Evidence that enolase-phosphatase 1 exacerbates early cerebral ischemia injury and blood–brain barrier breakdown by enhancing extracellular matrix destruction and inhibiting the interaction between ADI1 and MT1-MMP

Enolase-phosphatase 1 (ENOPH1) is a newly identified enzyme associated with stress responses and cell proliferation. Our previous study found that ENOPH1 mediates cerebral microvascular endothelial cell apoptosis under cerebral ischemia conditions. In this study, we systematically provide mechanistic insights into the regulation of ENOPH1 in blood-brain barrier (BBB) dysfuction induced by early ischemia. ENOPH1 knockout mice (ENOPH1 KO) and wild type (WT) mice were exposed to transient middle cerebral artery occlusion (tMCAO) for 90 min followed by 3 h of reperfusion in vivo, and brain microvascular endothelial cell lines (bEnd.3 cells) were exposed to oxygen-glucose deprivation (OGD) in vitro. BEnd.3 cells were transfected with ENOPH1 shRNA to knockdown ENOPH1 expression. Brain ischemic damage and nerve function was assessed with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and neurological scores. BBB permeability and tight junction (TJ) protein and adherens junction (AJ) proteins expression were analyzed by FITC-dextran staining, western blotting and coimmunofluorescence. The MMP-2/9 activity was analyzed by gelatin zymography. Differential protein expression was assessed by quantitative proteomics. The interaction between ADI1 and MT1-MMP was measured by coimmunoprecipitation assay and coimmunofluorescence. Knockout of ENOPH1 ameliorated cerebral ischemic injury, decreased BBB permeability, inhibited the activity of MMP-2/9, upregulated the expression of TJ/AJ proteins and reversed extracellular matrix destruction after ischemia in vivo. Mechanistic studies have shown that ENOPH1 silencing enhanced the interaction between ADI1 and MT1-MMP by promoting the nuclear translocation of ADI1 to inhibit MT1-MMP in bEnd.3 cells after OGD and decreasing the expression of Tnc and Fn1 to inhibit ECM degradation. Our results reveal that ENOPH1 increases the activity of MMP-2/9, then promotes TJ protein and extracellular matrix degradation, and eventually destroys the stability of the BBB. Therefore, ENOPH1 is a new therapeutic target for ischemic stroke.