Suppression of HIF2 signalling attenuates the initiation of hypoxia-induced pulmonary hypertension

Cheng-Jun Hu, Jens M. Poth, Hui Zhang, Amanda Flockton, Aya Laux, Sushil Kumar, Brittany McKeon, Gary Mouradian, Min Li, Suzette Riddle, Steven C. Pugliese, R. Dale Brown, Eli M. Wallace, Brian B. Graham, Maria G. Frid, Kurt R. Stenmark
European Respiratory Journal 2019 54: 1900378; DOI: 10.1183/13993003.00378-2019

Abstract

Most published studies addressing the role of hypoxia inducible factors (HIFs) in hypoxia-induced pulmonary hypertension development employ models that may not recapitulate the clinical setting, including the use of animals with pre-existing lung/vascular defects secondary to embryonic HIF ablation or activation. Furthermore, critical questions including how and when HIF signalling contributes to hypoxia-induced pulmonary hypertension remain unanswered.

Normal adult rodents in which global HIF1 or HIF2 was inhibited by inducible gene deletion or pharmacological inhibition (antisense oligonucleotides (ASO) and small molecule inhibitors) were exposed to short-term (4 days) or chronic (4–5 weeks) hypoxia. Haemodynamic studies were performed, the animals euthanised, and lungs and hearts obtained for pathological and transcriptomic analysis. Cell-type-specific HIF signals for pulmonary hypertension initiation were determined in normal pulmonary vascular cells in vitro and in mice (using cell-type-specific HIF deletion).

Global Hif1a deletion in mice did not prevent hypoxia-induced pulmonary hypertension at 5 weeks. Mice with global Hif2a deletion did not survive long-term hypoxia. Partial Hif2a deletion or Hif2-ASO (but not Hif1-ASO) reduced vessel muscularisation, increases in pulmonary arterial pressures and right ventricular hypertrophy in mice exposed to 4–5 weeks of hypoxia. A small molecule HIF2 inhibitor (PT2567) significantly attenuated early events (monocyte recruitment and vascular cell proliferation) in rats exposed to 4 days of hypoxia, as well as vessel muscularisation, tenascin C accumulation and pulmonary hypertension development in rats exposed to 5 weeks of hypoxia. In vitro, HIF2 induced a distinct set of genes in normal human pulmonary vascular endothelial cells, mediating inflammation and proliferation of endothelial cells and smooth muscle cells. Endothelial Hif2a knockout prevented hypoxia-induced pulmonary hypertension in mice.

Inhibition of HIF2 (but not HIF1) can provide a therapeutic approach to prevent the development of hypoxia-induced pulmonary hypertension. Future studies are needed to investigate the role of HIFs in pulmonary hypertension progression and reversal.

Abstract

Activation of HIF2 by hypoxia initiates vascular cell proliferation and recruitment of inflammatory cells at early stages of PH development through HIF2-dependent transcription of genes involved in these pathways in pulmonary vascular cells http://bit.ly/2lFwTGM

Introduction

Pulmonary hypertension due to lung disease and/or hypoxia (group 3 pulmonary hypertension) affects nearly 1 million people worldwide, making it the second largest group of patients suffering from this disease. Unfortunately, none of the currently approved drugs for group 1 pulmonary arterial hypertension have been shown in randomised controlled trials to benefit patients with group 3 pulmonary hypertension, emphasising the need for a better understanding of disease mechanisms as they might aid in the discovery of new therapies.

There is abundant evidence supporting the central involvement of hypoxia inducible factors (HIFs) in chronic hypoxia-induced pulmonary hypertension [1–11]. However, we believe the experimental approaches used in most previously published studies may not recapitulate the clinical settings. For example, some group 3 pulmonary hypertension development is due to hypoxia exposure in post-natal humans and animals with normal pulmonary circulation. It is important to note that except for two studies in which Hif1a deletion in smooth muscle cells (SMCs) or endothelial cells (ECs) was initiated in adult mice [10, 11], in all other studies Hif1a or Hif2a deletion was initiated in developing embryos [1–9]. It has been well established that all these HIF knockout or activation models (prolyl-4-hydroxylase domain 2 (Phd2) knockout) exhibit vascular defects in developing embryos and likely in adult mice derived from these embryos [12–17]. Also, due to the essential role of HIF in development, most published studies use cell-type-specific Hifa knockout models [2–7, 10, 11]. We believe such cell-type-specific Hifa knockout approaches may have shortcomings for the purpose of determining the general function of Hifa genes in a disease such as pulmonary hypertension where interactions between multiple cell types (ECs, SMCs and fibroblasts) are clearly necessary for disease progression and a specific gene may have different or opposite functions in different cell types involved in the disease.

Besides inducible deletion, pharmacological inhibition could be an important tool to study the general role of HIF in pulmonary hypertension development in normal adult animals. Given the potential important role of HIF proteins and particularly HIF2 in pulmonary hypertension, successful pharmacological inhibition could also lay an important foundation for potential pulmonary hypertension treatment. Recent studies have led to small molecules that specifically block HIF2 (but not HIF1) activity [18, 19]. These inhibitors have also been shown to exhibit strong antitumor activity in vivo and in vitro [18, 20]. Additionally, antisense oligonucleotides (ASOs) to HIF1 and HIF2 have also been developed, and shown to attenuate HIF expression in vivo and to abrogate HIF-mediated disease pathology [21].

In the study presented here, we sought to re-evaluate the role of HIFs in hypoxia-induced pulmonary hypertension using approaches that may better recapitulate the clinical settings. Specifically, HIF activity is inhibited by inducible gene deletion and pharmacological inhibitors in which global (not cell-type-specific) HIF1 or HIF2 activity inhibition is initiated in normal adult animals without any predisposed diseases resulting from embryonic deletion of HIFs. In addition, we sought to better understand the underlying molecular mechanisms concerning how HIFs promote pulmonary hypertension development, by studying the role of HIF in early as well as late stages of pulmonary hypertension development.

Methods

See the supplementary material for full details.

Statistics

In general, data is reported as mean±sem. Statistical differences were evaluated by two-way ANOVA as well as the unpaired two-tailed t-test. Figure legends specify the statistical analysis used for the data in each panel. p<0.05 was considered statistically significant. Group size (n) is reported in the corresponding figure legends. Data were logged using Excel (Microsoft, Redmond, WA, USA), graphing and calculations were performed using Prism (GraphPad, La Jolla, CA, USA), and figures were designed using PowerPoint (Microsoft).

