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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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BMP9 prevents apoptosis and promotes monolayer integrity in BOECs with and without BMPR-II mutations(a) Quantification of apoptotic (Annexin-V+/PI−) control (n=5 individuals) and BMPR2 mutation-bearing BOECs (n=6 individuals) after culture with or without BMP9 (5 ng/mL) for 16 hours prior to the addition of TNFα (10 ng/mL) and cyclohexamide (20 μg/mL) for 6 hours. (b–c) Immunoblotting for (b) phosphorylated JNK and (c) cleaved caspase-3 in control and BMPR2 mutation-bearing BOECs with or without BMP9 pre-treatment and apoptotic stimulus. Representative of five immunoblots. All blots were re-probed for α-tubulin as a loading control. (d–f) Permeability of (d) control or (e) BMPR2 mutation-bearing BOEC monolayers to HRP, assessed as a measure of colorimetric absorbance after incubation periods ranging from 30 minutes to 2 hours with or without BMP9 (20 ng/mL) and/or LPS (400 ng/mL) (f) Quantification of monolayer permeability at 2 hours post-LPS. (n=4 individuals per group). AU: Arbitraty units. ***P<0.001, **P<0.01, *P<0.05, 1-way ANOVA, repeated measures Tukey’s post test for Control or BMPR2 Mutation BOECs; ###P<0.001, ##P<0.01, 1-way ANOVA, Tukey’s post test for all groups. Mean +/− SEM. (g) Representative immunofluorescence images of PAECs stained for VE-cadherin following 24 hour culture with or without BMP9 (10 ng/mL) and/or LPS (400 ng/mL). Scale bars 10 μm.
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Figure 3: BMP9 prevents apoptosis and promotes monolayer integrity in BOECs with and without BMPR-II mutations(a) Quantification of apoptotic (Annexin-V+/PI−) control (n=5 individuals) and BMPR2 mutation-bearing BOECs (n=6 individuals) after culture with or without BMP9 (5 ng/mL) for 16 hours prior to the addition of TNFα (10 ng/mL) and cyclohexamide (20 μg/mL) for 6 hours. (b–c) Immunoblotting for (b) phosphorylated JNK and (c) cleaved caspase-3 in control and BMPR2 mutation-bearing BOECs with or without BMP9 pre-treatment and apoptotic stimulus. Representative of five immunoblots. All blots were re-probed for α-tubulin as a loading control. (d–f) Permeability of (d) control or (e) BMPR2 mutation-bearing BOEC monolayers to HRP, assessed as a measure of colorimetric absorbance after incubation periods ranging from 30 minutes to 2 hours with or without BMP9 (20 ng/mL) and/or LPS (400 ng/mL) (f) Quantification of monolayer permeability at 2 hours post-LPS. (n=4 individuals per group). AU: Arbitraty units. ***P<0.001, **P<0.01, *P<0.05, 1-way ANOVA, repeated measures Tukey’s post test for Control or BMPR2 Mutation BOECs; ###P<0.001, ##P<0.01, 1-way ANOVA, Tukey’s post test for all groups. Mean +/− SEM. (g) Representative immunofluorescence images of PAECs stained for VE-cadherin following 24 hour culture with or without BMP9 (10 ng/mL) and/or LPS (400 ng/mL). Scale bars 10 μm.

Mentions: BOECs from healthy controls and individuals with PAH-associated mutations in BMPR2 were used as a surrogate for PAECs to determine the efficacy of BMP9 therapy in the context of BMPR2 mutations. Although mutation-bearing BOECs displayed an increased susceptibility to apoptosis when compared to cells isolated from healthy control subjects (Fig. 3a), BMP9 blocked JNK phosphorylation (Fig. 3b) and apoptosis in both control and mutation-bearing BOECs (Fig. 3a, c), supporting the benefit of this approach, even in the context of BMPR-II deficiency.


