Fluid Shear-stress Increases TRPV4-Expression in Human Endothelial Cells in an Experimental in Vitro Model

Abstract

Introduction: Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is a non-selective cation channel that tunes the function of different tissues including the vascular endothelium, lung, chondrocytes, and neurons. In vascular collateral growth regularly seen in induced limb ischemia TRPV4 expression in endothelial cells is significantly increased. As a consequence induction of TRPV4 expression could be used for augmentation of arteriogenesis. This study aimed to determine the importance of fluid shear-stress on TRPV4 expression in endothelial cells. To avoid further investigations in small animal models an in vitro model using arterial endothelial cells was to be established. Methods: Human umbilical artery endothelial cells (HUAEC) were harvested and cultivated on special slides and exposed to a certain level of fluid shear-stress (20 Dyn/cm2) for 0.5, 1, 4, 8, 10, 12, 18 and 24 hours. Messenger RNA (mRNA) abundance was determined in HUAEC by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry in dependency to length of fluid shear-stress exposure. Results: The establishment and validation of an in vitro model using cultivated HUAEC to investigate the effect of fluid shear stress on TRPV4 expression was successful. The extent of endothelial TRPV4 mRNA expression showed a direct correlation to length of fluid shear-stress exposure with a peak after 8 hours of shear-stress exposure. After 8 hours endothelial mRNA expression was significantliy increased in comparison to control endothelial cells without fluid shear-stress stimulation (p= 0.041 in 8h FSS vs. Control). Fluid shear stress exposure of 10, 12, 18 and 24 hours showed decreasing TRPV4 mRNA expression in endothelial cells and no differences to control cells with fluid shear-stress stimulation extending to more than 12 hours. Conclusion: TRPV4 showed a significantly increased expression in fluid shear-stress stimulated HUAEC. The established in vitro model seems suitable to investigate further impacts on fluid shear-stress stimulated HUAEC, e.g. additional pharmacological TRPV4 activation to enhance arteriogenesis, as TRPV4 could be a possible candidate for the development of new therapeutic concepts in chronic limb ischemia. Disclosure: Nothing to disclose