Results

HIF1α is dispensable in establishing hypoxia-induced pulmonary hypertension at 5 weeks in adult mice

To delete the Hif1a gene in adult mice globally, Hif1afl/fl;UbcCreERT+ and Hif1afl/fl;UbcCreERT− (control) mice were injected with tamoxifen for 5 days (see the supplementary material for full details of transgenic mice and genotyping). Mice were then allowed to rest for 1 week before exposure to either hypobaric hypoxia (PB=370 mmHg; simulated altitude of ∼18 000 ft (∼5500 m)) or normobaric normoxia (PB=740 mmHg) for 5 weeks (figure 1a). After 5 weeks of hypoxia exposure, readout parameters were measured and animals were euthanised. The efficiency of Hif1a deletion in UbcCreERT+ mice under hypoxic conditions is 80% (supplementary table S1). Hypoxia exposure induced increases of haematocrit (Hct) (figure 1b), right ventricular systolic pressure (RVSP) (figure 1c) and right ventricular hypertrophy, as indicated by increases in the Fulton index (ratio of the weight of the right ventricle to the weight of the left ventricle and septum) (figure 1d) and in the ratio of the weight of the right ventricle to body weight (weightRV/weightBody) (figure 1e), in both Hif1afl/fl;UbcCreERT− and Hif1afl/fl;UbcCreERT+ mice. Hypoxia also similarly reduced body weight and increased heart rate in both UbcCreERT+ and UbcCreERT− animals compared with normoxic animals (supplementary table S2). Thus, our data demonstrate that Hif1a expression is dispensable for development of pulmonary hypertension and right ventricle hypertrophy in normal adult mice after 5 weeks of hypoxia.

FIGURE 1
FIGURE 1

Global hypoxia inducible factor 1α (Hif1a) is dispensable in establishing hypoxia-induced pulmonary hypertension at 5 weeks in adult mice. TMX: tamoxifen; Hct: haematocrit; UbcCre: fusion protein consisting of Cre recombinase and the human oestrogen receptor binding domain under control of a ubiquitously active ubiquitin C gene promoter, in which Cre recombinase is activated upon TMX exposure; RVSP: right ventricular systolic pressure. a) Experimental setup: TMX was injected into mice daily during week 1 to activate Cre and delete Hif1a, then all mice were moved to sea level for 1 week, followed by exposing mice to either normoxia (PB=740 mmHg) or hypoxia (simulated altitude of ∼18 000 ft (∼5500 m); PB=370 mmHg) for 5 weeks. After 5 weeks of hypoxia exposure, readout parameters were measured and animals were euthanised. b) Hct levels, c) RVSP, d) Fulton index and e) ratio of the weight of the right ventricle to bodyweight (weightRV/weightBody). Animal numbers in each group under normoxia or hypoxia can be found in the Fulton index panel (d). Due to technical difficulties, we were not able to obtain readings of Hct and/or RVSP for some mice, thus the animal numbers for these data were typically less than the animal numbers for the Fulton index in this and other figures of this study. ***: p<0.001, difference between hypoxia versus normoxia in the same genotype (or treatment) group. Statistical analysis by two-way ANOVA.

Mice with global genetic deletion of HIF2α do not survive long-term hypoxia

To investigate the role of Hif2a in hypoxia-induced pulmonary hypertension development in normal adult mice, adult Hif2afl/fl;UbcCreERT+ and Hif2afl/fl;UbcCreERT− mice were similarly treated with tamoxifen and exposed to normoxia or hypoxia, as we did for Hif1a mice. The efficiency of Hif2a deletion in hypoxic Hif2afl/fl;UbcCreERT+ mice at the end of the experiment was 71% (supplementary table S1). While there were no differences in survival and pulmonary hypertension development among Hif2afl/fl;UbcCreERT−, Hif1afl/fl;UbcCreERT+ and Hif1afl/fl;UbcCreERT− mice (figures 1 and 2a), all Hif2afl/fl;UbcCreERT+ mice died within 4 weeks of hypoxia exposure (figure 2a). These data indicate that global Hif2a (but not Hif1a) deletion is not compatible with survival of mice exposed to chronic hypoxic conditions.

FIGURE 2
FIGURE 2

Global Hif2a deletion is incompatible for mouse survival under hypoxic conditions, while global partial Hif2a deletion diminishes hypoxia-induced pulmonary hypertension development at 5 weeks in adult mice. UbcCre: fusion protein consisting of Cre recombinase and the human oestrogen receptor binding domain under control of a ubiquitously active ubiquitin C gene promoter, in which Cre recombinase is activated upon tamoxifen exposure; Hct: haematocrit; RVSP: right ventricular systolic. Mice were treated with tamoxifen and exposed to normoxia or hypoxia as described in figure 1a. a) Kaplan–Meier curve for survival of Hif2afl/fl;UbcCreERT+ mice during exposure to hypoxia compared with survival of Hif1afl/fl;UbcCreERT+ and Hif2afl/fl;UbcCreERT− mice during exposure to hypoxia. n>9 mice in each group were used for this experiment. b–e) Haemodynamics of Hif2afl/wt;UbcCreERT+ and Hif2afl/wt;UbcCreERT− mice after 5 weeks of exposure to normoxia or hypoxia: b) Hct, c) RVSP, d) Fulton index and e) ratio of the weight of the right ventricle to body weight (weightRV/weightBody). ***: p<0.001, difference between hypoxia versus normoxia in the same genotype (or treatment) group; ##: p<0.01, difference between genotypes or treatments under hypoxic conditions. Statistical analysis shown here is two-way ANOVA analysis (note in (c) there is a significant decrease in RVSP under hypoxia in UbcCre+ animals analysed by the unpaired t-test: p<0.05).

