Published online before print January 19, 2009, doi: 10.1161/CIRCULATIONAHA.108.821504
(Circulation. 2009;119:566-576.)
© 2009 American Heart Association, Inc.
Vascular Medicine |
Altered Bone Morphogenetic Protein and Transforming Growth Factor-β Signaling in Rat Models of Pulmonary Hypertension
Potential for Activin Receptor-Like Kinase-5 Inhibition in Prevention and Progression of Disease
From the Department of Medicine (L.L., A.C., X.Y., M.S., P.D.U., N.W.M.), University of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, Cambridge, United Kingdom, and College of Pharmacy (D.-K.K.), Ewha Womans University, Seoul, Korea.
Correspondence to Professor Nicholas W. Morrell, Division of Respiratory Medicine, Department of Medicine, Box 157, Addenbrooke’s Hospital, Hills Rd, Cambridge CB2 2QQ, United Kingdom. E-mail nwm23{at}cam.ac.uk
Received June 13, 2008; accepted November 13, 2008.
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Abstract |
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Background— Recent genetic studies have highlighted the role of the bone morphogenetic protein (BMP)/transforming growth factor (TGF)-β signaling pathways in the pathogenesis of familial pulmonary arterial hypertension (PAH). It remains unclear whether alterations in these pathways contribute to other forms of pulmonary hypertension and to what extent these changes can be exploited for therapeutic intervention.
Methods and Results— We studied BMP/TGF-β signaling in 2 rat models of PAH due to chronic hypoxia and monocrotaline. In both models, there was a significant reduction in lung BMP type IA receptor and BMP type II receptor mRNA expression, although these changes were more pronounced in the monocrotaline model. This was accompanied by a reduction in lung levels of phospho-Smad1/5 and Id (inhibitor of DNA binding) gene expression in the monocrotaline model. In contrast, we observed increased TGF-β activity, again more marked in the monocrotaline model, as evidenced by increased phospho-Smad2/3 and increased expression of TGF-β–regulated genes. Immunohistochemistry revealed increased TGF-β1 expression in pulmonary artery smooth muscle cells and macrophages surrounding remodeled pulmonary arteries in monocrotaline rats. Inhibition of activin receptor-like kinase-5 signaling in vivo with the selective small-molecule inhibitor IN-1233 prevented PAH, right ventricular hypertrophy, and vascular remodeling after monocrotaline injection and inhibited the progression of established PAH in this model. No significant effect was observed in hypoxic PAH. In vitro studies confirmed that TGF-β stimulated migration of distal rat pulmonary artery smooth muscle cells and that this effect was inhibited by IN-1233.
Conclusions— Disruption of BMP/TGF-β signaling is more pronounced in the monocrotaline model of PAH than in the chronic hypoxia model. Increased TGF-β activity is associated with greater macrophage recruitment with monocrotaline treatment. Inhibition of TGF-β signaling via activin receptor-like kinase-5 prevents development and progression of PAH in the monocrotaline model and may involve inhibition of pulmonary artery smooth muscle cell migration.
Key Words: hypertension, pulmonary • hemodynamics • hypoxia • pathology • vessels
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Introduction |
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Idiopathic pulmonary arterial hypertension (PAH) is a rare disease characterized by a marked increase in pulmonary arterial pressure and right ventricular (RV) hypertrophy (RVH).1 The increase in pulmonary vascular resistance is due to adventitial, medial, and intimal thickening that results from fibroblast, smooth muscle, and endothelial cell proliferation.2 Recruitment and migration of smooth muscle–like cells to small precapillary pulmonary arteries is also a feature common to diverse forms of PAH.3 Without treatment, progression of pulmonary hypertension leads to RV failure and death.
