Inhibition of microRNA-29b reduces murine abdominal aortic aneurysm development (original) (raw)
miR-29b is significantly downregulated and negatively correlated with its target genes during aneurysm development in 2 murine models of AAA. Using the porcine pancreatic elastase (PPE) infusion model in 10-week-old male C57BL/6J mice (14), we investigated the expression of the miR-29 family and its putative target genes at multiple time points during infrarenal AAA development.
B-mode ultrasound imaging performed 3, 7, 14, 21, and 28 days after PPE infusion demonstrated the expected progressive increases in abdominal aortic diameter (AAD) in elastase-infused (ELAST) mice compared with those in saline-infused control (sham) mice (Figure 1A and Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI61598DS1). Ex vivo diameters at necropsy (after 7, 14, and 28 days) were analyzed through direct measurement; the diameters closely correlated with AADs obtained with ultrasound (data not shown).
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.
Infrarenal aortic tissue was harvested at days 7, 14, and 28. Interestingly, miR-29b was the only miR-29 family member that was significantly downregulated at all 3 time points. miR-29c and miR-29a trended downward at all time points, but the only significant difference compared with sham-operated mice was miR-29c at 7 days (Figure 1B).
The miR-29 family is transcribed as 2 bicistronic primary miRs, located on 2 different murine chromosomes: primary-miR (pri-miR) 29b-1/29a is located on chromosome 6, and pri-miR-29b-2/29c is located on chromosome 1. Both pri-miRs were significantly downregulated (between –1.55 to –1.84 fold; P < 0.05) at all 3 time points (days 7, 14, and 28) during AAA expansion in the PPE model compared with those in sham-operated mice. These combined results suggest some level of posttranscriptional degradation of miR-29a and miR-29c when compared with miR-29b.
In situ hybridization (ISH) indicated that miR-29b expression was diminished throughout the aneurysmal aortic wall of ELAST mice compared with that of sham and untreated controls (Figure 1C), particularly in the adventitial layer. Collagen gene expression (Col1a1, Col3a1, Col5a1) was negatively correlated with miR-29b expression, and both effects peaked at day 14 (Figure 1D). Eln was significantly upregulated at 14 days after infusion but not at the other 2 time points. Notably, Fbn1 was not differentially regulated at any of the 3 time points, suggesting additional transcriptional modulators (Figure 1E).
We used a second established model of AAA formation (this one was suprarenal) with AngII infusion in 10-week-old Apoe–/– male mice (15) to confirm that aneurysm-related regulation of miR-29b and its ECM-related target genes was not exclusive to the PPE model. Compared with that in sham-operated mice, the AAD was significantly increased in the AngII group from day 7 through day 28 (Figure 2A and Supplemental Table 2). As expected, the mortality rate through day 28 was significantly (P < 0.01) higher in AngII mice (26%), due to aortic rupture and dissection, when compared with that of saline controls (0%). miR-29b was the only member of the miR-29 family that was significantly downregulated at 2 different time points during aneurysm development (days 14 and 28) in AngII mice compared with saline-infused control mice (Figure 2B). In this model, miR-29b expression was negatively correlated with expression of Col1a1, Col3a1, Col5a1, Eln, and Fbn1 (Figure 2, C and D). However, only the 3 collagen-encoding genes were significantly upregulated at both days 14 and 28. Eln was significantly upregulated after 14 days, and Fbn1 expression remained unaffected.
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.
As in the PPE model, both pri-miRs (pri-miR-29b-1/29a and pri-miR-29b-2/29c) were significantly downregulated at both time points (–1.57 and –1.64 fold after 14 days, respectively; –1.62 and –1.54 fold after 28 days, respectively; P < 0.05) in AngII mice compared with those in sham-operated mice.
miR-29b regulates fibrosis in cultured human aortic cell lines. Given the importance of various vascular cell types in AAA development, and to confirm that miR-29b is also expressed in human aortic cells, we performed in vitro experiments using non-growth arrested human aortic smooth muscle cells (hASMCs) and human aortic adventitial fibroblasts (hAFBs). Cells were treated with TGF-β1, a known regulator of miR-29b, and profibrotic stimulus in different cell lines, such as cardiac fibroblasts (8), tubular kidney cells (7), and hepatocytes (6).
