The central theme driving our Program Project derives from the hypothesis that engagement of Receptor for Advanced Glycation Endproducts (RAGE) by its signal transduction ligands, Advanced Glycation Endproducts (AGEs), and S100/calgranulins, members of a family of proinflammatory cytokines, triggers vascular stress, thereby leading to sustained injury and failure of reparative mechanisms. AGEs and S100/calgranulins accumulate in the tissues in diabetes, but, as well, in states of vascular stress in euglycemia, consequent to perturbation by key factors highly-relevant to human cardiovascular disease, such as hyperlipidemia and arterial injury, hypoxia and ischemia/reperfusion injury. In the first years of our Program Project, substantial progress has been made in generating, testing and validating key tools required for ongoing dissection of the role of RAGE in vascular dysfunction; homozygous RAGE (0) mice and transgenic mice bearing cell-specific targetted expression of a RAGE transgene in which the cytosolic domain has been deleted, thereby imbuing a """"""""dominant negative"""""""" effect in vitro and in vivo, are protected against the adverse impact of distinct forms of vascular stress. Significant progress has been made in identifying the signal transduction effector molecules recruited directly upon ligand engagement of the cytosolic domain of the receptor; recent studies in a yeast two-hybrid assay demonstrate interaction of this domain with a newly-identified member of the diaphanous family. Diaphanous proteins, first identified in Drosophila, are critical intracellular """"""""bridges"""""""" of cell signalling and modulation of the actin cytoskeleton in mammalian cells, as specific domains of these molecules engage downstream effectors such as the Rho GTPases, thereby impacting on downstream cascades, such as Nuclear Factor (NF)-kappaB; and mechanisms linked to cellular motility; two key facets of RAGE biology. Prompted by compelling evidence supporting central roles for RAGE in vascular dysfunction, our ongoing goal will be to analyze the contribution of RAGE to mechanisms linked to vascular injury imparted by hyperlipidemia, hypoxia and ischemia/reperfusion. RAGE cytosolic domain and diaphanous-like protein wild-type and mutant constructs will be generated and their sites of interaction delineated; in addition, their impact on vascular cell properties upon induction of stress will be rigorously examined. These endeavors will shed light on the utility of RAGE blockade as a strategy to protect the stressed vasculature from irreversible injury in human cardiovascular disease.
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