Global partial HIF2α gene deletion diminishes development of hypoxia-induced pulmonary hypertension at 5 weeks in adult mice

To prevent lethality, we generated Hif2awt/fl;UbcCreERT+ and Hif2awt/fl;UbcCreERT− mice, and then treated them with tamoxifen and exposed them to hypoxia for 5 weeks. The efficiency of Hif2a deletion in hypoxic Hif2awt/fl;UbcCreERT+ mice at the end of the experiment was 36% (supplementary table S1). Cre-negative animals displayed increases in Hct (figure 2b), RVSP (figure 2c), Fulton index (figure 2d) and weightRV/weightBody ratio (figure 2e) in response to hypoxia. While there were still significant increases in these parameters in hypoxia-exposed Hif2awt/fl;UbcCreERT+ mice, the level of increase for some parameters was significantly reduced in comparison with Cre-negative littermates (figure 2d and e). Thus, our data, generated from normal adult mice, demonstrate that reduction of Hif2a attenuates development of pulmonary hypertension induced by 5 weeks of hypoxia exposure.

Knockdown of Hif2a utilising ASOs significantly reduces development of hypoxia-induced pulmonary hypertension at 5 weeks in adult mice

To further investigate the role of Hif2a in hypoxia-induced pulmonary hypertension in normal adult mice and to examine whether targeting Hif2a therapeutically in adult mice under hypoxic conditions would be beneficial or lead to lethality, we used an ASO approach. The effectiveness and feasibility of Hif2a-ASO in reducing Hif2a expression in adult mice maintained under normoxia was tested in a pilot experiment, which showed an up to 90% reduction of Hif2a mRNA levels in lungs, spleens, kidneys and livers in mice treated with Hif2a-ASO two times per week for 2 weeks (figure 3a, left), without significant changes of Hif1a mRNA in all organs examined (figure 3a, right). Furthermore, Hif2a-ASO also reduced expression of the HIF2 target gene Epo1, but not the HIF1 target gene Pgk1, in kidneys (figure 3b).

FIGURE 3
FIGURE 3

Knockdown of Hif2a but not Hif1a utilising antisense oligonucleotides (ASOs) significantly reduces development of hypoxia-induced pulmonary hypertension at 5 weeks in adult mice. Hct: haematocrit; RVSP: right ventricular systolic; α-SMA: α-smooth muscle actin. a, b) Testing the effectiveness and specificity of ASOs in a pilot experiment. Wild-type C57BL/6J mice were treated either with injections of an ASO targeting Hif2a mRNA (Hif2a-ASO) or equal volumes of 0.9% NaCl (control) at days 1, 4, 8 and 11. At day 12, mice (n=3) were sacrificed and multiple organs were collected for RNA preparation. a) Levels of Hif1a and Hif2a mRNA were quantified by quantitative real-time PCR in the indicated organs. b) Levels of Pgk1 (a HIF1 target gene) and Epo1 (a HIF2 target gene) in kidneys from mice targeted with NaCl or Hif2a-ASO were quantified by quantitative real-time PCR. c) Experimental setup. In week 2, mice were kept at sea level and began to receive injections of control (ctrl), Hif1a-ASO or Hif2a-ASO (two injections per week on Monday and Thursday). Starting week 3, mice were exposed to either normoxia (sea level) or hypoxia (simulated altitude of ∼18 000 ft (∼5500 m)) for 5 weeks, at which two injections per week were maintained. d–k) End-point measurements for the experimental animals in (c): d) Hct, e) RVSP, f) Fulton index and g) ratio of the weight of the right ventricle to body weight (weightRV/weightBody). h–j) α-SMA-positive pulmonary vessels. Scale bar: 200 μm (top row); 100 μm (bottom row). k) Summary of Hct, RVSP, Fulton index and weightRV/weightBody for mice targeted with control-ASO or Hif1a-ASO under normoxia or hypoxia. *: p<0.05; ***: p<0.001, difference between hypoxia versus normoxia in the same genotype (or treatment) group; #: p<0.05; ##: p<0.01; ###: p<0.001, difference between genotypes or treatments under hypoxic conditions; ¶¶¶: p<0.001, difference between genotypes or treatments under normoxic conditions. Statistical significance as determined by the t-test (a, b, j and k) or two-way ANOVA (d–g).

To determine the general role of HIF in hypoxia-induced pulmonary hypertension development, mice were injected with Hif2a-ASO or a control-ASO (unspecific, no known target) for 1 week with two injections per week and then exposed to either normoxia or hypoxia for 5 weeks during which two injections of ASO per week were maintained (figure 3c). The efficiency of Hif2a deletion in hypoxic mice targeted with Hif2a-ASO was 72% at the end of the experiment (supplementary table S1). We did not observe any lethality in Hif2a-ASO-treated mice under hypoxia. However, hypoxic Hif2a-ASO-treated mice exhibited a trend towards increased weight loss (supplementary table S4). Further studies also revealed that levels of circulating catecholamines (epinephrine and norepinephrine) were reduced by Hif2a-ASO treatment, as were heart rate, cardiac output and maximal pressure increase dP/dtmax (supplementary figure S2b–e). In addition, two animals died during readout procedures. Taken together, these findings suggest an increased fragility in these mice.

Hct in Hif2a-ASO-treated mice was lower, both under normoxia and under hypoxia (figure 3d). Under hypoxic conditions, Hif2a-ASO-treated mice also exhibited a reduction in RVSP (figure 3e), Fulton index (figure 3f) and weightRV/weightBody ratio (figure 3g) compared with hypoxic control-ASO mice. Consistent with haemodynamic data, hypoxia-exposed Hif2a-ASO-treated animals exhibited a marked reduction in fully muscularised vessels in the lungs compared with hypoxia-exposed control-ASO mice (figure 3h–j).

However, Hif1a-ASO reduced Hif1a mRNA by 60% in lung tissues (supplementary table S1), but it had no effect on Hct, RVSP or the Fulton index in hypoxic mice (figure 3k), similar to the lack of effect observed in inducible Hif1a knockout mice (figure 1).

