Role Of High Glucose-induced BAG5 Expression In Pro-angiogenesis In Retinal Pigment Epithelial Cells

Objective: Diabetic retinopathy(DR)is the most common microvascular complication of diabetes mellitus with the highest risk of blindness,constituting the leading cause of visual impairment and blindness in people aged 24-64 years.As the number of people with diabetes increases worldwide,DR has become one of the causes of serious threats to human visual health.DR is a diabetic complication with no significant vision loss in the early stage.Over time,chronic hyperglycemia causes a breakdown of the blood-retinal barrier,aggravating neuroretinal damage and leading to cloudy or blurred vision,as well as increased neovascularization in the retina and increased vascular permeability leading to the increase in neovascularization and vascular permeability in the retina leads to hemorrhage and macular edema,which eventually leads to blindness.Therefore,the study of the molecular mechanisms underlying the development of diabetic retinopathy,especially the triggering mechanisms of blood-retinal barrier breakdown and neovascularization,is essential for the discovery of effective diagnostic and therapeutic approaches.BAG5 is a member of the Bcl-2-associated athanogene(BAG)family of antiapoptotic genes and is the only family member containing multiple BAG structural domains and no other structural domains,consisting of five BAG structural domains that make up the entire protein.interaction between BAG5 and heat shock proteins HSP70/HCP70 has a dual effect.Under normal physiological conditions BAG5 accelerates HSP70/HSC70-mediated protein refolding.However,in pathological states overexpression of BAG5 may inhibit HSP70 chaperone protein action and interfere with the protein refolding process.In Parkinson’s disease,BAG5 exerts both neuroprotective effects and promotes neuronal damage as well as the development of neurodegenerative diseases.And in tumors,BAG5 exerts both pro-cancer and anticancer effects.Although the role of BAG5 is uncertain and contradictory,BAG5 is involved in regulating biological processes such as cell proliferation,cell migration andinvasion,tumor growth and chemotherapy resistance.Recent studies on BAG5 have focused on tumor and neurodegenerative diseases,and its relationship with diabetic retinopathy has not been reported.In this research,we found that BAG5 protein expression was upregulated in retinal pigment epithelium(RPE)cells induced by high glucose,and the specific causes and mechanisms of its action remain to be elucidated.The purpose of this paper is to investigate the reasons for the upregulation of BAG5 expression induced by high glucose,and the specific mechanisms involved in regulating the development of EMT and pro-angiogenesis in RPE cells.Methods: 1.Single cell sequencing data were analyzed to find differentially expressed genes and PROGENy(Pathway Resp Onsive GENes for activity inference)pathway enrichment analysis,and cells were scored for cell signaling pathway activity after sorting by differentially expressed genes.2.Western blot assay was performed to detect protein expression levels of BAG5 in ARPE19 cells cultured in high and normal glucose cultures by Western blot assay.3.Gene transcript levels of BAG5 in ARPE19 cells cultured in high and normal glucose cultures were detected by RT-q PCR.4.ARPE19 cell lines with stable knockdown of BAG5 were constructed by infecting cells with custom-made lentivirus.5.BAG5 synonymous mutant plasmid and CK2α plasmid were constructed and transfected into BAG5 knockdown ARPE19 cells,respectively.6.The migration ability of ARPE19 cell lines cultured in high and normal glycemia was examined by Transwell assay.7.The pro-angiogenic ability of ARPE19 cell lines cultured in high and normal glycemia was examined by tubule formation assay.8.The pro-angiogenic ability of ARPE19 cell lines cultured in high and normal glycemia was examined by Western blot assay to detect the expression level of EMT markers in each group of ARPE19 cell lines cultured in high and normal glycemia.9.The expression level of VEGFA in each group of ARPE19 cell lines cultured in high and normal glycemia was detected by RT-q PCR assay.10.The stability of CK2α in ARPE19 cells with BAG5 knockdown was detected by cycloheximide protein assay.11.The BAG5 promoter activity was detected by Dual-luciferase reporter gene assay.12.The expression levels of transcription factors Tcf7l2,Trp53,Nr3c1 in ARPE19 cellscultured in high and normal glycemia were detected by RT-q PCR assay.Results: 1.The activity of EMT-related pathway and VEGF-related pathway was higher in BAG5-expressing retinal cells and RPE cells.2.High glucose induction increased the migratory ability and pro-angiogenic ability of ARPE19 cells.3.BAG5 expression was upregulated in ARPE19 cells induced by high glucose.4.In ARPE19 cells,knockdown of BAG5 reduced their migratory ability and pro-angiogenic ability.5.Restoration of BAG5 expression in ARPE19 cells restored cell migratory ability and pro-angiogenic ability.6.BAG5 upregulated CK2α expression by promoting the stability of CK2α,increasing the migratory ability and pro-angiogenic ability of ARPE19 cells.7.Transcriptional activation of the-1500~-1000 region of the BAG5 promoter under high glucose induction led to upregulation of BAG5 expression.Conclusion: BAG5 expression was upregulated in ARPE19 cells cultured with high glucose,and high expression of BAG5 was closely associated with enhanced cell migration and pro-angiogenic capacity.This paper suggests that BAG5 promotes the occurrence of EMT in RPE as well as plays a pro-angiogenic role by regulating the stability of CK2α.