背景:血管内皮生长因子(VEGF)信号通路在糖尿病视网膜病变(DR)的进展中起重要作用。DR患者中许多关键功能蛋白的糖基化修饰过程异常。然而,目前尚不清楚异常N-糖蛋白是否可能参与DR进展.
方法:进行玻璃体液的糖蛋白质组分析。蛋白质和N-糖蛋白的水平通过蛋白质印迹和凝集素印迹证实,分别。通过CCK-8和Transwell测定检测细胞活力和迁移效率。采用流式细胞仪检测细胞凋亡水平和活性氧种类。丙二醛,酶联免疫吸附法检测超氧化物歧化酶活性和VEGF含量。通过GST下拉法检测金属蛋白酶1(TIMP-1)与N-乙酰葡糖胺转移酶V(GnT-V)的相互作用。苏木精和伊红染色以及用异硫氰酸荧光素-葡聚糖测定法染色的脉络膜和视网膜平板染色用于体内功能研究。
结果:我们发现DR大鼠和高糖(HG)诱导的人视网膜色素上皮细胞系ARPE-19中N-糖基化上调。HG抑制ARPE-19细胞活力,促进细胞凋亡和氧化应激(OS),但是这些效果在kifunensine治疗中被逆转了,GnT-V敲低和TIMP-1突变。此外,GnT-V结合TIMP-1以促进TIMP-1的N-糖基化。GnT-V过表达抑制ARPE-19细胞活力,促进细胞凋亡,OS和VEGF释放,这些效应被TIMP-1突变逆转。有趣的是,GnT-V的过表达促进视网膜微血管内皮细胞(RMEC)的血管生成,但由于TIMP-1突变而受到推崇,VEGF-A治疗可最终增强。最后,GnT-V敲除减轻DR进展。
结论:结果表明,GnT-V通过激活VEGF信号通路,增加TIMP-1的N-糖基化水平,促进RMECs血管生成和ARPE-19细胞损伤,为DR的预防提供了新的理论依据。
BACKGROUND: Vascular endothelial growth factor (VEGF) signaling pathway plays an important role in the progression of diabetic retinopathy (DR). The glycosylation modification process of many key functional proteins in DR patients is abnormal. However, the potential involvement of abnormal N-glycoproteins in DR progression remains unclear.
METHODS: Glycoproteomic profiling of the vitreous humor was performed. The level of protein and N-glycoprotein was confirmed by Western blot and Lectin blot, respectively. The cell viability and migration efficiency were detected by CCK-8 and Transwell assay. Flow cytometry was conducted to analyze the level of cell apoptosis and reactive oxygen specie. Malondialdehyde, superoxide dismutase activity and VEGF content were detected by Enzyme linked immunosorbent assays. The interaction of metalloproteinase 1 (TIMP-1) with N-acetylglucosamine transferase V (GnT-V) was detected by GST pull-down. Hematoxylin and eosin staining and choroidal and retinal flat mount stained with fluorescein isothiocyanate-Dextran assay were used for functional research in vivo.
RESULTS: We found that N-glycosylation was up-regulated in DR rats and high glucose (HG)-induced human retinal pigment epithelium cell line ARPE-19. HG-induced inhibited the viability of ARPE-19 cells and promoted cell apoptosis and oxidative stress (OS), but these effects were reversed with kifunensine treatment, GnT-V knockdown and TIMP-1 mutation. Additionally, GnT-V binds to TIMP-1 to promote N-glycosylation of TIMP-1. Over-expression of GnT-V inhibited the viability of ARPE-19 cells and promoted cell apoptosis, OS and VEGF release, which these effects were reversed with TIMP-1 mutation. Interestingly, over-expression of GnT-V promoted retinal microvascular endothelial cells (RMECs) angiogenesis but was revered with TIMP-1 mutation, which was terminally boosted by VEGF-A treatment. Finally, knockdown of GnT-V relieved DR progression.
CONCLUSIONS: The findings indicate that GnT-V can promote RMECs angiogenesis and ARPE-19 cells injury through activation VEGF signaling pathway by increasing TIMP-1 N-glycosylation level, which provides a new theoretical basis for the prevention of DR.