Faculty Bibliography

Heightened expression of the S100 calcium-binding protein, S100A4/Mts1, is observed in pulmonary vascular disease. Loss of serotonin (5-hydroxytryptamine [5-HT]) receptors or of the serotonin transporter (SERT) attenuates pulmonary hypertension in animals, and polymorphisms causing gain of SERT function are linked to clinical pulmonary vascular disease. Because 5-HT induces release of S100beta, we investigated the codependence of 5-HT receptors and SERT in regulating S100A4/Mts1 in human pulmonary artery smooth muscle cells (hPA-SMC). 5-HT elevated S100A4/Mts1 mRNA levels and increased S100A4/Mts1 protein in hPA-SMC lysates and culture media. S100A4/Mts1 in the culture media stimulated proliferation and migration of hPA-SMC in a manner dependent on the receptor for advanced glycation end products. Treatment with SB224289 (selective antagonist of 5-HT1B), fluoxetine (SERT inhibitor), SERT RNA-interference, and iproniazid (monoamine oxidase-A inhibitor), blocked 5-HT-induced S100A4/Mts1. 5-HT signaling mediated phosphorylation (p) of extracellular signal-regulated kinase 1/2 (pERK1/2), but pERK1/2 nuclear translocation depended on SERT, monoamine oxidase activity, and reactive oxygen species. Nuclear translocation of pERK1/2 was required for pGATA-4-mediated transcription of S100A4/Mts1. These data provide evidence for a mechanistic link between the 5-HT pathway and S100A4/Mts1 in pulmonary hypertension and explain how the 5-HT1B receptor and SERT are codependent in regulating S100A4/Mts1.

PURPOSE: The receptor for advanced glycation end products (AGEs) has been implicated in the pathogenesis of diabetic complications. This study was conducted to characterize the role of the RAGE axis in a murine model of nonproliferative diabetic retinopathy (NPDR). METHODS: The retinas of hyperglycemic, hyperlipidemic (HGHL, apolipoprotein E(-/-) db/db) mice were examined for the development of early retinal vascular lesions of NPDR and compared to littermates at 6 months of age. Neural function was assessed with electroretinography. Immunohistochemistry, real-time RT-PCR, autofluorescence, and ELISA studies were used to localize and quantify the AGE/RAGE axis. Soluble RAGE, a competitor of cellular RAGE for its ligands, was administered to assess the impact of RAGE blockade. RESULTS: Early inner retinal neuronal dysfunction, manifested by prolonged latencies of the oscillatory potentials and b-wave, was detected in hyperglycemic mice. HGHL mice exhibited accelerated development of acellular capillaries and pericyte ghosts compared with littermate control animals. AGEs were localized primarily to the vitreous cavity and internal limiting membrane (ILM) of the retina, where they were intimately associated with the footplates of RAGE-expressing Muller cells. AGE accumulation measured by ELISA was increased within the retinal extracellular matrix of hyperglycemic mice. AGE fluorescence and upregulation of RAGE transcripts was highest in the retinas of HGHL mice, and attenuation of the RAGE axis with soluble RAGE ameliorated neuronal dysfunction and reduced the development of capillary lesions in these mice. CONCLUSIONS: In early diabetic retinopathy, the RAGE axis, comprising the cellular receptor and its AGE ligands, is amplified within the retina and is accentuated along the vitreoretinal interface. Antagonism of the RAGE axis in NPDR reduces neurovascular perturbations, providing an important therapeutic target for intervention

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