Controlling VSMC Proliferation and Migration

描述（由申请人提供）：血管平滑肌细胞（VSMC）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。在过去的十年中，如何在体内防止VSMC增殖、迁移和随后的再狭窄仍然是广泛研究的主题。我们令人兴奋的初步数据表明，药物或遗传激活的AMP激活蛋白激酶（AMPK）能够抑制VSMC增殖和新生内膜增生在体内。对小鼠VSMC的流式细胞仪分析显示AMPKa 2的缺失增加了VSMC从G1期到S期的转变。与这一发现相一致，细胞周期抑制剂p27 Kip 1（p27）在AMPKa 2敲除（KO）小鼠VSMC中显著下调，但在AMPKa 1-KO VSMC中没有。此外，我们发现p27 Kip 1的失调不是由于p27 Kip 1 mRNA水平，而是由于Skp 2的高表达，Skp 2是泛素E3连接酶的一个亚基，通过STAT结合在Skp 2启动子中。从机制上讲，我们发现S期激酶相关蛋白2（Skp 2），一种p27的E3泛素连接酶，在AMPKa 2-KO VSMC中升高，并负责增加p27的降解。AMPK依赖性抑制VSMC增殖和随后的再狭窄的最确凿证据是，AMPKa 2-KO小鼠颈动脉中导丝损伤诱导的新生内膜增生显著大于AMPKa 1-KO或野生型（WT）动物。因此，本申请的中心假设是AMPKa 2的缺失增加Skp 2（p27的E3连接酶）和Skp 2介导的p27降解，以产生异常的VSMC增殖和迁移，这是新生内膜增生和再狭窄发展中的关键事件。这一假设将在三个特定的目标进行测试：目标#1是建立在异常VSMC增殖和迁移AMPKa 2失活引起的p27的核心作用。目的#2是确定AMPKa 2缺失引起的Skp 2上调是否以及如何引起AMPKa 2-KO VSMC中p27降解和增强的细胞增殖和迁移。在最后一个目标中，我们将建立Skp 2和p27在体内新生内膜增生中的中心作用。将使用体外和体内技术、功能获得/丧失和药理学/遗传学方法的组合来实现研究目的。该项目的完成将为AMPK、p27和Skp 2是否是对抗与糖尿病、再狭窄、动脉粥样硬化和癌症等常见疾病相关的血管损伤的潜在治疗靶点提供新的见解。

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.