EPAS1 was first found by Tian (Tian et al., 1997) in 1997 as a transcription factor exclusively expressed in vascular ECs of the umbilical cord that regulates vascularization and response to hypoxia. The following year, Tian (Tian et al., 1998) further reported that EPAS1 is essential for the maintenance of cardiac output and circulating catecholamine levels from the endothelium under hypoxic conditions during embryonic development. During early embryonic development, as shown in Figure 3, HIF-2α is maintained at a high level in the chromaffin cells of the Organ of Zuckerkandl, the major source of fetal catecholamines. Homozygous EPAS1-deficient mice failed to survive in the mid-gestational stage owing to profound bradycardia and circulatory failure. Additionally, HIF-2α plays an indispensable role in angiogenesis compared to HIF-1α, which mainly activates the glucose metabolic pathways (Wang et al., 2005). Peng (Peng et al., 2000) found that EPAS1-deficient embryos failed to form large vessels or seal intact structures in the yolk sac, indicating improper vascular remodeling during vasculogenesis. Vadive (Vadivel et al., 2014) reported arrested growth of pulmonary vessels and alveoli after EPAS1 expression and HIF-1α was reduced by dominant-negative adenovirus-mediated gene transfer or chetomin. Mice with EC-specific EPAS1 deletion showed increased vascular permeability with ultrastructural abnormalities despite normal vascular anatomic development, resulting in PH(Skuli et al., 2009). Furthermore, EPAS1 regulates angiogenesis in various solid malignancies of the gastrointestinal and genitourinary tracts, such as colonic/hepatic/pancreatic cancer and breast/ovarian/prostate/renal carcinomas (Blancher et al., 2000; Palayoor et al., 2003; Zhang and Rigas, 2006; Osada et al., 2007; Bertout et al., 2008; Imamura et al., 2009; Menrad et al., 2010) by promoting ECs germination, migration, and adherens junctions (Majmundar et al., 2010; Kovacic et al., 2012; Park et al., 2013).

Vascular endothelial growth factor (VEGF) is believed to be the primary target of HIF-2α during angiogenesis. Dumpa (Dumpa et al., 2019) found that caffeine therapy for premature apnea may reduce the incidence of bronchopulmonary dysplasia by enhancing VEGF and angiopoietin expression through the HIF-2α pathway and restoring pulmonary microvasculature and alveolarization in the adult lung. Weidemann (Weidemann et al., 2010) reported that retinal astrocyte-derived VEGF driven by EPAS1 was a key mediator of vascular proliferation in a model of hypoxia-induced retinopathy. Turner (Turner et al., 2002) also revealed consistency in cellular VEGF mRNA and HIF-2α protein levels, indicating the pro-angiogenic role of HIF-2α exertion on the HRE sequences of VEGF and VEGF receptors (VEGFR) and promotion of transcription under hypoxic conditions.

Etiologies triggering excessive erythropoiesis include acute hemorrhage, adaptation to high altitude, or pathological hypoxic conditions such as congenital cyanotic heart diseases and chronic lung diseases (Semenza, 2022). HIF-2α promotes erythropoiesis by regulating erythropoietin (EPO) transcription (Figure 3), a glycoprotein highly expressed in perisinusoidal cells in the liver during embryonic development and produced by interstitial fibroblasts in the kidney during adulthood (Wang et al., 1995; Haase, 2010). HIF-2α recognizes and binds to the core sequence 5′-TACGTGCT-3′ of the EPO HRE sequence, which activates transcription and mediates the cellular response to the hypoxic microenvironment (Hussain et al., 2008). In a study by Gruber M (Gruber et al., 2007), both newborn and adult rats with EPAS1 knockout presented with anemia or severe pancytopenia with suppressed EPO production, indicating the pro-erythrocytosis effect of HIF-2α by transcriptionally regulating EPO production (Scortegagna et al., 2003; Scortegagna et al., 2005). Toshiharu Y (Yamashita et al., 2008) illustrated, however, that HIF-2α governs erythropoiesis by specifically regulating vascular adhesion molecule-1(VCAM-1) expression in ECs to maintain the hematopoietic microenvironment compared to normocytic anemia in mice with EPAS1 knockdown.

