Sphingosine 1 Phosphate and Embolic Stroke

描述(改编自申请人S摘要)： 血栓形成和栓塞症是大约80%的人类 中风是发病率和死亡率的主要原因，也是重大的经济问题 损失。这个应用程序将探索一种假设，即一个新的系统 调节脑血管张力(鞘氨醇-1-磷酸及其代谢 酶和受体)可以在药物上操纵，以便 有效缩小大鼠模型栓塞性脑梗塞面积。鞘氨醇-1-磷酸 (S1P)由活化的血小板释放，并与G蛋白偶联 受体分别命名为EDG-L、EDG-3、EDG-5、EDG-6和EDG-8。我们发现这个 脂质是一种强有力的大脑动脉收缩因子，但不是外周动脉的收缩因子。 其作用可能由EDG-3受体介导。初步证据显示 这种大脑的选择性可能是由高的SIP引起的 外周动脉中不受S1P影响的磷酸酶水平。 提出了两个目标来确定该新系统的哪些组件可以 目标是缩小梗塞面积。在第一个目标中，我们将在体外使用 准备测试Rho激酶是必需的假说 S1P引起的收缩(Y-27632可抑制)。我们将在以下方面进行扩展 初步数据显示，S1P磷酸酶(S1PP)高表达 外周动脉(而不是脑动脉)的mRNA与高[32P]S1P平行 具有与S1PP相对应的抑制谱的降解活性。 携带正义和反义S1PP重组腺病毒的研究 将确定S1PP是缺少S1P中介的原因 外周动脉收缩。 第二个目标将检验干扰SIP信令的假设 主要通过血流依赖机制减少梗塞面积。这将是 首先通过苏拉明阻断EDG-3受体和通过抑制 Rho与Y-27632的激活剂。同样，通过治疗大鼠来阻断S1P的合成 对于鞘氨醇激酶抑制剂，二甲基鞘氨醇(DMS)也应该 减少梗塞面积(将使用激光散斑分析评估血流 和[14C]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-5354C)的作用，不含 PKC抑制活性，将进一步确立S1P介导的作用 栓塞性卒中时的收缩。最后，我们将检验假设 神经鞘磷脂水平在血栓后高于细丝闭塞后， 因此，在后一种模式中，DMS保护应该不那么明显。 总之，这些研究将探索一种新系统的意义，它 似乎是大脑选择性的，并可能成为治疗的有效靶点 中风疗法。

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.