2.1 Bone Morphogenetic Protein Receptor Type 2

Bone morphogenetic protein receptor type 2 (BMPR2) gene plays an important role in PAH (Figure 2A) [21, 22]. Loss of BMPR2 enhances the proliferation response of endothelial cells to bone morphogenetic protein (BMP) 9 by delaying the phosphorylation of Smad proteins and extending the induction of inhibitor of DNA binding [23]. The reduction of BMPR2 mediated by tumor necrosis factor-α (TNF-α) induced BMP6 signaling, which preferentially activates ALK2 (activin receptor-like kinase 2)/ACTR-IIA (activin a receptor type 2A) signaling axis and upregulates SRC proto-oncogene, nonreceptor tyrosine kinase-family kinases. Both pathways induce notch receptor (NOTCH) 2 while inhibiting NOTCH3, thereby promoting the proliferation of PASMCs in heritable pulmonary arterial hypertension (HPAH) [24]. BMPR2 deficiency resulting from a BMPR2 mutation leads to the upregulation of the phosphorylated extracellular signal-regulated kinase–phosphorylated mitogen-activated protein kinase (MAPK)–SMAD2/3 signaling pathway. This, in turn, increases the levels of arrestin beta 2 (ARRB2). As a consequence, ARRB2 elevates catenin beta (CTNNB1) by enhancing phosphorylated AKT serine/threonine kinase 1 activity, which promotes PASMC proliferation via ATP-binding cassette (ABC) activation. Simultaneously, this process reduces the contractility of PASMCs by downregulating ras homolog family member A (RhoA), Rac family small GTPase 1, and transgelin [25]. Additionally, the BMPR2 mutation reduces BMPR2 levels and decreases IL (interleukin)-15Rα surface presentation by affecting the trans-Golgi network, which reduces natural killer (NK) cell numbers and exacerbates PH [26]. In PAH-PASMCs with BMPR2 loss, β-catenin drives an increase in aldehyde dehydrogenase 1 family member A3 gene expression, resulting in elevated levels of acetyl coenzyme A (acetyl CoA) in the nucleus. This rise in acetyl CoA, in conjunction with lysine acetyltransferase 2B (KAT2B), leads to the acetylation of lysine 27 on histone 3 (H3K27) at the β-catenin/TCF (T-cell factor)/LEF1 (lymphoid enhancer-binding factor 1) binding site of the nuclear transcription factor Y subunit α (NFYA) gene, thereby increasing the expression of NFYA. As a result, NFYA binds to KAT2B and stimulates cellular proliferation and glucose metabolism by promoting the expression of genes associated with the cell cycle and glucose metabolism, including cyclin B1, cyclin A2 (CCNA2), and dihydrolipoamide dehydrogenase [27].

2.2 Caveolin-1

Caveolin-1 (Cav-1), encoded by CAV1, is a component of the caveolae in the cell membrane [28], which has been shown to be associated with many lung diseases, including PH (Figure 2B) [29]. Inducible nitric oxide synthase and NADPH oxidase 1 (NOX1) colocalize with Cav-1 to produce the oxidant molecule peroxynitrite, which inhibits the transient receptor potential cation channel subfamily V member 4 through cysteine modifications on Cav-1. This inhibition reduces calcium influx mediated by this channel, which diminishes vasodilation and ultimately contributes to PH [30]. The peroxisome proliferator-activated receptor (PPAR) γ agonist GW1929 activated PPARγ in a Cav-1-dependent manner, leading to increased phosphorylation of p38 and ERK1/2, which promoted apoptosis and inhibited the proliferation of PASMCs. Additionally, GW1929 significantly reduced the hypoxia-induced upregulation of Cav-1, demonstrating a negative feedback mechanism in this pathway [31]. Additionally, there are associations between Cav-1 and BMPR2 in the pathogenesis of PH. Vascular injury triggers the shedding of microvesicles and small apoptotic bodies from epithelial cells, which enhances transforming growth factor-β (TGF-β) production by macrophages and subsequently upregulates pSMAD2/3. This shedding also leads to the depletion of Cav-1, resulting in reduced expression of BMPR2, ultimately contributing to vascular remodeling and the development of PAH [32]. Caveolae associated protein 1 (Cavin-1) competes with BMPR2 for binding to the caveolin scaffolding domain of Cav-1 at the cell membrane. Consequently, increased levels of Cavin-1 or the knockdown of Cav-1 facilitate the translocation of BMPR2 from the membrane to the cytoplasm. This translocation inhibits the phosphorylation of Smad1/5/9, promoting pulmonary vascular remodeling and ultimately contributing to the development of PH [33]. In the lungs of mice infected with Schistosoma mansoni, hyperphosphorylation of Cav-1, along with reduced expression of Cav-1 and BMPR2, was observed. This condition led to increased apoptosis and vascular remodeling in the lungs [34].

2.3 Serotonin Transporter

The serotonin transporter (SERT) gene plays a critical role in the genetic landscape of PH, affecting disease onset, severity, and progression through its promoter polymorphisms and pathway interactions. In both familial and idiopathic forms of PAH, the SERT promoter polymorphism modulates PH risk. The long (L) allele, linked to increased SERT transcription, is associated with earlier onset in familial PAH (FPAH), particularly among patients with BMPR2 mutations, suggesting a genetic interaction between SERT and BMPR2 that may accelerate PAH onset in FPAH [35]. However, while the L allele has been linked to FPAH onset, it has not shown a significant effect on survival rates in either FPAH or idiopathic PAH (IPAH) cases [35]. Furthermore, SERT polymorphisms are not associated with the risk of portopulmonary hypertension in patients with advanced liver disease [36]. Genetically, SERT contributes to PH progression by interacting with the platelet-derived growth factor receptor β (PDGFRβ), which promotes PASMC proliferation. Experimental studies have shown that SERT transactivates PDGFRβ in serotonin-stimulated PASMCs, indicating a genetic predisposition that impacts platelet-derived growth factor (PDGF) signaling pathways in PH [37]. Inhibiting or genetically downregulating SERT attenuates PDGF-stimulated PASMC proliferation, with evidence suggesting that the presence of the PDZ motif in SERT is essential for PDGF-induced Akt phosphorylation, underscoring a specific genetic requirement for SERT's regulatory impact on PH [38]. SERT also appears to play a significant role in the higher prevalence of PAH observed in females [38]. In female SERT-overexpressing (SERT+) mice, microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses have shown upregulation of genes associated with PAH, including CCAAT/enhancer binding protein β (CEBPβ), cytochrome P450 family 1 subfamily B member 1 (CYP1B1), and Fos proto-oncogene, AP-1 transcription factor subunit (FOS). Protein expression of these genes was also elevated, indicating a genetic predisposition toward PAH in females [39]. In PASMCs from IPAH patients, similar upregulation of CEBPβ, CYP1B1, and FOS was observed, suggesting parallels in human PAH. This sex-related genetic susceptibility is further supported by findings that 17β-estradiol promotes PAH development in female SERT+ mice through upregulation of SERT, tryptophan hydroxylase-1 (TPH1), and the 5-HT(1B) receptor. Ovariectomy or inhibition of TPH1 or the 5-HT(1B) receptor significantly reduces this PAH phenotype, highlighting estrogen's genetic influence on serotonin pathways in PAH [39]. While studies on SERT allele distributions (LL, LS, SS) in PAH patients compared with the general population have shown no significant differences [40], a meta-analysis suggests that the LL genotype in the SERT L/S polymorphism may increase the risk of developing IPAH, as individuals with this genotype are at higher risk than those with the SS genotype [41].

