CD137 signaling promotes the formation of plaque calcification via inhibiting the fusion of autophagy and lysosomal in Apo E-/- mice / 中华心血管病杂志

ABSTRACT

Objective@#To investigate whether CD137 signaling promoted the formation of atherosclerotic plaque calcification by inhibiting the fusion of autophagosome and lysosome.@*Methods@#(1) In vivo, CD137 agonist antibody and anti-CD137 antibody were used to stimulate and inhibit the CD137 signaling, respectively. Fifteen Apo E-/- mice were randomly divided into three groups control group (intraperitoneal injection of IgG2b 200 µg) , CD137 agonist group (intraperitoneal injection of CD137 agonist antibody 200 µg) , anti-CD137 group (pretreatment with 200 µg anti-CD137 antibody for 24 hours, then injection of CD137 agonist antibody) . (2) In vitro, primary culture of mouse aortic VSMCs obtained through adherence methods for tissues explants. The cells was divided into three groups control group, agonist-CD137 group (CD137 agonist antibody 10 μg/ml) , and anti-CD137 group (pretreatment with 10 μg/ml anti-CD137 antibody for 60 minutes, then incubated with 10 μg/ml CD137 agonist antibody) . Von kossa staining was used to detect the calcification in the cell and plaque. Immunohistochemical staining was used to observe the expression of LC3B, Beclin 1 and p62 which are associated with autophagy. The levels of autophagy related protein (LC3) , Beclin 1, p62, and the expression of Runx2 and bone morphogenetic protein 2, which is associated with osteogenic differentiation in the VSMCs, were determined by Western blot. The autophagy flow of each group was detected by fluorescence microscopy. The autophagy was observed by transmission electron microscope in vivo and in vitro.@*Results@#(1) In vivo, the calcified plaque area in CD137 agonist group was significantly larger than that in the control group (3.01%±0.45% vs. 0.27%±0.06%, P<0.01) , and calcified plaque area in anti-CD137 group was significantly smaller compared with that in the CD137 agonist group (1.23%±0.39% vs. 3.01%±0.45%, P<0.05) . Immunohistochemical staining showed that the expression of early autophagy marker protein LC3B and Beclin 1 were significantly upregulated in CD137 agonist group and anti-CD137 group than in control group, and the highest expression was observed in CD137 agonist group (P<0.05) . The expression of advanced autophagy marker protein p62 was higher in the CD137 agonist group than in the anti-CD137 group (P<0.05) . (2) In vitro, the ratio of autophagy related protein LC3 Ⅱ/Ⅰ and p62 protein expression were significantly higher in CD137 agonist group and anti-CD137 group than in control group (P<0.01) , while the expression of p62 protein was significantly higher in CD137 agonist group than that in anti-CD137 group (P<0.05) . In the cell calcification inducing experiment, the expression of BMP-2 and Runx2 protein was significantly higher in CD137 agonist group than that in control group (P<0.01) , but the levels of BMP-2 and Runx2 protein were lower in anti-CD137 group than in CD137 agonist group (P<0.05) .@*Conclusion@#Our results indicate that activation of CD137 signaling can promote the formation of atherosclerotic plaque calcification by inhibiting the fusion of autophagosome and lysosome.