Critical effects of epigenetic regulation in pulmonary arterial hypertension

Abstract

Pulmonary arterial hypertension (PAH) is characterized by persistent pulmonary vasoconstriction and pulmonary vascular remodeling. The pathogenic mechanisms of PAH remain to be fully clarified and measures of effective prevention are lacking. Recent studies; however, have indicated that epigenetic processes may exert pivotal influences on PAH pathogenesis. In this review, we summarize the latest research findings regarding epigenetic regulation in PAH, focusing on the roles of non-coding RNAs, histone modifications, ATP-dependent chromatin remodeling and DNA methylation, and discuss the potential of epigenetic-based therapies for PAH.

Introduction

Pulmonary arterial hypertension (PAH) is a fatal disease, characterized by a progressive increase in pulmonary artery pressure that is accompanied by pulmonary vascular remodeling, increased vasomotor tone and compensatory right ventricular hypertrophy, leading to heart failure and death. Five categories of PAH have been defined according to clinical, etiological, and hemodynamic features, but share common pathogenesis [1]. The vascular remodeling can be extensive, involving medial hypertrophy and formation of neointima and plexogenic lesions; moreover, these changes, individually and collectively, are associated with excessive cell proliferation, apoptosis resistance, phenotypic transition, metabolic shifts, and the recruitment of circulating inflammatory cells [2, 3].

Researchers are just beginning to elucidate the myriad of molecular factors underlying the causes and consequences of vascular remodeling. Endothelial cells (ECs) appear to be involved in the earliest stages, exerting direct modulating effects on other cells, such as pulmonary artery smooth muscle cells (PASMCs), fibroblasts and even other ECs, through paracrine signaling [4]. The initiation of different signaling pathways under these conditions can support sustained vasoconstriction and proliferation, as well as the anti-apoptotic phenotype. EC signaling is also capable of recruiting pro-inflammatory cells, which in the pulmonary arteries then sustain the inflammatory microenvironment and result in increased vascular wall thickness and muscularization of the small pulmonary arterioles [5–7]. The physiological imbalances related to PAH pathogenesis have known associations with genetic susceptibilities and non-homeostatic responses in gene regulation that occur via epigenetic modification [8].

The term epigenetics in its contemporary usage emerged in the 1990s, but for some years after it was applied with somewhat variable intent [9]. A consensus definition of the concept of epigenetic trait as a “stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence” was formulated at a Cold Spring Harbor meeting in 2008. During this time, intensive investigations sought to uncover the multitude of epigenetic mechanisms that contribute to normal physiology as well as pathogenesis of a wide array of human diseases, from metabolic dysfunctions to infectious diseases; as a result the beneficial and detrimental roles of chromatin activation, DNA methylation, histone modification and non-coding RNA (ncRNA) regulation have been extensively studied.

Accumulating evidence supports the hypothesis that epigenetic changes are involved in PAH [10, 11]. Furthermore, various epigenetic mechanisms have been confirmed to exert a profound influence on PAH, including miR deregulation, DNA methylation and histone modification [5]. Gaining a detailed understanding of the general and specific epigenetic principles in PAH may be a key for developing new and more effective treatments for PAH.

Herein, we review the latest advances in the understanding of epigenetic regulation in PAH, focusing on the role of ncRNAs, histone modifications, ATP-dependent chromatin remodeling, and DNA methylation and we discuss the potential for epigenetic-based therapy for PAH.

ncRNAs involvement in PAH

Around 98% of all RNA transcripts do not possess protein coding capability [12]. It is now clear that many, if not all, of them regulate individual steps of gene expression, including transcription, RNA processing and translation [13]. The ncRNAs can be broadly classified according to their size, representing the long ncRNAs (lncRNAs), which are longer than 200 nucleotides (nts) and can range up to hundreds of thousands of nts in length, and the small ncRNAs, which are less than 200 nts in length; the latter classification includes the much more recently discovered micro (mi) RNAs, which are ~22 nts in length [14].

In this section, we will discuss the effects and underlying mechanisms of miRNAs and lncRNAs on PAH pathogenesis. The information for the particular miRNAs and lncRNAs discussed herein are summarized in Table 1.

Table 1.

