To Explore The Effect And The Related Molecular Mechanism Of Rh-GDF-15on Hypoxia-induced Apoptosis And Angiogenesis Of Human Umbilical Vein Endothelial Cells(HUVECs)

Objective：To explore the effect of rh-GDF-15on hypoxia-induced apoptosis and angiogenesis ofhuman umbilical vein endothelial cells (HUVECs) and the related molecular mechanism.Methods:HUVECs were obtained from cord blood of healthy cesarean delivery, isolated andcultured in vitro. We used the chemical hypoxia method which mimicked the myocardialischemia condition in vivo. The mRNA levels of interested genes were detected byRT-PCR, the proteins levels by Western blot, and secretion levels of GDF-15fromHUVECs by ELISA. The mRNA, protein and supernatant levels of GDF-15in HUVECswere determined at different time-points（0、2、6、12、24、36、48h）after hypoxia; thedose effect (0、10、20、50ng/ml) of rh-GDF-15on HUVECs apoptosis and angiogenesiswere determined after hypoxia for24h. At the same, the mRNA and protein expressions ofp53, Bcl-2, VEGF and VEGF receptor2(VEGFR2) were determined. Then the expressionof HIF-1α was detected in the cytoplasm of HUVECs with or without pretreatment ofrh-GDF-15(50ng/ml) after hypoxia for24h. The cytoplasm and nuclear protein levels ofHIF-1α were determined in HUVECs at different time-points（0、12、24、48h）of hypoxiawith rh-GDF-15（50ng/ml）or with PBS pretreatment. Under the condition of normaloxygen supply and24h of hypoxia, the tube formation was compared among groups oftreatment with PBS、rh-GDF-15（50ng/ml）、HIF-1αsiRNA and HIF-1αsiRNA+rh-GDF-15（50ng/ml）; the effect of rh-GDF-15（50ng/ml）on the mRNA and protein levels of VEGFand protein expressions of VEGFR2were detected in hypoxia (24h)-induced HUVECs wascompared between before and after pretreatment with HIF-1αsiRNA; The expression of Sirtl and Mdm2in HUVECs induced by rh-GDF-15（0or50ng/ml）was determined at thecondition of hypoxia and norexia, respectively; the cytoplasm and nuclear protein levels ofp53in HUVECs at different time-points of hypoxia were detected after pretreatment withh-GDF-15（50ng/ml）or with nutlin-3（the special Mdm2antagonist）; theco-immunoprecipitation of p53and Mdm2after hypoxia for24h, the cytoplasm and nuclearprotein levels of p53in different hypoxic time points（0、2、6、12、24h）and the expressionof HIF-1α and VEGF after hypoxia for6h were respectively determined in HUVECs withpretreatment of rh-GDF-15（50ng/ml）, nutlin-3(10μmol/L) or rh-GDF-15（50ng/ml）plusnutlin-3.Results:1、the mRNA level of GDF-15in HUVECs at hypoxia for2h was significantlyupregulated compared with that at hypoxia for0h、6h、12h、24h、36h and48h, GDF-15mRNA was gradually decreased with the prolonged hypoxia (Relative mRNA levels for2hhypoxia0.77±0.08VS hypoxia for0h、6h、12h、24h、36h and48h,0.22±0.04、0.33±0.09、0.35±0.04、0.29±0.09、0.19±0.06、0.15±0.04，p＜0.05); the protein levels of GDF-15inHUVECs at hypoxia for2h and6h were significantly upregulated compared with that athypoxia for0h、12h、24h、36h and48h. GDF-15protein level was also gradually decreasedwith the prolonged hypoxia.(Relative protein levels for2h and6h hypoxia,0.36±0.04、0.30±0.00VS hypoxia for0h、12h、24h、36h and48h,0.06±0.00、0.15±0.02、0.10±0.03、0.09±0.02、0.06±0.01，p＜0.05); the supernatant levels of GDF-15in HUVECs wasupregulated at hypoxia for2h, and gradually decreased with the prolonged hypoxia(Relative GDF-15levels for2h hypoxia,11.93±1.55VS3.43±0.76、4.73±0.76、4.6±1.3、4.0±0.56、3.43±1.04，p＜0.05).2、24h hypoxia-induced HUVECs apoptosis was significantly reduced afterpretreatment with rh-GDF-15in a dose-dependent way. The apoptotic index for HUVECsinduced by rh-GDF-15(0ng/mL、10ng/mL、20ng/mL、50ng/mL) were22.0±3.16、17.8±3.90、15.6±5.68and13.4±3.58, respectively. The cell apoptosis was graduallydecreased with the increased dosage of rh-GDF-15. The apoptotic index showed significantdifference between group pretreated with rh-GDF-15（50ng/mL）and group not withrh-GDF-15(13.4±3.58VS22.0±3.16，p＜0.05). 