Curr Atheroscler Rep (2016) 18:15 DOI 10.1007/s11883-016-0567-4
GENETICS (A. MARIAN, SECTION EDITOR)
Epigenetics and Peripheral Artery Disease Jonathan Golledge 1,2 & Erik Biros 1 & John Bingley 3,4 & Vikram Iyer 1,2,4 & Smriti M. Krishna 1
# Springer Science+Business Media New York 2016
Abstract The term epigenetics is usually used to describe inheritable changes in gene function which do not involve changes in the DNA sequence. These typically include non-coding RNAs, DNA methylation and histone modifications. Smoking and older age are recognised risk factors for peripheral artery diseases, such as occlusive lower limb artery disease and abdominal aortic aneurysm, and have been implicated in promoting epigenetic changes. This brief review describes studies that have associated epigenetic factors with peripheral artery diseases and investigations which have examined the effect of epigenetic modifications on the outcome of peripheral artery diseases in mouse models. Investigations have largely focused on microRNAs and have identified a number of circulating microRNAs associated with human peripheral artery diseases. Upregulating or antagonising a number of
This article is part of the Topical Collection on Genetics * Jonathan Golledge [email protected]
1
The Vascular Biology Unit, Queensland Research Centre for Peripheral Vascular Disease, College of Medicine & Dentistry, James Cook University, Townsville, QLD 4811, Australia
2
Department of Vascular and Endovascular Surgery, The Townsville Hospital, Townsville, QLD, Australia
3
Vascular Surgery Unit, Mater Hospital Brisbane, South Brisbane, QLD, Australia
4
Department of Surgery, University of Queensland, Brisbane, Australia
microRNAs has also been reported to limit aortic aneurysm development and hind limb ischemia in mouse models. The importance of DNA methylation and histone modifications in peripheral artery disease has been relatively little studied. Whether circulating microRNAs can be used to assist identification of patients with peripheral artery diseases and be modified in order to improve the outcome of peripheral artery disease will require further investigation. Keywords Epigenetics . Peripheral artery disease . Abdominal aortic aneurysm . microRNAs . DNA methylation
Introduction The term peripheral artery disease (PAD) has been used to describe a group of clinical problems caused by narrowing, occlusion or aneurysm formation in the peripheral arteries [1, 2]. This review will focus on two particular aspects of PAD, namely lower limb occlusive disease, which usually results from atherosclerosis and associated thrombosis, and abdominal aortic aneurysm ( A A A ) [1 – 4 ] . We l l - e s t a b l i s h e d r i sk fa c t o r s f o r atherosclerosis-associated lower limb PAD include smoking, older age, diabetes, hypertension and dyslipidemia [1, 3]. These are also risk factors for AAA with the notable exception of diabetes which is reported to be protective against AAA development and growth [2, 4–6]. Family history is recognised as a risk factor for both atherosclerosis-associated lower limb occlusive artery disease and AAA [4, 7•]. It is now recognised that many environmental risk factors can modify genetic factors, such as gene expression, through dynamic changes within the individual. These modifications frequently occur
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through epigenetic mechanisms. This review describes current work being undertaken to understand the relevance of epigenetics to patients with PAD.
association of microRNAs with human PAD presence and the assessment of the effect of modulating microRNAs on the outcome of PAD in rodent models.
Epigenetics and Their Relevance to PAD
The Association of microRNAs with Human AAA
Epigenetics describes inheritable changes in gene function which do not involve changes in the DNA sequence (Fig. 1) [8–10]. Commonly described protein-, DNAand RNA-mediated epigenetic mechanisms include histone modifications such as acetylation, DNA methylation and RNA interference (RNAi) such as microRNAs [8–10]. Many of the risk factors for lower limb occlusive disease and AAA, such as smoking, diabetes and ageing, have been implicated in promoting these diseases by stimulating epigenetic changes [8–10]. Some circulating markers associated with PAD have also been implicated in promoting epigenetic changes [1, 10]. For example, high circulating concentrations of homocysteine have been shown to alter DNA methylation patterns [10]. Despite the proposed importance of epigenetic alternations in PAD, relatively few studies have examined the association of epigenetic changes with human PAD. Most studies have focussed on microRNAs [11–13]. The investigations have included studies examining the
The majority of the studies examining the association of microRNA expression with vascular disease have focused on patients with AAA (Tables 1 and 2). Table 1 summarises studies which have looked at the expression of microRNAs in biopsies of human AAA in comparison to control biopsies obtained from organ donors, postmortems, the non-dilated neck of the AAA or the ascending aorta during aortic valve replacement [14•, 15, 16•, 17, 18•, 19, 20]. These studies have suggested a large range of different microRNAs which are differentially expressed in AAA biopsies. Few microRNAs have been identified as consistently differentially expressed in repeated studies. miR-21 is an example of a microRNA reported as upregulated within the tissue of AAA in two independent studies [17, 19], but it has also been reported to be upregulated in the atheroma of patients with lower limb occlusive artery disease and thus may not be specific for AAA [20]. The inconsistencies between studies are not surprising given the variation in control samples used,
Fig. 1 Suggested epigenetic mechanisms implicated in the pathogenesis of abdominal aortic aneurysm and lower limb occlusive peripheral artery disease. This is a schematic overview of three major epigenetic mechanisms and their potential regulatory role in gene expression relevant to AAA and PAD. microRNAs can downregulate gene expression by mRNA degradation or preventing mRNA translation. The histone modifications include addition of chemical marks such as acetylation, phosphorylation and ubiquitination that regulate gene expression by affecting the chromatin structure. DNA methylation is the
addition of methyl groups within a cytosine (C) followed by a guanine (G) within the promoter region of a gene. Hypermethylation within the promoter region has been usually reported to result in gene silencing. Alteration of these epigenetic mechanisms could potentially perturb the artery wall homeostasis resulting in smooth muscle cell apoptosis or proliferation, phenotype switching, extracellular matrix degradation, inflammation, cell migration and angiogenesis, contributing to AAA or PAD development and progression. Abbreviations: AAA, abdominal aortic aneurysm; PAD, peripheral arterial disease
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the different sampling methods, the range of assessments used to measure microRNA expression, the small sample sizes employed and the dynamic nature of epigenetic changes (Table 1). Most studies used whole aortic biopsies although in one study aortic media alone was examined and in another study laser microdissection of adventitial tertiary lymphoid tissue was performed [14•, 15]. Overall, the reports suggest that a large range of microRNAs are differentially expressed in AAA samples as might be expected from the marked histological changes seen in end-stage AAA, such as dense inflammation and loss of smooth muscle cells and extracellular matrix proteins. Whether these findings simply reflect changes in the cell types present within the aortic wall or reflect important changes in the resident cells themselves is not resolved. A number of other studies have examined the association of circulating microRNAs with AAA presence using whole blood, plasma or serum [14•, 16•, 17, 18•, 21••] (Table 2). In general, these studies have identified Table 1