Clinical Perspective p 576
Some 6% to 10% of people with idiopathic PAH have a further affected family member.4 Mutations in the bone morphogenetic protein (BMP) type II receptor (BMPR-II) are now known to underlie at least 70% of cases of familial PAH.5,6 BMPR-II is a constitutively active serine-threonine kinase belonging to the transforming growth factor-β (TGF-β) superfamily of receptors.7 Mutations in the TGF-β type I receptor, activin receptor-like kinase (ALK)-1, cause hereditary hemorrhagic telangiectasia but have also been found in some cases of severe PAH,8 which highlights the potential importance of BMP/TGF-β signaling pathways in the pathobiology of PAH. Evidence also suggests that BMPR-II9 and BMP type IA receptor (BMPR-IA)10 expression is reduced in the lungs of patients with idiopathic PAH, which suggests that dysfunction of BMP signaling might underlie other forms of PAH. Dysfunctional Smad1/5 signaling is a feature of pulmonary artery smooth muscle cells (PASMCs) isolated from patients with BMPR-II mutations and patients with idiopathic PAH without identifiable mutations in BMPR-II.11 The involvement of reduced BMP signaling in diverse forms of PAH is also supported by recent observations demonstrating a reduction in BMP signaling in a rat model of pulmonary hypertension.12
Isoforms of TGF-β are multifunctional cytokines with well-recognized roles in the cellular and molecular processes that contribute to pulmonary vascular remodeling2; however, the majority of data on this subject are from in vitro studies. The contribution of TGF-β signaling to PAH pathogenesis in vivo is less clear. Previous reports suggested increased expression of TGF-β isoforms in the pulmonary arteries of patients with idiopathic PAH13 and in the lungs of rats with monocrotaline-induced PAH.14 Furthermore, TGF-β levels are increased in the lungs of sheep with pulmonary hypertension induced by air embolism.15 In contrast, more recent reports have concluded that TGF-β signaling is reduced in the monocrotaline model.16
BMPs and TGF-β isoforms bind to distinct heterodimeric complexes of type I and type II receptors at the cell surface.17 In both cases, ligand binding leads to activation of the type I receptor by the constitutively active type II receptor, which leads to phosphorylation of downstream signaling intermediaries termed Smad proteins. BMPs signal via Smads 1, 5, and 8, whereas TGF-β typically signals via Smads 2 and 3 in mesenchymal cells.18 These receptor-activated Smads then complex with the common partner Smad, Smad4, to enter the nucleus and regulate transcription of target genes in a highly cell- and context-specific manner. BMP/Smad1 signaling and TGF-β/Smad3 signaling have been shown to exert opposite and antagonistic effects on cell function in endothelial cells19 and in the process of epithelial to mesenchymal cell transition.20 Thus, it is conceivable that these pathways exert opposing effects in the context of pulmonary vascular remodeling.
Here, we characterized the signaling and gene expression downstream of BMP and TGF-β receptors in 2 widely used rat models of PAH due to monocrotaline and chronic hypoxia (CH). We determined that reduced BMP and enhanced TGF-β signaling was a feature of both models, but particularly the monocrotaline model. We further found that inhibition of TGF-β/Smad3 signaling with a small molecular inhibitor of ALK-5 could prevent and halt progression of PAH in the monocrotaline model but not that due to CH. The increased activity of TGF-β in the monocrotaline model was associated with increased macrophage infiltration in monocrotaline. In vitro studies suggest that a potential mechanism of action of ALK-5 inhibition includes inhibition of TGF-β1–driven smooth muscle cell migration.
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Methods |
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Chemicals
A selective ALK-5 inhibitor, 3-((4-(6-methylpyridin-2-yl)-5-(quinolin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1233), and its water-soluble phosphate salt form were supplied by the College of Pharmacy, Ewha Womans University, Seoul, Korea, and used for in vitro and in vivo experiments, respectively.