Non-coding RNAs involved in PAH development

ncRNA	Expression in PAH	Function	Target mRNA and signaling pathway	Models	References
miRNAs
miR-let-7g	↓	Promote PASMC proliferation and pulmonary vascular remodeling	C-myc-Bmi-1-p16 signaling	Rat-hypoxia	[87]
miR-9	↑	Promote PASMC phenotypic switch (inhibit SMC differentiation)	HIF1α-miR-9	Rat-hypoxia	[61]
miR-17/92	↑	Promote EC injury; Induce PASMC proliferation and reduce PASMC apoptosis	p21, STAT3-miR-17/92-BMPR2 pathway, HIF-1α and MFN2	Mouse-hypoxia Rat-MCT, hypoxia Human-PAH	[29, 67, 89, 90, 105]
miR-20a	↑	Promote PASMC proliferation and migration; inhibit PASMC differentiation	PRKG1, BMPR2	Mouse-hypoxia HPASMC-hypoxia	[49, 50]
miR-21	↑	Promote PAEC and PASMC proliferation, migration; vasoconstriction and pulmonary vascular remodeling	PDCD4, SPRY2, PPARα, WWP1, SATB1, PTEN, BMPR2 and RhoB	Mouse-hypoxia Rat-MCT Human-PAH HPASMC/HPAEC-hypoxia	[30, 52, 53, 72]
miR-23a	↑	Promote PASMC phenotypic transformation	HIF1α-miR-23a	Rat-hypoxia HPASMC-hypoxia	[62]
miR-26b	↓	Promote PASMC proliferation and pulmonary vascular remodeling	CTGF and CCND1	Rat and PASMC-MCT	[88]
miR-27a	↑	Promote PAEC proliferation	PPARγ-ET-1	Mouse-hypoxia HPAEC-hypoxia	[40]
miR-27b	↑	Promote vasoconstriction and remodeling	PPARγ-Hsp90-eNOS	Rat-MCT HPAEC	[41]
miR-29	↑	Regulate energy metabolism and promote pulmonary vascular remodeling	PPARγ	Human-PAH Bmpr2 mutant Mice-16αOHE	[54]
miR-34a	↓	Promote PASMC proliferation and migration; Reduce SMC apoptosis	PDGFA	Rat-hypoxia HPASMC-hypoxia	[73]
miR-103/107	↓	Promote PASMC proliferation and pulmonary vascular remodeling	HIF-1β	Rat-hypoxia	[70]
miR-124	↓	Promote PASMC and fibroblast proliferation and migration	SMCs: NFATc1 CAMTA1 and PTBP1; Fibroblasts: MCP-1, PTBP1, Notch1/PTEN/FOXO3/p21 and p27 signaling	Mouse-hypoxia Human and calve PAH	[82, 93]
miR-126	↓	Promote PAEC proliferation, migration and angiogenesis	Spred-1	Human-PAH, plexiform lesions	[43, 44]
miR-130/301	↑	Promote PASMC and PAEC proliferation	PPARγ-STAT3-miR-204-Src signaling, CDKN1A(p21) (SMC); PPARγ-apelin-miR-424/503-FGF2 signaling (EC)	Human-PAH Mouse-hypoxia	[42, 46, 71]
miR-135a	↑	Pulmonary vascular remodeling	BMPR2	Mouse-OVA and PM	[55]
miR-138	↑	Reduce PASMC apoptosis; Promote HMVEC dysfunction	S100A1; HIF-1α-miR-138-Mst1-akt signaling	Rat-hypoxia PASMC-hypoxia HMVEC-hypoxia	[36, 37, 63]
miR-140-5p	↓	Promote PASMC proliferation, migration and pulmonary vascular remodeling	Smurf1-BMP signaling	Human-PAH Rat-MCT, sugen5416/hypoxia	[56]
miR-143-3p	↑	Promote PASMC and PAEC migration (crosstalk) and pulmonary vascular remodeling		Mouse-hypoxia Human and calve PAH	[58]
miR-145	↑	Promote development of PAH	KLF4, KLF5, Smad4, Smad5	Mouse-hypoxia, BMPR2 deficient Human heritable and idiopathic PAH, BMPR2 mutation	[44]
miR-143/145	↓	Promote PASMC phenotypic switch and pulmonary vascular remodeling	BMP4, TGF-β, myocardin	Human-PAH, plexiform lesions	[44, 59]
miR-150	↓	Poor survival		Human-PAH plasma	[23]
miR-190	↑	Enhance vasoconstriction and Ca2+ influx in PASMCs	KCNQ5	Rat-hypoxia	[86]
miR-193	↓	Promote PASMC proliferation	LOXs and IGF1R	Mouse-hypoxia Rat-MCT	[74]
miR-199a-5p	↑	Promote vasoconstriction	Smad3	Rat-MCT	[31]
miR-204	↓	Promote cell survival, proliferation and apoptosis resistance, calcified lesion	SHP2-Src/STAT3 axis and PARP1-NFAT/HIF1α signaling pathway, RUNX2	Human-PAH, plexiform lesions Mouse-hypoxia Rat-MCT, sugen5416/hypoxia	[44, 83–85]
miR-206	↓	Promote PASMC proliferation; reduce apoptosis	Notch3 and HIF-1α/Fhl-1 pathway	Mouse and rat-hypoxia	[68, 69]
miR-210	↑	Reduce PASMC apoptosis Regulate cell metabolism	HIF-1α-miR-210-E2F3 signaling; ISCU1/2-Iron-sulfur (Fe-S) clusters	Mouse-sugen5416/hypoxia PASMC-hypoxia	[64, 65]
miR-214	↑	Regulate right ventricular hypertrophy	PTEN	Mouse and rat-sugen5416/hypoxia	[106]
miR-223	↓	Promote PASMC proliferation and migration; reduce apoptosis	PARP1, RhoB, MLC2 and IGF-1R	Human-PAH Rat-MCT, hypoxia Mouse-hypoxia	[75–77]
miR-322	↑	Promote PASMC proliferation and migration	BMPR1a and Smad5 (HIF-1α-miR-322-BMP-Smad signaling pathway)	Mouse and rat-hypoxia PASMC-hypoxia	[66]
miR–328	↓	Promote vasoconstriction and remodeling; reduce SMC apoptosis	CaV1.2 and IGF1R	Rat-hypoxia	[78]
miR-424/503	↓	Promote PAEC and PASMC proliferation (crosstalk) and pulmonary vascular remodeling	Apelin/MEF2-miR-424/503-FGF2, FGFR1 pathway	Rat-MCT, sugen5416/hypoxia	[45, 107]
lncRNAs
MALAT-1	↑	Promote EC proliferation, migration and vessel growth	Cell cycle regulators (CCNE1,CCNA2, p21) and XBP1,	Mouse Retinal Angiogenesis Model EC-hypoxia	[100, 101]
MANTIS	↓	Regulate angiogenesis	Interact with BRG1	Human-PAH Rat-MCT	[102]
lncRNA-p21		Promote SMC and fibroblast proliferation and neointimal hyperplasia; inhibit SMC apoptosis	P53, Thy-1	Human-coronary artery disease Mouse-ARDS and lung fibroblasts—LPS	[103, 104]

Cell types: PAEC, pulmonary artery endothelial cell; PASMC, pulmonary artery smooth muscle cell

Target genes/proteins: ACE, angiotensin-converting enzyme; BMPR, bone morphogenetic protein receptor; BRG1, Brahma-related gene 1, CAMTA1, calmodulin-binding transcription activator 1; CaV1.2, L-type calcium channel subunit alpha 1C; CCNA2, cyclin A2;CCND1, cyclin D1; CCNE1, cyclin E1; CTGF, connective tissue growth factor; E2/F3, transcription factor E2F3; Elk-1, ETS domain-containing protein 1; FGF2, fibroblast growth factor 2; FGFR1, fibroblast growth factor receptor 1; Fhl-1, four and a half LIM domains protein 1; FOXO3, forkhead box O3; HIF, hypoxia-inducible factor; IGF1R, insulin growth factor 1 receptor; ISCU, iron-sulfur cluster assembly enzyme; KCNQ5, potassium voltage-gated channel subfamily KQT member 5 protein; KLF4, Kruppel-like factor 4; KLF5, Kruppel-like factor 5; LOXs, lipoxygenases; LPS, lipopolysaccharide; MCP-1, monocyte chemotactic protein-1; MCT, monocrotaline; MLC2, myosin light chain of myosin II; MFN2, Mitofusin 2; Mst 1, macrophage stimulating protein 1; NFAT, nuclear factor of activated T cells; Nkx2.5, NK2 transcription factor related, locus 5; Notch1, neurogenic locus notch homolog 3 protein 1; Notch3, neurogenic locus notch homolog protein 3; p21, cyclin-dependent kinase inhibitor 1; p27, cyclin-dependent kinase inhibitor 1B; PARP1, poly(ADP-ribose) polymerase-1; PDCD4, programmed cell death protein 4; PDGFA, platelet-derived growth factor receptor alpha; PPAR, peroxisome proliferator–activated receptor alpha; PPARγ, peroxisome proliferator-activated receptor-gamma; PRKG1, protein kinase, cGMP-dependent, type I; PTBP1, polypyrimidine tract-binding protein 1; PTEN, phosphatase and tensin homolog; RhoB, Ras homolog gene family, member B; RUNX2, Runt-related transcription factor 2; S100A1, S100 calcium-binding protein A1; SATB1, special AT-rich sequence-binding protein-1; SHP, src homology region 2 domain-containing phosphatase; Smad, Sma and Mad-related protein; SMURF1, SMAD-specific E3 ubiquitin protein ligase 1; Sp-1, specificity protein 1; Spred-1, Sprouty-related protein-1; SPRY2, Sprouty homolog 2; Src, src kinase; STAT3, signal transducer and activator of transcription 3; TGF-β, transforming growth factor β; Thy-1, thymocyte differentiation antigen-1; WWP1, WW domain-containing protein 1; XBP1, X box-binding protein 1

