Sphingosine-1-phosphate-activated telomerase in stroke

描述(申请人提供)：中风是死亡和残疾的主要原因，并涉及脑血管。血管内皮细胞(EC)的凋亡性死亡、血管生成的失败和侧支血管的生长不足会加重卒中的预后。在脑内皮细胞中，鞘氨醇-1-磷酸(S1P)激活Akt和端粒酶，抑制细胞凋亡，诱导增殖。在小鼠缺氧缺糖和大脑中动脉闭塞(MCAO)后，神经元，可能还有胶质细胞，是S1P合成酶(鞘氨醇激酶2，SPK2)上调的来源。我们推测，缺血后，神经元/神经胶质细胞来源的S1P通过端粒酶对EC发挥抗凋亡和促血管生成作用。两(2)个特定的目标将检验这一假设，即神经元/胶质细胞来源的S1P激活EC S1P1受体、Akt和端粒酶，保护EC免受凋亡和诱导新生血管形成。目的1(体外)证实和表征EC对S1P的端粒酶反应(激活和上调)，并验证神经细胞和神经胶质细胞SPK2产生的S1P诱导端粒酶激活从而介导EC保护和增殖的假说。我们认为S1P1受体、Akt和热休克蛋白90(HSP90)在这些效应中起关键作用。目的2(在体内)将显示S1P受体激动剂前体药物FTY720增加脑内皮细胞端粒酶的表达，并扩大初步研究结果，表明该药在MCAO前后应用可减少梗塞面积。利用在EC特异启动子控制下表达显性阴性Akt的转基因小鼠，或缺乏端粒酶催化亚单位或RNA模板的小鼠，我们将检验FTY720的保护作用与EC中Akt和端粒酶的激活有关，并且这种作用不依赖于端粒酶的催化活性(FTY720应该在RNA模板敲除中保护端粒酶)。本项目将研究一种新的系统，能够在中风后提供EC功能的长期改善，并成为中风治疗的新靶点。

DESCRIPTION (provided by applicant): Stroke is a major cause of death and disability and brain blood vessels have been implicated. Stroke outcome is worsened by apoptotic death of endothelial cells (EC), failure of angiogenesis and insufficient growth of collateral vessels. In brain EC, sphingosine-1 -phosphate (S1P) activates Akt and telomerase, suppresses apoptosis and induces proliferation. Neurons, and possibly glial cells, are a source of S1P in ischemia because the S1P synthesizing enzyme (sphingosine kinase 2, SPK2) is upregulated in neurons following oxygen glucose deprivation and middle cerebral artery occlusion (MCAo) in mice. We hypothesize that following ischemia, neuron/glia-derived S1P exerts anti-apoptotic and pro-angiogenic effects on EC via telomerase. Two (2) specific aims will test the hypothesis that neuron/glia-derived S1P activates EC S1P1 receptors, Akt and telomerase, protecting EC from apoptosis and inducing neovascularization. Aim 1 (in vitro) will confirm and characterize the telomerase responses (activation and up-regulation) to S1P in EC, and test the hypothesis that S1P produced by neuronal and glial SPK2 induces telomerase activation, thereby mediating EC protection and proliferation. We propose that S1P1 receptors, Akt and Heat-Shock Protein 90 (HSP90) play a key role in these effects. Aim 2 (in vivo) will show that the S1P receptor agonist prodrug FTY720 increases telomerase expression in brain EC and extend preliminary findings showing that this agent decreases infarct size when administered before or after MCAo. Using transgenic mice expressing dominant negative Akt under the control of an EC-specific promoter, or mice lacking either the catalytic subunit or the RNA template of telomerase, we will test the hypothesis that the protective effect of FTY720 is related to Akt and telomerase activation in EC, and that this effect does not depend on the catalytic activity of telomerase (FTY720 should protect in RNA template knockouts).This project will study a novel system that can provide long-lasting improvement in EC function following stroke and serve as a new target for stroke therapy.