The term ‘epigenetics’ refers to heritable, reversible DNA or histone modifications that affect gene expression without modifying the DNA sequence. Epigenetic modulation of gene expression also includes the RNA interference mechanism. Epigenetic regulation of gene expression is fundamental during development and throughout life, also playing a central role in disease progression. The transforming growth factor β1 (TGF-β1) and its downstream effectors are key players in tissue repair and fibrosis, extracellular matrix remodelling, inflammation, cell proliferation and migration. TGF-β1 can also induce cell switch in epithelial-to-mesenchymal transition, leading to myofibroblast transdifferentiation. Cellular pathways triggered by TGF-β1 in thoracic ascending aorta dilatation have relevant roles to play in remodelling of the vascular wall by virtue of their association with monogenic syndromes that implicate an aortic aneurysm, including Loeys–Dietz and Marfan's syndromes. Several studies and reviews have focused on the progression of aneurysms in the abdominal aorta, but research efforts are now increasingly being focused on pathogenic mechanisms of thoracic ascending aorta dilatation. The present review summarizes the most recent findings concerning the epigenetic regulation of effectors of TGF-β1 pathways, triggered by sporadic dilative aortopathy of the thoracic ascending aorta in the presence of a tricuspid or bicuspid aortic valve, a congenital malformation occurring in 0.5–2% of the general population. A more in-depth comprehension of the epigenetic alterations associated with TGF-β1 canonical and non-canonical pathways in dilatation of the ascending aorta could be helpful to clarify its pathogenesis, identify early potential biomarkers of disease, and, possibly, develop preventive and therapeutic strategies.

DILATIVE AORTOPATHY OF THE ASCENDING AORTA

The term aortopathy’ defines a group of disorders characterized by dilatation, aneurysms, dissection and tortuosity of the aorta. Aortopathies may be sporadic, syndromic or familial non-syndromic [1]. An aortic aneurysm is defined as a localized or diffuse dilatation of the aorta with a diameter at least 1.5-fold greater than the expected normal size [2].

Demographic studies have indicated an epidemiological heterogeneity of thoracic and abdominal aortic aneurysms (AAAs), with about 9% of the population aged ≥65 years affected by AAAs [3], whereas the incidence of thoracic ascending aortic aneurysms (TAAs) is estimated to be 5.9/100000 per year, a rate markedly lower but increasing by virtue of advances in imaging methods and screening [4].

Different rates of aneurysm incidence in the abdominal and thoracic aorta are related mainly to marked heterogeneity at a molecular and structural level, which has been well reviewed elsewhere [5], e.g. it has been highlighted that matrix metalloproteinase (MMP)-2 and MMP-9 play a role in both TAAs and AAAs, but are produced by different cell types and with different timing during progression of the aneurysm [5,6]. Heterogeneity in intracellular signalling pathways in TAAs versus AAAs has also been described. In addition, the thoracic aorta and the abdominal aorta exhibit different susceptibilities to atherosclerosis and hence to macrophage infiltration, with the atherosclerotic plaque having a greater role to play in AAAs than in TAAs [4].

The multilevel heterogeneity of the thoracic and abdominal aorta in health and disease can be ascribed to their different embryological origin, because the thoracic ascending aorta is formed by cells from the neural crest, whereas the abdominal aorta precursor cells originate in the mesoderm [7]. The aortic regions above and below the diaphragm also exhibit differences in segmental growth patterns during embryonic development, leading to intrinsic structural differences. Although several studies and reviews have focused on the progression of AAAs, the present review summarizes the most recent findings concerning the epigenetic regulation of effectors of TGF-β1 pathways triggered in sporadic dilative aortopathy of the thoracic ascending aorta.

In patients with a tricuspid aortic valve (TAV), TAAs are characterized by severe elastin fragmentation, cystic medial necrosis, medial fibrosis and inflammation [8] (Figure 1). Moreover, our previous studies revealed an increased thickness of the intimal layer in TAAs than in healthy thoracic aortas, together with a thicker adventitia, appearing as highly cellularized and with several vasa vasora of variable diameters compared with healthy aortas [9].

Immunohistochemical analysis of α-SMA expression and distribution

Figure 1
Immunohistochemical analysis of α-SMA expression and distribution

This analysis shows the expression and distribution in the convexity of dilated ascending aortas from (B) BAV and (C) TAV patients, and in (A) non-dilated healthy ascending aorta from an organ donor. Red fluorescent staining: α-SMA; blue fluorescent staining: Hoechst 33258 nuclei counterstaining; green fluorescent staining: natural autofluorescence emitted by elastic fibres. Media layer, 20× magnification.

Figure 1
Immunohistochemical analysis of α-SMA expression and distribution

This analysis shows the expression and distribution in the convexity of dilated ascending aortas from (B) BAV and (C) TAV patients, and in (A) non-dilated healthy ascending aorta from an organ donor. Red fluorescent staining: α-SMA; blue fluorescent staining: Hoechst 33258 nuclei counterstaining; green fluorescent staining: natural autofluorescence emitted by elastic fibres. Media layer, 20× magnification.

In addition to intrinsic factors responsible for aortic dilatation, the wall of a dilated aorta is submitted to increased wall tension and a change of flow from laminar to turbulent at the dilated site. In addition to probable involvement in the progression and complications of the dilative process, these haemodynamic factors have also been advocated as primary determinants of at least some forms of aortopathy (‘post-stenotic dilatations’) [10]. In particular, the haemodynamic factors prompt altered shear stress on the endothelial layer, a phenomenon that can dysregulate the phenotype of smooth muscle cells (SMCs), thus contributing to aneurysm progression and rupture. Consistent with this observation, we revealed the loss of smoothelin, a specific marker of the SMC contractile phenotype, in TAAs, together with the increased expression of the ED-A isoform of fibronectin (ED-A FN1) and of α-smooth muscle actin (α-SMA) (Figure 1), known to be a prerequisite for differentiation of myofibroblasts (MFs) [11]. In addition, SMC apoptosis contributes to the weakening of the aortic wall during TAA progression and, together with the increased proteolytic activity in the extracellular matrix (ECM), can ultimately lead to vessel rupture [12]. It is interesting that some studies demonstrated that aortic stenosis and regurgitation were associated with distinct alterations of the wall of TAAs, giving further support to a relevant role for haemodynamic factors [13].

Dilatation of the aortic root and ascending aorta is frequently encountered in patients with congenital heart disease. In the present review we focus in particular on the dilative aortopathy of thoracic aortas associated with either a TAV or a bicuspid aortic valve (BAV), the most common adult congenital heart defect, occurring in 0.5–2% of the general population, with a higher prevalence in male patients [14]. Concomitant aortic dilatation is seen in 80% of patients with a BAV [15], where it occurs at least 10–15 years earlier in life than in patients with a TAV, whose aortic distension seems more related to age and hypertension [8].

A BAV can be heritable, but its cause is not yet well understood [16]. It results from abnormal cusp formation during valvulogenesis, in which adjacent cusps fuse into a single cusp. The two major morphological types of BAV are the right–left fusion (RL, the most prevalent–69–85%) and the right non-coronary (RN) fusion. Aortic dilatation rates vary according to the pattern of BAV cusp fusion, with greater rates of aortic sinus and ascending aorta dilatation associated with the RL phenotype [17].

The involvement of distinct factors in the onset of TAAs in patients with a BAV rather than a TAV is well recognized. Different from TAV patients, the ascending aorta of BAV patients generally shows non-inflammatory loss of vascular SMCs, with multifocal apoptosis and medial degeneration [8]. In addition, the collagen and elastin content is similar in BAV and TAV patients, but the overall ECM architecture of the BAV aorta is striking, because, in contrast to TAAs in TAV patients, where fibres appear to be organized randomly, in BAV patients they exhibit a highly oriented fibre architecture reminiscent of the structure observed for a normal aorta with respect to distribution of the fibre angle alignment [18] (Figure 1).

