Faculty Bibliography

The interaction of glucose-modified and inflammation-promoting ligands with the receptor for advanced glycation end products (RAGE) is emerging as a central mechanism contributing to the diverse complications of diabetes. These ligands, particularly in oligomeric form, bind to RAGE and transduce intracellular signals. The consequences of this interaction, as elucidated in cultured cells and animal models, include upregulation of inflammatory and tissue-degradative pathways. Pharmacologic antagonism of RAGE may hold promise for the treatment of diabetic complications

PURPOSE: The importance of VEGF in stimulating neovascular age-related macular degeneration (AMD) is well-recognized, but the initiating factors that induce local upregulation of VEGF remain unclear. The current study was conducted to test the hypothesis that activation of RAGE (receptor for advanced glycation end products [AGEs]) by its ligands, including AGEs, amyloid-beta peptide (Abeta), and S100B/calgranulins, some of which are known components of drusen and Bruch's membrane deposits, modulate secretion of VEGF by retinal pigment epithelial (RPE) cells. METHODS: ARPE-19 cells were used for all experiments. The cells were transfected with constructs encoding a signal transduction mutant of human RAGE to assess the RAGE-dependence of intracellular signaling. VEGF secretion and gene expression were assessed by ELISA and quantitative real-time PCR. SDS-PAGE and size exclusion chromatography were performed to analyze the structural changes of S100B after oxidation of its thiol groups under denaturing and nondenaturing conditions, respectively. NF-kappaB activation was assessed via electrophoretic mobility shift assay (EMSA). The impact of the NF-kappaB inhibition was assessed by using parthenolide. RESULTS: ARPE-19 cells basally secreted VEGF under normal cell culture conditions. Immobilized ligands of RAGE increased VEGF secretion in a RAGE-dependent manner. In contrast, soluble AGE-BSA, fresh Abeta, and S100B were less effective in increasing VEGF secretion. Studies with Abeta demonstrated that oligomeric and surface-immobilized forms of Abeta, but not soluble monomeric forms of Abeta, were effective upregulators of VEGF secretion via RAGE. Oxidation of S100B's thiol groups resulted in the formation of oligomers that displayed distinct RAGE biological activity compared with the simple dimeric form. RAGE-mediated upregulation of VEGF secretion by ARPE-19 cells was largely dependent on NF-kappaB, as indicated by studies with parthenolide. CONCLUSIONS: Immobilized or oligomerized ligands for RAGE induce RPE cells to increase VEGF secretion. NF-kappaB plays a central role in RAGE-dependent RPE secretion of VEGF. In AMD, activation of the RAGE axis in RPE cells may contribute to upregulation of VEGF, potentially inciting or propagating neovascular macular disease.

BACKGROUND: As a link between periodontal infections and an increased risk for vascular disease has been demonstrated, we assessed the ability of the Gram-negative periodontal pathogen Porphyromonas gingivalis to modulate properties of endothelial cells linked to inflammation and proatherogenic pathways. METHODS AND RESULTS: Primary human aortic endothelial cells (HAEC) were infected with either P. gingivalis strain 381 or its non-invasive fimbriae-deficient mutant, DPG3, and incubated with U-937 monocytes, or Jurkat T cells. P. gingivalis-infected HAEC demonstrated significantly increased adhesion of immune cells compared to non-infected cells or those infected with DPG3. Heat-killed bacteria had no effect on mononuclear cell adhesion and P. gingivalis LPS had only a minimal effect. P. gingivalis infection significantly increased HAEC expression of VCAM-1, ICAM-1 and E-selectin, and enhanced production of IL-6, IL-8 and MCP-1. CONCLUSION: These data demonstrate that live invasive P. gingivalis 381 elicits a pro-atherogenic response in HAEC.

The receptor for advanced glycation endproducts (RAGE) has complex roles in the immune/inflammatory response. RAGE is expressed on monocytes/macrophages, T and B lymphocytes, and dendritic cells. Previous studies illustrated that homozygous RAGE-/- mice subjected to overwhelming bacterial sepsis displayed normal clearance of pathogenic bacteria and significantly increased survival. In this issue of Critical Care, Lutterloh and colleagues confirm these findings and provide evidence that blocking antibodies to RAGE afford similar protection in mice, even when administration of anti-RAGE is delayed by 24 hours. Furthermore, these authors illustrate that deletion of RAGE is remarkably protective in mice infected with the intracellular pathogen Listeria monocytogenes. In this Commentary, we consider these findings and propose possible mechanisms by which RAGE exacts a heavy toll on the host in response to polymicrobial sepsis and L. monocytogenes.

OBJECTIVE: Receptor for advanced glycation end products (AGEs) (RAGE) plays a central role in the process of plaque rupture in diabetic patients. Recently, it has been reported that RAGE may be downregulated by improving glycemic control. In contrast, despite being well known that RAGE may be induced in human vessels in a glucose-independent fashion, also by myeloperoxidase (MPO)-dependent AGE generation, no data exist regarding the possibility of a pharmacological modulation of glucose-independent RAGE generation. Thus, the aim of this study was to characterize the effect of simvastatin on the expression of RAGE and RAGE-dependent plaque-destabilizing genes in human atherosclerotic plaques. METHODS AND RESULTS: Seventy type 2 diabetic patients with asymptomatic carotid artery stenosis (>70%) were randomized to American Heart Association (AHA) step 1 diet plus simvastatin (40 mg/d) or AHA step 1 diet alone for 4 months before endarterectomy. Plaque expression of MPO, AGEs, RAGE, NF-kappaB, COX-2, mPGES-1, matrix metalloproteinase (MMP)-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, procollagen 1, and interstitial collagen was analyzed by immunohistochemistry and Western blot; zymography was used to detect MMP activity. Plaques from the simvastatin group had less (P<0.0001) immunoreactivity for MPO, AGEs, RAGE, p65, COX-2, mPGES-1, MMP-2, and MMP-9, lipids and oxLDL; reduced (P<0.0001) gelatinolytic activity; increased (P<0.0001) procollagen 1 and collagen content; and fewer (P<0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells. Of interest, RAGE inhibition by simvastatin, observed not only in plaque sections but also in plaque-derived macrophages, was reverted by addition of AGEs in vitro. CONCLUSIONS: This study supports the hypothesis that simvastatin inhibits plaque RAGE expression by decreasing MPO-dependent AGE generation. This effect in turn might contribute to plaque stabilization by inhibiting the biosynthesis of PGE2-dependent MMPs, responsible for plaque rupture.

