Faculty Bibliography

The products of nonenzymatic glycation and oxidation of proteins and lipids, the advanced glycation end products (AGEs), accumulate in a wide variety of environments. AGEs may be generated rapidly or over long times stimulated by a range of distinct triggering mechanisms, thereby accounting for their roles in multiple settings and disease states. A critical property of AGEs is their ability to activate receptor for advanced glycation end products (RAGE), a signal transduction receptor of the immunoglobulin superfamily. It is our hypothesis that due to such interaction, AGEs impart a potent impact in tissues, stimulating processes linked to inflammation and its consequences. We hypothesize that AGEs cause perturbation in a diverse group of diseases, such as diabetes, inflammation, neurodegeneration, and aging. Thus, we propose that targeting this pathway may represent a logical step in the prevention/treatment of the sequelae of these disorders

OBJECTIVE: The interaction of advanced glycation end products (AGEs) with their main receptor RAGE in endothelial cells induces intracellular generation of reactive oxygen species (ROS) and the expression of vascular cell adhesion molecule (VCAM)-1. We investigated the role of distinct sources of ROS, including the mitochondrial electron transport chain, NAD(P)H oxidase, xanthine oxidase, and arachidonic acid metabolism, in AGE-induced VCAM-1 expression. METHODS AND RESULTS: The induction of ROS and VCAM-1 by AGEs in cultured human umbilical vein endothelial cells was specifically blocked by an anti-RAGE antibody. The inhibition of NAD(P)H oxidase by apocynin and diphenylene iodonium, and of the mitochondrial electron transport system at complex II by thenoyltrifluoroacetone (TTFA), significantly inhibited both AGE-induced ROS production and VCAM-1 expression, whereas these effects were potentiated by rotenone and antimycin A, specific inhibitors of mitochondrial complex I and III, respectively. The inhibition of Cu/Zn superoxide dismutase inhibited both ROS and VCAM-1 induction, indicating that H2O2 by this source is involved as a mediator of VCAM-1 expression by AGEs. CONCLUSIONS: Altogether, these results demonstrate that ROS generated by both NAD(P)H-oxidase and the mitochondrial electron transport system are involved in AGE signaling through RAGE, and indicate potential targets for the inhibition of the atherogenic signals triggered by AGE-RAGE interaction.

In the AD brain, there are elevated amounts of soluble and insoluble Abeta peptides which enhance the expression of membrane bound and soluble receptor for advanced glycation end products (RAGE). The binding of soluble Abeta to soluble RAGE inhibits further aggregation of Abeta peptides, while membrane bound RAGE-Abeta interactions elicit activation of the NF-kappaB transcription factor promoting sustained chronic neuroinflammation. Atomic force microscopy observations demonstrated that the N-terminal domain of RAGE, by interacting with Abeta, is a powerful inhibitor of Abeta polymerization even at prolonged periods of incubation. Hence, the potential RAGE-Abeta structural interactions were further explored utilizing a series of computational chemistry algorithms. Our modeling suggests that a soluble dimeric RAGE assembly creates a positively charged well into which the negative charges of the N-terminal domain of dimeric Abeta dock

Cardiovascular disease represents the major cause of morbidity and mortality in patients with diabetes mellitus. The impact of cardiac disease includes increased sensitivity of diabetic myocardium to ischemic episodes and diabetic cardiomyopathy, manifested as a subnormal functional response of the diabetic heart independent of coronary artery disease. In this context, we were to our knowledge the first to demonstrate that diabetes increases glucose flux via the first and key enzyme, aldose reductase, of the polyol pathway, resulting in impaired glycolysis under normoxic and ischemic conditions in diabetic myocardium. Our laboratory has been investigating the role of the polyol pathway in mediating myocardial ischemic injury in diabetics. Furthermore, the influence of the aldose reductase pathway in facilitating generation of key potent glycating compounds has led us to investigate the impact of advanced glycation end products (AGEs) in myocardial ischemic injury in diabetics. The potent impact of increased flux via the aldose reductase pathway and the increased AGE interactions with its receptor (RAGE) resulting in cardiac dysfunction will be discussed in this chapter

Many studies have suggested that the expression of RAGE (receptor for advanced glycation end products) is upregulated in human tissues susceptible to the long-term complications of diabetes. From the kidneys to the macrovessels of the aorta, RAGE expression is upregulated in a diverse array of cell types, from glomerular epithelial cells (podocytes) to endothelial cells, vascular smooth muscle cells, and inflammatory mononuclear phagocytes and lymphocytes. Although RAGE was first described as a receptor for advanced glycation end products (AGEs), the key finding that RAGE was also a signaling receptor for proinflammatory S100/calgranulins and amphoterin, led to the premise that even in euglycemia, ligand-RAGE interaction propagated inflammatory mechanisms linked to chronic cellular perturbation and tissue injury. Indeed, such considerations suggested that RAGE might even participate in the pathogenesis of type 1 diabetes. Our studies have shown that pharmacological and/or genetic deletion/mutation of the receptor attenuates the development of hyperglycemia in NOD mice; in mice with myriad complications of diabetes, interruption of ligand-RAGE interaction prevents or delays the chronic complications of the disease in both macro- and microvessel structures. Taken together, these findings suggest that RAGE is 'at the right place and time' to contribute to the pathogenesis of diabetes and it complications. Studies are in progress to test the premise that antagonism of this interaction is a logical strategy for the prevention and treatment of diabetes

