Transcription Factors,Transcriptional Coregulators,and Epigenetic Modulation in the Control of Pulmonary Vascular Cell Phenotype: Therapeutic Implications for Pulmonary Hypertension (2015 Grover Conference Series)

FoxO TFs

FoxO TFs belong to a family of transcriptional regulators characterized by a conserved DNA-binding domain termed the “forkhead box.” ³⁶ FoxO proteins mainly act as potent transcriptional activators by binding to the conserved consensus core recognition motif TTGTTTAC.^37,38 FoxOs, when present in the nucleus and bound to promoters that contain the FoxO consensus motif, can act as transcriptional activators and repressors. In mammals, four FoxO isoforms have been identified: FoxO1, FoxO3, FoxO4, and FoxO6. Despite some redundant functions, genetic loss of individual FoxOs does result in specific phenotypes, suggesting divergent roles of FoxO isoforms. ³⁹ FoxOs control various cellular responses, including proliferation, apoptosis, DNA repair, and metabolism, and they have been implicated in vascular structural maintenance.^40,41 FoxO activity is tightly controlled by posttranslational modifications (PTMs), such as phosphorylation, acetylation, methylation, ubiquitination, and binding of protein partners. These modifications alter transcriptional activity, DNA binding, subcellular localization, and protein stability. For example, phosphorylation of FoxO1 at Thr24 and Ser256 create binding motifs for the 14-3-3 scaffolding proteins, causing FoxO1 exclusion from the nucleus and promoting their cytoplasmic ubiquitination and degradation.^37,42

At the 2015 Grover Conference, one of us (SSP) presented evidence that among the FoxO family, FoxO1 is centrally involved in the hyperproliferative and apoptosis-resistant phenotype of pulmonary arterial SMCs (PASMCs), which represents a hallmark of PH. We observed that, in pulmonary vessels and PASMCs of human and experimental PH lungs, FoxO1 expression is downregulated and FoxO1 is inactivated via phosphorylation and nuclear exclusion. Importantly, stimulation of human PASMCs by growth factors and inflammatory cytokines typically involved in the pathogenesis of PH, such as PDGF (platelet-derived growth factor), IGF-1 (insulin-like growth factor), TNF-α (tumor necrosis factor alpha), and interleukin 6, led to downregulation of FoxO1 phosphorylation at Ser256 and Thr24, nuclear exclusion and inactivation, suggesting that FoxO1 is one of the central downstream TFs regulated by several PH stimuli. This postulated central role of FoxO1 in the pathogenesis of pulmonary arterial hypertension (PAH; i.e., group 1 PH) is further supported by in vitro and in vivo loss-of-function studies. ³¹ These data indicate that depletion of FoxO1 specifically in SMCs is sufficient to induce PH and also synergizes with the hypertensive effects of hypoxia, resulting in more severe PH. Reconstitution of PASMC FoxO1 activity by an adenovirus carrying a phosphorylation-deficient FoxO1 mutant reversed the features of PAH both in vitro and in vivo. Transfection of PASMCs with FoxO1 fully normalized growth factor–driven abnormalities in proliferation, migration, and apoptosis in vitro. In monocrotaline (MCT)-induced PAH, inhalation of the adenovirus (after full establishment of disease) markedly reduced RV systolic pressure, pulmonary vascular resistance, and RV hypertrophy—the central clinical features of this disease. In parallel, adenovirus-treated lungs showed reversal of vascular remodeling and normalization of vascular cell hyperproliferation and cell cycle abnormalities to favor the quiescent PASMC phenotype. ³¹

SSP's group also demonstrated that the anti-neoplastic drug paclitaxel has a similar ability to reverse the PAH PASMC phenotype in vitro and the clinical features of established experimental PAH in vivo. Both intravenous and inhaled paclitaxel markedly improved hemodynamics, RV hypertrophy, RV function, and vascular remodeling in various experimental PAH models. The finding that several of these pathways converge on FoxO1 as a central downstream effector molecule in hyperproliferative PASMCs offers new options for therapeutic intervention. The preclinical evidence for the efficacy of this approach will have to be assessed further in clinical trials.

Importantly, understanding of other PTMs on FoxOs, such as acetylation and ubiquitination, in the PH disease setting and the impact of FoxOs on other cellular processes driving PH, such as metabolism and inflammation, is essential. ¹³ In addition, the effects of FoxOs and FoxO-activating therapies directly on RV hypertrophy and failure should be studied.^13,43 Further characterizing the genes and gene networks regulated by FoxO1 in PAH-PASMCs, Savai et al. ³¹ found that FoxO1-regulated genes are associated with cell cycle control (cyclin D1, p27), apoptosis (BCL6, GADD45), and bone morphogenetic protein (BMP) signaling (BMP receptor 2 [BMPR2], ID1, ID3). However, the microarray analysis also revealed changes in several genes that are not prototype target genes of FoxO TFs. An attractive possibility to explain these changes would be that FoxO has effects on chromatin properties more generally. Emerging evidence indicates that FoxO, similar to FoxA, has the capacity to bind to and remodel compacted chromatin. ⁴⁴ Thus, in addition to regulating the expression of specific genes, FoxO PTMs, similar to histone PTMs, may regulate overall chromatin structure and participate in the epigenetic regulation of gene expression, which require deeper investigations.