2.4 Aquaporin-1

Research shows that aquaporin-1 (AQP1) variants are present in two unrelated families with PAH, one hereditary and one associated with scleroderma, identified from a cohort of 300 patients [42]. A family affected by HPAH was identified, with all three siblings carrying a novel missense variant of AQP1 (c.273C>G; p.Ile91Met) [43]. Whole-genome sequencing in PAH patients identified rare variants in AQP1, along with SOX17 (SRY-box transcription factor 17) and ATP13A3 (ATPase 13A3), suggesting a significant role for AQP1 in PAH genetics. Familial segregation of AQP1 mutations with PAH further underscores its involvement in the disease's molecular mechanisms [44]. In mice, Aqp1 deficiency attenuates vascular remodeling in hypoxic PH by reducing abnormal proliferation and migration of PASMCs, while also protecting lung endothelial cells from apoptosis [45]. In hypoxic PH, AQP1 is expressed in PASMCs and plays a critical role in migration. Hypoxia selectively increases AQP1 protein levels and intracellular calcium concentration ([Ca²⁺]і) in PASMCs. Blockade of calcium entry and AQP1 silencing inhibit this hypoxia-induced migration, indicating that AQP1’s genetic expression and function are pivotal in PASMC responses to hypoxia in pulmonary vascular remodeling [46]. AQP1 is essential for proliferation and migration in PASMCs, with its C-terminal tail being crucial for these processes. The EF-hand motif is not required for AQP1's role in hypoxia-induced responses in PASMCs [47]. In hypoxic PH, AQP1 enhances PASMC proliferation and migration by upregulating β-catenin levels, which in turn increases the expression of β-catenin targets like c-Myc and cyclin D1. This mechanism requires the AQP1 COOH-terminal tail, as deletion of this region abolishes the effects on β-catenin and its target genes [48]. Additionally, increased expression of AQP1 in PASMCs from preclinical PH models contributes to apoptosis resistance by modulating B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX) expression ratios. Elevating AQP1 levels in control PASMCs also conferred resistance to apoptosis, indicating a crucial role for AQP1 in regulating cell fate in PH [49]. In a Taiwanese idiopathic and heritable PAH cohort, some patients carried BMPR2 gene variants, while others had AQP1 and other PAH-related gene variants, suggesting a potential link between these genetic factors in the pathogenesis of PAH [50]. In PAH, silencing of BMPR2 in human pulmonary microvascular endothelial cells leads to reduced AQP1 expression at the mRNA, protein, and functional levels. This finding suggests that decreased BMPR2 may contribute to AQP1 dysfunction, highlighting a potential mechanism involved in PAH pathogenesis [51]. Silencing the AQP1 gene increased the expression of BMP9 and BMP10 in human pulmonary microvascular endothelial cells, indicating a regulatory role for AQP1 within the BMP signaling pathway. Furthermore, exogenous BMP9 administration restored decreased AQP1 protein levels, highlighting its potential therapeutic implications in PAH [52]

2.5 ATPase 13A3

A study identified ATPase 13A3 (ATP13A3) variants as one of the most frequent genetic contributors to PAH in Asian cohorts, alongside BMPR2 and growth differentiation factor 2 (GDF2) [53]. Rare variants in the ATP13A3 gene have been validated as contributing to approximately 2.7% of PAH cases, alongside other channelopathy genes [54]. A multistep genetic screening identified ATP13A3 as a novel candidate gene associated with PAH through its involvement in lung vascular remodeling [55]. Additionally, gene panel diagnostics identified disease-causing variants in ATP13A3 in families with at least two PAH patients [56]. Whole-genome sequencing in PAH cases revealed a significant overrepresentation of rare variants in ATP13A3, suggesting its potential role in the disease's genetic basis [44]. In a study of childhood-onset PAH, whole exome sequencing revealed a novel heterozygous mutation in the ATP13A3 gene as a genetic risk factor [57]. In a case involving a 21-month-old patient, biallelic mutations in the ATP13A3 gene were linked to malignant progression of PAH, prompting a high-risk Potts shunt procedure [58]. Biallelic variants in the ATP13A3 gene are associated with autosomal recessive childhood-onset PAH, characterized by high mortality and treatment resistance. The findings suggest a likely semidominant, dose-dependent inheritance for ATP13A3 in PAH cases [59]. In a study of multiple sclerosis patients treated with interferon beta 1a (IFN-β 1a), a nonsense variant in the ATP13A3 gene was identified in one patient who developed PAH. This suggests that genetic predisposition involving ATP13A3 may increase the risk of PAH in patients undergoing IFN-β 1a therapy, warranting further investigation [60]. ATP13A3 is identified as a crucial polyamine transporter in endothelial cells, where its deficiency leads to impaired polyamine homeostasis and endothelial dysfunction, contributing to PAH. Additionally, specific PAH-associated ATP13A3 variants demonstrate loss-of-function effects, and mice with an ATP13A3 variant developed a PAH phenotype, highlighting its role in disease pathogenesis [61].

2.6 SRY-Box Transcription Factor 17

SOX17 mutations are significantly associated with PAH, showing familial segregation with the disease and suggesting a key role in its heritability [44, 62–64]. Pathogenic variants in SOX17, though less common than other genes associated with HPAH, have been identified in patients, highlighting its role in disease susceptibility [65]. A novel heterozygous mutation in SOX17 (c.379C>T; p.Gln127*) was identified in a consanguineous family with autosomal-dominant PAH, exhibiting complete penetrance and loss of function [66]. In a case study of four Japanese patients with PAH, SOX17 mutations were identified, supporting SOX17 as a novel causative gene for PAH [67]. SOX17 has been identified as a novel risk gene for PAH, with rare deleterious variants contributing to 3.2% of PAH cases with CHD and 0.7% of idiopathic/familial PAH cases. These variants, particularly in the conserved HMG-box domain, implicate SOX17 and its targets in the development of pulmonary vasculature, underscoring its importance in PAH pathogenesis [68-70]. Moreover, genome-wide association studies identified a locus near SOX17 associated with increased risk for PAH, showing enhancer activity that affects SOX17 regulation in endothelial cells. Risk variants near SOX17 indicate that impaired SOX17 function is a significant factor in PAH, extending beyond rare mutations and impacting gene expression through common genetic variations [71, 72]. Another genome-wide association study found that PAH-related stimuli upregulate SOX17-mediated NAMPT promoter activity in endothelial cells, which contributes to vascular remodeling in PAH [73]. SOX17 expression is significantly downregulated in patients with IPAH, and its deficiency in endothelial cells exacerbates PH via E2F1 (E2F transcription factor 1)-mediated dysfunctions [74]. SOX17 deficiency is associated with increased PAH risk, with the genetic variant rs10103692 linked to reduced plasma citrate levels in PAH patients. Additionally, the downregulation of SOX17 by the pathological estrogen metabolite 16α-hydroxyestrone may contribute to PAH development, linking sexual dimorphism and SOX17 genetics in PAH [75, 76]. In mice, Sox17 deficiency in pulmonary endothelial cells promotes the development of PAH under hypoxia by enhancing hypermuscularization and inflammation, while also increasing hepatocyte growth factor/mesenchymal–epithelial transition factor signaling [77]. The biomimetic model of PAH reveals a critical BMPR2–SOX17–prostacyclin (PGI2) signaling axis, highlighting SOX17's role in endothelial-smooth muscle cell interactions and the remodeling process [78]. A study identified SOX17 as a crucial transcription factor mediating BMP9-induced semaphorin 3G expression, linking it to endothelial function and vascular regulation [79]. SOX17 plays a crucial role in maintaining endothelial function and vascular homeostasis in PH by regulating exosomal miRNAs that mitigate endothelial injury and promote vascular health [80]. Genetic risk variants upstream of the SOX17 promoter impair transcription factor binding, leading to reduced SOX17 expression in human PAECs (HPAECs) and contributing to endothelial dysfunction in PAH. A study identified homeobox A5 (HOXA5) and retinoic acid receptor-related orphan receptor-α as key transcription factors involved, with potential drug compounds capable of reversing the negative transcriptomic effects associated with SOX17 dysregulation [81]. In pediatric-onset PAH, rare deleterious variants in SOX17 are significant genetic contributors, distinct from adult-onset PAH, and associated with developmental origins of the disease [82-85].

