Thrombin-induced Signaling in Vascular Smooth Muscle

血管平滑肌细胞（SMC）过度增殖迁移在动脉粥样硬化斑块的形成中起重要作用。α -凝血酶是特别感兴趣的，因为它涉及介导血管病理在几种不同的动物模型。也有令人信服的证据表明α -v- β -3整合素参与血管愈合反应；挑战在于了解它们在调节受损动脉中SMC增殖和迁移中的作用。在我实验室的研究中，我们发现α -凝血素诱导的人主动脉SMC增殖被α -v- β -3拮抗剂部分抑制，α -v- β -3拮抗剂阻断α -凝血素诱导的JNK1激活。此外，我们已经确定了α -凝血酶治疗后SMC中发生的α - β -3整合素依赖的细胞质事件，这为g蛋白偶联受体和整合素的整合信号提供了见解。特别是，非肌肉肌球蛋白- a (NM-A)，一种与SMC增殖有关的细胞骨架蛋白，在用α -凝血酶治疗大鼠主动脉SMC （RASMC）后，与α -v - β -3整合素和局灶粘连（FAK）迅速相关。NM-A提供了一个独特的调控位点，因为在两种不同的构象之间存在平衡：折叠状态下肌凝蛋白不能组装成细丝（即10S构象）和扩展构象，促进细丝的组装（即6S）。6S和10S之间的平衡对肌球蛋白轻链（MLC）的磷酸化状态敏感。因此，受par -1介导的信号通路刺激的MLC磷酸化，可能在G蛋白偶联受体激活的整合素介导的信号通路中发挥重要的调节作用。在拟进行的研究中，我们将验证以下假设：NM-A与α -v - β -3整合素、FAK和肌动蛋白细胞骨架的诱导关联是α -凝血素诱导SMC中c-jun nh2末端激酶1 （JNK1）激活所必需的，并受PAR-1激活RhoA和肌球蛋白轻链磷酸化的调节。我们提出的研究将为血管生物学中两个至关重要的问题提供见解：1)α -凝血酶诱导的SMC生长的调控和2)G蛋白偶联受体和整合素信号的收敛。这些研究也将通过增加我们对α -v - β -3整合素如何调节血管愈合的理解，为血管疾病患者有效治疗的发展做出贡献。

Excessive proliferation of migration of vascular smooth muscle cells (SMC) plays a major role in the development of atherosclerotic plaques. Alpha-thrombin is of particular interest because it has been implicated in mediating vascular pathology in several different animal models. There is also compelling evidence that alpha-v-beta-3 integrins are involved in vascular healing responses; the challenge has been to understand their role in regulating SMC proliferation and migration in the injured artery. In studies from my laboratory, we found that alpha-thrombin-induced proliferation of human aortic SMC was partially inhibited by alpha-v- beta-3 antagonists and that alpha-v-beta-3 antagonists block alpha- thrombin-induced JNK1 activation. Furthermore, we have identified alphav-beta-3 integrin-dependent cytoplasmic events that occur in SMC in response to treatment with alpha-thrombin which provide insight into the integration signals from G-protein-coupled receptors and integrins. In particular, non-muscle myosin-A (NM-A), a cytoskeletal protein implicated in SMC proliferation, rapidly associates with alpha-v beta-3 integrins and with focal adhesion (FAK) following treatment of rat aortic SMC (RASMC) with alpha-thrombin. NM-A provides a unique site for regulation as an equilibrium exists between two distinct conformations: a folded state in which myosin can not assemble into filaments (i.e. 10S conformation) and an extended conformation that promotes the assembly of filaments (i.e. 6S). Equilibration between 6S and 10S is sensitive to the phosphorylation state of myosin light chain (MLC). Thus phosphorylation of MLC, which is stimulated by PAR-1-mediated signaling, could play an important regulatory role in integrin-mediate signaling in response to activation of G protein-coupled receptors. In the proposed studies, we will test the following hypothesis: The inducible association of NM-A with alpha-v beta-3 integrins, FAK and the actin cytoskeleton is required for alpha-thrombin-induced activation of c-jun NH2-terminal kinase-1 (JNK1) in SMC and is regulated by PAR-1 activation of RhoA and phosphorylation of myosin light chain. The studies we propose will provide insight into two critically important questions in vascular biology: 1) regulation of alpha-thrombin-induced SMC growth and 2) convergence of G protein-coupled receptor and integrin signaling. These studies will also contribute to the development of effective treatments for patients with vascular disease by increasing our understanding of how alpha-v beta-3 integrins regulate vascular healing.