Endothelial HIF-2α contributes to severe pulmonary hypertension due to endothelial-to-mesenchymal transition

Abstract

Pulmonary vascular remodeling characterized by concentric wall thickening and intraluminal obliteration is a major contributor to the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Here we report that increased hypoxia-inducible factor 2α (HIF-2α) in lung vascular endothelial cells (LVECs) under normoxic conditions is involved in the development of pulmonary hypertension (PH) by inducing endothelial-to-mesenchymal transition (EndMT), which subsequently results in vascular remodeling and occlusive lesions. We observed significant EndMT and markedly increased expression of SNAI, an inducer of EndMT, in LVECs from patients with IPAH and animals with experimental PH compared with normal controls. LVECs isolated from IPAH patients had a higher level of HIF-2α than that from normal subjects, whereas HIF-1α was upregulated in pulmonary arterial smooth muscle cells (PASMCs) from IPAH patients. The increased HIF-2α level, due to downregulated prolyl hydroxylase domain protein 2 (PHD2), a prolyl hydroxylase that promotes HIF-2α degradation, was involved in enhanced EndMT and upregulated SNAI1/2 in LVECs from patients with IPAH. Moreover, knockdown of HIF-2α (but not HIF-1α) with siRNA decreases both SNAI1 and SNAI2 expression in IPAH-LVECs. Mice with endothelial cell (EC)-specific knockout (KO) of the PHD2 gene, egln1 (egln1^EC-/-), developed severe PH under normoxic conditions, whereas Snai1/2 and EndMT were increased in LVECs of egln1^EC-/- mice. EC-specific KO of the HIF-2α gene, hif2a, prevented mice from developing hypoxia-induced PH, whereas EC-specific deletion of the HIF-1α gene, hif1a, or smooth muscle cell (SMC)-specific deletion of hif2a, negligibly affected the development of PH. Also, exposure to hypoxia for 48-72 h increased protein level of HIF-1α in normal human PASMCs and HIF-2α in normal human LVECs. These data indicate that increased HIF-2α in LVECs plays a pathogenic role in the development of severe PH by upregulating SNAI1/2, inducing EndMT, and causing obliterative pulmonary vascular lesions and vascular remodeling.

Keywords: endothelial cell; intimal lesion; prolyl hydroxylase domain-containing protein; pulmonary arterial hypertension.

Fig. 1.

Endothelial-to-mesenchymal transition (EndMT) and cell proliferation are enhanced in lung vascular endothelial cells (LVECs) isolated from patients with idiopathic pulmonary arterial hypertension (IPAH). A: real-time RT-PCR analysis of SNAI1, SNAI2, PECAM1, CDH5, ACTA2, S100A4, and FN1 (normalized to GAPDH expression) in LVECs isolated from healthy subjects (n = 7) and IPAH patients (n = 6). B: representative images showing immunofluorescence in normal and IPAH-LVECs stained for SNAI1, PECAM1, and TAGLN. Nuclei counterstained with Hoechst. Scale bar, 100 μm. C: summarized data showing the mean fluorescence intensity for each antigen in normal and IPAH-LVECs (n = 3 per group; for each experiment, we analyzed three fields of view with 10 regions of interest per field of view). D: representative images showing nuclear staining (left, Hoechst, blue), 5′ethnyl-2′-deoxyuridine (EdU) staining (middle, AlexaFluor, red), and overlay images (right, overlay, purple) of normal and IPAH-LVECs. Scale bar, 100 μm. E: summarized data showing the percentage of cells with EdU incorporation (left) and the percentage of total cell number (right) in normal and IPAH-LVECs (n = 3 per group; for each experiment, we analyzed 3 fields of view with 6 regions of interest per field of view). F: real-time RT-PCR analysis of MKI67 (normalized to GAPDH expression) in normal (n = 7) and IPAH (n = 6) LVECs. Values are median ± CI. *P < 0.05; **P < 0.01; ***P < 0.001 vs. normal.

Fig. 2.