2.7 Kallikrein 1

Kallikrein 1 (KLK1) is identified as a novel candidate risk gene for IPAH, linked to a later mean age of onset and relatively moderate disease phenotypes. This discovery underscores KLK1’s role in vascular hemodynamics and inflammation, suggesting its potential as a therapeutic target in PAH [86]. However, KLK1 is classified as having limited evidence for a causal relationship with PAH, suggesting that while it may contribute to the disease, more research is needed to establish its definitive role. Consequently, caution should be exercised in interpreting variants identified in KLK1 during genetic testing for PAH [87].

2.8 Vascular Endothelial Growth Factor Receptor 2

While vascular endothelial growth factor receptor 2 (KDR) is expressed in endothelial cells within plexiform lesions in PH, indicating its role in the disease's pathophysiological changes [88], Fawn-Hooded rats (FHR) raised at Denver's altitude showed comparable KDR expression to that of normal Sprague–Dawley and Fischer rat strains. This suggests that KDR is not directly implicated in the reduced pulmonary arterial density observed in this model of PH [89]. In previous studies, KDR was identified as a candidate gene associated with PAH, specifically IPAH based on the presence of rare deleterious variants [84, 87, 90]. A study identified heterozygous, high-impact, likely loss-of-function variants in the KDR gene that were strongly associated with a reduced carbon monoxide transfer coefficient and an older age at diagnosis of PAH [91]. In mice, the lack of endothelial nitric oxide synthase (eNOS) and exposure to bleomycin led to decreased expression of phosphorylated VEGFR2 (KDR), suggesting that the disruption of the VEGF (vascular endothelial growth factor) signaling pathway contributes to PAH and impaired alveolar development. This highlights the importance of KDR in the vascular remodeling and pathophysiological processes associated with PH in the context of bronchopulmonary dysplasia [92]. In a study investigating the genetic basis of PAH, researchers found that mutations in the KDR gene were associated with severe PAH in two families, characterized by low diffusing capacity for carbon monoxide (D_LCOc) and interstitial lung disease. This evidence supports the classification of KDR as a newly identified gene implicated in the development of PAH, particularly in cases presenting with radiological lung parenchymal disease [93]. The conditional deletion of the KDR gene, which encodes VEGF receptor-2, in mice, led to mild PH under normoxia that worsened under hypoxia, characterized by increased pulmonary arterial wall thickness and vessel occlusion [94].

2.9 T-Box Transcription Factor 4

Studies identify T-box transcription factor 4 (TBX4) as a potential risk gene for PH [95-105]. Research indicates that TBX4-related PAH tends to have a milder progression, with late diagnosis being the sole predictor of negative outcomes in its hereditary forms [106]. In a case report, the identification of a microdeletion at 17q22q23.2 highlights the haploinsufficiency of the TBX4 gene, which may play a significant role in the observed phenotypic features, particularly PH [107, 108]. A de novo missense mutation in the TBX4 gene (p.E86Q) was identified in a deceased newborn with severe acinar dysplasia of the lungs, suggesting a novel association between TBX4 mutations and this condition [109]. An intronic TBX4 variant near the splice site of exon 3 led to significantly reduced TBX4 expression, associated with a spectrum of cardiopulmonary symptoms in affected family members. This case highlights the variable expressivity of TBX4 mutations, linking them to both severe neonatal lung disease and milder adult phenotypes [110]. TBX4 mutations were identified in a notable proportion of children with childhood-onset PAH, while only a few mutations were found in adults with PAH, suggesting a stronger association between TBX4 and childhood-onset PAH [85, 97, 111–116]. Additionally, many patients with TBX4 mutations exhibited previously unrecognized small patella syndrome (SPS) [111]. Moreover, in a study of 40 children with IPAH or familial PAH, three mutations in the TBX4 gene were identified, highlighting its role in the genetic architecture of pediatric PAH. TBX4 mutations were associated with more severe disease at diagnosis, though the overall outcomes for mutation carriers were similar to those of noncarriers [117]. In a study, rare variants in the TBX4 gene, including six deletions at the 17q23 locus and 12 likely damaging mutations, were identified as associated with a new form of developmental lung disease in children with PH. These variants contribute to severe, often biphasic PH, along with other congenital anomalies, highlighting TBX4's significant role in pediatric PH [118]. In another study, a paternally inherited heterozygous missense variant in the TBX4 gene (c.1198G>A, p.Glu400Lys) was identified in a newborn with severe PH and other developmental abnormalities, suggesting its likely deleterious role. The findings indicate that TBX4 variants, in conjunction with CTNNB1 mutations, may synergistically contribute to a more severe phenotype in lethal lung developmental diseases [119]. In the case of a deceased newborn with PH and interstitial emphysema, two de novo heterozygous recurrent copy-number variant deletions were identified, including one affecting the TBX4 gene at 17q23.1q23.2. The study also detected seven novel regulatory noncoding single nucleotide variants upstream of TBX4, indicating a complex interplay between noncoding and coding variants that may influence lung development and contribute to lethal lung developmental disorders [120]. According to a study, the rs3744439 variant in the TBX4 gene was significantly associated with susceptibility to PH, with its AG genotype exclusively found in healthy controls and absent in PH patients [121]. A study suggests that TBX4’s involvement alongside known PAH-related genes like BMPR2 indicates its potential significance in the genetic landscape of PAH in the Middle East and North Africa region [122]. Variants in the TBX4 gene were identified as potential genetic causes of PAH in a subset of Japanese patients, disrupting its regulation of fibroblast growth factor (FGF) 10 and leading to insufficient lung morphogenesis. These findings suggest that TBX4 variants contribute to PAH pathogenesis through impaired pulmonary vascular development and endothelial dysfunction [123]. A preclinical study found that TBX4 is epigenetically reactivated in PAH, contributing to vascular remodeling, and silencing TBX4 showed potential therapeutic effects in rodent models [124].

