Objectives To assess the effects of vascular endothelial growth factor-C(VEGF-C)/vascular endothelial growth factor receptor-3(VEGFR-3)signaling on nitric oxide(NO)production and inducible nitric oxide synthase(iNOS)e...Objectives To assess the effects of vascular endothelial growth factor-C(VEGF-C)/vascular endothelial growth factor receptor-3(VEGFR-3)signaling on nitric oxide(NO)production and inducible nitric oxide synthase(iNOS)expression in human osteosarcoma MG63 cells and the subsequent impact on the proliferation of human umbilical vein endothelial cells(HUVECs).Methods MG63 cells were treated with VEGF-C alone(VEGF-C group),VEGF-C+iNOS inhibitor aminoguanidine(AG;AG group),and VEGF-C+VEGFR-3 inhibitor MAZ51(MAZ51 group);untreated MG63 cells were used as controls.NO production was evaluated by a colorimetric method involving nitrate reductase.Meanwhile,mRNA and protein levels of iNOS were examined by reverse transcription polymerase chain reaction(RT-PCR)and Western blot.To explore the effect of VEGF-C/VEGFR-3/iNOS signaling of MG63 cells on proliferation of HUVECs,we set up six groups:HUVECs,HUVECs+MG63,HUVECs+VEGF-C,HUVECs+MG63+VEGF-C,HUVECs+MG63+VEGF-C+AG,and HUVECs+MG63+VEGF-C+MAZ51 groups.The proliferation of HUVEC cells was assessed by Cell Counting Kit-8(CCK-8),5-ethynyl-2'-deoxyuridine(EdU)incorporation assay,and proliferating cell nuclear antigen(PCNA)expression quantitation.Results VEGF-C treatment enhanced iNOS expression at both gene and protein levels(mRNA:LSD-t=4.152,P<0.01;protein:LSD-t=3.486,P<0.01)and increased NO release of MG63 cells(LSD-t=3.774,P<0.01);treatment with either AG or MAZ51 decreased these effects(mRNA:LSD-t=9.183,P<0.001;LSD-t=8.639,P<0.001;protein:LSD-t=5.170,P<0.001;LSD-t=7.25S,P<0.001;NO production:LSD-t=10.326,P<0.001;LSD-t=l0.540,P<0.001).Interestingly,co-incubation of HUVECs with MG63 cells and/or VEGF-C significantly promoted HUVEC proliferation(EdU:LSD-t=5.374,P<0.001;LSD-t=2.984,P<0.05;LSD-t=8.526,P<0.001;PCNA:LSD-t=9.267,P<0.001;LSD-t=5.515,P<0.001;LSD-t=14.873,P<0.001).The proliferation effects of HUVEC induced by MG63 cells and VEGF-C attenuated by the treatment of AG(EdU:LSD-t=10.770,P<0.001;PCNA:LSD-t=19.94O,P<0.001)or MAZ51(EdU:LSD-t=6.950,P<0.001;PCNA:LSD-t=14.001,P<0.001).Conclusion In human osteosarcoma MG63 cells,activation of VEGFR-3 by VEGF-C promotes iNOS expression and NO production,which subsequendy induces HUVEC proliferation.展开更多
基金the Natural Science Fundation of Shanxi Province(201801D121220).
文摘Objectives To assess the effects of vascular endothelial growth factor-C(VEGF-C)/vascular endothelial growth factor receptor-3(VEGFR-3)signaling on nitric oxide(NO)production and inducible nitric oxide synthase(iNOS)expression in human osteosarcoma MG63 cells and the subsequent impact on the proliferation of human umbilical vein endothelial cells(HUVECs).Methods MG63 cells were treated with VEGF-C alone(VEGF-C group),VEGF-C+iNOS inhibitor aminoguanidine(AG;AG group),and VEGF-C+VEGFR-3 inhibitor MAZ51(MAZ51 group);untreated MG63 cells were used as controls.NO production was evaluated by a colorimetric method involving nitrate reductase.Meanwhile,mRNA and protein levels of iNOS were examined by reverse transcription polymerase chain reaction(RT-PCR)and Western blot.To explore the effect of VEGF-C/VEGFR-3/iNOS signaling of MG63 cells on proliferation of HUVECs,we set up six groups:HUVECs,HUVECs+MG63,HUVECs+VEGF-C,HUVECs+MG63+VEGF-C,HUVECs+MG63+VEGF-C+AG,and HUVECs+MG63+VEGF-C+MAZ51 groups.The proliferation of HUVEC cells was assessed by Cell Counting Kit-8(CCK-8),5-ethynyl-2'-deoxyuridine(EdU)incorporation assay,and proliferating cell nuclear antigen(PCNA)expression quantitation.Results VEGF-C treatment enhanced iNOS expression at both gene and protein levels(mRNA:LSD-t=4.152,P<0.01;protein:LSD-t=3.486,P<0.01)and increased NO release of MG63 cells(LSD-t=3.774,P<0.01);treatment with either AG or MAZ51 decreased these effects(mRNA:LSD-t=9.183,P<0.001;LSD-t=8.639,P<0.001;protein:LSD-t=5.170,P<0.001;LSD-t=7.25S,P<0.001;NO production:LSD-t=10.326,P<0.001;LSD-t=l0.540,P<0.001).Interestingly,co-incubation of HUVECs with MG63 cells and/or VEGF-C significantly promoted HUVEC proliferation(EdU:LSD-t=5.374,P<0.001;LSD-t=2.984,P<0.05;LSD-t=8.526,P<0.001;PCNA:LSD-t=9.267,P<0.001;LSD-t=5.515,P<0.001;LSD-t=14.873,P<0.001).The proliferation effects of HUVEC induced by MG63 cells and VEGF-C attenuated by the treatment of AG(EdU:LSD-t=10.770,P<0.001;PCNA:LSD-t=19.94O,P<0.001)or MAZ51(EdU:LSD-t=6.950,P<0.001;PCNA:LSD-t=14.001,P<0.001).Conclusion In human osteosarcoma MG63 cells,activation of VEGFR-3 by VEGF-C promotes iNOS expression and NO production,which subsequendy induces HUVEC proliferation.
