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. 2013 May;228(1):69-79.
doi: 10.1016/j.atherosclerosis.2013.02.023. Epub 2013 Feb 28.

Beneficial effects of quinoline-3-carboxamide (ABR-215757) on atherosclerotic plaque morphology in S100A12 transgenic ApoE null mice

Ling Yan  1 , Per Bjork, Radu Butuc, Joseph Gawdzik, Judy Earley, Gene Kim, Marion A Hofmann Bowman
Affiliations

Beneficial effects of quinoline-3-carboxamide (ABR-215757) on atherosclerotic plaque morphology in S100A12 transgenic ApoE null mice

Ling Yan et al. Atherosclerosis. 2013 May.

Abstract

Objective: There is an emerging widespread interest in the role of damage-associated molecular pattern molecules (DAMP) S100A8, S100A9 and S100A12 in cardiovascular and other diseases. In this study we tested the efficacy of ABR-215757, a S100 protein binding immuno-modulatory compound to stabilize atherosclerosis in transgenic ApoE null mice that express the human pro-inflammatory S100A12 protein within the smooth muscle cell (SM22α-S100A12).

Methods: Twelve-week old S100A12 transgenic/ApoE(-/-) and WT/ApoE(-/-) mice were treated with ABR-21575 for 5 weeks and were analyzed 4 month later.

Results: Surface plasmon resonance analysis demonstrated that S100A12 interacts with ABR-215757 in a zinc dependent manner in vitro. In vivo, ABR-215757 administration reduced features of advanced plaque morphology resulting in smaller necrotic cores, diminished intimal and medial vascular calcification, and reduced amount of infiltrating inflammatory cells. ABR-215757 normalized aortic expression of RAGE protein and normalized experimentally-induced delayed hypersensitivity. The effect of ABR-215757 was more prominent in ApoE(-/-) mice expressing S100A12 than in ApoE(-/-) animals lacking expression of human S100A12 protein.

Conclusion: Our data suggest that S100A12 is important for progression of atherosclerosis and can be targeted by the small molecule ABR-215757. The specific binding of quinoline-3-carboxamides to S100A12 attenuates S100A12-mediated features of accelerated murine atherosclerosis.

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Conflict of interest statement

Disclosures: PB is employed by Active Biotech, which is developing quinolines for commercial purposes. All other authors have no conflict of interest.