Atherosclerosis, restenosis, and the consequences of ischemia are the major causes of morbidity and mortality worldwide. Elucidation of key contributing pathways in animal models of ischemia-reperfusion injury, atherosclerosis, and restenosis consequent to vascular injury may lead to great interest in determining if blocking these pathways could prevent vascular disease in human subjects. This review details the evidence that the protein kinase C (PKC) beta/early growth response-1 axis plays a central role in the response to both acute and chronic vascular stresses in animal models and also indicates the clinical implications of a specific inhibitor of PKCbeta, ruboxistaurin (LY333531)

Oxidative and carbonyl stress leads to generation of N(epsilon)-carboxymethyllysine-modified proteins (CML-mps), which are known to bind the receptor for advanced glycation end products (RAGE) and induce nuclear factor (NF)-kappaB-dependent proinflammatory gene expression. To determine the impact of CML-mps in vivo, RAGE-dependent sustained NF-kappaB activation was studied in resection gut specimens from patients with inflammatory bowel disease. Inflamed gut biopsy tissue demonstrated a significant up-regulation of RAGE and increased NF-kappaB activation. Protein extracts from the inflamed zones, but not from noninflamed resection borders, caused perpetuated NF-kappaB activation in cultured endothelial cells, which was mediated by CML-mps including CML-modified S100 proteins. The resulting NF-kappaB activation, lasting 5 days, was primarily inhibited by either depletion of CML-mps or by the addition of sRAGE, p44/42 and p38 MAPKinase-specific inhibitors. Consistently, CML-mps isolated from inflamed gut areas and rectally applied into mice caused NF-kappaB activation, increased proinflammatory gene expression, and histologically detectable inflammation in wild-type mice, but not in RAGE-/- mice. A comparable up-regulation of NF-kappaB and inflammation on rectal application of CML-mps was observed in IL-10-/- mice. Thus, CML-mps generated in inflammatory lesions have the capacity to elicit a RAGE-dependent intestinal inflammatory response.

The receptor for advanced glycation end products (RAGE) has been implicated in the pathogenesis of numerous conditions associated with excessive inflammation. To determine whether RAGE-dependent signaling is important in the development of intestinal barrier dysfunction after hemorrhagic shock and resuscitation (HS/R), C57Bl/6, rage(-/-), or congenic rage(+/+) mice were subjected to HS/R (mean arterial pressure of 25 mmHg for 3 h) or a sham procedure. Twenty-four hours later, bacterial translocation to mesenteric lymph nodes and ileal mucosal permeability to FITC-labeled dextran were assessed. Additionally, samples of ileum were obtained for immunofluorescence microscopy, and plasma was collected for measuring IL-6 and IL-10 levels. HS/R in C57Bl/6 mice was associated with increased bacterial translocation, ileal mucosal hyperpermeability, and high circulating levels of IL-6. All of these effects were prevented when C57Bl/6 mice were treated with recombinant human soluble RAGE (sRAGE; the extracellular ligand-binding domain of RAGE). HS/R induced bacterial translocation, ileal mucosal hyperpermeability, and high plasma IL-6 levels in rage(+/+) but not rage(-/-) mice. Circulating IL-10 levels were higher in rage(-/-) compared with rage(+/+) mice. These results suggest that activation of RAGE-dependent signaling is a key factor leading to gut mucosal barrier dysfunction after HS/R.

BACKGROUND:Multiple studies have demonstrated a link between periodontal infections and vascular disease. Porphyromonas gingivalis, a major periodontal pathogen, has been shown to adhere to and invade endothelial cells. OBJECTIVE:In order to dissect mechanisms underlying these observations, we assessed the role of P. gingivalis infection in modulating properties of endothelial cells linked to atherothrombosis. METHODS:Primary human aortic endothelial cells (HAEC) were infected with either P. gingivalis 381 or its non-invasive fimbriae-deficient mutant, DPG3. Markers of coagulation and thrombosis were assessed 8 h and 18 h postinfection in cell lysates and supernatants. RESULTS:Infection with P. gingivalis 381 significantly enhanced tissue factor expression and activity, and suppressed levels of tissue factor pathway inhibitor. Furthermore, P. gingivalis infection decreased levels and activity of tissue plasminogen activator, and enhanced plasminogen activator inhibitor-1 antigen and activity. Consistent with an important role for bacterial adhesion/invasion in this setting, infection with DPG3 failed to induce procoagulant properties in HAEC. Most of the above effects of P. gingivalis 381 were more apparent at the later time point (18 h postinfection). This suggests that P. gingivalis infection, rather than having an immediate and direct effect, might activate pathways that, in turn, trigger endothelial procoagulant mechanisms. CONCLUSIONS:Taken together these data demonstrate for the first time that infection with a periodontal pathogen induces procoagulant responses in HAEC.