Effects Of CD137-CD137L Signal On Endothelial Cell Tube Formation And Angiogenesis Of Aortic Ring Via Activating Autophagy

ObjectivesTo explore whether the CD137-CD137L signal influences the endothelial cell tube formation and sprouts of aortic ring via activating endothelial autophagy.Methods1.Constructed the ex-vivo model of C57BL/6J mice to observe the following indicators under the inverted phase contrast microscope:(1)The effect of CD137-CD137L on the sprouts of aortic ring;(2)The effect of CD137-CD137L on the sprouts of aortic ring after intervention of autophagy process.2.To culture the human umbilical vein endothelial cells(HUVECs)on gelled basement membrane extract to observe the following indicators under the inverted phase contrast microscope:(1)The effect of CD137-CD137L on endothelial cell tube formation;(2)The effect of CD137-CD137L on the endothelial cell tube formation after intervention of autophagy process.3.Autophagy markers LC3 Ⅱ/Ⅰ and P62 proteins were detected by applying Western blot after the intervention of the CD137-CD137L signal.4.Autophagy was detected by using mRFP-GFP-LC3 adenovirus.Using mRFP-GFP-LC3 adenovirus to transfect the endothelial cells and observe the number of autophagosomes and autolysosomes under fluorescence microscopy by intervention of the CD137-CD137L singal.5.Transwell assay was adopted to measure the number of migratory cells mediated by the CD137-CD137L signal after intervention of autophagy process.6.CCK-8 kits was used to detected the CD137-CD137L signal mediated endothelial cell proliferation ability after intervention of autophagy process.Results1.Compared with the control group,the total tube length was increased(P<0.05)and the mean length of sprouts was significantly increased(P<0.01)in the agonist-CD137 group;Compared with the agonist-CD 137 group,the total tube length of HUVECs and the mean length of sprouts in the anti-CD137 group was significantly decreased(P<0.01).2.Compared with the control group,the relative expression of LC3 Ⅱ protein expression was increased(P<0.05),the relative expression of autophagy substrate protein P62 was decreased(P<0.05)and the number of autophagosomes and autolysosomes were both obviously increased in the agonist-CD 137 group(P<0.01)when compared with the control group.Compared with the agonist-CD137 group,the relative expression of LC3 II protein was decreased(P<0.05),relative expression of P62 protein was increased(P<0.05)in the anti-CD137 group.Compared with the agonist-CD137 group,the number of autophagosomes and autolysosomes were both obviously decreased in the anti-CD 137 group(P<0.01).3.Applied 3-MA(5mM)to inhibited autophagy before activating the CD137-CD137L signal,the total tube length of HUVECs was decreased(P<0.05)and the mean length of sprouts were significantly decreased(P<0.01)in 3-MA group when compared with the agonist-CD137 group;The proliferation and migration ability of endothelial cells were both sharply decreased in 3-MA group(P<0.01)when compared with the agonist-CD137 group.ConclusionCD137-CD137L signal may influence te endothelial tube formation and aortic ring angiogenesis via activating endothelial autophagy.