Bone morphogenetic protein receptor type II deficiency and increased inflammatory cytokine production. A gateway to pulmonary arterial hypertension

Abstract

Rationale: Mutations in bone morphogenetic protein receptor type II (BMPR-II) underlie most cases of heritable pulmonary arterial hypertension (PAH). However, disease penetrance is only 20-30%, suggesting a requirement for additional triggers. Inflammation is emerging as a key disease-related factor in PAH, but to date there is no clear mechanism linking BMPR-II deficiency and inflammation.

Objectives: To establish a direct link between BMPR-II deficiency, a consequentially heightened inflammatory response, and development of PAH.

Methods: We used pulmonary artery smooth muscle cells from Bmpr2(+/-) mice and patients with BMPR2 mutations and compared them with wild-type controls. For the in vivo model, we used mice heterozygous for a null allele in Bmpr2 (Bmpr2(+/-)) and wild-type littermates.

Measurements and main results: Acute exposure to LPS increased lung and circulating IL-6 and KC (IL-8 analog) levels in Bmpr2(+/-) mice to a greater extent than in wild-type controls. Similarly, pulmonary artery smooth muscle cells from Bmpr2(+/-) mice and patients with BMPR2 mutations produced higher levels of IL-6 and KC/IL-8 after lipopolysaccharide stimulation compared with controls. BMPR-II deficiency in mouse and human pulmonary artery smooth muscle cells was associated with increased phospho-STAT3 and loss of extracellular superoxide dismutase. Chronic lipopolysaccharide administration caused pulmonary hypertension in Bmpr2(+/-) mice but not in wild-type littermates. Coadministration of tempol, a superoxide dismutase mimetic, ameliorated the exaggerated inflammatory response and prevented development of PAH.

Conclusions: This study demonstrates that BMPR-II deficiency promotes an exaggerated inflammatory response in vitro and in vivo, which can instigate development of pulmonary hypertension.

Keywords: bone morphogenetic protein receptor type II; cytokine; inflammation; lipopolysaccharide; pulmonary hypertension.

Figure 1.

Bone morphogenetic protein receptor type II (Bmpr2)–deficient mice produce more IL-6 and KC after exposure to LPS. (A and B) Expression of (A) IL-6 messenger RNA (mRNA) and (B) KC mRNA in the lungs of wild-type (Bmpr2^+/+) and Bmpr2^+/− mice at baseline and 24 hours after exposure to LPS (100 μg/kg). KC is the mouse analog of IL-8. These data are normalized to the data of a wild-type mouse at baseline. Five to 10 mice have been used per arm. (C and D) Levels of (C) IL-6 and (D) KC in the sera of wild-type and Bmpr2^+/− mice at baseline, 3 hours, and 24 hours after exposure to LPS. Seven to 11 mice have been used per arm. (E) Representative immunoblot showing levels of pSTAT3 (phosphorylated signal transducer and activator of transcription 3) and total STAT3 from the lungs of wild-type and Bmpr2^+/− mice exposed to LPS. Lungs from four mice have been used per arm. (F) Densitometry for E normalized to total STAT3. *P ≤ 0.05, **P  ≤ 0.01, ***P  ≤ 0.001. NS = not significant.

Figure 2.