Rat Models of Pulmonary Hypertension
Male Sprague-Dawley rats (6 to 7 weeks old; weight 250 to 300 g) were used throughout the study. All protocols and surgical procedures were approved by the local animal care committee. For induction of PAH due to CH, groups of rats were maintained in a normobaric hypoxic chamber (FIO2 10% O2) for up to 21 days. In the monocrotaline model, animals received a single subcutaneous injection of monocrotaline (60 mg/kg). To characterize the BMP/TGF-β pathways during the development of PAH, animals were euthanized at 2, 7, and 21 days immediately after hemodynamic assessment. Rats were exsanguinated, and the lungs were removed for further analysis. Half of the lungs from all groups were fixed in situ in the distended state by infusion of 10% buffered formalin into the pulmonary artery (at 25 mm Hg pressure) and trachea for 1 minute and then were placed in 4% paraformaldehyde before being embedded in paraffin. The remaining lungs were immediately frozen in liquid nitrogen for protein and RNA isolation. To determine the effect of ALK-5 inhibition on the development of PAH, rats received IN-1233 phosphate (24.67 mg · kg–1 · d–1, equivalent to 20 mg · kg–1 · d–1 IN-1233) or vehicle continuously via intraperitoneal osmotic minipumps. The day after surgery, animals either received monocrotaline or were maintained in CH for 21 days, at which point they were anesthetized for hemodynamic assessment and lung tissue collection. In other groups of monocrotaline-treated rats, the ability of IN-1233 to prevent the progression of established pulmonary hypertension was tested. Animals were given monocrotaline 60 mg/kg. At 3 weeks, animals received daily intraperitoneal injections of IN-1233 (24.67 mg/kg) or vehicle for an additional 2 weeks before measurement of hemodynamics and RVH.
Hemodynamic Evaluation and RVH
At specific time points, rats were anesthetized for hemodynamic assessment. Body weight was recorded, and right-heart catheterization was performed to measure pulmonary arterial pressure, as described previously.21 To assess the extent of RVH, the heart was removed, and the RV free wall was dissected from the left ventricle plus septum (LV+S) and weighed separately; the degree of RVH was determined from the ratio RV/(LV+S).
Pulmonary Vascular Morphometry
To determine the degree of muscularization of small pulmonary arteries, lung tissue sections were stained with anti-smooth muscle 
-actin. At least 20 arteries accompanying alveolar ducts were identified per tissue section. Arteries were scored according to whether they were completely muscular, partially muscular, or nonmuscular.
Immunohistochemistry
Immunostaining for TGF-β1 and the macrophage marker CD68 was performed in control rat lungs and after 21 days of exposure to monocrotaline or hypoxia. Monoclonal mouse anti-rat CD68 and rabbit polyclonal anti-TGFβ1 (both from Santa Cruz Biotechnology, Santa Cruz, Calif) were detected with a StreptABC peroxidase technique (Vector Laboratories, Peterborough, United Kingdom) as described elsewhere.9
Western Blotting
Frozen lung tissue was homogenized in lysis buffer (250 mmol/L Tris-HCl, pH 6.8; 4% SDS, 20% vol/vol glycerol and 1 Roche EDTA-free protease inhibitor cocktail) and sonicated for approximately 1 minute, then centrifuged for 15 minutes at 15 000g. The protein concentration was determined with the Bio-Rad Lowry assay (Bio-Rad Laboratories, Hemel Hempstead, United Kingdom), with BSA used as the standard. An equal amount of protein (70 µg) from each sample was diluted with 5X sample loading buffer and boiled for 5 minutes. The protein suspensions were separated on a 10% gel (BMPR-II) or a 12% gel (phospho-Smad 1/5 and phospho-Smad 3), transferred to a nitrocellulose membrane, and incubated with blocking buffer. To determine the phosphorylation of specific Smads, membranes were incubated with a specific rabbit monoclonal antibody to either phospho-Smad1/5 (1:1000; Cell Signaling Technology, Inc, Beverly, Mass) or phospho-Smad3 (1:1000; Cell Signaling Technology, Inc) overnight at 4°C. For BMPR-II, a mouse monoclonal antibody to BMPR-II (1:250; BD Transduction Laboratories, Franklin Lakes, NJ) was used. Blots were then incubated with an appropriate horseradish peroxidase–conjugated antibody and enhanced chemiluminescence reagent (Amersham Bioscience, Little Chalfont, United Kingdom). To confirm equal loading, blots were incubated with an anti--tubulin antibody or β-actin antibody (Sigma, Dorset, United Kingdom).
