Epigenetic inheritance underlying pulmonary arterial hypertension: a new challenge for network medicine

Abstract

In pulmonary arterial hypertension (PAH), the Warburg effect (glycolytic shift) and mitochondrial fission are determinants of phenotype alterations characteristic of the disease, such as proliferation, apoptosis resistance, migration, endothelial-mesenchymal transition, and extracellular matrix stiffness. Current therapies, focusing largely on vasodilation and antithrombotic protection, do not restore these aberrant phenotypes suggesting that additional pathways need be targeted. The multifactorial nature of PAH suggests epigenetic changes as potential determinants of vascular remodeling. Transgenerational epigenetic changes induced by hypoxia can result in permanent changes early in fetal development increasing PAH risk in adulthood. Unlike genetic mutations, epigenetic changes are pharmacologically reversible, making them an attractive target as therapeutic strategies for PAH. This review offers a landscape of the most current clinical, epigenetic-sensitive changes contributing to PAH vascular remodeling both in early and later life, with a focus on a network medicine strategy. Furthermore, we discuss the importance of the application (from morphogenesis to disease onset) of molecular network-based algorithms to dissect PAH molecular pathobiology. Additionally, we suggest an integrated network-based program for clinical disease gene discovery that may reveal novel biomarkers and novel disease targets, thus offering a truly innovative path toward redefining and treating PAH, as well as facilitating the trajectory of a comprehensive precision medicine approach to PAH.

Introduction

Pulmonary arterial hypertension (PAH) is a severe, progressive, and incurable vascular disease that occurs in several forms: idiopathic, heritable, drug-induced, and associated with other conditions, such as connective tissue diseases, congenital heart disease, portal hypertension, and HIV or schistosomiasis.^1–3 PAH is clinically heterogeneous and is characterized by progressive dyspnea, an increase in right ventricular (RV) afterload and dysfunction, RV-pulmonary artery uncoupling, and right-sided heart failure, ultimately leading to circulatory collapse.^1–3 Contemporary registry data (except COMPERA) indicate that the average age of PAH patients at time of diagnosis is in the early 50 years.

During the initial stage of PAH, the symptoms (e.g., difficulty breathing, fatigue) are common to many other medical conditions, often resulting in a delayed diagnosis until more severe symptoms arise.⁴ Despite advances in understanding pathways underlying PAH, the mortality rate remains high (15% at 1 year).² Endothelial dysfunction is the main hallmark of (early) PAH and reflects complex changes in endothelial cells (ECs), smooth muscle cells (SMCs), and vascular wall fibroblasts (Figure 1). Phenotypic alterations leading to vascular remodeling include: 1) proliferation of ECs to form plexogenic lesions, 2) vasoconstriction, 3) dysregulated angiogenesis, 4) proliferation and migration of SMCs, 5) endothelial-to-mesenchymal transition, 6) thrombosis, 7) extracellular matrix (ECM) stiffening, and 7) inflammation (Figure 1).^5–8 Recently, a metabolic imbalance similar to the Warburg effect was correlated with a cancer-like (hyper-proliferative) phenotype of vascular cells, representing a perturbation of two fundamental pathways, glucose metabolism and mitochondrial oxygen sensing.^9–10 In essence, the Warburg effect is a persistent cellular predilection for glycolysis rather than mitochondrial glucose oxidation despite an abundance of available oxygen. As a result, a state of pseudo-chronic hypoxia (PCH) then shifts the pulmonary vasculature to glycolysis-induced medial hypertrophy while avoiding mitochondrial apoptosis.^9–10

Figure 1: Hallmarks of PAH pathobiology.

Genetic susceptibility, mainly LOF in BMPR2 gene, epigenetic factors, such as DNA methylation, histone modifications, and non-coding RNAs, as well as a variety of environmental risk factors, such as pathogens, ROS, and inflammation are the main trigger factors of the endothelial dysfunction. This letter leads to progressive reduction of vascular tone and arterial remodeling underlying PAH onset. Plexogenic lesions, thrombosis, and fibrosis are the phenotypic hallmarks of right ventricular failure.

