Mitomycin C Induces Apoptosis in Cultured Corneal Fibroblasts Derived From Type II Granular Corneal Dystrophy Corneas

Abstract

Purpose: : The corneal stromal dystrophies related with transforming growth factor- beta (TGF-bera) show abnormal protein deposits in the stroma. The clinical figures of MMC treated granular corneal dystrophy II (GCDII) showed exacerbated figures. Our previous study demonstrated that TGF beta-induced protein (TGFBIp) are internalized and degraded by cultured corneal fibroblasts. This present study investigated the effect of MMC on cell viability and apoptosis in cultured fibroblasts to find out the mechanism of recurrence of dystrophies after PTK with MMC.

Methods: : Keratocytes were obtained from normal cornea or heterozygote or homozygote GCD II patients after lamellar or penetrating keratoplasty. To measure cell viability, corneal fibroblasts were incubated with 0, 0.01, 0.02, or 0.04% MMC for 3, 6, or 24 h, and then tested using LDH and MTT assays. To measure apoptosis, cells were analyzed using FACS analysis and annexin V staining. Bcl-xL and Bax mRNA expressions were measured using reverse transcription polymerase chain reaction (RT-PCR) assays.

Results: : MTT and LDH assays showed that MMC reduced cell viability in all 3 cell types in a dose- and time-dependent manner. FACS analysis and annexin V staining showed MMC caused apoptosis, with GCD II homozygote cells being most affected. RT-PCR analysis showed MMC caused decreased Bcl-xL mRNA expression and increased Bax mRNA expression in all cell types, with GCD II homozygote cells being the most affected.

Conclusions: : Increased apoptosis of keratocyte by the MMC was observed in GCDII corneal fibroblast. It may be the cause of exacerbation of GCDII by reducing the number of functional keratocyte.

Keywords: cornea: stroma and keratocytes • cornea: clinical science • apoptosis/cell death