Real-Time Reverse-Transcription Polymerase Chain Reaction
mRNA expression of BMPR-II was evaluated by real-time quantitative polymerase chain reaction. Frozen lung tissue was homogenized and total RNA extracted. Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, Calif) according to the manufacturer’s instructions. DNA from each RNA sample was removed by TURBO DNA-free TURBO DNase treatment and removal reagents (Ambion, Foster City, Calif). Reverse transcription was then performed with the StrataScript first-strand synthesis system (Stratagene, La Jolla, Calif). Synthesized complementary DNA was amplified by a standard polymerase chain reaction protocol with iQ SYBR green supermix (Bio-Rad) and rat-specific primers (supplemental Table I). Parallel amplifications with primers for β-actin were performed. Cycling conditions were as follows: 3 minutes of preincubation at 95°C, 30 seconds of denaturation at 95°C, 30 seconds of annealing at 58°C, and 30 seconds of extension at 72°C for 50 cycles with iCycler (Bio-Rad). The fluorescent product was detected at the end of each cycle. Product specificity was confirmed by agarose gel electrophoresis and routinely by melting-curve analysis. Real-time polymerase chain reaction data were analyzed by use of iCycler software (Bio-Rad). The ratio of a specific gene to β-actin was calculated in each sample.
Migration Assay
Rat PASMCs were isolated from small pulmonary arteries, as described previously.22 Cells were made quiescent for 48 hours and then seeded onto fibronectin-coated transwell inserts (VWR International, West Chester, Pa) at a density of 30 000 cells/well. The inserts were placed into a 24-well plate. Each well contained either 0.1% FBS/DMEM alone or with the addition of TGF-β1, IN-1233, or TGF-β1 plus IN-1233. The cells were incubated for 4 to 6 hours at 37°C/5% CO2. The cells in the upper well were removed with a cotton swab, and the migrated cells on the lower surface of the membrane were fixed with methanol for 4 minutes, stained with Quick-Diff red (Reagena, Toivala, Finland) for 1 minute, and stained with Quick-Diff blue (Reagena) for 3 minutes. The cells on the lower surface were counted at high-power magnification (x20). Multiple fields (4) were counted per well and averaged for each condition studied.
Statistical Analysis
Data are presented as mean±SE. Data between groups were compared with a 2-tailed t test or 1-way ANOVA followed by Tukey’s honestly significant difference test, whichever was appropriate. P<0.05 was considered statistically significant.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Time Course of Pulmonary Hypertension Development With Monocrotaline and Hypoxia
Hemodynamic studies in rats exposed to monocrotaline or CH over 21 days demonstrated differences in the onset of elevation of pulmonary arterial pressure. Hypoxic exposure resulted in elevations of mean pulmonary artery pressure and RVH as early as 2 days, whereas monocrotaline exposure did not result in significant elevations in these indices until after day 7 (Figure 1A and 1B). In contrast, the time course of vascular remodeling, as assessed by muscularization of small pulmonary arteries, was similar in both models. Significant increases in the percent of muscularized arteries were only observed at the 3-week time point (Figure 1C and 1D).
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Reduced BMPR-II Signaling in Rat Models of Pulmonary Hypertension
Exposure to monocrotaline and CH led to a reduction in the expression of BMPR-II mRNA in rat lungs by day 21 (Figure 2A and 2B). Interestingly, there was a trend toward an initial increase in BMPR-II mRNA levels in the monocrotaline animals. The expression of BMPR-IA mRNA was also reduced in both models, with an initial increase in expression levels at the day 2 time point (supplemental Figure I). Immunoblotting for BMPR-II revealed that BMPR-II protein expression was also reduced at day 21 in the monocrotaline model, and to a lesser extent in CH (Figure 2C through 2F). Furthermore, expression of phospho-Smad1/5 was also reduced by day 21 in the monocrotaline rat lung, whereas the reduction in CH was less marked (Figure 2C, 2D, 2G, and 2H). To assess the transcriptional activity of BMP/Smad signaling in both models, we determined the time course of Id1 gene expression. Monocrotaline, but not CH, led to a significant reduction in the mRNA expression of Id1 by day 21 (Figure 3A and 3B).