Hypoxic inducible factors (HIFs)

HIFs are TFs and key regulators of the molecular response to hypoxia. The targets of HIFs include genes controlling vascularization, cellular proliferation, migration, and metabolism.^45,46 HIFs have therefore been strongly implicated in the pathogenesis of various forms of PH. Studies have shown that mice that are homozygous for either of the HIF isoforms (complete deletion of HIF1α or HIF2α is embryonic lethal) exhibit attenuated pulmonary pressures and vascular remodeling after experimental exposure to chronic hypoxia.^16,47 Further, recent studies show that conditional deletion of HIF1α in SMCs ameliorates the degree of remodeling in chronically hypoxic mice.^48,49 In addition, Kapitsinou et al. ⁵⁰ have recently shown that endothelial HIF2α is required for the development of increased PAP in chronically hypoxic mice. These studies strongly support the importance of better understanding the role of HIFs in the pathogenesis of PH, which includes how they interact with other transcriptional factors, transcriptional coactivators, and repressors.

HIFs are heterodimeric TFs composed of an α-subunit (HIF1α, HIF2α, or HIF3α) and a β-subunit (HIF1β, also called the arylhydrocarbon receptor nuclear translocator [ARNT]). HIF is absolutely required for HIF-target gene activation under hypoxia, because knockdown of HIF1α and/or ARNT significantly reduces or completely blocks hypoxic induction of HIF-target genes. However, recent reports demonstrate that other TFs are also required for HIF-target gene expression. These factors have been found to physically interact with HIF1α or HIF2α in transcriptional complexes on HIF-target gene promoters, demonstrating the possibility that some of these factors may be components of what have been termed HIF enhanceosomes. ⁵¹ The finding that HIFs interact with a diverse variety of cotranscriptional activators suggests a mechanism by which hypoxia activates HIF, as well as a number of physiologically and pathologically relevant signaling pathways, to induce HIF-target gene expression.^51–53 In addition, there are examples in which it has been shown that other TFs function to selectively enhance expression of HIF1α or HIF2α target genes, demonstrating that distinct HIF1α or HIF2α enhanceosome complexes exist and are composed of different sets of HIF-specific cotranscriptionally activating factors on the promoters of HIF-target genes.

Incorporation of multiple factors in enhanceosome complexes allows regulation of target gene transcription in a way that is highly sensitive to a variety of stimuli and can result in controlled levels of target gene expression under a variety of conditions. In addition, these complexes can allow for cooperation between factors acting within the same or overlapping signaling pathways and allow for cooperative or synergistic activation of target gene transcription in a context-specific manner (Fig. 1). ⁵¹

It is noteworthy that HIF transcriptional effects are modulated by epigenetic factors described in more detail below. It is now clear that HIF activates its target gene expression by recruiting histone acetylases CBP and p300 via their C- and N-terminal activation domains. ⁵⁴ Of importance in the similarity between many of the identified HIF cotranscriptional activators is their ability to bind and recruit the general coactivator proteins CBP and p300. It has been suggested, in fact, that HIF-dependent CBP and p300 recruitment contributes less to total CBP/p300 bound to HIF-target gene promoters than other coactivators, such as STAT3. These findings raise important questions for the future as to the specific functions of HIFs versus those of coactivators in transcriptional activation of HIF-target genes in PH and a myriad of other diseases including cancer. ⁵¹

Transcription corepressor RBPJ/CSL

The Notch signaling pathway is a versatile regulator of cell fate decisions and plays an essential role for vascular development. Notch signaling is activated upon cell-to-cell contact as a result of interactions between Notch receptors and their ligands (Delta or Jagged). ⁵⁵ Upon ligand binding, the Notch receptors undergo proteolytic processing, which involves ADAM metalloproteases and the γ-secretase complex, resulting in the intracellular domain of Notch (NICD) being released from the cell membrane and shuttling to the nucleus. This process activates the major downstream nuclear target for Notch, the CSL family of DNA-binding factors (CBF1/RBP-Jκ in mammals, Su(H) in Drosophila and Lag-1 in Caenorhabditis elegans). In the absence of active Notch, RBPJ is thought to be primarily a transcriptional repressor that exists in complexes with corepressors. ⁵⁶ When bound to the active NICD, RBPJ recruits a coactivator complex, including a mastermind homolog (MAML1–3 in mammals), activates transcription of genes containing RBPJ binding sites, and drives a complex transcriptional program with phenotypic effects.^57,58

