Bone Morphogenetic Protein 4 Promotes Pulmonary Vascular Remodeling in Hypoxic Pulmonary Hypertension
http://www.100md.com
循环研究杂志 2005年第9期
the Departments of Cell and Developmental Biology (D.B.F., H.S.B., M.P.d.C.), Medicine (A.A., D.J.Y., A.P., M.P.d.C.), Pathology (S.M., J.E.J.), Biostatistics (Y.S.)
Pediatrics (H.S.B.), Vanderbilt University School of Medicine, Nashville, Tenn.
Abstract
We show that 1 of the type II bone morphogenetic protein (BMP) receptor ligands, BMP4, is widely expressed in the adult mouse lung and is upregulated in hypoxia-induced pulmonary hypertension (PH). Furthermore, heterozygous null Bmp4lacZ/+ mice are protected from the development of hypoxia-induced PH, vascular smooth muscle cell proliferation, and vascular remodeling. This is associated with a reduction in hypoxia-induced Smad1/5/8 phosphorylation and Id1 expression in the pulmonary vasculature. In addition, pulmonary microvascular endothelial cells secrete BMP4 in response to hypoxia and promote proliferation and migration of vascular smooth muscle cells in a BMP4-dependent fashion. These findings indicate that BMP4 plays a dominant role in regulating BMP signaling in the hypoxic pulmonary vasculature and suggest that endothelium-derived BMP4 plays a direct, paracrine role in promoting smooth muscle proliferation and remodeling in hypoxic PH.
Key Words: bone morphogenetic proteins endothelial cells hypoxic pulmonary hypertension signaling pathways Smad vascular remodeling vascular smooth muscle cell proliferation
Introduction
Chronic hypoxia is the most common underlying cause of secondary pulmonary hypertension (PH) in humans.1 This is associated with the development of fixed defects in the pulmonary vasculature, including medial wall thickening and muscularization of the peripheral vasculature, both mimicked by a rodent model of hypoxic PH in rats and mice.2,3 Numerous studies using these experimental models provide evidence of an imbalance in the secretion of vasoactive agents and mitogens in the pulmonary vasculature, leading to structural changes in the pulmonary vasculature.4eC6
Genetic studies in patients with familial primary pulmonary hypertension (FPPH) have identified mutations in 1 of the 3 known type II bone morphogenetic protein receptors (BMP-RII), BMPR2.7eC9 BMP-RII is a member of the transforming growth factor type family of receptors that acts downstream of the BMP family of ligands.10,11 BMP ligands interact with 2 classes of transmembrane receptors, termed type I receptors, (ALK2, 3, and 6), and the type II receptors (BMP-RII and Act-RIIA and -IIB). Ligand binding induces type IeCreceptor phosphorylation by type II receptors, leading to activation of downstream signaling including the classical Smad1/5/8 and the alternative p38 and extracellular signaleCregulated kinase, mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase C pathways.10,11 Evidence of a role for this signaling pathway in hypoxic PH was shown in 2 recent in vivo studies. By manipulating BMP-RII signaling through 2 different approaches, these studies demonstrated that BMP-RII can promote opposite effects on the pulmonary vasculature under different conditions.12,13
To explore the mechanisms mediating these effects, we looked at the regulation BMP ligands and downstream signaling in a mouse model of hypoxia-induced PH. One of these ligands, BMP4, is widely expressed in the adult mouse lung and is upregulated by hypoxia in vivo. These changes are of functional significance as heterozygous null Bmp4 mutant mice show a reduction in hypoxic pulmonary vascular smooth muscle cell (VSMC) proliferation and remodeling, leading to a significant delay in the development of PH following exposure to hypoxia. These findings suggest that BMP4 plays an important role in promoting hypoxic pulmonary vascular remodeling, suggesting a more complex role for BMP signaling in the development of PH.
Materials and Methods
Quantification and Localization of BMP-Signaling Components
To assess Bmp4 expression in the Bmp4lacZ/+ mouse, a modified -galactosidase assay on trachea-perfused lungs was performed, as described.14 Immunohistochemistry was performed using citrate antigen retrieval, biotinylated secondary antibodies with Vectastain Elite ABC amplification (Vector), and developed with diaminobenzidine (Sigma) or 3-amino-9-ethylcarbazole reagents (Zymed). Primary antibodies used for both Western blotting and immunohistochemistry included rabbit polyclonal antibodies for phospho-Smad1/5/8 (Upstate, catalogue No. 06702; Cell Signaling, catalogue No. 9511) and Id1 (Santa Cruz, catalogue No. SC488), monoclonal anti-BMP4 antibody (Santa Cruz, catalogue No. SC12721), and a -actin monoclonal antibody (Sigma, catalogue No. A2228) for normalization of all Westerns. Quantitative RT-PCR (Q-RT-PCR) analysis of lung RNA was performed using a Light Cycler (Roche) and primers crossing exons for Bmp2, 4, 5, 6 and 7 and -actin (Table I in the online data supplement available at http://circres.ahajournals.org). Values are expressed as fold-change in the ratio of Bmp2, 4, 5, 6, or 7/-actin mRNA copies.
