to play a key role during early stages of Alzheimer's disease (AD) by perturbing synaptic function and cognitive processes. Receptor for advanced glycation end products (RAGE) has been identified as a receptor involved in Abeta-induced neuronal dysfunction. We investigated the role of neuronal RAGE in Abeta-induced synaptic dysfunction in the entorhinal cortex, an area of the brain important in memory processes that is affected early in AD. We found that soluble oligomeric Abeta peptide (Abeta42) blocked long-term potentiation (LTP), but did not affect long-term depression, paired-pulse facilitation, or basal synaptic transmission. In contrast, Abeta did not inhibit LTP in slices from RAGE-null mutant mice or in slices from wild-type mice treated with anti-RAGE IgG. Similarly, transgenic mice expressing a dominant-negative form of RAGE targeted to neurons showed normal LTP in the presence of Abeta, suggesting that neuronal RAGE functions as a signal transducer for Abeta-mediated LTP impairment. To investigate intracellular pathway transducing RAGE activation by Abeta, we used inhibitors of stress activated kinases. We found that inhibiting p38 mitogen-activated protein kinase (p38 MAPK), but not blocking c-Jun N-terminal kinase activation, was capable of maintaining LTP in Abeta-treated slices. Moreover, Abeta-mediated enhancement of p38 MAPK phosphorylation in cortical neurons was reduced by blocking antibodies to RAGE. Together, our results indicate that Abeta impairs LTP in the entorhinal cortex through neuronal RAGE-mediated activation of p38 MAPK

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

OBJECTIVE: The receptor for advanced glycation end products (RAGE) has been proposed to participate in the innate and adaptive immune responses. RAGE can induce production of pro-inflammatory cytokines and chemokines, as well as neutrophil chemotaxis in a manner that may be suppressed or stimulated by soluble, truncated forms of RAGE including the soluble form of RAGE (sRAGE) and endogenous secretory RAGE (esRAGE). The objective of this study was to determine whether intra-amniotic infection/inflammation (IAI) is associated with changes in the amniotic fluid concentration of sRAGE and esRAGE. STUDY DESIGN: Amniotic fluid (AF) was retrieved from patients in the following groups: 1) mid-trimester (14-18 weeks of gestation; n=68); 2) term not in labor (n=24); 3) term in labor (n=51); 4) preterm labor and intact membranes (n=124); and 5) preterm PROM (n=80). Intra-amniotic infection and inflammation were defined as the presence of a positive amniotic fluid culture for microorganisms and an AF interleukin-6 concentration >or=2.6 ng/mL, respectively. The AF concentration of sRAGE and esRAGE were determined using specific and sensitive ELISAs which measured total immunoreactive sRAGE and esRAGE, respectively. Patients were matched for gestational age at amniocentesis to compare the AF concentration of sRAGE and esRAGE in patients with and without IAI. Non-parametric statistics were used for analysis and a P<0.05 was considered significant. RESULTS: 1) Patients at term not in labor had higher median AF concentrations of sRAGE and esRAGE than those in the mid-trimester (P<0.001 for both comparisons) and those at term in labor (P=0.03 and P=0.04, respectively); 2) patients with preterm labor and intact membranes with intra-amniotic infection/inflammation (IAI) had higher median AF concentrations of sRAGE and esRAGE than those without IAI (P=0.02 and P=0.005, respectively); 3) similarly, patients with preterm PROM with IAI had higher median AF concentrations of sRAGE and esRAGE than those without IAI (P=0.03 and P=0.02, respectively). CONCLUSION: Intra-amniotic infection/inflammation is associated with increased amniotic fluid concentrations of sRAGE and esRAGE. Changes in the amniotic fluid concentration of sRAGE and esRAGE may represent part of the immune response to intra-amniotic infection/inflammation.

Receptor for advanced glycation end products (RAGE) is an activation receptor triggered by inflammatory S100/calgranulins and high mobility group box-1 ligands. We have investigated the importance of RAGE on Ag priming of T cells in murine models in vivo. RAGE is inducibly up-regulated during T cell activation. Transfer of RAGE-deficient OT II T cells into OVA-immunized hosts resulted in reduced proliferative responses that were further diminished in RAGE-deficient recipients. Examination of RAGE-deficient dendritic cells did not reveal functional impairment in Ag presentation, maturation, or migratory capacities. However, RAGE-deficient T cells showed markedly impaired proliferative responses in vitro to nominal and alloantigens, in parallel with decreased production of IFN-gamma and IL-2. These data indicate that RAGE expressed on T cells is required for efficient priming of T cells and elucidate critical roles for RAGE engagement during cognate dendritic cell-T cell interactions

Activation of the receptor for advanced glycation endproducts (RAGE) by its multiple ligands can trigger diverse signaling pathways with injurious or pro-survival consequences. In this study, we show that Rage mRNA and protein levels were stimulated in the mouse brain after experimental stroke and systemic hypoxia. In both cases, RAGE expression was primarily associated with neurons. Activation of RAGE-dependent pathway(s) post-ischemia appears to have a neuroprotective role because mice genetically deficient for RAGE exhibited increased infarct size 24 h after injury. Up-regulation of RAGE expression was also observed in primary neurons subjected to hypoxia or oxygen-glucose deprivation, an in vitro model of ischemia. Treatment of neurons with low concentrations of S100B decreased neuronal death after oxygen-glucose deprivation, and this effect was abolished by a neutralizing antibody against RAGE. Conversely, high concentrations of exogenous S100B had a cytotoxic effect that seems to be RAGE-independent. As an important novel finding, we demonstrate that hypoxic stimulation of RAGE expression is mediated by the transcription factor hypoxia-inducible factor-1. This conclusion is supported by the finding that HIF-1alpha down-regulation by Cre-mediated excision drastically decreased RAGE induction by hypoxia or desferrioxamine. In addition, we showed that the mouse RAGE promoter region contains at least one functional HIF-1 binding site, located upstream of the proposed transcription start site. A luciferase reporter construct containing this RAGE promoter fragment was activated by hypoxia, and mutation at the potential HIF-1 binding site decreased hypoxia-dependent promoter activation. Specific binding of HIF-1 to this putative HRE in hypoxic cells was detected by chromatin immunoprecipitation assay.