In addition to EPO production, HIF-2α stimulates erythropoiesis by participating in intestinal iron absorption, which is in high demand during erythropoiesis and critical for oxygen-carrying red blood cell maturation under hypoxic conditions. Anderson (Anderson et al., 2011) demonstrated that mice with intestinal disruption of EPAS1 (EPAS1 ^ΔIE) showed decreased serum iron levels and expression of iron absorption genes, such as divalent metal transporter 1 (DMT1) and Duodenal cytochrome b (Dcytb), in phenylhydrazine-induced erythropoiesis. In contrast, an iron responsive element (IRE) is found in the 5′untranslated region of the EPAS1 sequence, and the translation of the HIF-2α protein can be repressed by the binding of iron regulatory proteins to the 5′IRE of EPAS1 during iron deficiency (Percy et al., 2007). Additionally, elevated HIF-2α levels during hypoxia suppress hepcidin expression by inducing hepatic EPO production, leading to enhanced intestinal iron uptake and release from internal stores (Liu et al., 2012). Taken together, these data demonstrate the net regulation among EPAS1, erythropoiesis, and iron metabolism.

Metabolism is essential for the maintenance of biological activities, and metabolic reprogramming is a believed hallmark of cellular dysfunction of different etiologies (Faubert et al., 2020). Transformation from mitochondrial oxidative phosphorylation (OXPHOS) to cytoplasmic glycolysis is a major adaptive change in response to insufficient oxygen availability (Shaw, 2006). Unlike HIF-1α, which is considered the mediator of OXPHOS to glycolysis in hypoxic environments (Miska et al., 2019), limited evidence has shown the role of EPAS1 in glycolysis and OXPHOS balance. Farsijani (Farsijani et al., 2016) demonstrated that HIF-2α increased the expression of glycolytic enzymes including hexokinase 2(Hk2), glucose transporter 1 (GLUT1), aldolase C (Aldoc), phosphoglycerate mutase 1 (Pgam1), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), pyruvate kinase M2 (Pkm2), and lactate dehydrogenase A (Ldha), and promoted the synthesis of EPO in renal tubular epithelial cells during hypoxia in a VHL-dependent manner (Figure 3). Early elevated and later downregulated EPAS1 are required for pluripotency induction of induced pluripotent stem cells (iPSC) by glycolysis reprogramming (Mathieu et al., 2014).

In addition to glycolysis, accumulated HIF-2α during chronic hypoxia impairs fatty acid β-oxidation in hepatocytes and ultimately leads to steatosis by suppressing the expression of carnitine palmitoyl transferase 1 (CPT-1) and proliferator-activated receptor-γ coactivator-1α (PGC-1α), rate-limiting enzymes for the β-oxidation pathway, which can be rescued after EPAS1 knockdown (Liu et al., 2014). Similarly, Li (Li et al., 2021) revealed that HIF-2α transcriptally suppresses CPT-1 expression and prevents NLPR3 inflammasome activation, ultimately alleviating insulin resistance in chronic metabolic diseases. Qu (Qu et al., 2011) demonstrated that mice with liver-specific disruption of Vhl achieved EPAS1 overexpression and presented steatosis accompanied by pro-inflammatory and fibrogenic cytokine overexpression. The latest finding by Mooli (Mooli et al., 2022) suggest that HIF-2α augments G-protein-coupled receptor 40 (GPR40) and 120 (GPR120) expressions in intestinal lipid-sensing L cells and induces GLP-1 secretion, regulating the lipid metabolism network. Moreover, constitutive activation of EPAS1 impairs both fatty acid β-oxidation and lipogenesis by downregulating associated genes, whereas it promotes gene expression within the lipid storage and gluconeogenesis pathways (Rankin et al., 2009).