Transforming growth factor-β1 (TGF-β1) induces EndMT in normal LVECs to a level similar observed in IPAH-LVECs. A: representative images (panels at left) and summarized data (panel at right) showing cell length in normal LVECs treated with vehicle or TGF-β1 (10 ng/ml) and IPAH-LVECs at day 1 or day 7 (n = 3 per group). Scale bar, 30 µm. Values are median ± CI. ***P < 0.001 vs. vehicle; ^###P < 0.001 vs. TGF-β1. B: representative images (panels at left) showing immunofluorescence in normal LVECs treated with vehicle or TGF-β1 (10 ng/ml) for 7 days and IPAH-LVECs stained for PECAM1 and summarized data (panel at right) showing the fraction of adherens junctions that are discontinuous with adjacent cells (n = 3 per group; for each experiment, we analyzed 3 fields of view with 10 regions of interest per field of view). Yellow arrows highlight the continuous and discontinuous adherens junctions. C: representative images (panels at left) and summarized data (panels at right) showing the mean fluorescence intensity in normal LVECs treated with vehicle or TGF-β1 (10 ng/ml) for 7 days and IPAH-LVECs stained for SNAI1, PECAM1, vimentin (VIM), and TAGLN (n = 3 per group; for each experiment, we analyzed 3 fields of view with 10 regions of interest per field of view). Cells stained with secondary antibody only were used as a negative control. Nuclei counterstained with Hoechst. Red scale bars, 100 μm; white scale bars, 20 μm. D: real-time RT-PCR analysis of SNAI1, SNAI2, PECAM1, CDH5, ACTA2, and FN1 (normalized to GAPDH expression) in normal LVECs treated with vehicle or TGFβ1 (10 ng/ml) for 7 days (n = 3 per group). Values are median ± CI. *P < 0.05; **P < 0.01; ***P < 0.001 vs. vehicle.

Fig. 3.

Expression of prolyl hydroxylase domain protein 2 (PHD2) is decreased, and hypoxia-inducible factor 2α (HIF-2α) is increased in pulmonary arterial (PA) endothelium and LVECs from patients with IPAH. A: real-time RT-PCR analysis of PHD1/2/3 (normalized to GAPDH expression) in LVECs from normal subjects (n = 7) and patients with IPAH (n = 6). B: representative images (panels at left) showing immunohistochemical staining for HIF-2α in distal pulmonary arteries and summarized data (panel at right) in normal subjects and IPAH patients. Scale bars, 50 μm. C: representative Western blot images showing the protein expression level of HIF-2α (left) and summarized data (right) in LVECs from normal subjects and IPAH patients (n = 3 per group). D: representative images (left) and summarized data (right) showing the mean fluorescence intensity in normal and IPAH-LVECs stained for HIF-2α (green) (n = 3 per group; for each experiment, we analyzed 3 fields per view; 10 regions of interest per field of view). Nuclei counterstained with Hoechst (blue). Scale bars, 20 μm. Values are means ± SE **P < 0.01; ***P < 0.001 vs. normal. E: representative Western blot images showing the protein expression level of HIF2α (left) and summarized data (right) in normal human LVECs treated with a PHD2 inhibitor (FG-4592) at a concentration of 100 μM or vehicle (0.01% DMSO) for 4 h (n = 3 per group). All blots were reprobed for β-actin as a loading control. Values are means ± SE ***P < 0.001 vs. vehicle.

Fig. 4.

HIF-2α, but not HIF-1α, regulates SNAI1/2 expression in LVECs isolated from IPAH patients. A: representative Western blot images showing the protein expression level of HIF-1α (left) and summarized data (right) in LVECs from seven normal subjects and six IPAH patients as well as in pulmonary arterial smooth muscle cells (PASMCs) from four normal subjects and four IPAH patients. ND, not detectable. B: representative Western blot images showing the protein expression level of HIF-2α (left) and summarized data (right) in normal (n = 7) and IPAH (n = 6) LVECs as well as in normal (n = 4) and IPAH (n = 4) PASMCs. Values are means ± SE; *P < 0.05; ***P < 0.001 vs. normal. C: real-time RT-PCR analysis of HIF-1α and HIF-2α (normalized to GAPDH expression) in LVECs isolated from IPAH patients transfected with control siRNA (Cont-siR), HIF-1α siRNA (HIF-1α-siR), and HIF-2α (HIF-2α-siR) siRNA for 4–6 h (n = 4 per group). D: representative Western blot images showing the protein expression level of SNAI1 and SNAI2 (top) and summarized data (bottom) in IPAH-LVECs transfected with control, HIF-1α, and HIF-2α siRNA for 4–6 h (n = 4 per group). Values are means ± SE; **P < 0.01; ***P < 0.001 vs. Cont-siR. E: representative Western blot images showing the protein expression level of HIF-1α and HIF-2α (panels at left) and summarized data (panels at right) in normal human PASMCs and human LVECs exposed to normoxia (Nor) or hypoxia (Hyp) for 48–72 h (n = 5 per group). All blots were reprobed for β-actin as a loading control. Values are means ± SE; *P < 0.05; ***P < 0.001 vs. normoxia.

