Pulmonary artery hypertension (PAH) is a progressive and lethal disease caused by multiple pulmonary vascular disorders. Patients with PAH eventually died from right heart failure. Endothelial dysfunction, sustained vasoconstriction and vascular remodeling of pulmonary arterioles are the major characteristics of PAH, all of which act to narrow the blood vessels and increase pulmonary vascular resistance (PVR) and pulmonary artery pressure (PAP). Most patients with PAH have an insidious onset initially with no specific symptoms, however, with the further increase of PAP, asthma, chest pain, dizziness occur. Severe cases of right heart failure include edema of the lower extremities, enlargement of the liver, and even ascites or pleural effusion. Once the symptoms of right heart failure appear, the prognosis of patients is poor. At present, the drugs targeting PAH in clinical usage are suboptimal. In general, the median survival rate after diagnosis of idiopathic pulmonary hypertension is only 2–3 years. Most importantly, PH cannot be measured by conventional sphygmomanometers and most PH is found by echocardiography after patients are already exhibiting severe symptoms, which delays treatment. Therefore, it is critical that earlier methods of PAH diagnosis are developed.

In pathological conditions such as PAH, one of the common phenomenon is oxygen utilization rate decrease. Pulmonary hypertension caused by lung disease and/or hypoxia (WHO group 3 PH) has a large population, which cannot be ignored. A large amount of evidence supports the crucial role of hypoxia inducible factors (HIFs) in chronic hypoxia induced PH (Brusselmans et al., 2003; Kim et al., 2013; Cowburn et al., 2016; Dai et al., 2016). HIFs are a class of transcription factors sensitive to oxygen concentration, which are mainly responsible for regulating the adaptability of the body to hypoxia (Prabhakar and Semenza, 2012). HIFs, including HIF-1 and HIF-2 are involved in the regulation of cell proliferation, migration, and pulmonary vascular remodeling in PH (Voelkel and Tuder, 2000; Brusselmans et al., 2003; Prabhakar and Semenza, 2012). More importantly, HIF1α and HIF2α, particularly HIF2α in pulmonary artery endothelial cells, plays a major role in the development of PAH (Skuli et al., 2009; Kim et al., 2013; Ball et al., 2014; Dai et al., 2016; Kapitsinou et al., 2016). HIFs are regulated by HIF-prolyl hydroxylases (PHDs) through the hydroxylation of conserved proline residues. Hydroxylated HIFs are then degraded by the proteasome via the von Hipel-Lindau ubiquitination complex. Under hypoxia, the hydroxylation reaction is attenuated allowing HIFs to escape degradation, which leads to their nuclear translocation, heterodimerization with HIF1β, and increased expression of hypoxia-induced genes (Bishop and Ratcliffe, 2015). PHD2 is the most important isoenzyme under normoxic conditions, and is involved in various hypoxia-affected processes, such as angiogenesis and cardiac function. We, and others, have already reported that the Phd2^EC–/– mice, the endothelial conditional knockout mice, developed spontaneous PH with severe pulmonary vascular remodeling and occlusive pulmonary vascular lesions under normoxic conditions (Dai et al., 2016; Kapitsinou et al., 2016; Tang et al., 2018), while heterozygotes, Phd2^EC–/– mice developed mild PH symptoms. Significant progress has also been made on the mechanism of HIF2α in the pathogenesis of PAH and it appears that HIF2α causes PH by inducing endothelial-to-mesenchymal transition (Endo-MT), a phenomenon of endothelial cells transforming into fibroblast phenotype, which exacerbates pulmonary vascular lesions (Tang et al., 2018).

Preclinical studies have also begun to evaluate small molecule HIF2α inhibitors as potential candidates for PAH treatment. The transcriptional activity of HIF2α is activated by binding with ARNT while Gardner and his colleagues have found that the hydrophobic structure in the PAS-B domain of HIF2α that can be inactivated by specific small molecule entry, which leads to the dissociation of the HIF2α and ARNT heterodimer. Ultimately the DNA-binding capacity of HIF2A is abolished (Scheuermann et al., 2009). A series of HIF2α inhibitors for cancer therapy have been produced using this method, including PT2385, PT2399, and PT2576. The advantages of high specificity and oral high bioavailability make them of high clinical value. These HIF2α inhibitors show high safety and effectiveness in both animal experiments and clinical trials, which can significantly inhibit tumor growth in patients with metastatic clear cell renal cell carcinoma (ccRCC), with proven safety and high dose tolerance (Chen et al., 2016; Cho et al., 2016).

