Oxidative stress and proteolysis in the lens. Effects of anti-inflammatory drugs

Abstract: Oxidative stress has been implied in formation of age-related cataract. The objectives of this thesis were to study redox regulation of proteolysis and oxidative/antioxidative effects of anti-inflammatory substances in human lens epithelial cells (HLEC) and intact mouse lenses.Human lens epithelial cells and intact mouse lenses were exposed to the non-steroid anti-inflammatory substances (NSAIDs) indomethacin, diclofenac, celecoxib and acetylsalicylic acid (ASA) as well as to the glucocorticoid dexamethasone. HLEC were oxidatively stressed by H2O2. Apoptosis was studied by Hoechst staining of cell nuclei and caspase-3 activity. Cells were assayed for changes in superoxide production using dihydroethidium, for alterations in peroxide production using dichlorofluorescin diacetate and for glutathione variations using monochlorobimane. Mitochondrial depolarisation was measured with the potential-sensitive dye JC-1. Morphology was studied by transmission electron microscopy (TEM). Proteolytic activity of calpain, the proteasome and acid lysosomal proteases was measured using fluorogenic substrates. Low concentrations of NSAIDs/ASA protected against H2O2-induced apoptosis in HLEC whereas higher concentrations were toxic. Low concentrations of NSAIDs/ASA reduced superoxide and peroxide production as well as GSH depletion in oxidatively stressed HLEC. No protection of NSAIDs/ASA against H2O2-induced mitochondrial membrane potential depolarisation could be seen. Dexamethasone significantly increased apoptosis, but had no effect on superoxide production, GSH-levels or mitochondrial membrane potential. Only the highest concentration of dexamethasone (100 µM) showed an increase in peroxide production. TEM revealed multilayering of cells, vacuole formation and appearance of electron-dense multivesicular bodies in HLEC exposed to dexamethasone. A new method for measuring proteolysis in intact mouse lenses was established. Proteolytic activity was stimulated by increased intracellular calcium concentrations, as viewed by addition of ionomycin. The proteasome inhibitor lactacystin significantly decreased proteolysis whereas inhibitors of calpain and acid lysosmal enzymes did not. All three main peptidase activities of the proteasome in HLEC lysate were affected by incubation with reduced or oxidized glutathione (GSH, GSSG). The chymotrypsin-like and peptidylglutamyl peptidase-like activities responded similarly with stimulation upon addition of GSH, whereas the trypsin-like activity was inhibited. Addition of GSSG caused inhibition of all three peptidase activities, but simultaneous incubation with DTT reversed the inhibitory effect of GSSG. Oxidative stress induced by H2O2 significantly decreased the main peptidase activities of the proteasome in cultured HLEC. Intact mouse lenses incubated with H2O2 showed trends of decreased chymotrypsin-like proteasome activity as well as decreased GSH-levels.In conclusion, NSAIDs/ASA protects against apoptosis in oxidatively stressed HLEC by antioxidative actions. Steroid-exposed HLEC exhibit multilayering of cultured cells and increased apoptosis indicating disturbed differentiation/proliferation/migration as probable mechanisms behind steroid-induced cataract. Proteolysis in lens epithelial cells is strictly regulated by intracellular redox status.