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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 in human PAECs via BMPR-IIRepresentative immunoblots and densitometric analysis for (a) phosphorylated JNK (n=4) and (b) cleaved caspase 3 (n=3) in human PAECs cultured with or without BMP9 (5 ng/mL) for 16 hours prior to an apoptotic stimulus with TNFα (10 ng/mL) and cyclohexamide (20 μg/mL) (1-way ANOVA, Tukey’s post test). (c) Representative flow cytometry plots of human PAECs stained with Annexin-V and propidium iodide (PI) with or without BMP9 pre-treatment and apoptotic stimulus. (d) Quantification of apoptotic (Annexin-V+/PI−) PAECs (n=5); 1-way ANOVA, Tukey’s post test). (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 with or without BMP9 pre-treatment and apoptotic stimulus (n=6 for DH1 and siBMPR-II, n=5 for siCP; 1-way ANOVA for each siRNA group, Tukey’s post test). (g) Representative immunoblot and densitometric analysis of cleaved caspase-3 in PAECs following siRNA transfection with or without BMP9 pre-treatment and apoptotic stimulus. (n=3; 1-way ANOVA for each siRNA group, Tukey’s post test). All blots were re-probed for α-tubulin as a loading control. ***P<0.001, **P<0.01, * P<0.05. Mean +/− SEM.
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Figure 2: BMP9 prevents apoptosis in human PAECs via BMPR-IIRepresentative immunoblots and densitometric analysis for (a) phosphorylated JNK (n=4) and (b) cleaved caspase 3 (n=3) in human PAECs cultured with or without BMP9 (5 ng/mL) for 16 hours prior to an apoptotic stimulus with TNFα (10 ng/mL) and cyclohexamide (20 μg/mL) (1-way ANOVA, Tukey’s post test). (c) Representative flow cytometry plots of human PAECs stained with Annexin-V and propidium iodide (PI) with or without BMP9 pre-treatment and apoptotic stimulus. (d) Quantification of apoptotic (Annexin-V+/PI−) PAECs (n=5); 1-way ANOVA, Tukey’s post test). (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 with or without BMP9 pre-treatment and apoptotic stimulus (n=6 for DH1 and siBMPR-II, n=5 for siCP; 1-way ANOVA for each siRNA group, Tukey’s post test). (g) Representative immunoblot and densitometric analysis of cleaved caspase-3 in PAECs following siRNA transfection with or without BMP9 pre-treatment and apoptotic stimulus. (n=3; 1-way ANOVA for each siRNA group, Tukey’s post test). All blots were re-probed for α-tubulin as a loading control. ***P<0.001, **P<0.01, * P<0.05. Mean +/− SEM.

Mentions: As reported previously29, PAECs stimulated with TNFα in the presence of cyclohexamide exhibited elevated JNK phosphorylation as a part of an apoptotic signaling cascade (Fig. 2a). Pre-treatment of PAECs with BMP9 prevented JNK phosphorylation and blocked the induction of apoptosis, as demonstrated by immunoblotting for cleaved caspase-3 (Fig. 2b) and by flow cytometry for early apoptotic, Annexin-V+/PI− cells (Fig. 2c, d). Since BMP9 signals via complexes of the type I receptor ALK1 with either BMPR-II or ActR-II as the type II receptor30, we determined whether the anti-apoptotic effects of BMP9 were indeed BMPR-II dependent. siRNA knockdown of BMPR-II (Fig. 2e) eliminated the capacity of BMP9 to prevent apoptosis when compared to controls (Fig. 2f, g). Importantly, siRNA knockdown of Smad1 and Smad5 also partially blocked the anti-apoptotic effect of BMP9, reinforcing the importance of canonical Smad signaling in this process (Supplementary Fig. 6).


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 in human PAECs via BMPR-IIRepresentative immunoblots and densitometric analysis for (a) phosphorylated JNK (n=4) and (b) cleaved caspase 3 (n=3) in human PAECs cultured with or without BMP9 (5 ng/mL) for 16 hours prior to an apoptotic stimulus with TNFα (10 ng/mL) and cyclohexamide (20 μg/mL) (1-way ANOVA, Tukey’s post test). (c) Representative flow cytometry plots of human PAECs stained with Annexin-V and propidium iodide (PI) with or without BMP9 pre-treatment and apoptotic stimulus. (d) Quantification of apoptotic (Annexin-V+/PI−) PAECs (n=5); 1-way ANOVA, Tukey’s post test). (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 with or without BMP9 pre-treatment and apoptotic stimulus (n=6 for DH1 and siBMPR-II, n=5 for siCP; 1-way ANOVA for each siRNA group, Tukey’s post test). (g) Representative immunoblot and densitometric analysis of cleaved caspase-3 in PAECs following siRNA transfection with or without BMP9 pre-treatment and apoptotic stimulus. (n=3; 1-way ANOVA for each siRNA group, Tukey’s post test). All blots were re-probed for α-tubulin as a loading control. ***P<0.001, **P<0.01, * P<0.05. Mean +/− SEM.
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Related In: Results  -  Collection

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Figure 2: BMP9 prevents apoptosis in human PAECs via BMPR-IIRepresentative immunoblots and densitometric analysis for (a) phosphorylated JNK (n=4) and (b) cleaved caspase 3 (n=3) in human PAECs cultured with or without BMP9 (5 ng/mL) for 16 hours prior to an apoptotic stimulus with TNFα (10 ng/mL) and cyclohexamide (20 μg/mL) (1-way ANOVA, Tukey’s post test). (c) Representative flow cytometry plots of human PAECs stained with Annexin-V and propidium iodide (PI) with or without BMP9 pre-treatment and apoptotic stimulus. (d) Quantification of apoptotic (Annexin-V+/PI−) PAECs (n=5); 1-way ANOVA, Tukey’s post test). (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 with or without BMP9 pre-treatment and apoptotic stimulus (n=6 for DH1 and siBMPR-II, n=5 for siCP; 1-way ANOVA for each siRNA group, Tukey’s post test). (g) Representative immunoblot and densitometric analysis of cleaved caspase-3 in PAECs following siRNA transfection with or without BMP9 pre-treatment and apoptotic stimulus. (n=3; 1-way ANOVA for each siRNA group, Tukey’s post test). All blots were re-probed for α-tubulin as a loading control. ***P<0.001, **P<0.01, * P<0.05. Mean +/− SEM.
Mentions: As reported previously29, PAECs stimulated with TNFα in the presence of cyclohexamide exhibited elevated JNK phosphorylation as a part of an apoptotic signaling cascade (Fig. 2a). Pre-treatment of PAECs with BMP9 prevented JNK phosphorylation and blocked the induction of apoptosis, as demonstrated by immunoblotting for cleaved caspase-3 (Fig. 2b) and by flow cytometry for early apoptotic, Annexin-V+/PI− cells (Fig. 2c, d). Since BMP9 signals via complexes of the type I receptor ALK1 with either BMPR-II or ActR-II as the type II receptor30, we determined whether the anti-apoptotic effects of BMP9 were indeed BMPR-II dependent. siRNA knockdown of BMPR-II (Fig. 2e) eliminated the capacity of BMP9 to prevent apoptosis when compared to controls (Fig. 2f, g). Importantly, siRNA knockdown of Smad1 and Smad5 also partially blocked the anti-apoptotic effect of BMP9, reinforcing the importance of canonical Smad signaling in this process (Supplementary Fig. 6).

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