Current therapy targeting endothelial dysfunction involves five classes of drugs, prostanoid analogues, non-prostanoid IP receptor agonists, endothelin receptor antagonists, phosphodiesterase type 5 inhibitors, and soluble guanylyl cyclase stimulators, resulting in beneficial effects in many patients and/or leading to adverse or intolerable side effects in others.^11–13 The multifactorial nature of PAH indicates that epigenetic factors can bridge genetic (e.g., bone morphogenetic protein receptor type 2 gene, BMPR2) and environmental (oxidative stress, and inflammation) risk factors for disease.^14–19 Epigenetic mechanisms refer to heritable traits that modify gene expression without changing DNA sequence and strongly contribute to the pathogenesis of many cardiovascular diseases.^15,20−25 The main classes of epigenetic regulators are: DNA methylation, histone modifications, mRNA methylation, and a variety of noncoding RNA modifications (Figure 2).²⁶ Epigenetic information (epigenome) is transferred through both mitotic cell-to-cell and meiotic transgenerational inheritance, the latter thereby allowing the effects to persist for multiple generations.^20,25 The epigenetic hypothesis for PAH pathogenesis is supported by the behavior of human PAECs that maintain their in vivo characteristics (apoptosis-resistance and hyperproliferation) when placed in culture.²⁷ Additionally, epigenetic mechanisms can underlie the transgenerational transmission of environmentally acquired PAH risk factors, such as sensitivity to hypoxia.^28,29 Both genetic and epigenetic-sensitive alterations deregulate the network-based molecular architecture at the cellular level, thus leading to the pathogenesis of complex cardiovascular diseases.^15,20,25 Network medicine, an approach designed to understand human diseases from a molecular and phenotypic network point-of-view, is emerging from combining omics sciences, deep phenotyping, and potent quantitative algorithms, which can aid in identifying specific disease modules in the human interactome in order to improve diagnosis and personalized therapy.^30–36 This review is aimed at updating and discussing the most recent mitotic and meiotic clinical epigenetic pathways underlying PAH onset and their impact as discerned by network medicine analysis. Furthermore, we suggest an integrated program for clinical disease gene discovery, including network-based tools (cf. Haghighi et al.³⁷), in order to reveal new candidate genes that might be translated into diagnostic, prognostic, and predictive biomarkers, as well as drug targets for PAH precision medicine.

Figure 2: Basic epigenetic mechanisms.

The Figure shows the major epigenetic modifications: DNA methylation, histon modifications and RNA-based mechanisms such as microRNAs. The effect of epigenetic changes is to influence gene expression without modifying DNA sequence by remodeling the chromatin structure and/or targeting RNA messengers.

Epigenetic-sensitive clinical challenges in PAH primary prevention

To date, there are many pitfalls in the early diagnosis of PAH because many signs and symptoms do not appear until months and, at times, years after the onset of the disorder owing to the functional reserve of the pulmonary vasculature.³⁸ Indeed, it requires loss of 50–60% of the pulmonary vasculature before developing symptoms of the disorder, whereas loss of 50% of the vasculature does not result in pulmonary hypertension if the remainder of the vasculature is normal. Additionally, other pulmonary and cardio-pulmonary diseases, such as chronic obstructive pulmonary disease (COPD), heart failure, and asthma, mimic the signs and symptoms of PAH, thus complicating the differential diagnosis.³⁸ Imaging techniques, such as X-ray, computed tomography (CT), magnetic resonance imaging (MRI), as well as genetic screening, and echocardiography, help to identify PAH patients; however, invasive right heart catheterization remains the only method for diagnosing of the disease precisely.²⁸ Owing to the high mortality rate after diagnosis, there is clearly a need for innovative clinical experimental designs able to dissect the complex relationships among genetic background, epigenetic state, and environmental exposures in primary prevention of PAH. In fact, there is no approved therapy for use in primary prevention. Recently, the application of next-generation sequencing (NGS) platforms has substantially accelerated epigenomic research in this field, thus suggesting novel opportunities for both early and non-invasive detection of at-risk subjects. DNA methylation, histone modifications, mRNA modification, and noncoding RNA modification are the epigenetic regulators that modify chromatin and RNA affecting local transcription via selective expression or repression of specific molecular pathways.^15,21,26

In the past few decades, many investigators have demonstrated epigenetic tags as crucial players in vital cellular processes such as cell differentiation, morphogenesis, genomic imprinting, variability, and adaptability.²¹ Furthermore, epigenetic tags can be altered by inherited and acquired factors leading to different complex human disorders.^15,20–25 The epigenome is specific to the spatio-temporal status of an organism, but it is also dynamic and reversible responding to environment changes, such as nutrition, stress, toxins, exercise, and drugs.²¹ Unlike genetic mutations, epigenetic changes, and in particular DNA methylation, are pharmacologically reversible.²¹ This feature has encouraged many researchers to focus on epigenetic drugs (epidrugs) as a cornerstone in managing many cardiovascular disorders, including PAH.^{15,21- 25} Additionally, the epigenetic bases of selected disorders may provide novel biomarkers able to identify subjects prone to develop PAH at a very early stage, thus allowing timely and efficacious primary prevention.

