Protein Phosphatase Inhibitor-1 and vascular smooth muscle cell function

蛋白磷酸酶抑制剂1与血管平滑肌细胞功能

• 批准号: 7922464
• 负责人: Lahouaria HADRI
• 金额: $ 13.49万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7922464
• 关键词: Ablation; Adenoviruses; Affect; Angioplasty; Aorta; Apoptotic; Arterial Fatty Streak; Arterial Injury; Atherosclerosis; Blood Vessels; Ca(2+)-Transporting ATPase; Calcineurin; Calcium; Calmodulin; Carotid Artery Injuries; Cell Cycle; Cell Proliferation; Cell physiology; Cells; Coronary artery; Cyclic AMP-Dependent Protein Kinases; Cyclic AMP-Responsive DNA-Binding Protein; Dependovirus; Diet; Fatty acid glycerol esters; Future; Gene Expression; Gene Transfer; Goals; Growth; Heart; Homeostasis; Human; Immunofluorescence Immunologic; In Vitro; Injury; Knockout Mice; Mammary Arteries; Messenger RNA; Mitogen-Activated Protein Kinases; Modeling; Molecular; Mus; Pathway interactions; Percutaneous Transluminal Coronary Angioplasty; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Play; Principal Investigator; Process; Proliferating; Protein Isoforms; Protein Phosphatase 2A Regulatory Subunit PR53; Protein phosphatase; Proteins; Proteomics; Pulmonary Hypertension; Rattus; Reticulum; Role; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; SERCA2a; Sampling; Sarcoplasmic Reticulum; Signal Transduction Pathway; Small Interfering RNA; Smooth Muscle; Smooth Muscle Myocytes; Somatic Gene Therapy; Stents; TP53 gene; Techniques; Technology; Testing; Time; Tissue Sample; Transfection; Vascular remodeling; Western Blotting; abstracting; cell growth; human FRAP1 protein; immunocytochemistry; implantation; in vivo; in vivo Model; inhibitor/antagonist; injured; knock-down; migration; neointima formation; nuclear factors of activated T-cells; overexpression; phosphatase inhibitor; prevent; programs; protein phosphatase inhibitor-1; public health relevance; response; restenosis; transcription factor; vascular smooth muscle cell migration; vascular smooth muscle cell proliferation

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)

描述（申请人提供）：血管平滑肌细胞（VSMC）的生长和迁移是对动脉损伤的反应，这对于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。增殖与 VSMC 基因表达的改变相关，范围从静止/分化表型到增殖/去分化表型。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) 调节，该抑制剂由 PKA 磷酸化激活，并被蛋白磷酸酶 PP2A 和 PP2B（钙调神经磷酸酶）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。由于钙调神经磷酸酶强烈控制 VSMC 增殖，因此我们测试了 I-1 是否参与增殖相关途径。为了开展这个项目，我们使用了人冠状动脉平滑肌细胞和大鼠主动脉 VSMC，以及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动脉中层表达，在人动脉粥样硬化斑块中层表达下调；而与冠状动脉组织样本相比，增殖的人平滑肌细胞中 PP1 表达上调。实时 PCR 显示，增殖的人冠状动脉平滑肌细胞和增殖的 VSMC 中的 I-1 mRNA 降低了 1000 倍，表明存在增殖（合成）表型。腺病毒基因转移组成型活性 I-1 (I-1c) 和 siRNA-PP1 转染可抑制体外 VSMC 增殖和迁移。 I-1c 过表达增加了 Ser133 和下游转录因子 p53 和 p21 上的 CREB ​​磷酸化。在大鼠模型颈动脉损伤两周后获得的样本中，受损血管中缺乏I-1的表达，这一发现与新内膜增殖过程中的血管重塑一致。此外，I-1c 的腺病毒基因转移可防止颈动脉损伤模型中的新内膜增殖。总之，在VSMC中，磷酸酶抑制剂I-1是静止表型的标志物，并通过转录因子CREB参与控制VSMC增殖和迁移。尽管有前面描述的研究结果，但 I-1 在 VSMC 增殖和重塑中的作用尚不清楚。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的机制增殖和迁移；3) 定义血管损伤后体内 I-1 过度表达和消融的生理后果。定义 I-1 的机制及其生理后果，对于预防和逆转血管成形术后新内膜形成的治疗的分析和未来建议具有重要意义。 公共卫生相关性：蛋白磷酸酶抑制剂 1 和血管平滑肌细胞功能。经皮血管成形术或支架植入术后再狭窄的关键机制是血管平滑肌细胞（VSMC）的生长和迁移。 Inhibitor-1 是调节 VSMC 中钙循环的关键蛋白质，当 VSMC 增殖时，其会发生显着变化。我们的目标是重点关注抑制 VSMC 增殖和迁移并通过使用携带 I-1 的体细胞基因疗法增强再内皮化的策略，以提供预防再狭窄的治疗。 （摘要完）