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

Long L, Ormiston ML, Yang X, Southwood M, Gräf S, Machado RD, Mueller M, Kinzel B, Yung LM, Wilkinson JM, Moore SD, Drake KM, Aldred MA, Yu PB, Upton PD, Morrell NW - Nat. Med. (2015)

Bottom Line: However, selective targeting of this signaling pathway using BMP ligands has not yet been explored as a therapeutic strategy.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.

View Article: PubMed Central - PubMed

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

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.

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Pulmonary hypertension in Bmpr2+/R899X knock-in mice is reversed by BMP9(a) Assessment of right ventricular systolic pressure (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 with 4 weeks of BMP9 treatment (75 ng/day, i.p., n=10 (9 male, 1 female) for WT and n=11 (9 male, 2 female) for Bmpr2+/R899X; 1-way ANOVA, Tukey’s post test). (b) Right ventricular hypertrophy (Fulton index, ratio of RV weight over LV and septal weight) in the same animals as a (not significant). (c) Quantitative assessment of pulmonary arterial muscularization in the same groups as a. Displayed as a non-, partially- and fully-muscularized arteries as a percentage of total alveolar wall and duct arteries (n=12 (6 male, 6 female) for naïve WT and Bmpr2+/R899X mice; with BMP9 treatment n=3 (2 male, 1 female) for WT and n=5 (3 male, 2 female) for Bmpr2+/R899X; 1-way ANOVA, Tukey’s post test). (d) Representative images of immunohistochemical staining for smooth muscle α-actin in lung sections from WT and Bmpr2+/R899X mice with or without BMP9 treatment. (e) Assessment of RVSP and (f) RV hypertrophy in male 6 month-old WT (n=16), Smad1+/− (n=9), Bmpr2+/R899X (n=10) and compound Bmpr2+/R899X/Smad1+/− heterozygotes (n=10; 1-way ANOVA, Tukey’s post test). (g) Quantification of monolayer permeability at 2 hours post-LPS. (n=3; 1-way ANOVA, repeated measures Tukey’s post test for WT or Bmpr2+/R899X cells; #P<0.05, 1-way ANOVA, Tukey’s post test for all groups). AU: Arbitrary units. (h) Images of lungs isolated from mice injected with 2 mg/kg LPS or vehicle and either 36.79 ng/25g BMP9 or vehicle (all i.p.) 22 hours prior to the i.p. injection of Evans blue dye, which was delivered 2 hours prior to sacrifice. (i) Quantitative assessment of extravascular Evans blue dye in the lungs of the mice described in h (Control: n=7 (5 male, 2 female) for WT, n=9 (4 male, 5 female) for Bmpr2+/R899X; LPS: n=8 (5 male, 3 female) for WT, n=10 (6 male, 4 female) for Bmpr2+/R899X; LPS + BMP9: n=8 (4 male, 4 female) for WT, n=9 (5 male, 4 female) for Bmpr2+/R899X; 1-way ANOVA, Tukey’s post test for WT or Bmpr2+/R899X mice; #P<0.05, 1-way ANOVA, Tukey’s post test for all groups). ***P<0.001, **P<0.01, *P<0.05. Mean +/− SEM.
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Figure 4: Pulmonary hypertension in Bmpr2+/R899X knock-in mice is reversed by BMP9(a) Assessment of right ventricular systolic pressure (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 with 4 weeks of BMP9 treatment (75 ng/day, i.p., n=10 (9 male, 1 female) for WT and n=11 (9 male, 2 female) for Bmpr2+/R899X; 1-way ANOVA, Tukey’s post test). (b) Right ventricular hypertrophy (Fulton index, ratio of RV weight over LV and septal weight) in the same animals as a (not significant). (c) Quantitative assessment of pulmonary arterial muscularization in the same groups as a. Displayed as a non-, partially- and fully-muscularized arteries as a percentage of total alveolar wall and duct arteries (n=12 (6 male, 6 female) for naïve WT and Bmpr2+/R899X mice; with BMP9 treatment n=3 (2 male, 1 female) for WT and n=5 (3 male, 2 female) for Bmpr2+/R899X; 1-way ANOVA, Tukey’s post test). (d) Representative images of immunohistochemical staining for smooth muscle α-actin in lung sections from WT and Bmpr2+/R899X mice with or without BMP9 treatment. (e) Assessment of RVSP and (f) RV hypertrophy in male 6 month-old WT (n=16), Smad1+/− (n=9), Bmpr2+/R899X (n=10) and compound Bmpr2+/R899X/Smad1+/− heterozygotes (n=10; 1-way ANOVA, Tukey’s post test). (g) Quantification of monolayer permeability at 2 hours post-LPS. (n=3; 1-way ANOVA, repeated measures Tukey’s post test for WT or Bmpr2+/R899X cells; #P<0.05, 1-way ANOVA, Tukey’s post test for all groups). AU: Arbitrary units. (h) Images of lungs isolated from mice injected with 2 mg/kg LPS or vehicle and either 36.79 ng/25g BMP9 or vehicle (all i.p.) 22 hours prior to the i.p. injection of Evans blue dye, which was delivered 2 hours prior to sacrifice. (i) Quantitative assessment of extravascular Evans blue dye in the lungs of the mice described in h (Control: n=7 (5 male, 2 female) for WT, n=9 (4 male, 5 female) for Bmpr2+/R899X; LPS: n=8 (5 male, 3 female) for WT, n=10 (6 male, 4 female) for Bmpr2+/R899X; LPS + BMP9: n=8 (4 male, 4 female) for WT, n=9 (5 male, 4 female) for Bmpr2+/R899X; 1-way ANOVA, Tukey’s post test for WT or Bmpr2+/R899X mice; #P<0.05, 1-way ANOVA, Tukey’s post test for all groups). ***P<0.001, **P<0.01, *P<0.05. Mean +/− SEM.