Transgenerational epigenetic inheritance

Starting from the fetal origins of adult disease (FOAD) hypotheses, proposed by David Barker³⁹ and known as “fetal programming,” there is a growing evidence of the major role played by epigenetic factors during intrauterine life and the perinatal period.^39,40 In essence, the term “fetal programming” refers to a strong relationship between impaired fetal growth (lower birth weight) and an increased incidence of endothelial dysfunction, high blood pressure, dyslipidemia, and impaired glucose tolerance.³⁹ In accordance with this hypothesis, an altered in utero environment during sensitive periods of development may alter fetal physiological adaptations, thus increasing susceptibility to cardiovascular diseases in infancy and even in adulthood.^20,25,39 The late onset of such diseases in response to earlier transient insults indicated that developmental programming may have a key epigenetic component providing persistent memory of earlier environmental states. In accordance with developmental origin of PAH, maternal risk factors, such as preeclampsia, hypertension, obesity, and placental dysfunction, in the setting of epigenetic changes, lead to sustained disruption of vascular signaling pathways and growth, thereby increasing the risk for late-onset PAH.²⁸ Additionally, these environmental stressors may accelerate the decline of lung vascular bed density, thus shortening the timing of PAH onset.²⁸

Fetal or perinatal triggers of late-onset pulmonary hypertension have been demonstrated in humans. Early clinical evidence arose from a case-control study in which a transient hypoxic insult to the pulmonary circulation during the perinatal period led to a persistent vascular abnormality (likely a loss of nitric oxide synthesis) predisposing to a pathological high altitude-induced vasoconstrictor response in young healthy adults.²⁹ This evidence suggests that further investigation in survivors of perinatal PAH might reveal a high risk of developing this disorder later in life owing to transgenerational epigenetic inheritance.

Mitotic epigenetic inheritance

Here, we focus on the major, current epigenetic-sensitive mechanisms induced by environmental exposures during PAH onset. DNA methylation is one of the best studied epigenetic mechanisms generally associated with gene silencing.⁴¹ Transcription of DNA is repressed by the covalent addition of a methyl group at the C5 position on cytosine bases by DNA methyltransferase enzymes (DNMTs including DNMT1, DNMT3A, and DNMT3B). Methylation sterically hinders the binding of macromolecules for messenger RNA synthesis, thereby preventing gene expression.⁴¹ Mounting data suggest that an aberrant DNA methylation profile plays a key role in PAH vascular remodeling.^42–45

Using cultured PAECs from idiopathic (IPAH), heritable (HPAH), and control patients in another whole genome DNA methylation assay yielded somewhat different results.⁴² Although of different etiologic origins, data reported similar methylation profiles and similar gene expression patterns in both IPAH and HPAH compared to controls; however, significant DMRs mapped to the ATP-binding cassette 1 (ABCA1), adiponectin (ADIPOQ), and apolipoprotein A4 (APOA4) genes were detected comparing PAH patients and controls.⁴² In particular, ABCA1 was the major hypermethylated DMR suggesting that cholesterol metabolism is closely associated with PAH (Figure 3).⁴² In addition, ABCA1 downregulation could be a useful biomarker with which to discriminate PAH patients from controls.⁴²

Figure 3: The role of epigenetic memory for the induction of arterial remodeling in PAH.

Epigenetic changes represent the means by which environmental factors interact with the genome to alter gene expression. Following the persistence of pathological stimuli, epigenetic memory triggers stimulus-independent cancer-like alterations in ECs, SMCs, and Fs represented by aberrant transcription of pro-proliferative, pro-migratory, pro-ECM stiffness, and anti-apoptotic genes. This phenotypic switch of pulmonary vascular cells largely contributes to the development of complex vascular lesions during the progressive vascular remodeling process in PAH. Hyper-methylation of ABCA1, BMPR2 contributes to block vascular cells in mitotic phase. By increasing acetylation of PGC-1α, SIRT-1 HDAC down-regulation reduces mitochondrial mass and sustains cell proliferation. Hyperacetylation of extracellular CypA, as well as down-regulation of anti-oxidant SOD3 via class I HDAC3 up-regulation, contribute to oxidative stress underlying PAH phenotype. Up-regulation of HDAC4-5 by down-regulating MEF2 adds proliferative stimuli. Downregulation of circulating miR-1281, miR-124, miR-34a-3p, and lnc-RNA MEG3 plays a key role in the development of the proliferative phenotype. All of these epigenetic-sensitive changes might also contribute to transgenerational inheritance, in accordance with the developmental origins of PAH.