different differentially expressed microRNAs than the investigations examining tissue expression of microRNAs questioning whether these circulating markers are truly representative of AAA. Circulating microRNAs are very stable so they have been suggested as potential diagnostic tools. In one study, the potential of diagnosing AAA using the relative expression of plasma miR-191-3p, miR-4553p and miR-1281 was assessed [21••]. The investigators reported areas under the curve (AUC) for receiver operator characteristic (ROC) curves of 0.92–0.98 which suggests that these markers have potential to be used as diagnostics although this needs to be examined in much larger cohorts of individuals screened for AAA. The Association of microRNAs with Human Occlusive Lower Limb PAD A small number of studies have examined the association of microRNA expression with occlusive PAD using tissue
Examples of studies examining the association of tissue microRNAs with AAA and occlusive lower limb PAD
Reference
Cases
Controls
Sample
Screening
Differentially expressed miRNAs
14
AAA (open repair; n = 20)
Organ donors
Microarray assay for 850 miRNAs
15
AAA (open repair; n = 6)
Organ donors (n = 6)
Laser microdissection of adventitial tertiary lymphoid organs Aortic media
16
Large AAA (open repair; n = 19) Large AAA (open repair; n = 19)
Cadaveric donors (n = 10)
Aortic biopsies
Ascending aorta (at valve replacement; n = 7)
Aortic biopsies
16 selected miRNAs by real-time PCR miRNA microarray
18
Large AAA (≥50 mm, body, n = 10)
Large AAA (≥50 mm, neck, n = 10)
Aortic biopsies
Luminex’s FlexmiRTM microRNA Human Panel
19
Large AAA (open repair; n = 5)
Non-aneurysmal aortic biopsies from autopsy (n = 5)
Aortic biopsies
miRNA microarray
20
Atheroma samples from PAD patients (n = 51; “sclerotic intima”)
“normal intima” near edge of “sclerotic intima” from PAD patients (n = 51)
Tissue samples (site not stated)
Real time PCR of 13 miRNAs
Upregulated: miR-489-3p; downregulated: miR15a-3p and 30a-5p Upregulated: miR-516a; downregulated: miR1260 No significant difference in expression 77 upregulated: miR-126, 20a, 27a, 221, 222, 21, 155, 146a, 223, 124a and 29b and let-7 validated Upregulated: miR-155, 28, 30a-3p, 150, 302b∗, 93 and 99a; miR-96, 9, 105 and 33 only expressed in AAA body; miR-155 validated Upregulated: miR-181a*, 146a and 21; downregulated: miR133b, 331-3p, 133a, 30c-2* and 204; only downregulated miRNAs replicated miR-21, 130a, 27b and 210, and let-7f upregulated; miR-221 and 222 downregulated
17
miRCURY locked nucleic acid array
AAA abdominal aortic aneurysm, PAD lower limb occlusive peripheral artery disease, PCR polymerase chain reaction
Cases screened
Large AAA (≥55 mm; n = 15)
AAA (focal dilatation exceeding “normal” diameter by ≥50% on CT; n = 10)
AAA (not defined; n = 23)
Large AAA (≥50mm, n = 10)
AAA (n = 20)
PAD, chronic wounds and diabetes (n = 17)
PAD with intermittent claudication confirmed on imaging (n = 5)
16
21
17
18
14
22
23 Healthy controls (n = 6) not clear how PAD excluded
Diabetes but no PAD or chronic wounds (n = 20) not clear how PAD excluded
40 individuals (20 healthy controls and 20 PAD)
81 individuals (26 PAD and wounds; 12 PAD alone; 23 diabetes controls; 20 healthy controls)
Not applicable
Not applicable
Not applicable
120 individuals (60 healthy controls; 60 AAA)
200 individuals (40 healthy controls; 40 PAD; 40 small AAA; 40 large AAA and 40 repaired AAAs)
Validation group
TaqMan human microRNA array (362 miRNAs assessed)
Affymetrix miRNA microarray
Pooled plasma
Whole blood
Real time PCR
Selective real-time PCR for miRNAs upregulated in tissue microRNA array of 124 miRNAs
Affymetrix miRNA microarray
TaqMan human microRNA array (395 miRNAs assessed)
Screening technique
Plasma
Serum
Plasma
Pooled plasma
Whole blood
Sample screened
29 (19 downregulated and 10 upregulated); let-7e (FC1.80 AAA; FC-1.71 PAD), miR-15a (FC-2.24 AAA; FC-2.19 PAD), miR-196b (FC-2.26 AAA; FC-2.11 PAD) and miR-411 (FC5.90 AAA; FC4.52 PAD) validated; but similar pattern in PAD patients 151 miRNAs. miR-191-3p, 455-3p and 1281 most upregulated and confirmed in validation study. ROC curves had area under the curves of 0.9700, 0.9825 and 0.9206, respectively, for these miRNAs miR-126, 124a, 146a, 155, 223, 29b, 15a, and 15b downregulated miR-220, 10a and 23b only expressed in serum of AAA patients; upregulated miR155; downregulated miR141 Downregulated: miR-15a-3p (FC 0.5), 30a-5p (FC 0.8) Plasma miR-191 and 200b upregulated in patients with wounds (rather than PAD) compared to diabetes controls; these miRNAs correlated with markers of inflammation and wound size 53 (34 downregulated and 19 upregulated); 12 validated (Let-7e, miR-15b, 16, 20b, 25, 26b, 27b, 28-5p, 126, 195, 335, and 363); miR16, 363, and 15b had the
Differentially expressed miRNAs
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PAD (n = 17)
Healthy controls (no imaging reported; n = 12) PAD (age and sex matched and AAA excluded by CTA)
Healthy controls (AAA excluded by ultrasound; n = 10)
Healthy controls (n = 10) not clear how AAA excluded
Controls screened
Examples of studies examining the association of circulating microRNAs with AAA and occlusive lower limb PAD
Reference
Table 2
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PAD (n = 104) 20
PAD peripheral artery disease, AAA abdominal aortic aneurysm, PCR polymerase chain reaction, CT computed tomography, FC fold change or difference
Real time PCR of 13 miRNAs Serum
PAD (n = 40) 16
No PAD controls (n = 105)
Cases screened
Healthy controls (n = 40)
200 individuals (40 healthy controls; 40 PAD; 40 small AAA; 40 large AAA and 40 repaired AAAs) Not applicable
Whole blood
Selected miRNAs by real-time PCR
best predictive value with an area under the curve >0.92 (P < 0.001) Downregulated: let-7e (FC1.71), miR-15a (FC-2.19) and 196b (FC-2.11); Upregulated: miR-411 (FC4.52) miR-21, 130a, 27b and 210 upregulated; miR-130a, 27b and 210 associated with Fontaine stage
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Reference
Table 2 (continued)
Controls screened
Validation group
Sample screened
Screening technique
Differentially expressed miRNAs
Curr Atheroscler Rep (2016) 18:15
