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

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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.