Oxidative Stress in Keratoconus Is Evident in Tear Fluid and Stromal Cells and Alleviated in Cell Culture by Sulforaphane

Purpose: Keratoconus (KC) is a common eye disease characterized by progressive corneal thinning and steepening. Despite multiple treatment options, there is no definitive cure for KC. Previously we identified loss and dysregulation of nuclear factor erythroid 2-related factor 2 (NRF2) mediated antioxidant functions in stromal cells and extracellular matrix (ECM) in KC. Here we used tear fluid samples and cell culture models to investigate oxidative stress in KC. Methods: Primary human KC and donor (DN) stromal fibroblasts were exposed to hydrogen peroxide (H2O2) to induce oxidative stress and treated with sulforaphane (SFN) for antioxidant rescue. The fibroblasts were then assessed for NRF2 activation and apoptosis by measuring TXNRD1, HMOX1, NRF2, and GPX3 expression and caspase-3/7 activity. ML385 was used to inhibit NRF2 functions in DN fibroblast cultures followed by measurements of cell death (Caspase 3/7), proliferation (BrdU and Ki-67 labeling) and ECM deposition by immunohistology. Oxidative stress was directly assessed in KC and non-KC subjects by measuring malondialdehyde (MDA) and glutathione peroxidase 3 (GPX3) levels in the tear fluid. Results: H2O2-stressed KC fibroblasts displayed increased apoptosis and suboptimal NRF2 activation, which could be rescued with SFN. Conversely, DN fibroblasts treated with ML385 elicited KC-like cellular phenotypes, including decreased antioxidant response, reduced cell growth, myofibroblastic changes and poor ECM deposition. Compared to unaffected controls, KC patient tear fluid exhibited elevated levels of GPX3 and MDA, a byproduct of lipid peroxidation. Conclusions: NRF2-mediated anti-oxidative functions are dysregulated in KC. In the future SFN antioxidant treatments may be therapeutic in KC, while MDA and GPX3 may lead to promising biomarkers for diagnosis and severity predictions.