Pathological features of age-related macular degeneration such as the formation of extracellular deposits and neovascularization are frequently viewed as outcomes of compromising processes within retinal pigment epithelial cells, but the initiating circumstances are poorly understood. Here we tested the hypothesis that photooxidation events initiated by A2E, a blue light-excitable aging fluorophore of the retinal pigment epithelium, can set the stage for altered cellular signaling and changes in the expression of genes that can impact the extracellular milieu. Proteins modified by lipid peroxidation products (4-hydroxynonenal; malondialdhyde) and advanced glycation end products were detected at sites of blue light irradiation both in association with the cultured A2E-laden retinal pigment epithelial cells and within the fibronectin substrate on which the cells were grown. RAGE, the cell surface receptor that transduces the effects of advanced glycation end products, was also upregulated, and RAGE expression co-localized with the deposition of advanced glycation end products. Blue light triggered alterations in gene expression was also evidenced by elevations in both transcripts and protein for vascular endothelial growth factor, a potent angiogenic and permeability-enhancing factor. These findings indicate that cell associated and extracellular modification of proteins by lipid peroxidation products and advanced glycation end products together with increased expression of RAGE and vascular endothelial growth factor may be induced consequent to blue light illumination of A2E-burdened retinal pigment epithelial cells. Thus, photooxidative events that are not an immediate threat to retinal pigment epithelial cell viability may nevertheless elicit sustained perturbation that could ultimately alter neighboring tissues and impact retinal pigment epithelial cell function.

We tested the hypothesis that PKCbeta contributes to vascular smooth muscle cell (SMC) migration and proliferation; processes central to the pathogenesis of restenosis consequent to vascular injury. Homozygous PKCbeta null (-/-) mice or wild-type mice fed the PKCbeta inhibitor, ruboxistaurin, displayed significantly decreased neointimal expansion in response to acute femoral artery endothelial denudation injury compared with controls. In vivo and in vitro analyses demonstrated that PKCbetaII is critically linked to SMC activation, at least in part via regulation of ERK1/2 MAP kinase and early growth response-1. These data highlight novel roles for PKCbeta in the SMC response to acute arterial injury and suggest that blockade of PKCbeta may represent a therapeutic strategy to limit restenosis

The exquisite ability of the liver to regenerate is finite. Identification of mechanisms that limit regeneration after massive injury holds the key to expanding the limits of liver transplantation and salvaging livers and hosts overwhelmed by carcinoma and toxic insults. Receptor for advanced glycation endproducts (RAGE) is up-regulated in liver remnants selectively after massive (85%) versus partial (70%) hepatectomy, principally in mononuclear phagocyte-derived dendritic cells (MPDDCs). Blockade of RAGE, using pharmacological antagonists or transgenic mice in which a signaling-deficient RAGE mutant is expressed in cells of mononuclear phagocyte lineage, significantly increases survival after massive liver resection. In the first hours after massive resection, remnants retrieved from RAGE-blocked mice displayed increased activated NF-kappaB, principally in hepatocytes, and enhanced expression of regeneration-promoting cytokines, TNF-alpha and IL-6, and the antiinflammatory cytokine, IL-10. Hepatocyte proliferation was increased by RAGE blockade, in parallel with significantly reduced apoptosis. These data highlight central roles for RAGE and MPDDCs in modulation of cell death-promoting mechanisms in massive hepatectomy and suggest that RAGE blockade is a novel strategy to promote regeneration in the massively injured liver

OBJECTIVE: Chronic vascular inflammation may play a role in the development of macrovascular complications in diabetic patients. In this study, we examine the association of endothelial expression of two inflammatory mediators, receptor for advanced glycation end product (RAGE) and monocyte chemoattractant protein-1 (MCP-1), with type 2 diabetes using novel endothelial biopsy and RT-PCR techniques. RESEARCH DESIGN AND METHODS: Endothelial samples are obtained from the aorta of 12 patients with type 2 diabetes and 23 control subjects who underwent cardiac catheterization for chest pain syndrome or heart transplant follow-up. Endothelial cells are purified using magnetic beads with adsorbed CD146 antibody and subjected to RT-PCR analysis of RAGE and MCP-1 transcripts. The association of RAGE and MCP-1 expression with type 2 diabetes is assessed with chi(2) test and confirmed with in vitro experiments on human aorta endothelial cells. RESULTS: RT-PCR reveals gene expression patterns in patient-derived endothelial cells. Strong associations are observed between induction of RAGE mRNA and diabetes (P < 0.01) and between induction of RAGE and MCP-1 transcripts (P < 0.05). Treatment of cultured human aortic endothelial cells with S100b induces the expression of MCP-1 and RAGE transcripts. CONCLUSIONS: Endothelial cells can be harvested during cardiac catheterization and can be characterized with respect to molecular phenotypes under the influence of both genetic and environmental factors. Induction of RAGE and MCP-1 transcripts in patients with diabetes supports a role of chronic vascular inflammation in macrovascular complications.