SENP6-mediated DeSUMOylation Of VEGFR2 Enhances Its Cell Membrane Transport In Angiogenesis

Diabetes mellitus is a chronic metabolic disease in which pathological angiogenesis plays a significant role.Advanced glycation end products(AGEs)are significantly elevated in diabetic tissues and can affect vascular endothelial morphology and function.The VEGF-receptor 2(VEGFR2)signaling pathway is a key mechanism in the regulation of angiogenesis,and VEGFR2 activity can be altered by post-translational modifications.Small ubiquitin-related modifier(SUMO)is a type of post-translational protein modification that covalently binds to substrate proteins,and there have been few studies on SUMO-mediated VEGFR2 modifications.The current study investigated this using human umbilical vein endothelial cells(HUVECs)in conjunction with immunoblotting and immunofluorescence.AGEs increased the expression of sentrin/SUMO-specific protease 6(SENP6),which de-SUMOylated VEGFR2.And immunofluorescence test indicated a reduction in VEGFR2 accumulation in the Golgi and increased VEGFR2 transport from the Golgi to the cell membrane surface via the coatomer protein complex subunit beta 2(COPB2).VEGFR2 on the cell membrane was linked to VEGF generated by pericytes,triggering the VEGF signaling cascade.In conclusion,this study demonstrates that SENP6 regulates VEGFR2 trafficking from the Golgi to the endothelial cell surface.The SENP6-VEGFR2 pathway plays a important role in pathological angiogenesis induced by AGEs.Objective:The aim of this study was to investigate the role of SENP6-mediated VEGFR2 deSUMOylation and its intracellular transport in angiogenesis.Methods:HUVECs were used as the target of the experiment,and the expression level of SENP6 was detected by WB and qPCR;the co-localization level of VEGFR2 with Golgi was detected by immunofluorescence;the level of Golgi vesicle transport-related protein was detected by WB and qPCR;the cytoplasmic membrane VEGFR2 was extracted by the kit and its expression level was detected by WB;the expression level of pericytes VEGF was detected by WB and Elisa.Scratch,migration and tube formation assays were used to detect endothelial cell vascularization levels.Results:1.Down-regulation of SENP6 expression level attenuates the induction of endothelial cell angiogenesis by AGEs.The expression of SENP6 was detected by qPCR and WB,and we found that it was increased at 8 h with statistical differences.Futhermore,downregulated the expression of SENP6 gene with RNAi could weaken the angiogenesis caused by AGEs stimulation examined by migration and scratch tube forming experiments.2.DeSUMOylation of VEGFR2 mediated by SENP6 enhances its transport from the Golgi apparatus to the membrane.The SUMOylation modification of VEGFR2 was detected and showed that its SUMO2/3 modification level was reduced,demonstrating that VEGFR2 is deSUMOized under the stimulation of AGEs.What’s more VEGFR2 colocalization with Golgi apparatus was significantly reduced after AGEs stimulation by immunofluorescence test.While down-regulation of SENP6 could abolish those effects induced by AGEs.3.The vesicle transporter COPB2 enhance translocation of VEGFR2 to the cell membrane.HUVECs were incubated with AGEs for different times,then the cytoplasmic and nuclear proteins were extracted respectively.The results of western blot showed that the expression of VEGFR2 in the cell membrane was increased after AGEs stimulation.While down-regulated COPB2 expression could significantly reduce the expression of VEGFR2 in cell membrane.4.AGEs stimulate pericytes to secrete VEGF and promote endothelial angiogenesisPericytes were treated with different types of conditioned media to investigate the effects on VEGF expression in endothelial cells.There was an increase in total VEGF expression in pericytes,with a significant difference at 8 h.HUVECs were stimulated with different types of pericyte-conditioned media(PCM),and migration,wound healing,and tube formation assays were performed to demonstrate that AGEs induce VEGF secretion by pericytes and promote endothelial cell angiogenesis.Conclusion:1.AGEs promote angiogenesis by increasing SENP6 expression.2.SENP6 mediates the deSUMOylation of VEGFR2,which enhances its transport from the Golgi apparatus to the membrane via the vesicle transporter COPB2.3.AGEs stimulate pericytes to secrete VEGF and promote angiogenesis in endothelial cells.