Abstract
Purpose :
Previous studies have suggested that focal adhesion size is correlated with the stiffness of ECM, but it remains unclear how signaling downstream of focal adhesion formation modulates changes in corneal keratocyte morphology and mechanical activation.
Methods :
Polyacrylamide (PA) hydrogels of varying stiffness were fabricated on glass coverslips, functionalized with type I collagen, and used as substrata for two-dimensional (2D) cell culture. The gels were plated with primary corneal keratocytes (NRKs) in either serum-free media or media containing exogenous TGF-β1. In some experiments focal adhesion kinase (FAK) inhibitor (PF-573228) was also added to the culture media. Afterward, NRKs were fixed and stained for F-actin, as well as markers for myofibroblastic activation (α-SMA), contractility (pMLC), or focal adhesions (vinculin). Focal adhesion size and traction stresses exerted by NRKs were also measured.
Results :
Treatment with TGF-β1 elicited distinct cellular phenotypes when NRKs were cultured on gels of varying stiffness. Cells cultured on either stiff (10 kPa) PA gels or collagen-coated glass coverslips formed abundant stress fibers, exhibited elevated levels of α-SMA immunofluorescence, and exerted large traction forces. These cells also formed abundant focal adhesions distributed across the entire cell body. NRKs cultured on soft (1 kPa) substrata, however, exhibited behaviors more indicative of a quiescent phenotype, even in the presence of TGF-β1. They formed fewer stress fibers, retained a more elongated morphology, and exerted significantly lower traction forces, with small focal adhesions, localized primarily at the tips of cellular projections. pMLC immunofluorescence further revealed stiffness-dependent differences in subcellular contractility, with contractions localized in the tips of cellular projections in cells cultured on soft substrata. Traction force maps correlated strongly with these patterns of pMLC immunofluorescence. FAK inhibition blocked myofibroblast transformation and the associated phenotypic changes.
Conclusions :
Taken together, these data suggest that mechanotransductive signaling downstream of FAK activation is required for TGF-β1 induced myofibroblast transformation of corneal keratocytes.
This is a 2021 ARVO Annual Meeting abstract.