Abstract
Purpose :
The role of extracellular vesicles (EVs) in mediating cell-cell communication in the cornea has gained increased interest in recent years. The objective of this study was to determine the functional effects of EVs isolated from corneal epithelial cells on the stromal fibroblast response in vitro in the context of proliferation, migration, contractility, and myofibroblast differentiation.
Methods :
EVs were isolated from conditioned media from an immortalized human corneal epithelial cell line (hCE-TJ) using standard ultracentrifugation approaches. The size and morphology of isolated EVs were characterized using transmission electron microscopy and loaded with a fluorescent dye, PKH26, and monitored using confocal microscopy following application to human corneal fibroblasts (hCFs). Proliferation was evaluated using immunohistochemistry based on Ki67 expression. Contractility was assessed using rat-tail collagen hydrogels with suspended hCFs based on change in area of the gel over time. In addition, 3D constructs were generated by culturing hCFs on a 0.4µm-polycarbonate transwell membrane in complete fibroblast media (10% FBS in EMEM) in the presence of a stable Vitamin C-derivative (0.5mM) over 4 weeks. Constructs were isolated at 3 days post-EV application to assess expression of the myofibroblast marker, α-smooth muscle actin (αSMA).
Results :
hCE-TJ-derived EVs were endocytosed by hCFs within 12 hours post-application. By 24 hours, hCFs treated with EVs showed a modest, but not statistically significant, change in proliferation (% Ki67 positive cells ± SEM: controls = 16% ± 3%; EV-treated = 22% ± 5%) and migration (% change in area ± SEM: controls = 77% ± 11%; EV-treated = 94% ± 3%). A significant increase in contractility in hCF-collagen gel area was observed by 115 hours post-EV application compared to controls (relative gel area ± SEM: control = 0.36 ± 0.03; EV-treated = 0.25 ± 0.01) and EV-conditioned media (0.34 ± 0.02). The presence of stress fibers and αSMA expression were apparent in hCF constructs by 3 days following EV exposure.
Conclusions :
These results suggest that hCE-TJ-derived EVs promote a wound-healing phenotype by hCFs reminiscent of myofibroblasts with increased cell contractility.
This is a 2020 ARVO Annual Meeting abstract.