Treatment with TGF-β1 significantly decreased miR-29b expression in hAFBs but not in hASMCs (Figure 3A). miR-29b expression was then modified by transfecting with either an antagomir (anti-29b) to inhibit activity or a pre-miR (pre-29b) to enhance activity in both cell lines. Successful transfection (>50% of all cells) was confirmed by visual fluorescent microscopic analysis and FACS for labeled tag (data not shown).
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.
Modulation of miR-29b had a significant impact on collagen gene expression (COL1A1 and COL3A1) in both cell types, although the effect in hAFBs was more dramatic (Figure 3, B and C). Overexpression of miR-29b also inhibited expression of ELN in hASMCs (Figure 3C).
Since TGF-β1 preferentially altered miR-29b expression in hAFBs, we monitored collagen production after stimulation in order to confirm the profibrotic effect of miR-29b downregulation in this specific cell line. Collagen synthesis was increased in TGF-β1–treated cells compared with that in an untreated control group. Anti-29b exacerbated the effects of TGF-β1 treatment upon collagen synthesis. In contrast, pre-29b decreased collagen levels (Figure 3D).
In vivo modulation of miR-29b alters collagen gene expression, aneurysm progression, and aortic wall structure in the PPE model. We used FITC-labeled locked nucleic acid (LNA) anti–miR-29b to inhibit miR-29b (anti-29b) or GFP-labeled lentivirus for transduction with pre–miR-29b (lenti-pre-miR-29b; pre-29b) to study gain of function. Double immunofluorescence studies confirmed that anti-29b and pre-29b each successfully incorporated into the aortic wall (Figure 4A). Both miR-29b modulators coexpressed visually with the SMC marker, smooth muscle α-actin (SMA), as well as in the adventitial layer. However, localization of miR-29b modulators was almost exclusively limited to the site of injury (aneurysmal part of the abdominal aorta) in the PPE model. In noninjured suprarenal aortic segments from miR-29b–modulated mice, any green color appeared to be largely due to autofluorescence from elastic fibers or adventitial in nature, possibly delivered through vasa vasorum from the injured region. Successful inhibition and overexpression in vivo were furthermore confirmed by quantitative RT-PCR (qRT-PCR) measuring miR-29b expression in anti-29b– and pre-29b–transduced mice, in relation to that of a scrambled control miR (scr-miR; Supplemental Figure 1A).
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.
Enhancing expression of miR-29b with pre-29b after elastase treatment greatly augmented AAD growth, whereas inhibition with anti-29b inhibited AAD expansion (significant from day 7 to day 28; Figure 4B, Supplemental Figure 1B, and Supplemental Table 3). Interestingly, 4 mice died after receiving pre-29b (2 at day 9 and 1 each at 13 and 21 days), due to rupture of their greatly enlarged AAAs, an extremely uncommon event in the PPE-induced aneurysm model when using 10-week-old male C57BL/6 mice.
Anti-29 (10 mg/kg) significantly increased collagen (Col1a1, Col3a1) and Eln mRNA levels at days 7, 14, and 28 after elastase infusion compared with scr-miR (Figure 4, C–E). In contrast, treatment with lenti-pre-miR-29b (7.6 × 107 infection units/ml [IFU/ml]) in ELAST mice led to collagen and elastin gene normalization (nonsignificant compared with that of sham-operated mice) at all 3 time points after tail vein injection. Even scr-miR–injected mice had significantly higher expression of collagen genes and Eln compared with that of pre-29b mice (Figure 4, C–E).
Analogous to our double immunofluorescence results, anti-29b or pre-29b had minimal impact on target gene expression in the suprarenal (non-aneurysmal) abdominal aorta, suggesting that only the site of injury had significant uptake of the miR modulators (Figure 4F).
Polarized light microscopy of picrosirius red–stained aortic cross sections demonstrated substantial effects of anti-29b and pre-29b (compared with those of scr-miR) on PPE-induced aneurysm structure and composition at 28 days. Some enhancement of collagen deposition, indicating vascular fibrosis, was visible in scr-miR mice compared with that in sham-operated mice. This process became much more prominent in anti-29b–injected mice compared with that in the other 3 groups, whereas almost no fibrotic or proliferative response could be detected in the greatly dilated pre-29b–treated aortas (Figure 5A). Further, immunohistochemical analysis for SMA (SMC marker), Ki-67 (nonspecific marker of cell proliferation), and Mac-1 (monocyte/macrophage marker) revealed no major differences between miR-29b–modulated mice when compared with scr-miR (Supplemental Figure 1, C–E), with regards to smooth muscle cell proliferation or inflammatory cell infiltration. Cell proliferation (Ki-67–positive cells) appeared to be lower in the adventitial region in pre-29b–transduced mice, in conjunction with the limited fibrotic response, when compared with that in scr-miR– and anti-29b–transduced mice.
