Role of Plasminogen Activator Inhibitor-1 in Vascular Smooth Muscle Cell Stiffening and Senescence

纤溶酶原激活剂抑制剂 1 在血管平滑肌细胞硬化和衰老中的作用

• 批准号: 10259921
• 负责人: William P Fay
• 金额: --
• 依托单位: HARRY S. TRUMAN MEMORIAL VA HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-10-01 至 2025-09-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10259921
• 关键词: Actins; Adverse effects; Affect; Age; Aging; Alteplase; Arteries; Atherosclerosis; Atomic Force Microscopy; Biology; Blood; Blood Vessels; Blood coagulation; Blood flow; Cardiac; Cardiovascular Diseases; Cardiovascular Physiology; Cardiovascular system; Cell Adhesion; Cell Aging; Cell division; Cell physiology; Cells; Clinical; Clinical Trials; Cytoskeleton; Data; Disease; Drug Targeting; F-Actin; Fibrosis; Fluorescence Microscopy; Focal Adhesions; Generations; Genetic; Glycolysis; Goals; Heart; Heart failure; Hospitals; Hypertension; Inflammation; LDL-Receptor Related Protein 1; Link; Measurement; Mediating; Membrane Potentials; Missouri; Mitochondria; Mus; Myocardial Infarction; Myosin ATPase; Organ; Oxygen Consumption; Pathologic; Pathway interactions; Peripheral arterial disease; Pharmaceutical Preparations; Pharmacology; Phenotype; Plasminogen Activator Inhibitor 1; Play; Production; Protease Inhibitor; Proteins; Proteolysis; Reactive Oxygen Species; Research; Role; Scientist; Serpin Superfamily; Signal Pathway; Smooth Muscle Myocytes; Stress Fibers; Stretching; Stroke; Testing; Translating; Trees; Universities; Urokinase; Vascular Diseases; Vascular Smooth Muscle; Veterans; Work; arterial stiffness; base; blood pressure regulation; blood pump; burden of illness; cell motility; clinically relevant; cofilin; conditional knockout; dalton; experience; experimental study; fatty acid oxidation; fitness; in vivo; inhibitor; innovation; medical schools; member; military veteran; mitochondrial membrane; mouse model; multidisciplinary; normal aging; novel; novel strategies; prevent; recruit; respiratory; response; senescence; transcriptome sequencing; vascular inflammation

Vascular smooth muscle cells (SMCs) are present throughout the arterial tree and play a central role in cardiovascular physiology by regulating blood pressure and flow. Cardiovascular aging is characterized by pathologic changes in SMCs that contribute in a major way to high-burden diseases affecting US veterans, including hypertension, atherosclerosis, myocardial infarction, stroke, and peripheral artery disease. Two important phenotypic changes that occur in SMCs with aging are 1) stiffening, which produces hypertension and increases cardiac afterload, and 2) senescence, which is functionally defined as arrest of cell division and assumption of the senescence-associated secretory phenotype (SASP), a driver of vascular inflammation and fibrosis. Plasminogen activator inhibitor-1 (PAI-1), a member of the serpin superfamily of protease inhibitors, is the primary inhibitor of tissue-type plasminogen activator (t-PA) and urokinase (u-PA) and an important regulator of proteolysis and cell adhesion. PAI-1 expression increases with age and is associated with vascular fibrosis and generalized cell senescence. However, the specific effects of PAI-1 on SMC stiffening and senescence, as well as the mechanisms underlying them, are poorly understood. In preliminary studies involving pharmacologic and genetic modulation of PAI-1 expression in SMCs, we have shown that drug targeting of PAI-1 decreases SMC stiffness, assessed by atomic force microscopy (AFM), while also decreasing SMC cytoskeleton formation and enhancing activation of cofilin, which degrades filamentous (F)- actin. We also have demonstrated that PAI-1 promotes SMC senescence by a pathway involving the LDL receptor-related protein-1 (LRP1), while also dampening mitochondrial respiratory fitness, which is strongly linked to cell senescence. We have performed an RNA-sequencing analysis that has identified candidate signaling pathways mediating PAI-1’s effects on SMC stiffness and senescence. We also have generated mice with conditional knockout of PAI-1 in SMCs, which will enable us to study the significance of our findings in vivo. Based on our extensive preliminary data, we hypothesize that PAI-1 1) regulates SMC stiffness by controlling actin, myosin, and focal adhesion assembly in the cytoskeleton, and 2) promotes SMC senescence through adverse effects on mitochondrial energy substrate utilization and reactive oxygen species accrual. To test these hypotheses, we propose the following specific aims: 1) identify the intracellular signaling pathways by which PAI-1 regulates SMC stress fiber formation and stiffness, 2) determine the role of PAI-1 in regulating mitochondrial substrate utilization and reactive oxygen species accumulation in SMCs, probing underlying mechanisms, and 3) study the effects of pharmacologic and SMC-specific inhibition of PAI-1 on arterial stiffness and senescence in vivo. Several innovative strategies will be employed, including 1) AFM, 2) confocal fluorescence microscopy, 3) quantitative studies of PAI-1’s effects of F-actin, myosin, and focal adhesion assembly, 4) measurement of mitochondrial energy substrate utilization and membrane potential, and 5) novel murine models that enable quantification of the effects of systemic inhibition and SMC-specific deletion of PAI-1 on vascular stiffening and senescence in vivo. The experiments will be carried out by a highly experienced, multi-disciplinary team of scientists from the Truman VA Hospital, the University of Missouri School of Medicine, and the Dalton Cardiovascular Research Center. The significance of the proposed work is that it will 1) identify mechanisms by which PAI-1 and its pharmacological inhibition regulate key degenerative changes that occur in SMCs with aging, namely stiffening and senescence, and 2) translate these findings to the in vivo setting. The proposed experiments will yield important new information about the roles of PAI-1 and SMCs in vascular aging that are directly relevant to major cardiovascular diseases affecting the US veteran population. The studies also have great potential for transition to the clinical setting through the use of pharmacologic PAI-1 inhibitors.

