Application of Physiological and Pathological Wall Shear Stress to Endocardial Endothelial Cells Triggers Complex Signaling Pathways

Discrete subaortic stenosis (DSS) is a congenital heart disease characterized by the formation of a fibrotic membrane below the aortic valve. The underlying cellular mechanisms of this disease are currently unknown. As one of the distinguishing features of DSS is the elevated pressure gradient in the left ventricular outflow tract, it is theorized that the membrane formation is caused by elevated wall shear stress applied to the endocardial endothelial cells (EECs), triggering fibrosis. To relate shear stress to an EEC fibrotic phenotype, we applied fluid shear stress to EECs at physiological and pathological shear rates using a cone-and-plate device. Upon characterization of the EECs after the shear experiments, elevated shear stress triggered cell alignment as well as endothelial-to-mesenchymal transformation (EndMT) signaling pathways driven by upregulation of SNAI1 gene expression. The EECs were then treated with a small molecule inhibitor of Snail1 protein, CYD19, to attempt to attenuate EndMT signaling, and subsequently subjected to pathological shear stress. We found the Snail1 inhibitor did downregulate selected markers of EndMT signaling, although only transiently. Interestingly, the application of shear stress had a far greater effect on the EEC gene and protein expression in comparison to the Snail1 inhibition. Our findings are the first insight to EEC specific response to high shear stress. Further study should reveal the mechanisms that drive fibrosis and the formation of the DSS membrane.