Heterozygous loss of function (LOF) mutations harbored in the BMPR2 gene are the most important genetic risk factors for HPAH; however, they show an unexplained low penetrance.⁴³ By bisulfite sequencing, the DNA methylation profile of the BMPR2 promoter was performed on peripheral blood mononuclear cells from 12 HPAH patients and their unaffected relatives (controls).⁴³ Data showed that the BMPR2 promoter was hypermethylated in HPAH patients compared with controls (Figure 3). In particular, the wild-type allele showed significantly greater methylation compared with the aberrant allele.⁴³ This evidence suggested that the environment might promote hypermethylation of the wild-type allele, altering penetrance of LOF mutations.⁴³ In contrast, prior case-control studies reported no evidence of differential DNA methylation in PAH patients compared with controls.^44,45 No studies, to date, have investigated methylation patterns over time in patients with PAH or at risk for PAH.

Histone modification, including post-translational changes such as acetylation, methylation, phosphorylation, ubiquitination, and sumoylation, affects specific amino acid residues of histone tails; however, these mechanisms can also regulate gene expression by altering non-histone proteins.⁴⁶ The best studied of these reversible modifications are acetylation and methylation, which modify specific lysine residues on the amino-terminal tails of H3 and H4 histones and play crucial roles both in chromatin structure modeling and gene transcription. Histone acetylation and deacetylation are catalyzed by histone acetyl transferases (HATs) and histone deacetylases (HDACs), respectively.⁴⁶ In mammals, the major class of known HATs are Gcn5, P300, CBP, and PCAF, whereas HDACs are classified in four groups: class I, including HDAC 1, 2, 3, and 8; class II, including HDACs 4, 5, 7, 9, 6, and 10; class III, comprising the sirtuin (Sirt) family; and class IV, including HDAC 11. As compared to other classes, only the Sirt family requires a specific cofactor for activity, NAD+.⁴⁶ In response to certain specific stimuli, HATs can add acetyl groups to histone tails leading to a reduction in the positive charge and relaxed chromatin structure (open state); this latter change allows binding of transcriptional machinery, in order to promote gene expression.⁴⁶ By contrast, HDACs can remove certain acetyl groups resulting in a more closed chromatin conformation, which does not allow the binding of transactivator factors, thereby repressing gene expression.⁴⁶ The coordinate regulation of HATs and HDACs establishes the level of histone acetylation and, hence, contributes to the regulation of gene expression.⁴⁶ The final effect of histone methylation varies according to the specific methylated residue (lysines or arginines) and the number of added methyl groups.⁴⁶ Overall, they form a “histone code” that can dictate the transcriptional state of a gene.

A recent study reported that a failure of the silent mating type information regulator 2 homolog 1 (SIRT1) activity leads to the Warburg effect by increasing the amount of acetylated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in cultured PASMCs from PAH patients compared to controls.⁴⁷ Additionally, treatment with pharmacological activators of SIRT-1 by decreasing acetylated PGC-1α levels may counteract the hyperproliferation of PAH PASMCs, suggesting a promising therapy for the disease.⁴⁷

Using human PAECs, a persistent form of extracellular acetylated cyclophilin a (AcK-CYPA) could induce a PAH phenotype by increasing apoptosis, oxidative stress, and pro-inflammatory signals, such as vascular cell adhesion molecule 1 (VCAM1), intercellular adhesion molecule 1 (ICAM1), and nuclear factor kappa B (NF-KB) genes (Figure 3).⁴⁸ Furthermore, by using the cyclosporine A derivative CypA inhibitor MM284, targeted inhibition of AcK-CYPA was strongly associated with a lower rate of caspase-3/7-mediated apoptosis, inflammation, and oxidative stress both in ECs and VSMCs.⁴⁸ This preliminary observation suggests that reduction of AcK-CYPA levels could serve as a novel antioxidant strategy counteracting endothelial dysfunction; however, this issue deserves further investigation in in vivo models.⁴⁸ Using human lung tissue, downregulation of SOD3 via higher levels of class I HDAC3 was reported in PAH patients compared with controls (Figure 3).⁴⁹ Similar evidence was found by analyzing PASMCs isolated from the same case-control study.⁴⁹ In addition, cultured PASMCs treated with trichostatin A, a class I HDAC inhibitor (HDACi), showed increased expression of SOD3 and reduced cell proliferation in PAH patients compared with controls.⁴⁹ These data indicate SOD3 as a molecular mediator regulated by HDACi, and suggest a potentially useful novel anti-oxidant therapy.⁴⁹

