Abstract
Purpose :
During corneal wound healing, keratocytes located within the stroma are activated into a repair phenotype by the release of soluble growth factors, such as transforming growth factor-beta 1 (TGF-β1) and platelet-derived growth factor-BB (PDGF-BB). This process is often accompanied by an increase in tissue stiffness. Previous studies have shown that the TGF-β1-mediated myofibroblastic differentiation of corneal keratocytes is regulated by changes in stiffness, but it is unclear if the keratocyte response to other growth factors, such as PDGF-BB, is stiffness-dependent. Here, we used a polyacrylamide (PA) gel system to determine if changes in substratum stiffness modulate the proliferation and motility of primary corneal keratocytes cultured in the presence of PDGF-BB. We also investigated how the stiffness-dependent myofibroblastic differentiation of cultured keratocytes is influenced by treatment with both TGF- β1 and PDGF-BB.
Methods :
PA substrata with an elastic modulus of either 1 kPa (soft) or 10 kPa (stiff) were fabricated to mimic the mechanical properties of either normal or fibrotic corneal tissue, functionalized with type I collagen, plated with primary rabbit corneal keratocytes (NRKs), and cultured in defined serum-free media in either the presence or absence of TGF- β1 and/or PDGF-BB.
Results :
When treated with PDGF-BB, NRKs exhibited a more elongated morphology and a higher rate of proliferation on stiff, as opposed to soft, PA substrata. Using a freeze-injury assay, however, we did not observe stiffness-dependent differences in the PDGF-BB-driven motility of these cells. In the presence of TGF-β1, NRKs cultured on soft PA substrata exhibited low levels of myofibroblastic differentiation, as measured by alpha-SMA immunofluorescence and cellular traction forces. When treated with both TGF-β1 and PDGF-BB, however, increased levels of alpha-SMA immunofluorescence were observed on the soft PA substrata, and we no longer observed stiffness-dependent differences in myofibroblastic differentiation, suggestive of mechanotransductive cross-talk between the signaling pathways downstream of TGF-β1 and PDGF-BB.
Conclusions :
Taken together, these data highlight the importance of the combined biochemical and biophysical microenvironment on keratocyte behavior during corneal wound healing.
This work was supported by the NIH grants R01 EY030190 and P30 EY030413.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.