miRNAs

miRNAs silence gene expression by binding to the 3′-untranslated regions of messenger (m)RNAs and inhibiting translation or promoting degradation of the mRNAs [15, 16]. It is estimated that each miRNA expressed in a given cell may target about 100–200 mRNAs for down-regulation [17], and that about 60% of human protein coding genes are regulated by miRNAs. Interestingly, studies of miRNAs in PAH have suggested key roles in the pathogenic mechanism [18–20], making them novel candidates for therapeutic intervention [21]. Furthermore, a panel of circulatory miRNAs, including miR-126, miR-150, miR-193, and miR-204 appear to be promising biomarkers for PAH diagnosis and prognosis [22, 23]. These collective studies have also suggested that miRNAs may contribute to the development of PAH by regulating the cellular components known to be critically involved in the disease process, namely the PASMCs, and the pulmonary artery endothelial cells (PAECs) and fibroblasts.

miRNAs and endothelial dysfunction in PAH

The PAEC houses in innermost intimal layer of pulmonary vascular and immediate contact with the blood supply and able to detect changes in pressure, circulating factors and oxygenation. Proliferating and dysfunctional PAECs are considered as characteristic features of intimal thickening in PAH, and the targeting activities of miRNAs have been implicated in proliferation and apoptosis of these cell types through different signaling pathway.

BMPR2 signaling

Bone morphogenetic protein receptor (BMPR) 2 is a receptor for the transforming growth factor (TGF)-β/BMP superfamily, the loss of function has been linked to cellular pathophenotypes, including proliferation, cell survival [24, 25], endothelial dysfunction [26], repression of mitochondrial metabolism, and endothelial-to-mesenchymal transition and then leading to the development of PAH [27, 28]. miR-17/92 has been shown to mediate the endothelial injury that triggers PAH, itself being transcriptionally regulated by signal transducer and activator of transcription (STAT3) and targeting either BMPR2 directly to result in cell proliferation and apoptosis resistance [29]. The increased miR-21 is also known to target BMPR2 and to suppress Rho/Rho kinase activity, as shown in cultured human PAECs, in hypoxia, accompanying increased angiogenesis and vasoconstriction [30]. miR-199a-5p has also been reported as involved in PAH by targeting to the BMPR-Sma and Mad-related protein (Smad) signaling pathway; specifically, it was shown to be significantly increased in PAH rat models, while anti-miR-199a-5p was shown to increase nitric oxide (NO) level and decrease cytoplasmic Ca²⁺ level in PAECs, thereby decreasing the pulmonary artery pressure and right ventricular hypertrophy by attenuating the expression of Smad3, which was confirmed to be the target gene of miR-199a-5p [31].

HIFs signaling

Hypoxia-inducible factors (HIFs) are transcription factors that respond to decreased available oxygen in the cellular environment, or hypoxia. Active HIF transcription factors are comprised of an oxygen-regulated α-subunit and a constitutive β-subunit, and there are three HIF-α isoforms in humans, including HIF-1α, HIF-2α and HIF-3α [32]. Chronic hypoxia, or prolonged low oxygen exposure, is a key trigger for pulmonary vascular remodeling and PAH. The alteration of HIFs signaling in various PAH-relevant pathways contributing to vasocontraction, vascular cell proliferation, metabolic shifts and inflammation [33–35]. The transcription factor HIF-1α, a major mediator of these effects, modulates various miRNAs and reciprocally is regulated by miRNAs. The up-regulation of miR-138 has been shown to be HIF-1α-dependent and involved in hypoxia-induced EC dysfunction by regulating nitric oxide (NO) expression through S100A1 [36, 37]. miR-138 have also been shown to inhibit hypoxia-induced proliferation of endothelial progenitor cells [38].

PPARγ signaling

Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear hormone receptor that interacts with retinoid receptor (RXR) form heterodimer, upon binding with its ligand, triggering transcriptional activation of its target genes. The reduced expression of PPARγ has been shown to contribute to PAH progression by promoting proliferative and inhibiting apoptotic program in the pulmonary vasculature [39]. miR-27a was found to be increased under hypoxia, and to promote PAEC proliferation by regulating endothelin-1 (ET-1) expression through PPARγ [40]. miR-27b have also been shown to be up-regulated in monocrotaline (MCT)-induced PAH and inversely correlated with the levels of PPARγ, which was shown to mediate the disruption of endothelial nitric oxide synthase (eNOS) coupling to heat shock protein (Hsp)90 and the suppression of NO production associated with the PAH phenotype [41]. miR-130/301 was also up-regulated and this contributes to the proliferation of PAECs through the activation of PPARγ [42].

Related growth factors signaling

Growth factors play an important role in regulating a variety of cellular processes, such as cellular growth, proliferation and differentiation, through binding to specific receptors on the surface of their target cells. Lots of growth factors signaling pathway play important role in the formation of PAH. Some miRNAs have been shown to be down-regulated in PAH models and their participation in the pathogenesis is a promising topic of study. For instance, miR-126 was found to be down-regulated in plexiform lesions of patients with severe PAH, and contribute to the proliferation, migration of endothelial cells and angiogenesis by regulating several growth factors expression, such as vascular endothelial growth factor-A (VEGF-A) and fibroblast growth factor (FGF), through sprouty-related, EVH1 domain-containing protein-1 (Spred-1) [43, 44]. The down-regulation of miR-424 and miR-503 in hypoxia has been associated with, and proposed to result to, the FGF signaling (FGF2 and FGFR1) which is known to play a key role in regulating proliferation of PAECs and in maintaining pulmonary vascular homeostasis [45]. Bertero et al. [42] reported that miR-424/503-FGF2 signaling have also been regulated by up-regulated miR-130/301 in PAECs through the activation of PPARγ.

