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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 preferentially stimulates hPAECsVolcano plots of differentially expressed genes in PAECs treated with (a) 1 ng/mL BMP9, (b) 10 ng/mL BMP6 or (c) 10 ng/mL BMP2 versus control after fitting linear models and adjusting P values for multiple testing (FDR). Differentially expressed genes of the TGF-β pathway are highlighted in grey. Dashed lines represent an adjusted P value of 0.05 and a fold change of +/− 1x. (d) Heat map showing whole gene-set perturbation (test statistics) of regulated pathways in PAECs following BMP9 treatment. (e) Signaling pathway impact analysis (SPIA) to detect alterations in common signaling pathways in PAECs in response to BMP9 treatment. Corrected for false discovery rate. (f) Immunoblotting for phosphorylated Smad1/5/8, Id1, Id3, BMPR-II and β-actin in PAECs cultured with or without varying concentrations of BMP9, BMP4 and BMP6 ligand for 2 hours. Representative of 3 experiments. (g) Expression of BMPR2 in PAECs with or without treatment with 10 ng/mL BMP9 for 24 hours (n=3; Student’s t test). (h) Representative agarose gels for products from chromatin immunoprecipitation on lysates from HMEC-1 cells (top) without treatment, (middle) 72 hours after siRNA knockdown of SMAD1 or (bottom) following a 24 hour treatment with 1 ng/mL BMP9. (i) Luciferase activity in HMEC-1 lysates transfected with a luciferase reporter construct with or without an upstream 5kb portion of the BMPR2 promoter with or without mutation of the putative Smad binding region. Cells bearing the reporter construct were treated with 10 ng/mL BMP9 for 24 hours (n=3; 1-way ANOVA, Tukey’s post test). ***P<0.001, **P<0.01, *P<0.05. Mean +/− SEM.
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Figure 1: BMP9 preferentially stimulates hPAECsVolcano plots of differentially expressed genes in PAECs treated with (a) 1 ng/mL BMP9, (b) 10 ng/mL BMP6 or (c) 10 ng/mL BMP2 versus control after fitting linear models and adjusting P values for multiple testing (FDR). Differentially expressed genes of the TGF-β pathway are highlighted in grey. Dashed lines represent an adjusted P value of 0.05 and a fold change of +/− 1x. (d) Heat map showing whole gene-set perturbation (test statistics) of regulated pathways in PAECs following BMP9 treatment. (e) Signaling pathway impact analysis (SPIA) to detect alterations in common signaling pathways in PAECs in response to BMP9 treatment. Corrected for false discovery rate. (f) Immunoblotting for phosphorylated Smad1/5/8, Id1, Id3, BMPR-II and β-actin in PAECs cultured with or without varying concentrations of BMP9, BMP4 and BMP6 ligand for 2 hours. Representative of 3 experiments. (g) Expression of BMPR2 in PAECs with or without treatment with 10 ng/mL BMP9 for 24 hours (n=3; Student’s t test). (h) Representative agarose gels for products from chromatin immunoprecipitation on lysates from HMEC-1 cells (top) without treatment, (middle) 72 hours after siRNA knockdown of SMAD1 or (bottom) following a 24 hour treatment with 1 ng/mL BMP9. (i) Luciferase activity in HMEC-1 lysates transfected with a luciferase reporter construct with or without an upstream 5kb portion of the BMPR2 promoter with or without mutation of the putative Smad binding region. Cells bearing the reporter construct were treated with 10 ng/mL BMP9 for 24 hours (n=3; 1-way ANOVA, Tukey’s post test). ***P<0.001, **P<0.01, *P<0.05. Mean +/− SEM.

Mentions: We sought to identify a BMP ligand for therapeutic delivery that would selectively and efficiently enhance canonical BMP signaling in the endothelium while minimizing the potential for deleterious off-target effects, such as the calcification of soft tissue28. As an initial screen, we stimulated human pulmonary arterial endothelial cells (PAECs) with BMP9, BMP2 or BMP6 and assessed changes in gene transcription by microarray. Unlike BMP2 and BMP6, which induced negligible gene expression, BMP9 induced the differential regulation of 1883 genes (adjusted P value < 0.05), including several key components of canonical BMP signaling such as ID1, ID2 and BMPR2 (Fig. 1a–c). Generally applicable gene-set enrichment (GAGE) analysis for common signaling pathways and cellular processes revealed TGF-β signaling as the only pathway to be significantly (P=0.0012) upregulated by BMP9. However, BMP9 also downregulated other pathways, including apoptotic cell signaling (Fig. 1d; Supplementary Fig. 1). Signaling pathway impact analysis (SPIA) of common signaling pathways confirmed the TGF-β pathway as the only pathway to be enhanced in response to BMP9 treatment, supporting a central role for the canonical Smad pathway in the actions of BMP9 (Fig. 1e). We obtained similar results using blood outgrowth endothelial cells (BOECs) from healthy subjects, which, like PAECs, responded almost exclusively to BMP9 stimulation (Supplementary Fig. 2). Analysis of BMP9-induced gene expression for specific members of the TGF-β pathway revealed an enhancement of classical downstream targets of BMP signaling, whereas expression levels of TGF-β1 downstream targets, including PAI-1, Myc and CTGF, remained unchanged (Supplementary Fig. 3a). Of note, BMP9 did not alter the expression of a selected group of endothelial-derived paracrine factors and typical PASMC mitogens, including members of the PDGF and FGF families of cytokines (Supplementary Fig. 3b).


