Faculty Bibliography

Background: Intestinal fibrosis is a common and severe complication of inflammatory bowel disease (IBD) characterized by excessive deposition of extracellular matrix components (ECM). Inflamed colonic mucosa of patients with active IBD shows a significant increase in expression of the receptor for advanced glycation end products (RAGE), a member of the immunoglobulin superfamily of cell surface receptors. Several evidences in kidney, liver and lung fibrosis show how the increased myofibroblast activation and the consequent ECM accumulation are regulated by RAGE. Aim of this study is to investigate the involvement of RAGE i) in in vitro differentiation of human colonic fibroblasts (CCD-18 Co) and epithelial cells (HT29) into myofibroblasts and ii) in the development of the DSS-induced intestinal fibrosis in mice. Methods: Differentiation of CCD-18 Co and HT29 into myofibro-blasts was induced by 4 day of TGF-beta administration (1ng/mL and 10 ng/mL, respectively). Expression of alpha-SMA (ACTA2) gene and fibronectin (Fn-1) gene, as well as RAGE was measured by quantitative RT-PCR. Chronic colonic fibrosis was induced in C57BL/6 mice by successive 2,5% (w/v) DSS administration in drinking water for 6 weeks. After 6 weeks main parameters associated to a pro-fibrotic profile were assessed macroscopically (weight/length of the colon, edema, ulcers, adhesions, thickness, dilatation), histologically (inflammatory infiltrate, collagen deposition) and biologically (Tgf-beta1, Col1A1 and Fn-1 gene expression) Results: TGF-beta-induced myofibroblast activation was associated to a significant increase in the ACTA2 expression (65%, p< 0.01 for CCD-18 Co and 183.8%, p< 0.001 for HT29) and mRNA Fn-1 levels (75.5%, p<0.001 in CCD-18 Co and 122%, p< 0.001, in HT29) and correlated with a significant RAGE upregulation both in CCD-18 Co and HT29 (48%, p<0.005 and 1.47%, p<0.005, respectively). RAGE-/- mice showed a significant 23.8% decrease of total macroscopic score and 49% decrease of total microscopic score compared to WT mice. mRNA Tgf-beta1 expression was significantly increased 3.4 fold by the DSS administration in WT mice colon, whereas it was unchanged in RAGE null mice compared to mice receiving only tap water. Col1A1 and Fn-1 genes were upregulated in DSS-treated WT mice (5.52 folds, p= 0.137 and 53 folds, p= 0.0016, respectively). Lack of RAGE decreased 3.17 folds (p= 0.0341) Col1A1 expression and totally prevents the Fn-1 upregulation induced by DSS treatment Conclusions: The potential pro-fibrotic RAGE properties in IBD represent a new frontier for a better understanding of the mechanisms related to intestinal fibrosis and for the development of new therapeutic approaches

Diabetes exacerbates cardiovascular disease, at least in part via suppression of macrophage cholesterol efflux and levels of the cholesterol transporters, ATP binding cassette transporters A1 (ABCA1) and ABCG1. The receptor for advanced glycation end products (RAGE) is highly expressed in human and murine diabetic atherosclerotic plaques, particularly in macrophages. We tested the hypothesis that RAGE suppresses macrophage cholesterol efflux and probed the mechanisms by which RAGE downregulates ABCA1 and ABCG1. Macrophage cholesterol efflux to Apolipoprotein A1 (ApoA1) and High Density Lipoprotein (HDL) and reverse cholesterol transport to plasma, liver and feces were reduced in diabetic macrophages via RAGE. In vitro, RAGE ligands suppressed ABCG1 and ABCA1 promoter luciferase activity and transcription of ABCG1 and ABCA1 through PPARG-responsive promoter elements, but not through Liver X Receptor (LXR) elements. Plasma levels of HDL were reduced in diabetic mice in a RAGE-dependent manner. Laser capture microdissected CD68+ macrophages from atherosclerotic plaques of Ldlr-/- mice devoid of Ager (RAGE) displayed higher levels of Abca1, Abcg1 and Pparg mRNA transcripts vs. Ager-expressing Ldlr-/- mice, in a manner independent of glycemia or plasma levels of total cholesterol and triglyceride. Antagonism of RAGE may fill an important therapeutic gap in the treatment of diabetic macrovascular complications.

