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: 8255648
• 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/8255648
• 关键词: Ablation; Adenoviruses; Affect; Angioplasty; Aorta; Apoptotic; Arterial Fatty Streak; Arterial Injury; Atherosclerosis; Blood Vessels; Ca(2+)-Calmodulin Dependent Protein Kinase; Ca(2+)-Transporting ATPase; Calcineurin; Calcium; Carotid Artery Injuries; Cell Cycle; Cell Proliferation; Cell physiology; Cells; Coronary Arteriosclerosis; 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; Sampling; Sarcoplasmic Reticulum; Signal Transduction Pathway; Small Interfering RNA; Smooth Muscle; Smooth Muscle Myocytes; Somatic Gene Therapy; Stents; 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(VSMCs)的生长和迁移是对动脉损伤的反应，在动脉粥样硬化、肺动脉高压和经皮冠状动脉腔内成形术后再狭窄的过程中起关键作用。增殖与VSMCs基因表达的改变有关，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的主要调控因子，通过p53和p21参与细胞增殖的调控。PP1受磷酸酶抑制因子-1(I-1)的调节，后者被PKA磷酸化激活，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的增殖，我们测试了I-1是否参与了与增殖相关的通路。为了进行这一项目，我们使用了人冠状动脉平滑肌细胞和大鼠主动脉VSMCs，以及大鼠颈动脉损伤模型进行了体内研究。共聚焦免疫荧光和Western印迹分析表明，I-1蛋白在健康人冠状动脉和乳腺动脉中膜表达，在人动脉粥样硬化斑块中表达下调；而PP1在增殖中的人血管平滑肌细胞中表达上调。实时荧光定量聚合酶链式反应显示，I-1mRNA在增殖期的人冠状动脉平滑肌细胞和增殖期的血管平滑肌细胞中的表达降低了1000倍，表明这是一种增殖性(合成)表型。腺病毒介导的活性I-1(I-1c)和siRNA-PP1的转导可抑制VSMC的增殖和迁移。I-1c的过表达增加了Ser133及其下游转录因子p53和p21上CREB的磷酸化。在大鼠颈动脉损伤后2周的标本中，I-1在损伤的血管中没有表达，这一发现与新生内膜增生过程中的血管重塑相一致。此外，在颈动脉损伤模型中，腺病毒I-1c基因转移可以防止新生内膜的增殖。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的增殖和迁移的调控。尽管有上述发现，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，我们提出了以下具体目标：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调控VSMC增殖和迁移的机制；3)确定I-1在血管损伤后过表达和消融在体内的生理后果。明确I-1的作用机制及其生理后果，对于分析和提出未来预防或逆转血管成形术后新生内膜形成的治疗方案具有重要意义。 公共卫生相关性：蛋白磷酸酶抑制物-1与血管平滑肌细胞功能。血管平滑肌细胞(VSMC)的生长和迁移是经皮血管成形术或支架植入术后再狭窄的关键机制。抑制因子-1是一种调节VSMCs钙循环的关键蛋白，当VSMCs增殖时，它会发生显著的变化。我们的目标是专注于抑制VSMC增殖和迁移的策略，并通过使用携带I-1的体细胞基因治疗来加强再内皮化，以提供防止再狭窄的治疗方法。(摘要结束)