The Effect And Mechanism Of GLA Deficiency On Vascular Endothelial VE-cadherin Expression

Background:Fabry disease is an X-linked genetic disorder caused by mutations in the alpha-galactosidase A(GLA)gene,resulting in a deficiency of this lysosomal enzyme.Insufficient enzyme activity causes extensive accumulation of the substrate sphingolipids(mainly globular ceramides)in tissues and body fluids,leading to progressive multi-organ dysfunction.Vascular endothelial cells are one of the major cell types in which the substrate globular ceramide accumulates.Vascular complications in patients with Fabry disease include stroke,cardiovascular disease and renal failure.This is the leading cause of death in patients.GLA deficiency or stimulation of the substrate globular ceramide can induce changes in the expression and secretion of a variety of endothelium-derived molecules.This leads to a vascular pro-inflammatory state,hypercoagulable response and diastolic dysfunction.The vascular endothelium plays an important role in vascular permeability and maintenance of vascular homeostasis by acting as a semi-permeable barrier between blood and interstitial tissue.Impaired endothelial barrier function leads to vascular hyperpermeability,which promotes inflammatory responses and disease development and progression.However,little is known about the endothelial barrier function in Fabry disease.VE-cadherin is the major adhesion-linkage protein.Disruption of adherens junctions leads to vascular barrier dysfunction,which results in increased endothelial permeability.In this study,the mechanism of action of GLA deficiency on VE-cadherin expression in vascular endothelial cells was investigated by constructing an endothelial cell model with GLA knockdown.Objective:Explore the effect of GLA knockdown on vascular endothelial permeability.To identify the changes in expression of VE-cadherin,an adhesion-linked protein,in vascular endothelial cells during GLA knockdown and its regulatory mechanism.Methods:1.Investigating the effect of GLA knockdown on endothelial cell permeabilityTo investigate the effect of GLA knockdown on endothelial cell permeability,HUVECs were transfected with GLA siRNA for 24 h and the cells were inoculated in 24-well Transwell chambers.After the cells grew into a monolayer of endothelial cells,FITC-dextran was added to assess endothelial cell permeability at four time points of 1,2,3 and 4 h.2.Investigating the effect of GLA knockdown on vascular endothelial adhesion-linker protein VE-cadherin expressionRNA and proteins from GLA siRNA transfected HUVECs for 48 h were extracted for qRTPCR and Western blot to detect the expression level of VE-cadherin.The expression levels of VE-cadherin between endothelial cells were detected by cellular immunofluorescence staining.3.Investigating the effect of GLA knockdown on vascular endothelial transcription factor ERG expressionHUVECs were transfected with GLA siRNA for 48 h.Cellular RNA and proteins were collected,and the expression levels of transcription factor ERG were detected by qRT-PCR and Western blot assay.The expression level of ERG in endothelial cells was detected by cellular immunofluorescence staining.4.Validation of the regulatory effect of transcription factor ERG on VE-cadherin expressionERG overexpression plasmid and ERG siRNA were constructed and transfected into HUVECs for 48 h.After upregulating and knocking down the expression levels of ERG,the expression levels of ERG and VE-cadherin were detected using qRT-PCR and Western blot techniques.5.Validation of the role of transcription factor ERG on VE-cadherin expression in GLA knockdown endothelial cellsTo further verify the effect of transcription factor ERG on VE-cadherin expression in GLA knockdown endothelial cells.In this experiment,GLA siRNA was transfected in HUVECs transfected with ERG plasmid.The protein expression levels of ERG and VE-cadherin were detected by Western blot.GLA si-RNA-transfected HUVECs were collected for protein-DNA cross-linking.Chromatin fragments of appropriate length were obtained by cell lysis and ultrasonic fragmentation.The binding of transcription factor ERG to the-134/-118ETS site on VE-cadherin promoter and its changes were detected by chromatin immunoprecipitation assay(ChIP),Results:1.HUVECs transfected with GLA siRNA for 24 h were inoculated in 24-well transwell chambers.The fluorescence intensity of the lower chamber culture was measured after the addition of FITC-dextran.The results showed increased permeability of HUVECs at 1,2,3,and 4 h compared to the control group.2.GLA siRNA was transfected into HUVECs for 48 h.Both mRNA and protein levels of VE-cadherin were reduced compared to the control group.Immunofluorescence showed reduced intensity of VE-cadherin staining.3.GLA siRNA was transfected into HUVECs for 48 h.Both mRNA and protein levels of ERG were reduced compared to the control group.Immunofluorescence showed reduced intensity of ERG staining.4.ERG overexpression plasmid was transfected into HUVECs for 48 h after transfection.Both mRNA and protein levels of VE-cadherin were elevated compared to the control.Conversely,ERG siRNA was transfected into HUVECs for 48 h after transfection.The mRNA and protein levels of VE-cadherin were decreased compared to the control group.5.ERG overexpression plasmid was transfected into HUVECs for 36 h followed by GLA siRNA transfection into HUVECs for 36 h.The results showed that the down-regulation of VEcadherin by GLA knockdown was lost by ERG overexpression.ChIP results showed that ERG could bind to the-134/-118ETS site on the VE-cadherin promoter.after GLA knockdown,the binding of ERG to the-134/-118ETS site on the VEcadherin promoter was significantly reduced.Conclusions:1.GLA knockdown increased vascular endothelial cell permeability.2.GLA knockdown inhibited the expression level of VE-cadherin in vascular endothelial cells.3.GLA knockdown decreased the expression level of transcription factor ERG in vascular endothelial cells.4.The transcription factor ERG positively regulates VE-cadherin expression in vascular endothelial cells.5.GLA knockdown leads to downregulation of VE-cadherin expression by reducing the binding of transcription factor ERG to VE-cadherin promoter.