Selective enhancement of endothelial BMPR-II with BMP9 reverses pulmonary arterial hypertension

Journal name:
Nature Medicine
Volume:
21,
Pages:
777–785
Year published:
DOI:
doi:10.1038/nm.3877
Received
Accepted
Published online

Abstract

Genetic evidence implicates the loss of bone morphogenetic protein type II receptor (BMPR-II) signaling in the endothelium as an initiating factor in pulmonary arterial hypertension (PAH). However, selective targeting of this signaling pathway using BMP ligands has not yet been explored as a therapeutic strategy. Here, we identify BMP9 as the preferred ligand for preventing apoptosis and enhancing monolayer integrity in both pulmonary arterial endothelial cells and blood outgrowth endothelial cells from subjects with PAH who bear mutations in the gene encoding BMPR-II, BMPR2. Mice bearing a heterozygous knock-in allele of a human BMPR2 mutation, R899X, which we generated as an animal model of PAH caused by BMPR-II deficiency, spontaneously developed PAH. Administration of BMP9 reversed established PAH in these mice, as well as in two other experimental PAH models, in which PAH develops in response to either monocrotaline or VEGF receptor inhibition combined with chronic hypoxia. These results demonstrate the promise of direct enhancement of endothelial BMP signaling as a new therapeutic strategy for PAH.

At a glance

Figures

  1. BMP9 preferentially stimulates human PAECs.
    Figure 1: BMP9 preferentially stimulates human PAECs.

    (ac) Volcano plots of differentially expressed genes in PAECs treated with 1 ng/ml BMP9 (a), 10 ng/ml BMP6 (b) or 10 ng/ml BMP2 (c) versus medium-alone control after fitting linear models and adjusting P values for multiple testing. Differentially expressed genes of the TGF-β pathway are highlighted in red. Dashed lines represent an adjusted P value of 0.05 and a fold change of ±1×. (d) Heat map showing whole-gene-set perturbation of regulated pathways in PAECs following BMP9 treatment. A–D indicate individual PAEC lines. Sig, signaling. (e) SPIA to detect alterations in common signaling pathways in PAECs in response to BMP9 treatment. The probabilities were corrected for false discovery rate (FDR). PPert, probability of observing a total accumulated perturbation of the pathway more extreme than expected by chance; PNde, probability of obtaining a number of differentially expressed genes in the given pathway at least as large as the observed one. (f) Immunoblotting for phosphorylated Smads 1, 5 and 8 (pSmad1/5/8), Id1, Id3, BMPR-II and β-actin in PAECs cultured without or with the indicated concentrations of BMP9, BMP4 and BMP6 ligand for 2 h. The data shown are representative of three experiments. (g) Expression of BMPR2 in PAECs without or with treatment of 10 ng/ml BMP9 for 24 h (n = 3; Student's t-test). (h) Representative agarose gels showing amplification of the Smad-binding segment of the BMPR2 promoter from products of ChIP assays using lysates from HMEC-1 cells without treatment (top), 72 h after siRNA knockdown of Smad1 (middle) or after a 24-h treatment with 1 ng/ml BMP9 (bottom). pS1/5, phospho–Smads 1 and 5. IP, immunoprecipitation. (i) Luciferase activity in lysates from HMEC-1 cells transfected with a luciferase reporter construct without (PGL-3) or with an upstream 5-kb segment of the BMPR2 promoter without (BMPR2) or with (mut. BMPR2) mutation of the putative Smad-binding region. Transfected cells were treated with 10 ng/ml BMP9 for 24 h (n = 3; one-way ANOVA, Tukey's post hoc test). PGL-3, luciferase reporter vector alone. ***P < 0.001; NS, not significant. Error bars, mean ± s.e.m.

