Studies On The Role Of Axl Overexpression In HIF-1? Expression And Endothelium Tube Formation Under The Condition Of High Glucose And Hypoxia

Objective High glucose inhibits the expression of Hypoxia inducible factor-1? and angiogenesis. Axl regulates endothelium function and promote endothelium tube formation. However, whether axl involved in endothelium tube formation under the condition of high glucose and hypoxia is not clear. The objective was to observe endothelium tube formation and Axl expression and test Axl overexpression in HIF-la expression and endothelium tube formation under the condition of high glucose and hypoxia.Methods 1.The 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide solution was used to observe the effect of different glucose concentrations (5.5 mmol/1,11 mmol/1,25 mmol/1, and 30 mmol/l) and time (24 h,48 h and 72 h)on Human umbilical vein endothelial cells (HUVECs) and Eahy926 viability; 2. The 3-(4,5-dimethyl thiazol-2-yl) 2,5-diphenyltetrazolium bromide solution was used to observe the effect of different hypoxic time under high glucose on HUVECs and Eahy926.3.After find out the optimal condition of high glucose and hypoxia,the wound closure assay and tube formation assay under matrigel was performed to observe the endothelium migration and tube formation ability under the condition of high glucose and hypoxia; 4.Western blot was used to detect Axl and HIF-1? expression under the condition of high glucose and hypoxia; 5. Western blot was used to detect Axl expression after Eahy926 overexpressing Axl; 6.The experiment was divided into four groups, control group (negative virus), Axl overexpression, R428 group (Axl inhibitors R428+negative virus), Axl overexpression+R428 group. The tube formation assay under matrigel was performed to observe the endothelium tube formation ability of Eahy926 overexpression Axl.7. The mRNA and protein level of HIF-1? was detected by q-PCR and Western blot; 8. The cycloheximide, LY294002, PD98059 and Rapamycin was used to detect the molecular mechanism of overexpression Axl on HIF-1? expression.Results 1. The inhibit effect of high glucose on endothelium viability was concentration and time dependent. When supplemented with the concentration of 30 mmol/1 glucose for 48 h, the endothelium viability was significantly inhibited; 2. The effect of hypoxia on endothelium viability under high glucose was time dependent. Compared with high glucose group, the cell viability was inhibited in high glucose+hypoxia 12 h group and high glucose+hypoxia 24 h group; 3. The migration was in high glucose 24 h+hypoxia 6 h group was decreased when compared with hypoxia 6 h group in HUVECs and Eahy926(P <0.05), The tube formation in high glucose 24 h+hypoxia 6 h group was decreased when compared with hypoxia 6 h group in HUVECs and Eahy926,30.8%and 52.9% respectively (P<0.05); 4. The Axl protein expression was decrease in high glucose and hypoxia group compared with the hypoxia group in HUVECs and Eahy926,48.4%and 47.1% respectively (P<0.05); 5. The Axl protein was significantly increased after Eahy926 was transfected with Axl overexpressing virus.6. The total tubes was increased in Axl overexpression group than control group. The tubes was increased 66.8% compared with control group (P <0.05); 7. The qPCR showed that the mRNA of HIF-1? was unaffected by Axl overexpression; However, the protein level of HIF-1? was increased in Axl overexpression Eahy926 compared with control group. The protein level of HIF-1? increased% compared with control group (P<0.05); 8. When compared with Axl overexpression group, the HIF-1 a protein significantly decreased in Axl overexpression+ cycloheximide group. No diffrence was detect of HIF-1? protein between cycloheximide group and control group. The p-Akt and p-p70S6K was increased in Axl overexpression group.LY294002 and Rapamycin can abrogate overexpression Axl induced HIF-1? increase.Conclusion 1. The endothelium tube formation was inhibited under the the condition of high glucose and hypoxia; 2. Overexpression Axl rescued the negative effect of endothelium tube formation under the condition of high glucose and hypoxia; 3. The PI3K7Akt/mTOR/p70S6K pathway was involved in HIF-1? protein expression induced by Axl overexpression under the condition of high glucose and hypoxia.