or blood samples (Tables 1 and 2) [16•, 20, 22, 23••]. A number of the microRNAs identified, such as miR-21, have also been associated with AAA questioning the value of these markers in differentiating between patients with different types of vascular disease [17, 19, 20]. The majority of the studies performed have screened very small number of patients and then in some instances examined larger number of individuals to validate findings (Tables 1 and 2). The two largest studies included approximately 200 individuals (Table 2) [16•, 20]. One of the smaller studies examined blood from a total of 20 PAD patients and 20 healthy controls and reported that the use of groups of microRNAs, such as miR-16, miR-363 and miR-15b, can accurately differentiate between patients with intermittent claudication and healthy controls (AUC >0.92 for ROC curves) [23••]. Much larger studies are, however, needed to examine whether this excellent diagnostic potential can be reproduced. The Effect of Modifying microRNAs Within Mouse Models of AAA The effect of overexpressing or antagonising different microRNAs on the development of aortic aneurysm within mouse models has been studied by a number of investigators (Table 3) [24•, 25–27, 28••, 29]. The most widely studied has been miR-29b which has been independently reported to promote AAA development in three separate studies involving two different mouse models [25, 28••, 29]. miR-29b is known to control the expression of a number of key aortic extracellular matrix proteins including a large number of collagen isoforms, fibrillin-1 and elastin [28••]. miR-29b has also been shown to modulate a number of other genes implicated in aortic aneurysm formation such as matrix metalloproteinase-9 [28••]. Upregulation of miR-29b has been reported within the aortas of a number of mouse models of aortic aneurysm such as mice deficient in fibulin-4 and fibrillin-1, and the angiotensin-II induced model [29, 30]. In contrast, miR-29b downregulation has been reported during aneurysm induction using the elastase and angiotensin II infusion methods in another study [28••]. miR-29b upregulation has been reported in human thoracic and abdominal aortic aneurysm samples in some [17, 29] but not all studies [28••]. Antagonising miR-29b using locked nucleic acids or its antagomirs has been reported to limit aortic expansion in the angiotensin-II, elastase and fibrillin-1 deficient mouse models [25, 28••, 29, 30]. Manipulation of a number of other microRNAs has also been reported to limit AAA within mouse models, including overexpression of miR-21 and miR-24, and antagonism of miR-205 and miR-712 (Table 3) [24•, 26, 27]. The reported biological effects
miR-24
miR-29b
miR-712
miR-205
miR-21
miR-29b
miR-29b
24
25
26
26
27
28
29
AngII
AngII
Elastase and AngII
ApoE−/− male mice ApoE−/− male mice ApoE−/− and C57BL/6J
C57BL/6J
AngII
Elastase AngII
AngII
ApoE−/− male mice
C57BL/6J ApoE−/−
Elastase
AAA model
C57BL/6J
Mouse strain
Maximum AAD on ultrasound
Maximum AAD on ultrasound
Maximum AAD from histology of excised aortas Maximum AAD from histology of excised aortas Maximum AAD on ultrasound
Maximum AAD on MRI
Maximum AAD on ultrasound
AAA assessment
lentiviral vector overexpression of miR-21 locked nucleic acid anti-miR-29b lentiviral vector overexpression of miR-29b locked nucleic acid anti-miR-29
Anti-miR-205
Anti-miR-712
lentiviral vector overexpression of pre-miR-24, antimiR-24 or scrmiR antagomiR-29b
Intervention
NS
1 day after AAA induction
1 day after AAA induction
Days 1, 2 and 3 after starting AAA induction every 4 days after starting AngII infusion NS
1 day after AAA induction
Timing relative to AAA induction
Reduced AAA induction
Reduced AAA induction Enhanced AAA
Reduced AAA induction
Reduced AAA induction
Reduced AAA induction
Reduced AAA induction
Overexpressing miR-24 reduced AAA induction; Anti-miR-24 promoted AAA induction
Outcome
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AAA abdominal aortic aneurysm, AAD abdominal aortic diameter, ApoE−/− apolipoprotein E deficient, NS not stated, MRI magnetic resonance imaging, AngII angiotensin II
microRNAs
Examples of mouse studies examining the effect of microRNAs on AAA
Reference
Table 3
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included modifying proliferation of cells within the aortic wall, in addition to changes in inflammation and extracellular matrix remodelling [24•, 26, 27]. It is unclear, however, whether these findings in animal models are directly translatable to patients with AAA. HLI hind limb ischemia, FA femoral artery, LDI laser Doppler imaging, KO knockout, NS not stated, EC endothelial cell, TG transgenic, d days following ischemia induction
Slower perfusion recovery Slower perfusion recovery More rapid perfusion recovery Greater perfusion at d19 Slower perfusion recovery More rapid perfusion recovery Worse perfusion up to d14 Worse perfusion up to d22 Greater perfusion at d14 No effect prior prior 1 day prior 10 days after prior prior prior prior prior prior miR132/212 KO miR155 KO Gene silencing oligonucleotides miR126 microbubble liposomes antagomir-93 PremiR-93 106b∼25 KO EC-miR-15a TG antagomir-92a antagomir-126 LDI LDI LDI LDI LDI LDI LDI LDI LDI LDI Proximal FA transection Double FA ligation Double FA ligation Excision of FA Excision of FA Excision of FA FA ligation Excision of FA Excision of FA Electrocautery of FA 132/212 155 14q32 (329, 487b, 494 and 495) 126 93 93 106b∼25 cluster (106b, 93, 25) 15a 92a 126 31 32 33 34 35 35 36 37 38 39, 40
C57BL/6J C57BL/6J C57BL/6J ICR C57BL/6J BALB/cJ C57BL/6J NS C57BL/6J C57BL/6J
Perfusion assessment HLI model Mouse strain microRNAs Reference
Table 4
Examples of mouse studies examining the effect of microRNAs on hind limb ischemia
Intervention
Timing relative to ischemia induction
Outcome
Curr Atheroscler Rep (2016) 18:15
The Effect of Modifying microRNAs Within Animal Models of Hind Limb Ischemia Promotion of new vessel formation (angiogenesis) and stimulation of the expansion of existing collaterals (arteriogenesis) are coordinated by a group of signalling pathways, genes, growth factors and cells [1]. microRNAs, such as those listed in Table 4 [31, 32, 33••, 34–40], have been shown to control expression of genes implicated in controlling angiogenesis and arteriogenesis, such as vascular endothelial growth factor, fibroblast growth factor, insulin receptor substrate 1, E2F transcription factor 1, and progenitor cell recruitment [33••, 34, 35, 39]. The effect of promoting or antagonising a variety of different microRNAs has been reported to modify perfusion recovery in mouse models of hind limb ischemia, as summarised in Table 4 [31, 32, 33••, 34–40]. For example, enhancing miR-93 or mir-126 expression has been reported to improve hind limb blood supply recovery in different mice strains [34, 35]. Antagonism of miR-92a and a collection of microRNAs located on the chromosome region 14q32 (miR-329, miR-487b, miR-494 and miR-495) has been reported to promote hind limb re-perfusion following femoral artery ligation in C57BL6 mice [33••, 38]. The increased perfusion resulting from antagonism of these microRNAs was reported to be due to improved arteriogenesis evidenced by larger-diameter arterioles within the adductor muscle [33••]. Whether these approaches to improving limb blood supply can be brought into clinical practice is not yet clear since there are accepted deficiencies with current rodent models of limb ischemia (see a recent review [41]). Other Epigenetic Modifications and Aortic Aneurysm and Lower Limb Occlusive Artery Disease The association of the DNA methylation pattern with PAD has been relatively little studied. Ryer and colleagues recently examined the genome-wide DNA methylation profiles of mononuclear circulating cells in 20 patients that had AAAs in comparison to 21 age- and sex-matched controls [42•]. The investigators reported four genes with differential CpG island methylation, namely kelch-like family member 35 (KLHL35), calponin 2 (CNN2), serpin peptidase inhibitor clade B (ovalbumin) member 9 (SERPINB9) and adenylate
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cyclase 10 pseudogene 1 (ADCY10P1). It has also been suggested that the expression of microRNAs associated with occlusive lower limb PAD could be influenced by methylation within the promoter regions of DNA coding for microRNAs [42•]. The association of histone modifications with PAD has been little investigated. Vinh and colleagues investigated the effect of a histone deacetylase (HDAC) inhibitor on AAA induction within a mouse model [43]. They reported that the HDAC inhibitor metacept-1 inhibited AAA induction by angiotensin-II by downregulating matrix metalloproteinases. The importance of these findings in AAA and lower limb PAD pathogenesis is currently unclear.