A small molecule HIF2 inhibitor PT2567 significantly reduces development of hypoxia-induced pulmonary hypertension at 4 weeks in adult rats

To further address the safety of HIF2 inhibition and the role of HIF2 in initiation of hypoxia-induced pulmonary hypertension, we treated rats (which develop more severe pulmonary hypertension than mice) with a small molecule inhibitor (PT2567; Peloton Therapeutics, Dallas, TX, USA) that specifically blocks HIF2 activity [18, 19]. Exposure to 4 weeks of hypoxia led to pulmonary hypertension development in rats treated with the control reagent (0.5% methylcellulose/0.5% Tween-80), as demonstrated by increased mean pulmonary arterial pressure (mPAP) (figure 4a) and increased Fulton index (figure 4b). The HIF2 inhibitor PT2567 reduced mPAP (figure 4a) and attenuated right ventricle remodelling (figure 4b). To better understand the molecular mechanisms underlying HIF2's role in pulmonary hypertension development in adult animals, we examined multiple pathways and genes that are important in pulmonary hypertension development [3–7, 22–24]. Indeed, increased inflammatory cell accumulation (monocytes) (figure 4c, top panel), slightly increased numbers of proliferating cells (Ki-67) (figure 4c, middle panel) and increased tenascin C (TNC) expression (figure 4c, lower panel) were observed in hypoxia-exposed rats, which were reduced by the HIF2 inhibitor PT2567 in adventitial areas of pulmonary vessels from hypoxia-exposed rats. The changes in proliferation and TNC expression in hypoxic rats and rats treated with HIF2 inhibitor using immunostaining were confirmed by Ccna1 (cyclin A1, a marker for cell proliferation) and Tnc mRNA levels in lung tissues of the corresponding rats (figure 4c, middle and bottom right). However, the increased monocyte accumulation in hypoxic rats using immunostaining was not confirmed by their mRNA levels in lung tissues of the corresponding rats (figure 4c, top right), likely due to the possibility that macrophages/monocytes are attracted to pulmonary vessels, but the total cell numbers are not significantly altered in the whole lungs of rats exposed to hypoxia for 4 weeks. In addition, more muscularised vessels were observed in hypoxia-exposed rats, which were reduced by the HIF2 inhibitor PT2567 (figure 4d and e), data consistent with our Hif2a-ASO observations in mice. Furthermore, consistent with previous reports [3–7, 23], a number of functionally important genes in pulmonary hypertension such as Icam1, Sdf1, Arg1, Arg2, Ccnd1, Edn1, Pai1, Tgfa and Tsp1 exhibited increased levels in lungs of hypoxia-exposed rats (supplementary figure S3). The increase in the aforementioned genes, but not genes such as Adm, Glut1 and Ndrg1, was significantly reduced in hypoxia-exposed rats treated with the HIF2 inhibitor PT2567 (supplementary figure S3), suggesting that the first group of genes likely represents target (direct or indirect) genes unique to HIF2, while the HIF2 inhibitor-insensitive genes are likely to be regulated in a HIF2-independent manner at this time-point. These data indicate that HIF2 inhibition significantly reduces development of hypoxia-induced pulmonary hypertension, by preventing induction of genes involved inflammation (supplementary figure S3b) and cell signalling (proliferation and fibrotic responses) (supplementary figure S3c).

FIGURE 4
FIGURE 4

Small molecule hypoxia inducible factor 2 inhibitor PT2567 significantly attenuates development of hypoxia-induced pulmonary hypertension at 4 weeks in adult rats. mPAP: mean pulmonary arterial pressure; CCNA1: cyclin A1; TNC: tenascin C. Sprague Dawley male rats weighing 210–245 g (Charles River Laboratories, Wilmington, MA, USA) were housed in chambers under normoxia or hypoxic (simulated altitude of ∼18 000 ft (∼5500 m)) conditions for 4 weeks. Rats were dosed with vehicle methylcellulose (0.5%)/Tween-80 (0.5%) or PT2567 (300 mg·kg−1·day−1) beginning the day they were placed in chambers. After 4 weeks, end-point measurements for the experimental animals were conducted. a) mPAP. b) Ratio of the weight of the right ventricle to body weight (weightRV/weightBody). c) Representative images of pulmonary vessels stained with anti-macrophage/monocyte antibody clone ED-1 (top), anti-Ki-67 antibody (middle) or anti-TNC antibody (bottom). The levels of Cd68, Ccna1 and Tnc mRNAs in the whole lung tissues of indicated rats are also shown (right). d, e) α-SMA-positive pulmonary vessels. Scale bar: 100 µm. *: p<0.05; ***: p<0.001, difference between hypoxia versus normoxia in the same genotype (or treatment) group; ##: p<0.01; ###: p<0.001, difference between genotypes or treatments under hypoxic conditions; ¶¶: p<0.01, difference between genotypes or treatments under normoxic conditions. Statistical significance determined by two-way ANOVA (a, b) or the t-test (c–e).

HIF2 activity is required for increased accumulation of monocytes and increased cell proliferation observed in hypoxia-exposed adult rats at 4 days

Using both genetic and pharmacological approaches, our aforementioned experiments demonstrated that inhibition of HIF2 is able to attenuate the development of pulmonary hypertension at 4–5 weeks of hypoxia using normal adult mice and rats as models. Subsequent experiments are intended to address multiple critical remaining questions concerning how HIF2 promotes pulmonary hypertension initiation, but not to determine the general role of HIF2 in hypoxia-induced pulmonary hypertension. The first question we wanted to answer is at which stage of pulmonary hypertension development does HIF2 signalling have an impact? To help address this question, we performed short-term hypoxia experiments. Exposure to 4 days of hypoxia led to moderate increases of mPAP (figure 5a) and the Fulton index (figure 5b) in rats treated with control reagent, in which only the Fulton index was reduced by the HIF2 inhibitor PT2567 (figure 5b). Consistent with our previous findings [25, 26], in short-term hypoxia-exposed rats, changes in TNC expression were minimal (figure 5c), but there was a significant increase in accumulation of monocytes and cell proliferation (figure 5c). The HIF2 inhibitor PT2567 completely abolished these changes (figure 5c). The changes of Cd68, Ccna1 and Tnc expression in hypoxic rats and rats treated with HIF2 inhibitor examined by immunostaining were confirmed by their mRNA levels in lungs of the corresponding rats (figure 5c, right side). Examination of gene expression in lungs demonstrated that most HIF2-regulated genes (Icam1, Sdf1, Arg1, Arg2, Ccnd1, Edn1 and Pai1, but not Tgfa and Tsp1) that were observed in rats exposed to 4 weeks of hypoxia (supplementary figure S3) also exhibited induction in rats exposed to 4 days of hypoxia (supplementary figure S4). Importantly, the HIF2 inhibitor PT2567 reduced expression of these genes (supplementary figure S4). However, Pdgfb and Cxcr4 were induced, and Id1 was reduced, only in rats exposed to short-term hypoxia (supplementary figure S4). The effectiveness of the HIF2 inhibitor in preventing monocyte/macrophage accumulation and vascular cell proliferation, and the fact that changes in gene expression were largely overlapping in rats exposed to short-term hypoxia and to long-term hypoxia, support the idea that HIF2 activity is essential in initiating hypoxia pulmonary hypertension at a very early stage (4 days).

