The progression of pulmonary vascular disease often begins with the interplay between an initial pathogenic state and one or more triggering stimuli, referred to as the “multiple-hit hypothesis.” Two or more hits could comprise a genetic prediction coupled with an extra genetic factor (such as a mutation or polymorphism), epigenetic modification, comorbidity, stress, or environmental factors (Figure 2). When these factors align, a cascade of effects is set in motion, initiating vascular constriction, cellular proliferation, and a prothrombotic state to varying extents (11, 12). This intricate process culminates in PAH and its associated clinical manifestations. In PAH, pathological vascular remodeling results in distortions in macroscopic and microscopic structures of the pulmonary arterial vasculature. Studies indicate that PAH originates from the abnormal hyperproliferation of pulmonary vascular cells, resulting in neointima formation and narrowing of small distal pulmonary arterioles (13, 14). This luminal narrowing elevates pulmonary vascular resistance, exerting strain on the right ventricle, leading to right heart failure.

Various cell types present in the pulmonary arterial wall contribute to PAH vascular remodeling. Three distinct pathological vascular remodeling processes have been identified as contributors: the muscularization of distal pulmonary arterioles, medial hypertrophy, and neointima formation in proximal muscular arteries, along with the development of plexiform lesions (13, 15, 16).

The muscularization of distal pulmonary arterioles occurs due to the differentiation of progenitor cells into PASMCs, followed by subsequent proliferation (17–19). Progenitor cells involved in this process include stem cells, fibrocytes, or PAECs. Medial hypertrophy and neointima formation also result from differentiating progenitor cells into SMCs, followed by migration and replication in the proximal muscular arteries (20–22). Following muscularization and hypertrophy, there is also abnormal PAEC replication, and the formation of irregular endothelial channels results in plexiform lesions within the narrowed vessel lumen. The PASMCs and PAECs become more resistant to apoptosis, worsening the pathologic vascular remodeling and forming plexiform lesions (23–25).

Another cell type identified in the pathogenesis of PAH is the pulmonary pericyte. Located in perivascular regions, these cells play a crucial role in endothelial cell maturation, immune signaling, and modulation of vascular tone through their impact on cellular remodeling and immune responses (26). The involvement of pericytes in the pathogenesis of PAH has garnered significant research interest. A 2014 study by Ricard et al. revealed an increase in pericytes within the distal pulmonary arteries of human idiopathic PAH (IPAH) endothelial cells compared to in vitro control cells. This research underscored the significance of transforming growth factor-β in promoting the differentiation of pericytes into contractile smooth muscle-like cells in distal pulmonary arterioles (27).

Additionally, Yuan et al. demonstrated that PAH pericytes isolated from the lungs of PAH patients exhibited defective association with endothelial tubes in vitro, leading to smaller vascular networks as well as narrower endothelial tubes. This defect was attributed to an intrinsic issue in cell motility and polarization, impairing the ability of PAH pericytes to migrate toward vascular tubes and resulting in fewer endothelial-pericyte interactions. The study demonstrated that PAH pericytes are involved in in the loss of small vessels in PAH and suggested that therapeutic approaches targeting the restoring Wnt/PCP activity in these cells could help prevent vessel loss and promote small vessel regeneration in patients with this severe condition (28).

Furthermore, pyruvate dehydrogenase kinase 4 (PDK4) gene and protein expression were found to be significantly elevated in PAH pericytes. This elevation is associated with decreased mitochondrial metabolism, increased glycolysis rates, and hyperproliferation. Notably, lowering PDK4 levels restored mitochondrial function, decreased cell proliferation, and enhanced endothelial-pericyte interactions in PAH models (29).

Metabolic dysregulation plays a central role in PAH, marked by a transition from oxidative phosphorylation to glycolysis, a phenomenon termed the “Warburg effect” (30, 31). This shift leads to notable metabolic alterations, including increased cytoplasmic glycolysis and glutaminolysis, decreased oxidation of fatty acids and disrupted biogenesis of mitochondria (32, 33).