  2. BMP9 prevents apoptosis in human PAECs via BMPR-II.
    Figure 2: BMP9 prevents apoptosis in human PAECs via BMPR-II.

    (a,b) Representative immunoblots and densitometric analysis for phospho-JNK (n = 4) (a) and cleaved caspase-3 (n = 3) (b) in human PAECs cultured without or with BMP9 (5 ng/ml) for 16 h before an apoptotic stimulus with TNFα (10 ng/ml) and cycloheximide (CHX; 20 μg/ml). (c) Representative flow cytometry plots of human PAECs stained with annexin-V and propidium iodide (PI) without or with BMP9 pretreatment and apoptotic stimulus. (d) Quantification of apoptotic (annexin-V+ PI) PAECs (n = 5). (e) Validation of siRNA knockdown in PAECs by immunoblotting for BMPR-II following treatment with either Dharmafect 1 transfection reagent (DH1), siRNA for BMPR2 (siBMPR2) or a pooled siRNA control (siCP). (f) Cytometric quantification of apoptosis by staining for annexin-V and PI in PAECs treated with DH1 alone, siBMPR2 or siCP and cultured without or with BMP9 pretreatment and apoptotic stimulus (n = 6 for DH1 and siBMPR2; n = 5 for siCP; ANOVA for each siRNA group, Tukey's post hoc test). (g) Representative immunoblots and densitometric analysis of cleaved caspase-3 in PAECs following siRNA transfection without or with BMP9 pretreatment and apoptotic stimulus. (n = 3). All blots were reprobed for α-tubulin as a loading control. One-way ANOVA, Tukey's post hoc test used in a,b,d,f and g. ***P < 0.001, **P < 0.01, *P < 0.05. Error bars are mean ± s.e.m.

  3. BMP9 prevents apoptosis and promotes monolayer integrity in BOECs.
    Figure 3: BMP9 prevents apoptosis and promotes monolayer integrity in BOECs.

    (a) Quantification of apoptotic BOECs (annexin-V+ PI) isolated from either controls (n = 5) or from individuals with BMPR2 mutations (n = 6) after culture without or with BMP9 (5 ng/ml) for 16 h before the addition of TNFα (10 ng/ml) and cycloheximide (CHX; 20 μg/ml) for 6 h. (b,c) Immunoblotting for phosphorylated JNK (b) and cleaved caspase-3 (c) in control and BMPR2 mutation–bearing BOECs without or with BMP9 pretreatment and apoptotic stimulus. Each sample is from a single individual, and the results shown are representative of five individuals per group. All blots were reprobed for α-tubulin as a loading control. (d,e) Permeability of control (d) or BMPR2 mutation–bearing BOEC monolayers (e) to horseradish peroxidase, assessed by colorimetric absorbance after the indicated incubation periods, without or with BMP9 (20 ng/ml) and/or LPS (400 ng/ml). (f) Quantification of monolayer permeability at 2 h after the indicated treatments (n = 4 individuals per group). AU, arbitrary units. (g) Representative immunofluorescence images of PAECs stained for VE-cadherin following 24 h culture without or with BMP9 (10 ng/ml) and/or LPS (400 ng/ml). Arrows indicate loss of VE-cadherin staining. Scale bars, 10 μm. ***P < 0.001, **P < 0.01, *P < 0.05, one-way ANOVA, repeated-measures Tukey's post hoc test for control or BMPR2 mutation-containing BOECs; ###P < 0.001, ##P < 0.01, one-way ANOVA, Tukey's post hoc test for all groups. Error bars are mean ± s.e.m.

  4. Pulmonary hypertension in Bmpr2+/R899X knock-in mice is reversed by BMP9.
    Figure 4: Pulmonary hypertension in Bmpr2+/R899X knock-in mice is reversed by BMP9.

