MicroRNAs, fibrotic remodeling, and aortic aneurysms

Abstract

Aortic aneurysms are a common clinical condition that can cause death due to aortic dissection or rupture. The association between aortic aneurysm pathogenesis and altered TGF-β signaling has been the subject of numerous investigations. Recently, a TGF-β–responsive microRNA (miR), miR-29, has been identified to play a role in cellular phenotypic modulation during aortic development and aging. In this issue of JCI, Maegdefessel and colleagues demonstrate that decreasing the levels of miR-29b in the aortic wall can attenuate aortic aneurysm progression in two different mouse models of abdominal aortic aneurysms. This study highlights the relevance of miR-29b in aortic disease but also raises questions about its specific role.

The major disease affecting the aorta is the aneurysm, an abnormal widening or ballooning of an artery due to weakness in its wall. The most common location for aneurysms is the infrarenal segment of the aorta, and these are referred to as abdominal aortic aneurysms (AAAs). AAAs primarily affect men over the age of 65 years, and risk factors for AAAs include cigarette smoking, atherosclerosis, hypertension, and a family history of AAA (1). Although the epidemiology and heritability of the disease are well described, the relationship between the risk factors and the aneurysm formation is not well understood. For example, despite the atherosclerotic risk factors and concurrent risk for coronary artery disease in patients with AAAs, it is debated whether atherosclerotic processes directly contribute to aneurysm development or whether atherosclerosis in the aortic wall is merely a “bystander” condition. Other pathologic features of AAAs include chronic transmural inflammation, degradation of elastic fibers, and loss of SMCs (1).

The second most common location for aortic aneurysms is the ascending thoracic aorta, and these aneurysms present at a younger age and affect approximately twice as many men as women (2). Hypertension and congenital bicuspid aortic valve (BAV) are risk factors for thoracic aortic aneurysms (TAAs), and a genetic predisposition also contributes prominently to the etiology. Marfan syndrome (MFS) predisposes to TAAs and is caused by mutations in FBN1, which encodes fibrillin-1, a component of elastin-associated microfibrils (3). Studies of an established mouse model of MFS have suggested that defects in FBN1 lead to promiscuous activation of TGF-β as it is released from stores in the microfibrils (4). Other syndromes predisposing individuals to TAA caused by mutations in either the TGF-β receptor type I or II genes (TGFBR1 or TGFBR2, respectively) or SMAD3 have also implicated excessive activation of TGF-β signaling in aortic disease (5–7). TAAs inherited in families without syndromic features are due to mutations in genes encoding proteins involved in SMC contraction, including the SMC-specific isoforms of α-actin (ACTA2) and myosin heavy chain (MYH11), along with the kinase that controls SMC contraction (MYLK) (8–10). The aortic pathology of TAAs is characterized by elastic fiber fragmentation and loss, proteoglycan accumulation, and either focal or diffuse regions of SMC loss.

MicroRNA-29 and fibrosis

Individual microRNAs (miRs) can target numerous mRNAs and have been referred to as the “micromanagers” of cellular gene expression (11); they can orchestrate gene expression profiles associated with phenotypic changes in cells and disease progression. The miR-29 family of miRs contains 3 members (miR-29a, miR-29b, and miR-29c) that are encoded by two separate loci, giving rise to bicistronic precursor miRs (miR-29a/b1 and miR-29b2/c). This family has been demonstrated to target gene transcripts that encode ECM proteins involved in the fibrotic response, including those for type I collagen (COL1A1 and COL1A2), type III collagen (COL3A1), fibrillin-1, and elastin (ELN) (12), and is known to modulate gene expression during development and aging of the aorta (13, 14) and in the progression of aortic aneurysms (14, 15). Downregulation of miR-29 expression has been identified as part of the fibrotic response associated with liver (16) and kidney (17) fibrosis, fibrotic skin in systemic sclerosis (18), and cardiac fibrosis in response to ischemic insult (12). TGF-β represses miR-29 expression in cardiac fibroblasts, hepatic stellate cells, and dermal fibroblasts, thereby increasing expression of ECM target genes (12, 18, 19). Furthermore, analysis of mouse embryonic fibroblasts deficient in either Smad2 or Smad3 indicates that the TGF-β–driven decrease in miR-29 expression and increase in Col1a2 and Col3a1 expression is dependent on Smad3 and not Smad2 (20).

