An antiproliferative BMP-2/PPARγ/apoE axis in human and murine SMCs and its role in pulmonary hypertension
Georg Hansmann, Vinicio A. de Jesus Perez, Tero-Pekka Alastalo, Cristina M. Alvira, Christophe Guignabert, Janine M. Bekker, Stefan Schellong, Takashi Urashima, Lingli Wang, Nicholas W. Morrell, Marlene Rabinovitch
Georg Hansmann, Vinicio A. de Jesus Perez, Tero-Pekka Alastalo, Cristina M. Alvira, Christophe Guignabert, Janine M. Bekker, Stefan Schellong, Takashi Urashima, Lingli Wang, Nicholas W. Morrell, Marlene Rabinovitch
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Research Article Cardiology

An antiproliferative BMP-2/PPARγ/apoE axis in human and murine SMCs and its role in pulmonary hypertension

Abstract

Loss-of-function mutations in bone morphogenetic protein receptor II (BMP-RII) are linked to pulmonary arterial hypertension (PAH); the ligand for BMP-RII, BMP-2, is a negative regulator of SMC growth. Here, we report an interplay between PPARγ and its transcriptional target apoE downstream of BMP-2 signaling. BMP-2/BMP-RII signaling prevented PDGF-BB–induced proliferation of human and murine pulmonary artery SMCs (PASMCs) by decreasing nuclear phospho-ERK and inducing DNA binding of PPARγ that is independent of Smad1/5/8 phosphorylation. Both BMP-2 and a PPARγ agonist stimulated production and secretion of apoE by SMCs. Using a variety of methods, including short hairpin RNAi in human PASMCs, PAH patient–derived BMP-RII mutant PASMCs, a PPARγ antagonist, and PASMCs isolated from PPARγ- and apoE-deficient mice, we demonstrated that the antiproliferative effect of BMP-2 was BMP-RII, PPARγ, and apoE dependent. Furthermore, we created mice with targeted deletion of PPARγ in SMCs and showed that they spontaneously developed PAH, as indicated by elevated RV systolic pressure, RV hypertrophy, and increased muscularization of the distal pulmonary arteries. Thus, PPARγ-mediated events could protect against PAH, and PPARγ agonists may reverse PAH in patients with or without BMP-RII dysfunction.

Authors

Georg Hansmann, Vinicio A. de Jesus Perez, Tero-Pekka Alastalo, Cristina M. Alvira, Christophe Guignabert, Janine M. Bekker, Stefan Schellong, Takashi Urashima, Lingli Wang, Nicholas W. Morrell, Marlene Rabinovitch

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Figure 1

Antiproliferative effects of BMP-2 (A, C, D, and F), the PPARγ agonist rosiglitazone (Rosi; B), and apoE (E) on PDGF-BB–induced proliferation of human (A, B, C, and E) and murine (D and F) PASMCs.

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Antiproliferative effects of BMP-2 (A, C, D, and F), the PPARγ agonist r...
PASMCs were seeded at 2.5 × 104 cells per well of a 24-well plate in 500 μl of growth medium and allowed to adhere overnight. The cells were washed with PBS prior to the addition of starvation media (0.1% FBS) and incubated for 24 hours (murine PASMCs) or 48 hours (HPASMCs) and then stimulated with PDGF-BB (20 ng/ml) for 72 hours. BMP-2 (10 ng/ml), rosiglitazone (1 μM), and recombinant human apoE (1–10 μM) were added to quiescent cells 30 minutes prior to PDGF-BB stimulation. The PPARγ antagonist GW9662 (GW; 1 μM) was added 24 hours prior to the addition of BMP-2. Cells were finally washed twice with PBS, trypsinized, and counted in a hemacytometer (4 counts per well). Cell numbers in controls at time points 0 (CON) and 72 hours were not significantly different. A: shLacZi, HPASMCs transfected with short hairpin LacZi pLentivirus 6 (control); shBMP-RIIi, HPASMCs transfected with short hairpin pLentivirus 6 BMP-RIIi (i.e., BMP-RII–deficient PASMCs). D: Littermates, littermate control PASMCs; SMC PPARγ–/–, PASMCs isolated from SM22α Cre PPARγflox/flox mice. F: C57BL/6, control murine PASMCs; apoE–/–, PASMCs isolated from apoE-deficient mice. Bars represent mean ± SEM (n = 3 in A, D, and F; n = 4 in B and C; n = 6 in E; n = 12 in controls of A). *P < 0.05; **P < 0.01; ***P < 0.001 as indicated; ANOVA with Bonferroni’s multiple comparison test.