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 22␣ 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 ␤ 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. (Circ Res. 2010;106:145-154.)

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S100A12 is a small calcium binding protein that is a signal transduction ligand of the receptor for advance glycation endproducts (RAGE). S100A12, like RAGE, is expressed in the vessel wall of atherosclerotic vasculature, particularly in smooth muscle cells (SMC). While RAGE has been extensively implicated in inflammatory states such as atherosclerosis, the role of S100A12 is less clear. We tested the hypothesis that expression of human S100A12 directly exacerbates vascular inflammation. Several lines of Bl6/J transgenic mice (tg) expressing human S100A12 in SMC under control of the SM22a promoter were generated. Primary aortic SMC from tg and wild type (wt) littermates were isolated and analyzed for (i) proliferation using MTS/Formazan Assay and BrdU incorporation, (ii) oxidative stress using using flow cytometry with MitoSOX antibody, oxidative DNA damage using immunofluorescence microscopy with anti-8-oxo-dG antibody, and NF-kB activation measured by EMSA and (iii) cytokine expression measured by IL-6 ELISA. Furthermore, the aortas from tg and wt mice were examined. Results: Tg but not wt SMC expressed S100A12 protein. Tg SMC had a significant 1.9 to 2.7 fold increase in conversion of MTS into Formazan at 24–96 hours likely reflective of increased metabolic activity since BrdU incorporation into DNA was less in tg compared to wt SMC (4% vs 21% positive BrdU nuclei, p <0.05). Tg SMC showed significantly higher levels of mitochondrial generated ROS, nuclear staining for oxidative DNA damage which was not detected in the nuclei of wt SMC’s, and a 2.5 fold increase in NFkB activity. IL-6 production at baseline was higher in tg SMC’s (615 vs 213 pg/ml, p< 0.05) and increased dramatically after LPS treatment (10 ng/ml) in tg SMC’s (2130 vs 415 pg/ml). Histologic examination of the thoracic aorta at 10 weeks of age revealed increased collagen deposition in the aortic media with fragmentation and disarray of elastic fibers. In vivo ultrasound revealed a progressive dilation of the aortic arch from age 10 weeks to 16 weeks of age (1.27 to 1.60 mm, p<0.05) in tg but not in wt littermate mice (1.30 to 1.33 mm, p=0.1). These data reveal the novel finding that targeted expression of human S100A12 in SMC modulates oxidative stress, inflammation and vascular remodeling.

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