Figures

Figure 1
Figure 1. S100A12 and S100A9 interact differently with immobilized ABR-215757 and hRAGE
A. Binding to immobilized ABR-215757. Sensorgrams obtained after injection (1) of 100 nM S100A12 or S100A9 (positive control) for 3 min at 30 μL/min over amine-coupled immobilized ABR-215757 in the presence of 1 mM Ca2+ and 20 μM Zn2+. Sample buffer was pumped over the surface (2) and final regeneration was performed with a 60 s pulse of 3 mM EDTA (3). B. Binding of S100A12 and S100A9 after pre-incubation with ABR-215757. 100 nM S100A12 or S100A9 injected with 7.81-1000 μM ABR-215757 shown as the response during the late dissociation phase in % of response without competitor. Data were fit to a sigmoidal dose-response curve using GraphPad Prism. Fifty percent inhibition (IC50) was obtained at 80 μM and > 1 mM ABR-215757 for S100A9 and S100A12, respectively. C. Binding to immobilized hRAGE. RAGE was immobilized at a density of 3 kRU. Other conditions are as in A. D. Inhibition of RAGE binding with ABR-215757. IC50 values of 85 μM and > 1000 μM were calculated for S100A9 and S100A12 binding to RAGE which demonstrate that S100A12-hRAGE association is weakly displaced by ABR-215757 in solution. E/F. Effects of heparan sulfate (HS) on S100A12 and S100A9 binding to RAGE and ABR-215757. 100 nM S100A12 (■), S100A9 (◆) or S100A8/9 (▲) + HS was injected (3 min at 30 μL/min) over immobilized human RAGE (left panel) or ABR-215757 (right panel) in HBS-P containing 1 mM Ca2+ and 20 μM Zn2+. Displacement curves were fit to a sigmoidal dose-response model in GraphPad Prism for calculation of IC50. HS inhibited S100A9 binding to RAGE and ABR-215757 to 50% at 20 and 24 ng/mL, respectively, whereas inhibition of S100A8/9 required 96 and 44 times higher concentrations. S100A12 was not displaced even at the highest HS concentration, 5 μg/mL.
Figure 2
Figure 2. S100A12 interacts with mRAGE in a Zn2+-dependent manner similar to S100A9
A. Sensorgrams obtained after injection of 12.5-50 nM mRAGE over immobilized S100A12 (A) or S100A9 (B) in HBS-P containing 1 mM Ca2+ and 50 μM Zn2+. In C, signals at late association phase were plotted versus concentration of mRAGE. Bmax was calculated to 260 ± 27 and 162 ± 5 RU and KD to 60 ± 10 and 47 ± 3 nM for S100A12 and S100A9, respectively, after fit of curves to a 1:1 model in Graph Pad Prism. Recombinant human IgG1 Fc (from R&D Systems) demonstrated no binding to immobilized S100A12 or S100A9 (data not shown). D.S100A12 interacts with ABR-215757 and mRAGE in a Zn-dependent manner. 100 nM S100A12 was injected (2 min at 30 μL/min) over amine coupled mRAGE or ABR-215757 in a buffer containing 1 mM Ca2+ in the absence or presence of 3.125-200 μM Zn2+. Responses at late association phase were plotted versus Zn2+ concentration and show enhanced binding of S100A12 to both mRAGE and ABR-215757 in the entire concentration range. Even in the absence of Zn2+, S100A12 was able to associate with both mRAGE (51 RU) and ABR-215757 (195 RU).
Figure 3
Figure 3. Experimental design
Transgenic S100A12/ApoE-/- mice and WT/ApoE-/- littermates with established fatty streak atherosclerotic lesions were treated for 5 weeks with i.p injections of ABR-215757 or PBS, and analyzed 4 month later.
Figure 4
Figure 4. Treatment with ABR-21757 improves features of atherosclerotic plaque morphology
Representative images of innominated (brachiocephalic) artery lesions from S100A12/ApoE-/- (A-H) and WT/ApoE-/- mice (I-P) stained with Masson trichrome (A,E,I,M), Alizarin Red S (calcium phosphate in red, B,F,J,N), Verhoeff -Van Gieson (elastic fibers in black, C,G,K,O), and Hematoxin & Eosin (D, H,L,P). Original magnification, 10x, scale bar, 10 μm. Quantification of necrotic area within plaques (Q). Quantification of Alizarin Red stained area within plaques (R). Quantification of elastic fiber degradation (S); grade 1=intact fibers, grade 2=disruption of one fiber with intact neighboring fibers, grade 3=disruption of two or more fibers in direct contact, grade 4=disruption of all fibers from internal elastica lamina to external elastic lamina. Total plaque smooth muscle (SMC) content based on SMα-actin staining (T). * p<0.05, 10 animals per group.
Figure 5
Figure 5. Treatment with ABR-215757 reduces aortic dilatation
A. In vivo ultrasound for measurement of the diameter of the ascending aorta 2 cm above the aortic valve (AV, arrow) and at the origin of the brachiocephalic (BC) artery were quantified in B and C (n=5 male mice). D. M-Mode derived measurement of LV dimension at end-systole and end-diastole, and thickness of the LV posterior wall, and quantified in E and F.
Figure 6
Figure 6. ABR-215757 reduces vascular inflammation
Expression of markers of inflammatory cells (A) and inflammatory mediators (B) mRNAs as well as protein expression of S100 and RAGE (C) was analyzed in whole lysates prepared from the thoracic aorta of S100A12/ApoE-/- and WT/ApoE-/- mice immediately after 5 weeks of ABR-215757 or vehicle treatment. *p<0.05 vs. vehicle group, 10 animals per group.
Figure 7
Figure 7. ABR-215757 reduces local and systemic inflammation
Representative sections of the innominate artery stained with anti F4/80 (A-D) and anti CD3 (E-H) in S100A12/ ApoE-/- (left column) and in WT/ApoE-/- (right column) with control treatment (A, B, E, G), or treated with ABR-215757 (C, D, F, H). Scale bar =10μm. Inserts of serial sections stained with control non-immune IgG are shown in the right lower corner in A-D. Mouse spleen is shown as positive tissue control for F4/80 (I) and for CD3 (J) immunohistochemistry. Plaque macrophage content based on F4/80 staining (K) and number of infiltrating T cells based on CD-3 staining (L) were quantified in serial sections of atherosclerotic plaques in the innominate artery. Serum IL-6 (M) and serum amyloid A (N) was measured at study end. (n=10 animals for each group).
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