Thrombin-induced Signaling in Vascular Smooth Muscle

血管平滑肌细胞(SMC)的过度增殖和迁移在动脉粥样硬化斑块的形成中起重要作用。α-凝血酶尤其令人感兴趣，因为它在几种不同的动物模型中参与了血管病理的调节。也有令人信服的证据表明，α-v-β-3整合素参与了血管愈合反应；挑战在于了解它们在调节损伤动脉中的SMC增殖和迁移中的作用。在我实验室的研究中，我们发现α-凝血酶诱导的人主动脉SMC的增殖部分地被α-v-β-3拮抗剂抑制，并且α-v-β-3拮抗剂阻断了α-凝血酶诱导的JNK1的激活。此外，我们还鉴定了α-凝血酶治疗后在SMC中发生的依赖于α-β-3整合素的细胞质事件，这为深入了解G蛋白偶联受体和整合素的整合信号提供了线索。特别是，非肌肉肌球蛋白-A(NM-A)是一种与SMC增殖有关的细胞骨架蛋白，它与α-vβ-3整合素和α-凝血酶处理大鼠主动脉SMC(RASMC)后的局灶性粘连(FAK)迅速结合。NM-A为调控提供了一个独特的位置，因为在两种不同的构象之间存在着平衡：折叠状态下肌球蛋白不能组装成细丝(即10S构象)和延伸构象促进细丝组装(即6S)。6S和10S之间的平衡对肌球蛋白轻链(MLC)的磷酸化状态很敏感。因此，在整合素介导的G蛋白偶联受体激活的信号转导过程中，PAR-1介导的MLC磷酸化可能起着重要的调节作用。在拟议的研究中，我们将检验以下假设：NM-A与α-vβ-3整合素、FAK和肌动蛋白细胞骨架的诱导结合是α-凝血酶诱导SMC激活c-Jun NH2末端激酶-1(JNK1)所必需的，并受RhoA的PAR-1激活和肌球蛋白轻链的磷酸化调节。我们提出的研究将为血管生物学中的两个关键问题提供洞察力：1)调节α-凝血酶诱导的SMC生长；2)G蛋白偶联受体和整合素信号的融合。这些研究还将有助于通过增加我们对α-vβ-3整合素如何调节血管愈合的了解，为血管疾病患者开发有效的治疗方法。

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.