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: 8656748
• 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/8656748
• 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.

描述（由申请人提供）：血管平滑肌细胞（VSMC）的生长和迁移是对动脉损伤的反应，对动脉粥样硬化、肺动脉高压和经皮腔内冠状动脉成形术后再狭窄的过程至关重要。增殖与VSMC基因表达的改变相关，从静止/分化表型到增殖/去分化表型。蛋白磷酸酶1（PP 1）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。PP 1受磷酸酶抑制剂-1（I-1）的调节，I-1由PKA磷酸化激活，并受蛋白磷酸酶PP 2A和PP 2B（钙调神经磷酸酶）抑制。I-1在血管平滑肌细胞（VSMC）中表达，但似乎在收缩或舒张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，我们测试了I-1是否参与增殖相关途径。为了进行这个项目，我们使用人冠状动脉平滑肌细胞和大鼠主动脉VSMCs，和大鼠颈动脉损伤模型进行体内研究。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的介质层中表达，并且在人动脉粥样硬化斑块的介质中下调;而PP 1表达在增殖的人平滑肌细胞中上调，与冠状动脉组织样品相比。真实的时间PCR显示I-1 mRNA在增殖的人冠状动脉平滑肌细胞和增殖的VSMC中低1000倍，表明增殖（合成）表型。腺病毒介导的I-1c基因转染和siRNA-PP 1转染均能抑制VSMC的增殖和迁移。I-1c过表达增加了CREB在Ser 133和下游转录因子p53和p21上的磷酸化。在大鼠颈动脉损伤后2周的标本中，I-1的表达在损伤的血管中缺失，这一发现与新生内膜增生过程中的血管重塑一致。此外，腺病毒基因转移的I-1c，防止在颈动脉损伤模型中的新生内膜增殖。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VSMC增殖和迁移的控制。尽管有上述发现，I-1在VSMC增殖和重塑中的作用还不清楚。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调节VSMC增殖和迁移的机制，3）确定血管损伤后I-1过表达和消融的生理后果。定义I-1及其生理后果的机制，将是非常相关的分析和未来的治疗建议，以防止和逆转血管成形术后新生内膜形成。