MicroRNA-29 in Aortic Dilation: Implications for Aneurysm FormationNovelty and Significance
Rationale: Aging represents a major risk factor for coronary artery disease and aortic aneurysm formation. MicroRNAs (miRs) have emerged as key regulators of biological processes, but their role in age-associated vascular pathologies is unknown.
Objective: We aim to identify miRs in the vasculature that are regulated by age and play a role in age-induced vascular pathologies.
Methods and Results: Expression profiling of aortic tissue of young versus old mice identified several age-associated miRs. Among the significantly regulated miRs, the increased expression of miR-29 family members was associated with a profound downregulation of numerous extracellular matrix (ECM) components in aortas of aged mice, suggesting that this miR family contributes to ECM loss, thereby sensitizing the aorta for aneurysm formation. Indeed, miR-29 expression was significantly induced in 2 experimental models for aortic dilation: angiotensin II-treated aged mice and genetically induced aneurysms in Fibulin-4R/R mice. More importantly, miR-29b levels were profoundly increased in biopsies of human thoracic aneurysms, obtained from patients with either bicuspid (n=79) or tricuspid aortic valves (n=30). Finally, LNA-modified antisense oligonucleotide-mediated silencing of miR-29 induced ECM expression and inhibited angiotensin II-induced dilation of the aorta in mice.
Conclusion: In conclusion, miR-29-mediated downregulation of ECM proteins may sensitize the aorta to the formation of aneurysms in advanced age. Inhibition of miR-29 in vivo abrogates aortic dilation in mice, suggesting that miR-29 may represent a novel molecular target to augment matrix synthesis and maintain vascular wall structural integrity.
Age is one of the major risk factors for cardiovascular diseases. With increasing life expectancy, the prevalence of aging-associated cardiovascular diseases will even increase in the near future.1 One particular age-associated disease is abdominal aortic aneurysm formation, which affects approximately 9% of elderly men and has a high mortality rate.2 On the other hand, aneurysms in the ascending part of the thoracic aorta are less age-associated and are often the result of genetic defects involving extracellular matrix (ECM) components.3 On a mechanistic level, analysis of human pathological sections revealed that aneurysm formation and rupture are characterized by thinning of the vascular wall and blood vessel dilation.4 Decreased formation and/or increased degradation of ECM are believed to be the key pathophysiological processes leading to vascular wall thinning.5,6
MicroRNAs (miRs) have recently emerged as key regulators of several (patho-) physiological processes. MiRs are short noncoding RNAs that regulate protein expression by inducing degradation of the targeted mRNA or by blocking protein translation. Whereas various studies showed that specific miRs control vessel growth and cardiac function,7 the involvement of miRs in aortic wall pathologies are less well known.
Comprehensive methods are available as an Online Supplement at http://circres.ahajournals.org.
miRs Are Affected by Aging in the Aorta
miR and mRNA microarray expression profiles comparing aortas of aged with young mice revealed 18 miRs that are regulated (fold increase/decrease >1.5 and P<0.01) (Figure 1A and Online Table I). To establish which of these miRs affect mRNA expression changes, we used 2 distinct unbiased bioinformatics tools that use mRNA expression data to identify putative regulation by miRs. Both these tools, Sylamer8 and MirExTra,9 identified the miR-29 family (miR-29a, b, and c) to be the only 1 of the 18 regulated miRs to functionally affect mRNA levels (Figure 1B and 1C). The upregulation of the miR-29 family by age in aortic tissue was confirmed by real-time PCR (Figure 1D). The miR-29 family is transcribed as 2 bicistronic primary miRs (Figure 1E). We found that only the pri-miR-29b1/a cluster is transcriptionally induced by aging (Figure 1F), suggesting that miR-29 family members are increased by both transcriptional and post-transcriptional mechanisms. Together, these data indicate that miR-29 is the only age-regulated miRNA, which significantly affects mRNA expression levels in the aorta.
