Therapeutic Targeting of Vascular Remodeling and Right Heart Failure in Pulmonary Arterial Hypertension with a HIF-2α Inhibitor

Abstract

Rationale: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive vasoconstriction and obliterative vascular remodeling that leads to right heart failure (RHF) and death. Current therapies do not target vascular remodeling and RHF, and result in only modest improvement of morbidity and mortality.

Objectives: To determine whether targeting HIF-2α (hypoxia-inducible factor-2α) with a HIF-2α-selective inhibitor could reverse PAH and RHF in various rodent PAH models.

Methods: HIF-2α and its downstream genes were evaluated in lung samples and pulmonary arterial endothelial cells and smooth muscle cells from patients with idiopathic PAH as well as various rodent PAH models. A HIF-2α-selective inhibitor was used in human lung microvascular endothelial cells and in Egln1^Tie2Cre mice, and in Sugen 5416/hypoxia- or monocrotaline-exposed rats.

Measurements and main results: Upregulation of HIF-2α and its target genes was observed in lung tissues and isolated pulmonary arterial endothelial cells from patients with idiopathic PAH and three distinct rodent PAH models. Pharmacological inhibition of HIF-2α by the HIF-2α translation inhibitor C76 (compound 76) reduced right ventricular systolic pressure and right ventricular hypertrophy and inhibited RHF and fibrosis as well as obliterative pulmonary vascular remodeling in Egln1^Tie2Cre mice and Sugen 5416/hypoxia PAH rats. Treatment of monocrotaline-exposed PAH rats with C76 also reversed right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling; prevented RHF; and promoted survival.

Conclusions: These findings demonstrate that pharmacological inhibition of HIF-2α is a promising novel therapeutic strategy for the treatment of severe vascular remodeling and right heart failure in patients with PAH.

Keywords: cardiac fibrosis; hypoxia-inducible factor; obliterative vascular remodeling; pharmacological therapy; pulmonary arterial hypertension.

Figure 1.

Activation of HIF-2α (hypoxia-inducible factor-2α) signaling in lungs from patients with idiopathic pulmonary arterial hypertension (IPAH) and rodent PAH models. (A) Quantitative RT-PCR analysis showing increase in HIF2A but not HIF1A expression in the whole lungs of patients with IPAH compared with healthy donors. (B) Immunofluorescence staining against HIF-2α demonstrating upregulation of HIF-2α expression predominantly in endothelial cells (ECs) (vWF⁺). Arrows indicate HIF-2α (green)–positive ECs (red). Five normal donors and five patients with IPAH lung samples were analyzed. (C) Western blotting analysis demonstrating upregulation of HIF-2α in pulmonary arterial ECs (PAECs) but not pulmonary arterial smooth muscle cells (PASMCs) isolated from patients with IPAH compared with healthy donors. (D) Upregulation of HIF-2α protein levels in lungs from Sugen 5416/hypoxia (SuHx) rats compared with age- and sex-matched controls. Lung tissues from SuHx rats were collected 6 weeks after the 3-week SuHx challenge. (E) Increase in HIF-2α protein levels in lungs of monocrotaline (MCT)-exposed rats compared with controls. Lung tissues from MCT rats were collected 4 weeks after MCT exposure. Bars represent mean values. *P < 0.05 and **P < 0.01. (A and C–E) Mann-Whitney test. (B) Scale bar: 50 μm. A.U. = arbitrary units; Nor = normal healthy donors; n.s. = not significant; vWF = von Willebrand factor.

Figure 2.