Emerging evidence links the receptor for advanced glycation endproducts (RAGEs) to the pathogenesis of tissue damage in chronic metabolic and inflammatory diseases. In human subjects, multiple reports suggest that in the plasma/serum, circulating levels of distinct forms of soluble RAGEs may be biomarkers of the presence or absence, and the extent of chronic disease. These considerations prompt us to consider in this review, what are soluble RAGEs; how are they formed; what might be their natural functions; and may they serve as biomarkers of inflammatory and metabolic disease activity? In this brief review, we seek to address what is known and suggest new areas for scientific investigation to uncover the biology of soluble RAGEs.

We investigated treatment with a receptor for advanced glycation endproduct (RAGE) blocking antibody on angiogenic response to hind limb ischemia in diabetic mice. Streptozotocin treated C57BL/6 mice received either murine monoclonal anti-RAGE F(ab')2 intraperitoneally (n=10) or saline (n=9) for 9 weeks. Diabetic plus 10 non-diabetic C57BL/6 mice underwent left femoral artery ligation and 5 days later angiogenesis imaging with (99m)Tc-Arg-Gly-Asp (RGD) nanoSPECT/CT. Twenty-four days later, hind limb blood flow was measured with ultrasound, the mice were euthanized, and tissue was taken for immunohistochemistry. The angiogenic imaging signal in ischemic limbs was higher in RAGE-ab treated versus saline treated mice at day 5 (3.1+/-1.4 vs 1.68+/-0.35, p=0.02) and blood flow was higher at day 24 (1.49+/-0.5 vs 0.61+/-0.39, p=0.04). Immunohistochemistry of ischemic muscles showed greater capillary density in the RAGE-ab treated group versus the vehicle-treated group (p<0.001) (NS from non-diabetic mice). In conclusion, treatment with anti-RAGE F(ab')2 in diabetic mice improves neovascularization in the ischemic leg.

SCOPE: Advanced glycation end-products (AGEs) are endogenously produced and are present in food. N(epsilon) -carboxymethyllysine (CML) is an endothelial activator via the receptor for AGEs (RAGEs) and is a major dietary AGE. This work investigated the effects of a CML-enriched diet and RAGE involvement in aortic aging in mice. METHODS AND RESULTS: After 9 months of a control diet or CML-enriched diets (50, 100, or 200 mugCML /g of food), endothelium-dependent relaxation, RAGE, vascular cell adhesion molecule-1, and sirtuin-1 expression, pulse wave velocity and elastin disruption were measured in aortas of wild-type or RAGE(-/-) male C57BL/6 mice. Compared to the control diet, endothelium-dependent relaxation was reduced in the wild-type mice fed the CML-enriched diet (200 mugCML /g) (66.8 +/- 12.26 vs. 94.3 +/- 2.6%, p < 0.01). RAGE and vascular cell adhesion molecule-1 (p < 0.05) expression were increased in the aortic wall. RAGE(-/-) mice were protected against CML-enriched diet-induced endothelial dysfunction. Compared to control diet, the CML-enriched diet (200 mugCML /g) increased the aortic pulse wave velocity (86.6 +/- 41.1 vs. 251.4 +/- 41.1 cm/s, p < 0.05) in wild-type animals. Elastin disruption was found to a greater extent in the CML-fed mice (p < 0.05). RAGE(-/-) mice fed the CML-enriched diet were protected from aortic stiffening. CONCLUSION: Chronic CML ingestion induced endothelial dysfunction, arterial stiffness and aging in a RAGE-dependent manner.

Types 1 and 2 diabetes are on the rise worldwide. Although the treatment of hyperglycemia has benefitted from recent advances, aggressive efforts to maintain euglycemia may be fraught with risk, especially in older subjects or in subjects vulnerable to hypoglycemic unawareness. Hence, strategies to prevent and treat the complications of hyperglycemia are essential. In this review, we summarize recent updates on the biology of the receptor for advanced glycation endproducts (RAGE) in the pathogenesis of both micro- and macrovascular complications of diabetes, insights from the study of mouse models of obesity and diabetic complications, and from associative studies in human subjects. The study of the mechanisms and consequences of the interaction of the RAGE cytoplasmic domain with the formin, mDia1, in RAGE signal transduction, will be discussed. Lastly, we review the 'state-of-the-art' on RAGE-directed therapeutics. Tackling RAGE/mDia1 may identify a novel class of therapeutics preventing diabetes and its complications