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. 2011 Feb;31(2):337-44.
doi: 10.1161/ATVBAHA.110.217745. Epub 2010 Oct 21.

S100A12 in vascular smooth muscle accelerates vascular calcification in apolipoprotein E-null mice by activating an osteogenic gene regulatory program

Marion A Hofmann Bowman  1 , Joseph Gawdzik, Usama Bukhari, Aliya N Husain, Peter T Toth, Gene Kim, Judy Earley, Elizabeth M McNally
Affiliations

S100A12 in vascular smooth muscle accelerates vascular calcification in apolipoprotein E-null mice by activating an osteogenic gene regulatory program

Marion A Hofmann Bowman et al. Arterioscler Thromb Vasc Biol. 2011 Feb.

Abstract

Objective: The proinflammatory cytokine S100A12 is associated with coronary atherosclerotic plaque rupture. We previously generated transgenic mice with vascular smooth muscle-targeted expression of human S100A12 and found that these mice developed aortic aneurysmal dilation of the thoracic aorta. In the current study, we tested the hypothesis that S100A12 expressed in vascular smooth muscle in atherosclerosis-prone apolipoprotein E (ApoE)-null mice would accelerate atherosclerosis.

Methods and results: ApoE-null mice with or without the S100A12 transgene were analyzed. We found a 1.4-fold increase in atherosclerotic plaque size and more specifically a large increase in calcified plaque area (45% versus 7% of innominate artery plaques and 18% versus 10% of aortic root plaques) in S100A12/ApoE-null mice compared with wild-type/ApoE-null littermates. Expression of bone morphogenic protein and other osteoblastic genes was increased in aorta and cultured vascular smooth muscle, and importantly, these changes in gene expression preceded the development of vascular calcification in S100A12/ApoE-null mice. Accelerated atherosclerosis and vascular calcification were mediated, at least in part, by oxidative stress because inhibition of NADPH oxidase attenuated S100A12-mediated osteogenesis in cultured vascular smooth muscle cells. S100A12 transgenic mice in the wild-type background (ApoE+/+) showed minimal vascular calcification, suggesting that S100A12 requires a proinflammatory/proatherosclerotic environment to induce osteoblastic differentiation and vascular calcification.

Conclusions: Vascular smooth muscle S100A12 accelerates atherosclerosis and augments atherosclerosis-triggered osteogenesis, reminiscent of features associated with plaque instability.

