Macrovascular Dysfunction of Diabetes

描述(申请人提供)：我们的长期目标是研究胰岛素信号影响血管组织对其他更有效的有丝分裂原的细胞反应变化的机制。我们最近发现，胰岛素能增强血管平滑肌细胞(VSMC)和血管内皮细胞(EC)中PDGF和VEGF的作用。因此，我们想要确定这些细胞内的串扰机制，确定治疗药物的分子靶点，并有助于理解糖尿病血管疾病的病理生理学。我们假设，处于代谢性胰岛素抵抗状态的高胰岛素血症，通过增加前烯基基转移酶的活性，从而增加可用于有丝分裂原刺激的GTP负荷的前烯基化的RAS和Rho蛋白的量，从而增强更强大的丝裂原对血管系统的影响，导致夸大的细胞反应，导致动脉粥样硬化的发生和发展。我们将使用两个重要的工具来检验这一假说：(1)法尼基转移酶(FTase)和香叶基香叶基转移酶I(GGTase I)的显性负向亚基(DNFTa)突变体，以帮助确定确保信号通路与其他生长因子途径相联系的机制；(2)四环素诱导系统，以确定DNFTa在稳定转染的血管平滑肌细胞中的表达对胰岛素刺激ERK5途径有何影响。为了确定这些机制，我们确定了三个主要的具体目标：(1)DNFTa是否在胰岛素抵抗时抑制高胰岛素血症增强其他生长因子对大鼠主动脉血管平滑肌细胞RASMC的有丝分裂作用；(2)DNFTa是否在胰岛素抵抗时抑制高胰岛素血症增强其他生长因子对大鼠肺血管内皮细胞PAVEC的有丝分裂作用；以及(3)DNFTa是否在胰岛素抵抗时抑制胰岛素刺激的RASMC中ERK5的激活和转位。

DESCRIPTION (provided by applicant): Our long-term goal is to investigate the mechanisms by which insulin signaling effects changes in cellular responses of vascular tissue to other more potent mitogens. We have recently shown that insulin augments the actions of PDGF and VEGF in vascular smooth muscle cells (VSMC) and endothelial cells (EC). Thus, we would like to determine these mechanisms of cross-talk within these cells, determine molecular targets for therapeutic drugs and contribute to the understanding of the pathophysiology of vascular disease in diabetes. We hypothesize that hyperinsulinemia in the state of metabolic insulin resistance, augments the effects of more potent mitogens on the vasculature via increases in prenyltransferase activity, thereby increasing the amounts of prenylated Ras and Rho proteins that are available for mitogen-stimulated GTPloading, resulting in exaggerated cellular responses that lead to the development and progression of atherosclerosis. We will test this hypothesis using two important tools: (1) a dominant-negative mutant of the alpha-subunit (DNFTa) of farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) to help determine the mechanisms that link the insuring signaling pathway to other growth factor pathways, and (2) a tetracyline-inducible system to determine what effects does the expression of DNFTa in stably transfected vascular smooth muscle cells have upon insulin-stimulation of the Erk5 pathway. To determine these mechanisms we have defined three major Specific Aims: (1) Does DNFTa inhibit hyperinsulinemia's ability to potentiate the mitogenic effects of other growth factors in rat aorta vascular smooth muscle cells RASMC in the presence of insulin resistance; (2) Does DNFTa inhibit hyperinsulinemia's ability to potentiate the mitogenic effects of other growth factors in rat pulmonary aortic vascular endothelial cells PAVEC in the presence of insulin resistance; and (3) Does DNFTa inhibit insulin-stimulated phosphorylation, activation and translocation of Erk5 in RASMC in the presence of insulin resistance.