Repressive Effect And Mechanism Of Transthyretin In Neovascularization Of Diabetic Retinopathy

Objective The apoptosis of human umbilical vein endothelial cells has been reportedly induced by the protein transthyretin(TTR).In human ocular tissue,TTR is generally considered to be secreted mainly by retinal pigment epithelial cells(hRPECs);However,whether TTR affects the development of neovascularization in diabetic retinopathy(DR)remains unclear.This paper is to investigate biological effects and mechamism of TTR on the development of neovascularization under simulated DR condition associated with high glucose and hypoxia.Methods Human retinal microvascular endothelial cells(HRMECs)were cultured in normal and simulated DR environments with high glucose and hypoxia.The normal serum glucose concentration is approximately 5.5 mmol/L;thus,hyperglycemia was simulated with 25 mmol/L glucose,while hypoxia was induced using 200 ?mol/L CoCl 2.The influence of TTR on HRMECs and human retinal pigment epithelial cells(hRPECs)was determined by incubating the cells with 4?mol/L TTR in normal and abnormal media.A co-culture system was then employed to evaluate the effects of hRPECs on HRMECs.To understand the effects of TTR on HRMECs,cell proliferation was investigated under natural and DR conditions.Overexpression of TTR,an in vitro wound-healing assay,and a tube formation assay were employed to study the repression of TTR on HRMECs.Real-time fluorescence quantitative PCR(qRT-PCR)was used to study the mRNA levels of DR-related genes,such as Tie2,VEGFR1,VEGFR2,Angptl,and Angpt2.Results:Decreased HRMECs and hRPECs were observed under abnormal conditions,including high-glucose and hypoxic media.In addition,HRMECs were significantly inhibited by 4 ?mol/L exogenous TTR during hyperglycemic culture.During co-culture,hRPECs inhibited HRMECs in both the normal and abnormal environments.The proliferation of HRMECs was significantly decreased in the simulated hyperglycemic and hypoxic DR environments.The cells were further repressed by added exogenous or endogenous TTR only under hyperglycemic conditions.The in vitro migration and tube formation processes of the HRMECs were inhibited with TTR;furthermore,in the hyperglycemia and hyperglycemia/hypoxia environments,the levels of Tie2 and Angptl mRNA were enhanced with exogenous TTR,while those of VEGFR1,VEGFR2,and Angptl were repressed.Conclusions HRMECs growth is inhibited by exogenous TTR under simulated DR environments with high-glucose and hypoxic,particularly in the medium containing 25 mmol/L glucose.hRPECs,which manufacture TTR in the eye,also represses HRMECs in the same environment.TTR is predicted to inhibit the proliferation of HRMECs and neovascularization,The key genes for DR neovascularization,including Tie2,VEGFR1,VEGFR2,Angptl,and Angpt2,were regulated by TTR.Under DR conditions,TTR significantly represses neovascularization by inhibiting the proliferation,migration and tube formation of HRMECs.