A human lens model of cortical cataract: Ca2+-induced protein loss, vimentin cleavage and opacification

PURPOSE. Cortical cataract in humans is associated with Ca2+ overload and p rotein loss, and although animal models of cataract have implicated Ca2+-ac tivated proteases in this process, it remains to be determined whether the human lens responds in this manner to conditions of Ca2+ overload. The purp ose of these experiments was to investigate Ca2+-induced opacification and proteolysis in the organ-cultured human lens. METHODS. Donor human lenses were cultured in Eagle's minimum essential medi um (EMEM) for up to 14 days. The Ca2+ ionophore ionomycin was used to induc e a Ca2+ overload. Lenses were loaded with [H-3]-amino acids for 48 hours. After a 24-hour control efflux period, lenses were cultured in control EMEM (Ca2+ 1.8 mM), EMEM + 5 mu M ionomycin, or EMEM + 5 mu M ionomycin + 5 mM EGTA (Ca2+ <1 mu M). Efflux Of proteins and transparency were monitored dai ly. Protein distribution and cytoskeletal proteolysis were analyzed at the end of the experiment. Cytoskeletal proteins were isolated and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). West ern blot analyses were probed with anti-vimentin antibody (clone V9) and de tected by enhanced chemiluminescence. RESULTS. Lenses cultured under control conditions remained transparent for 14 days in EMEM with no added supplements or serum. The lenses synthesized proteins and had a low rate of protein efflux throughout the experimental p eriod. Ionomycin treatment resulted in cortical opacification, which was in hibited when external Ca2+ was chelated with EGTA. Exposure to ionomycin al so led to an efflux of [H-3]-labeled protein, amounting to 41% of the label ed protein over the 7-day experimental period, compared with 12% in ionomyc in + EGTA-treated lenses. Efflux was accounted for by loss from the lens so luble protein (crystallin) fraction. Western blot analysis of the cytoskele tal protein vimentin (56 kDa) revealed a distinct breakdown product of 48 k Da in ionomycin-treated lenses that was not present when Ca2+ was chelated with EGTA. In addition, high-molecular-weight proteins (similar to 115 kDa and 235 kDa) that cross-reacted with the vimentin antibody were observed in ionomycin-treated lenses. The Ca2+-induced changes were not age dependent. CONCLUSIONS. Human lenses can be successfully maintained in vitro, remainin g transparent for extended periods. Increased intracellular Ca2+ induces co rtical opacification in the human lens. Ca2+-dependent cleavage and cross-l inking of vimentin supports possible roles for calpain and transglutaminase in the opacification process. This human lens calcium-induced opacificatio n (HLCQ) model enables investigation of the molecular mechanisms of opacifi cation, and the data help to explain the loss of protein observed in human cortical cataractous lenses in vivo.