Increased Stress-Induced Generation of Reactive Oxygen Species and Apoptosis in Human Keratoconus Fibroblasts

Abstract

To determine whether keratoconus (KC) corneal fibroblast cultures have increased reactive oxygen species (ROS) production and are more susceptible to stress-related challenges. 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 (H2DCFDA) 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). 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. 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.