Beyond these general characteristics, BAV-associated aortic disease is characterized by large phenotype diversity and clinical variation, often leading to an individualized management of BAV patients [19]. Such heterogeneity of BAV-associated aortic disease could reflect the complexity of its underlying genetics, but possibly also the epigenetic mechanisms of regulation of gene expression, combined with the different contribution of haemodynamics in each single patient.

TGF-β1: ROLES AND MEDIATORS

Transforming growth factor β1 (TGF-β1) is a pleiotropic cytokine playing a key role in tissue repair and fibrosis, inflammation, cell proliferation and migration, as well as in ECM remodelling (Figure 2). Among their many activities, TGF-β1 and its mediators can also activate specific transcription, leading to epithelial-to-mesenchymal transition (EMT) and MF differentiation. MFs are of particular interest in dilative aortopathy, because they combine the ECM biosynthesis activity of fibroblasts with the cytoskeletal characteristics of contractile SMCs, including up-regulated α-SMA expression [20], due to the presence of TGF-β1-responsive, Smad-binding elements in the α-SMA promoter [21].

Interplay among the main molecular effectors of the TGF-β1 pathway in the vascular wall

Figure 2
Interplay among the main molecular effectors of the TGF-β1 pathway in the vascular wall

ECM stretching and proteolytic cleavage by various enzymes, including MMP-2 and MMP-14, can release the TGF-β1 homodimer from the latent complex composed of latency-associated protein (LAP) and LTBPs. MMP-14 also activates or up-regulates several MMPs, including MMP-2, through the mediation of different cytokines. MMPs can also be activated by biomechanical stimuli and reactive oxygen species (ROS). SOD3 is a well-known extracellular ROS scavenger. In the canonical pathway, TGF-β1 binds to TGF-βRII which is thus autophosphorylated and phosphorylates TGF-βRI [activin receptor-like kinase (ALK)5], the kinase activity of which in turn phosphorylates Smad2/3. Smad3 binds to Smad4 and translocates into the nucleus to regulate the expression of specific genes, including the profibrotic CTGF. One of the known alternative pathways is mediated by TGF-βRII/ALK1 and involves Smad1/5/8. Other non-canonical pathways involve different receptors, are independent of Smad signalling and can ultimately lead to up-regulation of proteolytic enzymes. ENG is a regulatory co-receptor for TGF-β1: it can be cleaved by MMP-14, resulting in sENG which can form inactive complexes with serum TGF-β1. Red asterisks highlight where there is major evidence for epigenetic changes affecting the TGF-β1 pathway in the context of dilative aortopathy. A detailed description of these epigenetic changes is included in the text. NO, nitric oxide.

Figure 2
Interplay among the main molecular effectors of the TGF-β1 pathway in the vascular wall

ECM stretching and proteolytic cleavage by various enzymes, including MMP-2 and MMP-14, can release the TGF-β1 homodimer from the latent complex composed of latency-associated protein (LAP) and LTBPs. MMP-14 also activates or up-regulates several MMPs, including MMP-2, through the mediation of different cytokines. MMPs can also be activated by biomechanical stimuli and reactive oxygen species (ROS). SOD3 is a well-known extracellular ROS scavenger. In the canonical pathway, TGF-β1 binds to TGF-βRII which is thus autophosphorylated and phosphorylates TGF-βRI [activin receptor-like kinase (ALK)5], the kinase activity of which in turn phosphorylates Smad2/3. Smad3 binds to Smad4 and translocates into the nucleus to regulate the expression of specific genes, including the profibrotic CTGF. One of the known alternative pathways is mediated by TGF-βRII/ALK1 and involves Smad1/5/8. Other non-canonical pathways involve different receptors, are independent of Smad signalling and can ultimately lead to up-regulation of proteolytic enzymes. ENG is a regulatory co-receptor for TGF-β1: it can be cleaved by MMP-14, resulting in sENG which can form inactive complexes with serum TGF-β1. Red asterisks highlight where there is major evidence for epigenetic changes affecting the TGF-β1 pathway in the context of dilative aortopathy. A detailed description of these epigenetic changes is included in the text. NO, nitric oxide.

TGF-β1 is synthesized as a precursor complexed in the ECM with latent TGF-β1-binding proteins (LTBPs) and adhesive matrix proteins, including FN1 and fibrillin 1 (FBN1). TGF-β1 is activated via proteolytic cleavage of LTBPs or through biomechanical stimuli. Activated TGF-β1 binds to a heterodimeric receptor complex (composed of TGF-β1 type I and II receptors) endowed with a serine/threonine kinase activity that is necessary for all responses to TGF-β1 [20]. In contrast, the TGF-β1 type III receptors, endoglin (ENG) and betaglycan, lack the kinase activity but can modulate TGF-β1 binding and signalling [22]. Moreover, the ENG extracellular domain can be cleaved by MMP-14 and circulate as soluble ENG (sENG), capable of sequestering TGF-β1 [23] (Figure 2). TGF-β1 receptors activate intracellular Smads, i.e. signal transducers from TGF-β1 to the nucleus, where they initiate the transcription of target genes (Figure 2). Different combinations of ligands, receptors and downstream effectors may result in complex patterns of TGF-β activity [23] (Figure 2).

TGF-β1 can be involved in both vascular ECM deposition and degradation, depending on the stimulation of canonical or non-canonical pathways. Canonical, Smad-dependent, TGF-β1 pathways induce the expression of genes involved in vascular remodelling and fibrosis, including, among others, the connective tissue growth factor (CTGF) (Figure 2), a marker for activated fibroblasts [24], and ECM proteins. The activation of canonical TGF-β1 pathways also leads to a decreased expression of MMPs and their inhibition by tissue inhibitors of metalloproteinases (TIMPs) [25]. Alternatively, the activation of non-canonical, Smad-independent, TGF-β1 pathways can induce ECM degradation through increased MMP activity.

Concentration is an important parameter which defines the biological effects of TGF-β1: at low concentrations, it stimulates endothelial cell (EC) and SMC proliferation and migration, whereas at high concentrations it inhibits these processes [26].

RELEVANCE OF THE TGF-β1 PATHWAY TO VASCULAR CELL PLASTICITY AND DILATATION OF THE THORACIC ASCENDING AORTA

The cell types that populate the wall of a human healthy thoracic aorta include SMCs, ECs, fibroblasts and stem/progenitor cells. Among them, SMCs are the focus of intense research by virtue of their uniqueness, because they retain a remarkable plasticity in adult tissues. SMC plasticity is characterized by the ability to reversibly shift in reaction to external stimuli from a differentiated and quiescent contractile phenotype, characterized by low migration and ECM synthesis rates and by a unique repertoire of contractile proteins (including α-SMA, smoothelin, calponin, SM22/transgelin, h-caldesmon and smooth muscle myosin heavy chain), to a proliferative, migratory phenotype, characterized by the synthesis of relatively few contractile proteins, high secretory activity of ECM proteins, and high proliferation and migration rates.

Several key transcription factors and regulatory cis elements play a role in modulation of the SMC phenotype. In particular, SMC genes are generally regulated by CC(A/T)6GG (or CArG) cis elements in their promoter or introns which bind the serum response factor (SRF), a transcription factor that can form a complex with the transcriptional co-activator myocardin and promote the SMC contractile phenotype, but can also regulate genes involved in cell proliferation when linked to co-factors such as Elk-1 and the Kruppel-like factor (KLF)-4 or KLF5 [27].