References
Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance 1.
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Conclusions: the Clinical Potential of Epigenetics in Patients with AAA and Lower Limb Occlusive Artery Disease
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As outlined above, the majority of investigations currently published have focused on the relevance of microRNAs to AAA and lower limb occlusive artery disease. Based on these studies, microRNAs may have potential as diagnostic aids in patients; however, larger studies are needed to examine whether the currently reported excellent sensitivity and specificity of these markers can be replicated. It also remains to be established whether concentrations of circulating microRNAs will be able to distinguish patients with different types of vascular disease. A large number of mouse studies suggest that upregulation and downregulation of selected microRNA has the potential to improve outcomes in AAA and lower limb occlusive artery disease. It is unclear, however, whether it is possible to translate findings from these mouse models to patients [41, 44, 45]. It also remains to be established whether administrating such gene expression modifying interventions would be safe in patients, particularly given that a single microRNA can control the expression of large number of genes. Phase 1 trials will be required in patients to address this. Other types of epigenetic modifications, such as DNA methylation and histone modification, have been little studied in relation to PAD.
Compliance with Ethical Standards Conflict of Interest Jonathan Golledge declares that he works for James Cook University & The Townsville Hospital and declares grant money to his institution from NHMRC, Queensland Government. Erik Biros, John Bingley, Vikram Iyer, and Smriti M Krishna declare that they have no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.
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Krishna SM, Moxon JV, Golledge J. A review of the pathophysiology and potential biomarkers for peripheral artery disease. Int J Mol Sci. 2015;16:11294–322. Golledge J, Norman PE. Pathophysiology of abdominal aortic aneurysm relevant to improvements in patients’ management. Curr Opin Cardiol. 2009;24:532–8. Golledge J. Lower-limb arterial disease. Lancet. 1997;350:1459– 65. Golledge J, Muller J, Daugherty A, Norman P. Abdominal aortic aneurysm: pathogenesis and implications for management. Arterioscler Thromb Vasc Biol. 2006;26:2605–13. Norman PE, Davis TM, Le MT, Golledge J. Matrix biology of abdominal aortic aneurysms in diabetes: mechanisms underlying the negative association. Connect Tissue Res. 2007;48:125–31. Golledge J, Norman PE. Atherosclerosis and abdominal aortic aneurysm: cause, response, or common risk factors? Arterioscler Thromb Vasc Biol. 2010;30:1075–7. Khaleghi M, Isseh IN, Bailey KR, Kullo IJ. Family history as a risk factor for peripheral arterial disease. Am J Cardiol. 2014;114:928– 32. This is one of the few studies to assess the association of family history with lower limb atherosclerosis associated occlusive disease and reported an odds ratio of 2.2. Krishna SM, Dear AE, Norman PE, Golledge J. Genetic and epigenetic mechanisms and their possible role in abdominal aortic aneurysm. Atherosclerosis. 2010;212:16–29. Krishna SM, Trollope AF, Golledge J. The relevance of epigenetics to occlusive cerebral and peripheral arterial disease. Clin Sci (Lond). 2015;128:537–58. Krishna SM, Dear A, Craig JM, Norman PE, Golledge J. The potential role of homocysteine mediated DNA methylation and associated epigenetic changes in abdominal aortic aneurysm formation. Atherosclerosis. 2013;228:295–305. Kloos W, Vogel B, Blessing E. MiRNAs in peripheral artery disease—something gripping this way comes. Vasa. 2014;43:163–70. Adam M, Raaz U, Spin JM, Tsao PS. MicroRNAs in Abdominal Aortic Aneurysm. Curr Vasc Pharmacol. 2015;13:280–90. Golledge J, Kuivaniemi H. Genetics of abdominal aortic aneurysm. Curr Opin Cardiol. 2013;28:290–6. Spear R, Boytard L, Blervaque R, Chwastyniak M, Hot D, Vanhoutte J, et al. Adventitial tertiary lymphoid organs as potential source of microRNA biomarkers for abdominal aortic aneurysm. Int J Mol Sci. 2015;16:11276–93. This study used laser microdissection to look at MicroRNAs expressed in human AAA associated lymphoid tissue. Cheuk BL, Cheng SW. Identification and characterization of microRNAs in vascular smooth muscle cells from patients with abdominal aortic aneurysms. J Vasc Surg. 2014;59:202–9. Stather PW, Sylvius N, Sidloff DA, Dattani N, Verissimo A, Wild JB, et al. Identification of microRNAs associated with abdominal aortic aneurysms and peripheral arterial disease. Br J Surg. 2015;102:755–66. This study identifies a number of circulating MicroRNAs associated with AAA and lower limb occlusive artery disease. Kin K, Miyagawa S, Fukushima S, Shirakawa Y, Torikai K, Shimamura K, et al. Tissue- and plasma-specific microRNA
Curr Atheroscler Rep (2016) 18:15 signatures for atherosclerotic abdominal aortic aneurysm. J Am Heart Assoc. 2012;1:e000745. 18.• Biros E, Moran CS, Wang Y, Walker PJ, Cardinal J, Golledge J. microRNA profiling in patients with abdominal aortic aneurysms: the significance of miR-155. Clin Sci (Lond). 2014;126:795–803. miR-155 was identified as upregulated within aortic biopsies of human AAA and also serum of patients that had AAAs. 19. Pahl MC, Derr K, Gäbel G, Hinterseher I, Elmore JR, Schworer CM, et al. MicroRNA expression signature in human abdominal aortic aneurysms. BMC Med Genomics. 2012;5:25. 20. Li T, Cao H, Zhuang J, Wan J, Guan M, Yu B, et al. Identification of miR-130a, miR-27b and miR-210 as serum biomarkers for atherosclerosis obliterans. Clin Chim Acta. 2011;412:66–70. 21.•• Zhang W, Shang T, Huang C, Yu T, Liu C, Qiao T, et al. Plasma microRNAs serve as potential biomarkers for abdominal aortic aneurysm. Clin Biochem. 2015;48:988–92. Three plasma microRNAs (miR-191-3p, miR-455-3p and miR-1281) were identified as having good sensitivity and specificity in identifying patients with AAA. 22. Dangwal S, Stratmann B, Bang C, Lorenzen JM, Kumarswamy R, Fiedler J, et al. Impairment of wound healing in patients with type 2 diabetes mellitus influences circulating microRNA patterns via inflammatory cytokines. Arterioscler Thromb Vasc Biol. 2015;35: 1480–8. 23.•• Stather PW, Sylvius N, Wild JB, Choke E, Sayers RD, Bown MJ. Differential microRNA expression profiles in peripheral arterial disease. Circ Cardiovasc Genet. 2013;6:490–7. This study identified a number of microRNAs with good diagnostic ability for lower limb arterial occlusive disease as judged by receiver operator characteristic curves. 24.• Maegdefessel L, Spin JM, Raaz U, Eken SM, Toh R, Azuma J, et al. miR-24 limits aortic vascular inflammation and murine abdominal aneurysm development. Nat Commun. 2014;5:5214. This study suggests that miR-24 inhibits AAA within a mouse model. 25. Zampetaki A, Attia R, Mayr U, Gomes RS, Phinikaridou A, Yin X, et al. Role of miR-195 in aortic aneurysmal disease. Circ Res. 2014;115:857–66. 26. Kim CW, Kumar S, Son DJ, Jang IH, Griendling KK, Jo H. Prevention of abdominal aortic aneurysm by anti-microRNA-712 or anti-microRNA-205 in angiotensin II-infused mice. Arterioscler Thromb Vasc Biol. 2014;34:1412–21. 27. Maegdefessel L, Azuma J, Toh R, Deng A, Merk DR, Raiesdana A, et al. MicroRNA-21 blocks abdominal aortic aneurysm development and nicotine-augmented expansion. Sci Transl Med. 2012;4: 122ra22. 28.•• Maegdefessel L, Azuma J, Toh R, Merk DR, Deng A, Chin JT, et al. Inhibition of microRNA-29b reduces murine abdominal aortic aneurysm development. J Clin Invest. 2012;122:497–506. This study confirms that miR-29b limits AAA within multiple mouse models. 29. Boon RA, Seeger T, Heydt S, Fischer A, Hergenreider E, Horrevoets AJ, et al. MicroRNA-29 in aortic dilation: implications for aneurysm formation. Circ Res. 2011;109:1115–9. 30. Merk DR, Chin JT, Dake BA, Maegdefessel L, Miller MO, Kimura N, et al. miR-29b participates in early aneurysm development in Marfan syndrome. Circ Res. 2012;110:312–24. 31. Lei Z, van Mil A, Brandt MM, Grundmann S, Hoefer I, Smits M, et al. MicroRNA-132/212 family enhances arteriogenesis after