Whereas EC dysfunction that can directly lead to intimal thickening and remodeling, probably equally important are the indirect signals capable of inducing PASMCs contraction, hypertrophy, and even hyperplasia. For instance, the down-regulation of miR-424 and miR-503 is known to play a key role in regulating proliferation of PAECs. And interestingly, the alteration of miR-424 and miR-503 in PAECs also contribute to the proliferation of PASMCs through PAEC-PASMC crosstalk [45]. The up-regulation of miR-130/301 in PAECs also link to the contractile function of PASMCs through the activation of endothelin-1 and its receptors [46].

miRNAs and PASMCs in PAH

Pulmonary vascular medial thickening, attributable mostly to PASMC proliferation and hypertrophy, is the main feature of pulmonary artery remodeling of PAH. PASMCs exhibit extraordinary plasticity in adult animals in response to a number of environmental cues. Under hypoxia, PASMCs undergo phenotypic switch from a contractile-differentiated to a proliferative/migratory-dedifferentiated phenotype [47]. The dedifferentiated smooth muscle cells are characterized by high rates of proliferation and migration, increased expression of the extracellular matrix (ECM) proteins and low expression of contractile proteins, which are comprised of smooth muscle actin-α (α-SMA), smooth muscle-myosin heavy chain (SM-MHC), calponin, caldesmon and sm22-α [48]. This process plays a major role in the development of vascular remodeling and PAH. Studies have shown that numbers of miRNAs dysregulated in PAH substantially contribute to its pathogenesis through targeting several signaling pathways, such as BMPR2, HIFs, PPARγ and NFAT signaling.

BMPR2 signaling

The expression of miR-20a was increased in lung, pulmonary artery and serum of hypoxia PAH modeled mice, and in human PASMCs exposed to hypoxia, thereby promoting the proliferation and migration of human PASMCs; the authors also showed that it inhibits the differentiation of PASMCs, which is otherwise accompanied by decreased BMPR2 [49] and cGMP-dependent protein kinase, type I(PRKG1) [50] expression. And the decreased PRKG expression has been demonstrated strongly related to the decrease expression levels of smooth muscle cell contractile markers in hypoxia [51]. miR-21 was also up-regulated in hypoxia-induced PASMCs, affecting proliferation and migration as well as pulmonary vascular remodeling and PAH by regulating multiple related gene targets, including BMPR2 [52, 53]. miR-29 has also been shown to be up-regulated in lung tissue of PAH patients and BMPR2 mutant mice, and anti-miR-29 had improvements in hemodynamic profile, histology, and markers of dysregulated energy metabolism [54]. Similarly, the marked increase in miR-135a induced by combined Th2 antigen (Ovalbumin, OVA) and urban particulate matter (PM) in mice has been shown to contribute the pulmonary artery remodeling by regulating BMPR2 [55]. The increased miR-199a-5p, involved in PAECs dysfunction, also contributed to PASMCs proliferation by targeting the BMPR-Sma3 [31]. The expression of miR-140-5p is reduced in patients with PAH and experimental models of PAH. This dysregulation of miRNA induces PASMC proliferation, migration and promote the development of PAH by allowing for activation of SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1)—a regulator of BMP signaling [56].

In other studies, miR-145 was shown to be up-regulated in lung tissue of patients with idiopathic and heritable PAH and also in the lungs of hypoxia or BMPR2-deficient mice, with miR-145 deficiency and anti-miR-mediated reduction shown to provide significant protection from the development of PAH [57]. Similarly, the marked increase of miR-143-3p in PAH models of calf and mice, and in samples from PAH patients, and in PASMCs exposed to TGF-β, causing a significant increase in PASMCs migration. The up-regulation of 143-3p in PASMCs also contribute to the migration of PAECs through PASMC-PAEC crosstalk [58]. Conversely, miR-143/145 were also found to be down-regulated in plexiform lesions of patients with severe PAH [44]. And interestingly, the down-regulation of miR-143/145 were also found in aortic artery SMCs, and cooperatively targeted a network of transcription factors, including Kruppel-like factor (Kfl)1, Klf5 and myocardin that regulates smooth muscle cell-specific genes’ expression, further regulate SMC fate and plasticity [59, 60].

HIFs signaling

Various miRNAs have been reported to be regulated by HIF-1α under hypoxic conditions. It has been demonstrated that miR-9 [61] and miR-23a [62] are both induced by hypoxia and subsequently involved in a hypoxia-induced phenotypic switch of the rat PASMCs. Specifically, the up-regulation of miR-9 and miR-23a were shown to be mediated by HIF-1α—a key transcription factor for hypoxia-induced gene transcription. Up-regulation of miR-138 under hypoxic conditions has been shown to be HIF-1α-dependent and plays a role in SMCs apoptosis and hypoxic pulmonary vascular remodeling by targeting macrophage stimulating protein (Mst) 1 [63]. Similarly, the marked increase of miR-210 under hypoxic conditions has also been shown to be HIF-1α-dependent and transcription factor E2F3, and iron-sulfur cluster assembly enzyme (ISCU)1/2 were identified as its targets responsible for PAH in PASMCs and in PAH mouse model [64, 65]. miR-322 has also been shown to be up-regulated in lungs of chronically hypoxic mice and rats, and in primary cultured rat PASMCs under hypoxic conditions has been shown to be HIF-1α-dependent, and that this change in miR expression promoted hypoxia-induced cell proliferation and migration by directly targeting BMPR1a and Smad5 [66].

The transcription factor HIF-1 has also been regulated by lots of miRNAs. Chen et al. [67] suggest that the increased expression of miR-17/92 in PASMCs under hypoxic conditions contributes to PASMC proliferation by directly targeting of PHD2 and then induce HIF-1α expression. Additionally, a plethora of other miRNAs down-regulated under hypoxic conditions has been considered as contributors to PAH. Among them, miR-206 has been found to be decreased both in animal models of PAH and in cultured PASMCs, with the dysregulation being shown to cause increased proliferation and reduced apoptosis of PASMCs likely due to the up-regulation of Notch3 [68] or by targeting HIF-1α [69]. miR-103/107 down-regulation was observed in remodeled intrapulmonary vascular in HPH rats and hypoxia-exposured PASMCs, and inversely correlated with the expression of HIF-1β, but not HIF-1α [70].

PPARγ signaling

Bertero et al. [42] first demonstrated that miR-130/301 was up-regulated in different PAH models and in patients with PAH, leading to sustainment of the PAH-PASMC pro-proliferative phenotype through the activation of PPARγ [42, 46] and cyclin-dependent kinase inhibitor(CDKN) 1 (alias p21) [71]. The authors subsequent findings suggested that down-regulation of miR-204 could result from the activation of miR-130/301 in PASMCs [42, 46]. PPARγ agonists (rosiglitazone) has reported to attenuate hypoxia-induced HPASMC proliferation, vascular remodeling and PAH through inhibition of hypoxia-induced miR-21 expression, which emerged as an important miRNA that contributes to PAH pathogenesis [72].

