Faculty Bibliography

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.

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.

Advanced glycation end products (AGEs) are proteins or lipids that become glycated after exposure to sugars. AGEs are prevalent in the diabetic vasculature and contribute to the development of atherosclerosis. The presence and accumulation of AGEs in many different cell types affect extracellular and intracellular structure and function. AGEs contribute to a variety of microvascular and macrovascular complications through the formation of cross-links between molecules in the basement membrane of the extracellular matrix and by engaging the receptor for advanced glycation end products (RAGE). Activation of RAGE by AGEs causes upregulation of the transcription factor nuclear factor-kappaB and its target genes. Soluble AGEs activate monocytes, and AGEs in the basement membrane inhibit monocyte migration. AGE-bound RAGE increases endothelial permeability to macromolecules. AGEs block nitric oxide activity in the endothelium and cause the production of reactive oxygen species. Because of the emerging evidence about the adverse effects of AGEs on the vasculature of patients with diabetes, a number of different therapies to inhibit AGEs are under investigation.

BACKGROUND: The accelerated incidence of colorectal carcinoma in individuals with inflammatory bowel disease suggests that cellular perturbation triggered by chronic inflammation is linked to the development of dysplasia and neoplastic transformation. To test the mechanistic links between these processes, we employed the following murine strains: (1) multiple intestinal neoplasia (Min) +/- mice, bearing a mutation in the adenomatous polyposis coli (APC) gene; (2) mice deficient in interleukin 10 (IL-10), which normally develop enterocolitis; and (3) Min +/-/IL-10 null mice, first developed in our laboratory. METHODS: Mice with either parental strain or the cross were sacrificed at time points ranging from 10 to 30 weeks of age. The small bowel and colon of 170 IL-10 null mice, 31 Min +/- mice, and 120 Min +/-/IL-10 null mice were examined microscopically. RESULTS: The number of flat adenomas was increased in the colons of the Min +/-/IL-10-/- mice, compared with the Min +/- mice (P = .0005). Neither colitis-type dysplasia nor carcinoma was increased in the Min +/-/IL-10 -/-, compared with the IL-10 null mice (P = .18). Mice deficient in IL-10 developed colitic-type dysplasia (P = .0001) or carcinoma (P = .0001) correlated with increasing inflammation. CONCLUSIONS: Breeding the Min +/- genotype into the IL-10 -/- background increased the incidence of colonic adenomas. Our studies demonstrate that acceleration of dysplasia and progression to invasion were associated with the degree of the inflammatory response in mice deficient in IL-10. These findings provide a novel system to dissect the pathways by which inflammatory mechanisms accelerate adenoma formation.

Diabetes is associated with an increase in circulating advanced glycosylation end products (AGEs) and the increased expression of the receptor for AGEs (RAGE). Inhibition of AGE/RAGE binding through the administration of soluble RAGE (sRAGE) has been shown to decrease neointimal hyperplasia. Peroxisome proliferator-activated receptor gamma (PPARgamma), which inhibits neointimal hyperplasia, has been shown to decrease RAGE expression in cultured endothelial cells. We hypothesized that PPARgamma agonists inhibit neointimal hyperplasia via down-regulation of RAGE in vivo. Pretreatment of rat aortic smooth muscle cells (SMCs) with PPARgamma agonist rosiglitazone significantly down-regulated RAGE expression and inhibited SMC proliferation in response to the RAGE agonist S100/calgranulins. In vivo studies showed that rosiglitazone decreased RAGE expression and SMC proliferation at 7 days following carotid arterial injury in both diabetic and nondiabetic rats. At 21 days following injury, neointimal formation was significantly decreased in both diabetic and nondiabetic animals that received rosiglitazone. To determine whether inhibition of neointimal formation by PPARgamma activation could fully be accounted for by its down-regulation of RAGE, we compared the results obtained in animals treated with sRAGE, PPARgamma activator, and sRAGE + PPARgamma activator. Consistent with PPARgamma working through its effects on RAGE, we found that the addition of PPARgamma activator to sRAGE did not result in any further decrease in neointimal formation. These data demonstrate for the first time that PPARgamma agonists inhibit RAGE expression at sites of arterial injury and suggest that down-regulation of RAGE by the PPARgamma activation inhibits neointimal formation in response to arterial injury.

