Inhibition of miR-155 attenuates abdominal aortic aneurysm in mice by regulating macrophage-mediated inflammation

Abstract

The aim of the present study was to identify abdominal aortic aneurysms (AAA)-associated miR-155 contributing to AAA pathology by regulating macrophage-mediated inflammation. Angiotensin II (AngII)-infused apolipoprotein E-deficient (ApoE-/-) mice and THP-1 cells model of miR-155 overexpression and deficiency were used in the experiments. The expression of miR-155 was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cytokines were evaluated using enzyme-linked immunoabsorbent assay (ELISA). Western blotting was used to measure the levels of MMP-2, MMP-9, iNOS, and monocyte chemoattractant protein (MCP)-1 proteins. Immunostaining and transwell were used to determine CD68, elastic collagen, proliferation, and migration of vascular smooth muscle cells (VSMCs). The results showed that miR-155 and cytokines were up-regulated in AAA patients or ApoE-/- mice. Overexpression of miR-155 enhanced MMP-2, MMP-9, iNOS, and MCP-1 levels, and stimulated the proliferation and migration of VSMCs. Meanwhile, inhibition of miR-155 had the opposite effect. In addition, histology demonstrated accumulation of CD68 and elastic collagen-positive areas significantly decreased in miR-155 antagomir injection group. In conclusion, the results of the present study suggest that inhibiting miR-155 is crucial to prevent the development of AAA by regulating macrophage inflammation.

Keywords: Abdominal aortic aneurysms (AAA); inflammation; macrophage; miR155.

Figure 1. The expression levels of miR-155

(A) QRT-PCR analysis of miR155 expression levels in patients with AAA (n=11), (B) in AAA model mice (n=10). (C) Analysis of serum IL-1, IL-6, and TNFα level in patients with AAA (n=11) and control (n=15) by ELISA. (D) QRT-PCR analysis of miR-155 levels in macrophage from peripheral blood of patients with AAA (n=11) and control (n=15). Data were presented as mean ± standard error; **P<0.01; AAA, abdominal aortic aneurysm; AAA body, maximum AAA dilatation; AAA neck, the macroscopically nondilated; AngII, AAA model mice; saline, control mice.

Figure 2. miR-155 exacerbated macrophage inflammasome activation in vitro

(A and B) QRT-PCR analysis of miR-155 and cytokines levels with supernatant of miR-155 mimic-transfered macrophage. (C) Western blot analysis of macrophage inflammatory protein level in transfected cells of miR-155 mimic and inhibitor NC. (D and E) QRT-PCR analysis of miR-155 and cytokines levels in supernatant of miR-155 inhibitor-transfered macrophage. (F) Western blot analysis of macrophage inflammatory protein level in transfected cells of miR-155 inhibitor and inhibitor NC. Each experiment was repeated three times with three replications. Data are mean ± SEM from two independent experiments, **P<0.01.

Figure 3. Macrophage-induced migration and proliferation of VSMC

(A and B) Representative images of proliferation and migration of VSMC in miR-155 mimic group and mimic NC group. (C and D) Representative images of proliferation and migration in miR-155 inhibitor group and inhibitor NC group; scale bar = 20 µm; **P<0.01. P-values were calculated by independent samples T test. On the right is quantification of the proliferation and migration of VSMC. (E) Expression of α-SMA and OPN protein in miR-155 mimic group and miR-155 inhibitor group. On the right is quantification of α-SMA and OPN protein. Each experiment was repeated three times with three replications. P-values were calculated by ANOVA with Tukey’s post-test; **P<0.01, ^##P<0.01.

Figure 4. miR-155 antagomir can block macrophage inflammasome ameliorated severity of AAA

(A) Representative images of HE staining; scale bar = 20 µm. (B) Representative images of EVG staining for elastic fiber collagen; scale bar = 20 µm. (C) Representative images of immunohistochemical staining for CD68+ macrophages; scale bar = 20 µm. (D) Western blot analysis of MMP-2, MMP-9, MCP-1, and TNFα in tissue of mice injected with miR-155 antagomir (n=10) and control (n=10). (E) Quantification of MMP-2, MMP-9, MCP-1, and TNFα. P-values were calculated by ANOVA with Tukey’s post-test; **P<0.01; ^##P<0.01.