Influence of riboflavin-UV-A illumination on the expression of pro- and anti-apoptotic markers in human corneal fibroblasts from healthy and keratoconus corneas.

PURPOSE: Corneal crosslinking (CXL) using riboflavin and ultraviolet A (UV-A) illumination is widely used to stabilize progressive ectatic corneal disorders such as keratoconus. However, the photochemical reaction generates oxidative stress and may induce apoptosis, while potential differences between healthy human corneal fibroblasts (HCF) and human corneal fibroblasts derived from keratoconus corneas (KC-HCF) remain insufficiently understood. This study examined in-vitro changes in pro- and anti-apoptotic markers in HCF and KC-HCF following riboflavin-UV-A illumination. METHODS: Cell cultures of HCFs (n = 5) and KC-HCFs (n = 5) were incubated with 0.01% or 0.1% riboflavin-dextran solutions (RF) for subsequent crosslinking and either placed in a dark chamber or illuminated with UV-A light (250 s, 375 nm, 2 J/cm2). The expression profiles of pro-apoptotic markers (BCL-2 homologous antagonist/killer (BAK), BCL-2 associated agonist of cell death protein (BAD), caspase-9 (CASP9) and cytochrome c (CYCS)) and the anti-apoptotic marker Baculoviral IAP Repeat Containing 5 (BIRC5) were analyzed using quantitative reverse transcription polymerase chain reaction (RT-PCR) (0.01%/0.1% RF; n = 5; for 2 h (h), 4 h, n = 3; for 24 h) and Western blot (0.1% RF; n = 4 for 24 h). Cell proliferation was assessed after 2 h, and ultrastructural changes were examined by transmission electron microscopy (TEM) after 24 h (0.1% RF; n = 1). RESULTS: Cell proliferation was significantly reduced in HCFs and KC-HCFs after 2 h in both treatment groups (0.01%/0.1% RF). Gene expression analysis showed a significant increase in BAK and BAD expression in KC-HCFs after 24 h with 0.01% RF, but not after 0.1% riboflavin-UV-A illumination. For CYCS and CASP9 gene expression levels remained unchanged for HCFs and KC-HCFs in both treatment groups. BIRC5 was significantly reduced in HCFs (0.01%/0.1% RF) and in KC-HCFs (0.1% RF). Protein expression analysis revealed a significant increase in BAK and CYCS after 24 h in HCFs and KC-HCFs, while CASP9 expression was significantly decreased. TEM showed some ultrastructural features of apoptosis after 24 h (0.1% RF). CONCLUSION: Riboflavin-UV-A illumination induced a time- and dose-dependent cellular response in both HCFs and KC-HCFs with KC-HCFs showing an increased sensitivity to photochemical stress.