Nitric oxide (NO) participates in many physiological and pathological processes. NO is generated inhumans by three NO synthases. Understanding their catalytic and regulatory mechanisms at themolecular 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 foruncoupled superoxide release, and the ratio of NO versus peroxynitrite formed. Each NOS likelyevolved to generate products and chemistries appropriate for specific circumstances in biology. Wehypothesize that NOS variants can be engineered (or have been created naturally)for specific biologicadvantage or disadvantage. We will test this by determining cellular consequences of NOS variantsengineered to generate superphysiological amounts of NO, and by characterizing NOS variants thatnaturally appear in the human population and may be associated with genetic predisposition towardcardiovascular disease.
Aim 1. Investigate efficacy of two 'super NO synthases' for inhibiting neointimalhyperplasia following vascular injury. We have created two NOS variants that generate up to 25times more NO compared to wild type enzyme. We will: (i) transfect the superNOS mutant genes intomammalian cells to test efficacy as superNO generators. (ii) Utilize viral delivery to test their efficacyfor generating therapeutic NO in our carotid-injury restenosis model. (iii) Determine how NOS genetransfer impacts oxidative/nitrative biomarkers in the injured vessels. (iv) Incorporate additionalmutations 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 iNOSand eNOS that contain amino acid substitutions resulting from SNP's in the protein-coding region oftheir genes. We will express, purify, and extensively characterize these NOS variants to determine howeach point mutation impacts enzyme function.
Aim 3. Test if the eNOS and iNOS SNP's are linked to the development of coronary arterydisease. The in vivo significance of NOS SNP's is largely unknown. We will: (i) Define the prevalence often SNPs that cause amino acid substitutions in iNOS and eNOS in individuals with and without CADfrom a cohort of well-characterized subjects. (ii) Test if NOS SNP's that alter enzyme function alsoserve to predict increased risk for cardiovascular disease. (iii) Test how NOS SNPs correlate withclinical markers of oxidative or nitrosative stress,
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