For analysis of protein expression in endothelial cells (ECs), primary mouse pulmonary microvascular ECs (mPMVECs) were recovered from wild-type and Bmp4lacZ/+ mutant mice, as described,15 and cultured up to P4 in EC medium (EGM-2; Clonetics). Temperature-sensitive, conditionally immortalized mPMVECs were isolated in the same manner from H-2Kb-tsA58 SV40 large T Ag transgenic mice16 and were characterized by Matrigel-based capillary formation and expression of endothelial-specific markers15 (online supplemental data section No. 1 and online Figure I). Following expansion, cells were maintained at 37°C in the absence of interferon- for 5 days, before serum starving and incubating in 1% oxygen. BMP4 protein was detected by Western blot in cell lysates and conditioned media after pull-down using heparin Sepharose beads. Additionally, other proteins were detected with rabbit polyclonal antibodies to phospho-Smad1/5/8, Id1, and hypoxia-inducible factor (HIF)-1 (Novus Biologicals, catalogue No. NB100449) and normalized to -actin.
Experimental PH
The Bmp4 mutant mice (Bmp4lacZ/+), bred on an ICR background, were a gift from Brigid Hogan (Duke University, Durham, NC).17 Eight to ten-week old heterozygous and wild-type littermates were exposed to 10% normobaric oxygen for 4 days, 3, and 5 weeks. Right ventricular systolic pressure (RVSP), heart rates, RV weights, and lung-tissue collection were performed as described in online supplemental data section No. 2. Experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee.
Analysis of medial wall thickening was performed as described in online supplemental data section No. 3, using a blind-coded analysis. For peripheral muscularization, sections were stained by double immunofluorescence using mouse antieC-SM actin (Sigma, catalogue No. A5228) and rabbit antieCvon Willebrand Factor (Dako, catalogue No. A0082). Peripheral vessels were defined as those small vessels distal to the terminal, muscularized bronchioles, and classified as fully muscularized, partially muscularized, or nonmuscularized.
Analysis of Cell Proliferation and Migration
Isolation of mouse pulmonary artery smooth muscle cells (mPASMCs) was performed as described previously.18 More than 95% of these cells expressed -SM actin and -SM actin by immunofluorescence and were used between P1 to P9. mPASMC proliferation was analyzed by 3H-thymidine incorporation performed in quadruplicate. After 48-hour serum starvation, media were treated with either BMP ligands, a 1:2 dilution of 24-hour 1% oxygen, or normoxic conditionally immortalized mPMVEC-conditioned media with or without recombinant Noggin (Alpha Diagnostics) or neutralizing anti-BMP4 monoclonal antibody (R&D systems, catalogue No. MAB757), and cells were cultured for an additional 24 hours at 1% oxygen while labeling with 3H-thymidine. Cell-migration assays were performed using 8 eol/L polycarbonate Transwell filter inserts (Costar) precoated with growth-factor reduced Matrigel (BD Bioscience). mPASMCs were washed and plated in serum-free media into the upper chamber, and the lower chamber was filled with serum-free media treated with the respective BMP ligands or conditionally immortalized mPMVEC-conditioned media, as described above. Plates were placed in 1% oxygen for 5 hours, fixed, and stained overnight with 2% crystal violet. After washing in PBS, cells in the upper chamber were removed using cotton buds and remaining cells counted in 5 randomly selected fields at x200 magnification for each Transwell, repeated in triplicate.
In vivo cell proliferation was determined by double immunofluorescence using rabbit polyclonal antieCproliferating cell nuclear antigen (PCNA) (Santa Cruz, catalogue No. SC7907), and monoclonal antieC-SM actin and nuclei were counterstained with 4',6-diamidino-2-phenylindole. Proliferation index was determined by counting the proportion of PCNA and actin-positive cells in 40 small/medium-sized arterioles accompanying muscularized airways.
