TRPV4 regulates matrix stiffness‐dependent activation of YAP/VEGFR2 signaling via Rho/Rho kinase/LATS1/2 pathway

Abstract

TRPV4 is a mechanosensitive ion channel in endothelial cells and shown to regulate physiological and pathological angiogenesis. We have recently shown that TRPV4 deletion and/or down regulation activates vascular endothelial growth factor receptor-2 (VEGFR2) via yes-associated protein (YAP) in human EC. However, the molecular mechanisms of TRPV4-dependant YAP/VEGFR2 activation are unknown. We have employed human microvascular endothelial cells (HMEC-1; EC) on ECM gels of varying stiffness that mimic normal and failing hearts (8 and 50 kPa) and examined nuclear translocation of YAP and disappearance of perinuclear VEGFR2, after siRNA knockdown of TRPV4. Next, we investigated the role of upstream and downstream regulators of YAP translocation by focusing on LATS-1, CYR61 and Rho/Rho Kinase, using phospho-specific antibodies (pYAP and pLATS) and pharmacological inhibitor of Rho kinase (Y-27632). siRNA knockdown of TRPV4 significantly increased nuclear translocation of YAP with concomitant disappearance of perinuclear-VEGFR2 in matrix stiffness-dependent manner. Further, we found increased Rho-GTP and decreased LATS-1 phosphorylation in TRPV4 knockdown EC compared to control siRNA treated EC. Furthermore, pharmacological inhibition of Rho kinase with Y-27632, increased phosphorylation of LATS1/2 kinase, inhibited nuclear translocation of YAP and VEGFR2 redistribution. Consistent with above results, YAP target genes CYR61 and CTGF mRNA expression levels were increased in a matrix stiffness-dependent manner in TRPV4 knockdown EC than the control siRNA treated EC. Our results suggest that TRPV4 regulates YAP/VEGFR2 activation in a matrix stiffness dependent manner during angiogenesis via Rho/Rho kinase/LATS1/2 pathway.