ChIP experiments revealed that a cultured human leukemia cell line (HL-60) treated with multiple HDACi (scriptaid, suberoylanilide hydroxamic acid, trichostatin, and valproic acid) reported low levels of RNA polymerase II, H3K4me3, H3K9ac, and p300 at the NOX promoter.⁵⁰ As a consequence, NOX1,2,4, and 5 isoforms and ROS levels were reduced in a dose-dependent manner, suggesting another route by which to counteract oxidative stress.⁵⁰ Downregulation of the myocyte enhancer factor 2 (MEF2) gene was associated with higher levels of class II HDAC4 and HDAC5 in PAECs from PAH patients compared to controls (Figure 3). ⁵¹ MEF2 is a transcription factor regulating other pulmonary vascular factors, such as microRNA (miR)-424 and miR-503, connexins 37 and 40, and krűppel like factors 2 and 4 (KLF2 and 4), involved in cell cycle progression.⁵¹ Treatment of PAECs with selective siRNA inhibiting class II HDACs led to restoration of MEF2 activity decreasing cell migration and proliferation.⁵¹

Micro-RNAs and long-non coding RNAs

A recent study has reported a novel regulatory axis involving phosphatidylinositol 3‐kinase (PI3K)-DMT1-miR-1281-HDAC4 participating in platelet‐derived growth factor BB (PDGFBB)-induced proliferation and migration of human PASMCs.⁵² Overexpression of DNM1 has been associated with hypermethylation of MIR-1281 and higher downstream levels of HDAC4 activating KLF 2 and 4, CCAAT/enhancer-binding protein homologous protein (CHOP), tribbles homolog 3 (TRB3), drosophila mothers against decapentaplegic protein 3 (SMAD3), vascular endothelial growth factor (VEGF), and glucose transporter-1 (GLUT-1) genes involved in PASMC remodeling.⁵² Lower levels of MIR-1281, then, could be a potential biomarker as well as a therapeutic target for PAH (Figure 3).⁵²

In ECs and fibroblasts, downregulation of miR-124 was correlated with overexpression of splicing factor polypyrimidine tract binding protein (PTBP1) in PAH patients respect with controls (Figure 3).^53–54 PTBP1 induced higher levels of pyruvate kinase muscle 2 (PKM2) as the key isoform producing pyruvate and ATP. Thus, the miR124-PTBP1 axis likely contributes to the Warburg effect, suggesting a target for innovative therapies.^53–54 Moreover, a link between the downregulation of miR-34a-3p and upregulation of mitochondrial dynamics protein of 49 and 51 kDa (MiD49 and MiD51) in both human PASMCs and PAECs from PAH patients was observed compared with controls (Figure 3).⁵⁵ MiD49 and MiD51 are crucial adaptor proteins for fission-mediating GTPase dynamin-related protein 1 (DRP1) activity, an essential regulator of mitochondrial fission (division).⁵⁵ Thus, the miR-34a-3p-MiD axis accelerating mitotic fission promotes a cancer-like phenotype and suggests further useful biomarkers for diagnosis and treatment of PAH.⁵⁵ As reported, miRNA studies in PAH have not been impressive owing in part to the wide variety of experimental conditions used by different investigators making comparisons and interpretation difficult. Based on the quality of the scientific studies of miR-1281, investigated by Li et al.⁵², we propose that this miRNA alone may be suitable for further observation at this time.

Downregulation of lncRNA maternally expressed gene 3 (MEG3) associated with overexpression of TP53 led to a more marked proliferative and migratory phenotype of human PAH PASMCs compared to controls (Figure 3).⁵⁶ Thus, MEG3 may also be a reasonable target for drug development for treating PAH.⁵⁶ Although there is no experimental evidence, the transgenerational effect of all these epigenetic changes has not been excluded; putative vertical transmission of such imprints may enable early intervention strategies to improve diagnosis and for primary prevention of PAH.