Related growth factors signaling

Wang et al. [73] has demonstrated that the down-regulation of miR-34a in rat distal PAs and HPASMCs is associated with the proliferation of PASMCs by enhancing platelet-derived growth factor receptor alpha (PDGFRA) expression. Decreased miR-193 expression has been observed in both the lung tissue and serum from patients with PAH as well as from rodent models of PAH [74]; this dysregulation of miRNA induces PASMC proliferation by allowing for activation of insulin growth factor 1 receptor (IGF1R) and lipoxygenases (LOXs). miR-223 has also been found to be down-regulated in patients and animal models of PAH, thereby significantly allowing PAH-PASMCs proliferation and resistance to apoptosis by targeting IGF-1and its receptor IGF-1R [75], poly(ADP-ribose) polymerase-1(PARP-1) [76], RhoB and myosin light chain of myosin II (MLC2) [77]. Similarly, the marked decrease in miR-328 induced by hypoxia has been shown to contribute to the pulmonary artery constriction and remodeling by regulating IGF-1R and L-type calcium channel-alpha 1C (CaV1.2) in hypoxic pulmonary hypertension [78].

NFAT signaling

The nuclear factor of activated T cells (NFAT), originally identified in T lymphocytes represents a family of Ca²⁺-dependent transcription factors comprising five isoforms: NFATc1 to -c4 and NFAT5. NFAT was activated and dephosphorylated by calcineurin, a Ca²⁺/calmodulin-dependent phosphatase, and then regulate diverse cellular process including growth and survival and are involved in the development of cancer and cardiovascular diseases [79]. It has been observed that NFAT family members are involved in the pathogenesis of PAH. NFAT has been shown to be up-regulated and activated in PAH patients, mouse model of PAH and hypoxia-treated PASMC, which is associated with increased cell proliferation and resistance to apoptosis by regulating membrane potential, Kv1.5 and increasing [Ca²⁺] influx [80, 81]. miR-124 has been shown to be down-regulated by hypoxia in human PASMCs and lung cells of hypoxia-induced PAH mouse model, consistent with the activation of nuclear factor of activated T cells (NFAT) during this process; in contrast, overexpression of miR-124 was shown to not only inhibit human PASMC proliferation, but to maintain its differentiated phenotype by repressing the NFAT pathway [82]. In line with those findings, down-regulated expression of miR-204 has also been found in human patient samples and in rodent models of PAH, the latter of which showed that rescue of miR-204 reverses the disease state [44, 83]. Various studies have begun to elucidate the mechanisms by which miR-204 might be involved in PAH, namely through activation of the PARP-1signaling pathway via regulating NFATc2 and HIF-1α [84], or through regulation of the Src/STAT3 axis [44, 83] and Runt-related transcription factor 2 (RUNX2) expression [85]. Another miRNA, miR-190, was reported to be significantly increased in the pulmonary artery and PASMCs under hypoxic conditions, and this differential expression was associated with the accompanying vasoconstriction responses and Ca²⁺ influx through targeting to the voltage-gated K(+) channel subfamily member, Kcnq5 [86].

Cyclin related signaling

miR-let-7g was down-regulated in remodeled pulmonary arteries of hypoxia PAH modeled rats, and in PASMCs exposed to hypoxia, with the dysregulation being shown to cause increased proliferation of PASMCs by regulating C-myc-Bmi-1-cyclin-dependent kinase inhibitor 2A(p16) signaling pathway [87]. miR-26b was also down-regulated in MCT induced PAH model rat, and in PASMCs exposed to MCT, and inversely correlated with the expression of connective tissue growth factor (CTGF) and cyclin D1 (CCND1),which were involved in pulmonary vascular remodeling by promoting PASMCs proliferation [88]. miR-17 was significantly increased in hypoxia or MCT induced PAH models, and the treatment of miR-17 inhibitor significantly decrease the proliferation of PASMCs, and right ventricular systolic pressure, and pulmonary vascular remodeling. The beneficial effects may be related to the up-regulation of cyclin-dependent kinase inhibitor 1 (p21) [89] and Mitofusin 2 (MFN2) [90]. Conversely, Bockmeyer et al. [44] showed that miR-17 expression was down-regulated in plexiform lesions of patients with severe PAH.

miRNAs and fibroblasts in PAH

The thickening of pulmonary vascular adventitia in PAH attributable to a significant increase in collagen and ECM protein deposition, proliferation of resident fibroblasts and possibly macrophages, as well as recruitment of circulating immune and progenitor cells [91]. Hypoxia can directly induce the differentiation of pulmonary artery adventitial fibroblasts into myofibroblasts, which likely play an important pathophysiological role in contributing to abnormalities of tone and structure of the pulmonary artery in PAH [92]. Wang et al. [93] demonstrated that miR-124 was decreased in hypertensive pulmonary adventitial fibroblasts, contributing to an epigenetically reprogrammed, highly proliferative, migratory and inflammatory phenotype via direct binding to polypyrimidine tract-binding protein 1 (PTBP1) and subsequent regulation of Notch1/phosphatase and tensin homolog/FOXO3/p21Cip1 and p27Kip1 signaling; moreover, it was shown that miR-124 expression was suppressed by histone deacetylases and that treatment of hypertensive fibroblasts with histone deacetylase inhibitors increased miR-124 expression and decreased proliferation and production of the monocyte chemotactic protein-1 (MCP-1).

lncRNAs

lncRNAs, non-protein coding transcripts longer than 200 nucleotides, represent an important component of animal genome regulation [94], exerting multiple developmental and cell-type-specific regulatory functions, and with greatly expanded numbers in multicellular animals and plants [95]. The lncRNAs regulate gene transcription under normal physiologic conditions through a variety of epigenetic, transcriptional and posttranscriptional mechanisms, such as splicing and epithelial-mesenchymal transition [96–98]. Recent research interest in the lncRNAs has segued into their potential roles in various pathogenic pathways, such as those underlying cardiovascular diseases, cancer and, more recently, PAH.

Comparative microarray analysis of lncRNAs and mRNAs in lung tissues from a hypoxia-induced pulmonary hypertension (HPH) rat model and a control group identified a total of 362 lncRNAs as significantly differentially expressed [99]. One of these lncRNAs, the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) which is known to control phenotypic switch of ECs and regulates vessel growth, was significantly increased under hypoxia. Moreover, genetic ablation of MALAT1 has been shown to inhibit proliferation of ECs and to reduce vessel growth [100]. Zhuo et al. [101] further indicated that the single nucleotide polymorphism (SNP) alteration from rs619586A to G in MALAT1 could directly upregulate X box-binding protein 1 (XBP1) expression, and consequentially inhibiting the vascular endothelial cells proliferation and migration in vitro, and decreasing the risk of PAH in Chinese people. LncRNA-MANTIS was down-regulated in patients with idiopathic pulmonary arterial hypertension (IPAH) and in rats treated with monocrotaline and the deletion or silencing of MANTIS inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress [102]. In a study of the neointimal hyperplasia observed in a model of carotid artery injury (which shows vascular remodeling similar to that in PAH), lncRNA-p21 expression was found to be decreased and linked to imbalance between proliferation and apoptosis of vascular smooth muscle cells [103]. Conversely, lncRNA-p21 expression showed a time-dependent increase in a mouse model of acute respiratory distress syndrome (ARDS) and in lipopolysaccharide-treated lung fibroblasts, subsequently contributing to elevated cell proliferation; moreover, the mechanism by which lncRNA-p21 promotes pulmonary fibrosis in ARDS was shown to involve inhibition of the expression of thymocyte differentiation antigen-1(Thy-1) through inhibition of acetylation of H3 and H4 at the Thy-1 promoter [104].