Since HIF2α is an important cause of pulmonary artery remodeling during PH, preclinical studies of HIF2α inhibitors for PH therapy have been evaluated. Published data shows that PT2567 significantly reduced hemodynamic parameters related to the development of PH in Sugen 5416/hypoxia- rats (Macias et al., 2020). These results suggest that HIF2α could be an early biomarker of PAH and that HIF2α inhibitors might provide a promising new approach for the treatment of PAH. There is evidence that HIF-2α and its target genes were upregulated in lung tissue and pulmonary artery endothelial cells (PAECs) from idiopathic PAH patients and a variety of rodent PH models (Dai et al., 2018; Tang et al., 2018). These findings suggest that HIF2α plays an important role not only in hypoxia-induced PH, but also in other types of PH. However, due to the heterogeneity of PH it is vital to specifically utilize the mots efficacious therapeutic agents. Therefore, in this study we aim to investigate the gene expression of HIF2α in PAH in order to determine if it was possible to identify a useful diagnostic method to differentiate PAH human subjects.

Because highly reproducible gene expression pattern can be acquired from the mechanism-associated gene sets related to PH development from transcriptional analysis (Abraham et al., 2010; Yang et al., 2012; Gardeux et al., 2014), We focused on the global gene expression pattern associated with the HIF2α rather than examining the expression abundance of individual genes. We compared the Gene Ontology (Bertout et al., 2009) gene set (Ashburner et al., 2000) expression pattern between wild type (WT) and Hif2a knockdown (KD) von Hippel-Lindau (VHL) deficient cells. The differentially expressed GO gene sets were deemed as HIF2α-mediated. We further validated the potential of the HIF2α-mediated gene sets as a possible diagnostic tool for PAH by analyzing the transcriptome data in lungs and PBMCs of PAH patients and healthy controls. To verify the ability of HIF2α-mediated gene sets for the identification of PAH, endothelial cell-specific Phd2 KO (Phd2^EC–/–) mice were generated, and their lung tissues were analyzed by transcriptome sequencing. Comparing the differences and similarities of gene expression and biological processes among WT, hypoxia and Phd2^EC–/– mice lung tissues we found that differentially expressed GO genes associated with PH were mediated by HIF2α. Together, these HIF2α-mediated gene sets were effectively able to differentiate PAH patients from controls.

PAH is a highly lethal vascular disease characterized by the medial remodeling in pulmonary arterioles and endothelial dysfunction, which leads to a progressive increase in PVR and pulmonary pressure (Rabinovitch, 2012). HIFs, especially HIF2α, has proved to play a significant role in the pathogenesis of PH. By deeply analyzing gene expression data from the GEO database, the present study was able to demonstrate that Hif2a knockdown regulates multiple biological processes associated with PAH, including but not limited to “blood vessel development” and “angiogenesis,” which were further shown to present among genes up-regulated in PAH patient lung tissues. More importantly, the 14 GO terms shared by Hif2a knockdown and up-regulated in PAH patient lung tissues show significantly better predictive power for PAH than random gene sets. Interestingly, 13 of the 19 down-regulated GO terms associated with Hif2a knockdown overlapped with the up-regulated GO terms in the peripheral blood mononuclear cells from PAH patients. By reason of the foregoing, these data indicate that HIF2α plays a critical role in PAH pathogenesis and HIF2α mediated gene sets can provide a distinctive and useful diagnostic method for distinguishing PAH human subjects. We investigated gene set expression profile rather than evaluating the expression level of individual genes for a compelling reason: mechanism-associated gene sets are highly reproducible and meaningful in transcriptome research (Abraham et al., 2010; Yang et al., 2012; Gardeux et al., 2014). Generally, single gene expression information is insufficient to unveil the underlying biological mechanisms. More importantly, most individual gene markers derived from genome-wide screening fail to be reproducible (Kern, 2012), because different genes jointly participating in a mechanism, such as a given GO term, may alternately be deregulated in different samples (Gardeux et al., 2014). Thus, the mechanistic gap between gene expression and transcriptome analysis can be effectively narrowed by mechanism-associated gene sets. Outliers appear in gene data sets of human CTR and PAH groups, which may be due to differences in their genetic background, or some of them have other underlying diseases. In clinical samples, it is often impossible to control intra group differences like animal models, which shows the advantages of animal model experiment.

HIF1α and HIF2α belong to a group of transcription factors, which regulate the transcription of most genes related to hypoxia adaptation (Hu et al., 2003). The degradation of HIFs depends on an oxygen dependent process. HIF1α and HIF2α are hydroxylated by PHDs, ubiquitinated and rapidly degraded through ubiquitin-proteasome under normoxia (Maxwell et al., 1999). However, under hypoxia, the decay of PHDs activity directly leads to the increase of HIF-α stability. This will facilitate their nuclear transposition, dimerization with ARNT and activation of their DNA-binding capacity. Then, the intracellular hypoxia adaptive regulation will be lunched (Hu et al., 2003). Some evidences show that HIF2α can act as an independent pathogenic factor during PAH. A mutation prevents HIF2α hydroxylation by PHDs, which accumulates HIF2α that will ultimately contribute to the development of PAH both in mice and human (Brusselmans et al., 2003; Tan et al., 2013). Current studies have shown that PHD2 expression was reduced in endothelial cells at the lesion sites of the pulmonary arteries in idiopathic PAH patients, and the loss of PHD2 in endothelial cells activates HIF2α rather than HIF1α (Dai et al., 2016). This is the reason that Phd2 KO mice lung tissues were chosen for experimental verification. These studies indicate the importance of the PHD2/HIF2α axis in PAH development.

