| Objective: By observing the abilities of proliferation,migration,tube formation(in vivo and in vitro)on human umbilical vein endothelial cells(HUVECs)treated with bevacizumab under the anoxic condition(<1% O2),to reveals its initiating factors that activated the HUVECs,and found out the mechanism of how the factor was up-regulated.Methods: Perform the experiment when the HUVECs was on good status(HUVECs grew to 60%-70%),according to previous Transwell experimental results,choose a middle high concentration of bevacizumab to simplify the experiment and fixed it to 100?g/ml,according to the result of cell morphology and determined by MTT,choose the best observation time point for 24 h after drug use.In vitro anoxic model(1%O2+5%CO2+94%N2)was established by using the Billups-rothenber device.The proliferation of HUVEC cells was detected by MTT assay.The migration of HUVEC was detected by Transwell.The tube-formation ability(in vivo and in vitro)was detected by the matrigel experiment.Using 400?l Matrigel embedded with HUVECs cells,VEGFA and different concentrations(0,10,100 ?g/ml)of bevacizumab,5-8 weeks-nu BALB/c mice was subcutaneously injected with the matrigel to monitor the tube formation in vivo,the mice was injected Intraperitoneally with different concentrations of bevacizumab(0,5,50 mg/kg)every 2 weeks to maintain the drug concentration of bevacizumab for 30 days,mice was sacrificed,the matrigel was fixed,the slides were then used for HE staining and immunohistochemical staining.The CD105(endoglin)and its downstream EMT(?-SMA,N-cadherin,twist,slug)and inflammatory factor(IL1B,CCL20)changes in protein levels was detected via western blot,Immunofluorescence of HUVECs treated with bevacizumab under hypoxia condition and immunohistochemistry of the matrigel separated from the matrigel were used to verify the CD105 expression.At the same time,the changes of TGF?1 and VEGFA in the supernatant of HUVECs were detected via ELISA assays.At the same time,the changes of smad1,smad5 and phosphorylated smad1/5 in protein levels were detected via western blot,and the m RNA levels of CD105,alk1,smad1 and smad5 were detected via RT-q PCR.In addition,HUVECs was treated with isotype Ig G1 to detect the effects of isotype Ig G1 on CD105.At the same time,the HIF-? protein expression was also detected via western blot to analyze the effects of bevacizumab on HIF-? under hypoxia condition.c-jun n-terminal kinase(JNK)inhibitor and JNK si RNA were used to explore the upstream mechanism of CD105 up-regulation,CD105 si RNA was used to detect the changes of downstream factors of CD105,like the EMT and inflammatory factors.And then we verified the change of CD105 and its up-regulation mechanism in mouse brain microvascular endothelial cells(b End.3 cells)and mouse retinal microvascular endothelial cells(MRMEC).Results: 1.High concentration of bevacizumab stimulation under hypoxia condition promoted HUVECs proliferation,and low concentration of bevacizumab inhibited the proliferation of HUVECs.2.There was no significant difference in the migration number of HUVECs,compared with the normoxia vehicle and the control group;under normoxia condition,high concentration of bevacizumab(80?g/ml)decreased the migration number of the HUVECs significantly.Interstingly,Compared with the control group,high concentration of bevacizumab.3.High concentrations of bevacizumab promoted the tube formation ability of HUVECs in vitro,compared with the control group,high concentration of bevacizumab significantly increased the length of the vascular branching length significantly(P < 0.001).4.The matrigel coated with high concentrations of bevacizumab and HUVECs formed more vascular tubular structure compared with control group,and HE staining showed the tubular structure of the high concentration of bevacizumab was of discontinuity,deformity,relative dispersion;the tubular structure formed in the low concentration bevacizumab group was a continuous,connective;the vascular tubular structure in the high concentration of bevacizumab was similar to the vessel co-option during the renal clear cell carcinoma expansion.5.The expression of CD105 in protein and m RNA levels was enhanced significantly by high concentration of bevacizumab under the hypoxic environment.At the same time,immunofluorescence results showed that HUVECs treated with high concentration of bevacizumab showed evidently higher CD105 expression compared with the control group.At the same time,the Ig G1 isotype failed to up-regulate CD105 expression.The expression of HIF-? in hypoxic environment was not affected by bevacizumab neither.6.The protein expression of CD105 downstream EMT and inflammatory factors were also increased significantly in HUVECs treated with high concentration of bevacizumab.Slug,twist,?-SMA,N-cadherin protein expression were evidently higher in the HUVECs treated with high concentration of bevacizumab compared with control group(P < 0.05);besides,the inflammatory factors such as CCL20 and IL1 B protein expression were significantly increased when treated with high concentration of bevacizumab under hypoxia condition.7.High concentrations of bevacizumab activated the TGF?1 pathways,when HUVECs was treated with high concentration of bevacizumab under hypoxia condition,the smad1,smad5,p Smad1/5 was increased significantly in protein levels,the smad5,ALK1 m RNA expression were also increased obviously via the RT-q PCR(P < 0.05).At the same time,TGF?1 concentration in the supernatant of HUVECs cells treated with high concentration of bevacizumab increased significantly.The aforementioned results showed that high concentration of bevacizumab activated TGF?1 pathway.8.High concentrations of bevacizumab activated JNK pathway,p JNK and JNK protein expression were significantly up-regulated in HUVECs treated with high dose bevacizumab,pre-stimulated HUVECs with JNK si RNA or JNK specific inhibitors abolished the up-regulation of CD105 caused by high concentration of bevacizumab.The results validated that the augment of CD105 was mediated by the JNK pathway.9.High concentration of bevacizumab increased the CD105 protein expression in MRMEC cells and b End.3 cells in hypoxia conditon and activated the smad1/5 pathways and JNK pathway,pre-treatment the mice endothelial cells with JNK inhibitor,CD105 augment in protein levels was abolished,which indicated that the up-regulation of CD105 was mediated by the JNK pathway in mice endothelial cells.10.CD105 si RNA was used to knock down the CD105 expression,when HUVECs was treated with CD105 si RNA,the expression of the EMT and inflammatory factors were significantly decreased,at the same time,the abilities of cell migration and proliferation were also down-regulated when treated HUVECs with CD105 si RNA.Conclusions: 1.High concentration of bevacizumab increased the abilities of HUVECs in migration,proliferation,tube formation(in vitro and in vivo),high dose bevacizumab stimulation or hypoxia stimulation alone failed to activate endothelial cells.2.TGF?1 pathways and JNK pathway were activated in HUVECs treated with high dose bevacizumab,the activation of the two pathway also increased the expression of CD105,CD105 and CD105 downstream factors(inflammatory and EMT factors)increased the abilities of HUVECs in migration,proliferation,tube formation.When CD105 expression was knocked down,the migration and proliferation abilities of HUVECs cells decreased,as well as the downstream factors of CD105.3.The up-regulation of CD105 was mediated by the JNK pathway,and the activation of endothelial cells was via the canonical HIF-? and VEGFA pathway.4.The inhibition of TGF? 1 and JNK pathway may reverse resistance to bevacizumab,but whether bevacizumab activated the JNK directly or through the activation of TGF?1 need further study. |
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