In vivo and in vitro effects of CD137 stimulation on vascular calcification in high fat diet fed ApoE-/- mice

Abstract

Objective: To investigate the effect and related mechanism of CD137 stimulation on aortic atherosclerotic plaque calcification in high fat diet fed ApoE-/- mice and on calcification of vascular smooth muscle cells (VSMCs). Methods: (1) ApoE-/- mice fed with high fat diet were randomly divided into 3 groups: CD137 activated group (treated by 200 μg CD137 agonist i. p. once per week for 6 weeks, n=5); CD137 inhibited group (anti-CD137 group: 200 μg anti-CD137 antibody + 200 μg CD137 agonist, i. p., once per week for 6 weeks, n=5) and control group (n=5). Von kossa staining was used to observe the calcification of the aortic plaque and VSMCs. Immunohistochemistry was used to observe the expression of BMP-2 and Runx2 which are known mediators of osteogenic differentiation. (2) The mouse aortic VSMCs were obtained by Patch-attaching method. The calcium content was measured by Methylthymol Blue complexone method. The mRNA expressions of bone morphogenetic protein 2 (BMP-2) and Runx2 were measured by real-time fluorescent quantitative PCR (RT-PCR). The protein levels of BMP-2, Runx2 of the VSMCs were determined by Western blot. Results: (1) In vivo, the plaque calcified area in ApoE-/- mice was significantly larger in CD137-agonist group than that in control group ((1.75±0.33)×104 μm2 vs. (0.23±0.07)×104 μm2,P<0.01), and this effect was significantly reduced by cotreatment with CD137-antagonist ((0.83±0.30)×104 μm2 vs. (1.75 ±0.33)×104 μm2,P<0.05). The levels of BMP-2 and Runx2 were all significantly upregulated in CD137-agonist group than in control group (both P<0.01), again, this effect was blocked by cotreatment with CD137-antagonist (P<0.05). (2) Consistent with the in vivo results, VSMCs calcification was also more serious in CD137-agonist group than in control group, which could be significantly attenuated by cotreatment with CD137-antagonist. In VSMCs, calcium content level in CD137-agonist group was higher than in control group ((0.001 3±0.000 2) mmol/mg protein vs. (0.000 7±0.000 1) mmol/mg protein, P<0.01), which could be significantly reduced by co-treatment with CD137-antagonist ((0.000 9±0.000 2) mmol/mg protein vs. (0.001 3±0.000 2) mmol/mg protein, P<0.01). The mRNA and protein levels of BMP-2 and Runx2 were significantly upregulated in CD137-agonist group compared with the control group (P<0.05), which could be significantly down-regulated by cotreatment with CD-137 antagonist (P<0.05). Conclusion: CD137 activation can promote vascular calcification in high fat diet fed ApoE-/- mice both in vivo and in vitro.