Statistical Analysis
For universal data analysis, the Student t test was applied for testing the unadjusted mean difference between the study groups. For multivariate data analysis, the general linear model (2-way ANOVA) with adjusted least-squares means was used for testing the adjusted mean difference between study groups. All tests of significance were 2-sided, and differences were considered statistically significant when the probability value was P<0.05. All data were expressed as means±SEM.
Results
BMP4 Is Selectively Upregulated in the Lungs of Hypoxic Mice
To evaluate the regulation of BMP ligands during hypoxic PH, we obtained lung-tissue samples from wild-type mice after 3 weeks of exposure to hypoxia. Bmp2, 4, 5, 6 and 7 mRNA levels were quantified using real-time RT-PCR. We saw a 2- to 3-fold increase in Bmp2 and Bmp4 mRNAs in the hypoxic mice compared with normoxic controls but no significant changes in mRNA levels for any of the other Bmp ligands (Figure 1A).
As BMP4 plays a vital role in lung branching and morphogenesis,19,20 we focused on the analysis of BMP4 in hypoxic PH. Bmp4 mRNA levels rose steadily at 4 days, peaked at 3 weeks, and returned to normoxic levels after 5 weeks of exposure to 10% oxygen (Figure 1B). Analysis of BMP4 protein expression confirmed that these changes in Bmp4 mRNA were associated with a significant increase in both mature secreted and preprotein forms of BMP4 in whole-lung lysates following exposure to hypoxia for 4 days (Figure 1C, lanes 1 through 12, and 1D). We used the Bmp4lacZ/+-reporter knock-in mice17 to localize Bmp4-dependent LacZ expression in the adult mouse lung. Using this mouse, we showed that LacZ is widely expressed in EC, VSMC, and epithelial compartments of the mouse lung (Figure 1E), with prominent expression in ECs seen both in muscularized and nonmuscularized vessels throughout the lung (Figure 1F and 1G).
Impaired BMP4 Signaling in Hypoxic Bmp4lacZ/+ Mutant Mice
We used heterozygous null Bmp4lacZ/+ mice to examine the functional role of BMP4 in hypoxic PH. Heterozygous null Bmp4lacZ/+ mutant mice exhibit minor renal, craniofacial, and skeletal phenotypes on different backgrounds,21 but we observed no evidence of structural defects in the lung or vasculature on the ICR background used for these studies. Q-RT-PCR in lung samples from Bmp4lacZ/+ mutant mice showed a 50% decrease in the basal expression of Bmp4 mRNA compared with wild-type mice, with no increase in Bmp4 expression following exposure to hypoxia at any of the time points tested (Figure 1B). Analysis of BMP4 protein confirmed that there was a loss of hypoxia-induced BMP4 expression in Bmp4LacZ/+ mutant mice, although basal expression was unchanged compared with wild-type mice (Figure 1C, lanes 13 through 24, and 1D).
We next examined the effects of hypoxia on the activation of two downstream components of the BMP-signaling pathway, the BMP-receptoreCactivated Smad proteins, Smad1, 5, and 8,10,11 and Id1, an inhibitory transcriptional cofactor that mediates a range of Smad-dependent responses in different cell types.22 Activation of Smad1/5/8 was detected using phospho-specific antibodies that recognize a specific C-terminal eCSSPVSP motif conserved in all 3 BMP-activated Smads but cannot be used to distinguish between phosphorylated forms of Smad1, 5, or 8. Western blots of lung lysates from wild-type mice exposed to hypoxia for 4 days demonstrated increased expression of Id1 associated with a minor, nonsignificant change in phospho-Smad1/5/8 (Figure 2A, lanes 1 through 12, and 2C and 2D), both of which were lost in the Bmp4lacZ/+ mutant mice (Figure 2A, lanes through 24, and 2B and 2C). Immunohistochemical analysis demonstrated a parallel increase in nuclear expression of phospho-Smad1/5/8 in ECs, VSMCs, and in bronchial and alveolar epithelial cells of hypoxic wild-type mice but was absent in hypoxic Bmp4lacZ/+ mutants (Figure 2D through 2G). Phospho-Smad1/5/8 staining was blocked by incubation with a specific phospho-peptide, and similar results were obtained using phospho-Smad1/5/8 antibodies from another commercial source (Cell Signaling; data not shown). Id1 was also induced in the pulmonary vasculature of hypoxic wild-type but not Bmp4lacZ/+ mutant mice (Figure 2H through 2K). Id1 was prominently expressed in the VSMCs of the hypoxic pulmonary vasculature. Specificity of this antibody was conformed in an earlier study showing absent staining in tumor vasculature from Id1-null mutant mice.23 Loss of hypoxia-induced vascular phospho-Smad1/5/8 and Id1 expression occurred despite the fact that Bmp2 mRNA was more highly upregulated in the lungs of hypoxic Bmp4lacZ/+ mutant than wild-type mice (Figure 2L).
