The Regulation Of Vegf And Fgf By Apelin-apj System

ObjectiveTo investigate the effects of apelin/APJ system on the proliferation of human umbilical vein endothelial cells (HUVECs) along with the production of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and other angiogenesis-related factors.Methods1. HUVECs culture:HUVECs were cultured in DMEM medium supplem-ented with 10% fetal calf serum at 37℃in an atmosphere of 5% CO2 in the presence or absence of apelin and PI3K inhibitor.The morphology of HUVECs was tested by phase-contrast microscope2. VEGF, FGF and other angiogenesis-related factors assay:After stimulation with apelin at different concentration for 3 days, HUVECs were lyzed to extract mRNA for RT-PCR assay of VEGF, FGF and other angiogenesis-related factors.3.Proliferation assay by MTT:HUVECs were seeded at the density of 1000 cells/well to 96 well plates. The cells were divided into five groups according to different stimuli conditions:①blank control group;②control group;③apelin group;④PI3K inhibitor group;⑤apelin+PI3K inhibitor group. The cells'OD value was detected by MTT assay after 48 hours of stimulation.4.Western blotting analysis of FGFR1 protein:HUVECs were cocultured with 10-7M apelin,25uM PI3K inhibitor or 10-7M apelin+25 uM PI3K inhibitor for 24 hours. HUVECs alone were used as the control. Western blotting was used to detect the expression of FGFR1 protein.Results1. HUVECs grew fast, arranged in "cobblestone"-like pattern, and displayed the morphology of larger polygonal cells. No overlapping growth was observed in the culture.2. mRNA of VEGF, FGF and other vascular factors could be detected in HUVECs. Within 1 hours of apelin stimulation, there were no significant changes in the target gene expression. After coculture with apelin for 4 hours or 8 hours, HUVECs expressed more APJ, VEGF, bFGF, FGFR1, MMP2/9 (P<0.05 or P<0.01) and less TIMP1/2 (P<0.05 or P<0.01) in an concentration-dependent manner. However, there was no significant different concerning these target gene expression between 4 hour stimulation and 8 hour stimulation.3. MTT analysis:①Apelin significantly induced the proliferation of HUVECs(P<0.01), while PI3K inhibitor inhibited HUVECs proliferation (P <0.05). There were no corelation between apelin-treated group and PI3K inhibitor-treated group (P>0.05).②There existed significant difference concerning the OD value among control group, apelin group, PI3K inhibitor group and apelin+PI3K inhibitor group (P<0.05 or P<0.01).4.Western blotting analysis:①Apelin significantly up-regulated FGFR1 protein expression in HUVECs (P<0.01) but PI3K inhibitor markedly down-regulated FGFR1 protein expression (P<0.05). However, there were no correlation between apelin-treated group and PI3K inhibitor-treated group (P> 0.05).②There existed significant difference concerning the FGFR1 expression among control group, apelin group, PI3K inhibitor group and apelin+PI3K inhibitor group (P<0.05 or P<0.01).ConclusionsAfter stimulation with Apelin, HUVECs were characterized by increased proliferation, up-regulation of APJ, VEGF, bFGF, FGFR1, MMP2/9 mRNA, and down-regulation of TIMP1/2 mRNA.We further confirmed that Apelin promoted FGFR1 protein expression.To investigate the effect of apelin on HUVECs, we demonstrated here that apelin-APJ system promotes the expression of VEGF, FGF and other angioge-nesis-related factors through the PI3K/akt signal. This effect may also be mediated by other signal transduction. Altogether, our study takes insights into the mechanisms by which apelin-APJ system affects angiogenesis.