Endothelial CYP2J2 Overexpression Restores The Inner Blood-Retinal Barrier Via ANXA1 Upregulation

Objective: The study aims to explore the role of endothelial CYP2J2 overexpression in rescuing the inner blood-retinal barrier after retinal ischemia-reperfusion(IR)injury.Another objective is to illuminate the effect of CYP2J2 overexpression on endothelial ANXA1 expression and the underlying regulatory mechanisms.Moreover,the essential role of endothelial ANXA1 in development of the murine retinal vasculature will also be clarified in this study.Methods: Retinal vascular barrier function and endothelial ANXA1 expression were examined after retinal IR in endothelial CYP2J2 overexpression rats and wildtype rats.In endothelial cells challenged with oxygen-glucose deprivation/reoxygenation(OGDR),the effect of CYP2J2 overexpression on endothelial permeability and endothelial junctions was investigated,accompanied by detecting the effect of CYP2J2 on ANXA1 expression.To determine the role of endothelial ANXA1 in development of the retinal vasculature,vascular formation and barrier function were examined in endothelial ANXA1 deletion(Anxa1ΔEC)mice in comparison with control mice.Furthermore,CYP2J2 metabolites EETs were administrated intravenously in Anxa1ΔEC mice and control mice to discover the role of ANXA1 in EETs-mediated protection of retinal vascular barrier.Results: Endothelial CYP2J2 overexpression ameliorated retinal vascular permeability and protected retinal ganglion cells(RGC)after IR injury.CYP2J2 overexpression mitigated oxidative stress in OGDR-treated endothelial cells and improved endothelial barrier and junctional organization.CYP2J2 elevated endothelial METTL3 levels,and accordingly promoted ANXA1 expression in a m~6A-dependent manner.Endothelial deletion of ANXA1 destroyed vascular stability and permeability during development of the retinal vascular without the effect on retinal vascularization.CYP2J2 metabolites EETs restored the inner blood-retinal barrier in an ANXA1-dependent manner.Intrinsically,EETs improved METTL3-mediated ANXA1 m6 A modification and ANXA1 expression.Conclusions: Endothelial CYP2J2 overexpression protects the inner blood-retinal barrier against retinal IR injury via ANXA1 upregulation.Endothelial ANXA1 plays a key role in maintaining retinal vascular stability and permeability during development.Part Ⅰ: Endothelial CYP2J2 Overexpression Restored the Integrity of the Inner Blood-Retinal Barrier via ANXA1 UpregulationObjective: The study aims to explore the effect and regulatory mechanisms of endothelial CYP2J2 overexpression on restoring the inner blood-retinal barrier after retinal IR.Methods: After retinal IR injury,the retinal vascular permeability,RGC survival and endothelial ANXA1 expression were detected in endothelial CYP2J2 overexpression rats and wildtype rats.Endothelial cells were transfected with adenovirus carrying human CYP2J2 gene,followed by analysis of endothelial tight junctions,adherens junctions and endothelial permeability in OGDR-treated CYP2J2 overexpression cells and control cells.Moreover,the effect of CYP2J2 overexpression on ANXA1 transcripts and protein levels was also examined.To explore the role of ANXA1 in CYP2J2-mediated protection of endothelial barrier,endothelial junctions and permeability were evaluated following ANXA1 inhibition by adenovirus transfection.Results: Endothelial CYP2J2 overexpression improved the inner blood-retinal barrier and RGC survival after IR injury.ANXA1 expression decreased in retinal vascular endothelial cells after retinal IR,whereas an increase of ANXA1 expression was detected in endothelial CYP2J2 overexpression rats than wildtype rats.In vitro experiments showed that CYP2J2 elevated ANXA1 protein levels in OGDR-treated endothelial cells without affecting the abundance of ANXA1 m RNA.CYP2J2 overexpression suppressed oxidative stress in endothelial cells,and then restored endothelial junctions and barrier function,which was counteracted by endothelial inhibition of ANXA1.Conclusions: Endothelial CYP2J2 overexpression counteracts the inhibition of endothelial ANXA1 expression induced by IR injury,and accordingly maintains the inner blood-retinal barrier.Part Ⅱ: Endothelial CYP2J2 Overexpression Regulates ANXA1 Expression via METTL3-Mediated m6 A ModificationObjective: The study aims to clarify how CYP2J2 regulates ANXA1 expression in endothelial cells.Methods: METTL3 was recognized as a mediator between CYP2J2 and ANXA1 through protein-protein interaction analysis.Endothelial cells were transfected with METTL3 to explore the effect of METTL3 overexpression on ANXA1 levels and ANXA1 m6 A abundance.Subsequently,METTL3 levels were examined in CYP2J2-overexpressed