TGF-β1 signalling is important for differentiation of SMCs into mature, contractile cells [28] through classic transcriptional activation pathways, e.g. it selectively induces the direct binding of Smad3 to the promoter of SM22/transgelin. Alternatively, Smad3 can interact with the SRF–myocardin complex, leading to the transcriptional activation of genes coding for contractile proteins [29].

TGF-β1 and its mediators have been attracting an increasing amount of attention in studies focusing on aortic aneurysms, in particular by virtue of their association with monogenic aortopathy syndromes.

TGF-β1 pathways could play divergent roles in AAAs and TAAs, because active TGF-β1 in thoracic aortas is associated with increased signalling and aneurysms, whereas in AAAs TGF-β1 can stabilize the already formed aneurysm and reprogramme the abdominal aorta through a decrease in MMPs and inflammation, and an increase in TIMPs [30]. This observation implies that the role of TGF-β1 in aneurysm progression cannot be generalized, because the activities of this cytokine can vary in distinct aortic regions. In the present review we look at the relevance of the TGF-β1 pathway specifically with regard to TAAs.

A dysregulation of TGF-β1 signalling in sporadic and syndromic forms of TAAs is often associated with mutations in genes belonging or related to the pathway itself. They include FBN1 (regulating TGF-β1 bioavailability), mutated in 90% of cases of Marfan's syndrome [31], TGF-β type I/II receptors (TGF-βRI/RII) in Loeys–Dietz syndrome [32], Smad3 in aneurysm–osteoarthritis syndrome [33] and TGF-β2, mutated in syndromic TAAs [34]. It is interesting that two polymorphisms in FBN1 identified in sporadic TAAs do not cause Marfan's syndrome but are correlated with plasma concentrations of TGF-β1 [35]. An association between mutated TGF-β3 and a syndromic aneurysm has recently been identified [36]. Of major interest, a dysregulation of TGF-β1 signalling can contribute to aneurysm pathogenesis through opposing mechanisms. Chronic TGF-β1 signalling in Marfan's syndrome leads to over-expression of contractile proteins in SMCs, contributing to their greater stiffness [37]. Authors conclude that this alteration may contribute to the aortic rigidity that precedes aneurysm formation in Marfan's syndrome. Conversely, another study conducted on TAA samples from patients with TGF-βRII mutations revealed that the consequent TGF-β1 signalling disruption leads to a lower expression of contractile proteins in SMCs and MFs [38]. Authors conclude that in this case the defective contractility of SMCs in the presence of disrupted TGF-β1 signalling can contribute to TAA progression.

Despite the evidence of an autosomal dominant pattern of BAV inheritance, with variable expression and incomplete penetrance in families, the genetic variants that cause a BAV or predict BAV-associated complications, such as aortopathy, remain largely unknown [39]. Only a few data are currently available for the genetic variations of the TGF-β1 pathway in BAV patients [40]. However, several studies revealed intrinsic differences between TAA samples from TAV and BAV patients with reference to TGF-β1 pathway expression/activation.

TGF-β1 has increased expression in dilated aortas in BAV and TAV patients, but a lower concentration and activity in dilated aortas of just BAV patients, presumably due to an increased sequestration of TGF-β1 in the ECM [41]. Our studies revealed an imbalance between the TGF-βRI and TGF-βRII subunits in TAAs from BAV patients, together with a positive correlation between TGF-βRII mRNA and aortic diameter, suggesting that patients with BAV stenosis and post-stenotic dilatation of the thoracic aorta may have an intrinsic defect of TGF-βRI expression [11]. This imbalance between the TGF-βRI and TGF-βRII subunits is consistent with findings obtained in a murine model of aortic aneurysm [42], and could determine a redirection towards non-canonical TGF-β1 pathways in BAV patients (see Figure 2). TAA samples from TAV and BAV patients show different alternative splicing fingerprints in the TGF-β1 pathway, including the impaired inclusion of the ED-A exon in FN1 mRNA of BAV patients [43]. The ED-A exon is included in a particular isoform of FN induced by TGF-β1 and known to be involved in MF differentiation. The impaired inclusion of the ED-A exon in BAV patients could be attributable to differences in TGF-β1 signalling [44].

The data that we have summarized support the role of TGF-β1 signalling in TAA progression and the relevance of the genetic/haemodynamic background. Novel studies, described below, suggest a relevant role also for epigenetics in the regulation of TGF-β1 signalling in the pathogenesis of TAAs in TAV and BAV patients.

EPIGENETICS: DEFINITION AND MAIN MECHANISMS

The term ‘epigenetics’ refers to the study of heritable, reversible DNA or histone modifications that affect gene expression without any variation in the DNA sequence. Epigenetic modulation of gene expression is fundamental during development and throughout life, regulating the differentiation and functionality of all cell types and allowing the cells to react quickly to environmental changes through a variation in chromatin accessibility [45]. However, it also plays a central role in the progression of several diseases.

Below we summarize the main histone, DNA and RNA interference (RNAi)-based mechanisms of epigenetic regulation of gene expression that can affect chromatin structure and organization, thus regulating the transition from silent eterochromatin to active euchromatin and vice versa [46] (Figure 3).

Schematic representation of the main mechanisms of epigenetic regulation of gene expression

Figure 3
Schematic representation of the main mechanisms of epigenetic regulation of gene expression

The RNAi mechanism, together with the modifications of genomic DNA and histone proteins in the nucleosome, can affect target mRNA concentration/translation or the chromatin structure and organization, respectively, regulating its transition from silent eterochromatin to active euchromatin and vice versa. (A) Main covalent post-translational modifications [acetylation (Ac), methylation (Me), phosphorylation (P) and ubiquitylation (Ub)] of N-terminal tails of the four core histones H2A, H2B, H3 and H4 (on lysine, arginine, serine and threonine residues). Different functions have been associated with each histone modification. (B) DNA modification through covalent methylation of cytosines located 5′ to guanines in CpG clusters. (C) Target-specific inhibition of mRNA translation through RNAi, mediated by miRNA binding to specific sequences in the 3′-UTR of target mRNA.

Figure 3
Schematic representation of the main mechanisms of epigenetic regulation of gene expression

The RNAi mechanism, together with the modifications of genomic DNA and histone proteins in the nucleosome, can affect target mRNA concentration/translation or the chromatin structure and organization, respectively, regulating its transition from silent eterochromatin to active euchromatin and vice versa. (A) Main covalent post-translational modifications [acetylation (Ac), methylation (Me), phosphorylation (P) and ubiquitylation (Ub)] of N-terminal tails of the four core histones H2A, H2B, H3 and H4 (on lysine, arginine, serine and threonine residues). Different functions have been associated with each histone modification. (B) DNA modification through covalent methylation of cytosines located 5′ to guanines in CpG clusters. (C) Target-specific inhibition of mRNA translation through RNAi, mediated by miRNA binding to specific sequences in the 3′-UTR of target mRNA.

Histone modifications

Double-stranded DNA is packaged into chromatin, the fundamental units of which are the nucleosome octamers, composed of a DNA segment of 147 bp wrapped around a protein core (including two copies of the histone proteins H3, H4, H2A and H2B). The N- and C-terminal histone tails protrude from the nucleosome core and have the potential to interact with adjacent nucleosomes and the linker DNA (Figure 3).

More than 60 histone post-translational modifications have been identified so far; however, the large majority of current information is about small covalent modifications, such as acetylation, methylation, ubiquitylation, phosphorylation, sumoylation and ADP-ribosylation [47,48]. These small histone modifications cross-talk and can regulate different functions, through the action of reader proteins [49,50].

Histone acetylation neutralizes the positive charge of lysine residues, interrupting the attraction between histone proteins and negatively charged phosphate groups in DNA, leading to a shift towards transcriptionally active chromatin. The histone acetylation status results from the balance between the activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs) [51]. Histone methylation takes place at arginine or lysine residues with mono-, di- or tri-methylated histones [52]. Compared with acetylation, which leads only to transcriptionally active chromatin, histone methylation can lead to active or silent states of chromatin. Histone methylation is regulated by the balanced activity of different histone methyltransferases and histone demethylases.

