Faculty Bibliography

The multi-ligand receptor RAGE was discovered on account of its ability to bind and transduce the cell stress-provoking signals of advanced glycation endproducts (AGEs). The finding that RAGE also bound pro-inflammatory molecules set the stage for linking RAGE and inflammation to the pathogenesis of diabetic macro- and microvascular complications. In this review, we focus on the roles of RAGE and its ligands in diabetes complications. We recount the findings from mice, rats, swine and human subjects suggesting that RAGE action potently contributes to vascular, inflammatory and end-organ stress and damage in types 1 and 2 diabetes. We detail the efforts to track ligands and RAGE in human subjects with diabetes to address if this axis may be a biomarker reflective of the state of the diabetic complications. Lastly, we suggest specific strategies to tackle AGE-ligand-RAGE interactions as potential therapeutic targets for diabetes and its complications.

PURPOSE: The receptor for advanced glycation end products (RAGE) contributes to multiple pathologies, including diabetes, arthritis, neurodegenerative diseases, and cancer. Despite the obvious need, no RAGE inhibitors are in common clinical use. Therefore, we developed a novel small RAGE antagonist peptide (RAP) that blocks activation by multiple ligands. EXPERIMENTAL DESIGN: RAGE and its ligands were visualized by immunohistochemical analysis of human pancreatic tissues, and siRNA was used to analyze their functions. Interactions between RAGE and S100P, S100A4, and HMGB-1 were measured by ELISA. Three S100P-derived small antagonistic peptides were designed, synthesized, and tested for inhibition of RAGE binding. The effects of the peptide blockers on NFkappaB-luciferase reporter activity was used to assess effects on RAGE-mediated signaling. The most effective peptide was tested on glioma and pancreatic ductal adenocarcinoma (PDAC) models. RESULTS: Immunohistochemical analysis confirmed the expression of RAGE and its ligands S100P, S100A4, and HMGB-1 in human PDAC. siRNA silencing of RAGE or its ligands reduced the growth and migration of PDAC cells in vitro. The most effective RAP inhibited the interaction of S100P, S100A4, and HMGB-1 with RAGE at micromolar concentrations. RAP also reduced the ability of the ligands to stimulate RAGE activation of NFkappaB in cancer cells in vitro and in vivo. Importantly, systemic in vivo administration of RAP reduced the growth and metastasis of pancreatic tumors and also inhibited glioma tumor growth. CONCLUSION: RAP shows promise as a tool for the investigation of RAGE function and as an in vivo treatment for RAGE-related disorders. Clin Cancer Res; 18(16); 4356-64. (c)2012 AACR.

Islet amyloid polypeptide (IAPP, amylin) is responsible for amyloid formation in type 2 diabetes and in islet cell transplants. The only known natural mutation found in mature human IAPP is a Ser20-to-Gly missense mutation, found with small frequency in Chinese and Japanese populations. The mutation appears to be associated with increased risk of early-onset type 2 diabetes. Early measurements in the presence of organic co-solvents showed that S20G-IAPP formed amyloid more quickly than the wild type. We confirm that the mutant accelerates amyloid formation under a range of conditions including in the absence of co-solvents. Ser20 adopts a normal backbone geometry, and the side chain makes no steric clashes in models of IAPP amyloid fibers, suggesting that the increased rate of amyloid formation by the mutant does not result from the relief of steric incompatibility in the fiber state. Transmission electronic microscopy, circular dichroism, and seeding studies were used to probe the structure of the resulting fibers. The S20G-IAPP peptide is toxic to cultured rat INS-1 (transformed rat insulinoma-1) beta-cells. The sensitivity of amyloid formation to the identity of residue 20 was exploited to design a variant that is much slower to aggregate and that inhibits amyloid formation by wild-type IAPP. An S20K mutant forms amyloid with an 18-fold longer lag phase in homogeneous solution. Thioflavin T binding assays, together with experiments using a p-cyanophenylalanine (p-cyanoPhe) variant of human IAPP, show that the designed S20K mutant inhibits amyloid formation by human IAPP. The experiments illustrate how p-cyanoPhe can be exploited to monitor amyloid formation even in the presence of other amyloidogenic proteins.