血管平滑肌细胞（SMC）存在于整个动脉树中，并在 通过控制血压和流动，心血管生理。心血管衰老的特征是 SMC的病理变化以主要方式促进影响美国退伍军人的高负荷疾病， 包括高血压，动脉粥样硬化，心肌梗塞，中风和周围动脉疾病。二 衰老的SMC中发生的重要表型变化是1）僵硬，这会产生高血压 并增加心脏后负荷，以及2）感应，在功能上定义为细胞分裂的停滞和 假设感应相关的秘书表型（SASP），是血管注射和 纤维化。纤溶酶原激活剂抑制剂1（PAI-1）是蛋白酶抑制剂的Serpin超家族的成员， 组织型纤溶酶原激活剂（T-PA）和尿激酶（U-PA）的主要抑制剂以及重要的 PAI-1表达随着年龄的增长而增加，并且与 血管纤维化和广义细胞感应。但是，PAI-1对SMC僵硬的特定影响 感应以及感应机制也很熟悉。在初步研究中 涉及SMC中PAI-1表达的药物和遗传调节，我们表明药物 PAI-1的靶向降低了SMC刚度，通过原子力显微镜（AFM）评估，同时也 减少SMC细胞骨架形成并增强Cofilin的激活，从而降解丝状（F） - 肌动蛋白。我们还证明了PAI-1通过涉及LDL的途径促进SMC的传感 受体相关蛋白1（LRP1），同时还原线粒体呼吸适应性，强烈 与细胞感应相关。我们进行了RNA测序分析，已确定了候选者 信号通路介导PAI-1对SMC刚度和感应的影响。我们也生成了 SMC中有条件敲除PAI-1的小鼠，这将使我们能够研究发现的重要性 体内。根据我们广泛的初步数据，我们假设PAI-1 1）通过 控制细胞骨架中的肌动蛋白，肌球蛋白和局灶性粘合剂组件，2）促进SMC感应 通过对线粒体能底物利用和活性氧的不利影响。到 测试这些假设，我们提出以下特定目的：1）确定细胞内信号通路 PAI-1调节SMC应力纤维形成和刚度，2）确定PAI-1在确定中的作用 线粒体底物的利用和活性氧在SMC中的积累，探测潜在的 机制，以及3）研究PAI-1药物和SMC特异性抑制对动脉的影响 体内刚度和感应。将执行几种创新策略，包括1）AFM，2） 共聚焦荧光显微镜，3）对F-肌动蛋白和局灶性PAI-1作用的定量研究 粘附组件，4）测量线粒体能底物利用率和膜电位的测量 5）可以量化系统性抑制和SMC特异性的效果的新型鼠模型 在体内删除PAI-1在血管僵硬和感受上的缺失。实验将由 来自杜鲁门VA医院的科学家经验丰富的多学科团队 密苏里州医学院和道尔顿心血管研究中心。意义的意义 拟议的工作是它将1）确定PAI-1及其药物抑制的机制 衰老，即僵硬和感应的SMC中发生的关键退化性变化，2）翻译 这些发现到体内设置。提出的实验将产生有关 PAI-1和SMC在血管衰老中的作用，与影响的主要心血管疾病直接相关 美国退伍军人人口。这些研究还具有通过过渡到临床环境的巨大潜力 使用药学PAI-1抑制剂。