    (a) Assessment of RVSP in 6-month-old, naive Bmpr2+/R899X mice (n = 21; 11 male, 10 female) and WT littermate controls (n = 23; 13 male, 10 female), or after 4 weeks of BMP9 treatment (75 ng/day, i.p.) of 6-month-old WT (n = 10; 9 male, 1 female) or Bmpr2+/R899X (n = 11; 9 male, 2 female) mice. (b) Right ventricular hypertrophy (Fulton index, ratio of right ventricular (RV) weight to left ventricular (LV) plus septal (S) weight) in the same animals as in a (no significant differences among groups). (c) Quantitative assessment of pulmonary arterial muscularization in the same groups as in a. Group sizes were: untreated WT mice (n = 12; 6 male, 6 female), untreated Bmpr2+/R899X mice (n = 12; 6 male, 6 female), BMP9-treated WT mice (n = 3; 2 male, 1 female) and BMP9-treated Bmpr2+/R899X mice (n = 5; 3 male, 2 female). Non-, partially and fully muscularized arteries as a percentage of total alveolar wall and duct arteries were scored. (d) Representative images of immunohistochemical staining for smooth muscle α-actin in lung sections from WT and Bmpr2+/R899X mice without or with BMP9 treatment. Scale bars, 100 μm. (e,f) Assessment of RVSP (e) and right ventricular hypertrophy (f) in male 6-month-old WT (n = 16), Smad1+/− (n = 9), Bmpr2+/R899X (n = 10) and Bmpr2+/R899X Smad1+/− (n = 10) mice. (g) Quantification of monolayer permeability at 2 h after LPS treatment (n = 3). Repeated measures ANOVA, Tukey's post hoc test for WT or Bmpr2+/R899X cells. #P < 0.05, one-way ANOVA for all groups. AU, arbitrary units. (h) Images of Evans blue dye staining in lungs isolated from WT and Bmpr2+/R899X mice injected with vehicle (control), LPS or LPS plus BMP9 before dye injection. (i) Quantitative assessment of Evans blue dye content in the lungs of the mice described in h. Group sizes were: no treatment, WT (n = 7; 5 male, 2 female); no treatment, Bmpr2+/R899X (n = 9; 4 male, 5 female); LPS, WT (n = 8; 5 male, 3 female); LPS, Bmpr2+/R899X (n = 10; 6 male, 4 female); LPS + BMP9, WT (n = 8; 4 male, 4 female); and LPS + BMP9, Bmpr2+/R899X (n = 9; 5 male, 4 female). One-way ANOVA for WT or Bmpr2+/R899X mice; #P < 0.05, one-way ANOVA for all groups. ***P < 0.001, **P < 0.01, *P < 0.05, one-way ANOVA, Tukey's post hoc test used in ac, eg and i. Error bars are mean ± s.e.m.

  5. BMP9 reverses established monocrotaline-induced pulmonary hypertension and prevents endothelial cell apoptosis in rats.
    Figure 5: BMP9 reverses established monocrotaline-induced pulmonary hypertension and prevents endothelial cell apoptosis in rats.

    (a,b) Assessment of right ventricular systolic pressure (RVSP) (a) and right ventricular (RV) hypertrophy (Fulton index) (b) in rats on a prevention protocol given vehicle (n = 6) or MCT (60 mg/kg, i.p.) and treated with BMP9 (n = 7, 600 ng/day, i.p.) or vehicle (n = 8) from days 0 to 21 after MCT treatment. (c) Quantitative assessment of pulmonary arterial muscularization in control rats or rats given MCT, followed by daily injections with saline or BMP9 in a prevention (days 0–21 post-MCT) or reversal (days 21–35 post-MCT) protocol. Non-, partially and fully muscularized arteries as a percentage of total alveolar wall and duct arteries were scored (n = 5 for each prevention group, n = 6 for each reversal group, n = 6 for control group); ANOVA for fully muscularized vessels. (d) Representative images of immunohistochemical staining for smooth muscle α-actin in lung sections from the rats described in c. Scale bars, 100 μm. (e,f) Assessment of right ventricular systolic pressure (RVSP) (e) and right ventricular hypertrophy (Fulton index) (f) in rats on a reversal protocol given vehicle (n = 4) or MCT (40 mg/kg, i.p.) (n = 39) and treated with BMP9 (n = 10, 600 ng/day) or vehicle (n = 9) from days 21 to 35 post-MCT (t-test on day 35 groups treated with saline or BMP9). (g) Representative images of immunohistochemical staining for von Willebrand factor (top) and cleaved caspase-3 (bottom) in lung sections from rats given vehicle (control) or MCT (60 mg/kg, i.p.), treated with BMP9 (600 ng/day, i.p.) or saline from day 0 and killed on day 5 after MCT treatment. Scale bars, 50 μm. (h) Quantification of cleaved caspase-3–positive endothelial cells in lung sections from the groups described in a, b and g and killed on days 2, 5 or 21 after MCT treatment (n = 11 for control, n = 6 for days 2 and 5 post-MCT, n = 5 for day 21 post-MCT). ***P < 0.001, **P < 0.01, *P < 0.05. One-way ANOVA, Tukey's post hoc test used in ac and h. Error bars are mean ± s.e.m.