Multiple biochemical, metabolic, and signal transduction pathways contribute to insulin resistance. In this review, we present evidence that the posttranslational process of protein glycation may play a role in insulin resistance. The posttranslational modifications, the advanced glycation end products (AGEs), are formed and accumulated by endogenous and exogenous mechanisms. AGEs may contribute to insulin resistance by a variety of mechanisms, including generation of tumor necrosis factor-alpha direct modification of the insulin molecule, thereby leading to its impaired action, generation of oxidative stress, and impairment of mitochondrial function, as examples. AGEs may stimulate signal transduction via engagement of cellular receptors, such as receptor for AGEs. AGE-receptor for AGE interaction perpetuates AGE formation and cellular stress via induction of inflammation, oxidative stress, and reduction in the expression and activity of the enzyme glyoxalase I that detoxifies the AGE precursor, methylglyoxal. Once set in motion, glycation-promoting mechanisms may stimulate ongoing AGE production and target tissue stresses that reduce insulin responsiveness. Strategies to limit AGE accumulation and action may contribute to the prevention of insulin resistance and its consequences.

Cardiovascular disease represents the major cause of morbidity and mortality in patients with diabetes mellitus. Studies by us and others have implicated increased flux via aldose reductase (AR) as a key player in mediating diabetic complications, including cardiovascular complications. Data suggest that increased flux via AR in diabetics perpetuates increased injury after myocardial infarction, accelerates atherosclerotic lesion formation, and promotes restenosis via multiple mechanisms. Most importantly, studies have shown that increased generation of reactive oxygen species due to flux via AR has been a common feature in animal models of diabetic cardiovascular disease. Taken together, these considerations place AR in the center of biochemical and molecular stresses that characterize the cardiovascular complications of diabetes. Stopping AR-dependent signaling may hold the key to interrupting cycles of cellular perturbation and tissue damage in diabetic cardiovascular complications.

Background: RAGE and its ligands have been implicated in the pathogenesis of I/R injury through pathways of inflammation and apoptosis. We hypothesized that RAGE-directed quantitative imaging of myocardial uptake of 99mTc-anti-RAGE F(ab')2 in a mouse model of I/R can detect RAGE expression in the myocardium and show quantitative differences between early (18-20 h) and later times (48 h) after reperfusion. Methods: Twenty-four wild-type (WT) mice underwent left anterior descending coronary artery (LAD) occlusion for 30 min followed by reperfusion for 18-20 h (n = 8) or 48 h (n = 12) and 3 WT mice underwent sham operation without coronary intervention. Twenty-seven mice (including shams) were injected with 19.98 +/- 1.78 MBq 99mTc anti-RAGE F(ab')2 and 5 h later, mice were injected with 6.14 +/- 2.0 MBq 201Tl and immediately underwent dual isotope SPECT/CT imaging. Four WT mice were injected with control nonspecific F(ab')2 and similarly imaged at 18-20 h. At the completion of imaging, hearts were removed, radioactivity counted, and sectioned for histology. Results: The uptake of 99mTc-anti-RAGE F(ab')2 in the ischemic zone from the scans as mean percentage injected dose (%ID) was significantly greater at 18-20 h (5.7 +/- 2.1 x 10-3%) compared to 48 h (1.4 +/- 1.1 x 10-3%; p < 0.001) following reperfusion. Disease and antibody controls showed no focal uptake in the infarct. Gamma well counting of the myocardium from experiments performed at 18-20 h and 48 h post reperfusion supported the quantitative scan data. Immunohistochemical staining of the myocardium showed greater caspase-3 and RAGE staining at 18-20 h vs. 48 h (p = 0.04 and p = 0.01, respectively). On dual immunofluorescence, RAGE colocalized mainly with injured cardiomyocytes undergoing apoptosis. Conclusion: RAGE expression in reperfusion injury can be imaged in-vivo using a novel 99mTc-anti-RAGE F(ab')2. This imaging approach may be useful in evaluating contribution of RAGE to myocardial ischemic injury in diseases with increased expression such as diabetes