Page 9 of 9 15 hindlimb ischaemia through modulation of the Ras-MAPK pathway. J Cell Mol Med. 2015;19:1994–2005. 32. Pankratz F, Bemtgen X, Zeiser R, Leonhardt F, Kreuzaler S, Hilgendorf I, et al. MicroRNA-155 exerts cell-specific antiangiogenic but proarteriogenic effects during adaptive neovascularization. Circulation. 2015;131:1575–89. 33.•• Welten SM, Bastiaansen AJ, de Jong RC, de Vries MR, Peters EA, Boonstra MC, et al. Inhibition of 14q32 microRNAs miR-329, miR-487b, miR-494, and miR-495 increases neovascularization and blood flow recovery after ischemia. Circ Res. 2014;115:696– 708. This study suggests that gene silencing oligonucleotides targeted on 14q32 expressed microRNAs stimulate arteriogenesis in a mouse model of hind limb ischemia. 34. Endo-Takahashi Y, Negishi Y, Nakamura A, Ukai S, Ooaku K, Oda Y, et al. Systemic delivery of miR-126 by miRNA-loaded Bubble liposomes for the treatment of hindlimb ischemia. Sci Rep. 2014;4: 3883. 35. Hazarika S, Farber CR, Dokun AO, Pitsillides AN, Wang T, Lye RJ, et al. MicroRNA-93 controls perfusion recovery after hindlimb ischemia by modulating expression of multiple genes in the cell cycle pathway. Circulation. 2013;127:1818–28. 36. Semo J, Sharir R, Afek A, Avivi C, Barshack I, Maysel-Auslender S, et al. The 106b∼25 microRNA cluster is essential for neovascularization after hindlimb ischaemia in mice. Eur Heart J. 2014;35: 3212–23. 37. Yin KJ, Olsen K, Hamblin M, Zhang J, Schwendeman SP, Chen YE. Vascular endothelial cell-specific microRNA-15a inhibits angiogenesis in hindlimb ischemia. J Biol Chem. 2012;287:27055– 64. 38. Bonauer A, Carmona G, Iwasaki M, Mione M, Koyanagi M, Fischer A, et al. MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice. Science. 2009;324: 1710–3. 39. van Solingen C, de Boer HC, Bijkerk R, Monge M, van OeverenRietdijk AM, Seghers L, et al. MicroRNA-126 modulates endothelial SDF-1 expression and mobilization of Sca-1(+)/Lin(−) progenitor cells in ischaemia. Cardiovasc Res. 2011;92:449–55. 40. van Solingen C, Seghers L, Bijkerk R, Duijs JM, Roeten MK, van Oeveren-Rietdijk AM, et al. Antagomir-mediated silencing of endothelial cell specific microRNA-126 impairs ischemia-induced angiogenesis. J Cell Mol Med. 2009;13:1577–85. 41. Krishna SM, Omer SM, Golledge J. Evaluation of the clinical relevance and limitations of current preclinical models of peripheral artery disease. Clin Sci. 2016;130:127–50. 42.• Ryer EJ, Ronning KE, Erdman R, Schworer CM, Elmore JR, Peeler TC, et al. The potential role of DNA methylation in abdominal aortic aneurysms. Int J Mol Sci. 2015;16:11259–75. This study looks as the association of DNA methylation patterns with AAA. 43. Hu W, Wang M, Yin H, Yao C, He Q, Yin L, et al. MicroRNA-1298 is regulated by DNA methylation and affects vascular smooth muscle cell function by targeting connexin 43. Cardiovasc Res. 2015;107(4):534–45. 44. Vinh A, Gaspari TA, Liu HB, Dousha LF, Widdop RE, Dear AE. A novel histone deacetylase inhibitor reduces abdominal aortic aneurysm formation in angiotensin II-infused apolipoprotein E-deficient mice. J Vasc Res. 2008;45:143–52. 45. Golledge J, Norman PE. Current status of medical management for abdominal aortic aneurysm. Atherosclerosis. 2011;217:57–63.
GENETICS (A. MARIAN, SECTION EDITOR)
Epigenetics and Peripheral Artery Disease Jonathan Golledge 1,2 & Erik Biros 1 & John Bingley 3,4 & Vikram Iyer 1,2,4 & Smriti M. Krishna 1
# Springer Science+Business Media New York 2016
Abstract The term epigenetics is usually used to describe inheritable changes in gene function which do not involve changes in the DNA sequence. These typically include non-coding RNAs, DNA methylation and histone modifications. Smoking and older age are recognised risk factors for peripheral artery diseases, such as occlusive lower limb artery disease and abdominal aortic aneurysm, and have been implicated in promoting epigenetic changes. This brief review describes studies that have associated epigenetic factors with peripheral artery diseases and investigations which have examined the effect of epigenetic modifications on the outcome of peripheral artery diseases in mouse models. Investigations have largely focused on microRNAs and have identified a number of circulating microRNAs associated with human peripheral artery diseases. Upregulating or antagonising a number of
This article is part of the Topical Collection on Genetics * Jonathan Golledge [email protected]
1
The Vascular Biology Unit, Queensland Research Centre for Peripheral Vascular Disease, College of Medicine & Dentistry, James Cook University, Townsville, QLD 4811, Australia
2
Department of Vascular and Endovascular Surgery, The Townsville Hospital, Townsville, QLD, Australia
3
Vascular Surgery Unit, Mater Hospital Brisbane, South Brisbane, QLD, Australia
4
Department of Surgery, University of Queensland, Brisbane, Australia
microRNAs has also been reported to limit aortic aneurysm development and hind limb ischemia in mouse models. The importance of DNA methylation and histone modifications in peripheral artery disease has been relatively little studied. Whether circulating microRNAs can be used to assist identification of patients with peripheral artery diseases and be modified in order to improve the outcome of peripheral artery disease will require further investigation. Keywords Epigenetics . Peripheral artery disease . Abdominal aortic aneurysm . microRNAs . DNA methylation
Introduction The term peripheral artery disease (PAD) has been used to describe a group of clinical problems caused by narrowing, occlusion or aneurysm formation in the peripheral arteries [1, 2]. This review will focus on two particular aspects of PAD, namely lower limb occlusive disease, which usually results from atherosclerosis and associated thrombosis, and abdominal aortic aneurysm ( A A A ) [1 – 4 ] . We l l - e s t a b l i s h e d r i sk fa c t o r s f o r atherosclerosis-associated lower limb PAD include smoking, older age, diabetes, hypertension and dyslipidemia [1, 3]. These are also risk factors for AAA with the notable exception of diabetes which is reported to be protective against AAA development and growth [2, 4–6]. Family history is recognised as a risk factor for both atherosclerosis-associated lower limb occlusive artery disease and AAA [4, 7•]. It is now recognised that many environmental risk factors can modify genetic factors, such as gene expression, through dynamic changes within the individual. These modifications frequently occur
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through epigenetic mechanisms. This review describes current work being undertaken to understand the relevance of epigenetics to patients with PAD.