2.10 Eukaryotic Translation Initiation Factor 2 Alpha Kinase 4

Research highlights TBX4 as a possible risk gene associated with PH [55, 125–132]. Recessive mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4) gene, in either homozygous or compound-heterozygous states, are strongly associated with PH, specifically in the context of pulmonary veno-occlusive disease (PVOD), where they disrupt EIF2AK4 function and contribute to disease progression [133, 134]. PVOD is an uncommon cause of PAH that can be categorized as idiopathic, heritable, drug-induced, radiation-induced, or associated with connective tissue disorders [135]. A sporadic PVOD patient with confirmed biallelic EIF2AK4 mutations displayed an atypical treatment response to PAH-targeted drugs, suggesting genetic heterogeneity in PVOD [136]. Pulmonary capillary hemangiomatosis (PCH) is a rare condition marked by abnormal capillary growth in the alveolar interstitium, exhibiting similarities to PVOD, both of which are associated with PH [137]. In a whole-exome sequencing study, two novel pathogenic variants in the EIF2AK4 gene (c.2137_2138dup and c.3358-1G>A) were identified in a patient diagnosed with PVOD/PCH [138]. While studies have associated EIF2AK4 genetic mutations with PVOD/PCH [139-148], in a large cohort study, biallelic EIF2AK4 mutations were identified in patients clinically diagnosed with idiopathic and heritable PAH, suggesting a misclassification of some cases that resemble PVOD/PCH. These patients exhibited distinct characteristics, such as a low carbon monoxide transfer coefficient (Kco) and earlier onset, underscoring the value of genetic testing for accurate diagnosis and management [149]. In a study, bi-allelic mutations in the EIF2AK4 gene were identified, including a novel nonsense mutation (c.1672C>T) and a documented deletion (c.560_564drlAAGAA), in two nonconsanguineous families with PH. These findings underscore the involvement of EIF2AK4 mutations in the diverse phenotypic expressions of PH, with differing clinical outcomes observed between symptomatic and asymptomatic individuals [150]. In a semiconductor chip-based next-generation sequencing study of PH patients, a homozygous EIF2AK4 variant (p.P1115L) was identified, highlighting its role in PH genetic susceptibility [151]. Also, in Iberian Gypsy families with severe PAH, homozygous EIF2AK4 mutation (c.3344C>T, p.P1115L) was identified, showing an aggressive phenotype and necessitating early lung transplantation [152]. In a study, a novel EIF2AK4 splice site mutation was identified alongside a BMPR2 mutation in family members with autosomal dominantly inherited HPAH, supporting a “second hit” model. The presence of both mutations led to manifest HPAH, suggesting EIF2AK4 as a potential modifier of PAH risk in conjunction with BMPR2 [153]. A study showed that the EIF2AK4 gene encodes general control nonderepressible 2 kinase (GCN2), which mediates pulmonary vascular remodeling and PAH. Notably, loss of GCN2 activity did not induce PVOD or PAH in mice, suggesting a nuanced role for EIF2AK4 in these conditions. GCN2 activation was linked to hypoxia-induced endothelin-1 expression, indicating its potential as a therapeutic target in PAH without EIF2AK4 mutations [154].

2.11 ATP Binding Cassette Subfamily C Member 8

ATP binding cassette subfamily C member 8 (ABCC8) is a member of the C branch of the ABC transporter superfamily, encoding sulfonylurea receptor 1 (SUR1), an ATP-sensitive potassium channel regulatory subunit [54, 62]. ABCC8 is identified as one of the most common causes of PAH, accounting for approximately 1% of cases [54]. In the US-PAH biorepository, which includes 2572 PAH cases subjected to whole exome sequencing, predicted deleterious missense variants in ABCC8 were identified in 28 patients [86]. The International PH Expert Panel employed a semi-quantitative scoring system developed through the NIH Clinical Genome Resource to classify the relative strength of evidence supporting gene-disease relationships for PAH based on genetic and experimental data, ABCC8 was categorized as having moderate evidence of clinical relevance to PAH [87].

Through exome sequencing of 99 pediatric and 134 adult PAH cases, 11 novel or rare missense mutations located in conserved regions of ABCC8 were identified: c.A214G (p.N72D), c.G558T (p.E186D), c.G718A (p.A240T), c.G2371C (p.E791Q), c.T2694+2G, c.G331A (p.G111R), c.C403G (p.L135 V), c.G2437A (p.D813N), c.G4414A (p.D1472N), c.C686T (p.T229I), and c.G3941A (p.R1314H). These mutations contribute to the loss of K_ATP channel functionality and are associated with PAH [155]. Additionally, genetic sequencing of 624 PAH cases from the Spanish national registry revealed 11 further variants in the ABCC8 gene [156]. Following computational and in vitro biochemical analyses, Lago-Docampo et al. [156] classified two variants as pathogenic (c.3288_3289del and c.3394G>A), six as likely pathogenic (c.211C>T, c.1429G>A, c.1643C>T, c.2422C>A, c.2694+1G>A, and c.3976G>A), and three as variants of unknown significance (c.298G>A, c.2176G>A, and c.3238G>A). A novel heterozygous missense variant 21 was observed in a 19-month-old male patient with PAH [57].

Mutations in ABCC8 may impair the K_ATP-independent effects of SUR1 that induce cellular apoptosis, potentially promoting the intimal proliferation or excessive growth of the intima observed in PAH. Pharmacological activation of SUR1 using three distinct activators: diazoxide, VU0071063, and NN414 results in vasorelaxation of pulmonary arteries, while inhibition of the SUR1/Kir6.2 channel is associated with vasoconstriction [157]. Moreover, metformin has been shown to selectively activate ABCC8, thereby restoring channel function [158].

2.12 Gamma-Glutamyl Carboxylase

Gamma-glutamyl carboxylase (GGCX), also known as vitamin K-dependent gamma-carboxylase, plays a critical role in the vitamin K cycle by catalyzing the gamma-carboxylation of glutamic acid residues in vitamin K-dependent proteins [159]. GGCX is involved in various biological processes, including inflammation, bone metabolism, blood coagulation, vascular calcification, and cell proliferation [160-162]. Recent studies have suggested a potential link between GGCX and PAH (Table 1). Mutations in GGCX have been classified by an international expert panel on PAH as having moderate evidence of clinical relevance to the disease [87]. Notably, Zhu et al. [86] conducted whole exome sequencing of 2572 PAH patients from a national biorepository, identifying 28 cases with GGCX variants. They hypothesized that these mutations may alter the inflammatory responses or cell proliferation in PASMCs or PAECs, both of which are hallmarks of PAH [86].

