目的:血管内皮生长因子受体(VEGFRs)已被证明在缺血性视网膜疾病中起关键作用,作为VEGFRs介导缺氧诱导的新生血管形成。不仅缺氧,缺血也会导致葡萄糖缺乏,然而,它对VEGFR信号和新生血管形成的影响很少被研究。生物信息学分析预测VEGFRs可能受O-GlcNAcylation的调控,而葡萄糖缺乏影响O-GlcNAcylation。
方法:在本研究中,我们单独使用低葡萄糖(LG)或与低氧(氧和葡萄糖剥夺,OGD)。细胞活力和凋亡率用于评估细胞生长特性。
结果:即使在存在蛋白质合成抑制剂的情况下,LG(2.8mmol/L)处理也会诱导VEGFR1,2,3的mRNA和蛋白质水平,环己酰亚胺(CHX),这表明VEGFR蛋白的增加与翻译后修饰部分相关。免疫沉淀分析显示,LG在VEGFR1,2中均降低了O-GlcNAc水平,但去-O-GlcNAc糖基化酶抑制剂恢复了O-GlcNAc水平。该抑制剂还消除了LG诱导的VEGFR2蛋白增加,而这种作用在蛋白酶体抑制剂的存在下并没有消失,MG132。在OGD条件下也观察到类似的结果。在LG和OGD条件下,VEGFR2敲低比VEGFR1、3敲低更显著地延缓hRMECs和HUVECs的生长。
结论:较低的葡萄糖抑制了VEGFR2中的O-GlcNAcylation,从而抑制其蛋白酶体降解;上调VEGFR2促进了缺血条件下血管内皮细胞的增殖。
OBJECTIVE: Vascular endothelial growth factor receptors (VEGFRs) have been demonstrated to play a critical role in ischemic retinal diseases, as VEGFRs mediate hypoxia-induced neovascularization. Not only hypoxia, ischemia also induces the deficiency of glucose, yet its effects on VEGFR signal and neovascularization have seldom been studied. Bioinformatics analysis predicted that VEGFRs may be regulated by O-GlcNAcylation, while glucose deficiency influences the O-GlcNAcylation.
METHODS: In this study, we treated human retinal microvascular endothelial cells with low glucose (LG) alone or in combination with low oxygen (oxygen and glucose deprivation, OGD). Cell viability and apoptosis rate were used to evaluate cell growth characters.
RESULTS: LG (2.8 mmol/L) treatment induced mRNA and protein levels of VEGFR1, 2, 3 even in the presence of the protein synthesis inhibitor, cycloheximide (CHX), suggesting that the increase in VEGFR proteins is partially associated with post-translational modifications. Immunoprecipitation analysis showed that O-GlcNAc level was decreased by LG in both VEGFR1, 2, but a de-O-GlcNAc glycosylase inhibitor restored the O-GlcNAc levels. This inhibitor also abolished the LG-induced increase in VEGFR2 protein, whereas this effect was not disappeared in the presence of the proteasome inhibitor, MG132. Similar results were also observed under OGD condition. VEGFR2 knockdown more significantly retarded the growth of hRMECs and HUVECs than VEGFR1, 3 knockdown under LG and OGD conditions.
CONCLUSIONS: A relatively low glucose suppressed O-GlcNAcylation in VEGFR2, whereby inhibiting its proteasome degradation; up-regulated VEGFR2 promoted the proliferation of vascular endothelial cells under ischemic condition.