This application is for a Mentored Research Scientist Development Award in Aging through which the candidate, an experienced clinical investigator, will learn state-of-the-art techniques in cell physiology, cell imaging and signaling under the mentorship of Dr. Brooke Mossman. The goal will be to determine whether oxidative stress associated with aging and/or high glucose contributes to alterations in vascular smooth muscle cell (VSMC) apoptosis or proliferation. The research plan will focus on age- and glycemia-related effects on activation of the redox-sensitive transcription factors Nuclear Factor kappa B (NF-kappaB) and Activator Protein-1 (AP-1), testing the hypotheses that 1) aging and/or high glucose will induce mitochondrial oxidative stress in VSMC; 2) aging is associated with dysregulation of NF-kappaB and AP-1 activation; and 3) an imbalance between VSMC apoptosis and proliferation, i.e. phenotypic endpoints regulated by redox- sensitive transcription factors, will contribute to the development of vascular disease.
Specific aims of the proposal will be to determine: 1) whether high glucose media causes mitochondrial oxidative stress in VSMC from old (22-24 mos) and young (6-8 mos) Fischer 344 rats using cell imaging of oxidative probes (dichlorofluorescin derivatives); 2) whether age, glucose or hydrogen peroxide (H2O2), conditions or agents associated with oxidative stress, alters mitochondrial gene integrity and expression that may influence cell function or viability; 3) whether VSMC from old compared to young rats exhibit differences in baseline or inducible redox-sensitive transcription factors by determining the effect of high glucose media or exogenously administered H2O2 on DNA binding and transcriptional activation of NF-kappaB and AP-1; and 4) whether aging and/or high glucose induces changes in the activation of NF-kappaB or AP-1 which are causally related to the development of apoptosis or proliferation in VSMC. In these latter studies, we will first quantitate the effect of different concentrations of H2O2 (as a positive control) and of glucose on apoptosis and proliferation. We will then block the separate pathways using transient transfection techniques with dominant negative mutant constructs to determine if the phenotypic expression can be altered. The data obtained will permit future studies aimed at interventions targeted to defined signaling pathways to attenuate the deleterious effects of oxidative stress in vivo. The long-term goal will be to apply the tools and approaches learned through the support of this award to examine the effect of oxidative stress and transcriptional regulation on muscle protein synthesis in vivo in humans. The mentor has an extensive background in the investigation of reactive oxygen species as mediators of cell injury and of signal transduction, and a successful track record of training undergraduate and graduate students, postdoctoral fellows, and faculty.
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