Mouse Arteries Predisposed to Neointimal Formation

该提案将研究1-磷酸鞘氨醇(S1P)是如何调节平滑肌细胞迁移的。 我们的初步数据显示，FVB小鼠的SMC对S1P有良好的迁移作用，并且迁移是 在鞘氨醇激酶抑制剂(SphK)存在的情况下被抑制。相比之下，C57BL/6小鼠的细胞 不受S1P和SphK抑制剂的刺激，实际上会刺激迁移。我们认为这些差异是 由于S1P受体的表达。FVB表达S1P1和S1P3，少量表达S1P2。相比之下，C57BL/6细胞 强表达S1P，降低S1P1和S1P3水平。 第一个目标是测量FVB和C57BL76小鼠动脉中循环S1P的水平 受伤后。S1P-激酶、S1P-磷酸酶活性及蛋白水解酶、磷酸酶的表达 裂解酶也将通过聚合酶链式反应进行测定。S1P也将在脂蛋白和脂蛋白中被检测到 耗尽的部分血浆。我们还将确定血浆S1P是否可以刺激动脉和血管中的事件 重要影响SMC迁移。第二个目标将确定SphK活性的丧失是否会影响SMC 在受损的小鼠动脉中的迁移。这些研究将使用SphK基因缺失的小鼠以及一种新的抑制剂来阻断 SphK活性。第三个目的将确定特定的S1P受体对SMC迁移的重要性。 使用siRNA对S1P1、S1P2和S1P3，以及S1P2和S1P3缺失细胞进行实验。这个 这些小鼠的动脉将受到损伤，并测量SMC迁移和新生内膜发育。 最终目标将决定S1P如何发出信号，从而调控迁移。这些研究将询问S1P信号 不同的S1P通过EGF受体发挥不同的作用。我们还将确定是否有必要使用PKB和RAC SMC迁移和对EGF受体的IF阻断将阻止SMC迁移和新生内膜形成 在受伤的动脉中。

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.