Differential Shear Forces on Endocardial Endothelial Cells Regulate a Fibrotic Spectrum in the Left Ventricular Outflow Tract

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PROJECT SUMMARY Discrete subaortic stenosis (DSS) is a congenital or acquired condition that accounts for ~10% of all cases of left ventricular outflow tract (LVOT) obstruction, and is characterized by a ring of fibrous tissue below the aortic valve. Current treatment is surgical removal of the obstruction, but the unpredictable recurrence and progression of DSS leads to multiple repeat surgeries and attendant morbidity into adulthood. Current theories postulate that altered LVOT geometry causes increased shear stress and ultimately fibrosis in DSS; however, little is known about the mechanism of DSS progression. Studies in vascular endothelial cells demonstrated increased inflammation and phenotypic changes in response to altered shear forces, but effects of shear are under-studied in endocardial endothelial cells (EEC). Additionally, resident fibroblasts are implicated in organ fibrosis, including cardiac tissue, in response to altered cytokine signaling and mechanical forces. We hypothesize that that altered shear forces induce an inflammatory response by EEC, which interacts with cardiac fibroblasts (CF) to govern a fibrotic phenotype that contributes to the pathophysiology of DSS, which we will address with three specific aims. AIM 1. Elucidate the mechanisms of how shear forces regulate EEC inflammatory phenotype.First, we shall utilize patient echo data and computational modeling to develop a bioreactor that resembles altered flows in DSS. Using this innovative system, we will then test the role of CD-31 mechanosensory signaling in EEC in response to altered shear forces and geometry. Lastly, we shall investigate the effects of altered shear forces on EEC interactions with inflammatory cells in propagating a pro-inflammatory environment. AIM 2. Determine EEC transduction of altered shear forces to govern fibrosis in the LVOT. We shall investigate EEC propensity towards endoMT in response to simulated DSS altered shear and immune cell interactions. We shall then investigate the direct and inflammatory-mediated effects of EEC mechanosensing on CF that produce a fibrotic ECM. Lastly, we shall study the effect of the stiffer environment induced by fibrosis on EEC-CF crosstalk, which may propagate the fibrotic response. AIM 3. Characterize the aggressive DSS phenotype using patient data to develop a predictive model. We shall first evaluate the ECM composition, remodeling profile and echo data that characterizes the aggressive forms of DSS in humans. We shall then use these data to develop a multivariate computational model that can be used to predict an aggressive phenotype, which will be validated and tested. This proposal will improve the care of children with DSS. With completion of these aims, innovative tools and new knowledge will emerge about the effects of shear force on EEC-CF and EEC-immune cell cross-talk. These findings have potential implications for any cardiovascular disease with altered flow associated with fibrosis.