Molecular mechanisms of the pathological angiogenesis in proliferative diabetic retinopathy.

在增殖性糖尿病视网膜病变（PDR）中，由于通过视网膜血管生成延伸到玻璃体腔中而形成的纤维血管组织的增殖而发生严重的并发症，诸如玻璃体出血和牵拉性视网膜脱离。纤维血管组织的形成需要眼内血管生成以及蛋白水解活性。至于血管生成活性，我们以前表明，在PDR中的血管生成的程度是密切相关的生产血管内皮生长因子的视网膜神经胶质细胞在纤维血管组织。在这项研究中，我们分析了PDR患者的玻璃体和纤维血管组织样本，重点是基质金属蛋白酶（MMP）物种，以指定负责PDR眼内蛋白水解活性的蛋白酶。采用夹心酶免疫分析法测定PDR和非糖尿病玻璃体视网膜疾病患者玻璃体样本中MMP-1、-2、-3、-7、-8、-9和-13的浓度。至e ...更多信息 明胶酶谱法测定玻璃体及纤维血管组织中MMP-2（proMMP-2）和MMP-9（proMMP-9）酶原的活化率。用免疫组化法检测MMP-2、膜型MMP（MT 1-MMP）、金属蛋白酶组织抑制剂（TIMP-2）和MMP-9在纤维血管组织中的表达和组织定位。逆转录-聚合酶链反应（RT-PCR）检测MT 1-MMP mRNA在纤维血管组织中的表达。在所检测的MMP中，PDR样品中MMP-2和MMP-9的水平显著高于对照。然而，在PDR玻璃体样品中proMMP-2和-9的活化比率较低，并且与对照组的那些没有显著差异。另一方面，在纤维血管组织中，发现proMMP-2和proMMP-9都高度活化。免疫组化显示MMP-2和MMP-9主要分布于血管内皮细胞和胶质细胞。此外，MMP-2与作为proMMP-2激活剂的MT 1-MMP和TIMP-2共定位。RT-PCR检测MT 1-MMP在纤维血管组织中的表达。根据这些数据，可以想象MMP-2和MMP-9是负责PDR眼中蛋白水解活性的酶。MMP-2与MT 1-MMP和TIMP-2在纤维血管组织中，特别是在胶质细胞中共定位，表明proMMP-2被胶质细胞局灶性激活。MMP-9作为活性形式存在于纤维血管组织中，也免疫定位于神经胶质细胞。因此，我们假设具有VEGF（血管生成活性）以及活性形式的MMP-2和MMP-9（蛋白水解活性）的视网膜神经胶质细胞在纤维血管组织形成中起核心作用。此外，为了分析proMMP-2和-9在PDR中的活化调节机制，我们建立了兔视网膜胶质细胞的体外培养体系。到目前为止，我们已经获得的数据，视网膜神经胶质细胞在缺氧，相当于在糖尿病视网膜的条件下，可以产生VEGF和激活proMMP-2。少

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