Aortic aneurysms and miR-29b

In this issue of the JCI, Maegdefessel et al. demonstrate that modulation of miR-29b expression affects aortic aneurysm progression in two mouse models of the disease (15). AAAs were induced in 10-week-old mice by infusing porcine pancreatic elastase into the infrarenal segment of the aorta for five minutes, which causes extensive destruction of the elastic lamellae and pronounced inflammation in the adventitial regions (21). AAAs were induced in the second model by infusing AngII into 10-week-old Apoe^–/– mice, which leads to aortic aneurysm formation in the suprarenal aorta, with associated elastin degradation and macrophage accumulation. In both models of aneurysm formation, Maegdefessel and colleagues showed that miR-29b expression was significantly downregulated with aneurysm progression over 21 days, with little to no change in miR-29a and miR-29c (15) Furthermore, expression of Col1a1 and Col3a1 increased over time, whereas Eln expression was only increased at 14 days in both mouse models. In both aneurysm models, increased expression of miR-29b and decreased collagen gene expression augmented aneurysm growth, whereas inhibition of miR-29b and increased collagen expression slowed aneurysm formation. Taken together, decreasing the expression of miR-29b beyond the normal decreases that accompany injury to aortic tissue was associated with enhanced expression of several ECM proteins and decreased expansion rates of aortic aneurysms (Figure 1). Expression of miR-29b was also assessed in the aortas of patients with large AAAs compared with that in donor control aortas. Despite the caveat that the control aortas were from substantially younger individuals than those from the patients with AAA (mean age, 33 years in the controls versus 64 years in the patients), miR-29 expression was decreased and COL1A1, COL3A1, and ELN expression was increased in the AAA aortas compared with that in control aortas.

Figure 1

Decreased expression of miR-29b and aortic aneurysm progression. AAAs were induced in 10-week-old mice by infusing porcine pancreatic elastase into the infrarenal segment of the aorta. miR-29b expression was significantly downregulated with aneurysm progression over 21 days, and expression of collagen genes (Col1a1 and Col3a1) increased. Administration of locked nucleic acid anti–miR-29b further decreased miR-29b levels and greatly increased expression of collagen genes, resulting in a reduction in aortic enlargement over time. Increasing the expression of miR-29b using a lentiviral vector (pre–miR-29b) increased expression of miR-29b and decreased collagen gene expression, leading to augmented aortic growth.

Maegdefessel and colleagues also provided some initial data to address which cells in the aorta altered expression of miR-29 (15). When adventitial fibroblasts explanted from the aortic arch of human donors were exposed to TGF-β1, they significantly decreased expression of miR-29b, whereas aortic SMCs did not, implicating adventitial fibroblasts as the cells responsible for the protective fibrotic response in the aorta. Cardiac fibroblasts, and not cardiomyocytes, have similarly been implicated in the fibrotic response in the heart with ischemic insult (12).

The role of miR-29 in aortic aneurysm formation was also recently investigated by Dimmeler and her colleagues but with different results (14). Using a mouse model of thoracic aortic disease, the FBLN4 hypomorphic mouse, they found that miR-29a, miR-29b, and miR-29c were increased in aortic aneurysms in the mutant mouse. Aortic disease in the FBLN4 mouse model is associated with evidence of increased TGF-β signaling, including increased nuclear phosphorylated Smad2 (pSmad2) and increased connective tissue growth factor (CTGF) and collagen deposition in the medial and adventitial layer (22). Therefore, it is surprising that miR-29b would be increased rather than decreased in the diseased aorta. Given that Maegdefessel and colleagues identified that adventitial fibroblasts rather than aortic SMCs responded to TGF-β to decrease miR-29b levels (15), the increased expression of the miR-29 family members with the FBLN4 mouse (14) may be due to the fact that the investigators removed the adventitial layer from the aortic tissues prior to analysis, therefore removing the adventitial fibroblasts. When the expression of the miR-29 family was analyzed in TAA tissues, including aortas from patients with TAAs and BAV, the investigators found increased miR-29b expression compared with that in control tissues (14). However, the methods used to process the human tissues were not provided, and whether the adventitia was removed is not known.