MiR-29 Is Induced in Aortic Dilation and Aneurysms
In the heart, the miR-29 family has been shown to control tissue fibrosis after acute myocardial infarction by targeting mRNA coding for ECM proteins such as collagens, fibrillin and elastin.10 All of these known targets of miR-29 were downregulated by age in the aorta (Figure 2A and Online Table II), including elastin, recently described to be crucially regulated by miR-29 in aortic development.11 Because reduced ECM expression is a hallmark of aneurysm formation,6,12 and age is the major risk factor for abdominal aortic aneurysm formation,2 we hypothesized that the induction of miR-29 may link aging to aneurysm formation. Therefore, we next studied the role of miR-29 in 18-month-old mice that were infused with angiotensin II (Ang-II) for 1 week to induce aortic dilation in vivo. Ang-II infusion significantly increased the expression of miR-29b in the aorta, whereas miR-29a and miR-29c were not affected (Figure 2B).
Because defects in ECM components also contribute to the formation of inherited aneurysms, we next evaluated whether miR-29 family members are also regulated in the genetically induced thoracic aortic aneurysms that develop due to ECM defects in Fibulin-4R/R knockdown mice.13 Fibulin-4R/R mice demonstrated a profound increase in expression of all 3 miR-29 family members in the aortic arch as compared to wild-type littermates (Figure 2C).
Finally, we analyzed the expression of miR-29a, b, and c in aortic biopsy samples obtained from a large series of patients with a thoracic aortic aneurysm undergoing aortic valve replacement surgery. The patients were divided into 2 groups, patients with a bicuspid aortic valve and patients with a tricuspid aortic valve, and were compared to control subjects without a thoracic aortic aneurysm, who underwent coronary bypass surgery (Online Table III). The expression of miR-29b is significantly increased in both aneurysm patient groups as compared to control subjects, whereas miR-29a and miR-29c are not affected (Figure 2D).
Inhibition of miR-29 In Vivo Prevents Ang II-Induced Dilation of the Aorta in Aged Mice
In order to assess whether miR-29 expression is causally linked to aortic dilation, we next designed LNA-modified antisense oligonucleotides14 (LNA-29) to silence the expression of miR-29 in vivo. LNA-29 dose-dependently inhibited miR-29a, b, and c expression (Figure 3A), whereas it did not affect the expression of its closest homologues (Online Figure I). LNA-29 significantly increased expression of the miR-29 targets Col1A1, Col3A1, and elastin in aortic tissue (Figure 3B, Online Figure I). To determine whether inhibition of miR-29 prevents aortic dilation, we treated 18-month-old mice with Ang-II and measured the diameter of the aorta proximal to the origin of the renal arteries by ultrasonography before treatment and 7 days after. LNA-29 potently inhibited the expression of miR-29 in the aorta, even during continuous Ang-II infusion (Online Figure II), and induced a concomitant increase in protein levels of the miR-29 target elastin (Figure 3C, Online Figure III). LNA-29 abrogated the Ang-II-mediated increase in aortic diameter, whereas the aorta significantly dilated in the PBS- and control LNAotreated groups, as compared to untreated mice (Figure 3D). Likewise, aortic dilation in Ang-II–infused 6-month-old ApoE−/− mice was reduced by LNA-29 treatment at 1 week (Online Figure IV and V). Blood pressure was unaffected by miR-29 silencing, indicating that the lack of aortic diameter increase in Ang-II/LNA-29 cotreatment is not due to indirect effects on blood pressure (Online Figure VI). MiR-29 is known to regulate fibrosis in the heart, liver, and kidney.10 However, LNA-29 did not affect cardiac function (data not shown) and liver or kidney fibrosis, as investigated by histological analysis (Online Figure VI).