In PAH patients, the increase in cytoplasmic glycolysis, observed in PAEC and PASMCs, compromises the efficiency of ATP generation via the mitochondrial tricarboxylic acid cycle (34–37). Further, PAH is characterized by reduced mitochondrial metabolism, evidenced by reduced mitochondrial abundance, oxygen consumption and mitochondrial DNA in PAEC and PASMC (38–41). This metabolic imbalance leads to compromised mitochondrial respiration elevated reactive oxygen species production, and a reduction in antioxidants like superoxide dismutase 1 and 2, exacerbating the progression of PAH (35, 38, 42).

The “Warburg effect” refers to the inhibition of pyruvate dehydrogenase, leading to decoupled glycolysis and decreased utilization of mitochondrial pyruvate in PAH (41, 43–45). Additionally, various cell types in PAH, such as PAECs, PASMCs, fibroblasts, macrophages, and myocytes of the right ventricle, exhibits reduced mitochondrial glucose oxidation and increased glycolysis (46). The altered metabolism in PAH, affecting glucose homeostasis and vascular remodeling, is associated with the downregulation of the transcription factor PPAR γ, a downstream target of BMPRII (47). Considering the regulatory role of mitochondrial products on transcription factors and epigenetic mechanisms, targeting the epigenetic and metabolism pathway holds promise as a strategy that could be used for PAH therapy (43, 48).

Although less understood, disturbances in fatty acid metabolism are observed in the pulmonary vascular system and right ventricle, resulting in PASMC proliferation, right ventricular steatosis, and lipotoxicity. This is attributed to triglyceride metabolites and reactive oxygen species resulting from elevated fatty acid delivery and synthesis, coupled with diminished oxidative mitochondrial capacity (49, 50).

Inflammation is a frequently noted phenomenon in PAH (51). Both the innate and adaptive immune systems are impacted by imbalances in inflammatory and immunological responses, which may be influenced by genetic and environmental factors. This anomaly is demonstrated by the perivascular infiltration of dendritic cells, mast cells, T and B lymphocytes, or macrophages situated in close proximity to pulmonary arteries (52).

PAECs and inflammatory cells play crucial roles as both are sources and targets of chemokines and cytokines, contributing to pulmonary vascular remodeling. Proinflammatory cytokines, interleukin 6 and interleukin 1β, directly influence smooth muscle, immune cells, and PAEC migration, proliferation, and differentiation (51, 53–55). In PAH, autoantibodies and local lymph follicles promote inflammation and immunological activation (56, 57). The alterations observed in PAECs and PASMCs in PAH patients create a synergistic effect, intensifying inflammation. Various cell types, including PAECs, PASMCs, myofibroblasts, and fibroblasts, exhibit a significant pro-inflammatory state, evident in the increased expression and release of inflammatory cell-associated cytokines, chemokines, and adhesion molecules, such as E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1, are seen. Deactivation of the Forkhead box O1 pathway and elevated levels of interleukins-1 and -6, leukotriene B4, leptin receptors, or tumor necrosis factor all contribute to harmful changes in the pulmonary vasculature via different signaling pathways (58–63).

Similarly, mutations in BMPRII result in heightened levels of proinflammatory cytokine levels, including interleukin 1β and interleukin 6 (51, 53, 64). The BMPR II loss of function results in an increase in fibroblast growth factor 2, enhanced mitogen-activated protein kinase activity, and elevated levels of interleukin 1β, and interleukin 6 and a reduction of vasoprotective peptide apelin in PAECs (53, 54, 65, 66). These cytokines can induce PAECs to produce more fibroblast growth factor 2 (55). They are also essential in coordinating proliferative responses in PASMCs and pulmonary artery fibroblasts because they secrete fibroblast growth factor 2 and interleukin-6 (53, 54, 66). Comparably, as demonstrated by both experimental PH models and human PAH, a disrupted signaling pathway of BMPR 2 can lead to the improper production of growth factors and proinflammatory responses in vascular cells (51, 64, 65, 67, 68).

The term “epigenetics” derived from the Greek word “Epi,” meaning on or above, refers to factors beyond the genetic code that modify DNA sequences, inducing phenotypic changes without altering the DNA base pair. Essentially, epigenetic factors influence the transmission and expression of traits not encoded in the genetic material. In humans, mounting evidence highlights the crucial role of epigenetic factors in cellular differentiation, survival, apoptosis, tumor suppression, and genomic imprinting. These factors are induced by environmental exposures and the complex interplay of gene–environment interactions, contributing to various disease states (69, 70). This emphasizes the essential role of epigenetic modifications in the formation of various pathological processes, including tumorigenesis, metabolic disorders, vasculopathy and PAH (71–75).

