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. 2010 Jan 8;106(1):145-54.
doi: 10.1161/CIRCRESAHA.109.209486. Epub 2009 Oct 29.

S100A12 mediates aortic wall remodeling and aortic aneurysm

Marion Hofmann Bowman  1 , Jeannine Wilk, Ahlke Heydemann, Gene Kim, Jalees Rehman, Joseph A Lodato, Jai Raman, Elizabeth M McNally
Affiliations

S100A12 mediates aortic wall remodeling and aortic aneurysm

Marion Hofmann Bowman et al. Circ Res. .

Abstract

Rationale: S100A12 is a small calcium binding protein that is a ligand of RAGE (receptor for advanced glycation end products). RAGE has been extensively implicated in inflammatory states such as atherosclerosis, but the role of S100A12 as its ligand is less clear.

Objective: To test the role of S100A12 in vascular inflammation, we generated and analyzed mice expressing human S100A12 in vascular smooth muscle under control of the smooth muscle 22alpha promoter because S100A12 is not present in mice.

Methods and results: Transgenic mice displayed pathological vascular remodeling with aberrant thickening of the aortic media, disarray of elastic fibers, and increased collagen deposition, together with increased latent matrix metalloproteinase-2 protein and reduction in smooth muscle stress fibers leading to a progressive dilatation of the aorta. In primary aortic smooth muscle cell cultures, we found that S100A12 mediates increased interleukin-6 production, activation of transforming growth factor beta pathways and increased metabolic activity with enhanced oxidative stress. To correlate our findings to human aortic aneurysmal disease, we examined S100A12 expression in aortic tissue from patients with thoracic aortic aneurysm and found increased S100A12 expression in vascular smooth muscle cells.

Conclusions: S100A12 expression is sufficient to activate pathogenic pathways through the modulation of oxidative stress, inflammation and vascular remodeling in vivo.

