Sphingosine-1-phosphate-activated telomerase in stroke

描述（由申请人提供）：中风是导致死亡和残疾的主要原因，与脑血管有关。内皮细胞（EC）凋亡死亡、血管生成失败和侧支血管生长不足使卒中预后恶化。在脑EC中，鞘氨醇-1 -磷酸（S1P）激活Akt和端粒酶，抑制细胞凋亡，诱导细胞增殖。神经元，可能还有神经胶质细胞，是缺血中S1P的来源，因为S1P合成酶（鞘氨酸激酶2，SPK2）在小鼠缺氧葡萄糖剥夺和大脑中动脉闭塞（MCAo）后在神经元中上调。我们假设缺血后，神经元/胶质来源的S1P通过端粒酶对EC发挥抗凋亡和促血管生成的作用。两个特定的目标将验证神经元/胶质来源的S1P激活EC S1P1受体、Akt和端粒酶，保护EC免于凋亡和诱导新生血管的假设。Aim 1（体外）将证实和表征端粒酶对EC中S1P的激活和上调反应，并验证神经元和胶质SPK2产生的S1P诱导端粒酶激活，从而介导EC保护和增殖的假设。我们认为S1P1受体、Akt和热休克蛋白90 （HSP90）在这些作用中起关键作用。目的2（体内）将显示S1P受体激动剂前药FTY720增加脑EC中端粒酶的表达，并扩展初步研究结果，表明该药物在MCAo之前或之后给药可减少梗死面积。利用在EC特异性启动子控制下表达显性负Akt的转基因小鼠，或缺乏端粒酶催化亚基或RNA模板的小鼠，我们将验证FTY720的保护作用与Akt和端粒酶在EC中的激活有关，并且这种作用不依赖于端粒酶的催化活性（FTY720应该在RNA模板敲除中起保护作用）。本项目将研究一种新颖的系统，可以提供脑卒中后EC功能的持久改善，并作为脑卒中治疗的新靶点。

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.