PAECs depend on expressing and silencing specific genes to maintain homeostasis. Epigenetic modifications can lead to alterations in these pathways, resulting in dysregulation of PAECs, increased proinflammatory cells, and ultimately, the formation of PAH (76). In PAH, these inheritable changes manifest via three major epigenetic mechanisms: DNA methylation, histone post-translational modification, and non-coding RNA regulation (Figure 3). These modifications are composed of writers, erasers, and readers, directly impact DNA transcription, chromatin packing, mRNA, and protein expression, thereby activating or inhibiting genes (77, 78). We will explore the major epigenetic modifications to understand their roles in the pathophysiology of PAH.

DNA methylation involves the covalent addition of a methyl group (-CH3) to adenosine or cytosine residues in CpG islands. The CpG sites are regions in the DNA with specific base pairing, commonly in the promoter regions, that can undergo methylation. Methylated cytosines act as attachment sites for methylated CpG-binding proteins (MeCP) that recognize and bind to methylated CpGs (79). Through the methylation of the CpG islands, proteins involved in gene expression and repression can be directly recruited. CpG methylation also promotes a dense winding of DNA around the histone core, resulting in a structural change (80). Methylation and demethylation are crucial for the regulation of gene expression, cellular differentiation, organ morphogenesis, cell reprogramming, X-chromosome inactivation, RNA splicing, transposon silencing, and DNA repair (79, 80). DNA methylation is mediated by a group of enzymes known as the DNA methyltransferases (DNMT), including DNMT 1, DNMT 2, DNMT3A, DNMT3B and DNMT 3 L (81, 82). In mammalian cells, DNMT1 is the primary methyltransferase implicated in the inheritance of DNA methylation. DNA methylation pattern from parents is copied into newly produced offspring DNA by DNMT1, which interacts with partly methylated DNA during DNA replication (79). DNMT1 exists in an inhibitory state, requiring binding with another protein, E3 ubiquitin-protein ligase (UHRF1). The UHRF1-DNMT complex attaches during the S phase of DNA replication (78). On the other hand, de novo DNA methylation requires Dnmt3a and Dnmt3b. They act directly on the CpG sites, adding methyl groups to form new genetic transcription. Methylated CpG islands also interact with methyl-CpG binding proteins (MBDs) via histone deacetylation, playing an essential role in gene silencing (83). Transcriptional repression happens through three mechanisms: (i) nucleosome compaction that obscures transcription factor binding sites, (ii) the recruitment of repressor proteins, and (iii) interaction with histone modification processes (84, 85). Hyper-or hypo-DNA methylation is susceptible to alteration by environmental factors. This phenomenon is noteworthy as it is the “second hit” required to unmask numerous disease conditions by creating a distinct synergistic effect with pathologic genetic mutations (86).

The ten eleven translocation (TET) proteins play a crucial role in regulating gene expression by promoting locus-specific reversal of DNA methylation. TET methylcytosine dioxygenases oxidizes 5-methylcytosine (5-mc) to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxycytosine (5-caC). These oxidized forms of cytosine enable DNA methylation through both active and passive mechanisms (87–89).

Tet-methylcytosine-dioxygenase-2 (TET2), an essential enzyme involved in DNA demethylation, has recently been linked to pathologic vascular remodeling, inflammation, and clonal hematopoiesis. In IPAH and other PAH-related conditions, TET2 expression was found to be significantly reduced in peripheral blood cells of these patients, making it a potential biomarker for PAH (90).

A study by Potus et al. reported an association between germline mutations in TET2 and PAH (91). Analyzing a PAH biobank of 2,572 cases, the study revealed numerous harmful germline and somatic variants of TET2. The data showed that 0.39% of PAH cases had TET2 abnormalities, with 75% being attributed to germline mutations and 25% to somatic mutations (91). Moreover, 86% of PAH patients exhibited significantly downregulated circulating TET2. These patients were typically older and responded to vasodilatory therapy.