association of microRNAs with human PAD presence and the assessment of the effect of modulating microRNAs on the outcome of PAD in rodent models.
Epigenetics and Their Relevance to PAD
The Association of microRNAs with Human AAA
Epigenetics describes inheritable changes in gene function which do not involve changes in the DNA sequence (Fig. 1) [8–10]. Commonly described protein-, DNAand RNA-mediated epigenetic mechanisms include histone modifications such as acetylation, DNA methylation and RNA interference (RNAi) such as microRNAs [8–10]. Many of the risk factors for lower limb occlusive disease and AAA, such as smoking, diabetes and ageing, have been implicated in promoting these diseases by stimulating epigenetic changes [8–10]. Some circulating markers associated with PAD have also been implicated in promoting epigenetic changes [1, 10]. For example, high circulating concentrations of homocysteine have been shown to alter DNA methylation patterns [10]. Despite the proposed importance of epigenetic alternations in PAD, relatively few studies have examined the association of epigenetic changes with human PAD. Most studies have focussed on microRNAs [11–13]. The investigations have included studies examining the
The majority of the studies examining the association of microRNA expression with vascular disease have focused on patients with AAA (Tables 1 and 2). Table 1 summarises studies which have looked at the expression of microRNAs in biopsies of human AAA in comparison to control biopsies obtained from organ donors, postmortems, the non-dilated neck of the AAA or the ascending aorta during aortic valve replacement [14•, 15, 16•, 17, 18•, 19, 20]. These studies have suggested a large range of different microRNAs which are differentially expressed in AAA biopsies. Few microRNAs have been identified as consistently differentially expressed in repeated studies. miR-21 is an example of a microRNA reported as upregulated within the tissue of AAA in two independent studies [17, 19], but it has also been reported to be upregulated in the atheroma of patients with lower limb occlusive artery disease and thus may not be specific for AAA [20]. The inconsistencies between studies are not surprising given the variation in control samples used,
Fig. 1 Suggested epigenetic mechanisms implicated in the pathogenesis of abdominal aortic aneurysm and lower limb occlusive peripheral artery disease. This is a schematic overview of three major epigenetic mechanisms and their potential regulatory role in gene expression relevant to AAA and PAD. microRNAs can downregulate gene expression by mRNA degradation or preventing mRNA translation. The histone modifications include addition of chemical marks such as acetylation, phosphorylation and ubiquitination that regulate gene expression by affecting the chromatin structure. DNA methylation is the
addition of methyl groups within a cytosine (C) followed by a guanine (G) within the promoter region of a gene. Hypermethylation within the promoter region has been usually reported to result in gene silencing. Alteration of these epigenetic mechanisms could potentially perturb the artery wall homeostasis resulting in smooth muscle cell apoptosis or proliferation, phenotype switching, extracellular matrix degradation, inflammation, cell migration and angiogenesis, contributing to AAA or PAD development and progression. Abbreviations: AAA, abdominal aortic aneurysm; PAD, peripheral arterial disease
Curr Atheroscler Rep (2016) 18:15
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the different sampling methods, the range of assessments used to measure microRNA expression, the small sample sizes employed and the dynamic nature of epigenetic changes (Table 1). Most studies used whole aortic biopsies although in one study aortic media alone was examined and in another study laser microdissection of adventitial tertiary lymphoid tissue was performed [14•, 15]. Overall, the reports suggest that a large range of microRNAs are differentially expressed in AAA samples as might be expected from the marked histological changes seen in end-stage AAA, such as dense inflammation and loss of smooth muscle cells and extracellular matrix proteins. Whether these findings simply reflect changes in the cell types present within the aortic wall or reflect important changes in the resident cells themselves is not resolved. A number of other studies have examined the association of circulating microRNAs with AAA presence using whole blood, plasma or serum [14•, 16•, 17, 18•, 21••] (Table 2). In general, these studies have identified Table 1
different differentially expressed microRNAs than the investigations examining tissue expression of microRNAs questioning whether these circulating markers are truly representative of AAA. Circulating microRNAs are very stable so they have been suggested as potential diagnostic tools. In one study, the potential of diagnosing AAA using the relative expression of plasma miR-191-3p, miR-4553p and miR-1281 was assessed [21••]. The investigators reported areas under the curve (AUC) for receiver operator characteristic (ROC) curves of 0.92–0.98 which suggests that these markers have potential to be used as diagnostics although this needs to be examined in much larger cohorts of individuals screened for AAA. The Association of microRNAs with Human Occlusive Lower Limb PAD A small number of studies have examined the association of microRNA expression with occlusive PAD using tissue
Examples of studies examining the association of tissue microRNAs with AAA and occlusive lower limb PAD
Reference
Cases
Controls
Sample
Screening
Differentially expressed miRNAs
14
AAA (open repair; n = 20)
Organ donors
Microarray assay for 850 miRNAs
15
AAA (open repair; n = 6)
Organ donors (n = 6)
Laser microdissection of adventitial tertiary lymphoid organs Aortic media
16
Large AAA (open repair; n = 19) Large AAA (open repair; n = 19)
Cadaveric donors (n = 10)
Aortic biopsies
Ascending aorta (at valve replacement; n = 7)
Aortic biopsies
16 selected miRNAs by real-time PCR miRNA microarray
18
Large AAA (≥50 mm, body, n = 10)
Large AAA (≥50 mm, neck, n = 10)
Aortic biopsies
Luminex’s FlexmiRTM microRNA Human Panel
19
Large AAA (open repair; n = 5)
Non-aneurysmal aortic biopsies from autopsy (n = 5)
Aortic biopsies
miRNA microarray
20
Atheroma samples from PAD patients (n = 51; “sclerotic intima”)
“normal intima” near edge of “sclerotic intima” from PAD patients (n = 51)
Tissue samples (site not stated)
Real time PCR of 13 miRNAs
Upregulated: miR-489-3p; downregulated: miR15a-3p and 30a-5p Upregulated: miR-516a; downregulated: miR1260 No significant difference in expression 77 upregulated: miR-126, 20a, 27a, 221, 222, 21, 155, 146a, 223, 124a and 29b and let-7 validated Upregulated: miR-155, 28, 30a-3p, 150, 302b∗, 93 and 99a; miR-96, 9, 105 and 33 only expressed in AAA body; miR-155 validated Upregulated: miR-181a*, 146a and 21; downregulated: miR133b, 331-3p, 133a, 30c-2* and 204; only downregulated miRNAs replicated miR-21, 130a, 27b and 210, and let-7f upregulated; miR-221 and 222 downregulated
17
miRCURY locked nucleic acid array