Notch signaling plays a critical role in controlling proliferation and differentiation of PASMCs. Upregulated Notch ligands and Notch3 receptors in PASMCs have been reported to promote the development of pulmonary vascular remodeling in patients with PAH and in animals with experimental PH. ²⁰ At the 2015 Grover Conference, one of us (JY) explained how hypoxia, both acute and chronic, regulates Notch signaling and how hypoxia-mediated Notch signaling regulates cytosolic free Ca²⁺ concentration ([Ca²⁺]cyt).^59,60

Because Notch3 and Notch3 NICD expression is also increased in hypoxia-induced PH (HPH) in mice, JY's group investigated the role of Notch signaling in the responses to acute and chronic hypoxia. Intriguingly, inhibition of Notch signaling with DAPT, a γ-secretase inhibitor (GSI), significantly attenuated the amplitude of acute alveolar hypoxia-induced increase in PAP in isolated perfused/ventilated mouse lungs. In addition, DAPT reversed the development of HPH in mice,^20,60 suggesting that Notch signaling is involved in the responses of the pulmonary vasculature to acute and chronic hypoxia.

Elevated [Ca²⁺]cyt is implicated in stimulating vascular SMC proliferation and inducing vasomotor tone and hence vasoconstriction. In PASMCs, [Ca²⁺]cyt can be increased by Ca²⁺ release from the intracellular stores and Ca²⁺ influx through plasmalemma Ca²⁺ channels. ⁶¹ In PASMCs, increased [Ca²⁺]cyt may occur by activation of various Ca²⁺-permeable channels, including (1) activation of receptor-operated (or ligand-gated) Ca²⁺ channels (ROCs) that are opened, independent of membrane potential, by agonist-mediated activation of membrane receptors; (2) activation of store-operated Ca²⁺ channels (SOCs) that are opened, independent of membrane potential, by depletion of Ca²⁺ from intracellular stores; (3) activation of voltage-dependent Ca²⁺ channels that are opened by membrane depolarization; (4) activation of inositol triphosphate (IP3) receptor– mediated Ca²⁺ release from the IP3-sensitive sarcoplasmic reticulum (SR); and (5) activation of ryanodine receptor–mediated Ca²⁺ release from ryanodine-sensitive SR. Previous work demonstrated that the resting [Ca²⁺]cyt is increased and that expression and activity of ROCs and SOCs are both upregulated in PASMCs isolated from patients with idiopathic PAH (IPAH).^62–64 Furthermore, it was found that extracellular Ca²⁺ induces a large increase in [Ca²⁺] cyt in IPAH PASMCs and functionally upregulated extracellular Ca²⁺-sensing receptor (CaSR) is involved in the enhanced Ca²⁺ influx and proliferation in IPAH PASMCs. ⁶⁵

Along similar lines, in HPH, increased expression of SOCs and enhanced store-operated Ca²⁺ entry (SOCE) was shown to contribute to increased [Ca²⁺]cyt.^64,66 SOCE is known to be important for cell proliferation and vascular remodeling in PH, and studies from JY's lab have demonstrated increased expression of several proteins involved in SOCE, such as canonical transient receptor potential (TRPC) channels (TRPC3, TRPC6), stromal interaction molecule 2 (STIM2), and Orai2, in patients with PH.^62,64,67,68 Understanding the molecular mechanisms that regulate [Ca²⁺]cyt and PASMC proliferation is critical in understanding the pathogenesis of HPH.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological response that optimizes the ventilation/perfusion ratio. ⁶⁹ In order to determine the involvement of Notch signaling in acute HPV, isolated perfused/ventilated mouse lungs and human PASMCs were treated with Notch ligand Jagged-1. Treatment with Jagged-1 resulted in a significant increase in PAP, demonstrating that Jagged-1 can mimic acute hypoxia to cause pulmonary vasoconstriction. In addition, interestingly, both short-term (15–30 min) and long-term (48 hours to days) activation of Notch by Jagged-1 enhances SOCE, which is closely associated with the level of NICD in PASMCs. Importantly, the short and rapid response to activation of Notch signaling in PASMCs can be explained by NICD functional interaction with TRPC6, leading to TRPC6 activation, enhanced SOCE, and an increase in [Ca²⁺]cyt in PASMCs. Moreover, employing several genetic and pharmacological approaches, Sylvester and colleagues ⁶⁹ elegantly demonstrated that TRPC6 is required for SOCE and acute HPV in mice and that inhibition of Notch signaling markedly attenuates hypoxia-enhanced SOCE and acute HPV. However, the marked decrease of acute HPV mediated by Notch inhibition is not observed in TRPC6-deficient (TRPC6^−/−) mice, confirming the noncanonical form of Notch signaling, that is, NICD-TRPC6 interaction under acute hypoxic exposure.^59,60