Histone phosphorylation takes place at serine, threonine and tyrosine residues, and its abundance can vary dramatically between interphase and mitosis [53].

Transcriptionally active and silent chromatin is characterized by distinct combinations of histone modifications, as has been well reviewed elsewhere [48], resulting from the balanced action of the enzymes listed above. Such combinations appear to fine-tune gene expression and regulate important cellular processes which, when dysregulated, can contribute to the genesis of a disease.

DNA methylation

DNA methylation is a reversible process resulting from the transfer of a methyl group from the co-factor S-adenosylmethionine (SAM) to carbon 5 of the cytosine ring to generate 5-methylcytosine groups (5mC), preferentially in regions where a cytosine is followed directly by a guanine in the DNA sequence, also known as CpG islands (Figure 3). The CpG islands are frequently found in regulatory regions of DNA, and in particular in the promoters of approximately 40% of human genes; their methylation can silence gene expression by: (i) direct interference, as the formation of 5mC can inhibit the specific binding of transcription factors to recognition sites in their respective promoters (e.g. cAMP response element-binding protein or CREB, hypoxia-inducible factor or HIF); and (ii) gene silencing, occurring by direct binding of specific transcriptional repressors to methylated DNA (e.g. methyl-CpG-binding proteins mCP1 and mCP2) [54].

The DNA-methylation profile derives not only from the direct action of enzymes such as DNA methyltransferases (DNMTs), but also from the indirect action of other proteins, including the CpG-binding proteins [55]. Mammalian cells express three DNMTs, namely, DNMT1, DNMT3a and DNMT3b. DNMT1 is a maintenance-type methyltransferase, helping in the copying of DNA-methylation profiles from parent strands to the daughter DNA strand during the replication process. DNMT3a and DNMT3b are important in new methylation, helping in the creation of new methylated patterns of DNA during development [56]. The DNMT activity is counterbalanced by the 10–11 translocation (TET) enzymes, implicated in DNA demethylation [57].

Changes in DNA-methylation profiles have been associated with several diseases, including fibrosis and aortopathy [54], as discussed further below.

Although DNA methylation and histone modifications are regulated by different enzymes and chemical processes, the two systems are linked in a cooperative regulation of gene expression. It appears that the relationship between these two epigenetic mechanisms can function in both directions, with histone methylation directing DNA-methylation patterns and DNA methylation acting as a template to facilitate histone modifications after DNA replication [58,59].

RNA interference

Epigenetics now also includes the endogenous RNAi mechanism, based on the mediation of miRNAs, i.e. single-stranded, non-coding, 21- to 23-nucleotide-long, RNA molecules that are transcribed from miRNA loci and operate mainly as post-transcriptional repressors of gene expression (Figure 3). The relatively recent development of next-generation sequencing (NGS) techniques allowed not only the genome-wide analysis of mRNA signatures, but also the identification of several types of non-coding RNAs (ncRNAs), with fundamental regulatory functions [60]. Among them, miRNAs operate through the binding to miRNA-response elements (MREs) within the 3′-UTRs of their target mRNAs. The formation of the miRNA/target mRNA hybrid induces the mRNA cleavage and/or its translational repression [61]. A single miRNA is capable of targeting multiple mRNAs, and a single mRNA may contain multiple miRNA-binding sites.

At present, about 2000 miRNAs have been identified as encoded by the human genome (see http://www.mirbase.org, release 21). Most human miRNAs are co-expressed with their host/target gene within gene introns, whereas others are transcribed independently of coding genes.

It is interesting that, in 2008, Mitchell et al. [62] revealed the presence of endogenous miRNAs in the circulation that are not cell associated. Such circulating miRNAs are exceptionally stable because they are protected from degradation by microvesicles, RNA-binding proteins or lipoproteins [63]. Circulating miRNAs are probably released by both circulating cells and damaged/diseased organs, holding great promise as biomarkers in disease detection, diagnosis and prognosis.

In addition to the interaction described above between DNA methylation and histone modifications, it is becoming increasingly clear that DNA methylation and/or histone modifications can also regulate the expression of some miRNAs, whereas other miRNAs can control the expression of DNMTs and HDACs, thus creating a quite complex circuit [64].

EPIGENETIC REGULATION OF VASCULAR CELL PLASTICITY AND INTERPLAY WITH THE TGF-β1 PATHWAY

As expected, several studies reveal that epigenetic mechanisms play a pervasive role in the SMC reaction to physiological and pathophysiological stimuli [27]. In reference to the specific interplay between epigenetic regulation of SMC plasticity and TGF-β1, it has been demonstrated that TGF-β1 can induce SMC differentiation, modulating the expression of SM22/transgelin in vitro, through not only the classic transcriptional activation described above, but also the induction of histone hyperacetylation of the SM22/transgelin promoter [65]. These data suggest that the modulation of histone acetylation is part of the molecular mechanisms of TGF-β1-mediated gene transcription in SMCs.

In addition to histone modifications, DNA methylation also seems to play a relevant role in SMC plasticity, especially in pathological settings, e.g. genomic DNA can be hypomethylated in human atherosclerotic lesions [66], consistent with a previous study by the same group focusing in particular on the methylation status of the gene coding for superoxide dismutase 3 (SOD3) during development of atherosclerosis in rabbits [67]. Some studies recently demonstrated a major role for the enzyme family TET, responsible for DNA demethylation, in the regulation of SMC plasticity. In particular, TET2 is both necessary and sufficient for SMC differentiation, whereas loss of TET2 is a cardinal feature of synthetic SMCs [68].

MiRNAs are critical modulators of vascular SMC function and plasticity. The importance of miRNA-mediated gene silencing in phenotype modulation of SMCs has recently been underscored by experiments in mice with smooth muscle-targeted knock-out of Dicer, resulting in late embryonic lethality at days 16–17 and impaired vascular SMC proliferation and contractility [69]. This effect was associated with a loss of actin stress fibres and was partly rescued by the over-expression of miR-145. This miRNA, together with miR-143, can inhibit the expression of KLF4 and KLF5, thus inducing a shift towards the SMC contractile phenotype [70]. The key role of the smooth muscle-enriched miR-143/-145 cluster for stretch-induced differentiation of SMCs has been further confirmed by studies on the portal vein of wild-type or miR-143/-145 knock-out mice [71].

MiR-21 can promote the differentiation of vascular SMCs in response to TGF-β1 and bone morphogenetic protein (BMP) stimulation through a decrease in programmed cell death 4 (PDCD4) expression [72].

A microarray-based study focusing on the process of human aortic SMC differentiation in vitro revealed that miR-26a exhibits the highest-ranked differential expression among miRNAs, with SMC differentiation promoted by under-expression of miR-26a and inhibited by its over-expression [73]. Of interest, additional experiments revealed an interplay between miR-26a and the TGF- β1 pathway, as miRNA inhibition enhanced Smad1 and Smad4 signalling.

In reference to ECs, DNA methylation has been identified in a swine model as a powerful epigenetic regulator of endothelial transcription associated with flow characteristics [74]. In this context, the constitutively expressed endothelial nitric oxide synthase (eNOS) is the most well-characterized example of an EC-specific gene that is regulated at the epigenetic level through its chromatin accessibility (the eNOS promoter is DNA hypomethylated and histone acetylated only in ECs) [75]; eNOS is also regulated by RNAi-based mechanisms, interacting with the methylation and acetylation profiles [76]. Similar epigenetic mechanisms of regulation of endothelial restricted genes have been described for von Willebrand's factor (vWF) and Notch4 [77].

