CRHR1 Mediates The S1PR2 Regulation On Brain Endothelial Permeability Under Oxdative Stress And The Underlying Mechanisms

Within the neurovascular unit?NVU?,the blood-brain barrier?BBB?is a highly specialized brain endothelial structure,which is essential for proper brain functions.The endothelium is responsible for reactive oxygen species?ROS?generation which occurs at the site of the BBB.Although the production increases in distinct dynamic patterns after permanent and reversible middle cerebral artery occlusion?MCAO?,ROS mediates oxidative stress which impairs endothelial barrier integrity and increases endothelial permeability.Activation of mitogen-activated protein kinase?MAPK?,including p38 and extracellular signal-regulated kinase?Erk?1/2,is implicated in ischemia-reperfusion?I/R?damage after transient MCAO?t MCAO?.The phosphorylation of cytosolic phospholipase A2?cPLA₂?by MAPK on Serine-505 increases its catalytic activity,leading to arachidonic acid?AA?release,which is involved in cerebral ischemia-induced oxidative injury and cerebrovascular permeability change.Sphingosine-1-phosphate?S1P?produced in vascular endothelium is a potent regulator of endothelial barrier integrity.And the primary S1 P receptors expressed in endothelial cells are sphingosine-1-phosphate receptor?S1PR?1,S1PR2 and S1PR3.In contrast to the protective effects of S1PR1 and S1PR3 activation on vascular integrity,S1PR2 inhibition reduces vascular permeability.More importantly,S1PR2 plays a critical role in the induction of cerebrovascular hyperpermeability after I/R injury and matrix metalloproteinase?MMP?-9 activation in brain endothelial cells.Corticotrophin-releasing hormone?CRH?and its family of related peptides play critical roles in regulating physiologic responses to multiple stressors,including stroke.Corticotrophin-releasing hormone receptor?CRHR?1 is a high-affinity G-protein coupled receptor for CRH.The antagonists of CRHR1 decrease infarction volume and brain swelling after permanent focal ischemia in rats.And CRHR1 deficient mice show reduced cerebral injury after focal ischemia,indicating that CRHR1 is the primary mediator of ischemic injury in stroke.CRH receptor protein has been shown to be expressed on brain microvessel endothelial cells isolated from rat and bovine brain,and stimulation with CRH increased intracellular cyclic adenosine monophosphate?c AMP?levels.These findings suggest that CRH could affect the structure and function of brain microvessel endothelial cells,regulating BBB permeability.Thus,CRHR1 may be involved in the modulation of cerebral endothelial barrier integrity.The aim of this study is to identify the regulation of cerebral endothelial permeability by S1PR2 and CRHR1 and their association under oxidative stress,and also to address the underlying mechanisms.Chapter 1 S1PR2 regulates brain endothelial permeability under oxdative stressBoth S1PR2 and cPLA₂ are implicated in the disruption of cerebrovascular integrity in experimental stroke.However,the role of S1PR2 in induction of cPLA₂ phosphorylation during cerebral ischemia-induced endothelial dysfunction remains unknown.The aim of this study was to investigate the role of S1PR2 in regulating oxidative stress-induced cerebrovascular endothelial barrier impairment and to explore the possible molecular mechanisms.Thus,to mimic the effect of oxidative stress on cerebrovascular endothelium in vitro and in vivo respectively,we exposed the b End3 cells to hydrogen peroxide?H2O2?and performed the permanent MCAO?p MCAO?mouse model.And pharmacological inhibition and ribonucleic acid?RNA?interference were used to assess the oxidative stress-induced endothelial permeability change and underlying mechanisms.In b End3 cells,cPLA₂ inhibitor CAY10502 as well as S1PR2 antagonist JTE013 profoundly suppressed H2O2-induced changes of paracellular permeability and ZO-1 localization.Both Erk1/2 and p38 are required for H2O2-increased cPLA₂ phosphorylation and endothelial permeability.Pharmacological and genetic inhibition of S1PR2 significantly suppressed their phosphorylation in response to H2O2.Using the p MCAO mouse model,we found JTE013 pretreatment markedly reduced Evans blue dye?EBD?extravasation and reversed the decrease in vascular endothelial-cadherin?VE-cadherin?,occludin,claudin-5 and CD31 expression in infarcted hemisphere.Furthermore,JTE013 pretreatment significantly attenuated neurological deficit,brain edema and infarction volume.Therefore,our findings suggest S1PR2 activation contributes to brain endothelial hyperpermeability via p38 and Erk1/2-dependent cPLA₂ phosphorylation under oxidative stress.Chapter 2 CRHR1 modulates brain endothelial permeability under oxdative stressCerebral ischemia-induced oxidative damage is involved in the BBB disruption.The blockade as well as gene knockout of CRHR1 confer neuroprotective effects against focal cerebral ischemia in rodents.The aim of this study was to explore the role of CRHR1 in regulating oxidative stress-induced cerebrovascular endothelial barrier disruption and underlying mechanisms.To mimic the effect of oxidative stress on cerebrovascular endothelium in vitro and in vivo,we exposed the b End3 cells to H2O2 and performed the t MCAO mouse model,respectively.And pharmacological blockade and RNA interference were used to assess the regulation of oxidative stress-induced endothelial permeability change by CRHR1 and underlying mechanisms.Here we show pharmacological antagonist as well as gene knockdown of CRHR1 profoundly inhibited H2O2-induced p38,Erk1/2 and cPLA₂ phosphorylation,and also suppressed H2O2-induced paracellular hyperpermeability and ZO-1 redistribution in b End3 cells.Using the t MCAO mouse model,we found CRHR1 antagonist NBI27914 pretreatment markedly reduced EBD extravasation and reversed the decrease in interendothelial junction protein expression in infarcted hemisphere.Further,NBI27914 pretreatment significantly decreased brain edema,infarction volume and neurological deficits after stroke.In hippocampus,CRHR1 knockdown attenuated EBD extravasation after 30 min MCAO with 7 days reperfusion.These findings demonstrate that CRHR1 activation may increase brain endothelial permeability via p38 and Erk1/2-dependent cPLA₂ phosphorylation under oxidative stress.Chapter3 CRHR1 is crucial for the regulation of brain endothelial permeability by S1PR2 under oxdative stressBoth S1PR2 and CRHR1 belong to the family of G-protein coupled receptors on the surface of endothelial cells,but the relationship between these two receptors during oxidative stress-induced cerebrovascular endothelial barrier impairment is still elusive.The aim of this study was to investigate the role of CRHR1 in the regulation of oxidative stress-induced cerebrovascular endothelial barrier impairment by S1PR2 and to explore the possible molecular mechanisms.Thus,to mimic the oxidative stress effect on cerebrovascular endothelium in vitro and in vivo respectively,we exposed the b End3 cells to H2O2 and performed the p MCAO mouse model.And pharmacological antagonist and agonist were used to address the oxidative stress-induced endothelial permeability change and underlying mechanisms.Here we report that CRH reversed S1PR2 antagoinst JTE013-induced decrease in H2O2-induced endothelial hyperpermeability and cPLA₂ phosphorylation in b End3 cells.After p MCAO in mice,CRH treatment counteracted JTE013-induced decrease in EBD extravasation in brains.Furthermore,CRH treatment counteracted JTE013-induced decreases in edema injury,infarct size,and neurological deficit after stroke.These data support a model in which CRHR1 mediates the regulation of brain endothelial permeability by S1PR2 under oxdative stress.