Controlling VSMC Proliferation and Migration

控制 VSMC 增殖和迁移

• 批准号: 9059320
• 负责人: MING-HUI ZOU
• 金额: $ 40.94万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2016-12-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9059320
• 关键词: 5' -AMP-activated protein kinase; AMP-activated protein kinase kinase; Abbreviations; Animals; Arterial Disorder; Atherosclerosis; Binding; Binding Proteins; Biological Assay; Blood Vessels; Bone Marrow; Bone Marrow Transplantation; CDK6-associated protein p18; Cardiovascular Diseases; Carotid Arteries; Cell Cycle; Cell Proliferation; Cells; Clinical; Coronary; Coronary artery; Cyclins; Data; Development; Diabetes Mellitus; Disease; Event; Fluorescence-Activated Cell Sorting; Genetic; Goals; Heart Transplantation; Hyperplasia; Immigration; Immunohistochemistry; In Vitro; Injury; Knock-out; Knockout Mice; Life; Malignant Neoplasms; Medial; Mediating; Messenger RNA; Molecular; Mus; NF-kappa B; Pathway interactions; Peripheral Vascular Diseases; Procedures; Proliferating; Proliferating Cell Nuclear Antigen; Research; Ribonucleosides; Role; S Phase; SKP2 gene; Serum; Skp1-Cullin-F-Box Proteins; Skp2 Proteins; Smooth Muscle Myocytes; Stents; Techniques; Testing; Therapeutic; Ubiquitin; Up-Regulation; cell motility; fetal; genetic approach; in vivo; inhibitor/antagonist; insight; loss of function; migration; multicatalytic endopeptidase complex; neointima formation; novel; prevent; promoter; restenosis; sensor; therapeutic target; tumor; ubiquitin-protein ligase; vascular smooth muscle cell migration; vascular smooth muscle cell proliferation

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.

描述（由申请人提供）：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动脉病变加速的主要原因。在过去的十年中，如何在体内预防VSMC的增殖、迁移和随后的再狭窄仍然是一个广泛研究的主题。我们令人兴奋的初步数据表明，amp活化蛋白激酶（AMPK）的药理或遗传激活能够抑制VSMC增殖和新生内膜增生。荧光活化细胞分选（FACS）分析小鼠VSMC显示AMPKa2的缺失增加了VSMC从G1期到S期的转变。与这一发现一致，细胞周期抑制剂p27Kip1 （p27）在ampka2敲除（KO）小鼠VSMC中显著下调，而AMPKa1-KO VSMC中不下调。此外，我们发现p27Kip1的失调不是由于p27Kip1 mRNA水平，而是由于Skp2的高表达，Skp2是泛素E3连接酶的一个亚基，通过STAT结合在Skp2启动子上。在机制上，我们发现s期激酶相关蛋白2 (Skp2)， p27的E3泛素连接酶，在AMPKa2-KO VSMC中升高，并负责p27的降解增加。ampk依赖性抑制VSMC增殖和随后的再狭窄的最确凿证据是，AMPKa2-KO小鼠颈动脉钢丝损伤诱导的新生内膜增生明显大于AMPKa1-KO或野生型（WT）动物。因此，该应用的中心假设是AMPKa2的缺失增加了p27的E3连接酶Skp2，以及Skp2介导的p27降解，从而产生异常的VSMC增殖和迁移，这是新生内膜增生和再狭窄发展的关键事件。这一假设将在三个具体目标中得到验证：目标1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是确定AMPKa2缺失导致Skp2上调是否以及如何导致AMPKa2- ko VSMC中p27降解和增强细胞增殖和迁移。在最后一篇文章中，我们将确定Skp2和p27在体内新生内膜增生中的核心作用。体外和体内技术、功能获得/丧失以及药理学/遗传学方法的结合将用于实现研究目标。该项目的完成将为AMPK、p27和Skp2是否是对抗与常见疾病（包括糖尿病、再狭窄、动脉粥样硬化和癌症）相关的血管损伤的潜在治疗靶点提供新的见解。