Expression of mutant BMPR-II in pulmonary endothelial cells promotes apoptosis and a release of factors that stimulate proliferation of pulmonary arterial smooth muscle cells
Mutations in the bone morphogenetic protein type II receptor gene (BMPR-II) are the major cause of heritable pulmonary arterial hypertension (PAH). Although both endothelial and smooth muscle cell BMPR-II dysfunction have been seen to contribute to pulmonary hypertension in vivo, little is known about the impact of BMPR-II mutation on the interaction between these two important cell types. We employed adenoviral vectors to overexpress wild type or mutant (kinase-deficient mutation, D485G) BMPR-II in human pulmonary arterial endothelial cells (PAECs). PAECs transfected with mutant BMPR-II demonstrated increased susceptibility to apoptosis. Conditioned media from PAECs transfected with mutant BMPR-II increased the proliferation of pulmonary arterial smooth muscle cells (PASMCs), when compared with conditioned media from PAECs transfected with wild-type BMPR-II. PAECs transfected with mutant BMPR-II released higher levels of TGF-β1 and FGF2 into the conditioned media than the wild-type BMPR-II-transfected cells. Conditioned media from PAECs expressing mutant BMPR-II also showed increased activation of luciferase activity in a TGF-β bioassay. The increased proliferation observed in PASMCs exposed to conditioned media from PAECs expressing mutant BMPR-II was inhibited by neutralizing the antibodies to TGF-β1, or small molecule inhibitors of ALK-5 (SD208) or FGFR1 (SU5402). We conclude that mutation in BMPR-II increases susceptibility to apoptosis of PAECs and leads to secretion of growth factors that stimulate the proliferation of PASMCs. These processes may contribute to the remodeling of pulmonary arteries observed in patients with familial or heritable PAH.
This project was funded by the British Heart Foundation (Program grant RG256 to NWM) and the European Commission, under the Sixth Framework Program (Contract No LSHM-CT-2005-018725, PULMOTENSION).
- 1.Runo JR, Loyd JE. Primary pulmonary hypertension. Lancet 2003;361:1533–44.
- 2.Lane KB, Machado RD, Pauciulo MW, Thomson JR, Phillips JA 3rd, Loyd JE, et al. Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension. Nat Genet 2000;26:81–4.
- 3.Deng Z, Haghighi F, Helleby L, Vanterpool K, Horn EM, Barst RJ, et al. Fine mapping of PPH1, a gene for familial primary pulmonary hypertension, to a 3-cM region on chromosome 2q33. Am J Respir Crit Care Med 2000;161:1055–9.
- 4.Machado RD, Aldred MA, James V, Harrison RE, Patel B, Schwalbe EC, et al. Mutations of the TGF-beta type II receptor BMPR2 in pulmonary arterial hypertension. Hum Mutat 2006;27:121–32.
- 5.Thomson JR, Machado RD, Pauciulo MW, Morgan NV, Humbert M, Elliott GC, et al. Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-beta family. J Med Genet 2000;37:741–5.
- 6.Atkinson C, Stewart S, Upton PD, Machado R, Thomson JR, Trembath RC, et al. Primary pulmonary hypertension is associated with reduced pulmonary vascular expression of type II bone morphogenetic protein receptor. Circulation 2002;105:1672–8
- 7.Morrell NW, Adnot S, Archer SL, Dupuis J, Jones PL, MacLean MR, et al. Cellular and molecular basis of pulmonary arterial hypertension. J Am Coll Cardiol 2009;54:S20–31.
- 8.Sakao S, Tatsumi K, Voelkel NF. Endothelial cells and pulmonary arterial hypertension: apoptosis, proliferation, interaction and transdifferentiation. Respir Res 2009;10:95.
- 9.Sakao S, Taraseviciene-Stewart L, Wood K, Cool CD, Voelkel NF. Apoptosis of pulmonary microvascular endothelial cells stimulates vascular smooth muscle cell growth. Am J Physiol Lung Cell Mol Physiol 2006;291: L362–8.
