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.)

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.

Background: ELDL is present in human atherosclerotic lesions and promotes foam cell formation in cultured macrophages and vascular smooth muscle cells (SMC). Here we study mechanism of ELDL uptake and its effects on SMCs. Methods and Results: Incubation of wild type murine aortic SMCs with 10 μg/ml ELDL (trypsin, cholesterol esterase modified) results in enhanced foam cell formation (analyzed by Oil Red O, lipid measurement) compared to SMCs incubated with acetylated LDL (500 μg/ml; -50%, p<0.01) and oxidized LDL (200 μg/ml; -75%, p<0.01). Inhibitors of macropinocytosis (50 μM LY294006, 2 μM wortmannin, and 3 mM amiloride) attenuated ELDL uptake (-50%, -50%, -100% respectively). In contrast, inhibitors of receptor mediated endocytosis (100 μM dynasore, 0.1 M Sucrose), and inhibitors of caveolae /lipid raft mediated endocytosis (5mM MBCD, 5 μM filipin) had no effect on ELDL uptake in SMCs. Moreover, ELDL incubation led to increased expression of scavenger receptor LOX1 (+ 3 fold, p<0.01) in wild type SMC’s, but not in SMC deficient in Receptor for AGE (RAGE-/-), while CD36 and SRA1 remained unchanged in both the SMCs. Importantly, RAGE-/- SMCs upon pretreatment with PI3K inhibitors that only partially inhibited macropinocytosis of ELDL in wild type SMCs, completely prevented ELDL uptake in RAGE-/- SMCs. Mechanistically, ELDL upregulates ROS (detected using H2DCFDA) and down regulates PIP3 (detected by pAkt immunoblotting) in wild type, but not in RAGE-/- SMCs. Since ROS is known to regulate macropinocytosis via increased Ca2+ levels, we tested Ca2+ channel inhibitor lacidipine (30 μM), and found complete inhibition of ELDL uptake in both, wild type and RAGE-/- SMCs. Lastly, we speculate that the fused structure of LDL in the ELDL preparation is preferentially activating RAGE, since oligomerization of ligands are known to increase RAGE signaling, and FPLC analysis demonstrated that ELDL consists mostly of fused LDL particles. Conclusions: ELDL is highly potent in inducing foam cells in aortic SMCs. ELDL endocytosis is mediated by RAGE-regulated, Ca2+ dependent macropinocytosis.

Background: Serum S100A12 and fibroblast growth factor (FGF) 23 are biomarkers for cardiovascular mortality in patients with chronic kidney disease (CKD) and are associated with left ventricular hypertrophy (LVH). FGF23 is induced in cultured cardiac fibroblasts in response to cytokines including IL-6, TNF-a, LPS and S100/calgranulins. Moreover, hBAC-S100 transgenic mice with CKD had increased FGF23 in valvular interstitial cells and exhibited LVH. The present study was designed to examine cardiac FGF23 expression in other murine models of LVH in the absence of CKD. Methods: Hearts from five groups of male mice were studied: (i) C57BL6/J with transgenic expression a bacterial artificial chromosome of the human S100/calgranulins (S1008/9 and S100A12, hBAC-S100), (ii) wild type littermates, (iii) LDLR-/- infused with saline (29 days, 0.9%), (iv) LDLR-/- infused with angiotensin (Ang) II (29 days, 1000 ng/kg/min), and (v) fibroblast specific depletion of angiotensin II type 1a receptor (AT1aR) (S100A4-Cre x AT1aR-/- x LDLR-/-) infused with AngII. Results: hBAC-S100, but not wild type littermate mice, developed significant LVH at 10 months by heart weight/body weight (5.9 ±1.1 mg/g vs. 4.2 ±0.8, p<0.04), decreased E/A ratio, and increased LVPW thickness, and associated with increased expression of FGF23 mRNA and protein in cardiac tissue lysates (2-4 fold increase). Similarly, Ang II induced significant LVH compared to saline infused LDLR-/- mice (6.1±1.3 vs. 3.6 ±0.9 mg/g, p<0.01), and associated with increased mRNA for hypertrophic genes (ANP, BNP, b-MHC, CTGF and Col1a1). However, there was no significant difference in FGF23 mRNA and protein between Ang II and saline infused mice. Cardiac hypertrophy was attenuated in AngII-infused mice with deficiency of AT1aR (S100A4-Cre+/-xAT1aRxLDLR-/-). In vitro, Ang II (100nM) did not induce FGF23 in valvular interstitial fibroblasts or myocytes. Summary: Transgenic expression of S100/calgranulins is sufficient to induce LVH in aged mice with normal renal function, and this is associated with FGF23 expression in cardiac interstitial fibroblasts. Future studies are needed to determine whether cardiac FGF23 promotes LVH in a paracrine manner. However, FGF23 does not play a role in Ang II-induced LVH.

Background: Enzyme modified LDL (ELDL) is present in human atherosclerotic lesions and is a major foam cell-forming modified LDL for murine vascular smooth muscle cells (SMC) as reported by us previously. Here we study ELDL and its effects on human coronary artery SMC (HCASMC) in vitro. Methods and Results: Incubation of HCASMC with 10 μg/ml ELDL (trypsin, cholesterol esterase modified) resulted in significant foam cell formation (analyzed by Oil Red O, lipid measurement) compared to HCASMC incubated with oxidized LDL (200 μg/ml; -75%, p<0.01) or native LDL (200 μg/ml; -50%, p<0.01). Whole genome gene expression (Illumina Bead Chip HT12v4, analyzed by DAVID v6.8 and IPA) of HCASMC treated with ELDL, oxLDL, LDL, and control (cell culture medium only) showed several top canonical pathways specifically induced by ELDL, together with activated upstream regulators including p38MAPK, NFkB. ERK. Upregulation of ANGPTL4 and BMP-2 -mRNA (22 and 2 fold respectively over native LDL) was verified by qRT-PCR and immunoblotting. ELDL-induced foam cells showed dose dependent (1-20 μg/ml ELDL) increase in migration in collagen coated trans well dishes, which was attenuated by Lacidepine, a known inhibitor of ELDL uptake in murine SMCs. Furthermore, rANGPTL4 also upregulated HCASMC migration dose dependently (1-5 μg/ml for 24 h) and was comparable with the migration induced by ELDL. However, Lacidipine had no effect on rANGPLT4 mediated migration, suggesting that ANGPLT-4 independently of ELDL uptake promotes migration of HCASMC. In calcification assays using MEM with 0.2% FCS and 1.5 mM phosphate, ELDL at 2.5 μg/ml induced more calcification native LDL (>25%, p<0.01, analyzed by alizarin red staining and organic extraction, and this was proceeded by increase in BMP-2 mRNA. Conclusions: ELDL is highly potent in inducing foam cells in cultured HCASMC. Whole genome expression and bioinformatics analysis indicate up-regulation of pathways linked to osteochondrogenic transformation. BMP-2 and ANGPTL4 are significantly upregulated in ELDL-induced HCASMC foam cells. These results point to the potential of ELDL to induce migratory and osteoblastic effects in HCASMC with potential implications in SMC migration and calcification in human atherosclerosis.