FIGURE 5
FIGURE 5

Small molecule hypoxia inducible factor 2 inhibitor PT2567 significantly attenuates early events in hypoxia-exposed adult rats at 4 days. mPAP: mean pulmonary arterial pressure; CCNA1: cyclin A1; TNC: tenascin C. Sprague Dawley rats were housed in chambers under normoxia or hypoxic conditions for 4 days and dosed with vehicle or PT2567 as described in figure 4. After 4 days, end-point measurements for the experimental animals were conducted. a) mPAP. b) Ratio of the weight of the right ventricle to body weight (weightRV/weightBody). c) Representative images of pulmonary vessels stained with anti-macrophage/monocyte antibody clone ED-1 (top), anti-Ki-67 antibody (middle) or anti-TNC antibody (bottom). The levels of Cd68, Ccna1 and Tnc mRNAs in lungs of indicated rats are also shown (right). Scale bar: 100 µm. *: p<0.05; ***: p<0.001, difference between hypoxia versus normoxia in the same genotype (or treatment) group; #: p<0.05; ###: p<0.001, difference between genotypes or treatments under hypoxic conditions. Statistical significance determined by two-way ANOVA (a, b) or the t-test (c).

Normal human pulmonary artery ECs display unique responses to acute hypoxia in a HIF2α-dependent manner

Our short-term in vivo hypoxia studies support the hypothesis that HIF2 activity is activated by hypoxia very early in one or multiple pulmonary vascular cells (ECs, SMCs and fibroblasts), initiating pulmonary hypertension development. Here, we attempted to determine the main cell type(s) whose activation by acute hypoxia may explain the observed events in rats exposed to 4 days of hypoxia (figure 5) by first comparing and contrasting the response of normal human pulmonary artery-derived ECs, SMCs and fibroblasts to acute hypoxia. We chose to use normal vascular cells as we were studying the function of HIF in pulmonary hypertension initiation. Acute hypoxia (1.5% for 16 h) significantly activated expression of several classic HIF target genes including ADM, CA9, GLUT1, NDRG1 and VEGFA in all three cell types, suggesting activation of these classical HIF target genes lacks cell type specificity (figure 6a). Hypoxic induction of the pro-inflammatory genes CXCR4, SDF1 and ICAM1 was most prominent in ECs (figure 6b). Additional unique responses of ECs to acute hypoxia was evidenced by reduced expression of ID1 and ID3, and increased expression of the growth factor TGFA, a ligand of EGFR (figure 6c), all of which can promote cell proliferation and survival. However, induction of APLN, EDN1, PDGFB and TSP1 was mainly observed in SMCs and fibroblasts, while ARG1 and ARG2 were not induced by acute hypoxia in any of the three cell types (figure 6c). These data support a unique role of ECs in response to acute hypoxia, to increase genes involved in inflammation and cell migration and proliferation. Thus, we determined if HIF2 is responsible for increased expression of inflammatory and proliferative genes in ECs under hypoxia. Interestingly, HIF2 inhibitor PT2567 completely abolished hypoxia-mediated changes of inflammatory (CXCR4, ICAM1 and SDF1) (figure 7b) and signalling/proliferation (ID1, ID3 and TGFA) (figure 7c) genes in control ECs, but only attenuated hypoxic induction of classical HIF target genes (figure 7a). The essential role of HIF2 (but not HIF1) in regulating inflammatory and signalling/proliferation genes in control ECs was further confirmed using a small interfering RNA (siRNA) approach as HIF2A siRNA (but not HIF1A siRNA) significantly attenuated changes of inflammatory and signalling/proliferation gene expression, induced by hypoxia in ECs (supplementary figure S5c and d). While the direct function of proteins such as ID1 and ID3 is likely intracellular, the increased production of genes/proteins such as stromal cell-derived factor 1 (SDF1) and tumour necrosis factor-α (TGFA) in ECs could also involve paracrine signalling. To assess the role of activated ECs in regulating other pulmonary vascular cells, we prepared conditional medium from normal ECs cultured under normoxia or hypoxia, in the presence or absence of a HIF2 inhibitor PT2567. We found that HIF2-mediated activation of control ECs also increased the expression of genes involved in cell proliferation (CCNE1 and CCNE2), pro-inflammation (CCL2) and anti-apoptosis (BCL2, BCL2L1 and BIRC5) in control SMCs. This suggests a role for HIF2 signalling in activating ECs, which promote SMC activation (supplementary figure S6). Thus, our studies support a hypothesis that activation of ECs by hypoxia in a HIF2-dependent manner (but not HIF1) activates ECs, also initiating proliferation of other vascular cells (SMCs) and recruitment of monocytes/macrophages via increased expression of inflammatory cytokines (SDF1) and growth factors (TGFA).

FIGURE 6
FIGURE 6

Normal human pulmonary artery vascular cells exhibit unique properties in response to acute hypoxia. FC: fold change; EC: endothelial cell; FB: fibroblast; SMC: smooth muscle cell. Normal human pulmonary artery vascular cells (ECs, FBs and SMCs; n=5 for each cell type) were cultured under normoxia (NX) or hypoxia (HX; 1.5% O2) for 16 h and then cells were collected for RNA preparation. The same set of genes examined in vivo was studied here. Results were from at least five different cell populations for each cell type, in which the result for a specific cell population was from three independent normoxia or hypoxia experiments here or other similar experiments in this paper. a) Classical hypoxia inducible factor target genes. b) Genes involved in inflammation. c) Genes involved in signalling. *: p<0.05; **: p<0.01; ***: p<0.001, difference between hypoxia versus normoxia in the same genotype (or treatment) group. Statistical significance determined by the t-test.

