Nitric oxide (NO) is a short-lived, free radical gas that accounts for the biologic properties of endothelium-derive relaxing factor (EDRF). My previous work focused on the physiological role of NO in the regulation of cardiovascular homeostasis. However, a deeper understanding of the role of NO in cardiovascular disease requires investigation of the pathways of NO synthesis within the vasculature. This proposal investigates an important molecular pathway of NO gene regulation in vascular smooth muscle cells. Nitric oxide (NO) is a potent vasodilator that contributes to the regulation of vascular tone. NO is synthesized from L-arginine by nitric oxide synthase (NOS). Two general classes of NOS have been identified: constitutive isozymes (neuronal (nc)NOS and endothelial (ec)NOS) that are present in tissues under basal conditions, and an inducible isozyme (iNOS) that requires cytokine or endotoxin activation for expression. We have demonstrated that iNOS mRNA levels are increased after cytokine stimulation in vascular smooth muscle cells. This upregulation of iNOS is mediated at the transcriptional level., and transforming growth factor (TGF)-beta1 but not dexamethasone downregulates iNOS mRNA after its induction by cytokines. The broad objectives of this proposal are to evaluate the regulation of smooth muscle cell iNOS by interleukin(IL)-1beta stimulation in cell culture and to investigate the mechanism(s) by which TGF-beta1 downregulates the vascular iNOS response to IL-1beta. Since the rate of gene transcription is regulated by the interaction of specific DNA sequences (cis-acting elements) and their cognate binding proteins (trans- acting factors), we plan to identify and characterize the cis-acting elements and trans-acting factors important in mediating IL-1beta induction of the iNOS gene in vascular smooth muscle cells. These specific elements may provide a target for the inhibition of the iNOS gene after it has been stimulated by cytokines. The Cardiovascular Biology Laboratory of the Harvard School of Public Health will serve as the site for the studies described in this proposal. The Laboratory encompasses 12,000 square feet and is equipped with instruments designed for research in cellular and molecular biology, and protein chemistry.
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