Substrate Rigidity Modulates Cell–Matrix Interactions and Protein Expression in Human Trabecular Meshwork Cells

Abstract

Extracellular matrix (ECM) composition, tension, and rigidity modulate cell-ECM interactions and have substantial impact on cell functions. The authors studied the effects of ECM rigidity on human trabecular meshwork (HTM) cells to assess ECM rigidity as a possible pathophysiologic factor in glaucoma. Trabecular meshwork cells derived from donor cornea rings and passaged three to seven times were plated on collagen-coated tissue culture plastic or polyacrylamide gels of different rigidity. Cell spreading and focal adhesions were assessed by immunofluorescence microscopy. Expression of focal adhesion kinase (FAK), alpha-smooth muscle actin (alpha-SMA), tubulin, alpha-B-crystallin and GAPDH, as well as phosphorylation of FAK and serum-induced activation of ERK, were studied by Western blot. The subcellular distributions of alpha-SMA and fibronectin were examined by confocal immunofluorescence microscopy. ECM rigidity modulated cell spreading and focal adhesion size. FAK activation and serum-induced ERK phosphorylation increased with rising substrate rigidity. Expression of alpha-SMA and recruitment of alpha-SMA to stress fibers were enhanced on rigid substrates, whereas myocilin and alpha-B-crystallin expression increased on soft substrates. The structure of fibronectin deposits differed on stiff and soft matrices. Extracellular matrix rigidity modulates cytoskeletal structures, protein expression patterns, signal transduction, and fibronectin deposition in HTM cells. ECM changes altering trabecular meshwork resiliency may therefore have significant effects on ocular outflow tract functions with implications in glaucoma.