Abstract Body: Background: Endothelial to mesenchymal transition (EndMT) is a fundamental differentiation program required for embryonic development and increasingly recognized as a driver of cardiovascular pathologies, including atherosclerosis and cardiac fibrosis. Although soluble mediators such as transforming growth factor β (TGF β) are well established inducers of EndMT, mechanical cues such as substrate stiffness, cyclic stretch, and shear stress also critically regulate this process. However, the mechanotransduction pathways that translate these mechanical signals into EndMT remain poorly defined. The mechanosensitive ion channel TRPV4 has emerged as an important regulator of cellular responses to mechanical stress and is a potential modulator of EndMT signaling.
Hypothesis: We hypothesized that TRPV4 mediates TGF β2 induced EndMT in endothelial cells through activation of downstream signaling pathways that promote mesenchymal differentiation.
Methods: EndMT was induced in HMEC 1 cells by treatment with TGF β1 or TGF β2. TRPV4 activity was blocked using the selective antagonist GSK2193874 (GSK2). EndMT progression was evaluated by measuring expression of the mesenchymal marker α smooth muscle actin (α SMA) and the endothelial markers CD31 and VE cadherin. TRPV4 protein levels and calcium influx were assessed following stimulation with the TRPV4 agonist GSK1016790A. Downstream signaling was examined by quantifying Smad2/3 phosphorylation, Rho GTP activation, and the expression of EndMT associated transcription factors, including Snail, Slug, Twist 1, SIP 1, and ZEB 1.
Results: TGF β2 induced a more robust EndMT response than TGF β1, as evidenced by increased α SMA expression and decreased CD31 and VE cadherin levels. TGF β2 also upregulated TRPV4 expression and enhanced TRPV4 dependent calcium influx. Pharmacological inhibition of TRPV4 with GSK2 significantly attenuated TGF β2–induced EndMT. Mechanistically, TGF β2 activated Smad2/3 phosphorylation, Rho signaling, and multiple EndMT related transcription factors. Notably, TRPV4 inhibition selectively reduced Smad2 phosphorylation, Rho activation, and Snail expression, with minimal effects on other transcription factors.
Conclusion: These findings identify TRPV4 as a key mediator of TGF β2–induced EndMT through the Smad2/Rho/Snail signaling axis. Targeting TRPV4 may therefore represent a promising therapeutic strategy for preventing EndMT driven vascular pathology.