3.1 RNA Methylation

Recent studies have underscored the pivotal role of RNA methylation, specifically N6-methyladenosine (m6A), in PH pathogenesis (Figure 3A) [175-179]. This type of methylation plays a crucial role in regulating mRNA stability, translation, and degradation, influencing various cellular processes, including proliferation, apoptosis, pyroptosis, and EndMT in the context of PH. Using bioinformatics analysis, Gao et al. [175] identified key m6A RNA methylation regulators, revealing that fragile X messenger ribonucleoprotein 1 and heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 are upregulated in IPAH, while leucine-rich pentatricopeptide repeat containing is downregulated. Experimental validation in human PASMCs (HPASMCs) indicates that m6A modification may significantly influence the immune microenvironment and progression of IPAH [175].

While RNA methyltransferase methyltransferase 3 (METTL3) is upregulated in response to hypoxia, promoting vascular remodeling by influencing the phenotypic switching of PASMCs [180, 181], research demonstrates that low sustained expression of METTL3 also facilitates PH by affecting the m6A modification of PH-related genes [181-184]. For instance, targeted deletion of METTL3 exacerbates hypoxia-induced PH and enhances PASMC phenotypic switching through decreased m6A modification and impaired processing of pri-miR-143/145. This leads to reduced levels of miR-143-3p and miR-145-5p, resulting in the upregulation of Krüppel-like factor 4 (KLF4), which further suppresses miR-143/145 transcription. This positive feedback loop sustains the contractile marker gene suppression and PASMC phenotype changes [181]. Additionally, in HPAECs, the global levels of m6A and METTL3 expression decrease during TNF-α- and TGF-β1-induced EndMT, promoting a shift toward mesenchymal markers and exacerbating pulmonary vascular remodeling and hypoxia-induced PH in mice. METTL3-mediated m6A modification of KLF2 is critical for maintaining endothelial characteristics; its overexpression counteracts EndMT by upregulating endothelial markers and downregulating mesenchymal markers, while mutations in the m6A site of KLF2 mRNA impair its protective effects [183]. Jiang et al. [184] showed that the downregulation of METTL3 in PH leads to decreased m6A methylation, promoting pyroptosis in PASMCs. Mechanistically, METTL3 modulates the stability of phosphatase and tensin homolog (PTEN) mRNA through m6A modification and interaction with the m6A reader protein insulin-like growth factor 2 mRNA-binding protein 2 [184].

The m6A reader protein YTH N6-methyladenosine RNA binding protein F1 (YTHDF1) promotes PH through the regulation of MAGE family member D (MAGED1) translation in an m6A-dependent manner [185]. Another study showed that YTHDF1 enhances Forkhead box M1 (FoxM1) translation efficiency, further driving hypoxia-induced PH [186]. Moreover, YTH N6-methyladenosine RNA binding protein C1 (YTHDC1)-mediated m6A modifications were found to induce the degradation of the lncRNA FOXF1 adjacent noncoding developmental regulatory RNA (FENDRR), a process that contributes to hypoxia-induced PH. FENDRR plays a key regulatory role by forming an RNA–DNA triplex at the promoter of dynamin-related protein 1 (DRP1), increasing promoter methylation and thereby reducing DRP1 transcription [187]. Additionally, YTHDF2 is upregulated in PASMCs during PH and promotes cell proliferation by stabilizing myeloid-associated differentiation marker (MYADM) mRNA in an m6A-dependent manner, while also inhibiting cyclin-dependent kinase inhibitor 1A (p21) [188]. In hypoxia-induced PH, the upregulation of AlkB homolog 5 (Alkbh5) is associated with a decrease in m6A modifications, affecting the stability of cytochrome P450 family 1 subfamily A member (CYP1A1) mRNA, and consequently impacting PASMC proliferation and migration [189].

Recent findings indicate that eukaryotic initiation factor 2α (eIF2α) is significantly upregulated in monocrotaline (MCT)-PAH rats, with its expression linked to m6A methylation. Elevated levels of METTL3, WT1-associated protein (WTAP), and YTHDF1 contribute to this m6A modification, promoting PASMC proliferation and exacerbating PAH. The eIF2α inhibitor GSK2606414 can reduce eIF2α expression and alleviate PASMC proliferation, suggesting a novel regulatory mechanism where m6A modification of eIF2α plays a critical role in PAH pathogenesis [190].

These mechanisms highlight how m6A-mediated RNA methylation promotes PH development by influencing the epigenetic regulation of critical genes under hypoxic conditions, suggesting that m6A modifications play a crucial role in modulating PH pathogenesis, particularly through hypoxia-related pathways.

3.2 DNA Methylation and Demethylation

DNA methylation is a vital epigenetic mechanism that plays a crucial role in regulating gene expression by adding a methyl group to cytosine residues within CpG dinucleotides. This process is primarily mediated by DNA methyltransferases (DNMTs), which control gene activity by either silencing or activating specific genetic regions. In mammalian cells, this process contributes to gene repression via various pathways, including hindering transcription factor binding, recruiting methyl-CpG binding proteins that interfere with transcriptional regulators, or altering chromatin structure to limit DNA accessibility. While the majority of CpG sites in healthy somatic cells are methylated, promoter regions, particularly CpG islands, tend to be protected from this modification [191-194]. On the other hand, DNA demethylation is the process of removing these methyl groups, which can lead to the reactivation of silenced genes. Demethylation can occur through passive mechanisms, such as during DNA replication when methylation patterns are not copied, or through active mechanisms involving specific enzymes like TET (ten-eleven translocation) proteins, which convert 5-methylcytosine to 5-hydroxymethylcytosine, eventually leading to complete demethylation [195-197]. Together, DNA methylation and demethylation are crucial in physiological processes such as embryonic development, genomic imprinting, and X-chromosome inactivation. However, aberrant methylation patterns, whether through hypermethylation or hypomethylation, have been implicated in a growing number of diseases, including PH. Emerging evidence suggests that dysregulated DNA methylation contributes to PH pathogenesis (Figure 3B).

Reduced representation bisulfite sequencing identified 631 differentially methylated CpG sites in circulating CD4⁺ T cells from PAH patients, highlighting the suppressor of cytokine signaling 3 as a key gene with hypomethylation associated with adverse hemodynamic parameters [198]. In human studies, DNA methylation patterns have been shown to differentiate between PVOD and PAH. For instance, increased methylation of the granulysin gene was observed in patients with PVOD but not in those with PAH, suggesting that DNA methylation signatures could serve as potential biomarkers for distinguishing between these two conditions [199]. In a study investigating PAH, it was found that DNA methylation significantly influences the expression of superoxide dismutase (SOD) 2, a key antioxidant enzyme located in mitochondria. The research demonstrated that levels of SOD2 are significantly reduced in PASMCs taken from individuals with PAH. It also showed similar findings in FHR, a strain known to be susceptible to spontaneous PAH, which serves as a valuable model for studying the disease [200, 201]. The reduction in SOD2 expression leads to increased activation of hypoxia-inducible factor-1 (HIF-1), resulting in elevated expression of voltage-gated potassium channels (Kv1.5), which are sensitive to oxygen levels. This cascade of changes impairs oxygen sensing and negatively affects redox status in both the cytoplasm and mitochondria [200]. Additionally, the suppression of SOD2 transcription and translation using small interfering RNA (siRNA) in normal PASMCs from Sprague–Dawley rats produces phenotypic features characteristic of PAH. Conversely, restoring SOD2 expression diminishes HIF-1 levels and reinstates Kv1.5 expression in PASMCs from FHR [201]. Genomic bisulfite sequencing has identified a specific hypermethylated CpG island in the promoter and enhancer regions of the SOD2 gene [201]. Notably, elevated DNA methylation levels correlate with increased expression of DNMT1 and DNMT3B in PASMCs from both PAH patients and FHR. Moreover, employing 5-aza-2'-deoxycytidine, a DNMT inhibitor, effectively reduces DNA methylation, thereby restoring SOD2 levels, decreasing cell proliferation, and promoting programmed cell death in PASMCs derived from FHR [201].