MiRNA-profiling experiments revealed that ECs are enriched in specific miRNAs, including, among others, let-7b, miR-16, miR-21, miR-23a, miR-29, miR-100, miR-126, miR-221 and miR-222, playing a role mainly in angiogenesis [78], and in vascular remodelling, e.g. through modulation of stromal cell-derived factor 1 expression [79]. Of major interest, other miRNA-profiling experiments revealed that EC treatment with TGF-β1 induces a marked silencing of miR-30a-3p, along with other members of the miR-30 family, leading to an impairment of the endothelial angiogenic response, with the mediation of mCP2, in turn acting on sirtuin 1 [80].

CURRENT EVIDENCE FOR EPIGENETIC MECHANISMS OF GENE-EXPRESSION REGULATION IN THORACIC DILATIVE AORTOPATHY

The large majority of data concerning the role of epigenetics in aortopathy have been obtained in AAAs, but there is also now increasing evidence for epigenetic alterations associated with TAAs.

In this section we summarize the most significant and recent findings concerning RNAi, DNA-methylation profile and histone modifications associated with dilative aortopathy in patients with a TAV or a BAV. The sections below focus further on the epigenetic regulation of TGF-β1 canonical and non-canonical pathways in the context of TAAs, and in other pathological settings that could be of potential interest for thoracic dilative aortopathy.

RNA interference

MiRNAs are implicated in the pathogenesis of several vascular diseases, including TAAs, mainly by virtue of their ability to participate in the modulation of SMC and EC phenotype [81]. Notably, increasing evidence also supports a role for miRNAs in mechanotransduction events determined by shear stress [82], implying that structural alterations in aortopathy could be mediated at least in part by modulation of gene expression through cognate miRNAs sensitive to cyclic biomechanical stress [83].

Although several studies performed a high-throughput analysis of miRNome in AAAs using arrays or sequencing techniques, only a few studies applied these techniques to TAAs [84], where differential quantitative real-time PCR has been preferred to analyse single, specific miRNAs.

Jones et al. [85] revealed a decrease in miR-1, miR-21, miR-29a, miR-133a and miR-486 in TAAs and a significant relationship between miRNA expression levels (miR-1, miR-21, miR-29a and miR-133a) and aortic diameter. The same group compared the expression of miR-1, miR-21, miR-29a, miR-133a, miR-143 and miR-145 in aortic samples from BAV and TAV patients with TAAs and from reference organ donors, revealing differences in miR-1 and miR-21 abundance between BAV and TAV aortic tissue samples, and a significant dysregulation of all the target miRNAs in TAV and/or BAV TAA samples versus reference donors [86].

Several studies concerning the role of miRNAs in TAAs focused on the miR-29 family (a, b and c), which directly targets at least 16 ECM genes, including collagen isoforms [collagen type 1α (COL1A)1, COL1A2 and COL3A1], FBN1 and elastin. In particular, among the members of the miR-29 family, many and contrasting data [85,87] have been obtained about the involvement and role of miR-29b in TAAs, as extensively described below.

In addition to studies focusing on single specific miRNAs, a larger miRNA microarray analysis has been conducted on RNA from pooled ascending aortas obtained from non-familial, non-syndromic, TAA patients with a TAV compared with matched control samples [84]. Of interest, in this study distinct experiments were performed for male and female patients to assess a possible sex effect on miRNA expression, revealing that, among the 99 dysregulated miRNAs in TAA patients, only 16 miRNAs were up- or down-regulated in both male and female patients, whereas the large majority of miRNA alterations in TAA patients was sex specific. Overall array data suggest a significant repression of the focal adhesion pathway in aneurysms and a potential miR-29b-mediated down-regulation of ECM proteins. Previous studies already suggested that vascular gene expression could be sex related, possibly due to the influence of sex hormones [88,89]. The data resulting from the above-described studies further support this hypothesis, but additional investigations on larger cohorts of patients are recommended.

Several other miRNAs have a well-established role in phenomena that are also key in TAA progression. They include miR-181b, known to be involved in vascular inflammation [90], miR-26a, a modulator of SMC differentiation [73], and miR-663, involved in SMC phenotypic switch [91]. Research focusing specifically on these and other miRNAs could unveil their role in dilative thoracic aortopathy.

DNA methylation

A DNA-methylation profile is stably inherited during mitosis in adult cells, and alterations can contribute to the pathogenesis of several cardiovascular diseases, e.g. global hypomethylation, together with decreased DNMT activity, occurs during an SMC shift from a contractile to a synthetic phenotype in human atheroslerotic lesions and in apolipopotein E knock-out mice [66]. These findings are in contrast with others derived from in vitro studies showing that a damaged collagen matrix causes overall hypermethylation in a discrete number of CpG sites proximal to genes related to SMC differentiation (including α-SMA), and that this effect is mediated by alterations in DNMT expression and localization [92]. The final result of this process is SMC dedifferentiation and proliferation.

Changes in DNA-methylation profiles have also been highlighted in human cardiac tissue submitted to hypoxia and are associated with tissue fibrosis and maintenance of profibrotic MFs [93]. In particular, the transcription factor HIF-1α, induced by hypoxia, stimulates the increase of DNMT expression, leading to DNA hypermethylation, in turn associated with up-regulated collagen and α-SMA expression. Consistently, the application of the DNA-methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) reduces MF differentiation in vitro. Other studies on rat lung fibroblasts confirmed that DNA methylation mediated by DNMTs is an important mechanism of regulation of α-SMA expression during MF differentiation, acting in particular on three CpG islands in its promoter [94]. Such a relationship between MF differentiation and DNA methylation could be of great interest in the context of TAA pathogenesis.

Regarding the analysis of DNA-methylation signatures associated with TAAs, Shah et al. [95] recently reported a different methylation profile in TAV and BAV patients submitted to surgery, with some genes (e.g. PTPN2, RIPK1) that were also differentially expressed, whereas others showed a different methylation profile, but not a different expression level, in BAV and TAV patients (e.g. TBX5, PRDM16). Additional investigations would be necessary to understand the relationship between the DNA-methylation status and the expression level for the different genes identified in this study.

Several studies have currently focused on the role of homocysteine in DNA methylation in the vasculature. Homocysteine is a sulfur-containing amino acid formed during methionine metabolism and able to affect the methylation status of the promoter of different genes in SMCs and ECs [96,97]. Some studies revealed that genome-wide hypomethylation is a common feature of genomic DNA in SMCs cultured with homocysteine [98]. Hyperhomocysteinaemia is a known risk factor for cardiovascular diseases [99], and it could also play a key role in TAA pathogenesis. Several studies already revealed that high levels of homocysteine increase the risk of AAAs [100], possibly due to alterations of the methylation profile in the abdominal aorta [101], but only a few indications are currently available about the role and risk associated with hyperhomocysteinaemia in TAAs. A pioneer study revealed that homocysteine concentration is significantly higher in both aortic tissue and serum of TAA patients (25 patients with a TAV and 2 with a BAV), and a negative correlation between tissue homocysteine and aneurysm diameter was detected, thus suggesting that this amino acid could play a role in TAA pathogenesis [102]. In addition, higher total plasma levels of homocysteine have been found in BAV patients with non-stenotic valve and dilated proximal aorta [103]. Moreover, patients with Marfan's syndrome and aortic dilatation who have severe cardiovascular manifestations are characterized by higher levels of serum homocysteine versus patients with mild manifestations, and homocysteine levels have been correlated with aortic dilatation [104]. Surprisingly, none of the studies conducted in TAA patients assessed the effect of hyperhomocysteinaemia on the methylation profile of genomic DNA; only a single study, through an indirect approach, suggested a possible role for hyperhomocysteinaemia in the regulation of gene expression through changes of DNA methylation in TAA patients [105]. Additional investigations are necessary for a clear demonstration of the interrelationship of homocysteine level and DNA-methylation profile in TAV/BAV patients with TAAs and disease progression.

