In vivo and in vitro effects of CD137 stimulation on vascular calcification in high fat diet fed ApoE-/-mice
10.3760/cma.j.issn.0253-3758.2016.10.010
- VernacularTitle:CD137-CD137L信号通路对小鼠动脉粥样斑块及血管平滑肌细胞钙化的影响
- Author:
Yao CHEN
1
;
Jinchuan YAN
;
Jiayi WENG
;
Zhongqun WANG
;
Cuiping WANG
;
Chen SHAO
Author Information
1. 江苏大学附属医院心内科
- Keywords:
Atherosclerosis;
Antigens,CD137;
Myocytes,smooth muscle;
Blood vessels
- From:
Chinese Journal of Cardiology
2016;44(10):879-884
- CountryChina
- Language:Chinese
-
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 eotreatment 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.
