Quiescent keratocytes fail to repair MMC induced DNA damage leading to the long-term inhibition of myofibroblast differentiation and wound healing

PURPOSE The purpose of this study was to determine the acute and long-term effects of mitomycin C (MMC) on quiescent rabbit corneal keratocytes regarding cell proliferation, myofibroblast differentiation and DNA repair. METHODS Quiescent keratocytes cultured in serum-free media were exposed to various concentrations of MMC and then treated with transforming growth factor-β (TGFβ). DNA damage was evaluated in both cultured keratocytes and live rabbit eyes following treatment with MMC. The long-term ability of quiescent keratocytes to repair MMC induced damage in vivo was evaluated in rabbits treated with MMC 2 months before 100 μm deep lamellar keratectomy (LK) injury. RESULTS MMC significantly blocked TGFβ-induced cell proliferation and myofibroblast differentiation in cultured quiescent keratocytes and altered the transcriptional regulation of macrophage chemotactic protein-1 (MCP-1) and alpha smooth muscle actin (αSMA). MMC also induced phosphorylation of the nuclear histone marker of DNA damage, γH2AX (a member of the H2A histone family), without induction of cell cycle entry or immediate DNA repair measured by Comet assay. In live rabbits, 0.2 mg/ml MMC significantly induced γH2AX nuclear immunostaining (p<0.05) throughout the cornea and corneas receiving 0.2 mg/ml MMC treatment 2 months before LK injury showed complete absence of any corneal scarring. CONCLUSIONS MMC induces DNA damage to quiescent corneal keratocytes, which remains unrepaired, resulting in abnormal cell replication and gene transcription that leads to long-term effects on corneal repair. Overall these findings suggest that there may be long-term and perhaps permanent consequences to the application of MMC as an anti-fibrotic therapy.