HPAH, which accounts for 25–30% of PAH cases, further underscores the role of DNA methylation in disease development [202]. Genetic studies have identified causal mutations in several genes associated with PAH, including the bone BMPR2, TBX4, and others [202]. Among these, mutations in the BMPR2 gene are recognized as significant risk factors for PAH, with a female penetrance of 42% and a male penetrance of 14% [125]. BMPR2 is crucial in the TGF-β signaling pathway, which is involved in cellular differentiation and bone formation [203]. Interestingly, Liu et al. [204] demonstrated that hypermethylation of the BMPR2 promoter leads to decreased expression of BMPR2 in patients with HPAH. This reduction in BMPR2 levels in PAECs promotes cell proliferation and vascular remodeling, which are key drivers of PAH progression. However, contrasting findings were reported by Poussada et al. [205], who found no evidence of BMPR2 methylation in PAH patients when investigating the methylation patterns of the BMPR2 promoter region in genomic DNA isolated from peripheral blood. These discrepancies may arise from variations in patient populations and the specific regions of the BMPR2 gene analyzed [17, 204, 205]. In an epigenome-wide association study, Ulrich et al. identified significant DNA methylation changes in PAH, highlighting three hypermethylated loci: cathepsin Z (CTSZ), component of oligomeric Golgi complex 6, and zinc finger protein 678, with CTSZ showing a correlation to reduced cathepsin Z mRNA levels [206]. Hypermethylation in CTSZ was associated with increased caspase-3/7 activity, implicating its role in endothelial apoptosis and pulmonary vascular remodeling. These findings suggest that epigenetic alterations, particularly in CTSZ, contribute to the molecular mechanisms underlying PAH, alongside the limited involvement of established PAH genes like BMP10 [206]. Fine particulate matter (PM_2.5) as an environmental pollutant is related to respiratory and cardiovascular diseases [207]. Chronic PM_2.5 exposure has been shown to induce PAH by causing mitochondrial dysfunction and phenotypic switching in HPASMCs, with DNA methylation affecting the hub gene ETS transcription factor ELK3 (ELK3) [207].

In a study, Yang et al. [208] demonstrated that global hypomethylation is linked to the loss of p21, a cyclin-dependent kinase inhibitor, in the pulmonary vasculature of fetal lambs exposed to long-term hypoxia at high altitudes. This hypomethylation-induced reduction in p21 may trigger excessive proliferation of PASMCs, contributing to pulmonary vascular remodeling and the development of PH in these newborn lambs [208]. Additionally, DNA methylation was reduced in the first intron of the endothelin-1 (ET-1) gene in pulmonary vascular endothelial cells (PVECs) and sperm of the first generation and also in the PVECs of the second generation. This demethylation facilitated the intergenerational transmission of endothelial dysfunction, perpetuating the PAH-like features in offspring [209]. TNF-α has been shown to significantly decrease DNA methylation in PASMCs without altering DNMT1 activity. The increased expression of growth arrest and DNA damage-inducible α suggests a role in mediating DNA demethylation, which ultimately leads to increased PASMC proliferation. This effect was reversible with a superoxide scavenger (tempol), linking oxidative stress to DNA hypomethylation and subsequent cellular effects [210].

Furthermore, the epigenetic modification of X chromosome inactivation in females likely impacts the expression of PAH-related genes, potentially contributing to the observed sex differences in the disease [211, 212]. Carman et al. [213] demonstrated that increased expression of the lncRNA X-inactive-specific transcript (Xist) in male PAECs is linked to altered DNA methylation at the Xist/Tsix (TSIX transcript, XIST antisense RNA) locus, contributing to PAH. This upregulation of Xist, driven by an intersectin-1s protein fragment (EHITSN), leads to decreased expression of the protective gene KLF2, suggesting that Xist-mediated changes in DNA methylation may play a critical role in the sex bias and pathogenesis of PAH in males [213].

Overall, DNA methylation and demethylation are crucial epigenetic mechanisms affecting both sporadic and hereditary forms of PH. Hypermethylation of protective genes leads to their silencing, resulting in increased oxidative stress, cell proliferation, and in some cases, apoptosis. Similarly, hypomethylation of key regulatory genes also contributes to these pathological effects. This dual role of methylation in promoting both abnormal cell growth and death underscores the potential for targeting epigenetic modifications as therapeutic strategies in PAH. Further research into these methylation patterns in diverse patient populations is essential for advancing personalized epigenetic therapies.

3.3 Histone PTMs

Histones are highly conserved proteins that serve as the primary constituents of chromatin structure. The histone octamer, composed of core histone dimers including H2A, H2B, H3, and H4 encases genomic DNA, forming nucleosomes that represent the basic repeating units of chromatin [214, 215]. The arrangement of DNA around this octamer influences chromatin structure and gene expression. PTMs of histones are fundamental in regulating gene expression by either promoting or inhibiting transcriptional activity. From a chemical perspective, histone modifications can be categorized into two main groups: one involving small organic substituents (acetylation, methylation, phosphorylation, deamination, and palmitoylation), and the other involving larger organic molecules (ubiquitylation, SUMOylation, biotinylation, glycosylation, and ADP-ribosylation) [216, 217]. While the most extensively studied PTMs include acetylation and methylation, several lesser-known modifications, such as glycosylation, biotinylation, SUMOylation, phosphorylation, ADP-ribosylation, and ubiquitination, also contribute to chromatin dynamic [218]. Recent studies have introduced a range of acylations such as propionylation, butyrylation, crotonylation, succinylation, malonylation, and 2-hydroxyisobutyrylation identified through mass spectrometry. These novel modifications provide a broader biochemical context that can influence gene promoters and transcriptional regions, similar to traditional acetylation [219].

Histone methylation and acetylation involve the addition of acetyl or methyl groups to specific lysine and/or arginine residues within the N-terminal domain, often referred to as the histone “tail,” which extends from the nucleosomal surface. This region is critical for modulating chromatin folding and the recruitment of regulatory proteins [219, 220].

Evidence indicates that PTMs play a crucial role in the pathogenesis of PH (Table 2) [17, 221–223]. Understanding these modifications is vital, as alterations in histone modifications may influence gene expression patterns associated with the disease, thereby offering potential avenues for therapeutic intervention.