Furthermore, the study revealed that TET2 depletion led to spontaneous development of PAH, as demonstrated by elevated right ventricular systolic pressure, increased total pulmonary resistance, reduced pulmonary artery acceleration time, adverse remodeling of the pulmonary vasculature, and inflammation (91). Further analysis of Tet2 hematopoietic conditional knockout (KO) mice and heterozygous Tet2+/− mice revealed that even partial Tet2 deletion could induce PAH, indicating a gene dose-effect response (91).

D’Addario et al. (92), examined differences in leukocyte expression of DNMT and TET and the severity of PAH among various ethnic groups. Their findings revealed that PAH patients had higher expression levels of DNMTs (3a and 3b) and TETs (2 and 3) compared to healthy controls, with notable ethnic variations (92). Specifically, the study found that TET2/TET3 expression levels were higher in Hispanic and African American patients diagnosed with scleroderma-associated and IPAH compared to Caucasians, whereas DNMT1 expression was downregulated in these patients (92). Additionally, the study found higher levels of inflammatory cytokines IL6 and CCL5 in Caucasian PAH patients compared to Hispanic/African American patients. The altered DNA methylation and reduced TET expression may be associated with elevated inflammatory cytokines and hematological disorders or malignancies.

Since the first histone methyltransferase was discovered in the 2000s, histone post-translational modification has been a great field of research interest in vascular biology (100–103). The nucleosome core particle of chromatin is made up of a histone protein octamer with 146 base pairs of DNA organized into a superhelix around it. The nucleosome is formed by two sets of histone octamers composed of H2A, H2B, H3, and H4 proteins, and is further condensed by linker DNA (104, 105). Histones are post-translationally modified in the histone tail, which consists of 10–30 amino acids at the N-terminal domain. Post-translational modification modulates chromatin folding and regulatory protein binding within the nucleosome to produce histone variants that modulate the transcription and expression of genes (106). Various histone post-translational modifications involve histone embryonic vascular development, remodeling, and various vascular disease pathologies (107).

The primary histone modifications of note include methylation and acetylation, processes where small organic molecules are added to the histone tail through chemical mechanisms. Other such modifications include phosphorylation, deamination, and palmitoylation. The second group of histone modifications involves the addition of larger organic molecules to the histone tail, including ubiquitylation, SUMOylation, biotinylation, glycosylation, and ADP-ribosylation (108, 109).

Histone methylation is a reversible post-translational modification involving two enzymes: histone methyltransferase (HMTs) as the writer and histone demethylases (HDMTs) as the eraser. Histone methylation involves the addition of -CH3 groups to the amino acids at the histone tail. This process tightly modulates gene expression depending on how many methyl groups and/or which amino acid is attached. The HMTs are classified into two groups: protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs) (110, 111). Multiple methyl groups may be added by this post-translational alteration, leading to monomethylation (me1), dimethylation (me2), and trimethylation (me3). The most common epigenetic modification observed in histone methylation is gene silencing via polycomb group proteins (PcG) (112). These proteins regulate cellular differentiation by inhibiting inappropriate cellular activation and proliferation. In mammals, PcG proteins organize into two main complexes: polycomb-repressive complex 1 (PRC1) and 2 (PRC2). The PRC1 is made up of the BMI1, RING1A/B, CBX, and PHC subunits (113). PRC1 functions by binding to nucleosomes, resulting in chromatin compaction (114). Conversely, PRC2 is composed of EZH2 or EZH1, EED, SUZ12, and RbAp46 (113–115). EZH2 is extensively researched as the catalytic subunit of PRC2, housing the SET domain crucial for tri-methylation of lysine 27 on histone H3 (H3K27me3), resulting in transcriptional inhibition (113). PcG-dependent gene silencing involves a synergist effect between PRC1 and PRC2 complexes. Once the PRC2 complex forms H3K27me3, it recruits PRC1 by binding the chromodomain of the PHC subunits (116). Chromodomains found within the HP1 proteins recognize di-and tri-methylated histones (H3K9), which results in the recruitment of heterochromatin protein 1 (HP1) to sites of repressed chromatin. Similarly, CBX proteins, containing a sequence structurally similar to the N-terminal of the chromodomain, are recruited to di-or tri-methylated H3K27, forming a multi-protein complex that regulates genes associated with development and differentiation. The CDY1 gene encodes a protein that includes both a chromodomain and a histone acetyltransferase catalytic domain that binds to H3K9me resulting in gene repression (112). Another chromodomain, CHD1, has been shown to recognize H3K4me, resulting in gene transcription. Independent of H3K27me3, EZH2, and other PRC2 subunits have been identified in the cytoplasm, where they modulate actin polymerization and cell proliferation of T lymphocytes and fibroblasts (117).