Histone modifications

Chromatin remodelling induced by histone modifications in vascular cells is also involved in several cardiovascular diseases, including restenosis [106]. HDACs play a crucial role in different medical conditions; among them, HDAC5 participates in the modulation of KLF2 transcriptional activation and in eNOS expression in ECs, thus contributing to endothelial dysfunction [107]. Other studies highlighted the important role of different groups of HDACs in the regulation of oxidative, inflammatory and proliferative responses of ECs to disturbed flow with oscillatory shear stress [108]. Consistently, HDAC inhibitors were revealed to be beneficial in several conditions, including experimental models of supraventricular arrhythmia, myocardial infarction, cardiac remodelling, hypertension and fibrosis [109].

Studies focusing on histone modifications in TAAs have been performed by Gomez et al. [110,111], revealing an autonomization of Smad2 from TGF-β1, associated with a basal increase in lysine acetylation and methylation of histone H3 tails in its transcription start site 1a, leading to Smad2 over-expression. It is interesting that these observations, confirmed in vitro are specific to cells from the tunica media of TAAs of different aetiologies (Marfan's syndrome, degenerative ascending aortic aneurysm and BAV) and lead exclusively to a dysregulation of Smad2 and its downstream signalling. The comprehension of the elements that can trigger the epigenetic modifications is a crucial point in research on the pathogenesis of aortic dilatation. The data obtained by Gomez et al. [110], in particular the cell-specific epigenetic activation of Smad2 in TAAs of different aetiologies, suggest that the genetic background is not the direct cause of Smad2 alteration, but it could trigger long-term environmental modifications, leading to epigenetic reprogramming. Additional experiments on TAA samples and aneurysmal SMCs demonstrated that HATs p300 and P300/CBP (CREB-binding protein)-associated protein play a major role in Smad2 promoter activation through histone acetylation [111]. The results of this study also suggest that other loci could be dysregulated in addition to the Smad2 promoter, and epigenome-wide analyses to identify regions exhibiting a differential histone acetylation in TAA samples versus controls would be of great interest.

miRNA PROFILING AND TGF-β1 INTERPLAY IN THORACIC DILATIVE AORTOPATHY

Several miRNAs have been identified as potential key players in the progression of AAAs [112]. In the present review we focus on miRNAs potentially implicated in progression of TAAs, with particular reference to those targeting the TGF-β1 pathway, as well as those that are affected by either TGF-β1 or biomechanical stimuli (Table 1).

Table 1
MiRNAs involved in epigenetic regulation of the main effectors of the TGF-β1 pathway and/or sensitive to biomechanical stimuli, selected on the basis of current literature
Final direct or indirect target gene miRNA Biological/pathological setting Notes on miRNA effect Reference 
ENG miR-15 family Overloaded heart in mice  [113
ENG miR-370 Human endometrioid ovarian cancer cells  [114
ENG miR-208a Volume overload-induced heart failure in rat  [115
ENG miR-208a Cyclic mechanical stretch in rat-cultured myoblasts The stretch-induced miR-208a is mediated by TGF-β1 and activates ENG expression [83
TGF-β1 miR-24 Cyclic mechanical stress in human trabecular meshwork cells miR-24 might play an important role in modulating the induction of TGF-β1 mediated by cyclic mechanical stress through direct targeting of furin, a subtilisin-like proprotein convertase known to play a major role in the processing of TGF-β1 [116
MMP-14 miR-24 Human coronary atherosclerotic plaques  [117
MMP-14 miR-133a Human lung cancer cell lines  [118
MMP-14 miR-133a Myocardial ischaemia–reperfusion in Yorkshire pigs  [119
MMP-2 miR-29b Hepatocellular carcinoma induced in mice  [120
Superoxide dismutase (SOD) 3 miR-21 Immortalized human bronchial epithelial cells; human umbilical vein endothelial cells miR-21 is induced in endothelial cells by shear stress; miR-21 modulates the levels of reactive oxygen species by targeting SOD3 in cancer cells [121
SOD3 miR-17Prostate cancer PC-3 cells  [122
CTGF miR-133a Diabetes-induced cardiac fibrosis in mice  [123
CTGF miR-133a Bladder wall remodelling caused by bladder outlet obstruction in rats miR-133 plays am antifibrotic functional role in modulating TGF-β1-induced bladder SMC phenotypic changes by targeting CTGF through the TGF-β–Smad3 signalling pathway [124
α-SMA, Smad2, Smad4 miR-27a-3p TGF-β1-treated human lung fibroblasts miR-27a-3p functions via a negative feedback mechanism in inhibiting lung fibrosis [125
Smad1, Smad4 miR-26a Human aortic SMCs and mice model of abdominal aortic aneurysm  [73
Forkhead box protein O4 (FOXO4), Smad3, urokinase plasminogen activator (uPA) miR-23b Human coronary SMCs, rat aortic SMCs, rat aortic ECs and rat carotid submitted to balloon angioplasty miR-23b acts as a regulator of vascular SMC phenotypic switch and its up-regulation inhibits SMC proliferation and migration  
Final direct or indirect target gene miRNA Biological/pathological setting Notes on miRNA effect Reference 
ENG miR-15 family Overloaded heart in mice  [113
ENG miR-370 Human endometrioid ovarian cancer cells  [114
ENG miR-208a Volume overload-induced heart failure in rat  [115
ENG miR-208a Cyclic mechanical stretch in rat-cultured myoblasts The stretch-induced miR-208a is mediated by TGF-β1 and activates ENG expression [83
TGF-β1 miR-24 Cyclic mechanical stress in human trabecular meshwork cells miR-24 might play an important role in modulating the induction of TGF-β1 mediated by cyclic mechanical stress through direct targeting of furin, a subtilisin-like proprotein convertase known to play a major role in the processing of TGF-β1 [116
MMP-14 miR-24 Human coronary atherosclerotic plaques  [117
MMP-14 miR-133a Human lung cancer cell lines  [118
MMP-14 miR-133a Myocardial ischaemia–reperfusion in Yorkshire pigs  [119
MMP-2 miR-29b Hepatocellular carcinoma induced in mice  [120
Superoxide dismutase (SOD) 3 miR-21 Immortalized human bronchial epithelial cells; human umbilical vein endothelial cells miR-21 is induced in endothelial cells by shear stress; miR-21 modulates the levels of reactive oxygen species by targeting SOD3 in cancer cells [121
SOD3 miR-17Prostate cancer PC-3 cells  [122
CTGF miR-133a Diabetes-induced cardiac fibrosis in mice  [123
CTGF miR-133a Bladder wall remodelling caused by bladder outlet obstruction in rats miR-133 plays am antifibrotic functional role in modulating TGF-β1-induced bladder SMC phenotypic changes by targeting CTGF through the TGF-β–Smad3 signalling pathway [124
α-SMA, Smad2, Smad4 miR-27a-3p TGF-β1-treated human lung fibroblasts miR-27a-3p functions via a negative feedback mechanism in inhibiting lung fibrosis [125
Smad1, Smad4 miR-26a Human aortic SMCs and mice model of abdominal aortic aneurysm  [73
Forkhead box protein O4 (FOXO4), Smad3, urokinase plasminogen activator (uPA) miR-23b Human coronary SMCs, rat aortic SMCs, rat aortic ECs and rat carotid submitted to balloon angioplasty miR-23b acts as a regulator of vascular SMC phenotypic switch and its up-regulation inhibits SMC proliferation and migration  

The expression of miR-378, a cardiomyocyte-abundant miRNA, has been demonstrated to inversely correlate with TGF-β1 release and activation during the development of cardiac fibrosis in mice, suggesting that in cardiomyocytes the expression of miR-378 protects the surrounding fibroblasts from activation by profibrotic stimuli through a paracrine mechanism [126]. A similar inverse correlation between miR-378 and TGF-β1 could also be hypothesized to occur in TAA progression, where TGF-β1 is known to be increased [11,41]. Among the miRNAs affected by TGF-β1, several data currently support the crucial role of the miR-29 family (a, b and c) in fibrosis and remodelling. In particular, miR-29b can induce global DNA hypomethylation targeting the DNMT genes. Consistently, a study conducted in human aortic SMCs demonstrated that miR-29b up-regulation is able to indirectly influence MMP-2/MMP-9 expression via DNMT3b down-regulation [127].