Hypoxia-inducible genes been transcriptional activated include vascular endothelial growth factor-a (VEGF-A), erythropoietin (EPO) and inducible nitric oxide synthase (iNOS) (Semenza and Wang, 1992; Melillo et al., 1995; Tuder et al., 1995). The adaptation to hypoxia mediated by HIFs is mainly achieved by regulating the protein content of α subunit. A hypoxic environment is a common occurrence in the lung in the pathological situation of PAH. As HIF-α signaling contributes to the endothelial dysfunction, vascular smooth muscle cells (VSMCs) proliferation, migration and tissue remodeling processes, it has the potential to become a therapeutic target.

HIF1α is widely expressed in the cells of most mammals, while HIF2α is restricted and highly expressed in the endothelial cells (Patel and Simon, 2008). The accumulation of HIF2α in ECs leads to increased ET-1 production, enhanced HIF1α activity in PASMCs during PAH, and the HIF target genes up-regulation (Shimoda and Laurie, 2014). It has been reported that Hif2a^+/– mice exhibit alleviated PAH pathological symptoms, including reduced right ventricular pressure and vascular remodeling (Brusselmans et al., 2003). The result of a statistical study showed that there is a correlation between HIF2α dysfunction and the decreased PAP in Tibetan natives (van Patot and Gassmann, 2011). Conversely, HIF2α overexpression is associated with development of PH in both humans (Gale et al., 2008; Formenti et al., 2011) and mice (Tan et al., 2013). Our GO analysis of PAH patient lung tissues samples further strengthens the important role of HIF2α in PAH. Comparing to the controls, 14 up-regulated GO gene sets (t-test: P < 0.05) in PAH patients overlapped with the down-regulated GO gene sets in Hif2a-KD cells (Figures 1, 2). As we can see in Figure 2, most of the GO terms up-regulated in PAH patient samples are associated with the function of HIF2α. PAH is a disease related to the dysregulation of the lung vascular system and we also identified the up-regulation of GO gene sets related to angiogenesis and blood vessel development (GO: 0001525, GO: 0045766, GO: 0001568). HIF2α has been shown to control genes involved in the regulation of angiogenesis and vascular development, and we identified two such gene among the genes significantly down-regulated in Hif2a-KD cells: vascular endothelial growth factor A (VEGFA) and sirtuin 1 (SIRT1). By binding its receptor VEGFR2, VEGFA regulates angiogenesis and vascular permeability through promoting the proliferation and survival of ECs, where the downstream signaling pathways include phosphoinositide 3 kinase (PI3K)/Akt, focal adhesion kinase (FAK), Rho family GTPases, nitric oxide (NO), and p38 mitogen−activated protein kinase (MAPK) (Claesson-Welsh and Welsh, 2013). Moreover, a recent study showed HIV protein transactivator of transcription (TAT) stimulated the proliferation of pulmonary artery SMCs (PASMCs) through increasing the expression VEGFA, and may be a potential therapeutic target for the treatment of HIV-associated PAH (Guo et al., 2018). SIRT1 is a NAD⁺-dependent deacetylase shown to play important roles in angiogenesis signaling both in vitro and in mice through controlling the acetylation of the forkhead transcription factor, Foxo1, which is highly expressed in ECs and mediates the expression of angiopoietin 2 (Ang2) (Potente et al., 2005, 2007). In addition, SIRT1 also regulates the proliferation of PASMCs by activating peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) (Zurlo et al., 2018). Thus, it is likely that uncontrolled activation of HIF2α induces the overexpression of its downstream genes, such as VEGFA and SIRT1, which causes dysregulated development of the lung vascular system and contributes to the development of PAH.

In conclusion, by deeply analyzing selected gene expression data deposited in the GEO database we were able to demonstrate that HIF2α-regulated gene sets significantly overlapped with gene sets up-regulated in PAH patient lung tissue samples and peripheral blood mononuclear cells. HIF2α, as a key regulatory molecule, plays a significant role in the pathogenesis of PAH and speculate that this methodology could provide a new application prospect in identifying PAH patients.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Guangzhou Medical University.

HT designed the project. JZ, LZ, YH, GC, and CB performed the experiments. YH, TZ, and HT wrote the manuscript with input from JW, WL, and SB. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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