Transient receptor potential vanilloid 4 modulates substrate stiffness mechanosensing and transcellular pore formation in human Schlemm’s canal cells

Pathological changes in the biomechanical environment of Schlemm’s canal (SC) inner wall cells, such as substrate stiffening and increased cellular stretch, are associated with ocular hypertension, a key risk factor for the development of glaucoma. Cell membrane stretch can trigger the activation of transient receptor potential vanilloid 4 (TRPV4) mechanosensitive ion channels, allowing calcium influx and initiating downstream signaling. However, the precise role of TRPV4 in SC cell mechanobiology remains unclear. Here, we demonstrate that sustained inhibition of TRPV4 activity modulates substrate stiffness mechanosensing to thereby affect the remodeling of the actin cytoskeleton and extracellular matrix of SC cells. This is accompanied by a reduction in cell stiffness and an increase in transcellular pore forming ability, potentially lowering outflow resistance and risk of ocular hypertension. Interestingly, acute activation of TRPV4 channels induces Ca²⁺ influx, increasing transcellular pore formation in SC cells. Notably, reduced TRPV4 mechanosensing is observed in glaucomatous SC cells, resulting in reduced transcellular pore forming ability. These findings suggest novel potential strategies based on targeting TRPV4 in SC cells for the treatment of ocular hypertension in glaucoma. Statement of significance This study identifies TRPV4 as a key mechanosensor in Schlemm’s canal (SC) endothelial cells, modulating cytoskeletal dynamics, extracellular matrix remodeling, cell stiffness, and transcellular pore formation – all of which are processes central to intraocular pressure (IOP) regulation/determination. Although TRPV4 in other ocular tissues has been implicated in IOP modulation, its role in SC cell mechanobiology remained undefined. Using tissue-mimetic hydrogels and pharmacological modulation, we demonstrate that TRPV4 activity governs SC cell responses to substrate stiffness and affects transcellular pore forming ability. Notably, these mechanosensory functions are diminished in glaucomatous SC cells. Our findings underscore the complexity of SC cell mechanobiology in glaucoma and suggest that TRPV4-targeted interventions may need to be tailored to disease-specific cellular contexts.