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Figures

Figure 1
Figure 1
Advanced calcification within the atherosclerotic lesions in the innominate artery (A) and medial calcification in the aortic arch (B and C) in 10-month-old S100A12/ApoE-null mice. A, Alizarin red stain shows large calcified nodules extending from the lumen (L) to internal elastic lamina in S100A12/ApoE mice (a) but not in advanced lesions of WT/ApoE-null littermates (d). Fibrofatty nodules (FFN) with cholesterol clefts are seen in both groups. S100A12/ApoE-null lesions showed layering of the lesions with presence of chondrocyte-like cells (marked *** in b and inset) and extensive erosion of the media (marked # # # in c) on Verhoeff-Van Giesen (VVG) stain. B, Alizarin red stain shows extensive medial calcification in the aortic arch of S100A12/ApoE-null mice, compared with calcifications located within the neointimal hyperplasia of a large plaque in aortic arch of WT/ApoE-null mice (C). With heavy vascular calcification, there was increased fragility of the vasculature in S100A12/ApoE-null mice during tissue processing, leading to artifacts (as seen in B).
Figure 2
Figure 2
A, Atherosclerotic lesion in the innominate artery of 3-month-old S100A12/ApoE-null mice (a to c) and of age-matched WT/ApoE-null littermates (d to f) shows atherosclerotic lesions in both groups and small areas of calcium deposition in S100A12/ApoE-null mice. B. Quantitative reverse transcription–polymerase chain reaction shows increased gene expression of Runx-2, BMP-2, Dmp-1, and BGLAP in aortae harvested from S100A12/ApoE-null mice. GAPDH expression was used as internal control. Data are expressed as mean±SEM (*P<0.001 and **P=0.05 versus WT).
Figure 3
Figure 3
S100A12 promotes calcification of VSMC cultured in conditioned media from primary mouse macrophages. A, Alizarin red S stain of VSMC harvested from the aortae of S100A12/ApoE-null mice and cultured in conditioned medium developed mineralized nodules (arrow) and cellular calcium deposits (arrowhead). B, Quantification of calcified nodules by counting 5 random ×10 power fields (*P<0.003 versus DMEM and WT). C, Total cell alizarin red staining (osteogenesis assay, Millipore) of VSMC cultured in conditioned media and treated with soluble RAGE (sRAGE) as indicated (**P<0.05 compared with bovine serum albumin [BSA]). D, Quantitative reverse transcription–polymerase chain reaction for gene expression of BMP-2, BGLAP, Dmp-1, and Runx-2 in S100A12 VSMC and WT VSMC that were cultured in DMEM or conditioned media and treated with sRAGE as indicated. GAPDH expression was used as internal control, and data are expressed as mean±SEM of fold increase compared with WT VSMC cultured in DMEM (***P<0.03 compared with baseline condition and with WT VSMC, **P=0.05 compared with S100A12-VSMC in conditioned media with BSA).
Figure 4
Figure 4
Effect of S100A12 on oxidative stress and ossification. A, 8-Isoprostane in the urine of WT/ApoE-null and S100A12/ApoE-null at 4 and 10 months (M) of age. (*P<0.05, **P<0.001). B, Intracellular hydrogen peroxide levels measured by 2′7-dichlorofluorescence diacetate (DCFDA) microscopy in cultured VSMC (*P<0.05, **P<0.001). Cond. indicates conditioned. C, Effect of apocynin and DPI on calcifying nodule formation in cultured VSMC from S100A12/ApoE-null aorta. Calcification nodules were quantified after staining with alizarin red (*P=0.05). D, Effect of apocynin and DPI on gene expression of Runx-2, BMP-2, BGLAP, and dentin matrix acidic phosphoprotein 1 (Drp-1) in VSMC from S100A12/ApoE-null aorta cultured in conditioned media (*P<0.001 compared with control dimethyl sulfoxide [DMSO]–treated cells).
Figure 5
Figure 5
S100A12 associates with Nox1. A, Ang II stimulated Nox-1 expression in rat VSMC (A7r5) in cells transiently transfected with S100A12-myc and in control cells. B, Membrane extracts from S100A12-myc or mock-transfected rat VSMC stimulated with and without Ang II were immunoprecipitated with antimyc IgG. Immunoblotting shows S100A12 in transfected cells (two left lanes) and Nox1 in S100A12-transfected and Ang II–stimulated VSMC (second lane from left). C, Membrane extracts were immunoprecipitated with α-Nox1 IgG or nonimmune rabbit IgG, followed by immunoblotting for S100A12. IB indicates immunoblotting; IP, immunoprecipitation.
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Comment in

  • Vascular calcification: it's all the RAGE!
    Towler DA. Towler DA. Arterioscler Thromb Vasc Biol. 2011 Feb;31(2):237-9. doi: 10.1161/ATVBAHA.110.220038. Arterioscler Thromb Vasc Biol. 2011. PMID: 21248279 Free PMC article. No abstract available.

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References

    1. Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2000;20:1262–1275. - PubMed
    1. Aikawa E, Nahrendorf M, Figueiredo JL, Swirski FK, Shtatland T, Kohler RH, Jaffer FA, Aikawa M, Weissleder R. Osteogenesis associates with inflammation in early-stage atherosclerosis evaluated by molecular imaging in vivo. Circulation. 2007;116:2841–2850. - PubMed
    1. Ding HT, Wang CG, Zhang TL, Wang K. Fibronectin enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular smooth muscle cells via ERK pathway. J Cell Biochem. 2006;99:1343–1352. - PubMed
    1. Foell D, Wittkowski H, Vogl T, Roth J. S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules. J Leukoc Biol. 2007;81:28–37. - PubMed
    1. Hofmann Bowman M, Wilk J, Heydemann A, Kim G, Rehman J, Lodato JA, Raman J, McNally EM. S100A12 mediates aortic wall remodeling and aortic aneurysm. Circ Res. 2010;106:145–154. - PMC - PubMed

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