Increased stress-induced generation of reactive oxygen species and apoptosis in human keratoconus fibroblasts

PURPOSE. To determine whether keratoconus ( KC) corneal fibroblast cultures have increased reactive oxygen species ( ROS) production and are more susceptible to stress-related challenges. METHODS. Normal (n = 9) and KC ( n = 10) stromal fibroblast cultures were incubated in either neutral- or low-pH conditions, with or without hydrogen peroxide. Catalase activities were measured with a fluorescent substrate assay. Superoxide and ROS/reactive nitrogen species (RNS) productions were determined with an amine-reactive green-dye assay and 2', 7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) dye assay, respectively. Cell viability was analyzed by a dye-exclusion assay. Caspase 3 activity was measured by a fluorochrome inhibitor of caspase (FLICA) assay. A cationic ( green) dye was used to measure the mitochondrial membrane potential (Delta Psi m). RESULTS. KC fibroblasts had increased superoxide and ROS/RNS production (6.2-fold, P < 0.001 and 1.8-fold, P < 0.001, respectively) and catalase activity ( P < 0.01) with higher concentrations of H2O2 compared with normal cultures ( P = 0.16). After a low-pH stress challenge, KC fibroblasts maintained higher ROS/RNS levels (3.3-fold, P < 0.02), showed higher caspase-3 activity (7.5-fold, P < 0.02) and decreased Delta Psi m ( 2.6-fold, P < 0.04), and had decreased cell viability (37%, P < 0.005 vs. 20%, P < 0.27) compared with normal fibroblasts. CONCLUSIONS. Under identical conditions, KC fibroblasts had increased basal generation of ROS/RNS and were more susceptible to stressful challenges (low-pH and/or H2O2 conditions) than were normal fibroblasts. In addition, the stressed KC fibroblasts possessed characteristics similar to those found in the intact KC corneas ( increased catalase activity, ROS production, and apoptosis). These properties may play a role in the pathogenesis of KC.