Precision medicine: from the application of omics sciences to network medicine in PAH

As most molecular factors exert their functions by interacting with other cellular components (interactome), any disease is rarely a consequence of a single gene defect; rather it likely reflects a perturbation of the complex molecular interaction network that governs the phenotype.^35,36 Innovative omics platforms are potent research tools with which to identify new molecular and clinical biomarkers, which may offer a more accurate diagnosis, early identification of patients at high risk, as well as targeted therapies and predictors of drug response for PAH (Figure 4).^57–60 However, there are still great challenges in translating omics findings in PAH clinical management owing to the low number of patients studied and a potentially high false discovery rate.⁵⁸ Thus, as integrative systems biology approach is absolutely necessary to discover novel alterations from genotype to phenotype levels, ideally defining the causative basis linking candidate genes and diseases (Figure 4); however, a large amount of data needs to be interpreted better before translating these concepts to the clinical setting. To achieve this aim, computational algorithms of large, system-wide, quantitative data sets are used to generate relevant molecular interaction pathways, such as metabolic and cell signaling networks.^32,34–36 In the era of network medicine, the analysis of topological properties of protein-protein interactions (PPIs), regulatory as well as co-expression networks can guide researchers into mapping biologically crucial molecular interconnections, which may subsequently be validated in biological samples (Figure 4).^32,34–36 All of these networks can be viewed as maps where disorders are represented with localized perturbation within a specific module (or pathway) of the interactome.^32,34–36 In essence, a network is a set of nodes and edges wherein the nodes are linked if there is a significant physical or functional interaction between them. In PPI networks (e.g., GenePanda, DIAMOnD, PRINCE, PRODIGE), the nodes are proteins that are linked to each other by physical interaction; in regulatory networks (e.g., MMI-Network and PANDA), the directed links represent regulatory relationships between a transcription factor and a gene; in co-expression networks (e.g., SWIM and WGCNA), genes with similar co-expression profiles are linked (Figure 4).^32,34–36 The final goal is both to increase the global knowledge of the interactome-related perturbations resulting in diseases and to translate computational data into real clinical applications.^32–36

Figure 4: A systems biology approach: from omics science to network-based tools in precision medicine.

Omics sciences offer high-throughput platforms monitoring diabetes onset at molecular level. Genomics (NGS, GWAS, DNA Chip) allows the whole genome or selected gene sequencing; transcriptomics (RNA Chip, RNA-seq) catalogues coding and non-coding RNAs; proteomics (immune assay, MS, protein CHIP) explores structure, function, and interactions of proteins, and epigenomics (MNase-seq, Dnase-seq, ATAC-seq, bisulfite sequencing, EWAS, e.g., meQTL, ChIP) investigated the complete set of chromatin modifications in a particular cell, tissue, or organism. The emerging bioinformatic network-based tools explore systematically PAH-associated modules and pathways to unravel novel candidate genes, the biological significance of PAH-associated polymorphisms, as well as drug targets and biomarkers. The main used algorithms are: 1) PANDA, Genomica (regulatory), and WGCNA (co-expression), at genomic level; 2) WGCNA (co-expression), at trascriptomic level; GenePanda, DIAMOnD, PRINCE, PRODIGE (PPIs), at proteomic level; MMI-network and IPA (regulatory), at epigenetic level. The challenge of network medicine is translate acquired knowledge into clinical practice in order to obtain a more accurate diagnosis, targeted treatment, and personalized therapy, thus ameliorating primary and secondary prevention of PAH.

A recent study involved an innovative network-based strategy to identify key genes regulated by aldosterone (ALDO) offering an explanation for the shift from adaptive to pathogenic fibrosis phenotypes.³⁰ In essence, proteins encoded by fibrosis genes have been mapped to the well-established human protein-protein interactome and the betweenness centrality (BC) analyses have emphasized the connectivity involving ALDO-related genes.³⁰ As a result, the SMAD3 target neural precursor cell expressed developmentally down-regulated 9 (NEDD9) is a critical node regulated by ALDO underlying the pathogenicity of enhanced collagen synthesis and resulting in (peri-vascular) fibrosis in PAH.³⁰ This finding suggests that NEDD9 ablation or inhibition may prevent fibrotic vascular remodeling and PAH onset.³⁰

Candidate regulatory targets for iPAH diagnosis have also been explored by performing a weighted gene co-expression analysis (WCGNA) algorithm and module-trait correlations based on a public microarray data set (GSE703).³¹ From this analysis, the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta (YWHAB) gene, playing a role in both mitogenic signalling and cell cycle pathways, was overexpressed and strongly associated with iPAH progression. This finding indicates that YWHAB might be a useful biomarker and therapeutic target for iPAH.³¹