AAA abdominal aortic aneurysm, PAD lower limb occlusive peripheral artery disease, PCR polymerase chain reaction
Cases screened
Large AAA (≥55 mm; n = 15)
AAA (focal dilatation exceeding “normal” diameter by ≥50% on CT; n = 10)
AAA (not defined; n = 23)
Large AAA (≥50mm, n = 10)
AAA (n = 20)
PAD, chronic wounds and diabetes (n = 17)
PAD with intermittent claudication confirmed on imaging (n = 5)
16
21
17
18
14
22
23 Healthy controls (n = 6) not clear how PAD excluded
Diabetes but no PAD or chronic wounds (n = 20) not clear how PAD excluded
40 individuals (20 healthy controls and 20 PAD)
81 individuals (26 PAD and wounds; 12 PAD alone; 23 diabetes controls; 20 healthy controls)
Not applicable
Not applicable
Not applicable
120 individuals (60 healthy controls; 60 AAA)
200 individuals (40 healthy controls; 40 PAD; 40 small AAA; 40 large AAA and 40 repaired AAAs)
Validation group
TaqMan human microRNA array (362 miRNAs assessed)
Affymetrix miRNA microarray
Pooled plasma
Whole blood
Real time PCR
Selective real-time PCR for miRNAs upregulated in tissue microRNA array of 124 miRNAs
Affymetrix miRNA microarray
TaqMan human microRNA array (395 miRNAs assessed)
Screening technique
Plasma
Serum
Plasma
Pooled plasma
Whole blood
Sample screened
29 (19 downregulated and 10 upregulated); let-7e (FC1.80 AAA; FC-1.71 PAD), miR-15a (FC-2.24 AAA; FC-2.19 PAD), miR-196b (FC-2.26 AAA; FC-2.11 PAD) and miR-411 (FC5.90 AAA; FC4.52 PAD) validated; but similar pattern in PAD patients 151 miRNAs. miR-191-3p, 455-3p and 1281 most upregulated and confirmed in validation study. ROC curves had area under the curves of 0.9700, 0.9825 and 0.9206, respectively, for these miRNAs miR-126, 124a, 146a, 155, 223, 29b, 15a, and 15b downregulated miR-220, 10a and 23b only expressed in serum of AAA patients; upregulated miR155; downregulated miR141 Downregulated: miR-15a-3p (FC 0.5), 30a-5p (FC 0.8) Plasma miR-191 and 200b upregulated in patients with wounds (rather than PAD) compared to diabetes controls; these miRNAs correlated with markers of inflammation and wound size 53 (34 downregulated and 19 upregulated); 12 validated (Let-7e, miR-15b, 16, 20b, 25, 26b, 27b, 28-5p, 126, 195, 335, and 363); miR16, 363, and 15b had the
Differentially expressed miRNAs
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PAD (n = 17)
Healthy controls (no imaging reported; n = 12) PAD (age and sex matched and AAA excluded by CTA)
Healthy controls (AAA excluded by ultrasound; n = 10)
Healthy controls (n = 10) not clear how AAA excluded
Controls screened
Examples of studies examining the association of circulating microRNAs with AAA and occlusive lower limb PAD
Reference
Table 2
15 Curr Atheroscler Rep (2016) 18:15
PAD (n = 104) 20
PAD peripheral artery disease, AAA abdominal aortic aneurysm, PCR polymerase chain reaction, CT computed tomography, FC fold change or difference
Real time PCR of 13 miRNAs Serum
PAD (n = 40) 16
No PAD controls (n = 105)
Cases screened
Healthy controls (n = 40)
200 individuals (40 healthy controls; 40 PAD; 40 small AAA; 40 large AAA and 40 repaired AAAs) Not applicable
Whole blood
Selected miRNAs by real-time PCR
best predictive value with an area under the curve >0.92 (P < 0.001) Downregulated: let-7e (FC1.71), miR-15a (FC-2.19) and 196b (FC-2.11); Upregulated: miR-411 (FC4.52) miR-21, 130a, 27b and 210 upregulated; miR-130a, 27b and 210 associated with Fontaine stage
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Reference
Table 2 (continued)
Controls screened
Validation group
Sample screened
Screening technique
Differentially expressed miRNAs
Curr Atheroscler Rep (2016) 18:15
or blood samples (Tables 1 and 2) [16•, 20, 22, 23••]. A number of the microRNAs identified, such as miR-21, have also been associated with AAA questioning the value of these markers in differentiating between patients with different types of vascular disease [17, 19, 20]. The majority of the studies performed have screened very small number of patients and then in some instances examined larger number of individuals to validate findings (Tables 1 and 2). The two largest studies included approximately 200 individuals (Table 2) [16•, 20]. One of the smaller studies examined blood from a total of 20 PAD patients and 20 healthy controls and reported that the use of groups of microRNAs, such as miR-16, miR-363 and miR-15b, can accurately differentiate between patients with intermittent claudication and healthy controls (AUC >0.92 for ROC curves) [23••]. Much larger studies are, however, needed to examine whether this excellent diagnostic potential can be reproduced. The Effect of Modifying microRNAs Within Mouse Models of AAA The effect of overexpressing or antagonising different microRNAs on the development of aortic aneurysm within mouse models has been studied by a number of investigators (Table 3) [24•, 25–27, 28••, 29]. The most widely studied has been miR-29b which has been independently reported to promote AAA development in three separate studies involving two different mouse models [25, 28••, 29]. miR-29b is known to control the expression of a number of key aortic extracellular matrix proteins including a large number of collagen isoforms, fibrillin-1 and elastin [28••]. miR-29b has also been shown to modulate a number of other genes implicated in aortic aneurysm formation such as matrix metalloproteinase-9 [28••]. Upregulation of miR-29b has been reported within the aortas of a number of mouse models of aortic aneurysm such as mice deficient in fibulin-4 and fibrillin-1, and the angiotensin-II induced model [29, 30]. In contrast, miR-29b downregulation has been reported during aneurysm induction using the elastase and angiotensin II infusion methods in another study [28••]. miR-29b upregulation has been reported in human thoracic and abdominal aortic aneurysm samples in some [17, 29] but not all studies [28••]. Antagonising miR-29b using locked nucleic acids or its antagomirs has been reported to limit aortic expansion in the angiotensin-II, elastase and fibrillin-1 deficient mouse models [25, 28••, 29, 30]. Manipulation of a number of other microRNAs has also been reported to limit AAA within mouse models, including overexpression of miR-21 and miR-24, and antagonism of miR-205 and miR-712 (Table 3) [24•, 26, 27]. The reported biological effects
miR-24
miR-29b
miR-712
miR-205
miR-21
miR-29b
miR-29b
24
25
26
26
27
28
29
AngII
AngII
Elastase and AngII
ApoE−/− male mice ApoE−/− male mice ApoE−/− and C57BL/6J
C57BL/6J
AngII
Elastase AngII
AngII
ApoE−/− male mice
C57BL/6J ApoE−/−
Elastase
AAA model
C57BL/6J
Mouse strain
Maximum AAD on ultrasound
Maximum AAD on ultrasound
Maximum AAD from histology of excised aortas Maximum AAD from histology of excised aortas Maximum AAD on ultrasound
Maximum AAD on MRI
Maximum AAD on ultrasound
AAA assessment
lentiviral vector overexpression of miR-21 locked nucleic acid anti-miR-29b lentiviral vector overexpression of miR-29b locked nucleic acid anti-miR-29
Anti-miR-205
Anti-miR-712
lentiviral vector overexpression of pre-miR-24, antimiR-24 or scrmiR antagomiR-29b
Intervention
NS
1 day after AAA induction
1 day after AAA induction
Days 1, 2 and 3 after starting AAA induction every 4 days after starting AngII infusion NS