Fig. 5.

Monocrotaline (MCT) treatment results in pulmonary hypertension at both 2- and 4-wk time points compared with controls. A: representative tracings (left) showing right ventricular pressure (RVP) and summarized data (right) showing peak right ventricular systolic pressure (RVSP) in control and treated (2 or 4 wk after MCT injection) rats. Scale bars, 0.2 s. B: summarized data showing RV hypertrophy defined by the Fulton index as a ratio [RV/(LV + S)] in control and MCT-treated rats (2 or 4 wk). Values are the median ± CI of individually tested rats (n = 6 per group). C: representative hematoxylin and eosin images of pulmonary artery (panels at left) and summarized data (panel at right) showing pulmonary artery thickness, as measured by the ratio of wall area to total vessel area in control and MCT-treated (2 or 4 wk) rats. Scale bars, 10 μm. D: representative angiograph images showing branches and junctions of the pulmonary arterial tree in the lungs of control and MCT-treated rats (4 wk). Insert represents the magnified area. E: summarized data showing the branch number, junction number, and total length of the pulmonary arterial tree measured from pulmonary angiograph images of control and MCT-treated (4 wk) animals. RVP, right ventricular pressure; RVSP, right ventricular systolic pressure; RV, right ventricle; LV, left ventricle; S, septum; PA, pulmonary artery; w, weeks. Values are the means ± SE of individually tested rat (n = 6 per group). **P < 0.01; ***P < 0.001 vs. control; ^#P < 0.05; ^###P < 0.001 vs. MCT 2w.

Fig. 6.

EndMT observed in the lung tissue from rats with MCT-PH. A: representative images showing immunofluorescence of lung tissues isolated from control and MCT-treated rats stained for Snai1, Pecam1, Tagln, and Acta2. Nuclei counterstained with Hoechst. Insert represents the magnified area. Red scale bars, 500 μm; white scale bars, 50 μm. B: summarized data showing the mean fluorescence intensity for each antigen in control and MCT-treated rat lung tissue (n = 3 per group; for each experiment we analyzed 5 fields of view per rat). C–E: real-time RT-PCR analysis of Snai1, Snai2, Pecam1, Cdh5, Ctnnd1, Acta2, Vim, Mki67, and Phd2 (normalized to Gapdh expression) in control and MCT-treated rat lung tissues (n = 4 per group). Values are median ± CI; *P < 0.05; **P < 0.01; ***P < 0.001 vs. control.

Fig. 7.

Deletion of egln1 in endothelial cells results in severe pulmonary hypertension. A: representative tracings (panels at left) showing RVP and summarized data (panel at right) showing peak RVSP in WT, egln1^EC+/−, and egln1^EC−/− mice (n = 10 per group). Scale bars, 0.2 s. B: summarized data showing RV hypertrophy defined by the Fulton index as a ratio [RV/(LV + S)] in WT, egln1^EC+/−, or egln1^EC−/− mice (n = 10 per group). C: representative hematoxylin and eosin images of small pulmonary arteries from WT mice or egln1^EC−/− mice. Scale bars, 10 μm. D: summarized data showing PA wall thickness, as measured by the ratio of wall area to total vessel area (left) and the number of PA with complete obliteration or plexiform lesion (right) in PA with a diameter less than 100 µm from WT mice or egln1^EC−/− mice (n = 10 per group). E: representative Western blot images (left) showing the protein expression level of Phd2 and summarized data (right) in WT and egln1^EC-/−murine lung tissue (n = 4 per group). F: representative gel images (left) and real-time RT-PCR analysis (right) of Phd2 (normalized to Gapdh expression) in WT, egln1^EC+/−, and egln1^EC−/− mouse lung tissue (n = 4 per group). G: representative images (left) and summarized data (right) showing immunofluorescence of lung ECs isolated from WT and egln1^EC+/− mice stained for Hif-2α and Cdh5 (n = 3 per group). Nuclei counterstained with Hoechst. Scale bars, 10 μm. H: representative Western blot images (left) showing the protein expression level of Phd2 and Hif-2α and summarized data (right) in lung ECs isolated from WT and egln1^EC+/− mouse lung tissue (n = 3 per group). All blots were reprobed for β-actin as a loading control. RVP, right ventricular pressure; RVSP, right ventricular systolic pressure; RV, right ventricle; LV, left ventricle; S, septum; PA, pulmonary artery; ND, not detectable; WT, wild-type; EC, endothelial cells. Values are means ± SE *P < 0.05; **P < 0.01; ***P < 0.001 vs. WT.