The impact of network-based approaches to primary prevention and diagnosis of PAH

Despite increasing progress in epigenomics, the field remains far from providing an understanding how, when, and where environmental insults perturb key epigenetic-sensitive mechanisms leading to PAH onset. It is well accepted that environmental stressors occur in utero; however, they have received little attention as predictors of disease in later stages of life. Thus, it is necessary both to identify innovative biomarkers and to monitor their variations at the fetal-perinatal stage, as well as during childhood, in older age, and over several generations (Figure 5). This comprehensive staging approach may provide earlier identification and management of at-risk subjects before the development of irreversible PAH, including in the neonatal and pediatric setting. For this reason, future PAH studies would greatly benefit from combining omics sciences and network-based tools to permit disease staging: 1) asymptomatic status despite increased genetic risk or structural abnormalities; 2) symptomatic status at time of diagnosis; and 3) patients with severe or end-stage disease. This schema suggests that implementation of empiric data from each stage of the illness could have important implications for improving the timing of the PAH diagnosis and its primary prevention (Figure 5). Collectively, these advances set the framework for a new and potentially transformative era in PAH in which preventive medicine and patient-specific therapies are a principal emphasis.

Figure 5: Primary prevention of PAH through genetic and epigenetic network-based approaches.

Genetic individual background is invariable during life, whereas epigenetic influences are dynamic and specific to the spatio-temporal status of an organism. Environmental changes can perturb crucial epigenetic-sensitive pathways involved in morphogenesis leading to PAH onset in later stages of life. In order to ameliorate primary prevention of PAH, it is necessary to combine genomics, epigenomics, proteomics, and network-based algorithms to identify innovative biomarkers mainly at the fetal-perinatal stage, as well as at the post-natal stage, during childhood, and in older age.

An integrated clinical network-based program for the discovery of PAH candidate genes

In this section, we suggest the application of an integrated program for clinical disease gene discovery [after Haghighi A. et al.³⁷], that might reveal novel PAH-associated genes (Figure 6). This workflow includes a computational pipeline (step 1), upstream analysis (step 2), and downstream analysis (step 3) with three major goals: 1) to discover robust genetic and epigenetic biomarkers associated with vascular remodeling, 2) to provide novel diagnostic and treatment options for patients, and 3) to promote clinical implementation of genomic medicine (Figure 6). The computational pipeline has four main stages for biomarker discovery: sample preparation, large-scale platforms, bioinformatics, and biomarker validation (terminating in downstream analyses).

Figure 6: An integrated clinical program for discovery of PAH-associated candidate genes.

TThis diagnostic research program, modified from Haghighi et al., (2018), offers an integrated approach to evaluate novel candidate genes in PAH onset and progression. The computational pipeline indicates all processing steps from sample preparation to biomarker validation by bioinformatic approaches. The second step is represented by upstream analysis that validate and prioritize candidate PAH-sensitive genetic and epigenetic variants. The third step includes downstream analysis that are designed to establish the causative effect between variants in candidate genes and PAH. Overall, the main goal is to integrate the findings about gene causality with the clinical management of PAH.

The upstream analyses are designed to produce validated and prioritized candidate genes, whereas the downstream analyses evaluate the possible causal effect between candidate genes and pathway determinants of PAH onset (Figure 6). In this way, the pipeline connects biomarker discovery with an established approach for evaluation and validation, as well as integrates the findings regarding gene causality with the clinical management plan (Figure 6). To provide an essential scientific background regarding PAH epigenetic-sensitive mechanisms, this strategy should be performed systematically across the lifespan, thus connecting fetal health, epigenetics, and PAH. In particular, there is a clear need for application in asymptomatic children at high-risk of PAH to provide an accurate screening test that may be used to guide strategies for disease prevention or onset delay. Furthermore, prospective clinical studies of children born to PAH mothers could assess the transgenerational influences of disease and evaluate fetal programming mechanisms inferred from preclinical and clinical studies.