- 10.Eddahibi S, Guignabert C, Barlier-Mur AM, Dewachter L, Fadel E, Dartevelle P, et al. Cross talk between endothelial and smooth muscle cells in pulmonary hypertension: Critical role for serotonin-induced smooth muscle hyperplasia. Circulation 2006;113:1857–64.
- 11.Sanchez O, Marcos E, Perros F, Fadel E, Tu L, Humbert M, et al. Role of endothelium-derived CC chemokine ligand 2 in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med 2007;176:1041–7.
- 12.Izikki M, Guignabert C, Fadel E, Humbert M, Tu L, Zadigue P, et al. Endothelial-derived FGF2 contributes to the progression of pulmonary hypertension in humans and rodents. J Clin Invest 2009;119:512–23.
- 13.Southwood M, Jeffery TK, Yang X, Upton PD, Hall SM, Atkinson C, et al. Regulation of bone morphogenetic protein signalling in human pulmonary vascular development. J Pathol 2008;214:85–95.
- 14.Yang X, Long L, Southwood M, Rudarakanchana N, Upton PD, Jeffery TK, et al. Dysfunctional Smad signaling contributes to abnormal smooth muscle cell proliferation in familial pulmonary arterial hypertension. Circ Res 2005;96:1053–63.
- 15.Van Waarde MA, van Assen AJ, Kampinga HH, Konings AW, Vujaskovic Z. Quantification of transforming growth factor-beta in biological material using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. Anal Biochem 1997;247:45–51.
- 16.Teichert-Kuliszewska K, Kutryk MJ, Kuliszewski MA, Karoubi G, Courtman DW, Zucco L, 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 2006;98: 209–17.
- 17.Rabinovitch M. EVE and beyond, retro and prospective insights. Am J Physiol 1999; 277:L5–12.
- 18.Song Y, Coleman L, Shi J, Beppu H, Sato K, Walsh K, et al. Inflammation, endothelial injury, and persistent pulmonary hypertension in heterozygous BMPR2-mutant mice. Am J Physiol Heart Circ Physiol 2008;295:H677–90.
- 19.Zhang S, Fantozzi I, Tigno DD, Yi ES, Platoshyn O, Thistlethwaite PA, et al. Bone morphogenetic proteins induce apoptosis in human pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2003;285:L740–54.
- 20.Morrell NW, Yang X, Upton PD, Jourdan KB, Morgan N, Sheares KK, et al. Altered growth responses of pulmonary artery smooth muscle cells from patients with primary pulmonary hypertension to transforming growth factor-beta(1) and bone morphogenetic proteins. Circulation 2001;104:790–5.
- 21.Yang J, Davies RJ, Southwood M, Long L, Yang X, Sobolewski A, 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 2008;102:1212–21.
- 22.Valdimarsdottir G, Goumans MJ, Rosendahl A, Brugman M, Itoh S, Lebrin F, et al. Stimulation of Id1 expression by bone morphogenetic protein is sufficient and necessary for bone morphogenetic protein-induced activation of endothelial cells. Circulation 2002;106:2263–70.
- 23.David L, Mallet C, Mazerbourg S, Feige JJ, Bailly S. Identification of BMP9 and BMP10 as functional activators of the orphan activin receptor-like kinase 1 (ALK1) in endothelial cells. Blood 2007;109:1953–61.
- 24.Upton PD, Davies RJ, Trembath RC, Morrell NW. 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 2009;284:15794–804.
- 25.McDonald PP, Fadok VA, Bratton D, Henson PM. Transcriptional and translational regulation of inflammatory mediator production by endogenous TGF-β in macrophages that have ingested apoptotic cells. J Immunol 1999;163:6164–72.
Address correspondence to: Prof. Nicholas W. Morrell, Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s Hospital, Box 157, Hills Road, Cambridge CB2 0QQ, United Kingdom. E-mail: firstname.lastname@example.org
Source of Support: British Heart Foundation (Program grant RG256 to NWM) and the European Commission, under the Sixth Framework Program (Contract No LSHM-CT-2005-018725, PULMOTENSION), Conflict of Interest: None declared.