Abstract
Purpose :
Our previous study has confirmed the presence of senescent fibroblasts in alkali-induced corneal neovascularization. Here we explored the role of fibroblast senescence in the dynamic process of corneal wound healing involving cell apoptosis, proliferation, migration and differentiation.
Methods :
Corneal wound healing model was performed with epithelial scrape in adult C57BL/6 mice. Mouse corneas were harvested for the fluorescence staining of TUNEL, Ki67 and α-SMA as the apoptosis, proliferation and myofibroblast markers. Cell senescence was confirmed by senescence associated β-galactosidase (SA-β-gal) staining, fluorescence staining, real-time qPCR, western blot and flow cytometry. In vitro cultured human corneal fibroblasts (HCFs) were treated with hydrogen peroxide for senescence induction and with TGFβ for myofibroblast differentiation. The response to growth factor PDGF-BB or bFGF and the ECM synthesis-related gene expression was compared among normal, senescent fibroblasts and myofibroblasts. Corneal cell senescence was further evaluated in mouse autologous penetrating keratoplasty (PKP) and rabbit photorefractive keratectomy (PRK) models.
Results :
Corneal keratocytes were found apoptosis (6 h) and proliferation (24 h) after epithelial scrape. The SA-β-gal staining was observed clearly in the anterior stroma at 3-5 days. The senescent cells displayed p16ink4a++Vimentin++α-SMA+ and represented corneal fibroblast origin. Hydrogen peroxide treatment induced the senescence of cultured HCFs that recapitulated the characteristics of in vivo senescent cells. The senescent fibroblasts showed the reduced proliferation capacity in response to PDGF-BB or bFGF. Moreover, the senescent fibroblasts assumed the increased expression of MMP3/13 and α-SMA, while decreased expression of fibronectin and collagen I. Cell senescence was also found in PKP and PRK animal models.
Conclusions :
Corneal keratocytes assume apoptosis, proliferation, migration and senescence during wound healing. The nonfibrotic senescent cells may play an important role in the fibrosis limitation of corneal stroma healing.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.