FIGURE 7
FIGURE 7

Hypoxia inducible factor-2 (HIF2) inhibitor PT2567 significantly attenuates altered production of genes involved in inflammation and signalling in normal pulmonary endothelial cells (ECs) in response to acute hypoxia. FC: fold change; DMSO: dimethyl sulfoxide. To determine if HIF2 activity is responsible for hypoxia-mediated gene expression changes in ECs, normal human pulmonary artery ECs (n=3) were cultured under normoxia (NX) or hypoxia (HX; 1.5% O2) for 16 h in the presence of DMSO (control) or different concentrations of HIF2 inhibitor PT2567, and then cells were collected for RNA preparation and quantitative real-time PCR. a) Select classical HIF target genes. b) Genes involved in inflammation that are significantly induced by hypoxia in ECs (figure 6b). c) Genes involved in signalling that are significantly altered by hypoxia in ECs (figure 6c). *: p<0.05; **: p<0.01; ***: p<0.001, difference between hypoxia versus normoxia in the same genotype (or treatment) group; #: p<0.05; ##: p<0.01; ###: p<0.001, difference between genotypes or treatments under hypoxic conditions. Statistical significance determined by the t-test.

Hif2a expression in ECs is required for hypoxia-induced pulmonary hypertension and vascular remodelling in mice

To confirm the essential role of EC Hif2a in pulmonary hypertension development in vivo and to better understand the role of EC HIF2 in pulmonary hypertension development, we generated and exposed EC Hif2a knockouts and their controls to normoxia or hypoxia for 5 weeks. As expected, Hif2afl/fl-EC-Cre− animals subjected to hypoxia displayed an increase in Hct (figure 8a) and pulmonary hypertension development (figure 8b–d). We observed a similar increase of Hct in the hypoxia-exposed Hif2afl/fl-EC-Cre+ mice, confirming previous reports that endothelial HIF2α is not required for hypoxia-induced erythropoiesis and erythrocytosis [4]. However, hypoxia-exposed Hif2afl/fl-EC-Cre+ mice exhibited no signs of pulmonary hypertension (figure 8b–d). The increase in the number of α-smooth muscle actin-positive pulmonary vessels in hypoxia-exposed Hif2afl/fl-EC-Cre− mice was also abolished in hypoxia-exposed Hif2afl/fl-EC-Cre+ mice (figure 8e and f). We also examined the same set of genes that we studied in rats treated with the HIF2 inhibitor (supplementary figure S3). As expected, genes induced by hypoxia in the lungs of control rats (supplementary figure S3) were partially overlapping with the genes induced in mice by chronic hypoxia (supplementary figure S7). The list included Ndrg1, Sdf1, Arg1 and Edn1. However, the only genes that were reduced in both hypoxia-exposed rats treated with HIF2 inhibitor (supplementary figure S3) and in mice with EC Hif2a knockout were Sdf1, Arg1 and Edn1 (supplementary figure S7). This indicates the particular importance of these genes in the development of hypoxia-induced pulmonary hypertension. Interestingly, we found that Acta1 and Myh7 gene expression was markedly reduced in the right ventricle of hypoxic Hif2afl/fl-EC-Cre+ mice (supplementary figure S8) compared with the right ventricle of hypoxic Hif2afl/fl-EC-Cre− mice, confirming reduced right ventricle remodelling.

FIGURE 8
FIGURE 8

Hif2a expression in endothelial cells (ECs) is required for the development of hypoxia-induced pulmonary hypertension. Hct: haematocrit; RVSP: right ventricular systolic pressure; α-SMA: α-smooth muscle actin. Hif2a knockout or Hif2a wild-type mice were exposed to either normoxia (sea level) or hypoxia (simulated altitude of ∼18 000 ft (∼5500 m)) for 5 weeks. a–f) End-point measurements in these mice: a) Hct, b) RVSP, c) Fulton index and d) ratio of the weight of the right ventricle to body weight (weightRV/weightBody). e, f) α-SMA-positive pulmonary vessels. Scale bar: 100 μm. *: p<0.05; **: p<0.01; ***: p<0.001, difference between hypoxia versus normoxia in the same genotype (or treatment) group; ##: p<0.01; ###: p<0.001, difference between genotypes or treatments under hypoxic conditions. Statistical significance determined by two-way ANOVA (a–d) or the t-test (f).

Discussion

Using both genetic and pharmacological approaches to inhibit global HIF1 or HIF2 activity, we found that inhibition of HIF2α (but not HIF1α) attenuates the development of pulmonary hypertension in normal adult animals exposed to chronic (4–5 weeks) hypoxia. Importantly, in addition to addressing the role of HIF1 and HIF2 in hypoxia-induced pulmonary hypertension development using normal adult animals, we also uncovered several novel roles of HIF2 in adult animals, including its requirement for animals to survive under chronic hypoxic conditions. Our in vitro studies (pulmonary vascular cell response to acute hypoxia), in combination with short-term hypoxia in vivo studies, support a hypothesis that EC HIF2 is “essential” in pulmonary hypertension initiation because only ECs can increase the production of diffusible cytokines (SDF1), which may recruit monocytes/macrophages and other blood/bone marrow-derived cells to lung vessels, at early stages of pulmonary hypertension development. This result is also consistent with an earlier study showing that EC-derived SDF1 contributes to pulmonary hypertension in PHD2-deficient mice [3]. Our studies also support the hypothesis that HIF2 in SMCs or fibroblasts is “not essential” in pulmonary hypertension initiation. This might be due to the fact that more than one cell type (among SMCs, fibroblasts and ECs) can induce the expression of the pulmonary hypertension-related genes such as APLN, EDN1, PDGFB, TGFA and TSP1. Thus, our studies contribute to a better understanding of the role of HIF2 in pulmonary hypertension initiation.

