Faculty Bibliography

Early growth response-1 (Egr-1) regulates expression of proinflammatory and procoagulant genes in acute cell stress. Experimental evidence suggested that Egr-1 transcripts were upregulated in human atherosclerotic plaques versus adjacent unaffected tissue. To test the impact of Egr-1 in chronic vascular stress, we examined its role in a murine model of atherosclerosis. Real-time PCR analysis of aortae retrieved from apoE-/- mice demonstrated increased Egr-1 transcripts in an age-dependent manner, compared with aortae retrieved from C57BL/6 control animals. Therefore, homozygous Egr-1-/- mice were bred into the apoE-/- background. Homozygous double-knockout mice (Egr-1-/-/apoE-/-) in the C57BL/6 background were maintained on normal chow diet. At age 14 and 24 weeks, atherosclerotic lesion area and complexity at the aortic root were strikingly decreased in mice deficient in both Egr-1 and apoE compared with mice deficient in apoE alone. In parallel, transcripts for genes regulating the inflammatory/prothrombotic response were diminished in Egr-1-/-/apoE-/- aortae versus apoE-/-. In vitro, oxidized low-density lipoprotein (OxLDL), a key factor inciting atherogenic mechanisms in the vasculature, upregulated Egr-1 expression in monocytes via the MEK-ERK1/2 pathway. We conclude that Egr-1 broadly regulates expression of molecules critically linked to atherogenesis and lesion progression

S100P is a member of the S100 protein family that is expressed in several malignant neoplasms. Currently the effects of this molecule on cell function are unknown. In the present study we investigated the biological effects and mechanisms of action of S100P using NIH3T3 cells. Expression of S100P in NIH3T3 cells led to the presence of S100P in the culture medium, increased cellular proliferation, and enhanced survival after detachment from the culture substrate or after exposure to the chemotherapeutic agent 5-flurouracil. The proliferation and survival effects of S100P expression were duplicated in a time- and concentration-dependent manner by the extracellular addition of purified S100P to wild-type NIH3T3 cells and correlated with the activation of extracellular-regulated kinases (Erks) and NF-kappaB. To determine the mechanisms involved in these effects, we tested the hypothesis that S100P activated RAGE (receptor for activated glycation end products). We found that S100P co-immunoprecipitated with RAGE. Furthermore, the effects of S100P on cell signaling, proliferation, and survival were blocked by agents that interfere with RAGE including administration of an amphoterin-derived peptide known to antagonize RAGE activation, anti-RAGE antibodies, and by expression of a dominant negative RAGE. These data suggest that S100P can act in an autocrine manner via RAGE to stimulate cell proliferation and survival.

Hepatic ischemia/reperfusion (I/R) injury associated with liver transplantation and hepatic resection is characterized by hepatocellular damage and a deleterious inflammatory response. In this study, we examined whether receptor for advanced glycation end product (RAGE) activation is linked to mechanisms accentuating inflammation on I/R in a murine model of total hepatic ischemia. Animals treated with soluble RAGE (sRAGE), the extracellular ligand-binding domain of RAGE, displayed increased survival after total hepatic I/R compared with vehicle treatment. TUNEL assay and histologic analysis revealed that blockade of RAGE was highly protective against hepatocellular death and necrosis on I/R; in parallel, proliferating cell nuclear antigen was enhanced in livers of mice treated with sRAGE. Rapid activation of p38, p44/42, stress-activated protein kinase and c-Jun N-terminal kinase mitogen-activated protein kinases, signal transducer and activator of transcription-3, and nuclear translocation of activator protein-1 was evident at early times on I/R. In the remnants of sRAGE-treated livers, however, activation of each of these signaling and transcription factor pathways was strikingly decreased. sRAGE-treated remnants displayed enhanced activation of nuclear factor kappaB, in parallel with increased transcripts for the proregenerative cytokine, tumor necrosis factor-alpha. In conclusion, these data suggest that RAGE modulates hepatic I/R injury, at least in part by activation of key signaling pathways linked to proinflammatory and cell death-promoting responses. We propose that blockade of this pathway may represent a novel strategy to attenuate injury in hepatic I/R and to facilitate regeneration.

The cardiovascular complications of diabetes represent the leading cause of morbidity and mortality in affected subjects. The impact of hyperglycemia may be both direct and indirect: indirect consequences of elevated blood glucose lead to generation of advanced glycation endproducts, the products of nonenzymatic glycation/oxidation of proteins/lipids that accumulate in the vessel wall, and are signal transduction ligands for Receptor for AGE (RAGE). Although enhanced in diabetes, AGE accumulation also occurs in euglycemia and aging, albeit to lower degrees, driven by oxidant stress and inflammation. In hyperglycemia, production of 3-deoxyglucosone, at least in part via the polyol pathway, provides an amplification loop to sustain AGE generation, oxidant stress, and vascular activation. Furthermore, recruitment of inflammatory cells bearing S100/calgranulins, also ligands for RAGE, augments vascular dysfunction. We hypothesize that activation of RAGE is a final common pathway that transduces signals from these diverse biochemical and molecular species, leading to cardiovascular perturbation. Ultimately, these pathways synergize to construct a scaffold on which the complications of diabetes in the vasculature and heart may be built. We propose that antagonism of RAGE will provide a unique means to dismantle this scaffold and, thereby, suppress initiation/progression of vascular disease and cardiac dysfunction that accompany diabetes and aging

