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.

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.

Activation of Swell1 in microglia suppresses neuroinflammation and reduces brain damage in ischemic stroke.

Cl- movement and Cl--sensitive signal pathways contributes to the survival and switch of inflammatory phenotype of microglia and are believed to play a key role in the inflammatory brain injury after ischemic stroke. Here, we demonstrated an important role of Cl- transmembrane transporter Swell1, in the survival and M2-like polarization of microglia in ischemic stroke. Knockdown or overexpression of Swell1 in cultured microglia inhibited or increased hypotonic-activated Cl- currents, respectively, and these changes were completely blocked by the volume-regulated anion channels (VRACs) inhibitor DCPIB. Swell1 conditional knock-in mice promoted microglia survival in ischemic brain region and resulted in significant reductions in neural cell death, infarction volume and neurological deficits following transient middle cerebral artery occlusion (tMCAO). Using gene manipulating technique and pharmacological inhibitors, we further revealed that Swell1 opening led to SGK1 (a Cl--sensitive kinase)-mediated activation of FOXO3a/CREB as well as WNK1 (another Cl--sensitive kinase)-mediated SPAK/OSR1-CCCs activation, which promoted microglia survival and M2-like polarization, thereby attenuating neuroinflammation and ischemic brain injury. Taken together, our results demonstrated that Swell1 is an essential component of microglia VRACs and its activation protects against ischemic brain injury through promoting microglia survival and M2-like polarization.

A Linarin Derivative Protects against Ischemia-Induced Neuronal Injury in Mice by Promoting Cerebral Blood Flow Recovery via KDELR-Dependent CSPG4 Activation.

The cerebral ischemic microvascular response and collateral circulation compensatory capacity are important for the outcome of ischemic stroke. Here, we sought to evaluate the effect of a linarin derivate 4'-benzylapigenin-7-ß-rutinoside (BLR) on neurological function and cerebral blood flow restoration in ischemic stroke. A mouse model of middle cerebral artery occlusion (30 min) with reperfusion (24 h) was used to mimic ischemic stroke in vivo, and 2,3,5-triphenyltetrazolium chloride (TTC) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, and immunofluorescence microscopy were used to assess the protective effects of BLR on infarct volume, neurological function, neuronal apoptosis, and inflammatory damage. Cerebral blood flow was assayed by laser speckle contrast imaging. Double immunostaining of GFAP-collagen IV and brain lucidification were performed to determine the protective effects of BLR on the disruption of brain vasculature. Differential gene expression was assessed by RNA sequencing. Coimmunoprecipitation and western blotting were used to explore the mechanism of BLR-induced neuroprotection. The results of in vivo experiments showed that BLR administration after reperfusion onset reduced infarct volume, improved neurological function, and decreased the neural cell apoptosis and inflammatory response in the ischemic brain, which was accompanied by increased cerebral blood flow and reduced detachment of astrocyte endfeet from the capillary basement membrane. The RNA sequencing data showed that BLR promoted the upregulation of extracellular matrix and angiogenesis pathway-related genes; in particular, BLR significantly increased the expression of the chondroitin sulfate proteoglycan 4 (CSPG4) gene, enhanced the membrane location of CSPG4, and promoted its downstream signaling protein expression, which is associated with KDEL receptor (KDELR) activation. In addition, activated KDELR further increased the phosphorylation of mitogen-activated protein kinases after BLR treatment. Taken together, our data showed that BLR could protect against ischemic brain injury and may serve as a new promising therapeutic candidate drug for ischemic stroke, and that KDELR might act as both a sensor and effector to activate CSPG4 to increase cerebral blood flow.