Controlling VSMC Proliferation and Migration

描述(申请人提供)：血管平滑肌细胞(VSMC)的增殖和迁移是心脏移植后冠状动脉支架内再狭窄和加速动脉病变的主要原因。如何在体内防止VSMC的增殖、迁移和随之而来的再狭窄仍然是过去十年来广泛研究的主题。我们令人兴奋的初步数据表明，AMP激活蛋白激酶(AMPK)的药理或基因激活能够抑制体内VSMC的增殖和新生内膜的增殖。荧光激活细胞分选分析显示，AMPKa2基因缺失可促进VSMC从G1期向S期过渡。与这一发现一致的是，细胞周期抑制因子p27Kip1(P27)在AMPKa2基因敲除(KO)的小鼠VSMC中显著下调，但在AMPKa1-KO VSMC中没有下调。此外，我们还发现p27Kip1的失控不是由于p27Kip1的mRNA水平，而是由于Skp2启动子中通过STAT结合的泛素E3连接酶的一个亚基Skp2的高表达。在机制上，我们发现p27的E3泛素连接酶S相蛋白2(Skp2)在AMPKa2-KO VSMC中升高，并与p27的降解增加有关。AMPK依赖抑制VSMC增殖和随之而来的再狭窄的最确凿证据是，在AMPKa2-KO小鼠中，钢丝损伤诱导的颈动脉新生内膜增生显著高于AMPKa1-KO或野生型(WT)动物。因此，这一应用的中心假设是，AMPKa2的缺失增加了p27的E3连接酶Skp2，以及Skp2介导的p27的降解，从而产生异常的VSMC增殖和迁移，这是新内膜增生和再狭窄发展的关键事件。这一假说将在三个特定的目标中得到验证：第一个目标是确定p27在AMPKa2失活引起的VSMC异常增殖和迁移中的核心作用。目的#2确定AMPKa2缺失上调Skp2是否以及如何导致AMPKa2-KO VSMC中p27的降解和促进细胞的增殖和迁移。在最后一个目标中，我们将确定Skp2和p27在体内新生内膜增生中的核心作用。将结合体外和体内技术、功能获得/丧失以及药理学/遗传学方法来实现研究目标。该项目的完成将为AMPK、p27和Skp2是否为对抗与常见疾病相关的血管损伤提供新的见解，包括糖尿病、再狭窄、动脉粥样硬化和癌症。

DESCRIPTION (provided by applicant): Vascular smooth muscle cell (VSMC) proliferation and migration are the major causes of coronary artery in- stent restenosis and accelerated arteriopathy following cardiac transplantation. How VSMC proliferation, migration, and consequent restenosis can be prevented in vivo remains a subject of extensive research in the last decade. Our exciting preliminary data suggest that pharmacological or genetic activation of AMP-activated protein kinase (AMPK) is able to suppress VSMC proliferation and neointimal hyperplasia in vivo. Fluorescence-activated cell sorting (FACS) analysis of VSMC from mice revealed that loss of AMPKa2 increased VSMC transition from G1 to S phase. Consistent with this finding, the cell cycle inhibitor, p27Kip1 (p27), was dramatically down-regulated in AMPKa2-knock out (KO) mouse VSMC but not AMPKa1-KO VSMC. In addition, we found that p27Kip1 deregulation was not due to p27Kip1 mRNA level but due to high Skp2 expression, a subunit of ubiquitin E3 ligase through the STAT binding in the Skp2 promoter. Mechanistically, we found that the S-phase kinase-associated protein 2 (Skp2), an E3 ubiquitin ligase for p27, was elevated in AMPKa2-KO VSMC and was responsible for increased degradation of p27. The most conclusive evidence for AMPK-dependent inhibition of VSMC proliferation and consequent restenosis was that wire injury-induced neointima hyperplasia in the carotid artery was significantly greater in AMPKa2-KO mice than in either AMPKa1-KO or wild type (WT) animals. Thus, the central hypothesis of this application is that loss of AMPKa2 increases Skp2, an E3 ligase for p27, and Skp2-mediated degradation of p27 to produce aberrant VSMC proliferation and migration, critical events in the development of neointimal hyperplasia and restenosis. This hypothesis will be tested in three specific aims: Aim #1 is to establish the central roles of p27 in aberrant VSMC proliferation and migration caused by AMPKa2 inactivation. Aim #2 is to determine if and how Skp2 up-regulation by AMPKa2 deletion causes p27 degradation and enhanced cell proliferation and migration in AMPKa2-KO VSMC. In the last Aim, we will establish a central role for Skp2 and p27 in neointimal hyperplasia in vivo. A combination of in vitro and in vivo techniques, gain-/loss-of-function, and pharmacologic/genetic approaches will used to accomplish the study objectives. The completion of this project will provide novel insights into whether AMPK, p27, and Skp2, are potential therapeutic targets for countering vascular damage associated with common diseases including diabetes, restenosis, atherosclerosis, and cancer.