Overexpression Of ?5?1 Integrin And Angiopoietin-1 Co-operatively Promote Blood-brain Barrier Integrity And Angiogenesis Following Ischemic Stroke

Objective We previously demonstrated that cross-talk between ?5?1 integrin and the angiopoietin-1(Ang1)/ Tie2 receptor plays an important role in regulating brain endothelial angiogenic responses in the ischemic penumbra following cerebral ischemic stroke(CIS).However,a recent study suggested that stimulation of the ?5?1 integrin also has the potential of increasing blood-brain barrier(BBB)permeability after CIS,raising doubt about whether ?5?1 integrin stimulation by itself will protect against ischemic injury.In light of these conflicting roles,the goal of this study was to evaluate the impact of co-overexpression of ?5 integrin and Ang1 on vascular remodeling and repair under cerebral ischemic conditions.Methods In vivo: AAV-control,AAV-Itg?5,AAV-Angl and AAV-Itg?5/Ang1 were injected into tail vein of male C57 Bl / 6 mice of 4-6 weeks old respectively to enhance the expression of corresponding genes,and to construct ischemia-reperfusion model.BBB injury was assessed by neurobehavioral tests,cerebral infarction volume assessment and Evans blue(EB)extravasation,the expression of related proteins detected by Immunofluorescence and Western blot.In vitro: BECs was transfected to overexpress ?5,Ang1 and both,then constructed an OGD/R model,Western blot and brain endothelial cell proliferation assay were used to detect the expression changes of related indicators at different times.Results Our results demonstrate that as compared to mock-transfected controls,overexpression of ?5 integrin alone didn't improve the outcomes in neurological score and size of infarct and caused worse BBB breakdown in the ischemic hemisphere,offsetting its beneficial angiogenic effects during the early stages of CIS.However,co-overexpression of ?5 integrin with Ang1 led to smaller infarcts and improved neurological deficits,which at the molecular level was underpinned by reduced BBB breakdown and increased expression of endothelial tight junction proteins in the ischemic penumbra during the early stages of CIS.Furthermore,co-overexpression of ?5 integrin and Ang1 synergistically promoted BEC proliferation during the early stage of CIS,resulting in increased blood vessel density at later stages.Positive effects of ?5 integrin and Ang1 co-overexpression on endothelial proliferation and tight junction protein expression were also confirmed in vitro.Conclusion Collectively,these data indicate that co-overexpression of Ang-1 and ?5 integrin in combination confers synergistic vascular protection against cerebral ischemic injury without the negative side effects on BBB permeability,suggesting a novel combinatorial approach for the treatment of CIS.