Adiponectin attenuates abdominal aortic aneurysm formation in hyperlipidemic mice

Abstract

Objective: Abdominal aortic aneurysms (AAA) are age-associated, life-threatening inflammatory dilations of the abdominal aorta. Human population studies have shown an association between obesity and AAA formation, but the molecular mechanisms underlying this connection remain largely unexplored. Adiponectin is an anti-inflammatory adipokine that is downregulated in obesity. In this study we evaluated the role of adiponectin in a model of AAA using apolipoprotein E/adiponectin double-knockout (Apoe(-/-)Apn(-/-)) mice.

Approach and results: Angiotensin II (Ang II)-infusion in male Apoe(-/-)Apn(-/-) mice led to a higher incidence of AAA and a significant increase of maximal aortic diameter compared with that of Apoe(-/-) mice (2.12 ± 0.07 mm vs. 1.67 ± 0.09 mm, respectively at 28 days). Adiponectin deficiency augmented the early infiltration of macrophages and increased the expression of pro-inflammatory factors in the dilated aortic wall. MMP-2 and MMP-9 activation was also augmented in the aorta of Apoe(-/-)Apn(-/-) mice compared to Apoe(-/-) mice. These data suggest that the downregulation of adiponectin could directly contribute to the elevated incidence of AAA observed in obese individuals.

Conclusions: Adiponectin attenuates Ang II-induced vascular inflammation and AAA formation in mice.

Keywords: Abdominal aortic aneurysm; Adiponectin; Inflammation.

Figure 1. Adiponectin deficiency exacerbates Ang II-induced AAA formation

A. Adipoq mRNA expression levels as determined by quantitative real time-PCR in white adipose tissues of Apoe−/− mice after 7 days of Ang II infusion. The value of sham is set as 1. Results are mean±SEM. n=8 in each group. *P<0.05 using Mann-Whitney U test. B, Native western blot of mouse serum using anti-adiponectin primary antibody. C, Incidence of Ang II-induced AAA in Apoe−/− mice (n=12) and Apoe−/− Apn−/− mice (n=10) after Ang II infusion for 4 weeks. *P<0.05 using Wilcoxon signed-rank test. D, Representative B-mode ultrasonographic images of maximum diameter of aorta at various time points Scale bars indicate 1mm. E, Maximum suprarenal abdominal aortic diameter quantified by ultrasonography at baseline, and at 7, 14, 21 and 28 days after Ang II infusion. Results are mean±SEM. n=12 in Apoe−/− mice and n=10 in Apoe−/− Apn−/− mice. **P<0.01, Apoe−/− mice vs. Apoe−/− Apn−/− mice using two-way ANOVA with repeated measures. F, Representative photographs of elastin-stained histologic section showing macroscopic features of aneurysms induced by Ang II at 28 days. These sections highlight the intramural involvement, but not differences in lumen size because a stabilizing substance was not infused prior to harvest to preserve the lumen’s architecture. Scale bars indicate 1mm.

Figure 2. Adiponectin deficiency increases macrophage content in AAA

A, Micrographs of representative F4/80 immunohistochemical staining (brown) of AAA in Apoe−/− mice and Apoe−/− Apn−/− mice at day 28 after Ang II infusion. Scale bars indicate 50µm. B, F4/80-positive macrophage content in the AAA vessel wall. Results are mean±SEM. n=4 in each group. **P<0.01 using Mann–Whitney U test. C, Quantitative real time-PCR analysis of the macrophage-specific transcripts Emr1 (F4/80) and Cd68 in suprarenal abdominal aortae at day 28 after Ang II infusion. Transcript levels of Gapdh, Actb and Rplp0 serves as an internal control. The value of gene expression in Apoe−/− mice at baseline is set as 1. Results are mean±SEM. n=8 in each group. *P<0.05, Apoe−/− mice vs. Apoe−/− Apn−/− mice using two-way ANOVA.

Figure 3. Adiponectin deficiency increases the expression of pro-inflammatory cytokines in the aneurysmal vascular wall

Quantitative real time-PCR analysis of the expression of pro-inflammatory cytokines in the aneurysmal suprarenal aorta (A) and the adjacent periaortic adipose tissue (B) at baseline and day 28 after Ang II stimulation. Transcript levels of Gapdh, Actb and Rplp0 serves as an internal control. The value of gene expression in Apoe−/− mice at baseline is set as 1. Results are mean±SEM. n=8 in each group. *P<0.05, **P<0.01, Apoe−/− mice vs. Apoe−/− Apn−/− mice using two-way ANOVA.

Figure 4. Adiponectin deficiency increases the accumulation of macrophages in the aortic wall in the very early phase

Quantitative real time-PCR analysis of the expression of pro-inflammatory cytokines and macrophage-specific markers in the suprarenal aorta at baseline and day 3 after Ang II stimulation. Transcript levels of Gapdh, Actb and Rplp0 serves as an internal control. The value of gene expression in Apoe−/− mice at baseline is set as 1. Results are mean±SEM. n=8 in each group. *P<0.05, Apoe−/− mice vs. Apoe−/− Apn−/− mice using two-way ANOVA.

Figure 5. Adiponectin deficiency increases MMP level in AAA

A, Representative gelatin zymography of aortic protein at baseline and day 28 after Ang II infusion. B, Quantification of total MMP level in aorta. The value of Apoe−/− mice at baseline is set as 1. Results are mean±SEM. n=4 in each group. *P<0.05, Apoe−/− mice vs. Apoe−/− Apn−/− mice using two-way ANOVA.