Pulmonary hypertension is observed in post-natal humans and animals with normal pulmonary circulation in response to chronic hypoxia stress. However, to study the role of HIF in pulmonary hypertension development, most published studies used models in which HIF deletion or HIF activation (in PHD2 knockout) was initiated in embryonic life [1, 3–8, 10]. We think results from such approaches need to be re-evaluated because 1) all these models are known to exhibit vascular defects in developing embryos and likely in adult mice derived from these embryos [12–17], and 2) there are examples demonstrating differences in phenotype (baseline as well as stressed) when gene deletion is initiated in the embryo versus in the adult [27–29]. For example, inhibition of monocyte chemoattractant protein 1 (MCP1) initiated in adult animals reduces hypoxia- or monocrotaline-induced pulmonary hypertension [27, 28]. In contrast, MCP1 or MCP1 receptor knockout mice (initiated in the embryo) exhibit spontaneous pulmonary hypertension [29]. Our data showing the effects of HIF2 inhibition in normal adult animals demonstrate the functional importance of HIF2 in pulmonary hypertension development, eliminating the concerns that HIF2's function in most previous reported studies is due to the defects in the lung vasculature and heart by Hif2a deletion or activation in developing embryos. Although we also used mice with constitutive EC-specific knockout of Hif2a, initiated in the embryo, we recognise that results from the EC Hif2a knockout study have limitations. However, the purpose of our EC Hif2a knockout study is not to determine the general function of HIF2 in hypoxia-induced pulmonary hypertension, but to provide a molecular explanation for the function of HIF2 in ECs in pulmonary hypertension development. Results from in vivo studies in these mice confirmed 1) results obtained from mice with an inducible, global knockout, 2) results from animals treated with two different inhibitors and 3) results from in vitro studies.

The role of HIF1α in the development of hypoxia-induced pulmonary hypertension is controversial: studies demonstrated either a partial amelioration of pulmonary hypertension [10, 11], or a temporary slowing of pulmonary hypertension progression [1] or even elevated PAPs [2], in mice with reduced Hif1a expression. Additional in vivo studies in which HIF1 is activated by genetic manipulation of PHDs or the von Hippel-Lindau (VHL) protein demonstrated HIF1α was not required for pulmonary hypertension development [3, 4, 9]. We speculate that the different results reported could be due to using hypoxia or pseudo-hypoxia (PHD or VHL knockout animals) approaches or deletion of Hif1a in different cell types or deletion of HIF1α initiated in embryos or adult mice. Our studies using global (not cell-type-specific) inhibition of HIF1α in normal adult animals (not initiated in embryo) and using hypoxia to activate HIF (not pseudo-hypoxia) do not support an indispensable role of HIF1α for pulmonary hypertension in animals after 4–5 weeks of hypoxia exposure. However, we cannot exclude a transient role of HIF1α in the earlier stage of pulmonary hypertension development, which we did not investigate. Indeed, a transient role of HIF1α in hypoxia-induced pulmonary hypertension has been reported [1]. Also, we cannot exclude disease-relevant HIF1α signalling in SMCs in vivo, as we did not confirm sufficient knockdown in this cell type in our ASO and inducible knockout systems. Lastly, our in vivo studies were performed using hypobaric hypoxia, while previous studies were performed using normobaric hypoxia. Since some physiological responses differ depending on the mode of hypoxia [30], the different experimental setting might also contribute to some of the differences between our findings and previous studies on the role of HIF1α in hypoxic pulmonary hypertension.

Another distinct feature of this study is to inhibit global, not cell-type-specific HIF activity for the purpose of determining HIF function in pulmonary hypertension development. Cell-type-specific knockout is a powerful method to understand the contribution of the targeted gene in a specific cell type to the disease. However, we believe conclusions derived solely from cell-type-specific knockout experiments may have shortcomings for the purpose of determining the general function of a gene in a disease such as pulmonary hypertension where interactions between multiple cell types are clearly necessary for disease progression and a specific gene may have different or opposite functions in different cell types involved in the disease. For example, caveolin-1 is reduced in pulmonary hypertension ECs and its reduction in ECs promotes pulmonary hypertension development [31]; however, caveolin-1 is overexpressed in pulmonary hypertension SMCs and its overexpression in SMCs also promotes pulmonary hypertension development [31, 32]. Using global HIF gene deletion or pharmacological inhibition, we concluded that HIF2 (but not HIF1) is important for hypoxia-induced pulmonary hypertension development. However, EC Hif2a deletion (figure 8) appears to be more effective than global HIF2 reduction (ASO and HIF2 inhibitor PT2567) in preventing development of hypoxia-induced pulmonary hypertension, suggesting the true role of HIF2 in hypoxia-induced pulmonary hypertension development is less important than reported in EC Hif2a deletion models.

Cell-type-specific knockout approaches may also miss the possible side-effects of therapeutic inhibition of a gene such as Hif2a, especially if Hif2a also plays important roles in other cell types for other processes. Indeed, complete global deletion of Hif2a is detrimental for survival of mice under chronic hypoxia, which is a novel and important finding. Although the underlying causes for this lethality and the different tolerance to chronic hypoxia between Hif2a knockout mice and mice/rats treated with Hif2a inhibitor are beyond the scope of this study, we speculate that mice with significant loss of Hif2a might have succumbed to cardiogenic shock under hypoxic conditions. This is based on our findings that Hif2a-ASO treatment reduced levels of circulating catecholamines that were significantly increased in hypoxia in control-ASO mice (supplementary figure S2b). Hif2a-ASO also abolished the increase in heart rate that was observed in control mice with hypoxia exposure (supplementary figure S2c), resulting in reduced cardiac output (supplementary figure S2d). Hif2a knockdown also resulted in lower dP/dtmax (right ventricle), a parameter of ventricular systolic function (supplementary figure S2e). These effects could be mediated by loss of Hif2a in heart tissue (supplementary figure S2a) or by loss in other organs (e.g. the adrenal glands). In conclusion, we speculate that lower Hct in Hif2a-ASO-treated animals, in addition to the observed changes in cardiac function, could result in critically low delivery of oxygen and finally death of Hif2a knockout mice. This interpretation of our data is consistent with previous studies demonstrating Hif2a-dependent changes in catecholamines [8], heart rate, and cardiac output and physiology [33–35]. Importantly, we did not observe changes in heart rate in mice with EC-specific Hif2a knockout, demonstrating that the observed reduction in RVSP in Hif2a-ASO-treated animals was not merely due to impaired cardiac function. In summary, our study suggests that we need to exercise caution, particularly for patients residing at high altitude, if a HIF2α inhibitor is going to be used in the clinic in the future.