3、The tube formation of HUVECs was totally inhibited on Matrial Matrix afterhypoxia for24h, and tubes became increased after pretreated with rh-GDF-15, which wasproportional to the dosage of rh-GDF-15(0ng/mL、10ng/mL、20ng/mL、50ng/mL), thenumbers of tube formation was significantly increased in HUVECs pretreated withrh-GDF-15(20ng/mL、50ng/mL) compared with that in rh-GDF-15untreated HUVECs(5.67±0.58、7.00±1.00VS1.00±1.00，p＜0.05).4、The expression of Bcl-2was increased by pretreating HUVECs withrh-GDF-15(0ng/mL、10ng/mL、20ng/mL、50ng/mL) and increased in a dose-dependentway. Bcl-2expression in rh-GDF-15（50ng/mL）treated HUVECs was significantlyenhanced compared with that in rh-GDF-15untreated HUVECs (0.79±0.22VS0.51±0.12，p＜0.05); as well as the expression of VEGF(0.67±0.06VS0.14±0.06，p＜0.05); however,the expression of p53was significantly decreased by rh-GDF-15（50ng/mL）compared withrh-GDF-15untreated HUVECs (0.38±0.06VS0.74±0.19，p＜0.05); but the mRNAexpression of VEGFR2was not changed.5、The cytoplasm expression of HIF-1α was significantly increased after hypoxia for2h(0.67±0.12VS0.38±0.10p0.05), which was gradually decreased with the prolonged hypoxia, andreduced to the level lower than that before hypoxia at12h of hypoxia. Although hypoxia inhibitedthe expression of HIF-1α was not reversed by rh-GDF-15pretreatment, but showedsignificant difference at the hypoxia time of24h、36h and48h compared with that in thesame hypoxia time without rh-GDF-15induction (0.49±0.13VS0.19±0.09，0.42±0.08VS0.12±0.05，0.37±0.10VS0.07±0.03，p＜0.05). After the pretreatment of rh-GDF-15,nuclear HIF-1α expression was significantly increased in HUVECs compared withrh-GDF-15untreated HUVECs, and showed significant difference at each time-point ofhypoxia (0.16±0.03VS0.04±0.02，0.33±0.02VS0.03±0.01，0.43±0.03VS0.05±0.02，0.29±0.02VS0.04±0.01，p＜0.05).6、Hypoxia-induced tube formation on Matrial Matrix was increased after preteatingHUVECs with rh-GDF-15, which could be suppressed by knocking down of HIF-1α, andsuch effect could not be reversed by increasing the dose of rh-GDF-15.7、The transcriptional and translational levels of VEGF were significantly lower inrh-GDF-15（50ng/ml）-pretreated HUVECs after knocking down of HIF-1α than that before HIF-1α siRNA treatment (0.19±0.02VS0.60±0.01，0.0800±0.01604VS0.1871±0.01694，p＜0.05); but rh-GDF-15（50ng/ml）-induced protein expression ofVEGFR2was not changed before and after HIF-1α siRNA treatment.8、The expressions of Sirtl and Mdm2was not changed in HUVECs pretreated withrh-GDF-15（50ng/ml）under the condition of normal oxygen supply; but Mdm2not Sirtlwas significantly increased after in HUVECs pretreated with rh-GDF-15（50ng/ml）underthe condition of hypoxia (0.6912±0.02732VS0.5405±0.05291，p＜0.05).9、The cytoplasm level of p53was upregulated but nuclear level of p53wasdownregulated in HUVECs pretreated with rh-GDF-15（50ng/ml）after hypoxia for24h;the effect was reversed by pretreating cells with nutlin-3.10、The immunopecipitation showed the interaction between p53and Mdm2inHUVECs after hypoxia for24h, which was enhanced in HUVECs pretreated withrh-GDF-15（50ng/ml）, and reduced in HUVECs pretreated with nutlin-3, also in HUVECspretreated with both nutlin-3and rh-GDF-15. p53-Mdm2interaction promoted p53degradation and enhanced the downstream HIF-1α and VEGF expressions, both of whichwere reduced after pretreating HUVECs with nutlin-3.Conclusions:1、GDF-15significantly inhibited hypoxia-induced endothelial apoptosis, andpromoted endothelial angiogenesis.2、GDF-15prevented hypoxia-induced increased expression of p53, and promotedp53nuclear export and protein ubiquitin degradation though activating Mdm2. Nucleardecreased p53would promoted HIF-1α nuclear translocation and tabilized the expressionof HIF-1α, thus activated the downstream HIF-1α/VEGF signaling pathway, promoted theformation of angiogenesis.