F7 - Functional Interplay Between TRPV4 and Piezo1 in Endothelial Mechanotransduction

Introduction: : Mechanotransduction in endothelial cells (ECs) in response to fluid shear stress (FSS) is essential for vascular integrity, inflammation regulation, and disease progression. Depending on its magnitude and pattern, FSS elicits distinct EC phenotypes—high, steady FSS promotes alignment with flow and vascular protection, while low or oscillatory FSS induces inflammation, perpendicular alignment, and dysfunction. The mechanosensitive ion channels Piezo1 and TRPV4 are key players in shear-induced calcium signaling and downstream processes such as nitric oxide synthesis, cytoskeletal remodeling, focal adhesion (FA) dynamics, and traction force (TF) generation. Although both channels respond to mechanical cues like FSS, their differential roles in modulating EC behavior remain unclear. As TFs provide a physical readout of mechanotransduction (1), we sought to investigate how Piezo1 and TRPV4 individually contribute to traction modulation and cell alignment under distinct shear flow conditions.

Materials and

Methods: : Human umbilical vein endothelial cells (HUVECs) were cultured on soft silicone gel substrates embedded with fluorescent beads to measure traction forces under shear stress using a custom polycarbonate flow chamber. Cells were seeded, stained, and transfected with siRNA targeting Piezo1 or TRPV4 before exposure to either high steady shear stress (HSS) or HSS following 60 minutes of low FSS preconditioning. Flow rates were calculated using a modified Poiseuille equation to achieve the desired shear levels. Time-lapse traction force microscopy and fluorescence imaging were used to capture bead displacement and cell alignment. Post-flow, cells were removed with trypsin to determine relaxed gel states, and traction orientation was quantified using an alignment parameter.

Results, Conclusions, and Discussions:: Piezo1 knockdown (KD) impaired the immediate traction force (TF) response under high steady shear stress (HSS), leading to a delayed and prolonged tension increase with minimal alignment to flow direction. Interestingly, under low-FSS preconditioning, Piezo1 KD cells exhibited a similar short-term TF pattern as control cells but showed faster relaxation and random alignment, indicating Piezo1’s role in facilitating rapid TF rise and directional adaptation under HSS. In contrast, TRPV4 KD cells showed a complete loss of the immediate TF rise under HSS and a delayed, gradual increase over time, along with impaired cell and TF alignment. TRPV4 KD also reduced short-term TF response under low FSS and accelerated long-term relaxation. TRPV4’s stronger impact may be linked to its involvement in β-catenin signaling and integrin activation. Together, both channels coordinate cytoskeletal tension and alignment behaviors, with TRPV4 acting upstream or in parallel with Piezo1 in regulating endothelial responses to dynamic flow environments.

Acknowledgements and/or References (Optional):: This work was funded by NIH 2R15GM135806.