Inhibition of HIF-2α (hypoxia-inducible factor-2α) activation in human lung microvascular endothelial cells (HLMVECs) by treatment with HIF-2α inhibitors. (A) Luciferase assay showing that HIF-2α inhibitors (H2A [HIF-2α antagonist 2] and HIF-2α translational inhibitor compound 76 [C76]) suppressed HRE (hypoxia response element) activity induced by PHD2 (prolyl hydroxylase-2) deficiency in HLMVECs. HLMVECs were cotransfected with HRE-Luc plasmid and pRL-TK plasmid as well as PHD2 siRNA (siPHD2) or control scrambled RNA (siCtl). Six hours after transfection, the cells were treated with either H2A (20 μM), C76 (20 μM), or vehicle (DMSO) for another 14 hours and then lysed for luciferase assay. (B) Representative Western blot demonstrating that C76 treatment reduced HIF-2α protein levels but not HIF-1α protein levels in PHD2-deficient HLMVECs. (C) Quantitative RT-PCR analysis showing inhibited expression of HIF-2α target genes by C76 treatment in PHD2-deficient HLMVECs. (D) A diagram showing the strategy of smooth muscle cell (SMC) treatment with conditioned medium from ECs. (E) 5-Bromo-2′-deoxyuridine (BrdU) immunostaining demonstrating C76 inhibition of idiopathic pulmonary arterial hypertension (IPAH) pulmonary arterial SMC (PASMC) proliferation induced by conditioned medium from PHD2-deficient HLMVECs. Forty-eight hours after transfection with either PHD2 siRNA or control, HLMVECs were treated with DMSO or C76 (20 μM) for 24 hours in serum-free medium. Conditioned medium from these HLMVECs was then added to human PASMCs for 12 hours. After 12 hours of incubation with BrdU (10 μM), SMCs were fixed and immunostained with anti-BrdU (green) for quantification of cell proliferation. Red arrows indicate BrdU-positive cells. Sample size: (A–C) n = 3, (E) n = 4. *P < 0.05, **P < 0.01, and ***P < 0.001; n.s. = not significant. (A–C and E) One-way ANOVA with Tukey post hoc analysis. (E) Scale bar: 50 μm. A.U. = arbitrary units.

Figure 3.

Pharmacological inhibition of HIF-2α (hypoxia-inducible factor-2α) inhibits severe pulmonary arterial hypertension (PAH) in Egln1^Tie2Cre mice. (A) Drastic decrease in right ventricular systolic pressure (RVSP) in C76 (compound 76)-treated Egln1^Tie2Cre (CKO) mice compared with vehicle-treated mice. Three-week-old Egln1^Tie2Cre mice exhibiting PAH were treated with either vehicle (0.5% DMSO) or C76 for 12 weeks. (B and C) Echocardiography measurement of pulmonary artery (PA) function showing normalization of PA acceleration time/ejection time (AT/ET) ratio in C76-treated Egln1^Tie2Cre mice. (D) Representative micrographs of Russell-Movat pentachrome staining, showing inhibited obliterative pulmonary vascular remodeling in C76-treated Egln1^Tie2Cre mice. Arrows indicate pulmonary vessels with severe remodeling. (E) Reduced occlusive lesions by C76 treatment in Egln1^Tie2Cre mice. Notably, there were no occlusive large vessels (>100 μm) in C76-treated Egln1^Tie2Cre mice. *P < 0.05, **P < 0.01, and ***P < 0.001. (A and C) One-way ANOVA with Tukey post hoc analysis for multiple group comparisons; and (E) Mann-Whitney test. (D) Scale bars: 50 μm. Br = bronchiole; V = vessel; Veh = vehicle; WT = wild type.

Figure 4.

HIF-2α (hypoxia-inducible factor-2α) inhibition prevents right heart failure and cardiac fibrosis and promotes survival of Egln1^Tie2Cre mice. (A) Reduction in right ventricular (RV) hypertrophy by C76 (compound 76) treatment in Egln1^Tie2Cre (CKO) mice. (B) Quantification of transverse heart showing reduced RV wall thickness during diastole in C76-treated Egln1^Tie2Cre mice. (C) Representative M-mode echocardiography micrographs showing reduced RV chamber size in C76-treated Egln1^Tie2Cre mice compared with vehicle-treated CKO mice. The ventricular chambers are indicated with white lines. (D) Normalization of RV fractional area change, indicative of RV contractility, by C76 treatment in Egln1^Tie2Cre mice. (E) Representative micrographs of trichrome staining showing marked decrease in cardiac fibrosis in the right heart of a C76-treated Egln1^Tie2Cre mouse. Blue indicates collagen deposition. (F) C76 treatment promoted survival of Egln1^Tie2Cre mice. ***P < 0.001. (A, B, and D) One-way ANOVA with a Tukey post hoc analysis for multiple group comparisons; and (F) log-rank (Mantel-Cox) test. (E) Scale bar: 100 μm. LV = left ventricle; RV FAC = right ventricular fractional area change; RV/(LV + S) = ratio of right ventricle to left ventricle plus interventricular septum; RV WTD = RV wall thickness during diastole; WT = wild type.

