EFFECT OF AGE AND GLYCEMIA ON TRANSCRIPTION FACTORS

这份申请是为老年研究科学家发展奖提供指导，候选人是一名经验丰富的临床研究员，将在布鲁克·莫斯曼博士的指导下学习细胞生理学、细胞成像和信号传递方面的最先进技术。其目标将是确定与衰老和/或高糖相关的氧化应激是否有助于血管平滑肌细胞(VSMC)的凋亡或增殖的改变。该研究计划将集中于年龄和血糖对氧化还原敏感型转录因子核因子-kappaB(NF-kappaB)和激活蛋白-1(AP-1)激活的影响，验证以下假说：1)衰老和/或高糖将诱导VSMC线粒体氧化应激；2)衰老与NF-kappaB和AP-1激活的失调有关；以及3)VSMC凋亡和增殖之间的失衡，即氧化还原敏感转录因子调控的表型终点，将有助于血管疾病的发展。该提案的具体目的将是确定：1)高糖介质是否导致老年(22-24个月)和年轻(6-8个月)Fischer 344大鼠的VSMC线粒体氧化应激；2)年龄、葡萄糖或过氧化氢(H_2O_2)与氧化应激相关的条件或试剂是否改变线粒体基因的完整性和表达，从而可能影响细胞功能或存活率；3)通过检测高糖介质或外源性H_2O_2对DNA结合和核因子-kappaB和AP-1转录激活的影响，老年大鼠和青年大鼠VSMC在基础或可诱导的氧化还原敏感转录因子方面是否存在差异；以及4)衰老和/或高糖是否诱导与VSMC的凋亡或增殖有关的核因子-kappaB或AP-1的激活变化。在后一项研究中，我们将首先量化不同浓度的过氧化氢(作为阳性对照)和葡萄糖对细胞凋亡和增殖的影响。然后，我们将使用带有显性负突变结构的瞬时转染技术来阻断分离的途径，以确定表型表达是否可以改变。所获得的数据将使未来的研究能够针对明确的信号通路进行干预，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所学到的工具和方法来检查氧化应激和转录调节对人类体内肌肉蛋白质合成的影响。该导师在研究作为细胞损伤和信号转导媒介的活性氧物种方面有广泛的背景，并在培训本科生和研究生、博士后研究员和教师方面有成功的记录。

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.