Protein Phosphatase Inhibitor-1 and vascular smooth muscle cell function

描述（由申请人提供）：血管平滑肌细胞（VSMCs）的生长和迁移是对动脉损伤的反应，对经皮冠状动脉成形术后动脉粥样硬化、肺动脉高压和再狭窄的过程至关重要。从静止/分化表型到增殖/去分化表型，VSMCs的增殖与基因表达的改变有关。蛋白磷酸酶1 （PP1）是转录因子CREB的主要调节因子，通过p53和p21强烈参与细胞增殖的控制。PP1受磷酸酶抑制剂-1 （I-1）调控，其受PKA磷酸化激活，受蛋白磷酸酶PP2A和PP2B（钙调磷酸酶）抑制。I-1在血管平滑肌细胞（VSMC）中表达，但似乎在收缩或松弛反应中不起重要作用。由于钙调磷酸酶强烈控制VSMC增殖，我们测试了I-1是否参与增殖相关途径。为了开展本项目，我们使用了人冠状动脉平滑肌细胞和大鼠主动脉VSMCs，并建立了大鼠颈动脉损伤模型进行体内研究。共聚焦免疫荧光和Western blot分析显示，I-1蛋白在健康人冠状动脉和乳腺动脉介质层表达，在人动脉粥样硬化斑块介质中表达下调；而与冠状动脉组织样本相比，增殖的人类平滑肌细胞中PP1的表达上调。Real - time PCR结果显示，在增殖的人冠状动脉平滑肌细胞和增殖的VSMCs中，I-1 mRNA的表达量降低了1000倍，表明其具有增殖性（合成）表型。腺病毒转移组成活性I-1 （I-1c）和转染siRNA-PP1可抑制体外VSMC的增殖和迁移。I-1c过表达增加了CREB对Ser133和下游转录因子p53和p21的磷酸化。在大鼠颈动脉损伤两周后获得的样本中，I-1在损伤血管中缺失表达，这一发现与新生内膜增殖过程中的血管重构一致。此外，在颈动脉损伤模型中，腺病毒I-1c基因转移可阻止新内膜增殖。综上所述，在VSMC中，磷酸酶抑制剂I-1是静止表型的标志，并通过转录因子CREB参与VSMC增殖和迁移的控制。尽管有先前描述的发现，但I-1在VSMC增殖和重塑中的作用尚未得到很好的理解。为了阐明I-1参与VSMC增殖的机制，我们提出以下具体目标：1)确定I-1是否是调节VSMC增殖的分子决定因素；2)确定I-1调节VSMC增殖和迁移的机制；3)确定血管损伤后体内I-1过表达和消融的生理后果。明确I-1的机制及其生理后果，将对分析和未来提出预防和逆转血管成形术后新内膜形成的治疗方法具有重要意义。

DESCRIPTION (provided by applicant): Growth and migration of vascular smooth muscle cells (VSMCs) are responses to arterial injury which are critical to the processes of atherosclerosis, pulmonary hypertension and restenosis after percutaneous transluminal coronary angioplasty. Proliferation is associated with alteration in gene expression of the VSMCs ranging from a quiescent/differentiated phenotype to a proliferating/dedifferentiated one. The protein phosphatase 1 (PP1), the main regulator of the transcription factor CREB, is strongly involved in the control of cell proliferation via p53 and p21. PP1 is regulated by phosphatase inhibitor-1 (I-1), which is activated by PKA phosphorylation and inhibited by protein phosphatase PP2A and PP2B (calcineurin). I-1 is expressed in vascular smooth muscle cells (VSMC), but does not appear to play a significant role in contractile or relaxant response. Since calcineurin strongly controls VSMC proliferation, we tested whether I-1 is involved in proliferation-related pathways. In order to conduct this project, we used human coronary artery smooth muscle cells and rat aortic VSMCs, and a rat carotid injury model for in vivo studies. Confocal immunofluorescence and Western blot analysis showed that I-1 protein is expressed in the media layer of healthy human coronary arteries and mammary arteries, and is down-regulated in the media of human atherosclerotic plaques; whereas PP1 expression is up-regulated in proliferating human smooth muscle cells compared to coronary artery tissue samples. Real time PCR showed that I-1 mRNA is 1000 fold lower in proliferating human coronary artery smooth muscle cells and in proliferating VSMCs, indicating a proliferative (synthetic) phenotype. Adenovirus gene transfer of constitutively active I-1 (I-1c) and transfection of siRNA-PP1 inhibited VSMC proliferation and migration in vitro. I-1c overexpression increased CREB phosphorylation on Ser133 and downstream transcription factors p53 and p21. In samples obtained two weeks after carotid artery injury in the rat model, the expression of I-1 was absent in the injured vessels, this finding is in concordance with vascular remodelling during neointimal proliferation. Furthermore, adenoviral gene transfer of I-1c, prevented neointimal proliferation in the carotid injury model. In conclusion, in VSMCs the phosphatase inhibitor I-1 is the marker of quiescent phenotype and is involved in the control of VSMC proliferation and migration via transcription factor CREB. Despite the previously described findings, the role of I-1 in VSMC proliferation and remodelling is not well understood. To elucidate the mechanisms by which I-1 is involved in VSMC proliferation, we propose the following specific aims: 1) determine if I-1 is a molecular determinant in modulating VSMC proliferation, 2) determine the mechanism by which I-1 regulates VSMC proliferation and migration and 3) define the physiological consequences of the overexpression and ablation of I-1 in vivo after vascular injury. Defining the mechanisms of I-1 and its physiological consequences, will be of great relevance in the analysis and future proposal of therapies to prevent and perhaps reverse neointima formation after angioplasty. PUBLIC HEALTH RELEVANCE: Protein Phosphatase Inhibitor-1 and vascular smooth muscle cell function. The key mechanisms of restenosis after percutaneous angioplasty or stent implantation are the growth and migration of vascular smooth muscle cells (VSMC). Inhibitor-1 is a critical protein that regulates calcium cycling in VSMCs, which significantly changes when VSMCs proliferate. Our goal is to focus on strategies that inhibit VSMC proliferation and migration and enhance re-endothelization by using somatic gene therapy carrying I-1 to provide a treatment that prevents restonosis. (End of Abstract)