Abstract：
We have previously demonstrated a rapid secretion of matrix metalloproteinase-2 (MMP-2) in the ischemic brain. Since Scube2 can interact with Sonic hedgehog (Shh) to maintain blood-brain barrier (BBB) integrity via regulating the interaction between brain capillary endothelial cells (ECs) and perivascular astrocytes, and it is also a substrate of MMP-2, we hypothesized that the secreted MMP-2 could degrade Scube2 and contribute to ischemic BBB disruption. Using an in vitro ischemic model of 90-min oxygen-glucose deprivation/3-h reoxygenation (OGD/R) and an in vivo mouse stroke model of 90-min middle cerebral artery occlusion (MCAO) with 3-h reperfusion, we established an important role of MMP-2-mediated Scube2 degradation in early ischemic BBB disruption. Exposure of C8-D1A cells and bEnd.3 cells to OGD/R increased MMP secretion in both cells, and C8-D1A cells appeared to secrete more MMPs than bEnd.3 cells. Co-IP and double-immunostaining revealed that Scube2 co-localized well with MMP-2 in C8-D1A cells and could be pulled down by MMP-2 antibodies. In MCAO mice, Scube2 protein showed a drastic reduction in ischemic brain tissue, which was accompanied by suppressed expression of Shh and its downstream molecules. Of note, specific knockdown of astrocytic Scube2 with AAV-shScube2 augmented MCAO-induced Shh suppression and exacerbated BBB leakage and inflammatory reactions in the ischemic brain. Last, incubation of bEnd.3 cells with conditioned medium derived from OGD-treated C8-D1A cells led to a significant inhibition of the Shh pathway in bEnd.3 cells and degradation of VE-cadherin and ZO-1. Inhibition of MMP-2 with SB-3CT or over-expression of Scube2 with plasmids in C8-D1A cells alleviated the above effect of C8-D1A cells-derived conditioned medium. Taken together, our data indicate that ischemia-induced secretion of MMP-2 may contribute to early BBB disruption in ischemic stroke via interrupting the shared Scube2-Shh pathway between brain capillary ECs and perivascular astrocytes.
摘要：
我们先前已经证明了缺血性脑中基质金属蛋白酶2（MMP-2）的快速分泌。由于Scube2可以与Sonichedgehog（Shh）相互作用，通过调节脑毛细血管内皮细胞（EC）和血管周围星形胶质细胞之间的相互作用来维持血脑屏障（BBB）的完整性，它也是MMP-2的底物，我们假设分泌的MMP-2可以降解Scube2并有助于缺血性BBB破坏。使用90分钟氧糖剥夺/3小时复氧（OGD/R）的体外缺血模型和90分钟大脑中动脉闭塞（MCAO）和3小时再灌注的体内小鼠中风模型，我们确立了MMP-2介导的Scube2降解在早期缺血性BBB破坏中的重要作用。C8-D1A细胞和bEnd.3细胞暴露于OGD/R会增加两种细胞的MMP分泌，C8-D1A细胞似乎比bEnd.3细胞分泌更多的MMP。Co-IP和双重免疫染色显示，Scube2与C8-D1A细胞中的MMP-2共定位良好，并且可以被MMP-2抗体拉低。在MCAO小鼠中,Scube2蛋白在缺血脑组织中显示出急剧下降，伴随着Shh及其下游分子的抑制表达。值得注意的是，用AAV-shScube2特异性敲除星形细胞Scube2可增强MCAO诱导的Shh抑制，并加剧缺血性脑中的BBB渗漏和炎症反应。最后,将bEnd.3细胞与源自OGD处理的C8-D1A细胞的条件培养基孵育导致bEnd.3细胞中Shh途径的显着抑制和VE-钙粘蛋白和ZO-1的降解。在C8-D1A细胞中用SB-3CT抑制MMP-2或用质粒过表达Scube2减轻了C8-D1A细胞来源的条件培养基的上述作用。一起来看,我们的数据表明,缺血诱导的MMP-2分泌可能通过中断脑毛细血管内皮细胞和血管周围星形胶质细胞之间共享的Scube2-Shh通路而导致缺血性卒中早期BBB破坏.
