Abstract
Purpose :
Following surgery or traumatic injury, corneal wound healing can cause a scarring response that impairs ocular function. This fibrosis is caused in part by myofibroblast differentiation of corneal keratocytes in response to transforming growth factor beta 1 (TGF-β1). Previous studies have shown that the changes in extracellular matrix (ECM) stiffness of can regulate this process, but it remains unclear how keratocytes sense changes in their physical microenvironment. Here, we used a polyacrylamide (PA) gel system to show that the subcellular patterning (and activation of signaling downstream) of focal adhesions underlies stiffness-dependent differences in myofibroblastdifferentiation of corneal keratocytes.
Methods :
Soft (1 kPa) and stiff (10 kPa) polyacrylamide (PA) hydrogels were fabricated on glass coverslips, functionalized with type I collagen, plated with primary corneal keratocytes (NRKs), and cultured in defined serum free media with or without exogenous TGF-β1. In some experiments, an inhibitor of focal adhesion kinase (PF-228) was also added to the media. Cells were fixed and stained for F-actin, as well as markers for myofibroblast activation (α-SMA), contractility (pMLC), or focal adhesions (vinculin). We also used traction force microscopy (TFM) to quantify NRK traction stresses.
Results :
Treatment with TGF-β1 elicited stiffness-dependent differences in the number, size, and subcellular distribution of FAs in cultured NRKs. On stiff substrata, cells exhibited large FAs distributed throughout the entire cell body, while on soft gels, the FAs were smaller and fewer in number, localized primarily to the distal tips of thin cellular extensions. Larger and increased numbers of FAs correlated with elevated cellular traction stresses. Inhibition of focal adhesion kinase (FAK) disrupted stiffness-dependent differences in contractility and myofibroblast differentiation of cultured NRKs. Further, in the presence of the inhibitor, we no longer observed stiffness-dependent differences in the subcellular patterning of FAs.
Conclusions :
Taken together, these data suggest that changes in the subcellular patterning of FAs, as well as the activation of FAK, have important implications for stiffness-dependent myofibroblast differentiation of corneal keratocytes during wound healing.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.