Stroke, Division of Clinical Neuroscience, University of Nottingham, Clinical Sciences Building, Hucknall Road, Nottingham, NG5 1PB, UK.
Eur J Neurosci. 2014 Jun;39(12):2119-28. doi: 10.1111/ejn.12552. Epub 2014 Mar 20.
Ischaemic injury impairs the integrity of the blood-brain barrier (BBB). In this study, we investigated the molecular causes of this defect with regard to the putative correlations among NAD(P)H oxidase, plasminogen-plasmin system components, and matrix metalloproteinases. Hence, the activities of NAD(P)H oxidase, matrix metalloproteinase-2, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA), and superoxide anion levels, were assessed in human brain microvascular endothelial cells (HBMECs) exposed to oxygen-glucose deprivation (OGD) alone or OGD followed by reperfusion (OGD + R). The integrity of an in vitro model of BBB comprising HBMECs and astrocytes was studied by measuring transendothelial electrical resistance and the paracellular flux of albumin. OGD with or without reperfusion (OGD ± R) radically perturbed barrier function while concurrently enhancing uPA, tPA and NAD(P)H oxidase activities and superoxide anion release in HBMECs. Pharmacological inactivation of NAD(P)H oxidase attenuated OGD ± R-mediated BBB damage through modulation of matrix metalloproteinase-2 and tPA, but not uPA activity. Overactivation of NAD(P)H oxidase in HBMECs via cDNA electroporation of its p22-phox subunit confirmed the involvement of tPA in oxidase-mediated BBB disruption. Interestingly, blockade of uPA or uPA receptor preserved normal BBB function by neutralizing both NAD(P)H oxidase and matrix metalloproteinase-2 activities. Hence, selective targeting of uPA after ischaemic strokes may protect cerebral barrier integrity and function by concomitantly attenuating basement membrane degradation and oxidative stress.
缺血性损伤会损害血脑屏障（BBB）的完整性。在这项研究中，我们研究了 NAD(P)H 氧化酶、纤溶酶原-纤溶酶系统成分和基质金属蛋白酶之间可能存在的相关性，以探讨这种缺陷的分子原因。因此，评估了人类脑微血管内皮细胞（HBMEC）在单独缺氧/葡萄糖剥夺（OGD）或 OGD 后再灌注（OGD+R）暴露下的 NAD(P)H 氧化酶、基质金属蛋白酶-2、尿激酶型纤溶酶原激活物（uPA）和组织型纤溶酶原激活物（tPA）的活性以及超氧阴离子水平。通过测量跨内皮电阻和白蛋白的旁细胞通量，研究了包含 HBMEC 和星形胶质细胞的 BBB 体外模型的完整性。OGD 无论是否再灌注（OGD±R）都会严重破坏屏障功能，同时增强 HBMEC 中的 uPA、tPA 和 NAD(P)H 氧化酶活性以及超氧阴离子释放。通过 NAD(P)H 氧化酶的药理学失活，通过调节基质金属蛋白酶-2 和 tPA 而不是 uPA 活性，减轻了 OGD±R 介导的 BBB 损伤。通过 cDNA 电穿孔 p22-phox 亚基对 HBMEC 中 NAD(P)H 氧化酶的过度激活证实了 tPA 在氧化酶介导的 BBB 破坏中的作用。有趣的是，uPA 或 uPA 受体的阻断通过中和 NAD(P)H 氧化酶和基质金属蛋白酶-2 活性来维持正常的 BBB 功能。因此，缺血性中风后 uPA 的选择性靶向可能通过同时减弱基底膜降解和氧化应激来保护脑屏障的完整性和功能。
