Nitric Oxide Synthases As Oxidative And Therapeutic Agents

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Nitric oxide (NO) participates in many physiological and pathological processes. NO is generated in humans by three NO synthases. Understanding their catalytic and regulatory mechanisms at the molecular level is critical for understanding their functions and potential use as therapeutic agents. Each NOS differs markedly in their rate of NO release, oxygen dependence profile, capacity for uncoupled superoxide release, and the ratio of NO versus peroxynitrite formed. Each NOS likely evolved to generate products and chemistries appropriate for specific circumstances in biology. We hypothesize that NOS variants can be engineered (or have been created naturally)for specific biologic advantage or disadvantage. We will test this by determining cellular consequences of NOS variants engineered to generate superphysiological amounts of NO, and by characterizing NOS variants that naturally appear in the human population and may be associated with genetic predisposition toward cardiovascular disease. Aim 1. Investigate efficacy of two "super NO synthases" for inhibiting neointimal hyperplasia following vascular injury. We have created two NOS variants that generate up to 25 times more NO compared to wild type enzyme. We will: (i) transfect the superNOS mutant genes into mammalian cells to test efficacy as superNO generators. (ii) Utilize viral delivery to test their efficacy for generating therapeutic NO in our carotid-injury restenosis model. (iii) Determine how NOS gene transfer impacts oxidative/nitrative biomarkers in the injured vessels. (iv) Incorporate additional mutations predicted to further increase efficacy of superNOS. Aim 2. Investigate the functional impact of specific single nucleotide polymorphisms (SNPs) that occur in endothelial and inducible NOS. There are five natural variants each of Human iNOS and eNOS that contain amino acid substitutions resulting from SNP's in the protein-coding region of their genes. We will express, purify, and extensively characterize these NOS variants to determine how each point mutation impacts enzyme function. Aim 3. Test if the eNOS and iNOS SNP's are linked to the development of coronary artery disease. The in vivo significance of NOS SNP's is largely unknown. We will: (i) Define the prevalence of ten SNPs that cause amino acid substitutions in iNOS and eNOS in individuals with and without CAD from a cohort of well-characterized subjects. (ii) Test if NOS SNP's that alter enzyme function also serve to predict increased risk for cardiovascular disease. (iii) Test how NOS SNPs correlate with clinical markers of oxidative or nitrosative stress,