Fig. 8.

EndMT observed in lung tissue of mice with endothelial-specific deletion of Phd2. A: representative images showing immunofluorescence of lung tissues isolated from WT (egln1^+/+) and egln1^EC−/− mice stained for Snai1, Snai2, Tagln, Acta2, and S100A4. Nuclei counterstained with Hoechst. Insert represents the magnified area. Red scale bars, 200 μm. White scale bars, 50 μm. B: summarized data showing the mean fluorescence intensity for each antigen in WT and egln1^EC−/− murine lung tissue (n = 3 per group; for each experiment, we analyzed 5 fields of view per mouse). C: real-time RT-PCR analysis of Snai1, Snai2, Pecam1, Cdh5, Acta2, and S100A4 (normalized to Gapdh expression) in WT (egln1^+/+) or egln1^EC−/− murine lung tissue (n = 4 per group). Values are median ± CI. *P < 0.05; **P < 0.01; ***P < 0.001 vs. WT.

Fig. 9.

Endothelial-specific deletion of hif2α attenuates hypoxia-induced pulmonary hypertension. Representative tracings showing RVP (A) and summarized data (B) showing peak RVSP in WT and hif2a^EC−/− mice (KO) in both normoxia and hypoxia for 3 wk. Scale bar, 0.2 s. C: summarized data showing right ventricle hypertrophy defined by the Fulton index as a ratio [RV/(LV + S)] in WT and hif2a^EC−/− mice under normoxic and hypoxic conditions. D: representative hematoxylin and eosin images of distal pulmonary arteries (left) and summarized data (right) showing PA wall thickness, as measured by the ratio of wall area to total vessel area, in WT and hif2a^EC−/− (KO) mice in normoxic and hypoxic conditions for 3 wk. Scale bars, 10 μm. Representative tracings showing RVP (E) and summarized data showing peak RVSP (F) and RV hypertrophy (G) in WT and hif2a^SM−/− mice (KO) under normoxia or hypoxia (for 3 wk). Representative tracings showing RVP (H) and summarized data showing peak RVSP (I) or RV hypertrophy (J) in WT and hif1a^EC−/− mice (KO) under normoxia or hypoxia (for 3 wk). Scale bar, 0.2 s. Nor, normoxia; Hyp, hypoxia; RVP, right ventricular pressure; RVSP, right ventricular systolic pressure; RV, right ventricle; LV, left ventricle; S, septum; PA, pulmonary artery; WT, wild-type; KO, knockout. Values are the median ± CI of individually tested mice (n = 10 per group). ***P < 0.001 vs. WT-Nor; ^###P < 0.001 vs. WT-Hyp.

Fig. 10.

Deletion of hif1a or hif2a in endothelial cells has no effect on acute hypoxia-induced pulmonary vasoconstriction. A: representative tracings showing pulmonary artery pressure (PAP) before, during (solid bar), and after superfusion of modified Kreb’s solutions with different K⁺ concentrations (10, 20, 30, 40, 60, 80, or 120 mM) and airway ventilation of hypoxia (Hyp) (solid bar; 1% O₂; shadowed) in isolated perfused/ventilated lungs from wild-type (WT) (top), hif1a^EC−/− (middle), or hif2a^EC−/− (bottom) mice. Scale bars, 3 min. B: summarized data showing dose-response curves of high K⁺-induced increases in mean PAP in isolated perfused/ventilated lungs from eight WT, four hif1a^EC−/−, or four hi2a^EC−/− mice. C: summarized data showing amplitudes of hypoxia-induced increases in mean PAP in isolated perfused/ventilated lungs from WT, hif1a^EC−/−, or hif2a^EC−/− mice. Values are means ± SE. No significant differences were seen between WT and hif1a^EC−/− or hif2a^EC−/− mice.