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 PPARγ^flox/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.

Cells were stimulated with PDGF-BB (20 ng/ml) or BMP-2 (10 ng/ml) as described in the legend for Figure 1. In separate experiments, we determined that neither of the solvents (DMSO, sterile water; both 1:10,000) influenced the results. Western immunoblotting and PPARγ DNA binding assays are described in Methods. For the PPARγ DNA binding assay, bars represent median ± SEM of triplicate measurements of 1 representative experiment of 2 (C) and 3 (D) independent experiments with similar results. For protein levels in cell fractions, bars represent mean ± SEM (n = 3–4). *P < 0.05; **P < 0.01 versus control; ANOVA with Dunnett’s post-hoc test.

Cells were preincubated with BMP-2 (10 ng/ml) for 30 minutes (A and B) or rosiglitazone (1 μM) for 24 hours (C), followed by PDGF-BB (20 ng/ml) stimulation for 10 minutes (A and B) or 5–60 minutes. (C) Western immunoblotting and PPARγ DNA binding assays are described in Methods and Figure 2. For protein levels in cell fractions (A) or cell lysates (C), bars represent mean ± SEM (n = 3 each). In C, all samples are compared with the DMSO control. For the PPARγ DNA binding assay (B), bars represent median ± SEM of triplicate measurements of 1 representative experiment of 3 independent experiments with similar results. *P < 0.05; **P < 0.01 versus control; ANOVA with Dunnett’s post-hoc test.

Control PASMCs were isolated from surgical resection specimens derived from patients undergoing lobectomy or pneumonectomy for suspected lung tumor. Additional peripheral pulmonary arteries (<1–2 mm external diameter) were obtained from a patient undergoing heart-lung transplantation for FPAH and known to harbor a mutation (W9X) in BMP-RII. The nature of the BMP-RII mutation, cell isolation, culture techniques, and cell counts are described in Methods and in Figure 1. HPASMCs were incubated for 48 hours in starvation media (0.1% FBS) and then stimulated with PDGF-BB (20 ng/ml) for 72 hours. BMP-2 (10 ng/ml) or rosiglitazone (1 μM) were added to quiescent cells 30 minutes prior to PDGF-BB stimulation. Bars represent mean ± SEM (n = 3). **P < 0.01; ***P < 0.001 as indicated; ANOVA with Bonferroni’s multiple comparison test. The number of PDGF-BB–stimulated cells was significantly higher than that of untreated control cells (P < 0.001).

(A) apoE protein expression in cell lysates (left) and apoE protein secretion in supernatant (right) induced by BMP-2 (10 ng/ml, 24 hours) and rosiglitazone (1 μM, 24 hours) were detected by immunoblotting as described in Methods (for cell lysates, densitometric values were corrected for equal loading using α-tubulin). For apoE secretion, the media of 3–4 cell culture flasks per condition were pooled and concentrated for the blots shown (representative of 2 independent experiments with similar results). (B) BMP-2–induced (10 ng/ml, 24 hours) upregulation of apoE in murine control PASMCs was reduced by half in PASMCs harvested from SM22α Cre PPARγ^flox/flox mice. PASMCs were isolated from 5 littermate control and 5 SM22α Cre PPARγ^flox/flox mice as described in Methods. PASMCs from each genotype were then pooled and subcultured prior to stimulation with BMP-2. The blot is representative of 2 independent experiments with similar results. For apoE protein levels in cell lysates (A), bars represent mean ± SEM (n = 3). *P < 0.05; **P < 0.01 versus control; unpaired 2-tailed t test.

(A and B) Genotyping of mice with targeted deletion of PPARγ in SMCs. (A) PCR reactions showing gain of a 300-bp knockout transcript and almost complete loss of the 700-bp wild-type transcript in PASMCs and aorta from SM22α Cre PPARγ^flox/flox mice. In the lung, which contains SMCs but also many other cell types, both transcripts are found in SM22α Cre PPARγ^flox/flox mice, whereas only the wild-type transcript is detected in littermate control mice. (B) Western immunoblotting of PASMC lysates isolated from both littermate and SM22α Cre PPARγ^flox/flox (SMC PPARγ^–/–) mice (n = 2 each) showed no detectable PPARγ protein expression when compared with control cells. (C) Both littermate control and SMC PPARγ^–/– PASMCs were stimulated with BMP-2 (10 ng/ml) for 5–60 minutes as described in the legend for Figure 1, and phospho-Smad 1/5/8 protein expression was detected by immunoblotting as described in Methods (densitometric values were corrected for equal loading using α-tubulin). Data for 1 of 2 representative experiments with similar results are shown.

Thirteen- to 15-week-old mice underwent RV catheterization, followed by organ harvest. (A) RVSP measurements, as described in Methods. (B) Right ventricular hypertrophy (RVH), measured as ratio of the weight of the RV to that of the LV plus septum (RV/LV+S), as described in Methods. (C) Muscularization of alveolar wall arteries (Musc. Arteries Alv. Wall), as described in Methods. (D) Representative photomicrographs of lung tissue (stained by Movat pentachrome) of 15-week-old mice showing a typical nonmuscular peripheral alveolar artery in a littermate control mouse. (E) A similar section in the SM22α Cre PPARγ^flox/flox (SMC PPARγ^–/–) mouse shows an alveolar wall artery surrounded by a rim of muscle. (F–I) Immunohistochemistry in serial lung tissue sections from littermate control (CON) and SMC PPARγ^–/– mice stained for α-SMA (F and G) and proliferating cell nuclear antigen (PCNA; H and I). Arrows in I indicate enhanced PCNA staining in PASMCs. See also Table 1. Bars represent mean ± SEM (n = 5). ***P < 0.001 versus control; unpaired 2-tailed t test.

This schema incorporates the findings described in our article and the literature to date as discussed. (A) BMP-2 inhibits SMC proliferation via PPARγ and apoE. apoE impairs PDGF-BB/MAPK signaling by binding to LDL receptor–related protein (LRP), thereby initiating endocytosis and degradation of the LRP/PDGFR-β/PDGF-B complex. PPARγ induces LRP and other growth-inhibitory/proapoptotic genes in SMCs and inhibits cell-cycle and other growth-promoting genes such as telomerase, cyclin D1, and retinoblastoma protein. Moreover, PPARγ induces phosphatases that can directly inactivate phospho-ERK. (B) BMP-RII dysfunction promotes SMC proliferation and survival in PAH. Heightened PDGF-BB signaling leading to SMC proliferation is a key clinical feature of PAH. Deficiency of both apoE and LRP enhances mitogenic PDGF-BB/MAPK signaling. Loss-of-function mutations in the BMP-RII gene will decrease endogenous PPARγ activity, leading to unopposed MAPK signaling, SMC proliferation and survival, and ultimately development of PAH.TF, transcription factor. (C) PPARγ agonists can rescue BMP-RII dysfunction and reverse PAH. PPARγ agonists such as rosiglitazone or pioglitazone might reverse SMC proliferation and vascular remodeling in PAH patients with or without BMP-RII dysfunction via induction of apoE and other growth-inhibitory/proapoptotic genes (as indicated) and through repression of growth-promoting genes (not shown).