Bromodomains and chromodomains are protein domains capable of identifying and attaching to modified amino acids on histone tails. The bromodomain and extra-terminal (BET) domain family are known as epigenetic readers and have been linked to numerous diseases, as evidenced by recent studies (118–122). This family includes BRD2, BRD3, BRD4, and testis-specific bromodomain proteins, all interacting with other proteins and acetylated histone tails through their tandem bromodomains 1 and 2. BET proteins regulate the transcription of genes involved in various biological processes such as cell division, apoptosis, and inflammation (123). Among these, BRD4 is extensively studied in current research on PAH.

BRD4 modifies the chromatin structure and facilitates the transcriptional activation of target genes by acting as a scaffold for transcription factors at promoters and super-enhancers (124). It stimulates proinflammatory endothelium genes such as IL-6 (interleukin-6), tumor necrosis factor-α, and monocyte chemoattractant protein-1 and is also linked to vascular remodeling (123, 125). This activation is further stimulated through a feedback loop (126). These proinflammatory cytokines have been shown to damage DNA and found to be increased in PAH (127, 128).

Furthermore, Meloche et al. found that, in comparison to controls, BRD4 was upregulated in the lungs, distal pulmonary arteries, and PASMCs of patients with PAH, indicating that BRD4 is dependent on miR-204 (124). Three key oncogenes overexpressed in PAH were reduced in expression by pharmacological suppression of BRD4 using JQ1 or siBRD4: nuclear factor of activated T cells, B-cell lymphoma 2, and survivin. Inhibiting this oncogenic signature resulted in a BRD4-dependent reduction in PAH-PASMC proliferation accompanied by an elevation in apoptosis, suggesting that BRD4 overexpression is pathologically associated with PAH and may be a target for therapy (124).

Therefore, through regulating gene expression, BRD4 is an essential regulator of the equilibrium between proliferation and apoptosis and is involved in post-DNA damage events, making it a potential target for the development of PAH therapy (129).

An additional post-translational modification of histones is the histone acetylation. It involves adding negatively charged cofactor acetyl groups to activate gene expression (131). Histone acetylation is catalyzed by histone acetyltransferase (HAT) enzymes, which transfer the acetyl groups to the positively charged lysine residues of the histone tail. The neutralization of the lysine residue weakens charge-dependent interactions between the histone and DNA, allowing for transcription. There are two types of HATs: Type A located in the nucleus and Type B located in the cytoplasm. They work together to acetylate histones for transcriptional activation and replication, respectively, before the chromatin is assembled (132). The most active HATs in mammals include HIV Tat interactive 60-kDa protein (Tip60), p300, CREB binding protein associated factor (PCAF), cAMP response-element binding protein (CREB), and CREB binding protein (CBP) (133). HATs like histone acetyltransferase 7 (KAT7) have been identified to regulate endothelial function in zebrafish embryos by initiating the transcription of vascular endothelial growth factors (VEGFs) (134).

Conversely, histone deacetylation uses histone deacetylases, or HDACs, to control the amount of histone acetylation. The four families of histone deacetylases (HDACs) are responsible for removing acetyl groups from histone proteins. (i) The nucleus contains class I HDACs (HDACs 1, 2, 3, and 8), (ii) The cytoplasm and nucleus express class II HDACs (HDACs 4, 5, 6, 7, 9, and 10), (iii) The nucleus contains class III HDACs (Sirtuins 1–7), and (iv) both the cytoplasm and the nucleus express class IV HDACs (HDAC 11) (135). The sirtuins (SIRT) regulate a number of processes, such as aging, stress damage, metabolism, transcription, and cell division (136). Overall, HDACs silence DNA transcription by tightening chromatin wrapping.