  6. BMP9 reverses established pulmonary hypertension in the Sugen-hypoxia rat model.
    Figure 6: BMP9 reverses established pulmonary hypertension in the Sugen-hypoxia rat model.

    (a) Schematic of the experimental design. Rats were given vehicle injections and maintained in normoxia (n = 4) or challenged with SU-5416 (20 mg/kg, i.p.) and 3 weeks of hypoxia (10% O2) before 5 weeks of normoxia and assessment at 8 weeks (n = 7) or at 11 weeks following daily treatment with saline vehicle (n = 11) or BMP9 (n = 11; 600 ng/day, i.p.). (b,c) Assessment of RVSP (b) and right ventricular hypertrophy (Fulton index) (c). Here and below: SU, SU-5416. (d) Quantification of non-, partially and fully muscularized arteries as a percentage of total alveolar wall and duct arteries (n = 4 for control, n = 6 for all other groups; one-way ANOVA for fully muscularized vessels). (e) Assessment of pulmonary arterial wall thickness as a percentage of luminal diameter (n = 4 for control, n = 6 for all other groups). (f) Quantification of neointimal lesion frequency (n = 3 for control, n = 6 for all other groups). (g) Quantification of cleaved caspase-3–positive endothelial cells in lung sections (n = 3 for control, n = 6 for all other groups). Error bars are mean ± s.e.m. (h) Representative images of neointima formation in rat lungs. Lung sections were stained for smooth muscle α-actin (SMA) or with elastic van Gieson (EVG) stain. Scale bars, 50 μm. ***P < 0.001, **P < 0.01, *P < 0.05. One-way ANOVA, Tukey's post hoc test used in bg.

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Author information

  1. These authors contributed equally to this work.

    • Lu Long,
    • Mark L Ormiston &
    • Xudong Yang
  2. These authors jointly directed this work.

    • Paul D Upton &
    • Nicholas W Morrell

Affiliations

  1. Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK.

    • Lu Long,
    • Mark L Ormiston,
    • Xudong Yang,
    • Stefan Gräf,
    • Janine M Wilkinson,
    • Stephen D Moore,
    • Paul D Upton &
    • Nicholas W Morrell
  2. Department of Pathology, Papworth Hospital, Papworth Everard, Cambridge, UK.

    • Mark Southwood
  3. University of Lincoln, School of Life Sciences, Lincoln, UK.

    • Rajiv D Machado
  4. Novartis Institutes for Biomedical Research, Basel, Switzerland.

    • Matthias Mueller &
    • Bernd Kinzel
  5. Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

    • Lai Ming Yung &
    • Paul B Yu
  6. Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.

    • Kylie M Drake &
    • Micheala A Aldred

Contributions

L.L. designed, performed and analyzed all in vivo and some in vitro and ex vivo experiments. M.L.O. designed, performed and analyzed multiple in vitro experiments, including array study, and some ex vivo experiments and wrote the manuscript. X.Y. designed, performed and analyzed multiple in vitro experiments and some ex vivo experiments. M.S. performed all histological analyses, including in vivo quantification of apoptosis. S.G. analyzed and interpreted array data. R.D.M., M.M. and B.K. designed and created the R899X knock-in mouse. L.M.Y. and P.B.Y. designed, performed and interpreted the mouse Sugen-hypoxia experiments and assessment of in vivo bone formation. J.M.W. performed in vitro three-dimensional tube formation assays. S.D.M. designed and performed collection and treatment of rat pulmonary arteries. K.M.D. and M.A.A. performed human and mouse NMD analysis. P.D.U. designed and supervised multiple experiments and performed in vitro assessment of VEGF-induced proliferation. N.W.M. conceived and supervised the study, designed experiments and wrote the manuscript.

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