Mentions: In order to determine whether exogenous administration of BMP9 would be beneficial in a mouse model of PAH that mimics the genetics of human disease, we created a knock-in mouse, bearing the human disease-associated R899X premature stop mutation in exon 12 of the endogenous Bmpr2 locus (Supplementary Fig. 8a). As reported for Bmpr2 mice31, mice homozygous for the R899X mutation were non-viable. Heterozygous Bmpr2+/R899X mice developed normally and displayed reduced BMPR-II protein (Supplementary Fig. 8b) and mRNA (Supplementary Fig. 8c), consistent with an R899X mutation that leads to mRNA degradation by nonsense mediated mRNA decay (NMD) in humans32. We confirmed NMD of the R899X transcript in both mouse and human smooth muscle cells bearing the mutant allele (Supplementary Fig. 8d–i). Bmpr2+/R899X mice exhibited normal right ventricular systolic pressures (RVSP) at 3 months of age (Supplementary Fig. 9a), but developed elevated RVSP by 6 months (Fig. 4a). While this increase in RVSP was not accompanied by right ventricular hypertrophy (Fig. 4b), 6 month-old Bmpr2+/R899X mice did exhibit enhanced muscularization of peripheral pulmonary arteries in the lung (Fig. 4c, d). Further characterization of left ventricular function demonstrated no significant differences in left ventricular end diastolic pressure or cardiac output between 6 month-old Bmpr2+/R899X and WT controls (Supplementary Table 1). In contrast, 6 month-old Bmpr2+/− mice did not exhibit elevated RVSP or RV hypertrophy in our hands (Supplementary Fig. 9e–f).


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

Long L, Ormiston ML, Yang X, Southwood M, Gräf S, Machado RD, Mueller M, Kinzel B, Yung LM, Wilkinson JM, Moore SD, Drake KM, Aldred MA, Yu PB, Upton PD, Morrell NW - Nat. Med. (2015)