1 day after AAA induction
Timing relative to AAA induction
Reduced AAA induction
Reduced AAA induction Enhanced AAA
Reduced AAA induction
Reduced AAA induction
Reduced AAA induction
Reduced AAA induction
Overexpressing miR-24 reduced AAA induction; Anti-miR-24 promoted AAA induction
Outcome
Page 6 of 9
AAA abdominal aortic aneurysm, AAD abdominal aortic diameter, ApoE−/− apolipoprotein E deficient, NS not stated, MRI magnetic resonance imaging, AngII angiotensin II
microRNAs
Examples of mouse studies examining the effect of microRNAs on AAA
Reference
Table 3
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Page 7 of 9 15
included modifying proliferation of cells within the aortic wall, in addition to changes in inflammation and extracellular matrix remodelling [24•, 26, 27]. It is unclear, however, whether these findings in animal models are directly translatable to patients with AAA. HLI hind limb ischemia, FA femoral artery, LDI laser Doppler imaging, KO knockout, NS not stated, EC endothelial cell, TG transgenic, d days following ischemia induction
Slower perfusion recovery Slower perfusion recovery More rapid perfusion recovery Greater perfusion at d19 Slower perfusion recovery More rapid perfusion recovery Worse perfusion up to d14 Worse perfusion up to d22 Greater perfusion at d14 No effect prior prior 1 day prior 10 days after prior prior prior prior prior prior miR132/212 KO miR155 KO Gene silencing oligonucleotides miR126 microbubble liposomes antagomir-93 PremiR-93 106b∼25 KO EC-miR-15a TG antagomir-92a antagomir-126 LDI LDI LDI LDI LDI LDI LDI LDI LDI LDI Proximal FA transection Double FA ligation Double FA ligation Excision of FA Excision of FA Excision of FA FA ligation Excision of FA Excision of FA Electrocautery of FA 132/212 155 14q32 (329, 487b, 494 and 495) 126 93 93 106b∼25 cluster (106b, 93, 25) 15a 92a 126 31 32 33 34 35 35 36 37 38 39, 40
C57BL/6J C57BL/6J C57BL/6J ICR C57BL/6J BALB/cJ C57BL/6J NS C57BL/6J C57BL/6J
Perfusion assessment HLI model Mouse strain microRNAs Reference
Table 4
Examples of mouse studies examining the effect of microRNAs on hind limb ischemia
Intervention
Timing relative to ischemia induction
Outcome
Curr Atheroscler Rep (2016) 18:15
The Effect of Modifying microRNAs Within Animal Models of Hind Limb Ischemia Promotion of new vessel formation (angiogenesis) and stimulation of the expansion of existing collaterals (arteriogenesis) are coordinated by a group of signalling pathways, genes, growth factors and cells [1]. microRNAs, such as those listed in Table 4 [31, 32, 33••, 34–40], have been shown to control expression of genes implicated in controlling angiogenesis and arteriogenesis, such as vascular endothelial growth factor, fibroblast growth factor, insulin receptor substrate 1, E2F transcription factor 1, and progenitor cell recruitment [33••, 34, 35, 39]. The effect of promoting or antagonising a variety of different microRNAs has been reported to modify perfusion recovery in mouse models of hind limb ischemia, as summarised in Table 4 [31, 32, 33••, 34–40]. For example, enhancing miR-93 or mir-126 expression has been reported to improve hind limb blood supply recovery in different mice strains [34, 35]. Antagonism of miR-92a and a collection of microRNAs located on the chromosome region 14q32 (miR-329, miR-487b, miR-494 and miR-495) has been reported to promote hind limb re-perfusion following femoral artery ligation in C57BL6 mice [33••, 38]. The increased perfusion resulting from antagonism of these microRNAs was reported to be due to improved arteriogenesis evidenced by larger-diameter arterioles within the adductor muscle [33••]. Whether these approaches to improving limb blood supply can be brought into clinical practice is not yet clear since there are accepted deficiencies with current rodent models of limb ischemia (see a recent review [41]). Other Epigenetic Modifications and Aortic Aneurysm and Lower Limb Occlusive Artery Disease The association of the DNA methylation pattern with PAD has been relatively little studied. Ryer and colleagues recently examined the genome-wide DNA methylation profiles of mononuclear circulating cells in 20 patients that had AAAs in comparison to 21 age- and sex-matched controls [42•]. The investigators reported four genes with differential CpG island methylation, namely kelch-like family member 35 (KLHL35), calponin 2 (CNN2), serpin peptidase inhibitor clade B (ovalbumin) member 9 (SERPINB9) and adenylate
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cyclase 10 pseudogene 1 (ADCY10P1). It has also been suggested that the expression of microRNAs associated with occlusive lower limb PAD could be influenced by methylation within the promoter regions of DNA coding for microRNAs [42•]. The association of histone modifications with PAD has been little investigated. Vinh and colleagues investigated the effect of a histone deacetylase (HDAC) inhibitor on AAA induction within a mouse model [43]. They reported that the HDAC inhibitor metacept-1 inhibited AAA induction by angiotensin-II by downregulating matrix metalloproteinases. The importance of these findings in AAA and lower limb PAD pathogenesis is currently unclear.
References
Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance 1.
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Conclusions: the Clinical Potential of Epigenetics in Patients with AAA and Lower Limb Occlusive Artery Disease
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As outlined above, the majority of investigations currently published have focused on the relevance of microRNAs to AAA and lower limb occlusive artery disease. Based on these studies, microRNAs may have potential as diagnostic aids in patients; however, larger studies are needed to examine whether the currently reported excellent sensitivity and specificity of these markers can be replicated. It also remains to be established whether concentrations of circulating microRNAs will be able to distinguish patients with different types of vascular disease. A large number of mouse studies suggest that upregulation and downregulation of selected microRNA has the potential to improve outcomes in AAA and lower limb occlusive artery disease. It is unclear, however, whether it is possible to translate findings from these mouse models to patients [41, 44, 45]. It also remains to be established whether administrating such gene expression modifying interventions would be safe in patients, particularly given that a single microRNA can control the expression of large number of genes. Phase 1 trials will be required in patients to address this. Other types of epigenetic modifications, such as DNA methylation and histone modification, have been little studied in relation to PAD.
Compliance with Ethical Standards Conflict of Interest Jonathan Golledge declares that he works for James Cook University & The Townsville Hospital and declares grant money to his institution from NHMRC, Queensland Government. Erik Biros, John Bingley, Vikram Iyer, and Smriti M Krishna declare that they have no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.
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