Sphingosine 1 Phosphate and Embolic Stroke

描述（改编自申请人？s abstract）： 血栓形成和栓塞是造成大约80%的人类死亡的原因。 中风是发病率和死亡率的主要原因， 损失这个应用程序将探索一个新的系统， 调节脑血管紧张度（鞘氨醇-1-磷酸，其代谢 酶和受体）可以被间接操纵， 有效地减少大鼠模型中栓塞性梗塞尺寸。鞘氨醇-1-磷酸 (S1P)由活化的血小板释放并结合G蛋白偶联 在一些实施方案中，所述受体被称为Edg-1、Edg-3、Edg-5、Edg-6和Edg-8。我们发现这 脂质是脑动脉而不是外周动脉的有效收缩剂， 可能由Edg-3受体介导的作用。初步证据显示 这种大脑的选择性可能是由高SIP 外周动脉中的磷酸酶水平不收缩到增加的S1 P。 提出了两个目标，以确定该新系统的哪些组件可以 旨在减少梗死面积。在第一个目标中，我们将使用体外 准备测试的假设，Rho激酶是必需的， S1 P诱导的收缩（Y-27632应抑制）。我们将扩展到 初步数据显示，S1 P磷酸酶（S1 PP）的高表达， 外周动脉中的mRNA，而不是脑动脉中的mRNA，被高[32 P] S1 P抑制， 降解活性，其抑制曲线与S1 PP的抑制曲线相对应。 最后，用携带正义和反义S1 PP构建体的腺病毒进行研究， 将确定S1 PP是负责缺乏S1 P介导的 外周动脉收缩。 第二个目标将测试干扰SIP信令的假设 主要通过血流依赖性机制减少梗死面积。这将 首先通过用苏拉明阻断Edg-3受体和通过抑制 Rho激酶与Y-27632。类似地，通过治疗大鼠来阻断S1 P的合成， 与鞘氨醇激酶抑制剂二甲基鞘氨醇（DMS）也应该 减少梗死面积（使用激光散斑分析评估血流 和[14 C]碘安替比林放射自显影），而不改变PKC底物。一个 其他鞘氨醇激酶抑制剂（F-12509和B-5354 C）的作用， PKC抑制活性，将进一步确立S1 P介导的作用 栓塞性中风中的收缩。最后，我们将测试假设， 鞘脂水平在凝块后比在细丝闭塞后更高， 因此，DMS保护在后一种模型中不太明显。 总之，这些研究将探讨一个新系统的意义， 似乎是选择性的，并可能是一个有用的治疗靶点， 中风治疗

DESCRIPTION(Adapted from applicant?s abstract): Thrombosis and embolism are responsible for approximately 80 percent of human stroke, a major cause of morbidity and mortality, and significant economic loss. This application will explore the hypothesis that a novel system regulating cerebrovascular tone (sphingosine-1-phosphate, its metabolizing enzymes and receptors) can be pharmacologically manipulated in order to effectively reduce embolic infarct size in a rat model. Sphingosine-1-phosphate (S1P) is released by activated platelets and binds to G-protein coupled receptors termed Edg-l, Edg-3, Edg-5, Edg-6, and Edg-8. We found that this lipid is a potent constrictor of cerebral, but not peripheral arteries, an effect likely to be mediated by Edg-3 receptors. Preliminary evidence shows that this cerebral selectivity might be accounted for by the high SIP phosphatase levels in peripheral arteries that do not constrict to added S1P. Two aims are proposed to determine which components of this novel system can be targeted to decrease infarct size. In the first aim, we will use in vitro preparations to test the hypothesis that Rho kinases are required for S1P-induced constriction (Y-27632 should inhibit). We will expand upon preliminary data by showing that the high expression of S1P phosphatase (S1PP) mRNA in peripheral, but not cerebral arteries, is paralleled by a high [32P]S1P degrading activity with an inhibition profile corresponding to that of S1PP. Finally, studies with adenovirus bearing sense and antisense S1PP constructs will establish that S1PP is responsible for the lack of S1P-mediated constriction in peripheral arteries. The second aim will test the hypothesis that interfering with SIP signaling reduces infarct size primarily by a blood flow-dependent mechanism. This will first be achieved by blocking Edg-3 receptors with suramin and by inhibiting Rho kinases with Y-27632. Similarly, blocking S1P synthesis by treating rats with the sphingosine kinase inhibitor dimethylsphingosine (DMS) should also decrease infarct size (blood flow will be assessed using laser speckle analysis and [14C]iodoantipyrine autoradiography), without altering PKC substrates. An effect of other sphingosine kinase inhibitors (F-12509 and B-5354C), devoid of PKC inhibiting activity, will further establish the role of S1P-mediated constriction in embolic stroke. Finally, we will test the hypothesis that sphingolipid levels are elevated after clot more than after filament occlusion, and therefore, DMS protection should be less apparent in the latter model. Together, these studies will explore the significance of a novel system, which appear to be cerebro-selective, and potentially a useful therapeutic target for stroke therapy.