Besides the DNMT family, several ECM components have also been validated as miR-29b targets, including elastin, FBN1 (involved in TGF-β1 storage in ECM) and collagen molecules [128]. Therefore, miR-29b could have multiple functions in remodelling during aortic dilatation. This hypothesis is supported by data obtained by Boon et al. [87], revealing the increase in the miR-29 family in the aortic tissue of 18-month-old mice versus young mice, associated with a significant down-regulation of ECM in aged mouse aortas, thus sensitizing the aorta to the formation of aneurysms in advanced age. More importantly, the same group also revealed that miR-29b expression is profoundly increased in biopsies of human TAAs from patients with either a BAV or a TAV. These findings are complemented by another interesting study showing that miR-29b is increased in the ascending aorta of a mice model of Marfan's syndrome, and is key to the pathogenesis of early aneurysm development by regulating aortic wall apoptosis and ECM abnormalities [129]. The same authors also demonstrated that TGF-β1 induces the de-repression of miR-29b through the reduction of active nuclear factor κB (NF-κB) in a murine model of Marfan's syndrome. Jones et al. [85] did not confirm the increased expression of miR-29b in TAV patients with TAAs, but revealed a substantial decrease in miR-29a levels compared with a control group consisting of heart donors and coronary artery bypass graft patients. Additional in silico and in vitro studies revealed that MMP-2 is a target of miR-29a and in vivo analyses confirmed a significant relationship between miR-29a decrease and MMP-2 abundance in human TAA samples.

Discrepancies among the expression data for the miR-29 family in TAAs are most probably due to disparities in sampling, concomitant risk factors and co-morbidities of the patients, the severity of aortic dilatation, and the potential inclusion of thrombus or atherosclerotic plaque during tissue extraction, affecting the composition of the miRNA population in aortic tissue. In particular, the severity of aortic dilatation could play a major role in the expression of miR-29 family members, because they could be up-regulated during the early phase of aortic dilatation and then down-regulated at later phases, with a negative feedback mechanism trying to limit aneurysm progression by an increase in tissue stiffness through collagen and elastin synthesis in the ECM, as also suggested elsewhere [81].

Jones et al. [85] also revealed the decrease of miR-133a in TAA samples versus control samples. Studies in different pathological settings identified TGF-β1 as a target of miR-133 [130]. A relationship between miR-133a and TGF-β1 could also be reasonably hypothesized in the context of TAAs, presumably contributing to the increased expression of TGF-β1 during TAA development. Studies are necessary to confirm this hypothesis.

Beyond the miRNA families described above, increasing evidence suggests a role for the cluster miR-143/-145 in not only physiological plasticity of SMCs, but also dilative thoracic aortopathy. This cluster is among the miRNAs that are released by vascular cells and can modulate processes in recipient cells [131]. In particular, the cluster miR-143/-145 is involved in an extracellular vesicle-mediated mechanism of communication between SMCs and ECs, with a final atheroprotective effect. These data are consistent with the finding that the miR-143/-145 cluster can be transferred from SMCs to ECs through thin membrane microstructures defined as tunnelling nanotubes. This transfer is triggered by TGF-β1 and leads to modulation of EC phenotype, with antiangiogenic effects and vessel stabilization [132]. MiR-145 is increased in TAA samples versus control samples and promotes media remodelling in aortic aneurysms through TGF-β1, as indicated by the concomitant increase in osteopontin and collagen III [133]. These results suggest an interplay between miR-145 and TGF-β1 in aneurysms and stimulate additional studies to verify whether the cluster miR-143/-145 or other miRNAs could also act as communication molecules among cells in the aortic wall during aortic dilatation, possibly under the control of TGF-β1 mediators and, if so, with which specific role.

HISTONE COVALENT MODFICATIONS AND TGF-β1 INTERPLAY IN THORACIC DILATIVE AORTOPATHY

A link between histone covalent modifications and the multiple activities played by TGF-β1 mediators has recently been highlighted in different pathological settings. In particular, several data are available for histone acetylation to play a role in TGF-β1-induced fibrosis and MF differentiation. Among the first observations, Glenisson et al. [134] revealed that HDAC4 is an essential epigenetic regulator of MF differentiation induced by TGF-β1, because HDAC4 silencing induces the expression of the 5′-TG–3′-interacting factor (TGIF) and of TGIF2, two homoeodomain proteins acting as endogenous inhibitors of the TGF-β1 signalling pathway.

As anticipated in a previous section, a major contribution to the analysis of histone epigenetic alterations associated with aneurysm progression and the TGF-β1/Smad2 signalling pathway has been provided by the study of Gomez et al. [110], demonstrating that the increase in H3K9/14 acetylation and the H3K4 methylation are involved in Smad2 over-expression in TAAs in syndromic and non-syndromic patients, in a cell-specific and transcription-start-site-specific manner. The epigenetic regulation of Smad2 leads to an autonomous activation of this factor with respect to the activity of both TGF-β1 receptors and the extracellular TGF-β1. These findings explain the lack of co-localization within the aneurysmal tissue between the TGF-β1 retained in the ECM and the activation of Smad2 [135], and underline the importance of chromatin dynamics in modulating SMC gene expression during aneurysm development.

HDAC inhibitors typically increase total cellular histone acetylation and activate the expression of susceptible genes. However, in large-scale genetic studies a significant number of genes are also down-regulated in the presence of HDAC inhibitors [136], presumably through mechanisms involving the increased acetylation of non-histone proteins [137], rather than through epigenetic regulation of gene expression. In this context, several studies reveal the antifibrotic actions of HDAC inhibitors, the molecular bases of which remain, however, not fully understood. It seems likely that HDAC inhibitors block fibrosis by multiple mechanisms, including the inhibition of fibroblast proliferation and/or migration, the induction of genes that suppress ECM production by fibroblasts, the suppression of proinflammatory stimuli for fibrosis and the blockade of EMT. In vitro studies indicated that the treatment of fibroblasts with the HDAC inhibitory trichostatin A (TSA), or the silencing of HDAC2, reduced the expression level of α-SMA, collagen and HDAC2 [138]. TSA induced the hyperacetylation of histones H3 and H4 in a dose-dependent manner and suppressed the opening of the α-SMA gene promoter, thus reducing its expression. TSA also inhibited TGF-β1-induced activation and translocation of Smad2/3 and rescued the TGF-β1-suppressed, Smad7 signalling pathway, with the inhibition of MF differentiation as the final effect. On this basis, it would be worthwhile establishing the effect of HDAC inhibitors in the context of dilative aortopathy of the ascending aorta and their interplay with TGF-β1.

An interesting study by Barter et al. [139] demonstrated that HDAC3 is required for activation of the extracellular signal-related kinase (ERK) and phosphoinositide 3-kinase (PI3K) signalling pathways by TGF-β1. However, HDAC inhibitors do not affect the TGF-β1-mediated phosphorylation or nuclear translocation of Smad transcription factors. The authors of this study [139] suggest a possible hypothesis as the basis of the selective action of HDAC inhibitors: a HDAC-dependent dynamic turnover of histone tail acetylation is necessary to modulate gene expression, as previously observed by others for the promoter of c-fos [140].