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Activation of TGF-β1 Signaling in Rat Models of Pulmonary Hypertension
Hypoxia did not significantly increase TGF-β1 mRNA expression at any time point (Figure 4A). In contrast, monocrotaline exposure led to a 7-fold increase in TGF-β1 mRNA expression by day 21 (Figure 4B). Expression of phospho-Smad3 in lung homogenates was also significantly increased only in monocrotaline lungs (Figure 4D and 4F) and not in CH (Figure 4C and 4E). To further assess the expression of known TGF-β1 targets, we determined the expression of plasminogen activator inhibitor-1 mRNA in lung homogenates. Plasminogen activator inhibitor-1 mRNA expression was markedly increased in monocrotaline lungs but not in hypoxia (Figure 4G and 4H).
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We further determined the mRNA expression of the TGF-β type II (TGF-βRII) receptor in both models. By day 21, TGF-βRII mRNA expression was significantly elevated in monocrotaline lungs but not in CH (Figure 5A). To determine the cellular origin of the increased TGF-β1 in the monocrotaline model, we performed immunohistochemistry for TGF-β1 and the macrophage marker CD68. This revealed larger numbers of macrophages in the monocrotaline lung than in control or CH lungs (Figure 5B). In addition, increased TGF-β1 immunostaining was observed in the walls of remodeled arteries and was associated with macrophages in the parenchyma (Figure 5B).
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ALK-5 Inhibition Inhibits the Development and Progression of Pulmonary Hypertension in the Monocrotaline Model
Having established that activation of TGF-β signaling is a feature of rat models of pulmonary hypertension, particularly in pulmonary hypertension due to monocrotaline exposure, we then determined whether inhibition of TGF-β1 signaling with the ALK-5 inhibitor IN-1233 could prevent the development of pulmonary hypertension in the monocrotaline and CH models. IN-1233 significantly inhibited the elevation of RV systolic pressure, RVH, and muscularization of peripheral arteries after 3 weeks in monocrotaline animals (Figure 6). In contrast, IN-1233 had no significant effect on these indices in CH rats (Figure 6).
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We next tested the ability of IN-1233 to prevent the progression of or reverse established pulmonary hypertension in the monocrotaline model. Treatment with IN-1233 or vehicle was begun 3 weeks after monocrotaline exposure, and hemodynamics and RVH were measured at 5 weeks. IN-1233–treated animals demonstrated a lower final RV systolic pressure and reduced RVH compared with vehicle-treated animals (Figure 7).
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ALK-5 Inhibition Reduced TGF-β1–Stimulated Gene Expression in Monocrotaline-Treated Rats
To confirm that IN-1233 was indeed inhibiting ALK-5 signaling in vivo, we examined the expression of phospho-Smad3 in lung homogenates and determined the expression of known genomic targets of TGF-β/ALK-5/Smad3 signaling, including plasminogen activator inhibitor-1 and TGF-β1 itself. IN-1233 significantly reduced the levels of phospho-Smad3 in lung homogenates from monocrotaline rats (Figure 8A and 8B). In addition, IN-1233 markedly suppressed the expression of TGF-β1 (Figure 8C) and plasminogen activator inhibitor-1 (Figure 8D) mRNA in monocrotaline rats. In contrast, treatment with IN-1233 had no effect on the suppression of BMPR-II mRNA in monocrotaline rats (Figure 8E).
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ALK-5 Inhibition Reduces PASMC Migration
Rat PASMCs were isolated from small peripheral pulmonary arteries. We first confirmed that these cells were competent for TGF-β1 signaling and confirmed the efficacy of IN-1233 as an ALK-5 inhibitor in vitro. Exposure of cells to TGF-β1 led to activation of phospho-Smad2 at 1 hour (supplementary Figure II). IN-1233 completely inhibited Smad2 phosphorylation in response to TGF-β1. In migration assays, we demonstrated that TGF-β1 stimulated the migration of PASMCs. Coincubation of PASMCs with IN-1233 completely inhibited TGF-β1–stimulated PASMC migration (Data Supplement Figure II).