Here, we demonstrate that vascular aging and age-associated pathologies induce a significant upregulation of the miR-29 family in the aorta of mice and humans. Whereas aging and genetically induced aneurysms were associated with increased expression of all family members, miR-29b was preferentially upregulated in Ang-II–induced aortic dilation in mice and in human thoracic aortic aneurysm biopsies. The upregulation of the miR-29 family in aging is consistent with a recent report, showing an increased expression of all miR-29 family members in an accelerated aging model in mice.15
MiR-29 targets several ECM proteins, which are known to play a key role in maintaining the integrity of the vascular wall. MiR-29 was additionally shown to induce apoptosis in cancer cells by targeting Mcl-1, an antiapoptotic Bcl-2 family member,16 and by augmenting p53 levels.17 Smooth muscle cell apoptosis is considered to favor aneurysm formation18 and, because miR-29 is highly expressed in smooth muscle cells (Online Figure V and VII), this mechanism may contribute to miR-29–mediated destabilization of the vascular wall. Interestingly, inhibition of miR-29 decreased the expression of matrix metalloproteinase MMP9 in the aorta (Online Figure VIII), which may additionally prevent further degradation of matrix proteins. However, whereas miR-29 inhibition also blocked the early dilation in the Ang-II-induced aneurysm model in ApoE−/− mice (Online Figure IV), it did not prevent aneurysm formation in this rapidly accelerated inflammatory model at 4 weeks suggesting that inhibition of miR-29 predominantly maintains the structural integrity of the vessel wall.
In conclusion, aging and miR-29 repress the expression of target genes that mainly encode ECM proteins and may, thus, sensitize the aorta for aneurysm formation (Figure 3E). Local inhibition of miR-29, eg, by drug-eluting balloons or stents, may provide a promising novel therapeutic approach to interfere with vascular aging by augmenting matrix synthesis.
Sources of Funding
The study was supported by a Rubicon fellowship of the Netherlands Organization of Scientific Research (NWO), the Deutsche Forschungsgemeinschaft (DFG) (Exc 147-1) and the Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz (LOEWE) centre (R.A.B.), the European Research Council (“Angiomirs,” S.D.), a ‘Lijfen Leven' grant and the Netherlands Genomics Initiative (P.v.H., J.E.), a European Molecular Biologie Laboratories (EMBL) Interdisciplinary Postdoc (EIPOD) Fellowship (M.V.), the European Union Integrated Project Heart Repair (S.D., N.R.) and the British Heart Foundation Centre for Research Excellence (N.R.). N.R. is a National Health and Medical Research Council Australia Fellow.
Reinier A. Boon, Andreas M. Zeiher, and Stefanie Dimmeler applied for a patent regarding this work.
We thank M. Muhly-Reinholz and D. Avaniadi for technical assistance.
In July 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.5 days.
This manuscript was sent to Ali J. Marian, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Non-standard Abbreviations and Acronyms
- Angiotensin II
- extracellular matrix
- locked nucleic acid
- real time PCR
- Received July 19, 2011.
- Revision received August 28, 2011.
- Accepted August 30, 2011.
- © 2011 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Advanced age is a major risk factor for developing aneurysms.
MicroRNA-29 inhibits expression of several extracellular matrix proteins.
What New Information Does This Article Contribute?
Advanced age induces microRNA-29 expression in the aorta and represses extracellular matrix expression.
MicroRNA-29 levels are increased in the aorta in animal models of aneurysm formation and patients with thoracic aneurysms.
Inhibition of microRNA-29 in vivo prevents aortic dilatation in mice.
Aging is a major risk factor for aneurysm formation. Because microRNAs play pivotal roles in various physiological processes, we assessed changes in the microRNA expression profile in the aorta in aged mice. MicroRNA-29, which is known to attenuate extracellular matrix expression, is induced in the aorta of old mice. Moreover, microRNA-29 is induced in mouse models of aortic aneurysm and human aneurysm biopsies. Inhibition of microRNA-29 prevents aortic dilation in aged mice. Thus, inhibition of microRNA-29 may constitute a promising treatment against aortic aneurysms.