The Protective Role Of TL1A In Regulating Blood-retinal Barrier Function In Nonproliferative Diabetic Retinopathy

Purpose To investigate the role of TL1 A in retinal vascular permeability during development of NPDR and to understand the underlying mechanisms.Methods In vitro:(1)Human retinal microvascular endothelial cells(HRMEC)and immortalized rat retinal microvascular endothelial cells(i RRMEC)were used to test the effect of high glucose(HG)on endothelial cell permeability.Moreover,the phosphorylation of VE-cadherin in response to HG were detected by western blot to evaluate the integrity of the endothelial cell monolayer.The expression of TL1 A in endothelial cells treated with HG were measured by RT-q PCR and Western blot.(2)Motif analysis was performed to identify the transcription factor binding sites in TL1 A promoter region.The transcriptional regulation of TL1 A by FOXP1 was further validated by Ch IP-q PCR,luciferase assay,and small interfering RNA(si RNA)knockdown experiments.(3)TL1A recombinant protein intervention was used to identify the protective effect of TL1 A on endothelial junctions.Hopping probe ion conductance microscopy(HPICM)was performed to detect endothelial cell junction integrity in vitro.(4)Co-immunoprecipitation was used to examine the interaction of death receptor 3(DR3)with SHP-1 and subsequent activation of its downstream signaling after TL1 A treatment.In vivo:(1)Diabetic retinal vascular permeability was evaluated by Evens blue staining and HE.The expression of TL1 A in rat retina was measured by western blot.(2)STZ diabetic and control rats(Wistar,8 weeks old)were given intravitreal injection of different doses of Ig G or TL1 A,then the integrity of blood-retinal barrier(BRB)was assessed by Fluorescence angiography(FA)combining indocyanine green angiography(ICGA),Miles assay,Immunohistochemistry(IHC)and Evens blue staining.Results In vitro:(1)HG treatment for 2 hours impaired the integrity of endothelial cell monolayer,induced phosphorylation of VE-cadherin at Tyr685,and deceased the expression of TL1 A in retinal endothelial cells(P<0.05).(2)Luciferase assay showed that transcriptional factor FOXP1 could negatively regulate the expression of TL1A(P<0.05).(3)Administration of 250ng/m L TL1 A showed protective effect on endothelial junction formation and endothelial cell permeability in vitro(P<0.05).Overexpression of TL1 A in i RRMEC showed similar results.(4)Mechanistic studies showed that TL1 A binding with DR3 inhibited Src phosphorylation,which in turn decreased VE-cadherin phosphorylation and protected the integrity of endothelial tight junctions.In vivo:(1)In STZ diabetic rats,TL1 A level in the retina was decreased at 2 weeks of DM onset,but was recovered afterwards.Intravitreal injection of 0.1ng TL1 A improved retinal vascular permeability in DR models without obvious side effects.Conclusions Hyperglycemia could reduce the level of TL1 A in endothelial cells,which may contribute to BRB function disruption in the early stage of DR.Early intervention by applying low dose of TL1 A could improve diabetic retinal vascular permeability.Altogether,these findings may lead to new therapeutic strategies for treating NPDR.