The Dimmeler group also found increased miR-29b expression in the aorta with AngII infusion (14), although their experiments differed from those of Maegdefessel et al. in that they used older mice (18 months) and a slightly lower dose of AngII. When miR-29 activity was inhibited, AngII-treated mice displayed increases in ECM gene expression and extraordinary reduction in aorta dilation (14). Therefore, these results correlate with the findings of Maegdefessel et al. in this issue of JCI.

miR-29 and missing pieces of the puzzle

From studies of development and aging, miR-29b has emerged as an important modulator of gene expression in the aorta. The studies of Maegdefessel et al. indicate that miR-29b expression is decreased with aortic aneurysm formation in mouse models of AAA, and the associated fibrotic responses attenuates aneurysm formation (15). The aortic wall weakens with increased diameter; therefore, the increased expression of collagens associated with decreased miR-29b expression provides additional tensile strength to the aortic wall. Since TGF-β modulates miR-29b expression, it may be a trophic factor responsible for decreased expression of miR-29b with aneurysm progression. Recently, Wang and colleagues showed greater frequency and severity of aortic disease with AngII infusion in wild-type C57BL/6 mice when TGF-β signaling was blocked using neutralizing antibodies (23). Partial but not complete improvement in the incidence and severity of aortic disease was found after manipulations of the immune response. TGF-β–induced decreases in miR-29b expression, leading to increased ECM protein production, may provide further protective effects of TGF-β. Therefore, the data by Maegdefessel et al. (15) indirectly provide further support for a protective role for TGF-β in these mouse models of aneurysm formation.

At the same time, these are puzzle pieces that do not yet provide a clear picture of the role of TGF-β signaling and miR-29 in thoracic aortic disease. Evidence of excessive TGF-β signaling, based on increased staining for nuclear pSMAD2 in aortic SMCs, has been identified in TAA tissue from patients with genetically triggered thoracic aneurysms and patients with thoracic aneurysms and BAV (4–6, 24). Therefore, the increased levels of miR-29b in a mouse model of TAA and human TAA tissue are difficult to reconcile in the face of evidence of increased TGF-β signaling. However, as mentioned above, the decrease in miR-29b could simply be due to not including the adventitial fibroblasts in the analyses, since these cells decrease expression of miR-29b more robustly in response to TGF-β than aortic SMCs.

An alternative explanation for increased pSMAD2 immunostaining in aortic tissues accompanied by increased miR-29b levels is a lack of response of aortic cells to TGF-β, leading to increased TGF-β production to compensate for this resistance. Relevant to this speculation is the fact that the TGFBR1 and TGFBR2 mutations causing thoracic aortic disease such as Loeys-Dietz syndrome (LDS) fall in the kinase domain of these receptors, and a subset of the mutations have been shown to disrupt kinase function critical for TGF-β signaling (25). Furthermore, some patients with thoracic aortic disease have frameshift mutations in SMAD3 predicted to cause haploinsufficiency (6, 7). Could the increased nuclear pSMAD2 immunostaining observed in the aortas of patients with SMAD3 mutations reflect increased shunting of TGF-β signaling through SMAD2 rather than SMAD3? Previous studies have indicated that decreased miR-29b levels in response to TGF-β are dependent on SMAD3 rather than SMAD2 (20), therefore the increased SMAD2 signaling would not compensate for the loss of SMAD3 signaling. It is also important to note that patients with TGFBR1, TGFBR2, or SMAD3 mutations have thin, translucent skin, with visible veins and scars that are atrophic and wide, findings that are suggestive of decreased expression of ECM proteins in the skin and poor myofibroblast contraction of scars and consistent with loss of TGF-β signaling. Incomplete transformation of dermal fibroblasts from patients with TGFBR2 mutations to myofibroblasts with exposure to TGF-β1 has been demonstrated, further supporting a loss of SMAD3 signaling in these cells (25).