3.4 Noncoding RNA

Nontranslating RNA molecules are commonly referred to as ncRNAs. Only about 2% of the human genome is responsible for coding proteins, while the remaining portion consists of ncRNAs [246]. Based on their roles, ncRNAs are categorized into two groups: functional ncRNAs, such as miRNAs, circular RNAs, and lncRNAs, and nonfunctional ones, sometimes called junk RNAs. Functional ncRNAs play a crucial role in regulating cellular pathways, thus influencing the onset and progression of various diseases. In recent decades, the functions of certain ncRNA types, particularly miRNAs and lncRNAs, have been extensively explored in the context of disease mechanisms, including PAH [17, 20, 247].

3.5 Potential Therapeutic Strategy for lncRNAs in PH

This comprehensive review highlights the intricate interplay among vital lncRNAs and their overlapped target genes (Figure S1) during the development and progression of PH. The various lncRNAs played intricate roles in the development and progression of PH, where some are upregulated while others are downregulated (Figure 4 and Table S2), which gives us a clue that these lncRNAs are changed almost simultaneously in PH. Hence, maintaining the balance of these lncRNAs will emerge as a novel potential therapeutic strategy for PH. Additionally, other therapeutic interventions, including targeting overlapped genes influenced by vital lncRNAs, employing stem cells or mitochondrial transplant strategies, or combining two or more of these strategies, may present a promising avenue for future research and clinical applications in the management of PH (Figure 5).

3.6 Translational Value and Challenges of lncRNAs in PH

Unlike protein-coding genes, lncRNAs exhibit high tissue and cell-type specificity [517, 518], making them ideal candidates for precision medicine. For instance, in PH, although both lncRNA H19 and MALAT1 exhibit higher pulmonary vascular tissue expression [519, 520], H19 is highly specific to PASMCs, driving their pathological proliferation and resistance to apoptosis [283], while MALAT1 is specific to PAECs, where it regulates endothelial dysfunction, angiogenesis, and inflammation [521-523]. By targeting these lncRNAs, therapies can modulate disease pathways with greater specificity, offering more personalized treatment options and potentially overcoming the limitations of current symptomatic treatments.

LncRNAs also hold significant promise in advancing the diagnosis, classification, risk stratification, and prognosis of PH. Altered levels of lncRNAs in the blood have been proposed as a potential novel diagnostic biomarker for PH [524, 525]. Unique lncRNA expression profiles in circulation could help categorize PH into different subtypes, offering valuable insights into the underlying disease mechanisms and informing treatment approaches. Additionally, these profiles could aid in risk stratification, particularly when used alongside other established biomarkers. Specifically, circulating lncRNAs such as H19 have been shown to differentiate PAH patients from controls, correlate with RV function, and predict long-term survival in two independent IPAH cohorts [282]. Moreover, H19 levels, when combined with N-terminal proBNP (NT-proBNP) levels or risk scores from the Registry to Evaluate Early and Long-term PAH Disease Management (REVEAL registry) and the 2015 European Pulmonary Hypertension Guidelines, help identify subgroups of patients with distinct prognoses [282]. For risk stratification, lncRNAs associated with disease severity or progression, such as those involved in hypoxia-responsive pathways, could help identify high-risk patients. Additionally, lncRNA signatures may serve as prognostic markers, aiding in the prediction of treatment outcomes and long-term survival [525].

Despite the aforementioned advantages, the clinical translation of lncRNA-based approaches is not without obstacles. The cell-type specificity and context-dependent expression of lncRNAs, while advantageous for targeted therapy, complicate their identification as universally effective targets. Delivery mechanisms for lncRNA-modulating agents, such as ASOs or siRNAs, face challenges like off-target effects, immunogenicity, and inefficient delivery to pulmonary tissues [526-528]. Moreover, preclinical models often fail to accurately predict drug efficacy in humans [529, 530], limiting the validation of therapeutic and diagnostic applications of lncRNAs in PH.

To overcome these barriers, future research should prioritize developing advanced delivery systems, such as lipid nanoparticles or viral vectors, to ensure precise and efficient targeting of lncRNAs in pulmonary tissues. Standardizing protocols for detecting and quantifying lncRNAs will enhance the reliability of diagnostic tools, facilitating early diagnosis and accurate risk stratification. Emerging technologies like single-cell transcriptomics and spatial profiling can provide a detailed understanding of the cell-specific and spatial roles of lncRNAs in PH.

4.1 Emerging Therapies and Novel Targets in PH Management

Current therapeutic targets for PH focus on PGI2, NO, ET-1, and TGF-β pathways. Approved treatments include endothelin receptor antagonists, PDE5Is, soluble guanylate cyclase (sGC) agonists, and PGI2 analogs and receptor agonists. Additionally, ongoing clinical studies are exploring drugs that target mechanisms involved in PH pathogenesis, such as BMP signaling, tyrosine kinase receptors, estrogen metabolism, ECM remodeling, angiogenesis, epigenetics, serotonin metabolism, and guanylate cyclase signaling.

Several clinical studies have investigated tyrosine kinase receptor inhibition as a therapeutic approach for PAH. Frost et al. [531] reported that imatinib mesylate, a tyrosine kinase inhibitor originally used for chronic myeloid leukemia, improved hemodynamics and right ventricular function in a PAH patient. The IMPRES trial (phase 3) evaluated imatinib in PAH patients receiving two or more therapies, finding a 32-m improvement in the 6-minute walk test (6MWT) and a 31.8% decrease in PVR, though adverse effects led to a 27% dropout rate. Additionally, subdural hematomas occurred in patients on anticoagulation [531]. Due to these safety concerns, the PIPAH trial is ongoing to determine a tolerable imatinib dose, with the exclusion of anticoagulated patients [532, 533]. Seralutinib, an inhaled tyrosine kinase inhibitor targeting PDGF receptors, is also under investigation. The Torrey Study (phase 2) assessed its efficacy in PAH patients, with endpoints including changes in PVR and 6MWT distance [532, 534]. The outcome shows that inhaled seralutinib significantly reduced pulmonary PVR compared with placebo in patients with PAH, meeting the primary endpoint. Cough was the most common treatment-emergent adverse event in both groups [535].

Heterozygous BMPR2 mutations are the most common genetic cause of PAH, contributing to 53–86% of familial and 14–35% of idiopathic cases [23, 202, 536, 537]. Recent studies highlight GDF2 mutations (BMP9), affecting 6.7% of IPAH cases, leading to reduced BMP9/BMP10 levels and dysfunction in pulmonary endothelial cells [44, 538] Sotatercept, an activin receptor type IIA fusion protein, restores BMPR2/SMAD1/5/8 signaling and counteracts PAH vascular remodeling [539]. In phase 2 (PULSAR) trials, sotatercept improved PVR, 6MWT distance, and NT-proBNP in patients [532, 540, 541], with adverse events like thrombocytopenia and telangiectasia. Additionally, in the phase 2a SPECTRA study, sotatercept significantly improved peak oxygen uptake, secondary endpoints including hemodynamics and 6-minute walk distance, and right ventricular function in patients with PAH after 24 weeks of treatment [542]. In the phase 3 STELLAR trial, sotatercept significantly improved exercise capacity, measured by the 6MWT, in patients with PAH on stable background therapy compared with placebo. The treatment group showed a median increase of 34.4 m versus 1.0 m in the placebo group, with a Hodges-Lehmann estimate difference of 40.8 m. Sotatercept also improved most secondary endpoints but was associated with increased rates of adverse events such as epistaxis, thrombocytopenia, and elevated hemoglobin [543]. In the STELLAR trial, 25.9% of participants developed antidrug antibodies (ADAs) against sotatercept, with 6.8% also showing neutralizing antibodies. However, ADA presence did not significantly impact the pharmacokinetics, efficacy, or safety of sotatercept in treating PAH [544].