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.
To confirm the gene expression results for Col3a1 (the most significantly regulated collagen gene), we performed immunohistochemical analysis in scr-miR– as well as anti-29b– and pre-29b–transfected mice 14 days after AAA induction (Figure 5B). As seen in Figure 5C, Col3a1 was significantly increased in the anti-29b aortic wall compared with that in the other 2 groups.
MMP activity is regulated through miR-29b expression. Using in situ zymography, we identified varying levels of MMP activity in anti-29b– and pre-29b–treated mice compared with those in scr-miR–treated mice. MMP activity appeared lower in anti-29b–treated mice compared with that in the other 2 groups at 14 days after AAA induction using PPE (Figure 5D). In contrast, MMP activity was increased in pre-29b–treated mice with large AADs. qRT-PCR confirmed that Mmp2 and Mmp9 expression was increased in mice with upregulated miR-29b (pre-29b) as compared with that in the other 2 groups (Figure 5, E and F). Mmp2 and Mmp9 expression was significantly decreased in anti-29b–treated mice at the same time point. A similar pattern of MMP2 and MMP9 regulation was observed in TGF-β1–stimulated and miR-29b–modulated hAFBs (Supplemental Figure 2, A and B).
In vivo modulation of miR-29b substantially affects AAA expansion and ruptures in the AngII model. In an effort to illustrate similar effects of miR-29b modulation in the second mouse model of AAA induction, the AngII infusion model, we modulated mice with either anti- or pre-29b (and compared them to those modulated with scr-miR) as described above for the PPE model. Further downregulation of miR-29b with anti-29b resulted in a significant decrease of AAA expansion after 28 days. Overexpression of miR-29b by pre-29b significantly increased the AAD 14 and 28 days after AAA induction with AngII (Figure 6A). Expression of Col1a1, Col3a1, and Eln was significantly altered after 14 days. After 28 days, only pre-29b significantly decreased target gene expression. No significant change of Col1a1, Col3a1, and Eln regulation was detectable 7 days after AAA induction (Figure 6, B–D). Ruptures occurred significantly more often in pre-29b–treated mice (63%) compared with those in scr-miR– (33%; P < 0.01) and anti-29b–transduced mice (20%; P < 0.01) until day 28. However, the overall effect of miR-29b modulation appeared to be more moderate compared with that in the PPE model. Interestingly, miR-29b expression in anti- and pre-29b–modulated mice was not altered as extensively as that in mice in the PPE study (Supplemental Figure 1A and Supplemental Figure 2C). A potential explanation for this may be due to the fact that the acute and localized injury induced in the PPE model likely leads to an increased uptake of miR modulators, whereas the intact aortic wall in the AngII model might result in limited uptake of anti- and pre-29b.
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.
Human patients with AAA display downregulated aneurysm tissue miR-29b and upregulated collagen mRNA levels. Human infrarenal aortic tissue samples from patients (n = 15) who underwent surgery for replacement of an enlarged abdominal aorta (AAD, 59–68 mm) corroborated our findings of downregulated miR-29b and increased collagen gene expression with aneurysm. We compared this group of patients with AAA (aged 64 ± 11 years) with a group of control organ donor patients without AAA (n = 5, mean age 33 ± 14 years) at time of explantation (n = 3 heart donors; n = 2 kidney donors). According to hospital documentation, all 15 patients with AAA were on similar medical therapy, which might have potentially influenced their aortic molecular milieu (beta blockers, either angiotensin receptor blocker or angiotensin-converting enzyme inhibitors, and statin therapy) at the time of surgical intervention. All patients were male, of mixed European descent, and nondiabetic. Similar to that in our preclinical animal models of AAA, qRT-PCR revealed that miR-29b was –2.3 ± 0.6 fold downregulated in diseased aortic tissue from patients with AAA. Further, miR-29b was the only member of the miR-29 family found to be significantly regulated in patients with AAA (Figure 6E). COL1A1, COL3A1, COL5A1, and ELN were also all significantly upregulated in AAA (Figure 6F), supporting the data from our murine AAA studies.