Mouse Arteries Predisposed to Neointimal Formation

该提案将研究鞘氨醇1-磷酸（S1 P）如何调节平滑肌细胞迁移。 我们的初步数据显示FVB小鼠的SMC对S1 P的迁移反应良好， 在鞘氨醇激酶抑制剂（SphK）的存在下抑制。总的来说，C57 BL/6小鼠的细胞是 而不是由S1 P刺激，SphK抑制剂实际上刺激迁移。我们认为这些差异是 由于S1 P受体的表达。FVB表达S1 P1和S1 P3，但S1 P2很少。C57 BL/6细胞 强烈表达S1 P2，S1 P1和S1 P3水平降低。 第一个目的是测量FVB和C57 BL 76小鼠动脉中的循环S1 P水平， 伤后S1 P-激酶和S1 P-磷酸酶的活性以及激酶、磷酸酶 和裂解酶也将通过PCR测定。还将测定脂蛋白和脂蛋白中的S1 P 耗尽的血浆我们还将确定血浆S1 P是否可以刺激动脉中的事件， 对SMC迁移有重要影响。第二个目标是确定SphK活性的丧失是否会影响SMC 在损伤的小鼠动脉中迁移。这些研究将使用SphK敲除小鼠以及一种新的抑制剂来阻断 SphK活性第三个目标将确定特定S1 P受体对SMC迁移的重要性。 将使用针对S1 P1、S1 P2和S1 P3以及S1 P2和S1 P3缺失细胞的siRNA进行实验。的 对这些小鼠的动脉进行损伤，并测量SMC迁移和新生内膜发育。 最终的目标将确定S1 P如何发出信号，从而调节迁移。这些研究会问S1 P是否发出信号 EGF的作用机制不同，S1 P的作用也不同。我们还将确定是否需要PKB和Rac， SMC迁移和EGF受体阻断剂可阻止SMC迁移和新生内膜的形成 受伤的动脉

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 S1P-kinase and S1P-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.