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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References
Morrell, N.W. et al. Cellular and molecular basis of pulmonary arterial hypertension. J. Am. Coll. Cardiol. 54, S20–S31 (2009).
Gaine, S.P. & Rubin, L.J. Primary pulmonary hypertension. Lancet 352, 719–725 (1998).
The International PPH Consortium et al. Heterozygous germline mutations in BMPR2, encoding a TGF-β receptor, cause familial primary pulmonary hypertension. Nat. Genet. 26, 81–84 (2000).
Deng, Z. et al. Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene. Am. J. Hum. Genet. 67, 737–744 (2000).
Thomson, J.R. et al. Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-β family. J. Med. Genet. 37, 741–745 (2000).
Atkinson, C. et al. Primary pulmonary hypertension is associated with reduced pulmonary vascular expression of type II bone morphogenetic protein receptor. Circulation 105, 1672–1678 (2002).
Long, L. et al. Altered bone morphogenetic protein and transforming growth factor-beta signaling in rat models of pulmonary hypertension: potential for activin receptor-like kinase-5 inhibition in prevention and progression of disease. Circulation 119, 566–576 (2009).
Hong, K.H. et al. Genetic ablation of the BMPR2 gene in pulmonary endothelium is sufficient to predispose to pulmonary arterial hypertension. Circulation 118, 722–730 (2008).
Reynolds, A.M., Holmes, M.D., Danilov, S.M. & Reynolds, P.N. Targeted gene delivery of BMPR2 attenuates pulmonary hypertension. Eur. Respir. J. 39, 329–343 (2012).
Reynolds, A.M. et al. Bone morphogenetic protein type 2 receptor gene therapy attenuates hypoxic pulmonary hypertension. Am. J. Physiol. Lung Cell. Mol. Physiol. 292, L1182–L1192 (2007).
Spiekerkoetter, E. et al. FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension. J. Clin. Invest. 123, 3600–3613 (2013).
Trembath, R.C. et al. Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia. N. Engl. J. Med. 345, 325–334 (2001).
Harrison, R.E. et al. Molecular and functional analysis identifies ALK-1 as the predominant cause of pulmonary hypertension related to hereditary haemorrhagic telangiectasia. J. Med. Genet. 40, 865–871 (2003).
Yeager, M.E., Halley, G.R., Golpon, H.A., Voelkel, N.F. & Tuder, R.M. Microsatellite instability of endothelial cell growth and apoptosis genes within plexiform lesions in primary pulmonary hypertension. Circ. Res. 88, E2–E11 (2001).
Teichert-Kuliszewska, K. et al. Bone morphogenetic protein receptor-2 signaling promotes pulmonary arterial endothelial cell survival: implications for loss-of-function mutations in the pathogenesis of pulmonary hypertension. Circ. Res. 98, 209–217 (2006).
Wilson, D.W. et al. Mechanisms and pathology of monocrotaline pulmonary toxicity. Crit. Rev. Toxicol. 22, 307–325 (1992).
Taraseviciene-Stewart, L. et al. Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J. 15, 427–438 (2001).
Burton, V.J. et al. Bone morphogenetic protein receptor II regulates pulmonary artery endothelial cell barrier function. Blood 117, 333–341 (2011).
Burton, V.J. et al. Attenuation of leukocyte recruitment via CXCR1/2 inhibition stops the progression of PAH in mice with genetic ablation of endothelial BMPR-II. Blood 118, 4750–4758 (2011).
Kim, C.W. et al. Anti-Inflammatory and Antiatherogenic Role of BMP Receptor II in Endothelial Cells. Arterioscler. Thromb. Vasc. Biol. 33, 1350–1359 (2013).
Yang, J. et al. Mutations in bone morphogenetic protein type II receptor cause dysregulation of Id gene expression in pulmonary artery smooth muscle cells: implications for familial pulmonary arterial hypertension. Circ. Res. 102, 1212–1221 (2008).
