Codependence of Bone Morphogenetic Protein Receptor 2 and Transforming Growth Factor-β in Elastic Fiber Assembly and Its Perturbation in Pulmonary Arterial Hypertension

Abstract

Objective: We determined in patients with pulmonary arterial (PA) hypertension (PAH) whether in addition to increased production of elastase by PA smooth muscle cells previously reported, PA elastic fibers are susceptible to degradation because of their abnormal assembly.

Approach and results: Fibrillin-1 and elastin are the major components of elastic fibers, and fibrillin-1 binds bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-β1 (TGFβ1). Thus, we considered whether BMPs like TGFβ1 contribute to elastic fiber assembly and whether this process is perturbed in PAH particularly when the BMP receptor, BMPR2, is mutant. We also assessed whether in mice with Bmpr2/1a compound heterozygosity, elastic fibers are susceptible to degradation. In PA smooth muscle cells and adventitial fibroblasts, TGFβ1 increased elastin mRNA, but the elevation in elastin protein was dependent on BMPR2; TGFβ1 and BMP4, via BMPR2, increased extracellular accumulation of fibrillin-1. Both BMP4- and TGFβ1-stimulated elastic fiber assembly was impaired in idiopathic (I) PAH-PA adventitial fibroblast versus control cells, particularly those with hereditary (H) PAH and a BMPR2 mutation. This was related to profound reductions in elastin and fibrillin-1 mRNA. Elastin protein was increased in IPAH PA adventitial fibroblast by TGFβ1 but only minimally so in BMPR2 mutant cells. Fibrillin-1 protein increased only modestly in IPAH or HPAH PA adventitial fibroblasts stimulated with BMP4 or TGFβ1. In Bmpr2/1a heterozygote mice, reduced PA fibrillin-1 was associated with elastic fiber susceptibility to degradation and more severe pulmonary hypertension.

Conclusions: Disrupting BMPR2 impairs TGFβ1- and BMP4-mediated elastic fiber assembly and is of pathophysiologic significance in PAH.

Keywords: BMPR2 receptor; TGF-beta-1; elastin; fibrillin-1; fibroblasts; hypertension, pulmonary; smooth muscle cells.

Figure 1. TGFβ1 increases elastin mRNA and protein and TGFβ1 and BMP4 increase extracellular fibrillin-1 in human pulmonary artery fibroblasts

Human pulmonary artery fibroblasts (PAF) isolated from unused donor lungs and used between passages 3–6 were stimulated with BMP4 (10ng/ml), TGFβ1 (2ng/ml), BMP4+TGFβ1, or vehicle (Con). (A, C) Fold change in mRNA of ELN and FBN1 was measured four and eight hours after stimulation. (B, D) Representative immunoblots above and densitometry below for elastin in cell lysates and fibrillin-1 in conditioned media 48h after stimulation. Beta-actin and Ponceau staining were used as loading controls for cell lysates and conditioned media respectively. Fibrillin-1 is normalized to Con. Bars represent Mean±SEM of n=3–5 independent experiments, *p<0.05, **p<0.01, ***p<0.001 vs. Con, by one-way ANOVA and post-hoc Bonferroni test. (E, F) PAF of donor controls were seeded in glass chamber slides and grown for four days to confluence. Cells were starved overnight, then stimulated every other day for seven days with vehicle (Con), BMP4, TGFβ1 or BMP4+TGFβ1. Elastic fibers were visualized by indirect immunofluorescence of elastin (E) and fibrillin-1 (F). Right, fluorescence intensities quantified by Image J software and normalized to cell number assessed by nuclei DAPI staining. Scale bar=100μm. Bars represent Mean±SEM of n=4 independent experiments, **p<0.01, ****p<0.0001 vs. Con; ^###p<0.001, ^####p<0.0001 vs. BMP4; ^&&&&p<0.0001 vs. TGFβ1, by two-way ANOVA and post-hoc Bonferroni test.

Figure 2. BMPR2 and fibrillin-1 are required for PAF elastic fiber formation

PAF of donor controls were transfected with siRNA oligonucleotides targeting BMPR2 or FBN1, or with non-targeting siRNA (siControl). Starting 24h after transfection, the cells were stimulated for 48h with BMP4 (10ng/mL), TGFβ1 (2ng/mL) or vehicle (Control). (A, B) Representative immunoblot above and densitometry below for elastin and BMPR2 in cell lysates (A) and fibrillin-1 in conditioned media (B) of PAF donor controls. β-actin and Ponceau were used as loading controls for cell lysates and conditioned media respectively. (C) To assess elastic fibers cells were stimulated every other day for seven days and then visualized by indirect immunofluorescence of elastin and quantified by Image J software. Note that reduction of BMPR2 and fibrillin-1 inhibited BMP4-induced elastic fiber formation, and reduced fibrillin-1 also inhibited TGFβ1-induced fiber formation. The elastic fibers induced by TGFβ1 appear more fragmented in cells treated with BMPR2 siRNA. Scale bar=100µm. Bars represent Mean±SEM of n=3 independent experiments, *p<0.05, ***p<0.001 vs. unstimulated Control; ^##p<0.01, ^###p<0.001 vs. non-targeting siRNA (siControl) by two-way ANOVA and post-hoc Bonferroni test.