These data reveal that TGF-β1 signalling pathways are affected by histone acetylation and by HDACs in different settings associated with fibrosis. These mechanisms remain to be demonstrated in dilative aortopathy of the ascending aorta.

CHANGES IN DNA METHYLATION AND TGF-β1 INTERPLAY IN THORACIC DILATIVE AORTOPATHY

Global data support a role for aberrant DNA methylation in sustained MF phenotype and fibrosis. Consequently, alterations in DNA methylation could play a major role in TAAs. As a matter of fact, only a few experimental and clinical data are currently available about the DNA-methylation profile in dilated thoracic aortas. A recent study performed tissue-specific, genome-wide, methylation profiling in BAV versus TAV patients, and identified novel genes associated with TAAs exhibiting a differential DNA-methylation profile [95]. In particular, Shah et al. [95] identified a group of both differently methylated and differently expressed genes (including PTPN22, RIPK1 and others) in TAV and BAV patients, and a group of differently methylated but not differentially expressed genes (including ACTA2, the gene coding for α-SMA, found to be hypomethylated in BAV versus TAV patients).

Several studies suggest a direct or indirect role for TGF-β1 pathways in the control of the methylation status of genes involved in fibrosis and remodelling. In particular, experiments on rat cardiac fibroblasts revealed that TGF-β1 stimulation can induce the expression and synthesis of collagen type 1 through a decrease in the percentage of DNA methylation across multiple CpG sites in the rat COL1A1 promoter, mediated by a decreased expression of DNMT1 and DNMT3a [141]. Similarly, TGF-β1 treatment inhibits DNMT1 and DNMT3a expression and subsequently induces α-SMA expression in rat lung fibroblasts [94]. These findings suggest that changes in the methylation profile of genes involved directly or indirectly in TGF-β1 pathways might contribute to the pathogenesis of TAAs in TAV and BAV patients.

CONCLUSIONS

Given the key role of miRNAs, histone modifications and DNA methylation in the regulation of gene expression, a more in-depth comprehension of the epigenetic alterations associated with dilative aortopathies of the thoracic ascending aorta could be of great help in clarifying their pathogenesis and, possibly, identifying preventive and therapeutic strategies. In particular, considering their relevant role in aortopathies, intense research should be focused on mechanisms of epigenetic regulation of the TGF-β1 canonical and non-canonical pathways in TAAs.

The divergent roles played by the TGF-β1 pathway in TAAs and AAAs should be carefully evaluated, in view of a potential therapeutic approach to aortic dilatation, because the recent findings described above suggest over-expression of TGF-β1 and prevention or control of the progression of AAAs, whereas this cytokine should be inhibited in the thoracic ascending aorta to control its dilatation [5].

It should also be considered that, on the basis of its pleiotropic nature, systemic interference with the epigenetic regulation of the TGF-β1 pathway could imply severe adverse events. The systemic administration of drugs targeting TGF-β1 pathways could also be ineffective at the aortic level due to low local drug concentration. Hence, local interference with the epigenetic regulation of the TGF-β1 pathway would be recommended both to reduce potential severe adverse events and to reach a high local concentration of therapeutic molecules.

Ongoing clinical trials in different pathological contexts (e.g. cancer) have revealed the feasibility of miRNA mimics or anti-miRNA applications as potential therapeutic tools [142], also suggesting the feasibility of a similar approach in the prevention or control of TAAs. However, an exhaustive comprehension of all the roles of single miRNAs in vascular homoeostasis and aortopathy is necessary before progressing to clinical application of anti-miRNAs or miRNA mimics in the context of TAAs.

A HDACi has been successfully administered to cultured human SMCs and in a murine model of AAAs, leading to a reduction in both MMP-2 and MMP-9 expression, and aneurysm incidence [143]. A similar approach could also be hypothesized in human TAAs, considering the role of MMPs in disease progression, if an eluting device or liquid/gel polymer were to be designed for local application to undilated or mildly dilated aortas, e.g. during surgery for dysfunctional aortic valve replacement.

Tissue and circulating miRNAs will probably attract considerable attention as not only therapeutic targets but also potentially non-invasive biomarkers of dilative aortopathy, because they could offer distinct advantages over other biomarkers. In particular, as nucleic acids, miRNAs can be amplified and detected with higher sensitivity and specificity than plasma proteins [63]. However, it should be considered that several drugs commonly used in cardiovascular patients, including statins, heparin and antiplatelet drugs, can affect the concentration and/or the enzymatic amplification of some miRNAs [144]. These confounding factors will require well-designed trials for biomarker validation to be set up, including appropriate cohorts of control individuals.

In addition, an increasing number of studies have identified a positive association between the DNA-methylation profile in peripheral blood leukocytes and that in cardiovascular diseases or predisposing conditions, thus suggesting DNA methylation as a potential biomarker of cardiovascular disease [145]. Future investigations will reveal whether there is a significant correlation between the methylation profile of genomic DNA in circulating cells and the risk of a TAA.

These findings support a role for epigenetic alterations not only as key players in TAA pathogenesis, with a potential role as therapeutic or preventive targets, but also as possible biomarkers of disease. This aspect is of particular interest, because ‘non-dimensional’ criteria for risk stratification of patients affected by aortopathy are needed, in particular in the presence of a BAV.

Finally, of interest for future studies could be the potential link between the inheritance of epigenetic signatures and their contribution to the pathogenesis of an aneurysm in patients with a BAV, a congenital malformation that is also inheritable.

AUTHOR CONTRIBUTION

All the authors collected and reviewed the relevant literature and wrote different sections of the manuscript.

FUNDING

This study was funded by the Italian Ministry of Health [FIRB 2012, protocol no. RBFR124FEN_003, to A. Della Corte].

Abbreviations

     
  • α-SMA

    α-smooth muscle actin

  •  
  • AAA

    abdominal aortic aneurysm

  •  
  • BAV

    bicuspid aortic valve

  •  
  • COL1A

    collagen type 1α

  •  
  • CREB

    cAMP response element-binding protein

  •  
  • CTGF

    connective tissue growth factor

  •  
  • DNMT

    DNA methyltransferases

  •  
  • EC

    endothelial cell

  •  
  • ECM

    extracellular matrix

  •  
  • EMT

    epithelial-to-mesenchymal transition

  •  
  • ENG

    endoglin

  •  
  • eNOS

    endothelial nitric oxide synthase

  •  
  • FBN

    fibrillin

  •  
  • FN

    fibronectin

  •  
  • HAT

    histone acetyltransferase

  •  
  • HDAC

    histone deacetylase

  •  
  • HIF

    hypoxia-inducible factor

  •  
  • KLF

    Kruppel-like factor

  •  
  • LTBP

    latent TGF-β1-binding protein

  •  
  • MF

    myofibroblast

  •  
  • MMP

    matrix metalloproteinase

  •  
  • NGS

    next-generation sequencing

  •  
  • RL

    right–left

  •  
  • RNAi

    RNA interference

  •  
  • sENG

    soluble endoglin

  •  
  • SMC

    smooth muscle cell

  •  
  • SOD

    superoxide dismutase

  •  
  • SRF

    serum response factor

  •  
  • TAA

    thoracic ascending aortic aneurysm

  •  
  • TAV

    tricuspid aortic valve

  •  
  • TET

    10–11 enzyme

  •  
  • TGF-β1

    transforming growth factor β1

  •  
  • TGIF

    5′-TG–3′-interacting factor

  •  
  • TIMP

    tissue inhibitor of metalloproteinase

  •  
  • TSA

    trichostatin A

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