Neural derived S1P and Endothelial Function 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、血管生成失败和侧支血管生长不足会使卒中预后恶化。在脑EC中，鞘氨醇-1-磷酸（S1P）诱导Akt/内皮型一氧化氮合酶（eNOS）活化，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，是缺血时S1P的来源，因为在小鼠的氧葡萄糖剥夺（OGD）和大脑中动脉闭塞后，神经元中的S1P合成酶（鞘氨酸激酶2，SPK2）上调。我们假设脑缺血后，S1P对EC具有抗凋亡和促血管生成的作用。三个具体目标将验证S1P激活EC S1P1受体和Akt/eNOS级联，保护EC免于凋亡和诱导新生血管的假设。Aim 1将证实和表征S1P对OGD后EC的保护作用，以及对这些细胞增殖的诱导作用。我们认为S1P1受体、Akt和eNOS在这些作用中起关键作用。Aim 2将扩展初步研究结果，表明神经元可能是卒中中S1P的相关来源，并且在Aim 1中观察到神经元来源的S1P可以介导脑EC的存活和增殖。通过使用特异性抑制剂和RNA干扰技术，我们将确定SPK2亚型的作用，以及神经元S1P是否通过S1P1受体介导上述作用。目的3将检测小鼠缺血后SPK2上调和S1P1/Akt/eNOS系统激活的体内意义。我们假设FTY720（一种由SPK2而非SPK1转化为S1P受体激动剂的前药）可以通过SPK2、S1P1和Akt/eNOS保护EC功能，增强EC增殖并诱导卒中后的新血管形成。该项目将研究一种新的系统，该系统可以长期改善脑卒中后的EC功能，并作为脑卒中治疗的靶点。

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 endothelial cell (EC) apoptosis, failure of angiogenesis and insufficient growth of collateral vessels. In brain EC, sphingosine-1-phosphate (S1P) induces Akt/endothelial nitric oxide synthase (eNOS) activation, suppresses apoptosis and induces proliferation. Neurons, and possibly glial cells, are a source of S1P in ischemia because a S1P synthesizing enzymes (sphingosine kinase 2, SPK2) is upregulated in neurons following oxygen glucose deprivation (OGD) and middle cerebral artery occlusion in mice. We hypothesize that following cerebral ischemia, S1P exerts antiapoptotic and pro-angiogenic effects on EC. Three specific aims will test the hypothesis that S1P activates EC S1P1 receptors and the Akt/eNOS cascade, protecting EC from apoptosis and inducing neovascularization. Aim 1 will confirm and characterize the protective effect of S1P on EC after OGD, as well as the induction of proliferation in these cells. We propose that S1P1 receptors, Akt and eNOS play a key role in these effects. Aim 2 will extend preliminary findings showing that neurons can be a relevant source of S1P during stroke and that neuron-derived S1P could mediate the survival and proliferation of brain EC observed in Aim 1. With the use of specific inhibitors, and RNA interference technology, we will determine the role of the SPK2 subtype, and whether neuronal S1P acts mediates the above effects via S1P1 receptors. Aim 3 will examine the in vivo significance of SPK2 up-regulation and the activation of the S1P1/Akt/eNOS system following ischemia in mice. We hypothesize that administration of FTY720 (a pro-drug converted to a S1P receptor agonist by SPK2, but not SPK1) will protect EC function, enhance EC proliferation and induce neovascularization following stroke via SPK2, S1P1 and Akt/eNOS. This project will study a novel system that can provide long-lasting improvement in EC function following stroke and serve as a target for stroke therapy.