Miyazono, K., Maeda, S. & Imamura, T. BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk. Cytokine Growth Factor Rev. 16, 251–263 (2005).
David, L., Mallet, C., Mazerbourg, S., Feige, J.J. & Bailly, S. Identification of BMP9 and BMP10 as functional activators of the orphan activin receptor-like kinase 1 (ALK1) in endothelial cells. Blood 109, 1953–1961 (2007).
Scharpfenecker, M. et al. BMP-9 signals via ALK1 and inhibits bFGF-induced endothelial cell proliferation and VEGF-stimulated angiogenesis. J. Cell Sci. 120, 964–972 (2007).
David, L. et al. Bone morphogenetic protein-9 is a circulating vascular quiescence factor. Circ. Res. 102, 914–922 (2008).
Frank, D.B. et al. Increased susceptibility to hypoxic pulmonary hypertension in Bmpr2 mutant mice is associated with endothelial dysfunction in the pulmonary vasculature. Am. J. Physiol. Lung Cell. Mol. Physiol. 294, L98–L109 (2008).
Song, Y. et al. Increased susceptibility to pulmonary hypertension in heterozygous BMPR2-mutant mice. Circulation 112, 553–562 (2005).
Kang, Q. et al. Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther. 11, 1312–1320 (2004).
Slowik, M.R. et al. Evidence that tumor necrosis factor triggers apoptosis in human endothelial cells by interleukin-1-converting enzyme-like protease-dependent and -independent pathways. Lab. Invest. 77, 257–267 (1997).
Upton, P.D., Davies, R.J., Trembath, R.C. & Morrell, N.W. Bone morphogenetic protein (BMP) and activin type II receptors balance BMP9 signals mediated by activin receptor-like kinase-1 in human pulmonary artery endothelial cells. J. Biol. Chem. 284, 15794–15804 (2009).
Beppu, H. et al. BMP type II receptor is required for gastrulation and early development of mouse embryos. Dev. Biol. 221, 249–258 (2000).
Machado, R.D. et al. Mutations of the TGF-β type II receptor BMPR2 in pulmonary arterial hypertension. Hum. Mutat. 27, 121–132 (2006).
Lechleider, R.J. et al. Targeted mutagenesis of Smad1 reveals an essential role in chorioallantoic fusion. Dev. Biol. 240, 157–167 (2001).
Reynolds, A.M., Holmes, M.D., Danilov, S.M. & Reynolds, P.N. Targeted gene delivery of BMPR-2 attenuates pulmonary hypertension. Eur. Respir. J. 39, 329–343 (2012).
Ciumas, M. et al. Bone morphogenetic proteins protect pulmonary microvascular endothelial cells from apoptosis by upregulating α-B-crystallin. Arterioscler. Thromb. Vasc. Biol. 33, 2577–2584 (2013).
Frump, A.L., Lowery, J.W., Hamid, R., Austin, E.D. & de Caestecker, M. Abnormal trafficking of endogenously expressed BMPR2 mutant allelic products in patients with heritable pulmonary arterial hypertension. PLoS ONE 8, e80319 (2013).
Valdimarsdottir, G. et al. Stimulation of Id1 expression by bone morphogenetic protein is sufficient and necessary for bone morphogenetic protein-induced activation of endothelial cells. Circulation 106, 2263–2270 (2002).
González-Núñez, M., Munoz-Felix, J.M. & Lopez-Novoa, J.M. The ALK-1/Smad1 pathway in cardiovascular physiopathology. A new target for therapy? Biochim. Biophys. Acta 1832, 1492–1510 (2013).
Prewitt, A.R. et al. Heterozygous null bone morphogenetic protein receptor type 2 mutations promote SRC kinase-dependent caveolar trafficking defects and endothelial dysfunction in pulmonary arterial hypertension. J. Biol. Chem. 290, 960–971 (2015).
Nasim, M.T. et al. Molecular genetic characterization of SMAD signaling molecules in pulmonary arterial hypertension. Hum. Mutat. 32, 1385–1389 (2011).