Conclusions

The usual idea that signs of PAH are clinically relevant only during adult life and later age is gradually changing. Indeed, increasing evidence supports the concept that PAH is initiated through developmental in utero processes. Epigenetics and other unknown mechanisms underlying these developmental events are yet to be elucidated.²⁸ Epigenetic regulation plays a key role in clarifying the interaction between genes and environment in PAH occurrence. In addition, the plasticity of epigenetic changes may offer more efficacious therapeutic targets, improving current limited PAH management. The epigenetic hypothesis raised evidence of a cancer-like phenotype exhibited by ex vivo cultured PAH vascular cells, and supports the strong impact of environment in PAH onset and progression. As a major participant in disease pathogenesis, PASMCs were crucial to the discovery of novel mechanisms leading to vascular reprogramming. Hypermethylation of CpG islands located in onco-suppressor genes was strongly associated with PASMC hyperproliferation and opened a new field of investigation about the use of antiproliferative and/or oncological drugs in PAH.^42,61 Furthermore, the role of BRD4, a member of the bromodomain and extra terminal domain (BET) family binding acetylated lysine residues, was investigated both in calcification and remodeling processes in PAH-associated coronary heart disease.⁶² Investigators reported the miR-204-induced upregulation of BRD4 in the lungs, distal pulmonary arteries, and PASMCs isolated from PAH patients compared to controls, thus suggesting a crucial role in disease development.⁶² Importantly, in vivo evidence reported that pharmacological inhibition of BRD4 may improve the PAH phenotype by counteracting both vascular remodeling and interleukin-6 expression, thus indicating a potentially useful therapeutic strategy.⁶² Several lines of evidence also report that some epigenetic-sensitive mechanisms may have a useful role as drug targets for the design of novel anti-oxidant strategies in PAH management; however, trials of antioxidants based on the ASK-1 inhibitor (NCT02234141) was unsuccessful, and those of bardoxolone (NCT02036970 and NCT03068130) have not been impressive.

Although basic research clarified additional epigenetic pathways underlying PAH, translation of these studies into clinically useful biomarkers and new therapeutic agents remains slow, with no clear path as yet by which to move a drug or biomarker from the bench to the bedside. In the past decades, the dominant goal of researchers was the identification of single molecular defects that represent the cause of disease as well as select biomarkers for that disease, identifying risk and resilience factors, providing early detection, predicting clinical outcome, and specifying targets for trajectory-altering therapeutics. This reductionist approach has been somewhat successful and responsible for most of drugs currently used in PAH management. However, given the complexity of human disease, this single-gene centered approach led to many ineffective treatments and many weak biomarkers, the former with a large spectrum of adverse side effects, as in PAH. In the era of network medicine, a systematic approach focuses, rather, on investigating pathway-based causes of disease by analyzing many different types of networks: from the cellular-molecular level of protein-protein interactions to correlational studies of gene expression in biological samples.^32,33,35,36 Using potent genomic tools as well as biostatistics, bioinformatics, and dynamic systems analysis, both genetic and epigenetic-sensitive networks can now be explored in an integrative context opening up a new scenario for uncovering causes and identifying putative biomarkers for PAH prevention and treatment.

Highlights.

• Epigenetic changes can underlie the mitotic/meiotic inheritance of environmentally acquired risk factors for PAH.

• Epigenetic-sensitive alterations can deregulate the interactome flow leading to the vascular remodeling in PAH.

• The network-based analysis of the interactome might reveal the key hubs underlying PAH onset and development.

• Network medicine is a novel paradigm that may amplify opportunities for PAH personalized therapy.

Abbreviations

ABCA1

ATP-binding cassette 1

ATAC

assay for transposase accessible chromatin

BMPR2

bone morphogenetic protein receptor type 2

CYPA

cyclophilin a

DIAMOnD

DIseAse MOdule Detection

EC

extra-cellular

ECs

endothelial cells

ECM

extra-cellular matrix

EWAS

epigenome-wide association study

Fs

fibroblasts

GWAS

genome wide association study

HDAC

histone deacetylase

Lnc-RNA MEG3

long non-coding RNA maternally expressed gene 3

MEF2

myocyte enhancer factor 2

MiR

micro-RNA

MMI-network

miRNA-mediated interactions

MNase

micrococcal nuclease

MT

mitochondria

MS

mass spectrometry

NGS

next generation sequencing

PAH

pulmonary arterial hypertension

PGC1-α

peroxisome proliferator-activated receptor gamma coactivator 1-alpha

PPI

protein-protein interaction

PRINCE

PRIoritizatioN and Complex Elucidation

ProDiGe

Prioritization Of Disease Genes

ROS

radical oxygen species

seq

sequencing

SIRT1

silent mating type information regulator 2 homolog 1

SMCs

smooth muscle cells

SOD3

superoxide dismutase 3

TSS

transcriptional start site

meQTL

DNA methylation quantitative trait loci

ChiP

chromatin immunoprecipitation

PANDA

passing attributes between networks for data assimilation

SWIM

SWItchMiner

WGCNA

weighted correlation network analysis.