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Discussion |
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The present study has provided clear evidence for an alteration in BMP/TGF-β signaling in 2 rat models of PAH. Both the hypoxia and monocrotaline models were associated with a reduction in the expression of BMPR-II mRNA and protein by day 21; however, the reduction in BMP signaling was most evident in the monocrotaline model, because only in this model did we observe a reduction in lung phospho-Smad1/5 expression and a reduction in expression of the BMP target gene, Id1. In contrast, we observed increased activation of TGF-β signaling in the monocrotaline model, as evidenced by increased lung expression of TGF-βRII mRNA and phospho-Smad3 and the induction of TGF-β–responsive genes. No significant activation of the TGF-β/Smad3 pathway was observed in hypoxic animals. Furthermore, we used a highly selective small-molecule inhibitor of ALK-5, IN-1233, to determine whether inhibition of TGF-β signaling would show efficacy in the prevention of PAH induced by monocrotaline or hypoxia. Consistent with our findings of increased TGF-β/Smad3 signaling in the monocrotaline model, IN-1233 significantly prevented the elevation of pulmonary artery pressure, RVH, and peripheral remodeling of small pulmonary arteries in the monocrotaline model. IN-1233 was ineffective at preventing PAH in the CH rat, again consistent with the observed lack of activation of this pathway in this model. Having established that IN-1233 could prevent the development of PAH in the monocrotaline model, we went on to confirm that ALK-5 inhibition could prevent the progression of established monocrotaline-induced PAH and RVH when treatment was initiated 3 weeks after monocrotaline injection. Moreover, we confirmed that these effects are likely to be due directly to inhibition of the TGF-β/ALK-5/Smad3 signaling axis, because IN-1233 inhibited the activation of Smad3 and the transcription of TGF-β target genes in the lungs of monocrotaline rats.
IN-1233 is a highly selective ALK-5 inhibitor and has an IC50 of 34 nm (see supplementary Figure III for structure). It specifically inhibits TGF-β signaling via ALK-5 by acting as a competitive inhibitor of the ATP-binding site.23 It was recently shown that a closely related compound, IN-1130, inhibited TGF-β–driven renal fibrosis in a rat model of obstructive nephropathy24 and ameliorated experimental autoimmune encephalomyelitis in a mouse model.25
Similar approaches have been used to demonstrate proof-of-concept for inhibition of the TGF-β pathway in lung fibrosis.26 Recently, it was shown that another orally active small-molecule ALK-5 inhibitor, SD208, reduced the monocrotaline-induced increase in RV systolic pressure, but the effects on established PAH were modest, and no convincing evidence for in vivo inhibition of TGF-β–driven target genes was presented.27 The lack of effect of IN-1233 during CH was unexpected, because it was previously reported that a transgenic mouse expressing a dominant-negative TGF-βRII was protected from hypoxia-induced PAH.28 In addition to the species difference, the length of hypoxic exposure was 6 weeks in that study. In the present study, we provided clear evidence for activation of TGF-β signaling in the monocrotaline model that was inhibited in vivo by IN-1233. The lack of effect of IN-1233 in CH would appear to be due to the less impressive activation of TGF-β signaling, at least under the experimental conditions used here.