Drake, K.M. et al. Altered microRNA processing in heritable pulmonary arterial hypertension: an important role for Smad-8. Am. J. Respir. Crit. Care Med. 184, 1400–1408 (2011).
Han, C. et al. SMAD1 deficiency in either endothelial or smooth muscle cells can predispose mice to pulmonary hypertension. Hypertension 61, 1044–1052 (2013).
Hemnes, A.R. et al. Evidence for right ventricular lipotoxicity in heritable pulmonary arterial hypertension. Am. J. Respir. Crit. Care Med. 189, 325–334 (2014).
Geti, I. et al. A practical and efficient cellular substrate for the generation of induced pluripotent stem cells from adults: blood-derived endothelial progenitor cells. Stem Cells Transl. Med. 1, 855–865 (2012).
Allport, J.R. et al. Neutrophils from MMP-9- or neutrophil elastase–deficient mice show no defect in transendothelial migration under flow in vitro. J. Leukoc. Biol. 71, 821–828 (2002).
Yang, X. et al. Dysfunctional Smad signaling contributes to abnormal smooth muscle cell proliferation in familial pulmonary arterial hypertension. Circ. Res. 96, 1053–1063 (2005).
Smyth, G.K. Limma: linear models for microarray data. in Bioinformatics and Computational Biology Solutions using R and Bioconductor (eds. Gentleman, R., Carey, V., Huber, W., Irizarry, R. & Dudiot, S.) 397–420 (Springer, New York, 2005).
Luo, W., Friedman, M.S., Shedden, K., Hankenson, K.D. & Woolf, P.J. GAGE: generally applicable gene set enrichment for pathway analysis. BMC Bioinformatics 10, 161 (2009).
Tarca, A.L. et al. A novel signaling pathway impact analysis (SPIA). Bioinformatics 25, 75–82 (2009).
Luo, W. & Brouwer, C. Pathview: an R/Bioconductor package for pathway-based data integration and visualization. Bioinformatics 29, 1830–1831 (2013).
Harrington, E.O. et al. Role of protein kinase C isoforms in rat epididymal microvascular endothelial barrier function. Am. J. Respir. Cell Mol. Biol. 28, 626–636 (2003).
Waters, J.P. et al. In vitro self-assembly of human pericyte-supported endothelial microvessels in three-dimensional coculture: a simple model for interrogating endothelial-pericyte interactions. J. Vasc. Res. 50, 324–331 (2013).
Bidart, M. et al. BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain. Cell. Mol. Life Sci. 69, 313–324 (2012).
Moitra, J., Sammani, S. & Garcia, J.G. Re-evaluation of Evans Blue dye as a marker of albumin clearance in murine models of acute lung injury. Transl. Res. 150, 253–265 (2007).
Acknowledgements
This work was supported by grants from the British Heart Foundation RG/13/4/30107 (N.W.M.), CH/09/001/25945 (N.W.M.), PG/11/10/28724 (P.D.U. and N.W.M.) and FS/12/39/29653 (M.L.O.); a Fondation Leducq Transatlantic Network of Excellence Award (N.W.M. and P.B.Y.); US National Institutes of Health grants 5R01-AR057374 (P.B.Y.), 5K08-HL079943 (P.B.Y.) and R01-HL098199 (M.A.A.); a Howard Hughes Medical Institute Early Career Physician Scientist Award (P.B.Y.) and a UK National Institute for Health Research Healthcare Science Fellowship (M.S.). The UK National Institute for Health Research Cambridge Biomedical Research Centre and Cell Phenotyping Hub provided infrastructure support.
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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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Long, L., Ormiston, M., Yang, X. et al. Selective enhancement of endothelial BMPR-II with BMP9 reverses pulmonary arterial hypertension. Nat Med 21, 777–785 (2015). https://doi.org/10.1038/nm.3877
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DOI: https://doi.org/10.1038/nm.3877
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