(Verbatim from the application): The underlying pathologic basis in vascular disorders such as atherosclerosis and restenosis following injury is the migration of medial vascular smooth muscle cells (VSMC) to the intima where they proliferate and secrete abundant amounts of extracellular matrix proteins. The mechanism underlying the modulation of the phenotype of VSMC from specialized contractile cells to fibroproliferative cells is key to our understanding of the genesis of vascular diseases. A number of studies have underscored the importance of endothelial-derived nitric oxide (NO) in maintaining a relatively quiescent and differentiated VSMC phenotype both in vivo and in vitro. The enzyme that mediates these beneficial effects of endothelial-derived NO is the Type I cyclic GMP-dependent protein kinase (PKG Ia). However, the expression of PKG Ia is reduced in proliferating neointimal VSMC in vivo and in cells cultured in vitro where they modulate to the secretory phenotype. Since restoration of PKG expression in vitro restores the contractile phenotype, then understanding the mechanism regulating PKG Ia expression is key to understanding and attenuating phenotypic modulation. Recent studies from our laboratory have shown that inflammatory cytokines, NO donor drugs and cyclic nucleotide analogs suppress PKG Ia expression in cultured VSMC. Furthermore, a 500 bp promoter for PKG Ia responds to NO and cyclic nucleotide analogs with a decrease in activity as assessed by in vitro transfection assays. However, the NO/cyclic nucleotide-responsive elements in the PKG Ia promoter have not been defined nor has the molecular basis for tissue-specific suppression of PKG expression been analyzed. Therefore, the specific aims of this proposal are to characterize the elements in the PKG Ia promoter that mediate NO/cyclic nucleotide-mediated suppression of PKG expression, and to identify tissue-specific co-factors that mediate the effects of NO/cyclic nucleotide suppression of PKG expression in VSMC. These studies will be done using in vitro transfection assays to analyze the cis-acting elements in the cloned PKG Ia promoter and electromobility shift assays (EMSA) to define the transcription factors regulated by NO/cyclic nucleotides. Yeast two-hybrid methods will be used to identify and characterize specific binding proteins that interact with NO/cyclic nucleotide-regulated transcription factors in a tissue specific manner. These studies are the first to characterize the mechanisms regulating Type I PKG expression in mammalian cells, and will be critical for our understanding of the mechanisms underlying phenotypic modulation in VSMC. Ultimately, new approaches for understanding and treating vascular diseases can be undertaken based on this new knowledge.
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