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)

BMP9 prevents apoptosis and promotes monolayer integrity in BOECs with and without BMPR-II mutations(a) Quantification of apoptotic (Annexin-V+/PI−) control (n=5 individuals) and BMPR2 mutation-bearing BOECs (n=6 individuals) after culture with or without BMP9 (5 ng/mL) for 16 hours prior to the addition of TNFα (10 ng/mL) and cyclohexamide (20 μg/mL) for 6 hours. (b–c) Immunoblotting for (b) phosphorylated JNK and (c) cleaved caspase-3 in control and BMPR2 mutation-bearing BOECs with or without BMP9 pre-treatment and apoptotic stimulus. Representative of five immunoblots. All blots were re-probed for α-tubulin as a loading control. (d–f) Permeability of (d) control or (e) BMPR2 mutation-bearing BOEC monolayers to HRP, assessed as a measure of colorimetric absorbance after incubation periods ranging from 30 minutes to 2 hours with or without BMP9 (20 ng/mL) and/or LPS (400 ng/mL) (f) Quantification of monolayer permeability at 2 hours post-LPS. (n=4 individuals per group). AU: Arbitraty units. ***P<0.001, **P<0.01, *P<0.05, 1-way ANOVA, repeated measures Tukey’s post test for Control or BMPR2 Mutation BOECs; ###P<0.001, ##P<0.01, 1-way ANOVA, Tukey’s post test for all groups. Mean +/− SEM. (g) Representative immunofluorescence images of PAECs stained for VE-cadherin following 24 hour culture with or without BMP9 (10 ng/mL) and/or LPS (400 ng/mL). Scale bars 10 μm.
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Figure 3: BMP9 prevents apoptosis and promotes monolayer integrity in BOECs with and without BMPR-II mutations(a) Quantification of apoptotic (Annexin-V+/PI−) control (n=5 individuals) and BMPR2 mutation-bearing BOECs (n=6 individuals) after culture with or without BMP9 (5 ng/mL) for 16 hours prior to the addition of TNFα (10 ng/mL) and cyclohexamide (20 μg/mL) for 6 hours. (b–c) Immunoblotting for (b) phosphorylated JNK and (c) cleaved caspase-3 in control and BMPR2 mutation-bearing BOECs with or without BMP9 pre-treatment and apoptotic stimulus. Representative of five immunoblots. All blots were re-probed for α-tubulin as a loading control. (d–f) Permeability of (d) control or (e) BMPR2 mutation-bearing BOEC monolayers to HRP, assessed as a measure of colorimetric absorbance after incubation periods ranging from 30 minutes to 2 hours with or without BMP9 (20 ng/mL) and/or LPS (400 ng/mL) (f) Quantification of monolayer permeability at 2 hours post-LPS. (n=4 individuals per group). AU: Arbitraty units. ***P<0.001, **P<0.01, *P<0.05, 1-way ANOVA, repeated measures Tukey’s post test for Control or BMPR2 Mutation BOECs; ###P<0.001, ##P<0.01, 1-way ANOVA, Tukey’s post test for all groups. Mean +/− SEM. (g) Representative immunofluorescence images of PAECs stained for VE-cadherin following 24 hour culture with or without BMP9 (10 ng/mL) and/or LPS (400 ng/mL). Scale bars 10 μm.
Mentions: BOECs from healthy controls and individuals with PAH-associated mutations in BMPR2 were used as a surrogate for PAECs to determine the efficacy of BMP9 therapy in the context of BMPR2 mutations. Although mutation-bearing BOECs displayed an increased susceptibility to apoptosis when compared to cells isolated from healthy control subjects (Fig. 3a), BMP9 blocked JNK phosphorylation (Fig. 3b) and apoptosis in both control and mutation-bearing BOECs (Fig. 3a, c), supporting the benefit of this approach, even in the context of BMPR-II deficiency.

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.

Show MeSH
Related in: MedlinePlus