Chronic hypoxia causes vascular remodeling, which is central to the pathogenesis of HPH. ⁷⁰ As it was previously shown that Notch3 and SOCs (TRPC6, Orai1/2) are upregulated in PH induced by chronic hypoxia, it is interesting to decipher whether and how Notch signaling regulates SOCs or TRPC6 specifically in chronic hypoxic situations. Strikingly, JY's group found that Notch signaling activation transcriptionally upregulates TRPC6, Orai1/2, and STIM2 via increased binding of RBP-Jκ with NICD, leading to enhanced SOCE under chronic hypoxia. Finally, pharmacological inhibition of Notch signaling attenuates TRPC6 expression, hypoxia-enhanced SOCE, and the development of HPH.^59,60 These studies suggest a novel dual role for Notch signaling on TRPC6 via direct functional interaction with TRPC6 and transcriptional upregulation of TRPC6, thereby maintaining increased levels of [Ca²⁺]cyt, that plays an important role in both acute HPV and HPH. Furthermore, recent work from this group suggests that Notch signaling regulates not only [Ca²⁺]cyt but also Ca²⁺ influx via regulation of CaSR function. Activation of Notch signaling by Jagged-1 enhances CaSR function and thus regulates Ca²⁺ influx and [Ca²⁺]cyt. Together, these data demonstrate the essential role of Notch-RBPJ signaling in initiation and perpetuation of HPH.

Transcriptional coactivator PKM2

PKM1 and PKM2 are pyruvate kinase isoforms expressed in different types of cells and tissues. Pyruvate kinase regulates the final rate-limiting step of glycolysis by catalyzing the transfer of a phosphate group from phosphoenolpyruvate to adenosine diphosphate to produce pyruvate and adenosine triphosphate (ATP), ⁷¹ which suggests their major functions in glycolysis. In addition to its well-known role in glycolysis, PKM2 has also been reported to be involved in other cellular functions. Importantly, PKM2 has recently been found to translocate into the nucleus upon mitogenic and oncogenic stimulation.^72,73 In the nucleus, PKM2 functions as a transcriptional coactivator and a protein kinase that phosphorylates histones, highlighting the crucial role of PKM2 in the epigenetic regulation of gene transcription that is important for the G1-S phase transition and the “Warburg effect” (which states that most cancer cells produce energy by a high level of glycolysis, followed by lactic acid fermentation).^74,75 PKM2 functions as a coactivator that stimulates HIF-1 transactivation of target genes encoding GLUT1, LDHA, and PDK1 in cancer cells. ⁷⁶ In addition to the crucial role of PKM2 in the G1-S phase, it phosphorylates important cell cycle regulators, such as the spindle checkpoint protein Bub3, to regulate chromatid segregation and the mitotic checkpoint during mitosis, and myosin light chain 2 (MLC2, encoded by MYL2) to initiate cytokinesis, leading to enhanced and governed tumor cell proliferation. ⁷⁷ In line with its functions, PKM2 expression has been found to be increased in all cancer types examined.^78,79

At the 2015 Grover Conference, one of us (KS) briefly reviewed his group's recent findings regarding the metabolic state of pulmonary arterial adventitial fibroblasts (PAAFs) isolated from hypertensive calves and humans with IPAH, the metabolic functions of PKM2, and the mechanisms that regulate PKM2 expression and its glycolytic enzymatic activity in these cells. Previous studies from the group suggested that some of the earliest pathophysiological changes in PH are observed in the pulmonary artery adventitia and include robust proliferation of fibroblasts and accumulation of macrophages.^80–83 It is increasingly recognized that increased proliferation and inflammatory activation can occur in the context of changes in cellular metabolism toward aerobic glycolysis (the Warburg effect), and these metabolic changes have been reported in PH in pulmonary arterial endothelial cells (PAECs), PASMCs, and, most recently, PAAFs and have formed the basis for a “metabolic theory of PH.”^84,85 However, the mechanisms that link metabolic reprogramming to inflammatory and proliferative pathways in PH remain largely unknown.