Impaired Functional Responses to Chronic Hypoxia in Bmp4lacZ/+ Mice
To explore the functional significance of these changes in BMP4 expression, we induced PH by placing wild-type and Bmp4lacZ/+ mutant mice in hypoxic chambers for 4 days, 3 weeks, and 5 weeks. Whereas we saw very little to no change in RV pressure and hypertrophy after 4 days of hypoxia, there was a marked increase in RVSP and RV hypertrophy in wild-type mice after 3 weeks of hypoxia, both of which were significantly attenuated in Bmp4lacZ/+ mice (Figure 3A and 3B). Multivariate statistical analysis indicated that neither sex nor weight of the mice had significant confounding effects on these differences (online Table II). In addition, although there was a significant increase in hematocrit in hypoxic mice (normoxia versus hypoxia, P<0.001), there was no difference between genotypes (wild type versus Bmp4 mutant, P=0.4896). After 5 weeks of hypoxia, there was a consistent fall in RVSP in wild-type mice compared with 3 weeks. The explanation for this is unclear, but it may reflect adaptive changes associated with prolonged hypoxic exposure on this genetic background. The attenuation of hypoxia-dependent increase in RVSP at 5 weeks in the Bmp4lacZ/+ mice was less pronounced than after 3 weeks, and although the increase in RV weight was still attenuated in Bmp4lacZ/+ mutant mice, this protection was not statistically significant.
Pulmonary Vascular Remodeling
We analyzed the effects of the Bmp4 mutation on two parameters of vascular remodeling, medial wall thickening of muscular arteries, and muscularization of small, peripheral vessels.2 Whereas there was a significant increase in arterial wall thickness in wild-type mice after 3 weeks at 10% oxygen, these changes were absent in mice Bmp4LacZ/+ mutant mice (Figure 4A through 4G). In addition, there was a marked increase in the proportion of peripheral muscularized vessels in wild-type mice exposed to hypoxia, which was significantly reduced in the heterozygous Bmp4 mutants after 3 and 5 weeks of exposure to hypoxia, although the effect was less pronounced at 5 weeks (Figure 5A through 5E). Similar changes were observed by immunohistochemical analysis using horse radish peroxidaseeCconjugated antibodies to detect -SM actin expression in the lung periphery (Figure 5F through 5I).
Hypoxia-Induced VSMC Proliferation
To explore this further, we examined effects of the Bmp4 mutation on hypoxia-induced VSMC apoptosis and proliferation. As previous studies have shown that proliferative effects of hypoxia occur early in the development of PH,2,3 we examined VSMC proliferation and apoptosis after 4 days of hypoxia. Although we were unable to detect any effect of hypoxia on cellular apoptosis in wild-type or heterozygous Bmp4 mutant mice by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (data not shown), we did observe an increase in the proportion of PCNA-positive pulmonary VSMCs in hypoxic wild-type mice, which was reduced in the Bmp4lacZ/+ mutants (Figure 6A through 6J).
Hypoxia-Induced BMP4 Expression in Pulmonary Microvascular Endothelial Cells
We used conditionally immortalized mouse PMVECs to evaluate the potential origin and mechanisms regulating BMP4 expression in the hypoxic lung. When grown under hypoxic conditions, these cells secrete BMP4 into the cell-culture supernatant (Figure 7A, conditioned media). Similar results were obtained using primary wild-type mPMVECs (Figure 7B, lanes 1 through 4). This response was lost in mPMVECs derived from Bmp4lacZ/+ mutant mice (Figure 7B, lanes 5 through 8), indicating that loss of hypoxia-induced BMP4 expression in Bmp4lacZ/+ mutant mice occurs cell autonomously. In addition, analysis of BMP-dependent Smad/Id1 signaling in hypoxic conditionally immortalized mPMVECs demonstrated parallel changes in phospho-Smad1/5/8, Id1, and HIF-1 expression (Figure 7A, cell lysates).