The differential response of adventitial fibroblasts compared with that of SMCs shown by Maegdefessel and colleagues also raises important questions as to the specific roles of SMCs and adventitial fibroblasts in aortic disease progression. It has been shown that adventitial fibroblasts constitutively secrete numerous proinflammatory cytokines, particularly IL-6 and MCP-1, and expression of these genes is further upregulated in response to AngII stimulation (26). AngII also stimulates adventitial fibroblasts to recruit monocytes, and the recruited monocytes further activate fibroblast proliferation, adventitial thickening, and additional cytokine production (26). Investigating the role of TGF-β and miR-29 in this adventitial inflammatory and fibrotic response with AngII infusion is critical for our understanding of aortic disease progression.

Finally, miR-29 expression is altered with skeletal muscle differentiation; miR-29 expression is suppressed in myoblasts but increases during differentiation to facilitate myogenesis (27). Studies have identified that the adventitia contains progenitor cells that can differentiate into SMCs (28). Could miR-29 expression similarly be involved in the differentiation of the adventitial progenitor cells to SMCs in the ascending thoracic aorta with aneurysm progression? Although a glimpse of the role of TGF-β and miR-29 is revealed as pieces of the puzzle are identified, there is much more research to be done before this picture will be complete.

Acknowledgments

The author would like to thank Amy Reid, Christina Papke, and Callie Kwartler for helpful discussions. Work in the laboratory of the author is funded by the NIH, the John Ritter Research Program, the National Marfan Foundation, the Ehlers Danlos Syndrome Network, the Vivian L. Smith Foundation, and Genentech.

Footnotes

Conflict of interest: The author has received research funding from Genentech.

Reference information: J Clin Invest. 2012;122(2):490–493. doi:10.1172/JCI62204

See the related article at Inhibition of microRNA-29b reduces murine abdominal aortic aneurysm development.

