Elastin-Derived Peptides Promote Abdominal Aortic Aneurysm Formation by Modulating M1/M2 Macrophage Polarization

Abstract

Abdominal aortic aneurysm is a dynamic vascular disease characterized by inflammatory cell invasion and extracellular matrix degradation. Damage to elastin in the extracellular matrix results in release of elastin-derived peptides (EDPs), which are chemotactic for inflammatory cells such as monocytes. Their effect on macrophage polarization is less well known. Proinflammatory M1 macrophages initially are recruited to sites of injury, but, if their effects are prolonged, they can lead to chronic inflammation that prevents normal tissue repair. Conversely, anti-inflammatory M2 macrophages reduce inflammation and aid in wound healing. Thus, a proper M1/M2 ratio is vital for tissue homeostasis. Abdominal aortic aneurysm tissue reveals a high M1/M2 ratio in which proinflammatory cells and their associated markers dominate. In the current study, in vitro treatment of bone marrow-derived macrophages with EDPs induced M1 macrophage polarization. By using C57BL/6 mice, Ab-mediated neutralization of EDPs reduced aortic dilation, matrix metalloproteinase activity, and proinflammatory cytokine expression at early and late time points after aneurysm induction. Furthermore, direct manipulation of the M1/M2 balance altered aortic dilation. Injection of M2-polarized macrophages reduced aortic dilation after aneurysm induction. EDPs promoted a proinflammatory environment in aortic tissue by inducing M1 polarization, and neutralization of EDPs attenuated aortic dilation. The M1/M2 imbalance is vital to aneurysm formation.

Figure 1

Elastin-derived peptides polarize macrophages to a pro-inflammatory M1 phenotype. A) qPCR analysis of M1 and M2 phenotype markers after treatment of macrophages with IFN-γ and LPS (M1), IL-4 (M2), VGVAPG (1 μg/ml), or EDP (1 μg/ml) for 24 hours. GAPDH was used as an internal control. B) ELISA measurement of TNF-α levels from EDP- or VGVAPG-treated macrophage media. Statistics performed using ANOVA and Student’s t-tests. Data expressed as mean ± SEM. *, P < .05; **, P < .01 versus no treatment (No Tx).

Figure 2

M1 and M2 macrophage influence on aortic size six weeks after aneurysm induction. A) Schematic representation of method of BMDM isolation, injection, and aortic aneurysm induction. B) Representative image of protein expression of M1 (iNOS) or M2 (Ym1) markers of macrophages treated with IFN-γ (20 ng/ml) and LPS (100 ng/ml) or IL-4 (20 ng/ml) prior to injection. C) Aortic diameter percent increase six weeks after aneurysm induction with M1 or M2 macrophage injection (n = 5–9 per group). Statistics performed using ANOVA and Student’s t-tests. Data expressed as mean ± SEM. *, P < .05; **, P < .01. D) Representative VVG images of aortas six weeks after aneurysm induction with injection of M1, M2, or no macrophages (Control) (n = 3–4 per group). VVG image of aorta from a mouse that required sacrifice within one week after aneurysm induction. Arrows indicate sites of elastin fragmentation.

Figure 3

Active MMP-2 and MMP-9 levels are decreased in mice injected with M2 macrophages. Signal intensities of ProMMP-9, MMP-9, ProMMP-2, and MMP-2 were quantified using ImageJ software. Statistics performed using ANOVA and Student’s t-tests. Data are presented as mean ± SEM (n = 4–5 aortas per group). *, P < .05.

Figure 4

M1 and M2 macrophage influence on aortic size three days after aneurysm induction. A) Schematic representation of methods three days after aneurysm induction and macrophage injection. B) Aortic diameter percent increase three days after aneurysm induction with injection of M1, M2, or no macrophages (Control) (n = 5–6 per group). NaCl was used as sham control. C) Bar graph on left represents M1/M2 ratio of M1 (CD86) to M2 (CD206) positive macrophages (F4/80) from flow cytometry (n = 5–6 per group). Right bar graph shows proportion of F4/80⁺ macrophages found in tissue. D) Representative immunofluorescence and VVG stained images of aortas three days after surgery and M1 or M2 macrophage injection (n = 3 per group). Top panel shows aortas stained with a rat anti-mouse F4/80 antibody (red), cell nuclei stained with DAPI (blue); bottom panel shows VVG stained aortas. Arrows indicate sites of elastin fragmentation or abnormal structure. Statistics performed using ANOVA and Student’s t-tests. Data expressed as mean ± SEM. *, P < .05; **, P < .01.

Figure 5

BA4 attenuates aortic dilation and elastin degradation. A) Schematic representation of methods for weekly anti-EDP treatment. B) Percent increase in aortic diameter six weeks after aneurysm induction and treatment with IgG or BA4 (n = 7–8 mice per group). C) Representative gelatin zymogram of mouse aortic tissue six weeks after aneurysm induction. Signal intensities of MMP-2, ProMMP-2, and MMP-9 were quantified using ImageJ software. Statistics performed using ANOVA and Student’s t-tests. Data are presented as mean ± SEM (n = 4–5 aortas per group). *, P < .05; **, P < .01. C) Representative VVG staining of aortic tissue sections from NaCl, CaCl₂ IgG, and CaCl₂ BA4 treated mice (n = 3–4 aortas per group). Arrows indicate sites of elastin damage.

Figure 6

BA4 reduced macrophage migration, MMP production, and the ratio of M1/M2 markers in aortic tissue one week after aneurysm induction. A) Percent increase aortic diameter one week after aneurysm induction (n = 8–10 per group). B) Number of F4/80 positive cells in NaCl, CaCl₂-IgG, and CaCl₂-BA4 treated aortas (n = 7–8 per group). C) Representative gelatin zymogram of aortic tissue one week after aneurysm induction. Bar graphs represent signal intensities of ProMMP-9, MMP-9, ProMMP-2, and MMP-2 by quantification with ImageJ software (n = 3–4 per group). Data expressed as mean ± SEM. *, P < .05; **, P < .01. D) Fold change in mRNA expression of M1 and M2 associated markers from aortic tissue of mice treated with CaCl₂-IgG or CaCl₂-BA4 compared to NaCl-treated mice one week after aneurysm induction (n = 4 per group). GAPDH was used as internal control. Dashed line indicates NaCl control (fold change = 1). Statistics performed using ANOVA and Student’s t-tests. Data expressed as mean ± SEM. *, P < .05; **, P < .01 versus NaCl. †, P < .05; ††, P < .01 versus CaCl₂-IgG treatment.