Plasminogen Activator Inhibitor-1 (PAI-1) Increases Mitochondrial Membrane Potential In Vascular Smooth Muscle Cells And Coronary Arteries

Background: Plasminogen activator inhibitor- 1 (PAI-1) expression is increased in cardiometabolic diseases associated with mitochondrial dysfunction and atherosclerosis, including obesity and diabetes mellitus. However, the role of PAI-1 in triggering mitochondrial dysfunction in the vasculature is poorly defined. Mitochondrial membrane potential (ΔΨm) is as a central bioenergetic parameter controlling reactive oxygen species (ROS) generation. We investigated the roles of PAI-1 in controlling Ψm in human coronary artery smooth muscle cells (HCASMCs) and murine coronary arteries, as well as ROS production in the arterial wall. Methods: HCASMCs were cultured in high glucose (25 mM), normal glucose (4.6 mM), and isosmotic control (mannitol 25 mM) conditions (with and without PAI-039, a specific PAI-1 inhibitor) for 1 hour. JC-1 (5 μM), a ratiometric marker of mitochondrial membrane potential, was added and red/green fluorescent imaging was used to quantify ΔΨm. To evaluate effects of PAI-1 on ΔΨm and mitochondrial ROS production in the vessel wall, coronary artery segments were isolated from wild-type (WT) and PAI-1-deficient mice, pressurized ex vivo (100 cmH 2 O), loaded with JC-1 (5μM) and Mitosox (5 μM), then subjected to quantitative fluorescence imaging. Results: Compared to vehicle control, PAI-039 significantly decreased ΔΨm in HCASMCs under all experimental conditions (normal glucose: 2.08±0.15 vs . 1.47±0.04 AU; high glucose: 2.49±0.05 vs . 1.83±0.15 AU; and isosmotic control: 2.04±0.09 vs . 1.52±0.07 AU, n=4 for all groups, P<0.05 for all comparisons). The ΔΨm was significantly lower in coronary arteries from PAI-1-deficient mice vs. WT controls (0.85±0.03 vs . 1.09±0.09AU, n=6/group, P<0.05). Coronary arteries from PAI-1 knockout mice showed a statistically insignificant trend toward decreased ROS production vs . WT controls (6.4± 1.44 vs . 10.2±1.31 AU, n=6/group, P=0.07). Conclusion: Pharmacologic inhibition of PAI-1 decreases ΔΨm in HCASMCs. Endogenous expression of PAI-1 increases ΔΨm in coronary arteries, which is accompanied by a trend towards increased ROS production. These findings indicate that PAI-1 regulates mitochondrial function in vascular SMCs and by such effects may increase ROS generation in the vasculature.