Figure 5.

HIF-2α (hypoxia-inducible factor-2α) inhibition reverses severe pulmonary arterial hypertension in Sugen 5416/hypoxia (SuHx) rats. (A) Diagram showing the experimental timeline including C76 (compound 76) treatment in SuHx rats. (B) C76 treatment reduced right ventricular systolic pressure in SuHx rats. (C) Echocardiography measurements showing improvement in pulmonary artery (PA) diastolic function by C76 treatment in SuHx rats. (D–F) Representative images of Russell-Movat pentachrome staining (D) and vascular lesion quantification (E and F) demonstrating inhibited pulmonary vascular remodeling including reduction in occlusive lesions and PA wall thickness in C76-treated SuHx rats. Arrows point to pulmonary vessels with occlusive vascular remodeling. *P < 0.05, **P < 0.01, and ***P < 0.001. (E) Mann-Whitney test. (B, C, and F) One-way ANOVA with Tukey post hoc analysis for multiple group comparisons. (D) Scale bars: 200 μm. mpk = milligrams per kilogram; PA AT/ET = PA acceleration time/ejection time ratio; RVSP = right ventricular systolic pressure; V = vessel.

Figure 6.

Treatment with a HIF-2α (hypoxia-inducible factor-2α) inhibitor prevents right heart failure and cardiac fibrosis in Sugen 5416/hypoxia (SuHx) rats. (A) C76 (compound 76) treatment inhibited right ventricular (RV) hypertrophy. (B and C) Echocardiography demonstrating decreased RV wall thickness (B) and restored RV contractility (C) in C76-treated SuHx rats. (D) Representative micrographs and quantification of trichrome staining demonstrating reduction in RV fibrosis by C76 treatment in SuHx rats. Blue indicates collagen deposition. *P < 0.05, **P < 0.01, and ***P < 0.001. (A–C) One-way ANOVA with Tukey post hoc analysis for multiple group comparisons. (D) Mann-Whitney test. (D) Scale bar: 50 μm. RV FAC = right ventricular fractional area change; RV/(LV + S) = ratio of right ventricle to left ventricle plus interventricular septum; RV WTD = RV wall thickness during diastole.

Figure 7.

HIF-2α (hypoxia-inducible factor-2α) inhibition halts pulmonary arterial hypertension progression and promotes survival of monocrotaline (MCT) rats. (A) Diagram showing the experimental timeline for C76 (compound 76) treatment in MCT rats. (B–D) C76 treatment reduced right ventricular systolic pressure (B) and right ventricular (RV) hypertrophy (C) and normalized RV contractility (D). (E) Representative micrographs of Russell-Movat pentachrome staining of MCT rat lungs. Arrows indicate vascular lesions. (F) Quantification of pulmonary arterial wall thickness in MCT rat lungs. (G) Marked increase in survival of C76-treated MCT rats compared with vehicle controls. The rats were challenged with MCT at (B–F) 32 mg/kg or (G) 35 mg/kg. **P < 0.01, ***P < 0.001. (B–D and F) One-way ANOVA with Tukey post hoc analysis for multiple group comparisons; and (G) log-rank (Mantel-Cox) test. (E) Scale bar: 50 μm. RV FAC = right ventricular fractional area change; RV/(LV + S) = ratio of right ventricle to left ventricle plus interventricular septum; RVSP = right ventricular systolic pressure; V = vessel.