Biophysical properties of the corneal stroma influence apoptosis of fibroblasts

Abstract

Purpose : Corneal wounding and its healing processes alter the biophysical attributes of the corneal stroma which, in turn, modulate a wide array of fundamental stromal cell behaviors including differentiation of keratocyte to fibroblasts and myofibroblasts. Myofibroblasts play a critical role in the normal corneal wound healing process and are subsequently removed from the wound space through apoptosis. Excessive numbers and prolonged persistence of myofibroblasts have been associated with the development of corneal haze that can impair vision. Tumor necrosis factor α (TNF-α) has been implicated in promoting myofibroblast apoptosis, but a knowledge gap exists as to whether the biophysical attributes of the stroma modulate this critical process. In this study, we investigated how matrix stiffness impacts fibroblast apoptosis in response to the pro-apoptotic signaling molecule TNF-α.

Methods : Primary human corneal fibroblasts (HCFs) were cultured on tissue culture plastic (TCP) as well as compliant substrates which mimic the normal and wounded environment of the human cornea (25 and 75 kPa, respectively). HCFs were then treated with TNF-α (1 pg/mL) and their apoptotic response was determined by detection of activated caspase 3/7 using flow cytometry. Untreated HCFs were used as negative control for each group. For all the experiments, staurosporine (350 ng/mL), a well studied promoter of apoptosis, was used as a positive control.

Results : Flow cytometry documented that, in the presence of TNF-α, the percentage of apoptotic HCFs cultured on soft substrates (25 kPa hydrogels) was significantly higher than the HCFs cultured on 75 kPa hydrogels and TCP (46.27 ± 8.12 compare to 18.23 ± 3.63 and 7.79 ± 1.46, respectively).

Conclusions : These results indicate that substratum stiffness directly influences the response of the fibroblast to soluble pro-apoptotic molecules. In vivo, this speaks to the dynamic interplay between the biophysical attributes of the stroma and the soluble signalling environment in coordinating complex processes such as wound healing. Additionally, these findings suggest that modulation of stromal stiffness represents a therapeutic approach that could improve wound healing outcomes for patients.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.