(A-14) Matrix stiffness modulates TRPV4 activity in human Schlemm's canal cells

Introduction:: Schlemm’s canal (SC) inner wall endothelium participates in homeostatic aqueous humor (AH) outflow resistance regulation, and thereby influences intraocular pressure. Disruption of homeostasis in the outflow pathway, such as responses of cells to pressure changes due to extracellular matrix (ECM) stiffening, increases the risk of primary open-angle glaucoma. SC cells are subjected to significant biomechanical stresses (e.g., tension, fluid shear) which affect their function. Transient receptor potential vanilloid 4 (TRPV4) channels are an important biomechanical stress-activated Ca²⁺ entry pathway in multiple cell types, yet the precise role of TRPV4 in SC cells remains unclear. Here, we investigate how ECM stiffness modulates TRPV4 activity and its downstream signaling in SC cells.


Materials and Methods:: Primary SC cells were isolated from human eyes and characterized using accepted protocols. Soft substrates were fabricated by photocrosslinking gelatin methacryloyl (GelMA; 6% + 0.075% (w/v) LAP photoinitiator), and matrix stiffening was achieved by incorporating methacrylate-conjugated hyaluronic acid (HA; 0.25%). Fibronectin (FN) was used to coat hydrogels (5 μg/cm²) to mimic the native basement membrane of SC inner wall cells. Normal (n=3 strains) and glaucomatous (n=3) human SC cells were plated on hydrogels and stimulated with the TRPV4 inhibitor HC067047 (10 µM) or the TRPV4 activator GSK1016790A (100 nM) for 2 days. F-actin was visualized by phalloidin staining. α-smooth muscle actin (αSMA) and ECM protein (collagen I, collagen IV, and fibronectin) expression were determined by qRT-PCR, immunocytochemistry, and immunoblotting. Hydrogel and cell stiffnesses were measured by atomic force microscopy (AFM).

Results, Conclusions, and Discussions:: Soft hydrogel stiffness was 2.36 kPa, close to the stiffness of ECM in healthy SC; incorporating HA stiffened hydrogels to glaucomatous levels of 8 kPa. Substrate stiffening increased TRPV4 transcript levels (~1.31-fold, p< 0.01) and F-actin (~2.17-fold, p< 0.0001) in normal SC (nSC) cells. TRPV4 activation upregulated F-actin stress fibers in nSC cells grown on the soft substrate (~1.70-fold, p< 0.001) but not on the stiff substrate. TRPV4 inhibition decreased F-actin in nSC cells grown on the stiff substrate (~0.60-fold, p< 0.0001) but not on the soft substrate. TRPV4 modulation also influenced αSMA and ECM protein expression, and SC cell stiffness. Interestingly, TRPV4 modulation showed a stronger effect in nSC cells vs. glaucomatous cells.

Our data suggest that a stiffer substrate increases basal TRPV4 activity, leading to upregulation of SC cytoskeletal and ECM remodeling, as well as cell stiffness; we thus hypothesize that TRPV4 influences AH conductance across the SC inner wall endothelium.


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