Microenvironmental hypoxia is commonly observed in various systemic inflammatory diseases, such as atherosclerosis, diabetes mellitus, inflammatory bowel diseases (IBD), cancers, and PH. In adipocytes (Figure 3), EPAS1 overexpression upregulates pro-inflammatory cytokines, including interleukin (IL)-1β, monocyte chemotactic protein-1, and the nuclear factor κB (NF-κB) pathway (Lin et al., 2013). Mice with adipocyte-specific EPAS1 overexpression developed lethal cardiac hypertrophy, which is considered the molecular mechanism linking obesity and cardiomyopathy. Xue (Xue et al., 2013; Kerber et al., 2020) reported that highly activated EPAS1 in the intestinal epithelium of IBD patients and mice enhances tumor necrosis factor-α (TNF-α) expression and promotes experimental colitis by inducing TNF-α promoter activity through the myc-associated zinc-finger protein (MAZ) binding, sparing NF-κB pathway. Esophageal HIF-2α expression correlates with reflux esophagitis severity. Refluxed acidic bile salts stabilize epithelial HIF-2α promoting pro-inflammatory cytokine expression (Huo et al., 2017). In contrast, HIF-2α in renal endothelial cells suppresses inflammatory reactions and sustains recovery from ischemic kidney injury (Kapitsinou et al., 2014). The pro-inflammatory role of HIF-2α has also been reported in osteoarthritis (Inoue et al., 2015), hepatoma (Ahn et al., 2010), atopic dermatitis, and psoriasis (Tashiro et al., 2019).

Chronic hypoxia signaling plays a central role in the progression of pulmonary vascular remodeling, leading to irreversible PH and right heart failure. Investigators implied different cellular predominance of HIF-1 and HIF-2 in the development of PH. EPAS1 exclusively governs the ECs dysfunction compared with HIF-1α, whereas in SMCs and fibroblasts, both HIF-1α and EPAS1 orchestrate the pathogenesis of vascular remodeling (Waypa and Schumacker, 2019). Detailed aspects of EPAS1 in PH pathogenesis are listed as below (Figure 4).

As the primary initiation of pulmonary vasculopathy, different molecular mechanisms focusing on EC dysfunction, including a spatiotemporal imbalance between apoptosis and proliferation, impairment of migration, sprouting and angiogenesis, methanological abnormalities, and endothelial to mesenchymal transition (EndoMT), explain the development of PH. HIF-2α was significantly elevated in the lung tissues, especially in ECs from patients with idiopathic PH, activates the cellular growth pathways, including MAPK, PI3K-Akt, VEGF, and transforming growth factor-β, increases pulmonary artery EC proliferation as well as collagen synthesis, and stimulates fibrotic remodeling of the pulmonary vasculature (Rimon et al., 2008; Fijalkowska et al., 2010; Bryant et al., 2016; Dai et al., 2018). Upregulated HIF-2α in ECs increases arginase 1 transcription and competes with the endothelial NO complex enzyme for L-Arginine, decreasing NO synthesis, the predominant vasodilator, promoting vascular remodeling and PH progression (Xu et al., 2004; Girgis et al., 2005; Cowburn et al., 2016). Research (Macias et al., 2021) also shown that Arg2 enzyme activity is elevated and correlated with EPAS1 expression in vascular ECs derived from patients with PH. Using model of excessive accumulation of HIF-1 and HIF-2 proteins by inhibiting HIF prolyl hydroxylase domain-containing protein 2 (PHD2), investigators concluded that mice with EC knockout of PHD2 developed severe pulmonary hypertension and right ventricular failure in a HIF-2 dependent manner, despite excessive accumulations of both HIF-1 and HIF-2 proteins (Kapitsinou et al., 2016). Dai (Dai et al., 2016) reported that mice with EC and bone marrow hematopoietic cells knockout of PHD2 showed severe obliterative vascular remodeling and PH, by promoting SMCs proliferation through CXCL12 activation. HIF-2α (Tang et al., 2018) promotes ECs transition into mesenchymal myofibroblasts (EndoMT) by enhancing the expression of Snai1/Snai2, zinc-finger transcription factors family, and leads to remodeled pulmonary vasculopathy. EC-specific deletion of EPAS1 may reduce the expression of ECM proteins, fibronectin, integrin receptors, and ET-B, disrupt cellular overgrowth, block plexiform formation, and ultimately inhibit hypoxia-induced pulmonary vascular remodeling (Hu et al., 2019). Rodents exposed in chronic hypoxia plus inhibition of VEGFR signal by Sugen5416 present as severe pulmonary obliterative vasculopathy and ventricular failure, whereas selectively disrupting VEGFR2 Y949, the tyrosine at residual 949 of VEGFR2, prevents vascular permeability with subsequent myeloid cells infiltration and pulmonary arterioles muscularization (Zhou et al., 2022). HIF-2α regulates angiogenesis in ECs by promoting transcription of VEGF-A/C and VEGFR1/2 pathway, regardless VEGF-B and VEGFR3 regulated by HIF-1α(Downes et al., 2018). Although the correlation between HIF-2α and VEGF/VEGFR signal pathway in PH development is rarely reported, but the paradoxical influence in ECs and pulmonary vascular function is still required clarification.