Bone morphogenetic protein receptor type II (Bmpr2) deficiency is associated with increased IL-6 and KC/IL-8 production in mouse Bmpr2^+/− and human BMPR2mut pulmonary artery smooth muscle cells (PASMCs). (A and B) Expression of (A) IL-6 messenger RNA (mRNA) and (B) KC mRNA in mouse wild-type (Bmpr2^+/+) and Bmpr2^+/− PASMCs at baseline and after 4 hours of treatment with LPS at 1, 10, and 20 μg/ml. KC is the mouse analog of IL-8. The data are normalized to the data of wild-type cells at baseline. Each arm represents the result of three to nine independent experiments. (C and D) Secretion of (C) IL-6 and (D) KC by mouse PASMCs at baseline and after 8 hours of treatment with LPS (10 μg/ml). Each arm represents the result of four to six independent experiments. (E) Secretion of IL-6 by human PASMCs at baseline and after 8 hours of treatment with LPS (10 μg/ml). Each arm represents the result of five independent experiments. All ELISA data (C–E) are normalized to the number of cells per well. (F) Representative immunoblot for pSTAT3 (phosphorylated signal transducer and activator of transcription 3) in mouse wild-type and Bmpr2^+/− PASMCs after 4 hours of treatment with LPS (10 μg/ml). Data represent the result of six independent experiments. (G) Representative immunoblot for pSTAT3 in human wild-type and BMPR2mut PASMCs with and without LPS in the presence and absence of anti-IL-6 antibody. m = BMPR2mut; wt = wild-type. Data represent the result of seven independent experiments. (H) Densitometry for G. (I) Pro-proliferative effect of IL-6 on human BMPR2mut smooth muscle cells. Proliferation rates in response to cytokines are normalized to proliferation rates in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (FBS), as BMPR2mut PASMCs grow faster at baseline. The concentration of IL-6 and IL-8 used was 25 ng/ml. Data represent the results of three independent experiments. *P  ≤ 0.05. NS = not significant.

Figure 3.

The proinflammatory response associated with bone morphogenetic protein receptor type II (BMPR-II) deficiency is also associated with increased levels of superoxide. (A) Staining for superoxide species in wild-type (BMPR2wt) and BMPR-II mutant (BMPR2mut) pulmonary artery smooth muscle cells (PASMCs) treated with Dulbecco’s modified Eagle’s medium with 0.1% fetal bovine serum (negative control), LPS, or hydrogen peroxide (positive control) with dihydroethidium (pale blue), which reacts with superoxide to form ethidium (red). Slides have been counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to demonstrate the nucleus (bright blue). A is representative of three independent experiments. (B) Quantitation of red light produced by ethidium, using a separate plate-based assay with a luminometer detecting light at 605 nm. Counts have been normalized to the number of cells per well. Each arm represents the result of four or five independent experiments. SOD = superoxide dismutase. (C) Secretion of IL-6 by human BMPR2wt and BMPR2mut PASMCs at baseline and after 8 hours of treatment with LPS (10 μg/ml) in the absence and presence of tempol, a superoxide dismutase mimetic. Each arm represents the result of four or five independent experiments. IL-6 levels have been normalized to the number of cells per well. *P  ≤ 0.05, **P  ≤ 0.01. NS = not significant.

Figure 4.

Bone morphogenetic protein receptor type II (BMPR-II) deficiency is associated with reduced superoxide dismutase 3 (SOD3). (A) SOD3 expression in mouse wild-type and Bmpr2^+/− pulmonary artery smooth muscle cells (PASMCs) after 4 hours of exposure to vehicle or LPS. The data represent the results of five independent experiments. (B) Representative immunoblot showing secreted SOD3 from mouse wild-type and Bmpr2^+/− PASMCs after 4 hours of exposure to vehicle or LPS. The data represent the results of three independent experiments. (C) Densitometry for B. (D) SOD3 expression in human wild-type (BMPR2wt) and BMPR-II mutant (BMPR2mut) PASMCs after 4 hours of exposure to vehicle or LPS. The data represent the results of four independent experiments. (E) Representative immunoblot showing secreted SOD3 from human wild-type and BMPR2mut PASMCs after 4 hours of exposure to vehicle or LPS. The data represent the results of three independent experiments. (F) Densitometry for E. (G) SOD3 expression in the lungs of wild-type and Bmpr2^+/− mice at baseline and after exposure to LPS. These are normalized to a single randomly chosen heterozygous mouse at baseline. Each arm represents the results from 6 to 10 mice. (H) Correlation between IL-6 and SOD3 expression in the lungs of 10 wild-type and 9 Bmpr2^+/− mice exposed to LPS. Spearman’s ρ is shown. (I) Changes in SOD3 expression in mouse PASMCs after exposure for 48 hours to vehicle, LPS, trichostatin A (TSA, 0.4 μM), or LPS + TSA. The data represent the results of five independent experiments. Messenger RNA data are normalized to the wild-type at baseline (A, D, and I). *P  ≤ 0.05, **P  ≤ 0.01, ***P  ≤ 0.001. NS = not significant.

Figure 5.