The Ubiquitin Proteasome System (UPS) is essential for several cellular processes, such as protein transport and breakdown, transcription control, and DNA repair. All tissues of eukaryotic organisms have the 76-amino acid protein known as ubiquitin (165). The ubiquitination process involves adding a C-terminal group of ubiquitin to a lysine residue of the substrate, for example, the histone tail, resulting in epigenetic modification. Ubiquitination is a multistep ATP-dependent process mediated by three essential enzymes, E1, E2, and E3 enzymes, facilitating the attachment of ubiquitin monomers or chains to proteins (166). Similarly, the proteasome system, which is made up of the 20S core proteasome (CP) and the 19S regulatory particle (RP), is part of this process. The RP governs substrate protein binding and ubiquitination into the CP through its primary structure containing ATPase and its cap structure without ATPase. The process of ubiquitination and de-ubiquitination is reversible and highly controlled, as dysregulation in this complex has been linked to numerous medical conditions (167).

Another layer of epigenetic modifications is through non-coding RNAs (ncRNAs). They are typically classified into two groups based on size: small ncRNAs (<200 nucleotides) and long non-coding RNAs (lncRNAs) (>200 nucleotides). The small ncRNAs are made up of microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and small-interfering RNAs (siRNAs). Conversely, LncRNAs, consist of small nucleolar RNAs (snRNAs) and natural antisense transcripts. LncRNAs are gene transcripts that directly bind to non-coding regions of mRNAs to modify the expression of those genes (177).

miRNAs are small non-coding genetic transcripts with ~25 base sequence that regulate gene expression. Numerous miRNA genes have been found in eukaryotic organisms, making them one of the largest gene families (178). The regulatory network of miRNA activity is intricate, given that an individual miRNA can bind to and control the expression of numerous genes. Conversely, multiple miRNAs can bind to and regulate a single mRNA target (179). The RNA polymerase III (pol III) transcribes pri-miRNA, a long primary transcript from the nucleus to pre-miRNA. The pre-mRNA maturation occurs through a sequential process. The pre-mRNA, which is several hundred base pairs long, is processed by the RNAse III endonuclease Drosha into a smaller fragment of 60–70 nucleotides, which is then transported to the cytoplasm (180, 181). The pre-miRNA is then processed by another RNA endonuclease, Dicer, to produce a mature double-stranded miRNA. The mature miRNA is separated and incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC) (182). The incorporated miRNA plays a crucial regulatory role in various cellular processes, such as hematopoietic cell differentiation, cell proliferation, apoptosis, and organ development (179, 180). miRNAs have been shown to regulate critical signaling pathways involved in the proliferation and migration of both PASMCs and PAECs necessary in the pathogenesis of PAH.

Like miRNA, lncRNAs are processed by the RNA processing apparatus after being transcribed by RNA polymerase II or III. They may be multiexonic, 5′-capped, or polyadenylated. LncRNAs are in the nucleus or cytoplasm, where they regulate transcriptional and posttranscriptional gene expression (205). In the cytoplasm, lncRNAs compete with mRNAs for miRNA binding and hence reduce the mRNA-destabilizing potential of miRNAs (206). On the other hand, inside the nucleus, lncRNAs regulate gene expression at the epigenetic level by modulating transcription (205).

The impact of sex disparities on PAH is substantial, leading to influencing its prevalence, pathogenesis and treatment responses. Strikingly, there is epidemiological variation, with a 4:1 ratio of females to males, generating curiosity in how estrogen and its metabolites function in the pathogenesis of PAH. Therefore, exploring the therapeutic implications of estrogen inhibitors has gained attention (219–222).

Through estrogen receptors, estrogen signaling decreases BMPRII expression and increases PASMC proliferation in experimental models (223). Although females exhibit a higher incidence of PAH, paradoxically, a worse prognosis is associated with males. This discrepancy is likely attributed to the advantageous effects of estrogen on the right ventricle, commonly known as the “estrogen paradox,” thus making it more challenging to use drugs that target estrogen signaling therapeutically (224–226).

Estrogen is one of the sex hormones that directly affects the mitochondria’s functional dynamics in tissues related to PAH development. Estrogen-receptor-equipped mitochondria contribute to PAH by promoting nuclear respiratory factor-1 (NRF-1) transcription, which increases the amount of mitochondrial transcriptional factors (TFAM) (227). These mitochondrial processes are essential to the pathogenic mechanisms of PAH, since they affect vascular function and cause pulmonary vasoconstriction (228, 229). The role of mitochondria in vascular cells is, at least partially, responsible for the proliferative and antiapoptotic features seen in PAH (229).