Elegant work employing RNA-Seq transcriptome profiling and nuclear magnetic resonance– and mass spectrometry–based metabolomics analysis showed significant metabolic reprogramming to aerobic glycolysis in PH-fibroblasts (PH-fibs). Glycolytic changes included sugar phosphates, ¹³C-glycolysis signature, ¹³C-lactate, D-glucose and glucose uptake, glyceraldehyde 3-phosphate, pyruvate, and lactate production. In addition, PH-fibs were characterized by alterations of redox homeostasis, as indicated by increased amounts of glutathione (GSH) and glutathione disulfide (GSSG), decreased GSH/GSSG ratios, evidence of utilization of the NADPH (nicotinamide adenine dinucleotide phosphate)-generating pentose phosphate pathway, and increased phosphogluconate and ribose phosphate (K. Stenmark, unpublished data). These data provide strong evidence that in vivo and in vitro aerobic glycolysis is a metabolic adaptation in PAAFs in the setting of chronic hypoxia as well as in IPAH. Metabolic data here suggest that, in a Warburg-like fashion, incomplete glucose oxidation affords PH-fibs building blocks to support the anaplerotic reactions (amino acid anabolism, fatty acid synthesis, urea cycle, gluconeogenesis) necessary to sustain proliferation, counteract oxidative stress, and promote resistance to apoptosis.

Importantly, these authors found an isoform switch from PKM1 to PKM2 in PH-fibs. Knockdown of PKM2 decreases PH-fib proliferation and lactate production, providing a link between transcriptional regulation and the metabolic status of the cell. The PKM1-to-PKM2 switch (i.e., PKM2 expression) is regulated at the level of transcribed PKM pre–messenger RNA (mRNA) by splicing factors. Polypyrimidine tract–binding protein 1 (PTBP1) is upregulated in PH-fibs by microRNA 124 (miR124) and subsequently binds to splicing signals that flank PKM exon 9, repressing the inclusion of exon 9 and thus promoting an enhanced expression of the PKM2 isoform. Knockdown of PTBP1, miR124 mimics, and histone deacetylase (HDAC) inhibitors reverses the PKM2-PKM1 switch in PH-fibs and reduces lactate production, suggesting that epigenetic regulation is an important mechanism in controlling PKM splicing in PH.

Transcriptional corepressor CtBP1

In the context of increased glycolysis, where increases in NADH/NAD (ratio of reduced to total nicotinamide adenine dinucleotide) are frequently observed, the functions of the CtBP family members, traditionally considered transcriptional corepressors, must also be considered. CtBPs comprise a dimeric family of transcriptional repressors encoded by two paralogous genes (CTBP1 and CTBP2) that have major roles in animal cell development and cancer and have been suggested to be “sensors” of the metabolic state of cells that can modulate many aspects of cell phenotype. ⁸⁶ Overexpression of CtBP is observed in a number of cancers, including prostate, ovarian, colon, and breast.^74,87,88 Studies in cancer and the nervous system revealed that CtBPs promote cellular survival primarily through repression of Bcl-2 family members and other proapoptotic molecules (PERP, Bax, Bik, Puma, p21, and Noxa), as well as tumor suppressors (e.g., p16INK4a, p15INK4b).^86,88,89 Importantly, there is emerging evidence that CtBPs may also function to modulate the inflammatory response. ⁹⁰ Further, a recent genome-wide analysis in breast cancer provided a more comprehensive description of CtBP repression targets.^88,91 CtBP targets were categorized into three main categories: genes that regulate cell renewal and pluripotency, genes that control genome stability, and genes that regulate epithelial differentiation and prevent epithelial-to-mesenchymal transition, all processes of potential importance in PH pathogenesis.^88,91 At the 2015 Grover Conference, one of us (KS) presented evidence for CtBP1 activation and control of PAAF proliferation, apoptosis resistance, and inflammatory gene expression in PAAFs derived from animals and humans with PH.

TF TWIST1

TWIST1 was recently demonstrated to play a crucial role in the pulmonary vascular remodeling responsible for human and experimental PAH through a process called endothelial-to-mesenchymal transition (EndoMT).^34,92 Interestingly, BMPR2 expression negatively correlated with Twist1 expression. The Twist proteins (Twist1 and Twist2) are highly conserved developmental proteins with key roles for transcriptional regulation in mesenchymal cell lineages. They belong to the superfamily of basic helix-loop-helix (bHLH) proteins and exhibit bifunctional roles as both activators and repressors of gene transcription. The Twist proteins are expressed at low levels in adult tissues but may become abundantly reexpressed in cells undergoing malignant transformation. This observation prompted extensive research on the roles of Twist proteins in cancer progression and metastasis. Twist1 suppresses BMP-induced osteoblast differentiation, and downregulation of endogenous Twist1 enhances BMP signaling. Maximal inhibition of BMP signaling was observed when Twist1 was bound to E47, which markedly enhanced the stability of Twist1. Co-immunoprecipitation assays revealed that Twist1 formed a complex with Smad4 and HDAC. ⁹³

Very recent studies also indicate a novel role for Twist1 as a potential regulator of adipose tissue remodeling and inflammation. Several studies have suggested that Twist genes are important determinants of obesity, fat distribution, and remodeling capacity of different adipose depots. Moreover, Twist1 expression is strongly correlated with body mass index and insulin resistance in humans. ⁹⁴ Interestingly, mice overexpressing Twist1 mutants in cardiomyocytes developed pathological cardiac remodeling. Twist1 may play a growth-inhibitory role in the postnatal heart, and phosphorylation may release their inhibitory effects, leading to hypertrophy. ⁹⁵ Interestingly, the genes involved in oxidative phosphorylation and the Krebs (citrate) cycle were found with reduced levels in the hearts of mice overexpressing Twist1 mutants, suggesting that decreased metabolism may result in heart failure. Twist1 play also critical roles in diverse developmental systems, including myogenesis. Several experiments have demonstrated its role in inhibition of muscle cell differentiation. Overexpression of Twist1 can reverse muscle cell differentiation in the presence of growth factors. ⁹⁶ Hence, Twist1 is potentially involved in all pulmonary and systemic abnormalities found in PAH. Regarding EndoMT as responsible for neointima and plexiform lesion formation and distal neomuscularization, Hopper et al. ³⁵ also demonstrated that another TF might be involved. They showed in vitro that loss of BMPR2 in PAECs leads to heightened expression of the chromatin architectural factor high-mobility group AT-hook 1 (HMGA1), which increases the TF Slug. HMGA1 binds to DNA and may promote binding of additional pro-EndoMT TFs. ³⁵

MicroRNA dysregulation in the pathogenesis of PH

MicroRNAs are small ncRNAs that negatively regulate gene expression by binding to the 3′ untranslated region of target mRNAs, thereby promoting mRNA degradation or inhibiting translation. Profiling of the miR expression in rodent models of PH and in human pulmonary arteries from PAH patients indicated dysregulation of several miRs, including miR204, the mir17/92 cluster, the miR143/145 cluster, and others. ¹⁰³ To fine-balance these dysregulated miRs, different strategies, such as miR mimics or antagomirs, were employed and demonstrated to reverse the vascular remodeling in PH experimental models. Because of space limitations, we do not provide an extended analysis here, as the topic is covered in greater depth elsewhere.^103,106

Altered DNA methylation patterns in PH

DNA methylation is one of the best-studied epigenetic modifications occurring in mammals, almost exclusively on the 5′ position of the pyrimidine ring of cytosines in the context of CpG (5′-cytosinephosphateguanine-3′) dinucleotides. This DNA modification is mediated by DNA methyltransferases and generally results in tight packing of DNA and histones (heterochromatin) and in the long-term silencing of transcription. DNA methylation may result in gene silencing that can be propagated to daughter cells.

The influence of nongenetic factors in autoimmune and inflammatory diseases has been demonstrated in studies of genetically identical (monozygotic [MZ]) twins who show a variable degree of discordance with respect to different phenotypic traits, including susceptibility to these diseases. MZ twins show a concordance rate for different autoimmune diseases ranging from 12% for rheumatoid arthritis up to 70% for psoriasis. There is substantial epigenetic variation between MZ twin pairs. This variation increases with age and if twins live in different environments. These environment-host interactions might result in such severe epigenetic changes that the epigenetic homeostasis can no longer be maintained, leading to aberrant gene expression. Aberrant gene expression in specific cells of the immune system most likely contributes to the loss of immune tolerance, inflammation, and autoimmunity, which is characterized by the failure of an organism to recognize self-antigens. While a large number of candidate gene and genome-wide studies analyzing the DNA methylation profiles have been published for inflammatory and autoimmune diseases such as type 1 diabetes, multiple sclerosis, and systemic lupus erythematosus, ¹⁰⁷ evidence for the implication of DNA methylation modifications in PAH is currently scarce.

Archer's group ¹⁰⁸ has identified a methylation-induced attenuation of Sod2 (mitochondrial superoxide dismutase 2) expression as an epigenetic mechanism in an animal model of spontaneous and heritable PAH (fawn-hooded rats). This may be relevant for clinical PAH because SOD2 is also downregulated in human PAH. The epigenetic downregulation of SOD2 impairs H₂O₂-mediated redox signaling, activates HIF-1, and creates a proliferative, apoptosis-resistant state. Both the mechanism of SOD2 downregulation and its consequences parallel those discovered in human cancer. ¹⁰⁹

Moreover, DNA methylation for the measurement of a PAH marker has a number of attractive features. First, DNA molecules are very stable and, in contrast to mRNA and many proteins, can survive harsh conditions for long periods of time. Second, unlike proteins, nucleic acid can be amplified by polymerase chain reaction and related techniques, thus allowing measurements on small amounts of test sample. ¹¹⁰ Moreover, blood levels of most currently used protein biomarkers are rarely increased in the early stage of the disease. Consequently, most existing blood protein biomarkers are of little value in either screening for or aiding the early diagnosis of PAH. On the other hand, aberrant methylation of the promoter regions of multiple genes may exist in both early and advanced PAH. As proof of principle, FP's group^111,112 published an epigenetic study showing that DNA methylation alterations in the granulysin (GNLY) gene in explanted lungs and in peripheral blood mononuclear cells allow discrimination between pulmonary veno-occlusive disease (PVOD) patients (PVOD is a subgroup of PH with worse prognosis and risk of developing severe pulmonary edema with specific PAH therapy) and PAH patients.

At the 2015 Grover Conference, FP presented his ongoing work about the identification of a DNA methylation-based PAH signature. His group analyzed the promoters of protein-coding sequences, miRs, and long noncoding RNA in the genomic DNA from cultured PAECs isolated from IPAH and heritable PAH (BMPR2 mutation carriers) patients and controls. Enrichment analyses demonstrated that the differentially methylated promoters clustering in controls and PAH groups were enriched in pathways, networks, and functions related to metabolism, cardiovascular system development and function, cellular development, skeletal and muscular system development and function, cell morphology, cellular function and maintenance, cell death and survival, cell-mediated immune response, cellular development, cancer, and organismal injury and abnormalities (F. Perros, unpublished data). Further analysis of these data will enhance the discovery of novel regulatory pathways in PAH that require innovative therapy.

Alterations in histone levels in PAH

Linker histones (H1) have been found to play an important role in transcription, gene regulation, protein-protein interactions, and innate immunity and are associated with complexes to downregulate proinflammatory genes. Talati et al. ¹¹³ employed histology-based matrix-assisted laser desorption ionization–mass spectrometry (MALDI-MS) and identified significant increases, compared to controls, in two fragments of histone H1 in the pulmonary arteries from the explanted lungs of the IPAH patients. However, a reduction in the nuclear histone H1 and an increase in the cytoplasmic H1 were observed in IPAH cells. The decreased nuclear H1 contributes to the less compact nucleosomal pattern in IPAH, and this, in turn, contributes to the increase in nucleosomal repeat length and ultimately to changes in transcription. ¹¹³

Role of HDACs in PAH

Two different families of enzymes mediate the balance between the acetylated and deacetylated states of histone or nonhistone proteins: histone acetyltransferases and HDACs. The role of aberrant HDAC activity and histone acetylation has been strongly implicated in PAH pathogenesis on the basis of the promising therapeutic benefits observed upon the application of small-molecule HDAC inhibitors in different animal models of PH. Treatment with pan-HDAC inhibitors (suberoylanilide hydroxamic acid [vorinostat]) and class I selective inhibitors (MGCD0103, valproic acid) both attenuated and reversed the development of hypoxia- and MCT-induced PH in a rodent model.^114,115 Table 2 summarizes all research findings that reported the aberrant expression and activity of HDACs and the therapeutic effects of pan-HDAC inhibition. Moreover, Li et al. ⁸³ demonstrated that class I HDAC inhibition reverses stable proinflammatory phenotype of PH. This study suggests that the persistent proinflammatory phenotype exhibited by activated PAAFs was associated with abnormal activity of HDACs, specifically, class I HDACs. ⁸³ Importantly, catalytic inhibition of class I HDACs using the selective class I HDAC inhibitor apicidin significantly suppressed production of proinflammatory mediators by PH-fibs and caused a marked reduction in the ability of PH-fibs to induce monocyte migration and activation. ⁸³ Using Bmpr2^+/− mice, Soon and coworkers ¹²² recently demonstrated that BMPR2 deficiency promotes an exaggerated inflammatory response. In their study, BMPR2 deficiency in mouse and human PASMCs was associated with increased phospho-STAT3 and loss of extracellular superoxide dismutase (SOD3). Interestingly, the proinflammatory effect of constitutive BMPR2 haploinsufficiency could not be mimicked by short-term BMPR2 deficiency. The authors suggested that a potential mechanism is epigenetic modification of the promoter regions of genes as a consequence of chronic loss of BMPR2. Accordingly, they demonstrated that the loss of SOD3 expression in Bmpr2^+/− cells was reversed by incubation with trichostatin A.^122,123

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Table 2.

