Elucidating the effects of shear stress on endothelial cell morphology and the expression of ENaC.

Abstract

The epithelial sodium channel (ENaC) is important for blood pressure regulation through its function within the kidney and arteries, as it is a channel which facilitates the transportation of sodium into the intracellular environment of cells (Kashlan & Kleyman, 2011). ENaC has been reported in endothelial cells (ECs) as a prime candidate, facilitating endothelial function (Kusche-Vihrog et al., 2014a). ENaC is sensitive to mechanical forces such as laminar shear stress (LSS) and this may be important for endothelial-mediated-vascular function (Davies et al., 2005) In cardiovascular pathologies, LSS can become oscillatory (OSS), altering endothelial-mediated-vascular function, which may be associated with changes in ENaC (Davies, 2009). The aim of this project was to investigate the expression of ENaC and the change in morphology in endothelial cells, under shear stress with the addition of aldosterone and amiloride. Human endothelial cells (ECs) were cultured under LSS and OSS under shear stress magnitudes of 0, 5 and 10 dyns/cm2 for human umbilical vein endothelial cells (HUVECs) and 0, 10 and 15 dyns/cm2 for human aortic endothelial cells (HAECs), using a cell culture perfusion system. Endothelial morphology was assessed using ImageJ, Fiji and Icy software. ENaC subunits (α, β, γ and δ) at mRNA were quantified using RT-qPCR. HAECs demonstrated a change in morphology in response to increasing shear stress (SS) and patterns of OSS but not LSS. For HUVECs, a change in morphology was noted under OSS and LSS with corresponding changes to the EC morphology. This project was able to establish the presence of all four ENaC subunits expression in HAECs using mRNA, which has not been shown previously. ENaC expression in HUVECs was found to be increased with varying SS magnitudes and patterns. The treatments of aldosterone and amiloride suggested preliminary changes in ENaC expression and with SS combined, changes were observed in EC morphology. Taken together, results from this project indicate that EC morphology is affected by SS and in particular by the different SS magnitudes and patterns of LSS or OSS. The quantified changes noted in ENaC expression could suggest a potential role for ENaC within the endothelium as mechanosensor, regulating morphology and other downstream signalling i.e. nitric oxide production.