Influence of apolipoprotein E, age and aortic site on calcium phosphate induced abdominal aortic aneurysm in mice Yutang Wang, Smriti M. Krishna, Joseph.

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Influence of apolipoprotein E, age and aortic site on calcium phosphate induced abdominal aortic aneurysm in mice Yutang Wang, Smriti M. Krishna, Joseph Moxon, Tam Nguyen Dinh, Roby J. Jose, Hongyou Yu, Jonathan Golledge Atherosclerosis Volume 235, Issue 1, Pages 204-212 (July 2014) DOI: 10.1016/j.atherosclerosis.2014.04.033 Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 The effect of ApoE deficiency on infra-renal aortic dilatation (A–C) and aortic structure (D) in response to calcium phosphate administration. Representative images of the infra-renal aorta of 3 month old wild type (Ai and Aii) and ApoE−/− mice (Aiii and Aiv) at 2 weeks after the administration of saline (Ai and Aiii) or calcium phosphate (Aii and Aiv). (B) Box plot of the expansion of the infra-renal aorta in mice 2 weeks after administration of saline (open box) or calcium phosphate (filled box). Results are shown as median and interquartiles range (IQR). CaPO4: calcium phosphate; IRA: infra-renal aorta. *P < 0.05 between the two groups using Mann–Whitney U-test. (C) The expansion of the infra-renal aorta of C57BL/6 and ApoE−/− mice at 1, 2 or 4 weeks after administration of calcium phosphate. The results are shown as mean ± SEM. * Increase in aortic diameter was significantly greater in ApoE−/− mice 2 weeks after calcium phosphate administration compared to the following groups: ApoE−/− mice 1 week after calcium phosphate administration (P = 0.037); C57BL/6 mice 1 week after calcium phosphate administration (P = 0.003); and C57BL/6 mice 2 weeks after calcium phosphate administration (P = 0.002); but not C57BL/6 mice 4 weeks after calcium phosphate administration (P = 0.138). (D) Representative haematoxylin and eosin (H&E) micrographs of the infra-renal aorta of C57BL/6 mice (Di and Dii) or ApoE−/− mice (Diii and Div) 2 weeks after administration of saline (Di and Diii) or calcium phosphate (Dii and Div). The scale bar represented 500 μm in the left panels of Di-iv, and 50 μm in the right panels of Di-iv. Atherosclerosis 2014 235, 204-212DOI: (10.1016/j.atherosclerosis.2014.04.033) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 The effect of ApoE deficiency on calcification and macrophage infiltration in response to calcium phosphate administration. (A) Percentage of calcification area (positive birefringence for Alizarin red) in the infra-renal aorta of C57BL/6 or ApoE−/− mice 2 weeks after calcium phosphate administration (n = 5). Results are shown as mean ± SEM. *P = 0.03. (B) Representative immunohistochemistry micrographs showing CD68 (a macrophage marker) staining in infra-renal aortic sections of C57BL/6 (Bi) or ApoE−/− mice (Bii) 2 weeks after administration of calcium phosphate. (C) Bar graph of aortic macrophage staining in C57BL/6 or ApoE−/− mice 2 weeks after the administration of calcium phosphate. Results are shown as mean ± SEM. Atherosclerosis 2014 235, 204-212DOI: (10.1016/j.atherosclerosis.2014.04.033) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 The effect of ApoE deficiency on aortic elastin degradation (A and C) and apoptosis (B and D). Elastin, represented by black after staining with the Verhoeff–Van Gieson (EVG) technique, in sections of the infra-renal aorta of C57BL/6 control (Ai), C57BL/6 experimental (Aii), ApoE−/− control (Aiii), and ApoE−/− experimental mice (Aiv) at 2 weeks after administration of saline or calcium phosphate. Scale bar: 50 μm. * Represented lumen. (B) TUNEL staining in sections of the infra-renal aorta of C57BL/6 control (Bi), C57BL/6 experimental (Bii), ApoE−/− control (Biii), and ApoE−/− experimental mice (Biv) at 2 weeks after administration of saline or calcium phosphate. Scale bar: 50 μm. * Represented lumen. (C) Quantification of elastic lamellar degradation by EVG staining. Aortic wall elastic lamellar degradation was recorded under 40× magnifications from 2 to 3 aortic sections from each mouse (n = 5 in each group). Elastic lamellar degradation was graded as follows [29]: 1 – no elastic lamellar degradation, 2 – mild elastic lamellar degradation; 3 – moderate to severe elastic lamellar degradation; and 4 – severe elastic lamellar degradation. Sections from the infra-renal aortas of ApoE−/− mice that received calcium phosphate showed extensive elastic lamellar degradation within the tunica media layer at 2 weeks. Results are shown as mean ± SEM. *P = 0.02. (D) Bar graph of the percentage of TUNEL staining area in C57BL/6 and ApoE−/− mice 2 weeks after the administration of calcium phosphate. Results are shown as mean ± SEM. *P = 0.007. Atherosclerosis 2014 235, 204-212DOI: (10.1016/j.atherosclerosis.2014.04.033) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Correlation between the plasma lipids levels and the expansion of the infra-renal aorta 2 weeks after the perivascular application of calcium phosphate. Calcium phosphate was applied to the infra-renal aortas of 3 month old C57BL/6 (N = 5) and ApoE−/− (N = 5) mice and aortic expansion examined 2 weeks later. The plasma concentrations of total cholesterol (A), LDL/VLDL cholesterol (B) and HDL cholesterol (C) were measured. The correlation between the lipid concentrations and the expansion of the infra-renal aorta was analysed using Spearman correlation. Atherosclerosis 2014 235, 204-212DOI: (10.1016/j.atherosclerosis.2014.04.033) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Effect of age (A) and aortic site (B) on expansion of the infra-renal aorta in the calcium phosphate model. (A) Box plot of the expansion of the infra-renal aorta in 3 and 7 month old wild type mice 2 weeks after saline or calcium phosphate administration. Results are shown as median and interquartiles range (IQR). *P = 0.0001 between the two groups using Mann–Whitney U-test. (B) Box plot showing the maximal aortic diameters of the aortic arch (Arch), thoracic aorta (Thx), supra-renal aorta (SRA) and the mean maximal diameter (MMD, the average of the maximal diameter in the Arch, Thx and SRA) in 3 month old C57BL/6 mice 2 weeks after calcium phosphate (filled box) or saline application (open box). Results are shown as median and interquartiles range (IQR). *P = 0.03 between the two groups using Mann–Whitney U-test. Atherosclerosis 2014 235, 204-212DOI: (10.1016/j.atherosclerosis.2014.04.033) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions