SMII activity drives VSMC phenotype - ABSTRACT Vascular smooth muscle cells (VSMCs) are highly dynamic and contractile, playing an essential role in the physiologic regulation of blood flow. VSMCs exist on a functional spectrum between the physiologically differentiated contractile phenotype and the pathological dedifferentiated migratory phenotype. Within a lifetime, a single VSMC can transition back and forth along this phenotypic spectrum. VSMC migration is necessary for tissue wound healing in response to vascular injury, facilitated by VSMC loss of contractility and dedifferentiation. Misregulation of these transitions can be maladaptive, resulting in VSMC proliferation and migration, and contributing to pathologies such as hypertension and atherosclerosis. There is limited mechanistic knowledge of how the contractile machinery and its interplay with the mechanical environment regulate the transition between phenotypes. Smooth muscle myosin II (SMII) plays an essential role in VSMC contractility by assembling into bundles that bind and pull on the actin cytoskeleton. Our preliminary results establish a discernible difference in endogenous EGFP-labeled SMII filament kinetics among varying phenotypes of primary VSMCs. The dedifferentiated phenotype exhibits rapid SMII turnover while the differentiated phenotype has stabilized SMII bundles that show little exchange of monomers. We hypothesize that SMII-driven contractile activity is not simply correlated with VSMC phenotype, but that contractile activity is determinant in establishing VSMC phenotype. To test this hypothesis, we will modulate contractility using ex-vivo and hypertensive murine models, to investigate the role of SMII on VSMC phenotype in normal physiology and pathophysiological contexts. In Aim I, we will characterize the role of SMII in the transitions between phenotypes. We recently engineered a novel endogenously-tagged EGFP-SMII murine model, which allows us to directly observe and quantify stepwise changes in both the expression and activity of VSMC contractile machinery. By modulating SMII activity, we hypothesize we will be able to drive VSMCs from one phenotype to the other. Our initial findings suggest that expression of pathology-associated SMII motor domain variants in primary VSMCs results in an increase in filament exchange and a decrease in whole-cell contractility, consistent with dedifferentiation. In Aim II, we will investigate SMII during VSMC phenotype switching and vascular wall remodeling in vivo. Specifically, we will characterize the role of the entire cellular contractile machinery during the progression from acute to chronic hypertension, using our EGFP-SMII murine model with an angiotensin II infusion model of hypertension. By utilizing a novel EGFP-SMII murine model, we plan to unveil the role of SMII in phenotype transitions in pathophysiology. We hypothesize that by modulating SMII activity, we can drive VSMC phenotype.