Moreover, mitochondria possess the capacity to control the dispersion of reactive oxygen species originating from the mitochondria, impacting their distribution to the cytoplasm and plasma membrane. This, in turn, stimulates redox-sensitive targets like Hif-1 or the NFAT signaling pathway, ultimately resulting in the contraction of pulmonary arterial smooth muscle cells (229).

The transmission of mitochondrial DNA to offspring suggests that female mitochondria are responsive to the changes in their environment that are subsequently inherited by the next generation (230).

Emerging evidence indicates that estrogen may influence the epigenetic modifications involved in the pathophysiology of PAH through mechanisms involving miRNA and lncRNA. Research indicates that estradiol engages in various genomic and non-genomic processes via estrogen receptor alpha (ERα) and beta (ERβ) (231). ERα binds to regulatory regions of target genes, recruiting co-regulatory proteins, and inducing chromatin modifications that can enhance or suppress gene transcription. This enables estrogen to regulate the expression of various miRNAs implicated in PAH (232). In a study by Wallace et al., the researchers examined whether miRNAs expressed in PASMCs are affected by estrogens and play a role in PASMC proliferation. They found that estradiol downregulates the expression of miR-96, which regulates the 5-hydroxytryptamine 1B (5-HT1B) receptor, a mediator of pulmonary artery SMCs. Decreased miR-96 expression was observed in PASMCs of female BMPR II mutant mice and female PAH patients, leading to increased 5-HT1B expression and 5-HT-driven proliferation (233).

Additionally, a study by Mair et al. explored the role of sex, estrogen, and BMPR-II protein mutation in PAH. The study demonstrated that estrogen-driven suppression of mRNA, BMPR-II, and Smad1 signaling in PASMCs of non-PAH females contributed to a pro-proliferative phenotype in hPASMCs, potentially predisposing women to PAH. Furthermore, small interfering RNA (siRNAs) silencing of Smad1 revealed proliferative responses to BMP4 in male PASMCs, with estrogen decreasing messenger RNA and protein expression of Id genes, which are involved in BMPR-II signaling (234). While the role of estrogen in PAH pathophysiology is well-documented, the extent to which estrogen influences PAH and the right ventricle remains unclear.

Considering the heightened severity of PAH due to the interaction of intricate genetic and epigenetic factors, identifying novel therapeutic avenues by investigating the epigenetic mechanisms involved in PAH pathogenesis is of growing interest (86). Over the years, studies have highlighted the role of epigenetic modifications, such as altered DNA methylation and histone modification, in the pathogenesis of PAH (7–9). Most research on the epigenetics of PAH has been conducted in vivo, necessitating further studies to understand their implications in the clinical management of PAH. Current therapies for PAH target the imbalance between vasoactive and vasodilatory mediators in PAEC, aiming to mediate vasoconstriction through vasodilatory properties. These therapies target three key pathways and are classified into four classes: endothelin receptor antagonists, phosphodiesterase type 5 inhibitors, soluble guanylate cyclase stimulators, and prostacyclin analogs. These therapies are summarized in Figure 4, and some of are believed to modulate epigenetic modifications observed in PAH.

The complexity of PAH etiopathogenesis cannot be overstated, with its disease state involving multifactorial pathogenesis contributing to its variable expression. In recent years, growing attention has been directed toward the role of epigenetic mechanisms in PAH through both experimental and clinical studies. These investigations illuminate pathogenic mechanisms and uncover potential therapeutic and curative targets.

Epigenetic modifications are emerging as a critical factor in the pathogenesis of PAH, integrating various risk factors, including environmental exposures and modulating genetic expression (114). Despite significant progress in understanding epigenetics, the identification of clinically therapeutic epigenetic treatments remains challenging. Most of the identified epigenetic therapeutic targets have been tested in animal models.

Further research is crucial to enhance our understanding of epigenetic mechanisms, as robust data suggest that epigenetic modulators hold great promise as emerging targets for PAH therapy. Additionally, ongoing research will contribute to a better understanding of the safety profile of new treatments and their impact on other gene targets.