References

1. Thompson RW, Geraghty PJ, Lee JK. Abdominal aortic aneurysms: basic mechanisms and clinical implications. Curr Probl Surg. 2002;39(2):110–230.
   View this article via: PubMed CrossRef Google Scholar
2. Hiratzka LF, et al. 2010 ACCF/AHA/AATS/ACR/ASA/SCA/SCAI/SIR/STS/SVM Guidelines for the Diagnosis and Management of Patients With Thoracic Aortic Disease: Executive Summary. A Report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines, American Association for Thoracic Surgery, American College of Radiology, American Stroke Association, Society of Cardiovascular Anesthesiologists, Society for Cardiovascular Angiography and Interventions, Society of Interventional Radiology, Society of Thoracic Surgeons, and Society for Vascular Medicine. Circulation. 2010;121(13):e266–e369.
   View this article via: PubMed CrossRef Google Scholar
3. Faivre L, et al. Effect of mutation type and location on clinical outcome in 1,013 probands with Marfan syndrome or related phenotypes and FBN1 mutations: an international study. Am J Hum Genet. 2007;81(3):454–466.
   View this article via: PubMed CrossRef Google Scholar
4. Habashi JP, et al. Losartan, an AT1 antagonist, prevents aortic aneurysm in a mouse model of Marfan syndrome. Science. 2006;312(5770):117–121.
   View this article via: PubMed CrossRef Google Scholar
5. Loeys BL, et al. Aneurysm syndromes caused by mutations in the TGF-beta receptor. N Engl J Med. 2006;355(8):788–798.
   View this article via: PubMed CrossRef Google Scholar
6. van de Laar IM, et al. Mutations in SMAD3 cause a syndromic form of aortic aneurysms and dissections with early-onset osteoarthritis. Nat Genet. 2011;43(2):121–126.
   View this article via: PubMed CrossRef Google Scholar
7. Regalado ES, et al. Exome sequencing identifies SMAD3 mutations as a cause of familial thoracic aortic aneurysm and dissection with intracranial and other arterial aneurysms. Circ Res. 2011;109(6):680–686.
   View this article via: PubMed CrossRef Google Scholar
8. Guo DC, et al. Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections. Nat Genet. 2007;39(12):1488–1493.
   View this article via: PubMed CrossRef Google Scholar
9. Zhu L, et al. Mutations in myosin heavy chain 11 cause a syndrome associating thoracic aortic aneurysm/aortic dissection and patent ductus arteriosus. Nat Genet. 2006;38(3):343–349.
   View this article via: PubMed CrossRef Google Scholar
10. Wang L, et al. Mutations in Myosin light chain kinase cause familial aortic dissections. Am J Hum Genet. 2010;87(5):701–707.
    View this article via: PubMed CrossRef Google Scholar
11. Small EM, Olson EN. Pervasive roles of micro­RNAs in cardiovascular biology. Nature. 2011;469(7330):336–342.
    View this article via: PubMed Google Scholar
12. van Rooij E, et al. Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proc Natl Acad Sci U S A. 2008;105(35):13027–13032.
    View this article via: PubMed Google Scholar
13. Ott CE, et al. MicroRNAs differentially expressed in postnatal aortic development downregulate elastin via 3′ UTR and coding-sequence binding sites. PLoS ONE. 2011;6(1):e16250.
    View this article via: PubMed CrossRef Google Scholar
14. Boon RA, et al. MicroRNA-29 in aortic dilation: implications for aneurysm formation. Circ Res. 2011;109(10):1115–1119.
    View this article via: PubMed CrossRef Google Scholar
15. Maegdefessel L, et al. Inhibition of microRNA-29b reduces murine abdominal aortic aneurysm development. J Clin Invest. 2012;122(2):497–506.
    View this article via: JCI Google Scholar
16. Kwiecinski M, et al. Hepatocyte growth factor (HGF) inhibits collagen I and IV synthesis in hepatic stellate cells by miRNA-29 induction. PLoS One. 2011;6(9):e24568.
    View this article via: PubMed CrossRef Google Scholar
17. Wang B, et al. Suppression of microRNA-29 expression by TGF-beta1 promotes collagen expression and renal fibrosis [published online ahead of print November 17, 2011]. J Am Soc Nephrol. doi:10.1681/ASN.2011010055.
    View this article via: PubMed Google Scholar
18. Maurer B, et al. MicroRNA-29, a key regulator of collagen expression in systemic sclerosis. Arthritis Rheum. 2010;62(6):1733–1743.
    View this article via: PubMed CrossRef Google Scholar
19. Ogawa T, Iizuka M, Sekiya Y, Yoshizato K, Ikeda K, Kawada N. Suppression of type I collagen production by microRNA-29b in cultured human stellate cells. Biochem Biophys Res Commun. 2010;391(1):316–321.
    View this article via: PubMed CrossRef Google Scholar
20. Qin W, et al. TGF-beta/Smad3 signaling promotes renal fibrosis by inhibiting miR-29. J Am Soc Nephrol. 2011;22(8):1462–1474.
    View this article via: PubMed CrossRef Google Scholar
21. Daugherty A, Cassis LA. Mouse models of abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol. 2004;24(3):429–434.
    View this article via: PubMed CrossRef Google Scholar
22. Hanada K, et al. Perturbations of vascular homeostasis and aortic valve abnormalities in fibulin-4 deficient mice. Circ Res. 2007;100(5):738–746.
    View this article via: PubMed CrossRef Google Scholar
23. Wang Y, et al. TGF-beta activity protects against inflammatory aortic aneurysm progression and complications in angiotensin II-infused mice. J Clin Invest. 2010;120(2):422–432.
    View this article via: JCI PubMed CrossRef Google Scholar
24. Gomez D, et al. Syndromic and non-syndromic aneurysms of the human ascending aorta share activation of the Smad2 pathway. J Pathol. 2009;218(1):131–142.
    View this article via: PubMed CrossRef Google Scholar
25. Inamoto S, et al. TGFBR2 Mutations alter smooth muscle cell phenotype and predispose to thoracic aortic aneurysms and dissections. Cardiovasc Res. 2010;88(3):520–529.
    View this article via: PubMed CrossRef Google Scholar
26. Tieu BC, et al. Aortic adventitial fibroblasts participate in angiotensin-induced vascular wall inflammation and remodeling. J Vasc Res. 2011;48(3):261–272.
    View this article via: PubMed Google Scholar
27. Wang H, et al. NF-kappaB-YY1-miR-29 regulatory circuitry in skeletal myogenesis and rhabdomyosarcoma. Cancer Cell. 2008;14(5):369–381.
    View this article via: PubMed CrossRef Google Scholar
28. Hu Y, et al. Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice. J Clin Invest. 2004;113(9):1258–1265.
    View this article via: JCI PubMed Google Scholar