The hyperplastic proliferation of pulmonary arterial SMCs (PASMCs) is attributed to the core pathogenesis of pulmonary vascular remodeling. Different studies showed that HIF-1α is overactivated in PASMCs in PH, whereas EPAS1 is rarely reported (Dai et al., 2018). In the study of Xin Yi Chan (Chan et al., 2021), gain-of-function mutation of EPAS1 in SMCs increases the expression of fibrillin-1, fibronectin, and elastic fiber components elastin (ELN) via activating endothelin 1 (EDN1) transcription, which contributes to the contractility and stiffness of SMCs and development of pulmonary hypertension in mice. Another data support from Raghavan (Raghavan et al., 2012) pointed out that HIF-2α stimulated PASMCs proliferation by upregulating the expression of transcription factor Oct-4 (Firth et al., 2010). Conclusively, the effects of EPAS1 on PASMCs in PH are still undetermined and correlations among different vascular cells governed by HIF families pend further investigations.

Adventitial fibroblast activation and transition to myofibroblasts, the major origin of collagen and ECM synthesis, are believed to promote vasculopathy in PH. With inhibition of HIF-1α and EPAS1 using RNA interference technology, Eul (Eul et al., 2006) reported that the proliferative response of adventitial fibroblasts relies only on HIF-2α by activation of VEGF, VEGFR2, Phosphoglycerate kinase (PGK) and TGFβ, while the migratory response is correlated with both HIF-1α and HIF-2α. Moreover, mutation of the VHL gene at codon 200 results in cellular HIF-2α accumulation, further induces pulmonary fibroblasts activation with ECM overproduction, and, thereby promoting pulmonary fibrosis (Hickey et al., 2010).

Myeloid cells, especially the monocyte-macrophage lineage, are increasingly reported to contribute to PH pathogenesis (Lim et al., 2013; He et al., 2020). During the early stage of PH, circulatory monocytes are recruited to the pulmonary interstitial space, infiltrating as a pro-inflammatory phenotype and mediating EC dysfunction and PASMC proliferation (Tian et al., 2013; Florentin et al., 2018). In addition to HIF-1α, Wang (Wang et al., 2021) demonstrated that myeloid activation of PFKFB3 impels inflammatory macrophage differentiation via the HIF-2α pathway. HIF-2α upregulates macrophage colony-stimulating factor (M-CSF) expression and promotes monocyte perivascular infiltration and differentiation into inflammatory macrophages (Wang et al., 2018), secreting cytokines including IL-1β, IL-6, IL-8, IL-13, IL-18, and TNF-α, which are believed to be related to PH severity and prognosis (Groth et al., 2014). Interestingly, phenotypic alterations in anti-inflammatory/profibrotic macrophages participate in the late stage of pulmonary vascular remodeling. Few studies have revealed the role of EPAS1 in anti-inflammatory macrophages in PH. Li and colleagues (Li et al., 2021) argued that NLRP3 inflammasome activation of M1 could be suppressed by HIF-2α, thereby preventing insulin resistance. Takeda (Takeda et al., 2010) explained that EPAS1 induced by T helper 2 cytokines (IL-4) specifically promoted arginase 1 expression during M2 macrophage polarization. Despite 48% structural similarity, several data revealed the opposing regulatory roles of HIF-1α and HIF-2α on macrophages phenotype dominance which destine outcomes of inflammatory diseases and malignant tumor (Takeda et al., 2010; Eubank et al., 2011).