MMP‐2‐mediated Scube2 degradation promotes blood–brain barrier disruption by blocking the interaction between astrocytes and endothelial cells via inhibiting Sonic hedgehog pathway during early cerebral ischemia

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.