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Expression of matrix metalloproteinases and their inhibitors in experimental retinal ischemia-reperfusion injury in rats.
Matrix metalloproteinases (MMPs) are endopeptidases that degrade the extracellular matrix (ECM) and are involved in the pathogenesis of retinal degeneration along with tissue inhibitors of metalloproteinases (TIMPs). The present study examined the expression and activation of two specific members of MMPs (MMP-2 and MMP-9) and their related inhibitors (TIMP-1 and TIMP-2) in an experimental retinal ischemia-reperfusion injury. Retinal ischemia-reperfusion injury (RIRI) was induced in adult rats with a ligation method. After one hour of ischemia and a varied reperfusion time (0, 3, 6, 12, 24, 48 and 76 hr), the rat eyes were enucleated. Retinal extracts underwent zymographic analysis to measure the activity of MMP-2/9. The activity of TIMP-1 and TIMP-2 was measured by reverse zymography. The protein level was examined by Western blot. Immunohistochemistry analysis was undertaken to assess the anatomical distribution of MMP-9 in the retina after RIRI. The gelatinolytic activity of ProMMP-2 (72 kDa) was increased markedly at 6 hr after RIRI. ProMMP-9 (92 kDa) was not detected in the control specimens, while it appeared at 3 hr, increased markedly at 6 hr, and reached maximal levels at 24 hr after RIRI. The gelatinolytic activity found ian retinal extracts was shown to be inhibited by 10 m M EDTA and activated in vitro by a known metalloproteinase activator (4-aminophenylmercuric acetate (APMA)), indicating that these enzymes were of the metalloproteinase class. By western blot, MMP-2/9 levels increased parallel to protein activity level in zymography. No corresponding increase in TIMP-1 and TIMP-2 protein activity and protein level was detected by reverse zymography and western blot. Elevated levels of MMP-9 and its distribution in retina were confirmed by immunohistochemistry. Expression of MMP-9 was detected in the inner and outer segments of rat retina, and the level becomes stronger at 24 hr after RIRI. In this study, ProMMP-2 and ProMMP-9 were expressed and increased significantly, but their inhibitors (TIMP-1 and TIMP-2) remained relatively unaltered in ischemic retina after RIRI in rats. These results suggest that MMP-2 and MMP-9 may play an important role in the pathomechanism of retinal ischemic injury.
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