The role of estrogen in PAH has gained significant attention due to its higher incidence in women and the impact of certain estrogen metabolites on cell growth and pulmonary vascular remodeling. Clinical studies have explored the therapeutic potential of antiestrogen drugs traditionally used for breast cancer treatment, such as anastrozole and tamoxifen, in PAH patients. A clinical trial involving 18 male and female PAH patients treated with anastrozole (1 mg/day) for 12 weeks reported significant reductions in estradiol levels and a modest improvement in the 6MWT distance, increasing by 26 m compared with placebo. However, there were no significant changes in right ventricular function or quality of life, and invasive hemodynamics were not assessed [545]. In the phase 2 PHANTOM trial, anastrozole did not significantly improve the 6MWT compared with placebo in postmenopausal women and men with PAH after 6 months. The placebo-corrected treatment effect was −7.9 m, with no significant differences in adverse events between groups [546]. Tamoxifen, an estrogen receptor blocker, is also being assessed in a phase 2 trial focusing on its effects on PAH patients who are already receiving background therapy. The primary endpoint of this study is the change in tricuspid annular plane systolic excursion, with secondary endpoints including 6MWT distance and quality of life [532, 547]. These trials are critical in understanding the clinical implications of targeting estrogen signaling in PAH, despite the complexities and paradoxes surrounding estrogen's role in this disease.

Elafin, a potent inhibitor of elastases and matrix metalloproteinases, shows promise in preventing pulmonary vascular remodeling by maintaining ECM integrity. A phase 1 clinical trial assessing the safety, tolerability, and pharmacokinetics of elafin in healthy subjects has been completed, with plans for a phase 2 trial in severe PAH currently underway [532, 548].

The deficiency of eNOS contributes to reduced NO production in PAH, leading to endothelial dysfunction and vascular remodeling. Clinical studies have explored the therapeutic potential of EPCs transfected with eNOS (EPC–eNOS) in PAH patients. A phase 1 trial, known as PHACeT, demonstrated the safety of EPC–eNOS administration and significant improvements in the 6MWT at 1, 3, and 6 months, although no sustained hemodynamic benefits were observed [532, 549, 550]. Currently, the ongoing SAPPHIRE trial is investigating the efficacy of EPC–eNOS in PAH, with a focus on exercise capacity and RV function [532, 551]. These studies are essential in understanding the clinical implications of targeting eNOS and EPCs in the management of PAH.

Research into the role of epigenetics in PAH has highlighted BRD4 as a significant factor regulating gene expression associated with PAH pathogenesis [221, 552]. Based on promising preclinical findings, the 16-week pilot study APPRoAcH-p, initiated in 2018, evaluated the safety and hemodynamic effects of oral apabetalone, a bromodomain and extraterminal inhibitor, in PAH patients classified as functional classes 2 or 3 receiving standard therapy. The study has been completed and aimed to assess whether apabetalone could effectively improve hemodynamics and overall patient outcomes in this population [532, 553].

Bardoxolone methyl is a potential therapy for PAH that reduces oxidative stress and NF-κB activation by activating Nrf2, promoting antioxidant gene transcription, and enhancing oxidative phosphorylation [554]. The LARIAT phase 2 clinical trial showed interim improvement in patients with connective tissue disease-associated PAH (CTD-PAH) [555, 556]. This prompted two follow-up trials, CATALYST and RANGER, which were later terminated due to COVID-19 concerns [557, 558]. Further research is needed to explore bardoxolone's therapeutic role in CTD-PAH and other forms of PH.

Rodatristat ethyl is an orally bioavailable, reversible inhibitor of TPH, aimed at reducing peripheral serotonin production without affecting the central nervous system. Patients with PAH exhibit elevated free serotonin levels, contributing to pulmonary vasoconstriction and vascular remodeling [559]. Rodatristat ethyl has been tested in the completed ELEVATE2 clinical trial, a placebo-controlled, randomized study evaluating doses of 300 and 600 mg twice daily for 24 weeks [560]. The study found that Rodatristat ethyl (300 and 600 mg) significantly reduced PVR at week 24, with mean changes of 63.1% and 64.2%, respectively, compared with 5.8% for placebo. Adverse events occurred in 92% of the 300 mg group and 100% of the 600 mg group, with discontinuation rates of 11% for both RE doses. One death was reported in the 300 mg group [561].

In the phase 1 clinical trial, MK-5475, a selective inhaled sGC stimulator, was evaluated for safety and pharmacokinetics/pharmacodynamics in participants with PH associated with COPD (PH-COPD). The trial included 23 participants, with a median age of 68.5 years and 86.4% being male, who were randomized to receive either MK-5475 or a placebo. The results demonstrated a favorable safety profile, with similar rates of adverse events in both groups. While MK-5475 did not significantly affect arterial blood oxygenation or pulmonary blood volume, it resulted in a numerical reduction in PVR of −21.2% compared with −5.4% in the placebo group. These findings suggest potential efficacy for MK-5475 and support its further development as a treatment for PH-COPD [562].

4.2 Efficacy and Safety of Approved Pharmacological Therapies in PH

The KABUKI trial provided important insights into the efficacy and safety of edoxaban compared with warfarin in patients with CTEPH following reperfusion therapy. The study found that edoxaban was noninferior to warfarin in preventing worsening PVR over 48 weeks, with no incidents of symptomatic venous thromboembolism. Clinically relevant bleeding events occurred at similar rates in both groups, suggesting that edoxaban may serve as a viable alternative for anticoagulation in this patient population [563].

In the MERIT-1 trial, macitentan demonstrated a significant reduction in PVR, achieving 71.5% of baseline compared with 87.6% in the placebo group at week 16 among patients with CTEPH. The most common adverse events associated with macitentan included peripheral edema and decreased hemoglobin [564]. In contrast, the PASSION trial evaluated the efficacy of tadalafil in patients with heart failure with preserved ejection fraction and combined PH. This trial found no benefit in achieving the primary composite endpoint of heart failure hospitalization or all-cause mortality. Notably, the tadalafil group exhibited a significantly higher all-cause mortality rate. Due to medication supply issues, the trial was terminated early, and no significant improvements were observed in secondary endpoints [565]. Building on the efficacy of individual therapies, the A DUE study revealed that a fixed-dose combination of macitentan and tadalafil (M/T FDC) effectively reduced PVR by 29% compared with macitentan alone and by 28% compared with tadalafil alone. Although the M/T FDC group experienced three unrelated deaths and a higher rate of adverse events leading to treatment discontinuation, the findings support the combination as an effective option for simplifying treatment in PAH patients [566].