Mouse Arteries Predisposed to Neointimal Formation

描述（由申请人提供）：本提案将研究鞘氨醇1-磷酸（S1P）如何调节平滑肌细胞迁移。我们的初步数据表明，FVB小鼠的SMCs对S1P有良好的迁移反应，并且在鞘氨酸激酶抑制剂（SphK）的存在下，这种迁移被抑制。相比之下，C57BL/6小鼠的细胞不受S1P的刺激，SphK抑制剂反而刺激了迁移。我们认为这些差异是由于S1P受体的表达。FVB表达S1P1和S1P3，但S1P2表达较少。相比之下，C57BL/6细胞强烈表达s1p2，而S1P1和S1P3水平降低。第一个目的是测量FVB和C57BL76小鼠损伤前后动脉循环中S1P的水平。S1 p激酶和S1 p磷酸酶的活性以及激酶、磷酸酶和裂解酶的表达也将通过PCR检测。S1P也将在脂蛋白和血浆脂蛋白耗尽部分中测定。我们还将确定血浆S1P是否可以刺激动脉事件并重要地影响SMC迁移。第二个目的是确定SphK活性的丧失是否会影响损伤小鼠动脉中SMC的迁移。这些研究将使用SphK缺失小鼠以及一种新的抑制剂来阻断SphK的活性。第三个目标将确定特定的S1P受体对SMC迁移的重要性。实验将使用siRNA对S1P1、S1P2和S1P3以及S1P2和S1P3空细胞进行。这些小鼠的动脉将受到损伤，并测量SMC的迁移和新生内膜的发育。最终的目标是确定S1P信号是如何调控迁移的。这些研究将询问S1P信号是否通过EGF受体传递，以及不同的S1P是否有不同的作用。我们还将确定PKB和Rac是否是SMC迁移所必需的，以及阻断EGF受体是否会阻止受损动脉中SMC迁移和新生内膜的发育。

DESCRIPTION (provided by applicant): This proposal will examine how the sphingosine 1-phosphate (S1P) regulates smooth muscle cell migration. Our preliminary data show that SMCs of FVB mice migrate well in response to S1P and that migration is inhibited in the presence of a sphingosine kinase inhibitor (SphK). In total contrast, cells of C57BL/6 mice are not stimulated by S1P and the SphK inhibitor actually stimulates migration. We believe these differences are due to S1P receptor expression. FVB express S1P1 and S1P3 but little S1P2. In contrast C57BL/6 cells strongly express S1P2with reduced levels of S1P1 and S1P3. The first aim will measure circulating S1P levels in arteries of FVB and C57BL76 mice before and at times after injury. The activities of the S1 P-kinase and S1 P-phosphatase and expression of kinases, phosphatase and lyase will also be determined by PCR. S1P will also be determined in lipoprotein and in lipoprotein depleted fractions of plasma. We will also determine if plasma S1P can stimulate events in arteries and importantly affect SMC migration. The second aim will determine if loss of SphK activity influences SMC migration in injured mice arteries. These studies will use SphK null mice as well as a new inhibitor to block SphK activity. The third aim will determine the importance of specific S1P receptors for SMC to migrate. Experiments will be carried out using siRNA to S1P1, S1P2 and S1P3, and S1P2 and S1P3 null cells. The arteries of these mice will be subjected to injury and SMC migration and neointimal development measured. The final aim will determine how S1P signals and so regulates migration. These studies will ask if S1P signals via EGF receptor and if different S1P act differently. We will also determine if PKB and Rac are necessary for SMC migration and if blockade to the EGF receptor will prevent SMC migration and neointimal development in injured arteries.