Collectively, these lncRNA findings support the ongoing research interest in lncRNAs contributions to PAH, likely through regulation of cellular proliferation.

Chromatin remodeling in PAH

Chromatin remodeling is the enzyme-assisted process to facilitate access of nucleosomal DNA by remodeling the structure, composition and positioning of nucleosomes. Such remodeling is principally carried out by; (1) histone modifications by specific enzymes and (2) ATP-dependent chromatin remodeling [108]. Although remodeled chromatin is not always inherited, and not all epigenetic inheritance involves chromatin remodeling. Chromatin remodeling is essential to several important biological processes, including DNA replication and repair, chromosome segregation, embryonic development, apoptosis and cell-cycle progression.

Histone modification in PAH

The nucleosome, the fundamental unit of eukaryotic chromatin, is composed of four core histones (H2A, H2B, H3 and H4) surrounded by 146 bp of DNA. The linker histones (H1 and H5) bind the nucleosome at the entry and exit sites of the DNA, thus locking the DNA into place [109]. The H3 and H4 histones have long tails protruding from the nucleosome, which can be covalently modified on specific residues, catalyzed by histone-modifying enzymes. These tail modifications include methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, citrullination and ADP-ribosylation [110]. The posttranslational modifications of histone tails are important components of the regulatory genomic landscape in eukaryotes, and it has been confirmed that most of the well-studied histone modifications are involved in control of transcription.

Among the histones, modifications to H3 and H4 have been found to be largely conserved across the eukaryotes analyzed. Acetylation modifications involve the lysine residues and are coupled by histone acetyltransferases and removed by histone deacetylases (HDACs), which was classified into five subgroups: class I (HDAC 1, 2, 3, 8), class IIa (HDAC 4, 5, 7, 9), class IIb (HDAC 6, 10), class III (sirt1-7) and class IV (HDAC 11) [111]. Acetylation of lysine residues within nucleosomal histone tails also plays central roles for epigenetic control of gene expression, and is usually associated with active transcription. On the other hand, particular trimethylations of H3, trimethylation of H3 lysine 4 (H3K4Me3) [112] and trimethylation of H3 lysine 36 (H3K36Me3) [113], appear to be exclusively associated with active transcription. Some methylations of H3 and H4 are associated with repressed genes; these include trimethylation of H3 lysine 27 (H3K27Me3) [114], di- and trimethylation of H3 lysine 9 (H3K9Me2/3) [115] and trimethylation of H4 lysine 20 (H4K20Me3) [116].

Histone modifications act in diverse biological processes, such as gene regulation, DNA repair, chromosome condensation (mitosis) and spermatogenesis (meiosis) [117]. Consequently, they play important roles in various pathogeneses, and studies have begun to uncover their contribution to PAH as well. Several studies have demonstrated that early epigenetic changes, such as the maternal nutrient restriction consequence of increased histone acetylation, can result in a highly sensitive phenotype to hypoxia later in life, thereby causing more significant PAH or pulmonary vascular remodeling [118]. These events are similar to the effects of epigenetics in mitochondrial stress-induced longevity [119, 120].

Class I HDACs are dramatically elevated in the pulmonary arteries of humans with PAH and in the lungs and vessels from PAH animal models, and have been reported to contribute to pulmonary vascular remodeling by promoting smooth muscle cell proliferation [121]. Inhibition of class I HDACs alleviates the PDGF-induced smooth muscle cell proliferation and migration by inhibiting Akt phosphorylation and cyclin D1 expression [122]. Zhao et al. [123] showed that expression of HDAC1 and HDAC5 was increased in rats exposed to hypoxia and in human idiopathic PAH and that these differential expressions were associated with fibroblast proliferation and inflammatory cytokines’ expression by regulating p21, FOXO3 and survivin levels. The class I HDACs was also reported to regulate the activation of distinct adventitial fibroblasts and monocytes/macrophages, both of which are associated with changes in cytokine/chemokine expression and pro-inflammatory response activation [124]. In addition, IGF-1 expression is also regulated by class I HDACs and their inhibition (via apicidin treatment,) reduces chronic hypoxia-induced activation of IGF-1/pAKT signaling in lungs and attenuates neonatal hypoxia-induced PAH, as well as global DNA methylation [125].

HDACs have also been reported to regulate oxidative stress injury in PAH. Moreover, the HDAC inhibitors scriptaid have been reported to attenuate expression of the NADPH oxidases (Nox) in human lung fibroblasts and human lung microvascular endothelial cells (HLMVECs), which have been proposed to contribute to PAH and other cardiovascular diseases via blockade of the binding of both the RNA polymerase II and the histone acetyltransferase p300 to the Nox promoter regions and to decrease histone activation markers (H3K4me3 and H3K9ac) at these promoter sites as well [126]. Finally, histone deacetylation, specifically via class I HDAC3, has been shown to decrease superoxide dismutase 3 (SOD3) expression in PASMCs, and consequently it was proposed that HDAC inhibitors may protect PAH in part by increasing PASMC SOD3 expression [127].

The impaired myocyte enhancer factor 2 (MEF2) activities in PAH-PAECs has been shown to be regulated by 2 class IIa HDACs, namely HDAC4 and HDAC5. And selectively inhibition of class IIa HDACs led to restoration of MEF2 activity and its target expression in PAECs, decreased cell migration and proliferation, and rescue MCT and hypoxia-induced PAH [107].

Moreover, histone lysine methylation mediated by G9a—a key enzyme for histone H3 dimethylation at position lysine-9—is involved in cell proliferation, migration, contractility and global DNA methylation, companied by increased p21 expression, as shown in fetal PASMCs [128]. Our unpublished data have also revealed that H3K9me3 can also play an important role in smooth muscle cell proliferation and phenotypic switch, contributing to the development of HPH.

Our laboratory very recently identified an H3K4me3-dependent pathway that contributes to hypoxia pulmonary hypertension (HPH). EC-specific silencing of ASH2 and WDR5, two key components of the histone H3K4 methyltransferase complex, ameliorated HPH in mice, and this effect was enhanced by overexpression of ASH2 and WDR5, but dampened by their depletion. These events were associated with hypoxia-induced transactivation of cell adhesion molecules (CAMs), supporting establishment of a pro-inflammatory milieu that contributes to the pathogenesis of chronic hypoxia-induced pulmonary hypertension [129]. The ASH2 and WDR5 components were also found to activate endothelin (ET-1)-induced pro-inflammatory transcription in vascular smooth muscle cells, which contribute to vascular inflammation [130], and to induce the transactivation of ET-1 in vascular ECs [131]. Interestingly, the increased synthesis of ET-1 by human vascular ECs in response to hypoxia underlies the persistent vasoconstriction observed in patients with pulmonary hypertension [131].