The glycation and oxidation of proteins/lipids leads to the generation of a new class of biologically active moieties, the advanced glycation endproducts (AGEs). Recent studies have elucidated that carboxymethyllysine (CML) adducts of proteins/lipids are a highly prevalent AGE in vivo. CML-modified adducts are signal transduction ligands of the receptor for AGE (RAGE), a member of the immunoglobulin superfamily. Importantly, CML-modified adducts accumulate in diverse settings. In addition to enhanced formation in settings of high glucose, these adducts form in inflammatory milieu. Studies performed both in vitro and in vivo have suggested that the proinflammatory/tissue destructive consequences of RAGE activation in the diabetic/inflamed environment may be markedly attenuated by blockade of the ligand-RAGE axis. Here, we will summarize the known consequences of RAGE activation in the tissues and highlight novel areas for therapeutic intervention in these disease states

BACKGROUND: RAGE (receptor for advanced glycation end products [AGEs]) plays a role in diabetic atherosclerosis. Recently, we have demonstrated enhanced expression of cyclooxygenase-2 and PGE synthase-1 (COX-2/mPGES-1) in human symptomatic plaques, and provided evidence that it is associated with metalloproteinase (MMP)-induced plaque rupture. However, the specific transmembrane signaling pathway(s) influencing plaque COX-2/mPGES-1 expression is unknown. The aim of this study was to characterize RAGE expression in human plaques and to correlate it with the inflammatory infiltration, COX-2/mPGES-1 and MMP expression, and with clinical evidence of diabetes. METHODS AND RESULTS: Plaques obtained from 60 patients undergoing carotid endarterectomy were divided into diabetic and nondiabetic according to clinical evidence of type 2 diabetes. Plaques were subjected to analysis of RAGE, NF-kappaB, COX-2/mPGES-1, MMP-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, and collagen content by immunohistochemistry and Western blot, whereas zymography was used to detect MMP activity. Immunohistochemistry was used to identify CD68+ macrophages, CD3+ T-lymphocytes, smooth muscle cells (SMCs), and HLA-DR+ inflammatory cells. Diabetic plaques had more (P<0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells, more (P<0.0001) immunoreactivity for RAGE, activated NF-kappaB, COX-2/mPGES-1, and MMPs, increased (P<0.0001) gelatinolytic activity, reduced (P<0.0001) collagen content, and increased (P<0.0001) lipid and oxLDL content. Interestingly, RAGE, COX-2/mPGES-1, and MMP expression was linearly correlated with plasma level of HbA1c. CONCLUSIONS: In conclusion, this study demonstrates in humans that RAGE overexpression is associated with enhanced inflammatory reaction and COX-2/mPGES-1 expression in diabetic plaque macrophages, and this effect may contribute to plaque destabilization by inducing culprit metalloproteinase expression.

Angiotensin II (Ang II), a vasoactive peptide that is also considered a growth factor, has been implicated in both normal and diabetic cellular proliferation. We recently found that activation of janus kinase 2 (JAK2) is essential for the Ang II-induced proliferation of vascular smooth muscle cells (VSMCs) and that high glucose augments Ang II-induced proliferation of VSMCs by increasing signal transduction through activation of JAK2. Here, we demonstrate that S100B, a ligand for the receptor of advanced glycation end products (RAGEs), augmented both Ang II-induced tyrosine phosphorylation of JAK2 and cell proliferation in VSMCs in a receptor-dependent manner. We also found that S100B-RAGE interaction triggered intracellular generation of reactive oxygen species (ROS), VSMC proliferation, and JAK2 tyrosine phosphorylation via activation of phospholipase D (PLD)2. These results provide direct evidence for linkages between PLD2, ROS production, and S100B-RAGE-induced enhancement of Ang II-induced cell proliferation and activation of JAK2 in VSMCs.

OBJECTIVE: Because recent epidemiologic evidence suggests that periodontal infections may increase the risk of atherosclerosis and related events in humans, we assessed the impact of oral inoculation with the periodontal pathogen Porphyromonas gingivalis on atherogenesis in hypercholesterolemic apolipoprotein E-null mice. METHODS AND RESULTS: In the absence of alterations in distinct risk factors, P gingivalis infection exacerbated the early stages of atherogenesis in this model. Infected animals displayed evidence of local periodontal infection, as the severity of alveolar bone loss, the hallmark of periodontitis, was increased. Generalized activation of host inflammatory responses was evident in infected mice, as demonstrated by serum IgG response to P gingivalis and elevated levels of interleukin-6. P gingivalis DNA was localized in the aortic tissue from a limited number of infected mice but not in any noninfected controls. Infected mice displayed enhanced vascular activation, as suggested by increased aortic expression of vascular cell adhesion molecule-1 and tissue factor. CONCLUSIONS: Oral infection with P gingivalis accelerates early atherosclerosis. Thus, uncovering the underlying mechanisms is critical for the design of preventive and therapeutic strategies targeting atherosclerotic vascular disease and its sequelae.

Amyloid-beta peptide (Abeta) interacts with the vasculature to influence Abeta levels in the brain and cerebral blood flow, providing a means of amplifying the Abeta-induced cellular stress underlying neuronal dysfunction and dementia. Systemic Abeta infusion and studies in genetically manipulated mice show that Abeta interaction with receptor for advanced glycation end products (RAGE)-bearing cells in the vessel wall results in transport of Abeta across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-1), the latter mediating Abeta-induced vasoconstriction. Inhibition of RAGE-ligand interaction suppresses accumulation of Abeta in brain parenchyma in a mouse transgenic model. These findings suggest that vascular RAGE is a target for inhibiting pathogenic consequences of Abeta-vascular interactions, including development of cerebral amyloidosis