The Endothelial Prolyl-4-Hydroxylase Domain 2/Hypoxia-Inducible Factor 2 Axis Regulates Pulmonary Artery Pressure in Mice

Abstract

Hypoxia-inducible factors 1 and 2 (HIF-1 and -2) control oxygen supply to tissues by regulating erythropoiesis, angiogenesis and vascular homeostasis. HIFs are regulated in response to oxygen availability by prolyl-4-hydroxylase domain (PHD) proteins, with PHD2 being the main oxygen sensor that controls HIF activity under normoxia. In this study, we used a genetic approach to investigate the endothelial PHD2/HIF axis in the regulation of vascular function. We found that inactivation of Phd2 in endothelial cells specifically resulted in severe pulmonary hypertension (∼118% increase in right ventricular systolic pressure) but not polycythemia and was associated with abnormal muscularization of peripheral pulmonary arteries and right ventricular hypertrophy. Concurrent inactivation of either Hif1a or Hif2a in endothelial cell-specific Phd2 mutants demonstrated that the development of pulmonary hypertension was dependent on HIF-2α but not HIF-1α. Furthermore, endothelial HIF-2α was required for the development of increased pulmonary artery pressures in a model of pulmonary hypertension induced by chronic hypoxia. We propose that these HIF-2-dependent effects are partially due to increased expression of vasoconstrictor molecule endothelin 1 and a concomitant decrease in vasodilatory apelin receptor signaling. Taken together, our data identify endothelial HIF-2 as a key transcription factor in the pathogenesis of pulmonary hypertension.

FIG 1

EC-specific inactivation of Phd2 stabilizes HIF-1α and HIF-2α. (A) (Left) Genomic PCR analysis of DNA isolated from the lungs of ePhd2^−/− mutants and Cre⁻ control mice. Primers used amplified both the conditional (2-lox) and the recombined (1-lox) alleles. (Right) Phd2 transcript levels in whole lung homogenates from ePhd2^−/− mutants and Cre⁻ littermate controls (n = 7; P = 0.02). (B) (Top) Immunoblot analysis of HIF-1α and HIF-2α in lung nuclear extracts isolated from ePhd2^−/− and Cre⁻ mice. Ponceau S staining was used to assess for equal protein loading. Nuclear kidney extracts from a mouse treated with an oral PHD inhibitor served as a positive control (Ctrl). (Bottom) Pgk1, Ldha, and Glut1 mRNA levels in lungs from mutant mice compared to those from Cre⁻ littermate controls (n = 6). Bars represent mean values ± SEM. *, P < 0.05; **, P < 0.01.

FIG 2

Endothelial Phd2 inactivation results in premature mortality and pulmonary hypertension associated with right ventricular hypertrophy. (A) Shown are Kaplan-Meier survival curves for mutants and control mice. (B) The top panel shows body weights (BW) for ePhd2^−/− mice and Cre⁻ controls at 6, 8, and 12 weeks of age (n = 5 to 10). The middle panels depict ratios of heart or lung weight (W) to BW (heart, n = 8 or 9; lung, n = 3 or 4). The bottom panel shows the ratio of wet weight to dry weight for lungs from ePhd2^−/− mutants and littermate controls (n = 5 or 6). (C) Hemoglobin (Hb) concentration and hematocrit (Hct) levels in ePhd2^−/− mutants and controls (n = 5). Bars represent mean values ± SEM. *, P < 0.05; **, P < 0.01. (D) Fulton index [(RV/(LV+S)] (n = 5 to 7), right ventricular systolic pressure (RVSP) (n = 5 or 6), and systolic blood pressure (SBP) in ePhd2^−/− mice and Cre⁻ controls (n = 4 to 6). Bars represent mean values ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

Histopathological characterization of mice lacking endothelial Phd2. (A) Representative images of H&E-stained lungs from ePhd2^−/− mutants and Cre⁻ control mice at 10 weeks of age. (B) Representative images of αSMA-, F4/80-, and Ki67-stained lungs from ePhd2^−/− mice and littermate controls. Arrows identify αSMA^+ve arterioles (top), F4/80^+ve cells (middle), and Ki67^+ve cells (bottom). Graphs show quantification of muscularized vessel number/10 HPF, F4/80⁺ cell number/HPF, and Ki67⁺ cell number/HPF (n = 5 to 7). Graph bars represent mean values ± SEM. *, P < 0.05; ****, P < 0.0001. Scale bars indicate 50 μm.

FIG 4

Pulmonary hypertension in ePhd2^−/− mice is dependent on HIF-2. (A) Shown are HIF-1α (top) and HIF-2α (bottom) protein levels as detected by immunoblot analysis of nuclear pulmonary extracts from ePhd2^−/−Hif1a^−/− mice, ePhd2^−/− Hif2a^−/− mice, and their Cre⁻ littermate controls. Nuclear protein extracts from the kidney or liver of a PHI-treated mouse were used as positive controls (Ctrl). Ponceau staining was used to assess for equal protein loading. The graphs depict Pgk1 transcript levels in lungs from ePhd2^−/− Hif1a^−/− (n = 6) and ePhd2^−/− Hif2a^−/− (n = 5) mice. (B) RVSP and Fulton index in ePhd2^−/− Hif1a^−/− and ePhd2^−/− Hif2a^−/− mice. (C) Representative images of lungs stained for αSMA and quantification of muscularized vessels expressed as number/10 HPF in ePhd2^−/− Hif1a^−/−, ePhd2^−/− Hif2a^−/−, and control mice. Bars represent mean values ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bar, 50 μm.

FIG 5

Inactivation of endothelial Hif2a protects from the development of hypoxia-induced pulmonary hypertension. (A) Shown are RVSP and Fulton index measurements performed for eHif2a^−/− and Cre⁻ mice following chronic normobaric hypoxia (10% O₂ for 4 weeks) (n = 4 to 6). (B) Representative images of lungs stained for αSMA, with the graph depicting muscularized vessel number/10 HPF in eHif2a^−/− and Cre⁻ mice following chronic hypoxic exposure (10% O₂ for 4 weeks) (n = 4 to 6). (C) RVSP (n = 3 or 4) and Fulton index (n = 4) in transplanted WT mice subjected to chronic normobaric hypoxia (10% O₂ for 4 weeks). eHif2a^−/− to WT, WT mice transplanted with bone marrow cells from eHif2a^−/− mutants; WT to WT, WT mice transplanted with bone marrow cells from WT mice. Bars represent mean values ± SEM. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Scale bar, 50 μm.

FIG 6

HIF-2 regulates pulmonary Edn1 and Aplnr expression. Relative levels of Edn1, Apln, and Aplnr mRNAs in ePhd2^−/− (n = 8), ePhd2^−/− Hif1a^−/− (n = 5), ePhd2^−/− Hif2a^−/− (n = 6), and eHif2a^−/− (n = 4 to 6) mice following chronic normobaric hypoxia (10% O₂ for 4 weeks) and in their corresponding Cre⁻ littermate controls. 18S was used for normalization. Bars represent mean values ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 7

Hypoxia and pharmacological PHD inhibition induce Edn1 and suppress Aplnr. (Top) Relative levels of Edn1, Apln and Aplnr in mice subjected to acute normobaric hypoxia (8% O₂ for 2 days) compared to normoxic controls (n = 4 or 5). (Bottom) Edn1, Apln, and Aplnr transcript levels in mice treated with prolyl-4-hydroxylase inhibitor GSK1002083A compared to vehicle-treated controls (n = 4 or 5). 18S was used for normalization. Bars represent mean values ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.