<scp>TRPV4</scp> ‐Mediated Mechanosensing Regulates the Endothelial‐to‐Mesenchymal Transition: Implications for Atherosclerosis

Endothelial-to-mesenchymal transition (EndMT) is a crucial biological process where endothelial cells lose their specialized phenotype and acquire mesenchymal characteristics, such as enhanced motility and the ability to produce extracellular matrix components. This process serves dual roles in cardiovascular health and disease. In this study, we investigated the role of mechanosensitive Ca²⁺-permeant transient receptor potential vanilloid 4 (TRPV4) channels in matrix stiffness- and transforming growth factor β1 (TGFβ1)-induced EndMT. Using primary mouse wild-type aortic endothelial cells (aECs) and TRPV4-null aECs, we validated TRPV4 functionality through live Ca²⁺ influx detection in response to selective agonists and antagonists. Employing physiologically relevant hydrogels with varying stiffness that mimic healthy and diseased aortic tissue, we observed that genetic deletion of TRPV4 or its pharmacological inhibition suppressed intracellular matrix stiffening and cellular force generation as quantified by atomic force microscopy analysis and traction force microscopy analysis, respectively, and prevented matrix stiffness- and TGFβ1-induced EndMT. Further analysis revealed that the N-terminal residues 100-130 of TRPV4 are critical for intracellular matrix stiffening, traction force generation, MLC2 phosphorylation, and EndMT in aECs. Additionally, our findings demonstrated that TRPV4 regulates matrix stiffness-induced MLC2 activity, thereby modulating EndMT and cellular force generation, identifying a potential mechanism by which TRPV4 activity regulates EndMT in aEC. These results uncover a novel role for TRPV4-mediated mechanotransduction in regulating EndMT and suggest that TRPV4 could be a promising therapeutic target for addressing cardiovascular diseases.