EVIDENCE OF A ROLE FOR MATRIX METALLOPROTEINASES IN COLD PRESERVATIONINJURY OF THE LIVER IN HUMANS AND IN THE RAT

Previous studies have determined that proteases are important in cold preservation injury to the liver, The purpose of this study was to det ermine the role of matrix metalloproteinases (MMPs) in cold preservati on injury, Effluents were collected from rat livers after various peri ods of preservation either in Eurocollins solution or in University of Wisconsin (WV) solution, Effluents were also collected from 17 human donor livers stored in UW solution, To determine whether sinusoidal en dothelial cells released MMPs when placed in the cold, these cells wer e isolated from rat livers and cultured at 4 degrees C. Gelatin zymogr aphy, quantitative assay of gelatinolytic activity, immunoprecipitatio n, and Western blotting were used to identify metalloproteinases and t o measure their activity. Human and rat liver effluents contained gela tin-digesting bands on zymography. Their appearance was inhibited by s pecific metalloproteinase inhibitors and also by lactobionate, the maj or ingredient of UW solution. The most prominent bands in humans and t he rat appeared at approximately 72 kd and 92 kd, suggesting that they were the MMPs 72-kd gelatinase and 92-kd gelatinase, Supernatants of isolated rat sinusoidal endothelial cells stored in the cold contained similar bands, In the rat, the proteinases were present in both laten t and active forms, but, in humans, predominately the latent form was seen. In humans, there were four prominent bands in the gelatin zymogr aphy. By immunoprecipitation, two of the bands were identified as the 92-kd gelatinase and a dimer or polymer of 92-kd gelatinase. Using Wes tern blotting with a monoclonal antibody, a third band was identified as 72-kd gelatinase. In quantitative terms, gelatinolytic activity inc reased with time of cold storage in humans and in the rat. In the rat, gelatinolytic activity was greater when Eurocollins was the preservat ive than when UW solution was used, Taken together, these results indi cate an important role for MMPs in the injury produced by cold preserv ation of the liver.