ATP-dependent chromatin remodeling in PAH

Unlike the prokaryotic organisms, eukaryotic genes are wrapped by histones into individual nucleosomes to form chromatin. The process of unwrapping the high-order DNA structure requires a group of highly conserved proteins called chromatin remodeling complex. And there are at least five families of chromatin remodeling complexes in eukaryotes: SWI/SNF, ISWI, NuRD/Mi-2/CHD, INO80, and SWR1. Especially, ATP-dependent chromatin remodeling complexes could reposition (slide, twist or loop) nucleosomes along the DNA depend on the common ATPase domain and energy from the hydrolysis of ATP [133].

We have discovered that the Brahma-related gene 1 (Brg1) and brahma (Brm)—two key catalytic components of the mammalian chromatin remodeling complex—are induced in cultured ECs upon hypoxia challenge as well as in pulmonary arteries in an animal model of HPH. Brg1 and Brm can activate transcription of the CAMs by altering the chromatin structure surrounding the CAMs’ promoters, and EC-specific deletion of Brg1/Brm was found to ameliorate vascular inflammation and HPH in mice [134]. Brg1 and Brm can also be recruited onto the ET-1 promoter, causing subsequent modulation of ET-1 transactivation by impacting histone modifications [135]. Furthermore, Brg1 and Brm have been shown to be required for ET-1-dependent induction of pro-inflammatory mediators, performing this function by communicating with ASH2, and to be responsible for vascular inflammation inflicted by ET-1 [130]. Collectively, these data suggest that Brg1 and Brm provide the crucial epigenetic link to hypoxia-induced CAM induction and leukocyte adhesion that engenders endothelial malfunction and pathogenesis of HPH.

DNA methylation in PAH

DNA methylation occurs on cytosine residues in cytosine-phospho-guanine (CpG) regions of the genome and is essential for normal development [136]. The process is regulated by DNA methyltransferases (DNMTs), which add methyl groups to the DNA. The addition of the methyl group typically serves to repress gene transcription, and about 60–90% of all CpGs are methylated in mammals [137]. Studies have shown that DNA methylation plays an important role in the pathogenesis of PAH. For instance, Archer et al. [138] determined that the observed decreased expression of SOD2 in pulmonary arteries and plexiform lesions, which is responsible for the hyperproliferative PAH phenotype, is due to hypermethylation of CpG islands in the SOD2 gene; moreover, reversal of the methylation status via DNMT1 inhibition rescued the SOD2 expression and restored the ratio of proliferation to apoptosis. The epigenetic silencing of SOD2 was also reported to contribute to the normoxic activation of HIF-1α that is implicated in the Warburg effect of cancer cells and in PAH-PASMC proliferation [139, 140]. Interestingly, Nozik et al. [127] suggested that the decreased expression of SOD3 in patients with IPAH and animal models was not regulated by DNA methylation.

Hypoxia also significantly enhances the expression of DNMT3A in visceral smooth muscle cells, which is associated with the phenotypic switch of these cells [141]. IGF-1, which is regulated by HDACs, has also been shown to be controlled by DNA methylation, and the apicidin-mediated inhibition of HDACs decreases global DNA methylation levels in lungs [129], suggesting a role in PAH. A study applying the methylated DNA immunoprecipitation microarray to investigate the differential profile of the extrauterine growth restriction (EUGR)-induced PAH rat model showed that the hypermethylated genes are vascular development-associated and that the hypomethylated genes are late-differentiation-associated and related to signal transduction [142]. Thus, epigenetic dysregulation appears to be a strong mechanism for propagating the cellular memory of early postnatal events, causing changes in the expression of genes and long-term susceptibility to pulmonary hypertension. Nevertheless, Pousada et al. [143] reported that BMPR2, regulated by miRNAs and associated with the development of PAH, was not methylated in the promoter region in PAH patients and controls.

PAH Treatment basing on epigenetics

PAH is characterized by sustained vasoconstriction and progressive remodeling of pulmonary arteries [144]. Despite considerable advances in PAH treatment, this devastating disease still carries a prognosis worse than many cancers [2]. Current pharmacological treatments of PAH are primarily vasodilators, and offer a significant increase in survival, but there remains no cure other than transplantation, therefore, the new strategies and effective biomarkers used for assessing disease severity and response to treatment are urgently required [145]. We have summarized that epigenetic modification processes exert pivotal influences on PAH pathogenesis by regulating vasoconstriction and pulmonary vascular remodeling (Fig. 1). And some studies have already demonstrated that epigenetic-based therapy, such as inhibition of HDACs and restoration of miRNA expression can reverse PAH in different animal models. Additionally, multiple studies in cancers indicated that targeting of epigenetic regulation pathways is currently evolving as a major therapeutic strategy in the treatment of several cancers [146–148]. Thus, we summarize the latest research findings regarding epigenetic-based therapies for PAH, mainly focusing on histone modifications and non-coding RNAs.

Fig. 1.

Epigenetic regulation in the pathogenesis of pulmonary arterial hypertension. Several triggers, including general mutation, vascular injury and some stress, has been known risk factors and drivers of PAH pathogenesis. With alterations in epigenetic modification, such as non-coding RNAs dysregulation, histone acetylation and methylation, ATP-dependent chromatin remodeling and DNA methylation, the most common downstream molecular, signaling pathway, ion channels, metabolic, and cytokine production consequences in PAH are changed. As a consequence of this perturbed signaling, there is an increase in PAEC dysfunction, PASMC dedifferentiation, fibroblast and macrophage activation, which is associated with vascular cells proliferation, migration and hypertrophy, Immune cell recruitment and Inflammation, and collagen and ECM deposition. These changes drive vasoconstriction and both inward and outward vascular remodeling. This culminates in vasoconstriction and remodeling of the pulmonary vessels, which can result in increased pulmonary vascular resistance and right ventricular afterload, eventually leading to right heart failure and death. ECM extracellular matrix, PAEC pulmonary artery endothelial cells, PASMCs pulmonary artery smooth muscle cells

High levels of HDAC expression and activity are found in PAH, and research has shown the potential of HDAC inhibitors in repressing the development of PAH via induction of anti-inflammatory and anti-proliferative effects. Some recent studies have also shown that effectiveness of the HDAC inhibitors valproic acid (VPA) and suberoylanilide hydroxamic acid(SAHA)in hypoxia-induced models of PAH, is sufficient to support their development as a therapeutic strategy in PAH patients [122, 123]. This efficacy is based on their abilities to inhibit the highly proliferative phenotype of fibroblasts and PASMCs and down-regulate the expression of inflammatory cytokines, such as MCP-1 and interleukin-6 (IL-6). Coincidentally, both Lan group [132] and Chen group [126] showed that daily administration of VPA therapy prevented and partially reversed the development of combined MCT- and chronic hypoxia-induced severe PAH in rats, and decreased inflammation and proliferation in the remodeled pulmonary arteries. Class I HDAC inhibitors (MGCD0103) have also been shown to be capable of reducing pulmonary arterial wall thickening and maintained right ventricular function [121, 127]. Furthermore, selective inhibition of class IIa HDACs has been shown to rescue PAH by decreasing PAECs migration and proliferation of PAECs [107]. The information for the particular inhibitors of HDACs discussed herein has been summarized in Table 2.