We have previously shown that idiopathic and familial forms of PAH are associated with a reduction in lung protein expression of BMPR-II9 and reduced activation of Smad1/5 in concentric intimal lesions.11 A reduction in BMPR-II expression has been noted previously in the hypoxic29 and monocrotaline12 rat models and confirmed in the present study, which suggests that dysregulation of this signaling pathway may be a common finding in PAH due to diverse causes; however, the present study extended these findings to demonstrate that Smad1/5 signaling and Id gene expression is also reduced in monocrotaline-induced PAH. Moreover, the reduction in BMPR-II expression may contribute critically to PAH pathogenesis. In support of this, we previously reported that adenoviral delivery of BMPR-II attenuated the development of PAH in the hypoxic rat model.30
We,17 and others,31 have argued that one possible consequence of a reduction in BMPR-II signaling is a deleterious increase in TGF-β signaling. This may occur as a consequence of the cross talk between BMP- and TGF-β–activated Smad signaling pathways.19 We have previously shown that PASMCs isolated from patients with idiopathic PAH demonstrate an abnormal proliferative response to TGF-β.32 Increased TGF-β/Smad2 signaling has been noted in lung tissues of patients with idiopathic PAH.33 Moreover, we have shown that BMPs and TGF-β can antagonize each other in the regulation of cyclooxygenase expression34 and myoblast differentiation.35 Such antagonism has also been demonstrated in rat models of renal fibrosis.20 Taken together, the results of the present study add further support to the concept that a reduction in BMP signaling and increased TGF-β signaling may contribute to the fibroproliferative process in the context of pulmonary hypertension.
A pathological feature common to all forms of PAH is the appearance of smooth muscle in small, normally nonmuscular pulmonary arterioles.36 This is thought to be due to a combination of differentiation and migration of smooth muscle cells and fibroblast-like cells, pericytes, and intermediate cells. TGF-β is known to induce migration of fibroblasts and systemic vascular smooth muscle cells.37 Here, we confirmed that TGF-β stimulates migration of PASMCs from peripheral pulmonary arteries and that this effect could be inhibited by IN-1233 acting via inhibition of Smad2/3 phosphorylation. The inhibition of smooth muscle migration may contribute to the beneficial effects of IN-1233 in vivo. Presumably, rather different processes may be responsible for driving the appearance of smooth muscle in peripheral pulmonary arteries in hypoxia and monocrotaline, because IN-1233 had no effect on this process in hypoxia. The present data are consistent with the monocrotaline model being a TGF-β–responsive form of pulmonary hypertension. Immunohistochemistry localized the sites of increased production of TGF-β1 to pulmonary vascular smooth muscle and increased numbers of alveolar macrophages in the monocrotaline lung. Monocrotaline-induced pulmonary hypertension previously has been shown to be a macrophage-dependent process.38
We confirmed that there are important differences between the 2 commonly used rat models of PAH. The monocrotaline rat model appears to be more clearly regulated by TGF-β than the hypoxic rat model. The time course of the increase in pulmonary arterial pressure and RVH is also delayed in the monocrotaline model compared with hypoxia, in which presumably hypoxic pulmonary vasoconstriction would have elevated pulmonary arterial pressure immediately. These differences have been noted previously,39,40 but here, we provide evidence that the changes in the monocrotaline model may more closely reflect the alteration in BMP/TGF-β signaling in human familial or idiopathic PAH.
An interesting finding in the present study was that after 2 days of monocrotaline exposure, there was a tendency for lung BMPR-II and especially BMPR1A mRNA expression to increase. One possible explanation for this is that monocrotaline initiates an early inflammatory response, especially monocyte recruitment.38 Monocytes themselves express high levels of BMPR-II, and thus, they could be responsible for the increase that was seen.9 However, it was not until 21 days after monocrotaline treatment that an increase in pulmonary arterial pressure was observed, and this coincided with a dramatic reduction in both BMPR-II mRNA and BMPR-II protein expression in the monocrotaline lung.
In summary, we have provided evidence for a reduction in BMP signaling and a gain in TGF-β signaling in the monocrotaline rat model of PAH. Inhibition of TGF-β/ALK-5 signaling with IN1233 inhibited the development and progression of monocrotaline-induced pulmonary hypertension in this model. These studies provide proof-of-concept for approaches that manipulate TGF-β/BMP signaling in clinical pulmonary hypertension.
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Acknowledgments |
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Sources of Funding
This study was funded by a research grant from the Garfield Weston Trust and the British Heart Foundation.
Disclosures
None.
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References |
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*The first 2 authors contributed equally to this work.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.821504/DC1.
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