ICAM-1 Up-regulation Mechanism Of Endothelial Cells In High-glucose Culture Due To O-GlcNAc Glycosylation Modification And Validation Research Of Anti-angiogenic Peptide HRH

Purpose: To study the relationship between O-GlcNAc glycosylation and expression of inflammatory factors in diabetic retinopathy(DR)and its related regulatory mechanisms.To study the role of peptide HRH in inhibiting neovascularization in DR.Methods: Detection of inflammatory factors was made by protein array in Human Umbilical Vein Endothelial Cells(HUVECs)as normal and high glucose groups.HUVECs and RRCECs(Rat Retinal Capillary Endothelial Cells)were treated with 5 mmol/L(low glucose)or 30 mmol/L(high glucose)glucose for 24 hours and 48 hours,which were respectively detected by q RT-PCR,western blotting and ELISA.si RNA was employed to decline Sp1 expression.Besides,inhibitors of O-GlcNAc transferase(OGT)and O-GlcNAcase(OGA)were used to change O-GlcNAc levels in HUVECs and RRCECs.In addition,a phage shown library(Ph.D TM-12 phage shown library)was used to screen peptide.The anti-angiogenesis effect of the peptide HRH was further confirmed in vivo and vitro.Results: Compared to control conditions(5 mmol/L),high glucose(30 mmol/L)increased ICAM-1 protein levels.The OGT inhibitor was added to decrease O-GlcNAc and ICAM-1 expression.Conversely,the addition of the OGA inhibitor increased O-GlcNAc and ICAM-1 expression.Besides,cells including exogenous si Sp1 showed ICAM-1 expression decreasing significantly.At the same time,a novel peptide designated HRHTKQRHTALH(peptide HRH),which was isolated from the Ph.D TM-12 phage display library by VEGFR-Fc fusion protein as the bait,was reported.This peptide displays dose-dependently inhibit the proliferation of HUVECs stimulated by VEGF.The anti-angiogenesis effect of the HRH peptide was also checked in vivo by the chick chorioallantoic membrane(CAM)assay,which also showed dose-dependent.Besides,peptide HRH was proved to inhibit corneal neovascularization in alkali-burnt rat corneal model and suture-induced rat corneal model.Conclusion: The raising of ICAM-1 with hyperglycaemia is regulated by O-GlcNAc modification of Sp1.This result explains the mechanism of ICAM-1 in HUVECs and RRCECs.Understanding how these inflammatory factors are processed during diabetic retinopathy will be good for designing novel therapeutics to treat this condition.Further,the peptide HRH can inhibit angiogenesis both in vitro and in vivo.Consequently,the peptide HRH might be a promising lead peptide for the development of potential angiogenic inhibitors.