Biochemical and biophysical characterization of shear stress mediated vascular remodeling

Abstract

The overall focus of this thesis is to investigate the role of shear stress role in endothelial remodeling and angiogenesis with relation to nitric oxide (NO) and how Rho-kinase is involved in cellular migration and remodeling. Our hypothesis was that inclusion of shear stress would lead to endothelial migration and thereby angiogenesis upon increased release of nitric oxide. Rho-kinase inhibitor Y-27632 under shear stress regulates angiogenesis through NO mediated endothelial migration. We studied endothelial functions under shear stress, and static cell cultures keeping as reference control. In the first part of this research we have studied the characterization of the flow dynamics on endothelial migration and angiogenesis using cell based and Chorio-allantoic membrane (CAM) models. We observed EC migration in relation to actin rearrangements under shear stress. Functional studies of ECs proved that Y-27632 attenuation of pre-capillary formation is mediated through NO dependent pathway. Y-27632 reduced NO production levels under shear stress conditions. Migration of ECs considerably reduced with Y-27632 treatments under shear stress. Y-27632 (5and#956;M) strongly attenuated ring formation. Paternal changes were observed in EC monolayers and NO intensity clustering studies (k-nearest neighborhood (KNN) approach) with ECs treated under Y-27632 and shear stress. Similar inhibitory pattern observed under eNOS inhibitors L- NAME and L-NIO, confirms the inhibitory mode of Y- 27632. Immunofluorescent studies with phosphorylated-eNOS showed an altered localization pattern in the presence of Y-27632 under shear stress, showing a reduced cytoplasm to nuclear intensity. Rho-kinase signaling is involved in the shear-induced in vitro EC migration and pre-capillary formation. Ultimately, this research may help in understanding the dosage effects of Rho-kinase inhibitor, a popular therapeutic target for the vascular diseases and its link to hemodynamics. newline