We sought to study the presence of the receptor for advanced glycation endproducts (RAGE) and its ligands, advanced glycation endproducts (AGEs), S100/calgranulins and amphoterin (high mobility group box 1 protein; HMGB1), in the vitreous cavity and epiretinal membranes (ERMs) of eyes of patients with proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). Undiluted vitreous specimens were collected from 30 eyes of 30 patients undergoing pars plana vitrectomy for repair of retinal detachment (RD) secondary to PDR (n = 15) or PVR (n = 15). The vitreous samples obtained from 10 eyes undergoing macular hole repair were used as controls. Epiretinal membranes were obtained from eight eyes with PDR and from 10 eyes with PVR. The levels of AGEs in the vitreous were measured using ELISA. The vitreous levels of soluble RAGE (sRAGE), S100/calgranulins and amphoterin were measured using Western blot analyses. The localization of RAGE and its ligands in ERMs was determined with immunohistochemistry. The vitreous levels of sRAGE were significantly increased in both PDR and PVR (p < or = 0.05) compared to control vitreous. In both PDR and PVR, the vitreous levels of AGEs (p < or = 0.01), S100/calgranulins (p < or = 0.05), and amphoterin (p < or = 0.01) were also elevated compared to control eyes. Expression of RAGE was detected in six of eight ERMs from eyes with PDR and eight of 10 ERMs from eyes with PVR. Many cells expressing RAGE also expressed vimentin, suggesting a glial cell origin. Ligands for RAGE were also detected in ERMs, with AGEs detected in five eyes with PDR and eight eyes with PVR. Similarly, S100 and amphoterin ERM expression was observed in six eyes with PDR; these ligands were also expressed in ERMs from eyes with PVR (8 and 7 cases, respectively). We conclude that RAGE and its ligands are increased in the vitreous cavity of eyes with PDR and PVR and are present in ERMs of eyes with these proliferative retinal disorders. These findings suggest a role for the proinflammatory RAGE axis in the pathogenesis of proliferative retinal diseases.

BACKGROUND: The beneficial effects of reperfusion therapies have been limited by the amount of ischemic damage that occurs before reperfusion. To enable development of interventions to reduce cell injury, our research has focused on understanding mechanisms involved in cardiac cell death after ischemia/reperfusion (I/R) injury. In this context, our laboratory has been investigating the role of the receptor for advanced-glycation end products (RAGE) in myocardial I/R injury. METHODS AND RESULTS: In this study we tested the hypothesis that RAGE is a key modulator of I/R injury in the myocardium. In ischemic rat hearts, expression of RAGE and its ligands was significantly enhanced. Pretreatment of rats with sRAGE, a decoy soluble part of RAGE receptor, reduced ischemic injury and improved functional recovery of myocardium. To specifically dissect the impact of RAGE, hearts from homozygous RAGE-null mice were isolated, perfused, and subjected to I/R. RAGE-null mice were strikingly protected from the adverse impact of I/R injury in the heart, as indicated by decreased release of LDH, improved functional recovery, and increased adenosine triphosphate (ATP). In rats and mice, activation of the RAGE axis was associated with increases in inducible nitric oxide synthase expression and levels of nitric oxide, cyclic guanosine monophosphate (cGMP), and nitrotyrosine. CONCLUSIONS: These findings demonstrate novel and key roles for RAGE in I/R injury in the heart. The findings also demonstrate that the interaction of RAGE with advanced-glycation end products affects myocardial energy metabolism and function during I/R