Most published studies have evaluated the end-effect of HIF inhibition in animals exposed to chronic hypoxia without knowledge of the role of HIF in the early stage of pulmonary hypertension initiation. We performed studies in animals exposed to chronic as well as short-term hypoxia. We revealed that HIF2 is essential in several early events (macrophage recruitment and vascular cell proliferation) of pulmonary hypertension development, likely by activating genes such as those for Sdf1 (inflammation) and Tgfa (cell proliferation) in vivo.

To the best of our knowledge, this is the first time that the responses of the three primary normal human pulmonary vascular cell types (ECs, SMCs and fibroblasts) to acute hypoxia have been examined concurrently for a set of genes that have been reported [3, 4, 6, 9, 22–24] and demonstrated in our current studies (supplementary figures S3, S4 and S7) to be important for pulmonary hypertension development. Our studies lead to novel findings that the EC is the primary cell type that can be activated by short-term hypoxia (ID reduction), in a HIF2-dependent manner to produce inflammatory (SDF1) and growth promoting factors (TGFA) (figure 7), to activate other vascular cells (supplementary figure S6). Our findings from short-term in vivo and in vitro studies are consistent with previous studies by us and others that recruitment of bone marrow/blood-derived cells such as monocytes/macrophages is an early and critical event in pulmonary hypertension development [25, 36–39], and cytokines/chemokines including SDF1 are among the earliest inflammatory factors increased in the pulmonary arteries of hypoxia-exposed animals and whose increase precedes monocyte/macrophage accumulation [26]. Our in vitro studies also indicated that normal SMCs and fibroblasts can be acutely activated by hypoxia to produce factors such as APLN, EDN1 and TSP1 that have been shown to be involved in vessel constriction [4, 6]. While knockout of HIF2A in SMCs does not prevent hypoxic pulmonary hypertension [7], studies on the effect of HIF2A inhibition in fibroblasts, in combination with other pulmonary cell types, such as SMCs, are needed. Furthermore, our data indicated that there is no hypoxic induction of ARG1 and ARG2 in normal ECs, SMCs and fibroblasts (figure 6), although we consistently observed increased expression of Arg1 and Arg2 in lungs of chronic and short-term hypoxia-exposed rodents and whose expression is reduced by HIF2 inhibition (supplementary figures S3, S4 and S7). These data suggest that increased expression of genes such as those for ARG1 and ARG2 could be mainly from recruited cells.

The critical role of HIF2 in the development of hypoxia-induced pulmonary hypertension has raised significant interest in targeting HIF2 for the treatment of pulmonary hypertension patients. In fact, one study has already demonstrated a quite effective role of a HIF2 inhibitor in reversing pulmonary hypertension in animal models [40]. However, small animal models for pulmonary hypertension have well-recognised limitations [41]. More importantly, targets that have been demonstrated to be effective in animal models often fail in clinical trials [42, 43]. Thus, we believe further studies are needed before initiation of a HIF2 inhibitor clinical trial in humans. Accumulating data indicate that vascular cells established from pulmonary hypertension patients and large animals (cows) exhibit and maintain their unique phenotypes in vitro [44]. We believe these cells could provide an excellent platform to further determine the role of HIF2 for pulmonary hypertension treatment. In addition, although HIF1 is not essential in hypoxia-induced pulmonary hypertension development, the role of HIF1 in pulmonary hypertension disease maintenance is also possible.

In summary, our studies have demonstrated a positive role of HIF2 in pulmonary hypertension development in response to chronic hypoxia in normal adult animals. However, more research is needed to determine if HIF2 is truly a good pulmonary hypertension treatment target because HIF2 activity is likely to be one of the many factors that are required, acting in different cell types, at different stages, to initiate pulmonary hypertension [45]. In addition, there are data from cancer research that factors/pathways that initiate cancer often fail to be good treatment targets because there are many additional changes that occur during cancer progression.

Supplementary material

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Acknowledgements

Ionis Pharmaceuticals (Carlsbad, CA, USA) supplied Hif1-ASOs and technical support.

Footnotes

  • This article has supplementary material available from erj.ersjournals.com

  • Conflict of interest: C-J. Hu has nothing to disclose.

  • Conflict of interest: J.M. Poth has nothing to disclose.

  • Conflict of interest: H. Zhang has nothing to disclose.

  • Conflict of interest: A. Flockton has nothing to disclose.

  • Conflict of interest: A. Laux has nothing to disclose.

  • Conflict of interest: S. Kumar has nothing to disclose.

  • Conflict of interest: B. McKeon has nothing to disclose.

  • Conflict of interest: G. Mouradian has nothing to disclose.

  • Conflict of interest: M. Li has nothing to disclose.

  • Conflict of interest: S. Riddle has nothing to disclose.

  • Conflict of interest: S.C. Pugliese has nothing to disclose.

  • Conflict of interest: R.D. Brown has nothing to disclose.

  • Conflict of interest: E.M. Wallace has a patent “Compositions for Use in Treating Pulmonary Arterial Hypertension” pending to Peloton Therapeutics.

  • Conflict of interest: B.B. Graham reports grants from National Institutes of Health, during the conduct of the study.

  • Conflict of interest: M.G. Frid has nothing to disclose.

  • Conflict of interest: K.R. Stenmark reports personal fees for advisory board or steering committee work from Pfizer, New York, Actelion (Entelligence) and Janssen Research and Development, outside the submitted work.

  • Support statement: This work was supported by the US Dept of Defense (grant PR140977) and the National Heart, Lung, and Blood Institute (grant P0 HL014985). Funding information for this article has been deposited with the Crossref Funder Registry.

  • Received February 22, 2019.
  • Accepted August 28, 2019.

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Suppression of HIF2 signalling attenuates the initiation of hypoxia-induced pulmonary hypertension
Cheng-Jun Hu, Jens M. Poth, Hui Zhang, Amanda Flockton, Aya Laux, Sushil Kumar, Brittany McKeon, Gary Mouradian, Min Li, Suzette Riddle, Steven C. Pugliese, R. Dale Brown, Eli M. Wallace, Brian B. Graham, Maria G. Frid, Kurt R. Stenmark
European Respiratory Journal Dec 2019, 54 (6) 1900378; DOI: 10.1183/13993003.00378-2019
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