C-reactive protein deficiency ameliorates experimental abdominal aortic aneurysms

Figures

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  Figure 1. CRP expression is elevated in experimental aneurysmal aortas. H&E staining or CRP immunostaining using a rabbit anti-mouse CRP antibody or normal rabbit IgG as the negative control were performed on frozen sections from non-aneurysmal and aneurysmal aortas.

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  Figure 2. CRP deficiency suppresses experimental aneurysmal dilation. (A): Phenotype identification of CRP deficient mice. Phenotyping for CRP homozygotes (CRP deficient mice: CRP-/-), CRP heterozygotes (CRP+/-) and wild type (WT) mice using PCR assay. CRP expression levels in the livers of CRP-/- and WT mice were determined via qRT-PCR and Western blotting analyses. (B): Representative photographic images for infrarenal aortas of wild type and CRP-/- mice prior to and 14 days after the elastase infusion. AAAs were induced in male CRP-/- and its wild type control mice using intra-aortic infusion of PPE. Influence on AAAs were assessed via in situ measurements of maximal infrarenal aortic diameters. Dotted line indicates aortic expansion even after PPE solvent (PBS) pressed infusion (about 0.8 mm in our lab) (C, D): Maximal infrarenal aortic external diameters presented as absolute diameter on days 0 (baseline) and 14 after PPE infusion (C) or the percentage of diameters over baseline (D). n=10-11 mice per group. Two-ANOVA followed by two group comparison, **p<0.01 compared to wild type mice at same timepoint (C). Student t-test, p=0.06 compared to wild type mice (D). (E): Representative…

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  Figure 3. CRP deficiency alters aortic leukocyte accumulation. (A): Representative aortic immunostaining images for CD68+ macrophages, CD4+ T cells, CD8+ T cells and B220+ B cells from non-aneurysmal normal aorta (left panels) and aneurysmal (wild type: middle panels; CRP-/- mice: right panels). (B): Quantification of macrophage accumulation scores (media and interquartile) in WT and CRP-/- aneurysmal aorta. N=10-11 mice per group, Non-parametric Mann-Whitney, *P<0.01 compared to WT mice. (C–E): Quantification of different lymphocyte subsets of aortic leukocytes [mean ± standard deviation for positive cells/aortic cross-section (ACS)] from the aneurysmal aortas of WT and CRP-/- mice. No statistical difference in all lymphocyte subsets between two mouse strains.

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  Figure 4. CRP deficiency reduces aneurysmal aortic MMP2 expression. (A): Representative immunostaining images for MMP2 and MMP9 in non-aneurysmal normal or aneurysmal (WT and CRP-/-) aortas. (B, C): Quantification of MMP2 and MMP9 expression (mean ± standard for the percentage of positively stained area in total aortic cross section area) in wild type and CRP-/- aneurysmal aortas. Student’s t tests, n=10-11 mice per group, **p<0.01 compared to wild type mice.

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  Figure 5. CRP deficiency has no remarkable impact on angiogenesis in aneurysmal aortas. Frozen sections were prepared from the aortas of non-aneurysmal normal (upper left) and aneurysmal (wild type: upper right; CRP-/-: lower left) mice (n=10-11 mice per group) and stained with an anti-CD31 antibody to assess aneurysmal angiogenesis. Lower right panel: angiogenesis in wild and CRP-/- aneurysmal mouse aortas was quantitated as the number of CD31-positive neovessels per ACS. Data are mean ± standard deviation. Student’s t tests, no significant difference between two groups.

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  Figure 6. CRP deficiency impairs classic proinflammatory macrophage activation/polarization. Peritoneal macrophages from non-aneurysmal wild type (WT) and CRP-/- mice underwent the classic activation (conventionally known as M1 macrophages) in the presence of LPS (50 ng/ml) or vehicle alone for 6 hours. The mRNA expression levels of indicated classic activation macrophage marker genes were detected by using qRT-PCR. mRNA levels are presented as mean ± standard deviation fold changes relative to vehicle-treated WT macrophages. Two-way ANOVA followed two group comparison, **p<0.01 between two groups, n=6 biological repeats for each group.