Altered venous flow drives endothelial to mesenchymal transition in varicose veins by suppressing PIEZO1-KLF2 signaling.

Abstract

Varicose veins are characterized by disturbed hemodynamics due to blood reflux. We previously identified altered flow-driven endothelial-to-mesenchymal transition (EndMT) in varicose veins. Mechanosensors such as Piezo1 provide a molecular bridge between hemodynamic forces and cellular mechanoregulation in vasculature. We hence hypothesized that depletion of blood flow-sensing Piezo1 channels induces EndMT process during venous remodeling. Here, we analyzed 20 human varicose veins and 22 healthy saphenous veins for a comprehensive view of Piezo1 expression using qRT-PCR, western blot and immunohistostaining techniques. Our study reveals a significant loss of Piezo1 expression in varicose veins at both mRNA and protein levels. Our in vitro experiments using microfluidic flow channels showed that Piezo1 expression and calcium influx function are drastically affected in endothelial cells (ECs) exposed to oscillatory venous shear stress. Piezo1 depletion reduces KLF2, which triggers the EndMT program in venous ECs. Kinase inhibitor assays indicated that Piezo1-based calcium influx promotes CAMKII autophosphorylation and induces KLF2 expression via both MEK5/ERK5 and PI3K-AKT-FOXO pathways in venous flow. Piezo1 agonist Yoda1, at low doses mimicking venous flow, augmented KLF2 expression and prevented aberrant molecular EndMT reprogramming in cells exposed to oscillatory shear stress. Yoda1 was also effective in restoring calcium influx and stimulating angiogenesis by promoting endothelial tube formation hindered by Piezo1 depletion. Taken together, the endothelial-protective role of Piezo1 ion channels due to uniform hemodynamic flow is coupled to KLF2, a downstream mediator in the venous circulation. The Piezo1-KLF2 pathway can be utilized for identifying targeted therapeutic options in patients with varicose veins.