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 preferentially stimulates hPAECsVolcano plots of differentially expressed genes in PAECs treated with (a) 1 ng/mL BMP9, (b) 10 ng/mL BMP6 or (c) 10 ng/mL BMP2 versus control after fitting linear models and adjusting P values for multiple testing (FDR). Differentially expressed genes of the TGF-β pathway are highlighted in grey. Dashed lines represent an adjusted P value of 0.05 and a fold change of +/− 1x. (d) Heat map showing whole gene-set perturbation (test statistics) of regulated pathways in PAECs following BMP9 treatment. (e) Signaling pathway impact analysis (SPIA) to detect alterations in common signaling pathways in PAECs in response to BMP9 treatment. Corrected for false discovery rate. (f) Immunoblotting for phosphorylated Smad1/5/8, Id1, Id3, BMPR-II and β-actin in PAECs cultured with or without varying concentrations of BMP9, BMP4 and BMP6 ligand for 2 hours. Representative of 3 experiments. (g) Expression of BMPR2 in PAECs with or without treatment with 10 ng/mL BMP9 for 24 hours (n=3; Student’s t test). (h) Representative agarose gels for products from chromatin immunoprecipitation on lysates from HMEC-1 cells (top) without treatment, (middle) 72 hours after siRNA knockdown of SMAD1 or (bottom) following a 24 hour treatment with 1 ng/mL BMP9. (i) Luciferase activity in HMEC-1 lysates transfected with a luciferase reporter construct with or without an upstream 5kb portion of the BMPR2 promoter with or without mutation of the putative Smad binding region. Cells bearing the reporter construct were treated with 10 ng/mL BMP9 for 24 hours (n=3; 1-way ANOVA, Tukey’s post test). ***P<0.001, **P<0.01, *P<0.05. Mean +/− SEM.
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Related In: Results  -  Collection

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Figure 1: BMP9 preferentially stimulates hPAECsVolcano plots of differentially expressed genes in PAECs treated with (a) 1 ng/mL BMP9, (b) 10 ng/mL BMP6 or (c) 10 ng/mL BMP2 versus control after fitting linear models and adjusting P values for multiple testing (FDR). Differentially expressed genes of the TGF-β pathway are highlighted in grey. Dashed lines represent an adjusted P value of 0.05 and a fold change of +/− 1x. (d) Heat map showing whole gene-set perturbation (test statistics) of regulated pathways in PAECs following BMP9 treatment. (e) Signaling pathway impact analysis (SPIA) to detect alterations in common signaling pathways in PAECs in response to BMP9 treatment. Corrected for false discovery rate. (f) Immunoblotting for phosphorylated Smad1/5/8, Id1, Id3, BMPR-II and β-actin in PAECs cultured with or without varying concentrations of BMP9, BMP4 and BMP6 ligand for 2 hours. Representative of 3 experiments. (g) Expression of BMPR2 in PAECs with or without treatment with 10 ng/mL BMP9 for 24 hours (n=3; Student’s t test). (h) Representative agarose gels for products from chromatin immunoprecipitation on lysates from HMEC-1 cells (top) without treatment, (middle) 72 hours after siRNA knockdown of SMAD1 or (bottom) following a 24 hour treatment with 1 ng/mL BMP9. (i) Luciferase activity in HMEC-1 lysates transfected with a luciferase reporter construct with or without an upstream 5kb portion of the BMPR2 promoter with or without mutation of the putative Smad binding region. Cells bearing the reporter construct were treated with 10 ng/mL BMP9 for 24 hours (n=3; 1-way ANOVA, Tukey’s post test). ***P<0.001, **P<0.01, *P<0.05. Mean +/− SEM.
Mentions: We sought to identify a BMP ligand for therapeutic delivery that would selectively and efficiently enhance canonical BMP signaling in the endothelium while minimizing the potential for deleterious off-target effects, such as the calcification of soft tissue28. As an initial screen, we stimulated human pulmonary arterial endothelial cells (PAECs) with BMP9, BMP2 or BMP6 and assessed changes in gene transcription by microarray. Unlike BMP2 and BMP6, which induced negligible gene expression, BMP9 induced the differential regulation of 1883 genes (adjusted P value < 0.05), including several key components of canonical BMP signaling such as ID1, ID2 and BMPR2 (Fig. 1a–c). Generally applicable gene-set enrichment (GAGE) analysis for common signaling pathways and cellular processes revealed TGF-β signaling as the only pathway to be significantly (P=0.0012) upregulated by BMP9. However, BMP9 also downregulated other pathways, including apoptotic cell signaling (Fig. 1d; Supplementary Fig. 1). Signaling pathway impact analysis (SPIA) of common signaling pathways confirmed the TGF-β pathway as the only pathway to be enhanced in response to BMP9 treatment, supporting a central role for the canonical Smad pathway in the actions of BMP9 (Fig. 1e). We obtained similar results using blood outgrowth endothelial cells (BOECs) from healthy subjects, which, like PAECs, responded almost exclusively to BMP9 stimulation (Supplementary Fig. 2). Analysis of BMP9-induced gene expression for specific members of the TGF-β pathway revealed an enhancement of classical downstream targets of BMP signaling, whereas expression levels of TGF-β1 downstream targets, including PAI-1, Myc and CTGF, remained unchanged (Supplementary Fig. 3a). Of note, BMP9 did not alter the expression of a selected group of endothelial-derived paracrine factors and typical PASMC mitogens, including members of the PDGF and FGF families of cytokines (Supplementary Fig. 3b).

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