Inhibition of microRNA-29b reduces murine abdominal aortic aneurysm development

Abstract

MicroRNAs (miRs) regulate gene expression at the posttranscriptional level and play crucial roles in vascular integrity. As such, they may have a role in modifying abdominal aortic aneurysm (AAA) expansion, the pathophysiological mechanisms of which remain incompletely explored. Here, we investigate the role of miRs in 2 murine models of experimental AAA: the porcine pancreatic elastase (PPE) infusion model in C57BL/6 mice and the AngII infusion model in Apoe-/- mice. AAA development was accompanied by decreased aortic expression of miR-29b, along with increased expression of known miR-29b targets, Col1a1, Col3a1, Col5a1, and Eln, in both models. In vivo administration of locked nucleic acid anti-miR-29b greatly increased collagen expression, leading to an early fibrotic response in the abdominal aortic wall and resulting in a significant reduction in AAA progression over time in both models. In contrast, overexpression of miR-29b using a lentiviral vector led to augmented AAA expansion and significant increase of aortic rupture rate. Cell culture studies identified aortic fibroblasts as the likely vascular cell type mediating the profibrotic effects of miR-29b modulation. A similar pattern of reduced miR-29b expression and increased target gene expression was observed in human AAA tissue samples compared with that in organ donor controls. These data suggest that therapeutic manipulation of miR-29b and its target genes holds promise for limiting AAA disease progression and protecting from rupture.

Figures

Figure 1. miR-29 in AAAs induced by PPE infusion in mice.

(A) AAD (versus baseline in percentage) in PPE-induced (ELAST) AAA compared with that in sham-operated control mice (sham). (B) miR-29 family (miR-29a, miR-29b, miR-29c) expression in ELAST mice compared with that in sham-operated control mice. (C) ISH for miR-29b (purple chromogen) in control aortas (untreated), sham-operated mice, and ELAST mice 14 days after surgery (original magnification, ×200). (D and E) mRNA expression levels in ELAST mice compared with those in sham-operated mice for (D) Col1a1, Col3a1, and Col5a1 as well as (E) Eln and Fbn1. n = 5–8 for each treatment group and time point. Data are mean ± SEM. *P < 0.05 versus sham.

Figure 2. miR-29 and target gene expression in AngII-induced AAAs.

(A) Expansion of AADs (in percentage of baseline) in mice with AngII-induced AAA (ANGII; n = 27) compared with that in saline-infused controls (sham; n = 12). (B) miR-29 expression in mice with AngII-induced AAA compared with that in sham-operated mice. (C) Col1a1, Col3a1, and Col5a1 expression in mice with AngII-induced AAA compared with that in sham-operated mice. (D) Eln and Fbn1 expression in mice with AngII-induced AAA compared with that in sham-operated mice. Data are mean ± SEM. *P < 0.05 versus untreated.

Figure 3. miR-29b in vitro.

(A) miR-29b expression in TGF-β–stimulated hASMCs and hAFBs. (B) Expression levels of miR-29b target genes in Tgf-β–stimulated anti-29b– and pre-29b–transfected hAFBs. (C) Expression levels of miR-29b target genes in Tgf-β–stimulated anti-29b– and pre-29b–transfected hASMCs. (D) Soluble collagen assay to quantify collagen synthesis in Tgf-β–stimulated anti-29b– and pre-29b–transfected hAFBs. Data are mean ± SEM. *P < 0.05 versus untreated. #P < 0.05 versus scr-miR and untreated. †P < 0.05 versus scr-miR and anti-29b.

Figure 4. Effects of anti-29b and pre-29b in PPE-AAA.

(A) Double immunofluorescence detection of smooth muscle cell α-actin (SMA, red) and GFP/fluorescein isothiocyanate label (green) in the non-aneurysmal part of the suprarenal abdominal aorta and in the infrarenal site of injury (AAA) in anti-21– or pre-21–transduced mice with AAA (as single color and merged images; original magnification, ×200). (B) AAD (in percentage versus baseline) in scr-miR and anti-/pre-21 PPE-induced AAA. (C) Col1a1, (D) Col3a1, and (E) Eln expression in scr-miR– and anti- and pre-29b–transduced mice compared with that in sham-operated mice in the PPE model. (F) Col1a1, Col3a1, and Eln expression is not significantly different between scr-miR– and anti- and pre-29b–transduced mice in the non-aneurysmal suprarenal aorta in the PPE-AAA model 14 days after induction. n = 4–8 mice for each time point and group. Data are mean ± SEM. *P < 0.05 versus sham. #P < 0.05 versus scr-miR and sham. †P < 0.05 versus scr-miR and anti-29b.

Figure 5. Fibrosis in miR-29b–modulated mice with PPE-induced AAAs.

(A) Representative polarized light microscopy of picrosirius red–stained aortic cross sections (original magnification, ×40), illustrating aneurysm expansion and fibrosis (collagen fibers are bright yellow) in sham, scr-miR, and anti- and pre-29b mice 28 days after AAA induction with PPE. (B) Representative Col3a1 immunohistochemical images, demonstrating effects of pre-29b, scr-miR, and anti-29b 14 days after AAA induction with PPE (original magnification, ×200). (C) Quantification of Col3a1-positive cells in the intima/media region (n = 4 high-power fields of 3 different aortas per group) 14 days after AAA induction with PPE. (D) In situ zymography images to detect MMP activity in scr-miR– and anti- and pre-29b–transduced aortas 14 days after PPE-induced AAA in mice (original magnification, ×200). (E) Mmp2 and (F) Mmp9 expression in miR-29b–modulated mice with PPE-induced AAA (after 14 days). n = 4–8 mice per treatment group. Data are mean ± SEM. *P < 0.05 versus sham. #P < 0.05 versus scr-miR and anti-29b or pre-29b.

Figure 6. Effects of anti-29b and pre-29b in AngII infusion model and miR-29b regulation in human patients with AAA.

(A) AAD (in percentage versus baseline) in scr-miR and anti- and pre-29b AngII-induced. (B) Col1a1, (C) Col3a1, and (D) Eln expression in scr-miR– and anti- and pre-29b–transduced mice compared with that in sham-operated mice in the AngII model (n = 4–6 mice for each time point and group). (E) miR-29b is the only significantly downregulated miR in human AAA samples (n = 8) compared with tissue from control patients (n = 7) without AAA. (F) COL1A1, COL3A1, COL5A1, and ELN are significantly upregulated in human AAA tissue compared with non-aneurysmal aortic tissue. Data are mean ± SEM. *P < 0.05 versus sham or control patients. #P < 0.05 versus scr-miR and sham. †P < 0.05 versus scr-miR and anti-29.