title: [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)×104μm2vs. (0.23±0.07)×104μm2,P<0.01), and this effect was significantly reduced by cotreatment with CD137-antagonist ((0.83±0.30)×104μm2vs. (1.75 ±0.33)×104μm2,P<0.05). The levels of BMP-2 and Runx2 were all significantly upregulated in CD137-agonist group than in control group (bothP<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.
authors.affiliation: Department of Cardiology, Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.forename: J C
authors.initials: JC
authors.lastname: Yan
authors.forename: J Y
authors.initials: JY
authors.lastname: Weng
authors.forename: Z Q
authors.initials: ZQ
authors.lastname: Wang
authors.forename: C P
authors.initials: CP
authors.lastname: Wang
authors.forename: C
authors.initials: C
authors.lastname: Shao
mesh_terms: D000818:Animals; D001011:Aorta; D001057:Apolipoproteins E; D002118:Calcium; D002454:Cell Differentiation; D059305:Diet, High-Fat; D051379:Mice; D018345:Mice, Knockout; D010012:Osteogenesis; D058226:Plaque, Atherosclerotic; D012333:RNA, Messenger; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9; D015854:Up-Regulation; D061205:Vascular Calcification
chemical_list: D001057:Apolipoproteins E; D012333:RNA, Messenger; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9; D002118:Calcium