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Figures

Figure 1
Figure 1. Generation of S100A12 transgenic mice
(A) Human cDNA S100A12 was expressed under control of the smooth muscle promoter SM22α. (B) PCR showing TG+ mice. (C) Southern blotting revealed S100A12 incorporation. (D) Immunofluorescence microscopy of aortic frozen sections from WT mice (upper panel) and TG mice (lower panel) were stained with α-S100A12 IgG (red color), α-smooth muscle actin IgG (SMA, green color) and DAPI (blue color). (E) Immunofluorescence microscopy of cultured primary VSMC isolated from WT mice (left) and TG mice (right) were stained with α-S100A12 IgG (red color) and DAPI (blue color), Scale bar 10 μm. Immunoblotting with α-S100A12 IgG of 10 μg whole cell lysates from cultured VSMC shows no S100A12 expression in WT-VSMC (lane 1-3) and strong S100A12 expression in TG-VSMC at passage 3-8 (lane 4-6). (F) Immunoblotting with α-S100A12 IgG of lung and aortic tissue extracts reveals S100A12 protein expression in TG but in WT mice. (G) Immunoblotting with α-S100A12 IgG and α-βactin IgG of 10 μg tissue extracts from WT mouse aorta (lane 1), TG mouse aorta (lane 2), human healthy aorta from heart donors (lane 3-4), and human aorta from patients undergoing aortic root repair for cystic media necrosis (h aorta-TAA, lane 5-6). (H) Immunoblotting with α-S100A12 IgG and α-βactin IgG of whole cell lysate of cultured aortic SMC. SMC from human thoracic aneurysm at baseline (lane 1) and after LPS 1 μg/ml for 24 h (lane 2), SMC from human control aorta at baseline (lane 3) and after 1 μg/ml LPS for 24 h (lane 4), SMC from TG-aorta (lane 5) and SMC from WT-aorta (lane 6).
Figure 2
Figure 2. Characterization of aortic wall structure
(A-P) Histology of aortic sections obtained from the mid aortic arch from 10 week old WT mice (left column, Figure A, E, I and M), 10 week old TG mice (second column from left, Figure B, F, J, N), 16 week old WT mice (third column, Figure C, G, K, O) and 16 week old TG mice (right column, Figure D, H, L, P). Serial sections were stained using H&E, Masson’s Trichrome and Verhoeff-vanGiessen. The upper panel shows a 25x magnification of the mid aortic arch with the following structures: N=vagal nerve, E=esophagus, L Br= left bronchus, R Br= right bronchus. * marks areas of disrupted elastic fibers (scale bar 100μm in A-D). The lower three panel shows a 400x magnification (scale bar =10μm) from the fields marked in A-D. (Q) Medial elastin degradation in WT and TG mice were graded on VVG stained section of the aortic arch as following: grade 1: no disruption; grade 2: disruption of one elastic fiber with intact neighboring fibers; grade 3: disruption of two or more fibers in direct contact, grade 4: disruption of all elastic fibers from the internal elastic lamina to the external elastic lamina. (n=6 mice of each group, * p<0.005). (R) Collagen deposition in the proximal aortic arch was evaluated by measurement of the blue stained area on the MT slides (6 mice of each group measuring 5 random fields per slide, * p<0.005). (S) Aortic wall thickness was measured on a longitudinally cut section of the aortic arch stained with VVG (6 mice of each group, measuring thickness × 5 per slide, * p=0.01). (T) Myeloperoxidase activity. Aortic tissue were retrieved from TG and WT mice (n=3 per group and time point) and assayed for MPO activity, *p<0.05.
Figure 3
Figure 3. Abnormal aortic tissue composition
(A) Immunoblot from aortic tissues from TG and WT (n=3 for each group, *p<0.05). (B) Representative gelatin Zymogram from aortic tissue lysates from WT and TG mice (n=4 for each group for quantitation, *p<0.01). (C) Gelatin zymogram from concentrated supernatant of cultured aortic SMC harvested from WT and TG-aorta. The insert shows an immunoblot of lysed SMC using anti-MMP-2 IgG.
Figure 4
Figure 4. In vivo ultrasound of the aortic root of TG mice and WT littermate mice
(A) Baseline ultrasound measurements were performed at 10 weeks of age, followed by repeat ultrasound at 16 weeks (B) and 22 weeks of age (C). Ao (proximal aorta), PA (pulmonary artery), BC brachiocephalic trunc, aortic root dimensions were measured 0.5 mm proximal of the aortic valve, scale bar 1 mm. Male (D) and female (E) TG mice (n=4) showed significant dilatation of the aortic root at age 16 and 22 weeks compared to their respective gender-matched littermates (n=4 for each group, * p<0.01).
Figure 5
Figure 5. S100A12 induces IL-6 and nuclear translocation of pSmad2
(A) ELISA measuring IL-6 from cell culture supernatant from TG-VSMC and WT-VSMC cultures stimulated with 1 μg/ml LPS and soluble RAGE as indicated (*p < 0.001 compared to baseline conditions, and # p<0.05 compared to LPS stimulated condition) (B) Immunoblotting with α-S100A12 IgG using culture supernatant from TG-VSMC (lane 1, 2) and WT-VSMC (lane 3, 4) revealed S100A12 protein release into the cell culture supernatant only from transgenic VSMC after treatment with LPS. (C) IL-6 was also produced from aortic ring tissues (3 mm) after LPS exposure (* p=0.03). (D) IL-6 ELISA from mouse serum (*< 0.05). (E) Immunofluorescence microscopy of cultured primary VSMC isolated from WT mice (upper panel) and TG mice (mid and lower panel) were stained with α-pSmad2 IgG and DAPI, scale bar 20 μm. Pretreatment with neutralizing TGF-β (0.5μg/ml) is shown in the lower panel.
Figure 6
Figure 6. Effect of S100A12 on metabolic activity in cultured VSMCs
(A) Incubatin of cultured VSMC with BrdU showed reduced proliferatin. (B) VSMC were treated with MTS and TG-VSMC were shown to have increased metabolic activity. (C-D) Flow cytometry of cultured VSMC from WT and TG mice were stained with MitoTrackerGreen (C) and MitoSOX (D) consistent with increased mitochondrial content in TG-VSMC. (E) Immunofluorescence microscopy of cultured WT-VSMC (left) and TG-VSMC (right) with α-8-oxo-dG IgG (red color) and DAPI (blue color) showed an increase in α-8-oxo-dG staining (*p<0.005). (F) WT-VSMC and TG-VSMC were stimulated with BSA or recombinant S100A12, and stained with MitoSOX and analyzed by FACS for Mean Fluorescence Intensity. (G). WT-VSMC and TG-VSMC were co-cultured for 3 days prior to staining with MitoSOX dye (green color) and α-S100A12-IgG (red color). Fluorescence intensity was measured in TG-VSMC and WT-VSMC on 5 power fields (* p< 0.005).
Figure 7
Figure 7. S100A12 is expressed in VSMC in human aortic tissue with cystic media necrosis
(A) Pedigree of a family with familial thoracic aneurysms with the MYH11 mutation. Affected family members with aortic dissection and aortic valve replacement are marked with ν. (B-C) Three dimensional reconstruction of the computed tomography scans of the proximal aorta shows a 5.2 cm aneurysm distal the area of prior aortic repair. (D) Resected TAA aorta and control aortic tissue were stained using vVG, α-smooth muscle actin (SMA) and α-S100A12 antibodies. The tunica interna, media (MEDIA) and adventia (AD) are shown. (E) Colocalization of SMA and S100A12 is shown by immunofluorescence microscopy.
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Comment in

  • S100A12 links to thoracic aortic aneurysms.
    Daugherty A, Rateri DL, Lu H. Daugherty A, et al. Circ Res. 2010 Jan 8;106(1):13-5. doi: 10.1161/CIRCRESAHA.109.210757. Circ Res. 2010. PMID: 20056940 Free PMC article. No abstract available.

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