Bmp2 and Bmp4 exert opposing effects in hypoxic pulmonary hypertension

Abstract

The bone morphogenetic protein (BMP) type 2 receptor ligand, Bmp2, is upregulated in the peripheral pulmonary vasculature during hypoxia-induced pulmonary hypertension (PH). This contrasts with the expression of Bmp4, which is expressed in respiratory epithelia throughout the lung. Unlike heterozygous null Bmp4 mice (Bmp4(LacZ/+)), which are protected from the development of hypoxic PH, mice that are heterozygous null for Bmp2 (Bmp2(+/-)) develop more severe hypoxic PH than their wild-type littermates. This is associated with reduced endothelial nitric oxide synthase (eNOS) expression and activity in the pulmonary vasculature of hypoxic Bmp2(+/-) but not Bmp4(LacZ/+) mutant mice. Furthermore, exogenous BMP2 upregulates eNOS expression and activity in intrapulmonary artery and pulmonary endothelial cell preparations, indicating that eNOS is a target of Bmp2 signaling in the pulmonary vasculature. Together, these data demonstrate that Bmp2 and Bmp4 exert opposing roles in hypoxic PH and suggest that the protective effects of Bmp2 are mediated by increasing eNOS expression and activity in the hypoxic pulmonary vasculature.

Fig. 1.

Bone morphogenetic protein (BMP) signaling in Bmp2^+/− heterozygous null mice. A and B: RT-PCR for Bmp2 (A) and Bmp4 (B) mRNA expression in wild-type and Bmp2^+/− mouse lungs under normoxia, (wild type: n = 8; Bmp2^+/−: n = 6), 1 wk of hypoxia (wild type: n = 6; Bmp2^+/−: n = 6), or 3 wk of hypoxia (wild type: n = 5; Bmp2^+/−: n = 5). C and D: whole lung pSmad1/5/8 in wild-type and Bmp2^+/− mice detected by Western blot (C), quantified by densitometry (D). Data are expressed as means ± SE for wild-type mice (open bars) and Bmp2^+/− mice (filled bars). ANOVA with Bonferroni posttest, P < 0.05 vs. wild-type normoxia (a), wild-type 1 wk hypoxia (b), and wild-type 3 wk hypoxia (c).

Fig. 2.

Hypoxic pulmonary hypertension (PH) responses in Bmp2^+/− mice. A: representative right ventricular (RV) pressure tracings from wild-type and Bmp2^+/− mice exposed to normoxia or 3 wk hypoxia. B: RV systolic pressure (RVSP) in wild-type and Bmp2^+/− mice exposed to normoxia or 3 wk hypoxia. Mouse nos./group are indicated in Table 1. C: analysis of peripheral vessel muscularization in wild-type and Bmp2^+/− mice maintained under normoxic conditions or after 3 wk hypoxia. D: vascular smooth muscle cell (VSMC) proliferation in small, muscularized peripheral vessels (20–50 μm) after 1 wk of hypoxia. Because there are few small, peripheral muscularized vessels in normoxic lungs, studies were performed by comparing genotypes exposed to hypoxia only. E: VSMC proliferation in larger airway-associated arteries (50–200 μm) from mice maintained under normoxia or after 1 wk of hypoxia. Nos. analyzed are indicated in Table 2. Data are expressed as means ± SE for wild-type mice (open bars) and Bmp2^+/− mice (filled bars). ANOVA with Bonferroni posttest, P < 0.05 vs. wild-type normoxia (a), wild-type 1 wk hypoxia (b), and wild-type 3 wk hypoxia (c).

Fig. 3.

LacZ expression in 5′-Bmp2 BAC reporter mice. A–F: β-galactosidase staining of lung tissue sections from 5′-Bmp2 BAC LacZ reporter mice under normoxia (A), 1 wk of hypoxia (B), or 3 wk of hypoxia (C and D). Higher-power images of LacZ-expressing cells under normoxic conditions (E) and after 3 wk of hypoxia (F). LacZ staining is indicated in alveoli (thin black arrows), small vessels (<50 μm and distal to terminal bronchioles, thick black arrows), and larger vessels (50–200 μm associated with muscularized airways, clear block arrows). Black scale bars = 100 μm (A–D); white scale bars = 50 μm (E and F). G: quantification of LacZ-expressing cells. LacZ positive cells were counted and expressed as stained cells/unit. Data are expressed as means ± SE (n = 5/group). ANOVA with Bonferroni posttest, P < 0.05 vs. normoxic controls (a).

Fig. 4.

Endothelial nitric oxide synthase (eNOS) expression in Bmp2^+/− and Bmp4^LacZ/+ mouse lungs. A–D: Western blot and quantification of eNOS (A and B), phosphor-S239 VASP (A and C), and phospho-S157 VASP (A and D) in whole lung lysates from wild-type and Bmp2^+/− mice exposed to normoxia or 3 wk hypoxia. E: pulmonary eNOS mRNA expression assessed by RT-PCR under normoxic conditions (wild type: n = 8; Bmp2^+/−: n = 6) or after 3 wk hypoxia (wild type: n = 5; Bmp2^+/−: n = 5). F and G: Western blot and quantification for eNOS expression in intrapulmonary artery preparations (IPAs) isolated from normoxic or 3 wk hypoxic wild-type and Bmp2^+/− mice. H and I: eNOS protein expression and quantification in wild-type and Bmp4^LacZ/+ lungs. Data are expressed as means ± SE for wild-type (open bars), Bmp2^+/− (filled bars), and Bmp4^LacZ/+ (hatched bars) mice. ANOVA, with Bonferroni posttest, P < 0.05 vs. wild-type normoxia (a) and wild-type 3 wk hypoxia (c).

Fig. 5.

Regulation of eNOS by Bmp2. A: expression of endothelial cell (EC) markers vascular endothelial growth factor receptor (Vegfr) 2, eNOS, and VE-cadherin assessed by Western blot in wild-type IPAs after culture for 18 h. Lanes 1 and 2 are from two separate IPA preparations. B: eNOS and phospho-Smad1/5/8 in wild-type IPAs treated with BMP2 for 18 h. C: Western blot for eNOS and pSmad1/5/8 in pulmonary endothelial cells (PECs) treated with 10 ng/ml BMP2 over 4 h. D: BMP2-mediated effects on NOS activity in PECs assessed using a radiolabeled arginine conversion assay in PECs treated with 10 ng/ml of BMP2 for 1 or 4 h ±1 mM N^G-nitro-l-arginine methyl ester (l-NAME). Data are expressed as means ± SE. The assay was performed in triplicate and repeated with similar results. Results from one experiment are shown. ANOVA with Bonferroni posttest, P < 0.05 vs. untreated control (a) and 4 h BMP2 without l-NAME (b).

Fig. 6.

Regulation of eNOS by BMP2 and BMP4 in isolated IPA preparations. A: Western blot for eNOS expression in IPAs cultured ±50 ng/ml BMP2 or BMP4 for 18 h before lysis. B: quantification of eNOS blots by densitometry normalized to β-actin. Data are expressed as means ± SE. t-Test, P < 0.05 vs. untreated controls (a).