EFFECT OF AGE AND GLYCEMIA ON TRANSCRIPTION FACTORS

此应用程序是一个指导研究科学家发展奖在老化，通过候选人，经验丰富的临床研究者，将学习国家的最先进的技术在细胞生理学，细胞成像和信号的指导下博士。 我们的目标将是确定是否与衰老和/或高糖相关的氧化应激有助于血管平滑肌细胞（VSMC）凋亡或增殖的改变。 这项研究计划将集中在年龄和糖尿病对氧化还原敏感的转录因子核因子κ B激活的影响上（NF-kappaB）和激活蛋白-1（AP-1），验证以下假设：1）衰老和/或高糖将诱导VSMC中的线粒体氧化应激; 2）衰老与NF-kappaB和AP-1激活的失调相关; 3）VSMC凋亡和增殖之间的失衡，即氧化还原敏感性转录因子调节的表型终点，将有助于血管疾病的发展。 该提案的具体目标将是确定：1）高葡萄糖培养基是否会导致老年人VSMC的线粒体氧化应激（22-24个月）和年轻（6-8个月）Fischer 344大鼠使用氧化探针的细胞成像（二氯荧光素衍生物）; 2）是否年龄、葡萄糖或过氧化氢（H2 O2）、与氧化应激相关的条件或试剂，改变线粒体基因的完整性和表达，可能影响细胞功能或活力; 3）来自老年大鼠的VSMC与年轻大鼠相比是否表现出基线或诱导性氧化还原的差异。通过测定高糖培养基或外源H2 O2对DNA结合和转录的影响，NF-κ B和AP-1的活化;和4）老化和/或高葡萄糖是否诱导NF-κ B或AP-1活化的变化，其与VSMC中细胞凋亡或增殖的发展有因果关系。 在后面的这些研究中，我们将首先定量不同浓度的H2 O2（作为阳性对照）和葡萄糖对细胞凋亡和增殖的影响。 然后，我们将使用瞬时转染技术与显性阴性突变体构建体阻断单独的途径，以确定表型表达是否可以改变。 所获得的数据将允许未来的研究，旨在针对定义的信号通路的干预措施，以减轻体内氧化应激的有害影响。 长期目标将是应用通过该奖项的支持学到的工具和方法，以研究氧化应激和转录调控对人体体内肌肉蛋白合成的影响。 导师在活性氧作为细胞损伤和信号转导介质的研究方面具有广泛的背景，并成功培训了本科生和研究生，博士后研究员和教师。

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.