Table 2.

Inhibitors of HDACs involved in PAH development

Inhibitors	Targets HDACs	Function	Targets genes/proteins and Signaling pathway	Models	References
Apicidin	Class I	Inhibit fibroblast and monocytes/macrophages activation; attenuate right ventricular hypertrophy and pulmonary vascular remodeling	Inhibit pro-inflammatory cytokines and pro-fibrogenic mediators (TIMP1) expression; Inhibit IGF-1/pAKT signaling;	Human-PAH Rat-hypoxia Neonatal mouse-hypoxia Fibroblasts, PASMC and PAEC	[124] [125]
MC1855	Class I	Inhibit PASMC proliferation and migration	Inhibit Akt Phosphorylation and Cyclin D1 expression	Rat PASMC-PDGF	[122]
MC1568	I Class IIa	Inhibit PAEC proliferation and migration	Increase MEF2-miRNA-424/503, connexins 37/40, and Klf 2 and 4	Rat-MCT, sugen5416/hypoxia PAEC	[107]
MGCD0103	Class I	Inhibit PASMC proliferation pulmonary vascular remodeling	Increase FOXO3a, P27;SOD3	Rat-hypoxia PASMC-hypoxia	[121] [127]
SAHA	Class I, II and IV	Inhibit fibroblast and PASMC proliferation and inflammation	Increase p21 and FOXO3 levels, reduced the expression of survivin and cytokines	Human-PAH Rat-hypoxia Bovine vascular fibroblasts, PASMC-PDGF	[123] [124]
Scriptaid	Class I, II and IV	Reduce PAH	Reduce Nox transcription and ROS production	Rat-MCT HLF and HLMVEC	[126]
VPA	Class I	Inhibit fibroblast and PASMC proliferation and inflammation	Increase p21 and FOXO3 levels, reduced the expression of survivin and cytokines	Human-PAH Rat-hypoxia, MCT Bovine vascular fibroblasts, PASMC-PDGF	[123] [132]

Drugs: MCT, monocrotaline; SAHA, suberoylanilide hydroxamic acid; TSA, trichostatin A; VPA, valproic acid

Cell types: PAEC, pulmonary artery endothelial cells; PASMCs, pulmonary artery smooth muscle cells; HLF, human lung fibroblasts; HLMVEC, Human lung microvascular endothelial cells

Target genes/proteins: FOXO, forkhead box O; GF-1, insulin growth factor 1; KLF4, Kruppel-like factor; MEF2, myocyte enhancer factor; Nox, NADPH oxidase; p21, cyclin-dependent kinase inhibitor 1; p27, cyclin-dependent kinase inhibitor 1B; PDGF, platelet-derived growth factor; ROS, reactive oxygen species; SOD, superoxide dismutase; TIMP1, TIMP metallopeptidase inhibitor 1

Findings from recent studies are supportive for the clinical application of HDAC inhibitors as PAH treatment, and the underlying mechanism appears to involve regulation of Tregs in particular [149–151]. A study of this Treg-related protective mechanism against PAH conducted by Chu et al. [152] indicated that the mechanism is further associated with suppression of the inflammatory response, inhibition of (human) PASMC proliferation and regulation of the cell cycle. Yet, other studies have yielded some troubling findings, showing potential detrimental effects of therapeutically applied HDAC inhibitors, particularly on the right ventricle and in decreasing normal angiogenesis [153]. Thus, the therapeutic role of HDAC inhibitors in PAH remains controversial. Based on these studies; however, we can speculate that selective HDAC inhibitors might be beneficial for PAH if they can be delivered directly to the lungs, thereby avoiding the detrimental right ventricle effects.

Interestingly, miRNA mimics and antagonists (known as antogomiRs) have recently entered into clinical trials for patients with liver cancer [154] and hepatitis C [155]. Some mimics and antagomiRs have recently been applied to experimental PAH models [156, 157], and in one of those studies miR-204 expression rescue of the PAH-reduced miR-204 was shown to reverse the disease in rats [83]. Treatment with nebulized miR-140-5p mimic also prevents the development of experimental PAH in rats [56]. Similarly, inhibition of miR-17, which is up-regulated in PAH rats, was shown to improve PAH [89], antagomiR-135a injected into mice showed to reverse PAH [55] and anti-miR-199a-5p [31] and anti-miR-27b [41] were shown to overcome the PAH-related significant increase and promote the NO level and decrease the pulmonary artery pressure and the extent of right ventricular hypertrophy. Thus, while numerous studies have provided clear demonstrations of miRNAs’ association with the development of PAH, the clinical utility of these findings currently remains unclear. Nonetheless, the collective findings put forth the promise of RNAi therapy for PAH and support its ongoing investigation.

Conclusion and perspectives

The past two decades of PAH research have yielded a better understanding of the pathophysiological processes and new therapies that have emerged, but the effective prevention of PAH remains an unmet objective and diagnosis still occurs largely at the late stage and relies on invasive methods. The physiological imbalances, resulting from stress, are linked with non-homeostatic responses in gene regulation that occur via epigenetic modification. A significant amount of research studies have examined, and begun to elucidate, the myriad roles of miRNAs and HDACs in the development of PAH; however, other histone modifications, such as methylation and phosphorylation, as well as DNA methylation and mitochondrial epigenetics should be studied in the same manner, as they are likely to be important contributors to the pathogenesis of PAH and may represent manipulable targets of therapy. While some studies have already demonstrated that restoration of miRNA expression and inhibition of HDACs can reverse PAH in different animal models, no epigenetic-based therapy for PAH has yet reached clinical or preclinical development. There is a lacuna between the extent of findings from animal studies and those from humans, especially in relation to therapeutics, and further research is needed to fully understand the epigenetics of PAH. In all, epigenetic regulation presents a flicker of hope for new therapeutic strategies and targets of PAH in the future.

Compliance with ethical standards

Conflict of interest

The authors have declared that no conflict of interest exists.