Neuron-Dervied S1P and Endothelial Function 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、血管生成的失败和侧支血管的生长不足会加重中风的预后。在脑内皮细胞中，1-磷酸鞘氨醇(S1P)诱导Akt/内皮型一氧化氮合酶(ENOS)激活，抑制细胞凋亡，诱导细胞增殖。神经元，可能还有胶质细胞，是脑缺血时S1P的来源之一，因为S1P合成酶(鞘氨醇激酶2，SPK2)在小鼠缺氧缺糖(OGD)和大脑中动脉闭塞后上调。我们推测，脑缺血后，S1P对EC具有抗细胞凋亡和促血管生成的作用。三个特定的目标将检验这一假说，即S1P激活EC S1P1受体和Akt/eNOS级联反应，保护EC免受凋亡和诱导新生血管形成。目的1证实和表征S1P对OGD后内皮细胞的保护作用以及对这些细胞的增殖诱导作用。我们认为S1P1受体、Akt和eNOS在这些效应中起关键作用。目的2将扩展初步结果，表明神经元可能是卒中过程中S1P的相关来源，神经元来源的S1P可介导AIM 1中观察到的脑内皮细胞的存活和增殖。我们将利用特异性抑制剂和RNA干扰技术，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研究脑缺血后SPK2上调和S1P1/Akt/eNOS系统激活的体内意义。我们推测FTY720(一种被SPK2转化为S1P受体激动剂的前体药物，而不是SPK1)将通过SPK2、S1P1和Akt/eNOS保护EC功能、促进EC增殖和诱导新生血管形成。本项目将研究一种新的系统，该系统可以为中风后的EC功能提供长期的改善，并作为中风治疗的目标。

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.