endothelial cells to explore the regulation of METTL3 expression by CYP2J2.In oder to find out whether CYP2J2 regulated ANXA1 expression dependent on ANXA1 m6 A modification,ANXA1 proteins levels in response to CYP2J2 overexpression were examined in HEK293 T cells transfected with exogenous ANXA1 with or without the mutation of ANXA1 m6 A sites.Next,the investigation focused on the role of METTL3 in CYP2J2-mediated ANXA1 upregulation and endothelial barrier restoration.METTL3 expression was inhibited in CYP2J2-overexpressed endothelial cells followed by detecting ANXA1 levels and endothelial permeability.Results: In endothelial cells challenged with OGDR or oxidative stress,ANXA1 protein levels and ANXA1 m6 A abundance increased but ANXA1 m RNA levels remained unchanged in response to METTL3 transfection.Moreover,CYP2J2 promoted METTL3 expression as well as ANXA1 m6 A abundance,and thereby elevated ANXA1 protein levels.CYP2J2 overexpression enhanced exogenous ANXA1 expression,however following the mutation of ANXA1 m6 A sites,CYP2J2 overexpression had no effect on ANXA1 protein levels,which indicated that CYP2J2 regulated ANXA1 expression in an m~6A-dependent manner.Inhibition of METTL3 expression in CYP2J2-overexpressed endothelial cells abrogated the improvememt of ANXA1 levels and endothelial barrier.Conclusions: Endothelial CYP2J2 overexpression upregulates METTL3 levels,and thereby promotes ANXA1 expression in an m~6A-dependent manner.Part Ⅲ: Endothelial Deletion of ANXA1 Disrupts Retinal Vascular Stability and PermeabilityObjective: The study aims to illuminate the role of ANXA1 in development of the retinal vasculature.Methods: Following the establishment of endothelial ANXA1 knockout model,neonatal Anxa1ΔEC mice and control littermates were acquired to analyse the differences in the formation of retinal vasculature as well as retinal vascular stability and permeability.OCTA was performed on adult Anxa1ΔEC and control mice to assess the retinal blood flow and vascular density.Meanwhile,retinal vascular permeability and endothelial junctions were detected by immunofluorescence to determine the effect of endothelial ANXA1 deletion on the inner blood-retinal barrier.Results: There were no differences in retinal vascular radial growth,vascular density,the number of branching points and vascular sprouting between Anxa1ΔEC and control mice at postnatal day(P)5.Otherwise,P5 Anxa1ΔEC mice showed decreased diameters of retinal pre-capillary arterioles and post-capillary venules,as well as vascular instability.At P12,declined vascular density was detected along with interrupted endothelial VE-cadherin arrangement and vascular leakage in Anxa1ΔEC retinas.OCTA reported that the absence of ANXA1 inhibited retinal blood flow and vascular density.Compared with the controls,adult Anxa1ΔEC mice exhibited retinal vascular hyperpermeability and endothelial junctional disorganization,which suggested the dysfunction of the inner blood-retinal barrier.Conclusions: Endothelial ANXA1 deletion disrupts retinal blood flow,vascular stability and vascular barrier without the effect on the formation of retinal vasculature.Part Ⅳ: CYP2J2 Metabolites EETs Maintained Endothelial Barrier Via ANXA1 UpregulationObjective: The study aims to explore the protective effect of CYP2J2 metabolites EETs on endothelial permeability and the underlying regulatory mechanisms.Methods:(±)14(15)-EET-SI was administrated intravenously in Anxa1ΔEC and control mice suffered from retinal IR injury,followed by detecting retinal vascular permeability and endothelial VE-cadherin expression.Moreover,OGDR-stressed endothelial cells were treated with(±)14(15)-EET-SI to assess its effect on endothelial barrier function,METTL3 and ANXA1 protein levels and ANXA1 m6 A abundance.Subsequently,an EETs antagonist was added to CYP2J2-overexpressed endothelial cells in order to explore whether CYP2J2 improved METTL3 and ANXA1 expression as well as endothelial barrier function in an EETs-dependent manner.Results:(±)14(15)-EET-SI maintained retinal vascular barrier and promoted VE-cadherin expression in retinal IR model,which was counteracted by endothelial ANXA1 deletion.(±)14(15)-EET-SI protected against endothelial hyperpermeability induced by OGDR treatment,and also elevated METTL3 and ANXA1 protein levels and ANXA1 m6 A abundance.Furthermore,treatment of the EETs antagonist reversed the effect of endothelial CYP2J2 overexpression on METTL3 and ANXA1 expression as well as endothelial permeability.Conclusions: EETs restore retinal vascular barrier dependent on endothelial ANXA1 expression.EETs upregulate ANXA1 levels through METTL3-mediated m6 A modification,and thereby protect endothelial permeability.