Matrix metalloproteinase-mediated disruption of tight junction proteins in cerebral vessels is reversed by synthetic matrix metalloproteinase inhibitor in focal ischemia in rat
Matrix metalloproteinase-mediated disruption of tight junction proteins in cerebral vessels is reversed by synthetic matrix metalloproteinase inhibitor in focal ischemia in rat
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DOI：
10.1038/sj.jcbfm.9600375
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发表时间：
2007-04-01
影响因子：
6.3
通讯作者：
Rosenberg, Gary A.
中科院分区：
文献类型：
--
作者：
Yang, Yi;Estrada, Eduardo Y.;Rosenberg, Gary A.
再灌注过程中，基质金属蛋白酶 (MMP) 会破坏血脑屏障 (BBB)。最近描述了 Occludin 和 Claudins 是形成 BBB 的紧密连接蛋白 (TJP)。我们假设 BBB 的开放是由于 MMP 降解 TJP 所致。自发性高血压大鼠的大脑中动脉闭塞 90 分钟，再灌注 2、3 或 24 小时。通过免疫组织化学以及原位和凝胶酶谱法测量基质金属蛋白酶。实时聚合酶链反应 (PCR) 测量 MMP-2 和 -9、弗林蛋白酶、膜型 MMP (MT1-MMP)、occludin 和 claudin-5 的 mRNA。再灌注 3 小时后，梨状皮质中的 BBB 打开，MMP 抑制剂 BB-1101 (30 mg/kg) 阻止了打开。 3小时时，原位酶谱显示明胶酶活性。酶谱分析和 PCR 显示 MMP-2 的增加幅度大于 MMP-9。 MT1-MMP 和弗林蛋白酶的 mRNA 和免疫组织化学结果均增加，从而激活 MMP-2。 2 小时后，两个半球的 Claudin-5 和 occludin mRNA 表达均下降，在缺血侧 3 小时时，Western blot 中可看到这两种蛋白的片段； BB-1101 处理逆转了 TJP 的降解。 3小时的免疫组织化学显示内皮细胞裂隙内有碎片化的TJP。 24 小时时，原位酶谱显示明胶酶活性，凝胶酶谱显示 MMP-9 水平升高。先前在内皮细胞中看到的破坏的 TJP 出现在周围的星形胶质细胞中。我们的结果提供了直接证据，证明 MMP 通过降解 TJP 来打开 BBB，并且 MMP 抑制剂可防止 MMP 降解 TJP。
Matrix metalloproteinases ( MMPs) disrupt the blood-brain barrier (BBB) during reperfusion. Occludin and claudins are recently described tight junction proteins (TJPs) that form the BBB. We hypothesized that the opening of the BBB was because of the degradation of TJPs by the MMPs. Spontaneously hypertensive rats had a 90 mins middle cerebral artery occlusion with reperfusion for 2, 3, or 24 h. Matrix metalloproteinases were measured by immunohistochemistry and in situ and gel zymography. Real-time polymerase chain reaction (PCR) measured mRNAs of MMP-2 and -9, furin, membrane-type MMP (MT1-MMP), occludin, and claudin-5. There was opening of the BBB in the piriform cortex after 3 h of reperfusion, and an MMP inhibitor, BB-1101 (30 mg/kg), prevented the opening. At 3 h, in situ zymograms showed gelatinase activity. Zymography and PCR showed greater increases in MMP-2 than in MMP-9. There were increased mRNA and immunohistochemistry for MT1-MMP and furin, which activate MMP-2. Claudin-5 and occludin mRNA expression decreased at 2 h in both hemispheres with fragments of both proteins seen on Western blot by 3 h on the ischemic side; treatment with BB-1101 reversed the degradation of the TJPs. Immunohistochemistry at 3 h showed fragmented TJPs within the endothelial cell clefts. By 24 h, in situ zymography showed gelatinase activity and gel zymography showed elevated levels of MMP-9. Disrupted TJPs previously seen in endothelial cells appeared in the surrounding astrocytes. Our results provide direct evidence that MMPs open the BBB by degrading TJPs and that an MMP inhibitor prevents degradation of the TJPs by MMPs.