TPA Disrupts The Bbb Through Increasing The Inflammatory Response Mediated By Pericytes After Cerebral Ischemia

Objective: Recombinant tissue plasminogen activator(rt-PA)is an important drug for the treatment of patients with ischemic stroke,but recent studies had found that rt-PA could damage the blood brain barrier(BBB)by inducing inflammatory response.As an important component of neurovascular unit(NVU),pericytes play an important role in maintaining the integrity of the BBB,regulating the angiogenesis,regulating the inflammation and phagocytosis.At present,there is no relevant report on whether rtPA can damage BBB by affecting pericytes.The purpose of this study is to investigate whether rt-PA disrupts the BBB by increasing the inflammatory response induced by pericytes and the relevant mechanism.Method: 1.The construction of vitro BBB model: the mouse microvascular endothelial cells and the mouse microvascular pericytes were cocultured to construct the vitro BBB model,and the success of the construction of vitro BBB model was determined by measuring the TEER.2.The construction of cerebral ischemia/reperfusion model: in vivo the construction of MCAO model: the mice were inserted the monofilament through the left external carotid artery into the left middle cerebral artery of the mouse to block blood flow for 1h to construct the vivo ischemia/reperfusion model;In vitro the construction of OGD/R model: the pericytes were treated with hypoxia for 4h,after that,the pericytes were re-oxygenated for 12 h or 24 h under normal culture conditions.3.The test of the BBB function: in vivo,the water content of the brain tissue was measured.The ultrastructure of BBB was observed by transmission electron microscope and the FITC-dextran leakage was determined by immunofluorescence;In vitro BBB model,the TEER and the permeability of the FITC-dextran were determined.4.The determination of pericytes proliferation and survival rate: in vivo,immunofluorescence was used to measure the expression of ki67 on pericytes;In vitro experiment,MTT was used to measure the survival rate of pericytes.5.The determination of the inflammatory factor tumor necrosis factor-α(TNF-α),monocyte chemoattractant protein 1(MCP-1)expression level: in vivo,the expression of TNF-α,MCP-1 were determined by immunofluorescence;In vitro,the expression of TNF-α,MCP-1m RNA were determined by real-time quantitative PCR and the expression of TNF-α,MCP-1 were determined by enzyme-linked immunosorbent assay.6.Determination of the signaling pathway proteins platelet-derived growth factor receptor-b(PDGFR-b),transforming growth factor-b(TGF-b),p-Smad2/3: in vivo,the expression of PDGFR-b,TGF-b were determined by immunofluorescence;In vitro the expression of PDGFR-b,TGF-b,p-Smad2/3 were determined by western blot.7.The construction of PDGFR-b overexpression pericyte model: the e-PDGFR-b was transfected into pericytes to construct PDGFR-b overexpression model,and the transfection efficiency was determined by western blot.Results: 1.After cerebral ischemia,the administration of rt-PA increased the water content in the infarcted brain(P<0.05).Transmission electron microscopy also showed that the microstructure of the BBB in infarcted brain tissue was further disrupted after rt-PA treatment;The TEER decreased and the permeability increased of the vitro BBB model treated with rt-PA after OGD(P<0.05).2.The results of immunofluorescence and western blot showed that the expression of PDGFR-b,TGF-b,p-Smad2/3 on the pericytes were decreased treated with rt-PA after cerebral ischemia(P<0.05).3.The results of immunofluorescence showed that the expression of TNF-α and MCP-1 were increased treated with rt-PA after cerebral ischemia(P<0.05);Immunofluorescence also showed that TGF-b combined with rt-PA decreased the expression of TNF-α and MCP-1 compared with rt-PA treatment(P<0.05);ELISA showed that rt-PA treatment increased the expression of TNF-α,MCP-1 in pericytes after OGD/R(P < 0.05);ELISA also showed that TGF-b combined with rt-PA decreased the expression of TNF-α and MCP-1 compared with rt-PA treatment(P<0.05).4.Immunofluorescence results showed that etanercept(TNF-αinhibitor)combined with rt-PA reduced the leakage of FITC-dextran caused by rt-PA treatment(P < 0.05).5.ELISA results showed that rt-PA treatment did not decrease TNF-α and MCP-1 expression in PDGFR-b overexpression pericytes,but TPO427788 HCl administration reversed this effect(P < 0.05).6.The results also showed that rt-PA did not decrease the TEER of the PDGFR-b overexpressed mouse pericytes and endothelial cells coculture model,but TPO427788 HCl administration reversed the protective effect(P < 0.05).Conclusion: This study confirmed that rt-PA disrupt the BBB by inhibiting PDGFR-b-TGF-b-pSmad2/3 pathway and increasing the expression of TNF-α and MCP-1 on pericytes after cerebral ischemia.These results suggest that PDGFR-b-TGF-b-p-Smad2/3 pathway may provide a new therapeutic target for BBB injury caused by rt-PA treatment and provide the therapeutic drugs for rt-PA combined therapy.