Chronic LPS administration promotes pulmonary hypertension in bone morphogenetic protein receptor type II (Bmpr2)–deficient mice, which is prevented by tempol. (A) Right ventricular systolic pressures, (B) right ventricular weight index, (C) Fulton index [right ventricular weight/(left ventricular + septal weight)], and (D) heart rate in wild-type and Bmpr2^+/− mice exposed to LPS in the absence and presence of tempol. (E) Left ventricular systolic pressures in wild-type and Bmpr2^+/− mice exposed to LPS. For A–E, each arm represents the results from 5 to 15 mice. (F) Representative immunoblot showing p-STAT3 (phosphorylated signal transducer and activator of transcription 3) and total STAT3 levels in lungs from control and LPS-exposed wild-type and Bmpr2^+/− mice, with (G) densitometry. Each arm represents the results from four to seven mice. (H) Representative immunoblot showing superoxide dismutase 3 (SOD3) levels in lungs from control and LPS-exposed wild-type and Bmpr2^+/− mice, with (I) densitometry. The data represent results from four different mice per arm. *P  ≤ 0.05, **P  ≤ 0.01. LV = left ventricular weight; LVSP = left ventricular systolic pressure; NS = not significant; RV = right ventricular weight; RVSP = right ventricular systolic pressure; S = septum.

Figure 6.

Immunohistochemical and morphological features of LPS-induced pulmonary hypertension. (A) Immunohistochemical studies on lung sections containing at least one distal pulmonary artery from control wild-type mice (WT), control mice heterozygous for a null allele in bone morphogenetic protein receptor (Bmpr2^+/− mice) (Mut), wild-type mice receiving LPS for 6 weeks (WT+LPS), Bmpr2^+/− mice receiving LPS for 6 weeks (Mut+LPS), wild-type mice receiving LPS and tempol for 6 weeks (WT+LPS+Tempol), Bmpr2^+/− mice receiving LPS and tempol for 6 weeks (Mut+LPS+Tempol), wild-type mice receiving tempol for 6 weeks (WT+Tempol), and Bmpr2^+/− mice receiving tempol for 6 weeks (Mut+Tempol). Shown are hematoxylin and eosin (H&E), elastic van Gieson (EVG), smooth muscle actin (SMA), and Ki67 staining. Black arrows indicate positively stained cells for SMA and Ki67. Scale bars, 50 μm. The data represent results from four to six different mice per arm. (B) Morphometry showing increased muscularization in pulmonary arteries of Bmpr2^+/− mice compared with wild-type controls. This difference is further exaggerated by the administration of chronic LPS and ameliorated by coadministration of tempol. The data represent results from three to six different mice per arm. (C) Close-up of SMA and EVG staining in Bmpr2^+/− mice receiving LPS (Mut+LPS) and Bmpr2^+/− mice receiving LPS and tempol for 6 weeks (Mut+LPS+Tempol). The majority of the thickened walls of the small arteries of the Bmpr2^+/− mice exposed to LPS are due to hypertrophy of the medial smooth muscle cells as indicated by increased smooth muscle (indicated with a red arrow and stained brown by SMA). The EVG stains the elastic fibers comprising the inner and outer elastic lamina (stained blue/black and indicated with a red arrow) and highlights the medial layer of the vessel. *P  ≤ 0.05, **P  0.01. NS = not significant.

Figure 7.

Proposed sequence of events arising from loss of bone morphogenetic protein receptor type II (BMPR-II). When pulmonary artery smooth muscle cells (PASMCs) deficient in BMPR-II face a proinflammatory insult, they react by producing abnormally high levels of IL-6 and IL-8, and abnormally low levels of IL-10. The concomitant reduction in superoxide dismutase 3 (SOD3) associated with BMPR-II deficiency also predisposes these PASMCs to increased oxidative stress. The net result of this abnormal proinflammatory response is a sustained increase in STAT3 (signal transducer and activator of transcription 3) signaling, increased proliferation of PASMCs, and accumulation of DNA damage within the PASMCs. We hypothesize that this sequence of events leads to pulmonary artery muscularization and a pulmonary hypertensive phenotype. PAH = pulmonary arterial hypertension; TLR4 = Toll-like receptor 4.