Pulmonary hypertension in Bmpr2+/R899X knock-in mice is reversed by BMP9(a) Assessment of right ventricular systolic pressure (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 with 4 weeks of BMP9 treatment (75 ng/day, i.p., n=10 (9 male, 1 female) for WT and n=11 (9 male, 2 female) for Bmpr2+/R899X; 1-way ANOVA, Tukey’s post test). (b) Right ventricular hypertrophy (Fulton index, ratio of RV weight over LV and septal weight) in the same animals as a (not significant). (c) Quantitative assessment of pulmonary arterial muscularization in the same groups as a. Displayed as a non-, partially- and fully-muscularized arteries as a percentage of total alveolar wall and duct arteries (n=12 (6 male, 6 female) for naïve WT and Bmpr2+/R899X mice; with BMP9 treatment n=3 (2 male, 1 female) for WT and n=5 (3 male, 2 female) for Bmpr2+/R899X; 1-way ANOVA, Tukey’s post test). (d) Representative images of immunohistochemical staining for smooth muscle α-actin in lung sections from WT and Bmpr2+/R899X mice with or without BMP9 treatment. (e) Assessment of RVSP and (f) RV hypertrophy in male 6 month-old WT (n=16), Smad1+/− (n=9), Bmpr2+/R899X (n=10) and compound Bmpr2+/R899X/Smad1+/− heterozygotes (n=10; 1-way ANOVA, Tukey’s post test). (g) Quantification of monolayer permeability at 2 hours post-LPS. (n=3; 1-way ANOVA, repeated measures Tukey’s post test for WT or Bmpr2+/R899X cells; #P<0.05, 1-way ANOVA, Tukey’s post test for all groups). AU: Arbitrary units. (h) Images of lungs isolated from mice injected with 2 mg/kg LPS or vehicle and either 36.79 ng/25g BMP9 or vehicle (all i.p.) 22 hours prior to the i.p. injection of Evans blue dye, which was delivered 2 hours prior to sacrifice. (i) Quantitative assessment of extravascular Evans blue dye in the lungs of the mice described in h (Control: n=7 (5 male, 2 female) for WT, n=9 (4 male, 5 female) for Bmpr2+/R899X; LPS: n=8 (5 male, 3 female) for WT, n=10 (6 male, 4 female) for Bmpr2+/R899X; LPS + BMP9: n=8 (4 male, 4 female) for WT, n=9 (5 male, 4 female) for Bmpr2+/R899X; 1-way ANOVA, Tukey’s post test for WT or Bmpr2+/R899X mice; #P<0.05, 1-way ANOVA, Tukey’s post test for all groups). ***P<0.001, **P<0.01, *P<0.05. Mean +/− SEM.
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Figure 4: Pulmonary hypertension in Bmpr2+/R899X knock-in mice is reversed by BMP9(a) Assessment of right ventricular systolic pressure (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 with 4 weeks of BMP9 treatment (75 ng/day, i.p., n=10 (9 male, 1 female) for WT and n=11 (9 male, 2 female) for Bmpr2+/R899X; 1-way ANOVA, Tukey’s post test). (b) Right ventricular hypertrophy (Fulton index, ratio of RV weight over LV and septal weight) in the same animals as a (not significant). (c) Quantitative assessment of pulmonary arterial muscularization in the same groups as a. Displayed as a non-, partially- and fully-muscularized arteries as a percentage of total alveolar wall and duct arteries (n=12 (6 male, 6 female) for naïve WT and Bmpr2+/R899X mice; with BMP9 treatment n=3 (2 male, 1 female) for WT and n=5 (3 male, 2 female) for Bmpr2+/R899X; 1-way ANOVA, Tukey’s post test). (d) Representative images of immunohistochemical staining for smooth muscle α-actin in lung sections from WT and Bmpr2+/R899X mice with or without BMP9 treatment. (e) Assessment of RVSP and (f) RV hypertrophy in male 6 month-old WT (n=16), Smad1+/− (n=9), Bmpr2+/R899X (n=10) and compound Bmpr2+/R899X/Smad1+/− heterozygotes (n=10; 1-way ANOVA, Tukey’s post test). (g) Quantification of monolayer permeability at 2 hours post-LPS. (n=3; 1-way ANOVA, repeated measures Tukey’s post test for WT or Bmpr2+/R899X cells; #P<0.05, 1-way ANOVA, Tukey’s post test for all groups). AU: Arbitrary units. (h) Images of lungs isolated from mice injected with 2 mg/kg LPS or vehicle and either 36.79 ng/25g BMP9 or vehicle (all i.p.) 22 hours prior to the i.p. injection of Evans blue dye, which was delivered 2 hours prior to sacrifice. (i) Quantitative assessment of extravascular Evans blue dye in the lungs of the mice described in h (Control: n=7 (5 male, 2 female) for WT, n=9 (4 male, 5 female) for Bmpr2+/R899X; LPS: n=8 (5 male, 3 female) for WT, n=10 (6 male, 4 female) for Bmpr2+/R899X; LPS + BMP9: n=8 (4 male, 4 female) for WT, n=9 (5 male, 4 female) for Bmpr2+/R899X; 1-way ANOVA, Tukey’s post test for WT or Bmpr2+/R899X mice; #P<0.05, 1-way ANOVA, Tukey’s post test for all groups). ***P<0.001, **P<0.01, *P<0.05. Mean +/− SEM.
Mentions: In order to determine whether exogenous administration of BMP9 would be beneficial in a mouse model of PAH that mimics the genetics of human disease, we created a knock-in mouse, bearing the human disease-associated R899X premature stop mutation in exon 12 of the endogenous Bmpr2 locus (Supplementary Fig. 8a). As reported for Bmpr2 mice31, mice homozygous for the R899X mutation were non-viable. Heterozygous Bmpr2+/R899X mice developed normally and displayed reduced BMPR-II protein (Supplementary Fig. 8b) and mRNA (Supplementary Fig. 8c), consistent with an R899X mutation that leads to mRNA degradation by nonsense mediated mRNA decay (NMD) in humans32. We confirmed NMD of the R899X transcript in both mouse and human smooth muscle cells bearing the mutant allele (Supplementary Fig. 8d–i). Bmpr2+/R899X mice exhibited normal right ventricular systolic pressures (RVSP) at 3 months of age (Supplementary Fig. 9a), but developed elevated RVSP by 6 months (Fig. 4a). While this increase in RVSP was not accompanied by right ventricular hypertrophy (Fig. 4b), 6 month-old Bmpr2+/R899X mice did exhibit enhanced muscularization of peripheral pulmonary arteries in the lung (Fig. 4c, d). Further characterization of left ventricular function demonstrated no significant differences in left ventricular end diastolic pressure or cardiac output between 6 month-old Bmpr2+/R899X and WT controls (Supplementary Table 1). In contrast, 6 month-old Bmpr2+/− mice did not exhibit elevated RVSP or RV hypertrophy in our hands (Supplementary Fig. 9e–f).

Bottom Line: However, selective targeting of this signaling pathway using BMP ligands has not yet been explored as a therapeutic strategy.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.

View Article: PubMed Central - PubMed

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

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.

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Related in: MedlinePlus