Figure 3. Reduced PAF elastic fibers from IPAH and HPAH with BMPR2 mutation (BMPR2m) related to elastin and fibrillin-1

Elastic fiber formation by PAF from donor controls, IPAH and HPAH with a BMPR2 mutation (BMPR2m) analyzed as described in Figure 2. Fold change in ELN and FBN1 mRNA (A, C) measured four and eight hours following stimulation by BMP4 (10ng/ml), TGFβ1 (2ng/ml), or vehicle Control. Representative immunoblot and densitometry for elastin in the cell lysates and fibrillin-1 in the conditioned media (B, D) measured 48 hours after stimulation. (E) Elastic fibers were visualized by indirect immunofluorescence of elastin and quantified by Image J software. Scale bar=100µm. β–Actin and Ponceau were used as loading controls. Bars represent Mean±SEM of n=3 different cell lines (donor controls) per condition, *p<0.05, **p<0.001, ***p<0.001 vs. unstimulated (Control); ^#p<0.05, ^##p<0.01, ^###p<0.001 vs. Donor, by two-way ANOVA and post-hoc Bonferroni test.

Figure 4. Reduced PA elastin and fibrillin-1 in IPAH and HPAH pulmonary arteries with a BMPR2 mutation vs. donor control

Representative PAs in lung tissue sections from donor control, IPAH and HPAH with a BMPR2 mutation (BMPR2m) were immunostained for elastin (green) and fibrillin-1 (red) and quantified below. Note that in PAs at the level of the terminal bronchiolus from IPAH lungs the elastic laminae are fragmented associated with a reduction in fibrillin-1, and in the BMPR2m there is substantial loss of elastic laminae associated with a decrease in both fibrillin-1 and elastin. Fluorescence intensity of elastin fibers was quantified by ImageJ. Bars represent Mean±SEM for donor control (n=5), IPAH (n=4) or BMPR2m (n=4). ^#p<0.05, ^##p<0.01, ^###p<0.001 vs. Donor by 2-way ANOVA and post-hoc Bonferroni test. Scale bar=50µm.

Figure 5. Reduced fibrillin-1 and heightened susceptibility to elastic fiber degradation in Bmpr2/1a compound heterozygote mice

(A) Representative immunoblot and densitometric analysis of elastin and fibrillin-1 of three sets of four pooled main PAs from Bmpr2/1a (Het) and wild type (WT) mice. Proteins were assessed on a reducing gel and the bands designated by the arrows were quantified. n=3 pools of Het or WT. (B) Representative confocal images of internal elastic lamina of central PAs from Het and WT mice. Central PAs were incubated with vehicle (PBS, top row) or porcine pancreatic elastase (5ng/mL, middle). On the bottom are PAs from mice exposed to Sugen 5416 (SU) and hypoxia as described in (C) below. Note the increase in size and number of fenestrations in the Het vs. WT PAs at baseline, following elastase treatment, or after Sugen and hypoxia (arrows). (SU+ hypoxia). On the right, quantification of number and area of fenestrations per total area of elastin assessed in six separate fields per condition (n=5/group). Scale bar=30µm. (C–H) Het and WT mice were exposed to room air (normoxia), or 10% O₂ (hypoxia) for three weeks following subcutaneous injection of VEGF receptor blocker Sugen 5416. (C) Pulmonary artery acceleration time (PAAT) measured as described in “Methods”. n=5–6 (WT) or 5–8 (Het) mice. (D, E) Development of PAH assessed by the right ventricular systolic pressure (RVSP, D) and right ventricular hypertrophy (RVH; E) given by the Fulton index (weight of RV/left ventricle and septum, RV/LV+S). Bars indicate Mean±SEM of n=5–6 mice for normoxia and n=9–10 mice for hypoxia. (F) Representative images of PAs at the level of the terminal bronchiolus stained for elastin and fibrillin-1. Note the increased number of breaks in the elastic lamina of Het vs. WT. Scale bars=50µm, 10µm in the higher magnification panels, right column. n=3. (G) Representative images of arteries at the alveolar duct level stained for elastin and fibrillin-1 from the het and WT mice exposed to hypoxia and Sugen. Scale bars=20µm, and 5µm in the higher magnification panels, right column. (H) Quantification of number of vessels per mm² in normoxia and hypoxia. n=3. Bars represent Mean±SEM. In A, F and H, *p<0.05, **p<0.01, ***p<0.001 vs. WT by t-test; In B, C, D, E, ***p<0.001 and ****p<0.0001, Het vs. WT; ^#p<0.05, ^##p<0.01, ^###p<0.001 and ^####p<0.0001, SU+Hypoxia vs. Normoxia, same genotype, by two-way ANOVA and post-hoc Bonferroni test. In A, 3 sets of pooled PAs (n=4) from female (F) mice were used. For confocal measurements of elastin fenestrations, 3F and 2 males (M) in each genotype in each condition. PAAT, in RA, 3F, 2M of each genotype, and in Su+hypoxia 6M WT and 8M het. RVSP: in RA 3F and 3M of each genotype and in SU+/Hypoxia 9M WT and 10M hets. For RVH, 3M and 3F RA and 10M of each genotype in SU+Hypoxia.

Figure 6

Model of elastic fiber formation in control, IPAH and HPAH with a BMPR2 mutation. While TGFβ1 stimulates elastin mRNA, production of elastin and fibrillin-1 proteins are both largely BMPR2 dependent. TGFβ1 and BMP4 via BMPR2 increase fibrillin-1. In IPAH the elastic fiber formation is impaired due to reduced fibrillin-1 mRNA and protein and elastin mRNA, although some elastin protein is produced in response to TGFβ1. When there is a mutation in BMPR2, fibrillin-1 and elastin protein are both markedly decreased in response to both TGFβ1 and BMP4, leading to poorly assembled elastic fibers.