Abstract
Purpose :
Schlemm’s canal (SC) inner wall endothelium participates in homeostatic aqueous humor (AH) outflow resistance regulation, and thereby influences intraocular pressure. SC cells experience significant biomechanical stress (e.g., tension, fluid shear) which affects 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 TRPV4 activity regulates SC cell cytoskeletal and extracellular matrix (ECM) remodeling, and cell and cell-derived ECM stiffnesses.
Methods :
Donor-derived normal (n=4 strains) and glaucomatous (n=3) human SC cells were plated on coverslips/tissue-culture plastic and stimulated with the TRPV4 inhibitor HC067047 (HC06; 10 µM) for 2 d followed by the TRPV4 activator GSK1016790A (GSK101; 100 nM) for 2 d or 7 d. SPY555-actin-labeled Filamentous (F)-actin in SC cells was imaged using live-cell time-lapse (every 5 min; total imaging time = 1 h) confocal imaging. F-actin polymerization, α-smooth muscle actin (αSMA) expression, and ECM protein expression and remodeling were determined by qRT-PCR and immunocytochemistry. Cell-derived ECM were obtained by decellularization of cultures using 20 mM ammonium hydroxide and 0.05% Triton X-100. Cell and ECM stiffnesses were measured by atomic force microscopy (AFM).
Results :
Glaucomatous SC cells exhibited significantly lower TRPV4 transcript levels (~60% of normal SC cells, p<0.05). F-actin fibers in both normal and glaucomatous SC cells began to diminish 30 min after treatment with TRPV4 inhibitor HC06. TRPV4 inhibition also decreased expression of αSMA and ECM proteins (collagen I, collagen IV, and fibronectin), and cell size (p<0.01); HC06 showed a stronger effect on normal SC cells vs. glaucomatous cells. Cytoskeletal and ECM remodeling stimulated by HC06 was partially reversed by 2 d treatment with TRPV4 activator GSK101 and fully restored by TRPV4 activation for 7 d. Importantly, SC cell/ECM stiffness and cell contractility were significantly reduced by HC06 (p<0.001), and were rescued by GSK101.
Conclusions :
Our data suggest that TRPV4 regulates cytoskeletal and ECM remodeling in SC cells to control cell/ECM stiffness; we therefore hypothesize that TRPV4 will influence AH conductance through SC inner wall endothelium.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.