CD137-CD137L Signaling Promotes The Formation Of Plaque Calcification In ApoE^-/- Mice Through Regulation Of Autophagy

ObjectiveTo observe whether the CD137 signaling would affect the progression of atherosclerotic plaque calcification through inhibiting the fusion of autophagosome and lysosome.MethodsIn this study,we use ApoE^-/-mice and primary cultured mouse aortic VSMCs from adherence methods for tissues explants as a model.Thirty ApoE^-/-mice and primary VSMCs were randomly divided into three groups: control group: pre-stimulate by cytokine mixture（TNFα,INF-γ,IL-1β10ng/ml respectively）,agonist-CD137 group: control group + recombinant CD137 L protein;anti-CD137 group: control group + inhibitory CD137 antibody + recombinant CD137 L protein.Vonkossa staining was used to detect the calcification in the cell and plaque.Immunohistochemical staining was used to observe the expression of LC3 B,Beclin 1and p62 which are associated with autophagy.Western blot was used to determine the level of autophagy related protein（LC3）,Beclin 1,p62,and the expression of Runx2 and BMP-2 which are associated with osteogenic differentiation in the VSMCs.Fluorescence microscopy was adopted to detect the autophagy flow of each group.Flow cytometry was used to measure the autophagy levels of VSMC.Transmission electron microscope was adopted to detect the autophagosomes in vivo and vitro.Results（1）Western blot showed that the levels of autophagy-related protein（LC3II / I ratio）in mouse VSMCs increased in a time and concentration-dependent manner when CD137 singal was activated.The autophagy occurs highest when recombinant-CD137 L stimulate 6h at 10μg / ml.The ratio of LC3 II / I in agonist-CD137 group and anti-CD137 group was significantly higher than that in control group（9.41 ± 0.93 vs 1.61 ± 0.33;4.20 ± 0.55 vs 1.61 ± 0.33,P <0.01）,and the agonist-CD137 group was higher than the anti-CD137 group.The expression of p62 protein in agonist-CD137 group was higher than that in anti-CD137 group（2.76 ± 0.39 vs 2.16 ± 0.18,P <0.05）.（2）mRFP-GFP-LC3 adenovirus was used to mark autophagosomes,and the result of fluorescence microscopy showed that the total number of spots in the agonist-CD137 group was higher than that in the control group,and the yellow spots were significantly higher than those in the control group,and there were only few red spots,indicating that autophagy was enhanced and autophagy flux was blocked;The total number of spots in the anti-CD137 group was also higher than that in the control group.The average red spots of each cell were more than that of the agonist-CD137 group,while the yellow spots were less than those in the agonist-CD137 group,indicating that the autophagy flow was smooth.（3）We use flow cytometry to observe the level of autophagy.The result showed that the autophagy level of VSMCs in agonist-CD137 group was（77.3 ± 6.4%）,（72.5 ± 7.1%）in anti-CD137 group,63.0 ± 5.1% in control group,The level of autophagy in agonist-CD137 group and anti-CD137 group were both higher than that of the control group（P<0.05）,and the agonist-CD137 group was the highest.（4）The autophagosomes of ApoE^-/-mouse atherosclerosis plaques and smooth muscle cells were detected by transmission electron microscopy.The results showed that the number of autophagosomes in agonist-CD137 group was higher than that in anti-CD137 group,autophagy was less active than the anti-CD137 group.We also detected the autophagosomes in cultured VSMCs in vitro,and the result was consistent with that in the vivo,which showed that the number of autophagosomes in agonist-CD137 group was higher than that in anti-CD137 group,the number of autophagosomes was less.（5）In the cell calcification induction experiment,the expression of BMP-2 and Runx2 in agonist-CD137 group was higher than that in control group（3.39 ± 0.32 vs 1.25 ± 0.57,2.48 ± 0.17 vs 0.88 ± 0.08,P <0.01）.The expression of BMP-2 and Runx2 protein in anti-CD137 group was lower than that in agonist-CD137 group（2.42 ± 0.14 vs 3.39 ± 0.32,2.39 ± 0.15 vs 2.48 ± 0.17,P <0.05）.Vonkossa staining showed that the calcification of VSMC in agonist-CD137 group was more serious than the control group,and the cell calcification in anti-CD137 group was ligter than that in the agonist-CD137 group.（6）Vonkossa staining of the ApoE^-/-mouse atherosclerosis plaque showed that,the agonist-CD137 showed significant lumen structure disorder,intimal destruction,poor continuity,visible point of calcification,and the calcification area was increased（3.01±0.45 vs 0.27±0.06,P<0.01）.While the calcified area of anti-CD137 group was less than the agonist-CD137 group（1.23±0.39 vs 3.01±0.45,P<0.05）。The results of immunohistochemical staining showed that the expression of LC3 B and Beclin 1 in the agonist-CD137 group and the anti-CD137 group were significantly higher than those in the control group.The late marker protein p62 expression in the agonist-CD137 group was also higher than that in the anti-CD137 group.ConclusionsCD137 signaling may affect the progression of calcification of atherosclerosis plaque by inhibiting the fusion of the autophagosome and lysosome.