Molecular mechanisms of the pathological angiogenesis in proliferative diabetic retinopathy.

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In proliferative diabetic retinopathy (PDR), severe complications such as vitreous hemorrhage and tractional retinal detachment occur as the results of proliferation of fibrovascular tissue which is formed by the extension of retinal angiogenesis into the vitreous cavity. Formation of fibrovascular tissue requires the intra-ocular angiogenic as well as proteolytic activities. As concerns the angiogenic activity, we previously showed that the degree of angiogenesis in PDR is closely correlated with the production of VEGF by retinal glial cells in the fibrovascular tissue. In this study, we analysed vitreous and fibrovascular tissue samples from PDR patients, with focus on matrix metalloproteinase (MMP) species, to specify the proteinases responsible for the intra-ocular proteolytic activity in PDR. Sandwich enzyme immunoassays were used to measure concentrations of MMP-1, -2, -3, -7, -8, -9, and -13 in vitreous samples from patients with PDR and non-diabetic vitreoretinal diseases. To e … More valuate activation ratios of the zymogen of MMP-2 (proMMP-2) and -9 (proMMP-9) in the vitreous and fibrovascular tissue samples, gelatin zymography was performed. Production and tissue localization of MMP-2, membrane type 1-MMP (MT1-MMP), tissue inhibitor of metalloproteinases (TIMP)-2, and MMP-9 in fibrovascular tissues were examined by immunohistochemistry. mRNA expression of MT1-MMP in the fibrovascular tissues was determined by reverse transcription-polymerase chain reaction (RT-PCR). Among the MMPs examined, the levels of MMP-2 and -9 were significantly higher in the PDR samples than in the control. However, activation ratios of proMMP-2 and -9 in PDR vitreous samples were low and not significantly different from those of the control. On the other hand, in fibrovascular tissues, both proMMP-2 and -9 were found to be highly activated. Immunohistochemical study demonstrated the localization of MMP-2 and -9 in the endothelial cells and glial cells of fibrovascular tissues. In addition, MMP-2 was colocalized with MT1-MMP and TIMP-2 which are activators of proMMP-2. MT1-MMP expresison in fibrovascular tissues was also indicated by RT-PCR analysis. From these data, it is conceivable that MMP-2 and MMP-9 are the enzymes responsible for proteolytic activity in the eyes with PDR. Colocalization of MMP-2 with MT1-MMP and TIMP-2 in fibrovascular tissues, especially in glial cells, suggests the focal activation of proMMP-2 by glial cells. MMP-9, which exists in the fibrovascular tissue as the active form, is also immunolocalized to glial cells. Therefore, we hypothesize that retinal glial cells with VEGF (angiogenic activity) as well as active forms of MMP-2 and -9 (proteolytic activity) play the central role in fibrovascular tissue formation. Furthermore, in order to analyze the mechanisms to regulate the activation of proMMP-2 and -9 in PDR, we have established the in vitro culture system of retinal glial cells from rabbit retina. Until now, we have obtained the data that retinal glial cells under hypoxia which corresponds to the condition in diabetic retina could produce VEGF and activate proMMP-2. Less