Angiogenic factor response of rabbit corneal stroma cells to lipid hydroperoxide stress in vitro

Purpose. We previously reported a rabbit corneal model of neovascularization induced by linoleic acid hydroperoxide (18:2/LHP). To evaluate the effect of LHP on corneal stroma cell metabolism in vitro, we measured LHP transport, and propagation, gluthathione peroxidase (GSHPx), antioxidant status and angiogenic factors (TNF-α, VEGF). Methods. Corneal stroma cells were isolated from NZ rabbits and cultured in M-199 (15% FBS) to confluence. After incubation, LHP (1.05 × 10^-3 mol/L) was added and the concentration in cells assayed by TBARS, immunoassay (VEGF), WEGI 164 subclone 13 cytotoxicity assay(TNF-α), free radical scavenging ability in mmol Trolox equivalents (TE), and GSHPx activity using 1-chloro-2,4-dinitrobenzene. Results. Untreated cells had 6.92±1.01 nmol TBARS/mg CHOL, 9.74x10^-10TE/cell, 1.41±0.29 IU GSHPx, and constitutive levels of 581.2±312.7 pg VEGF, and 6.71±3.54 pg TNF-α/mg TP. Cell number remained relatively constant through 48 hr of incubation and then decreased. TBARS peaked at 1 hr (8.79 ± 1.61 nmol/mg CHOL), decreased at 6hr when antioxidant capacity was maximal, them increased again through 96 hr. GSHPx was initially suppressed but increased significantly at 72 hr. VEGF peaked at 1 hr and decreased to non detectable levels by 48 hr. TNF-α appeared in the media at 6 hr, but was not detected in cells. A later induction occurred in cells at 48 hr. Conclusions. This study demonstrates that corneal cells at confluence contain basal levels of antioxidants, VEGF, TNF-α, and GSHPx, which undergo changes during expose to LHP.