Histone deacetylases in PH: published evidence

Species	Model/tissues/cells	Summary	Reference
Rat	PAB; RV	Suppression of HDACs (TSA) worsens RV dysfunction after PAB	Bogaard et al. 116
Bovine	Hypoxia; adventitial fibroblasts	Alterations in class (I) HDAC1/2/3 expression and activity contribute to a proinflammatory phenotype of PH-fibroblasts and was attenuated by application of HDAC inhibitors (SAHA, apicidin)	Li et al. 83
Human/bovine/rat		Increased expression of HDAC1/4/5 in IPAH lung homogenates, HI PH lungs and RV; HDAC inhibition (SAHA, VPA) attenuated the development of HI PH and hyperproliferative phenotype of PH-fibroblasts and R-cells from hypertensive bovine pulmonary arteries	Zhao et al. 115
Human	IPAH; lungs
Bovine	Adventitial fibroblasts, R-cells
Rat	Hypoxia; lungs, RV
Rat	Hypoxia; RV, PASMCs	Selective class I HDAC inhibitors (MGCD0103 and MS-275) suppresses HI PH and RVH in a preclinical model of hypoxia-mediated PH	Cavasin et al. 114
Ovine (sheep)	Serum; PASMCs	Inhibition of class I and II HDACs by apicidin and HDACi VIII suppressed serum-induced proliferation and migration of newborn PASMCs	Yang et al. 117
Rat	PDGF; PASMCs	Sodium butyrate inhibits PDGF-induced proliferation and migration in PASMCs through Akt inhibition	Cantoni et al. 118
Rat	PDGF; PASMCs	Class I HDAC inhibitor (MC1855), but not class II HDAC inhibitor (MC1575), counteracts PDGF-induced proliferation and migration of PAH PASMCs	Galletti et al. 119
Bovine	Hypoxia; adventitial fibroblasts	Pan-HDAC inhibitors (SAHA, apicidin) rescued the HI downregulation of antiproliferative miR-124 expression in hypoxic adventitial fibroblasts	Wang et al. 120
Rat	MCT, SuHx-PH, PAB	Pan-HDAC inhibitor TSA has no therapeutic or beneficial effects in SuHx-PH	De Raaf et al. 121
Human	IPAH PAECs, MCT, SuHx-PH	Increased nuclear accumulation of class IIa HDACs HDAC4 and HDAC5 impaired MEF2 activity in PAH PAECs; pharmacological inhibition of class IIa HDACs using MC1568 rescued the impaired MEF2 transcriptional activity in PAH PAECs and also reduced PAH PAEC proliferation and migration; no evidence of myocardial fibrosis in MCT and SuHx groups upon HDAC inhibition	Kim et al. 33

Note: HDAC: histone deacetylase; HI: hypoxia-induced; IPAH: idiopathic PAH; MCT: monocrotaline; MEF2: myocyte enhancer factor; PAB: pulmonary artery banding; PAECs: pulmonary artery endothelial cells; PAH: pulmonary arterial hypertension; PASMCs: pulmonary artery smooth muscle cells; PDGF: platelet-derived growth factor; RV: right ventricle/ventricular; RVH: RV hypertrophy; SAHA: suberoylanilide hydroxamic acid; SuHX-PH: hypoxia+SU5416–induced PAH (SU5416 is a vascular endothelial growth factor receptor [VEGFR-2] inhibitor); TSA: trichostatin A; VPA: valproic acid.

Collectively, these findings also highlight the need for identification of deregulated isoforms in the PAH setting and to dissect the “histone code” that can mediate a unique cellular response, such as the proinflammatory/proproliferative PAAF phenotype, the hyperproliferative PASMC phenotype, and the angioproliferative PAEC phenotype. In order to elucidate the histone acetylation code underlying the PAH phenotype in human setting, SSP's group employed three approaches: (1) investigate the expression, regulation, and functional role of histone acetylation modifiers, (2) explore the therapeutic potential of isoform-selective modulation of deregulated HDAC isoforms in PAH disease phenotype reversal ex vivo and in vivo, and (3) employ an unbiased next-generation sequencing-based approach to identify the disease-specific epigenetic signatures (histone modification patterns and transcriptome profiles) and the regulatory network that facilitates the vascular cells to acquire and sustain the disease phenotype. Interestingly, aberrant gene and protein expression profiles of class I HDAC isoforms across a variety of cardiopulmonary samples from human PAH was documented. The screening also revealed an organ- and cell-specific expression pattern of these isoforms between IPAH patients and donors. The stable disease phenotype exhibited by ex vivo isolated IPAH PAAFs was associated with aberrant expression and activity of class I HDACs isoforms. Importantly, integrative analysis of vascular cell–specific epigenomics data provides insights into the mechanistic link between recruitment of histone modifiers and the regulatory network of histone modifications that cooperatively regulates aberrant transcriptional responses in PAH (S. S. Pullamsetti and P. Chelladurai, unpublished data).