Paracrine Signaling by BMP4 Mediates EC-VSMC Interactions
To determine whether hypoxia-induced BMP4 expression by ECs could induce paracrine effects on VSMCs, we isolated primary mPASMCs and cultured them in the presence of conditioned media from hypoxic conditionally immortalized mPMVECs. Two parameters of VSMC activation, cellular proliferation and migration, were analyzed under hypoxic conditions. In addition, to determine whether these effects were BMP dependent, experiments were performed both with and without the addition of saturating concentrations of the BMP antagonist Noggin, as previously described,23,24 or a commercially available BMP4-specific neutralizing antibody. Incubation of mPASMCs with conditioned media from conditionally immortalized mPMVECs exposed to 1% oxygen or normoxia for 24 hours demonstrated increased mPASMC proliferation and migration compared with baseline, and both responses were inhibited by addition of Noggin or the neutralizing BMP4 antibody inhibited these responses, indicating that they are BMP4 dependent (Figure 8A and 8B). Furthermore, BMP4 induced a dose-dependent increase in thymidine incorporation and migration that was only observed in early passage, cultured mPASMCs (Figure 8C through 8E). In contrast to BMP4, BMP2, which was also upregulated in the hypoxic lung, had opposite effects on cellular proliferation, but it promoted mPASMC migration (Figure 8D and 8E).
Discussion
These studies demonstrate that Bmp4-dependent LacZ expression is widely distributed in the mouse lung and that BMP4 mRNA and protein are upregulated during the early phases of hypoxic PH. This response was lost in the Bmp4 heterozygous null mutant mice, indicating that these mice provide a useful model to study the functional effects of hypoxia-induced BMP4 expression. Furthermore, whereas we showed that the related BMP family member Bmp2 is also upregulated in the hypoxic lung, decreased BMP4 expression in the Bmp4lacZ/+ mutants was associated with a dramatic decrease in phospho-Smad1/5/8 and Id1 expression in the hypoxic pulmonary vasculature. Therefore, we postulate that BMP4 plays a dominant role in regulating hypoxia-induced BMP signaling in the lung. In support of this, a reduction in hypoxic BMP4 expression in Bmp4 mutant mice attenuated pulmonary VSMC proliferation and delayed vascular remodeling associated with chronic hypoxia. This delay in pulmonary vascular responses to hypoxia correlates with the kinetics of Bmp4 expression during the progression of this disease. At stages where protection from the development of PH is greatest, we see the largest difference in Bmp4 mRNA levels between wild-type and Bmp4lacZ/+ mutant mice. Furthermore, these changes in VSMC proliferation after 4 days of hypoxia are not associated with physiological changes in RV pressure, suggesting that the dominant effect of the Bmp4lacZ/+ mutation is on vascular remodeling rather than hypoxic vasoconstriction. This indicates that BMP4 plays a critical role in regulating the early phases of hypoxic pulmonary vascular remodeling.
These findings were unexpected, as previous studies demonstrated that loss of normal BMP-RII signaling is associated with aberrant pulmonary vascular remodeling in patients with FPPH7eC9 and that interference with BMP-RII signaling in VSMCs gives rise to spontaneous PH in mice.12,13 As BMP-RII is 1 of the type II receptors responsible for mediating BMP4 signaling,10,11 we predicted that a reduction in BMP4 expression would exacerbate rather than attenuate vascular remodeling in hypoxic PH. However, these findings in hypoxic PH may bear no relation to the functional effects of BMPR2 mutations in patients with FPPH, which, by definition, excludes states of chronic hypoxia. Although this is possible, an alternative explanation is that our findings suggest that the dominant effects of BMP4 on the pulmonary vasculature are mediated through BMP-RIIeCindependent signaling pathways. Our observation that Bmp2 mRNA is upregulated in the hypoxic lung and that BMP2 can exert an opposite and growth inhibitory effect on VSMCs suggest that BMP2 could be signaling through BMP-RII and that this may be opposing the effects of BMP4 under normal physiological conditions. Recent studies using human PASMC cultures indicate that alterations in BMP-receptor stoichiometry associated with BMPR2 mutations can give rise to a switch from a growth inhibitory to a mitogenic response to BMP2 in patients with sporadic PPH.25 Additionally, there is recent in vitro evidence indicating that interference with normal type IIeCreceptor usage can lead to a net gain in signaling by certain BMP ligands through the use of other alternative type II receptors.26 These findings suggest that aberrant vascular remodeling could result from alterations in the balance in cellular responses to different BMP ligands through multiple type IIeCreceptor complexes, rather than a global loss of BMP responsiveness in patients with FPPH.
Although multiple mechanisms could be involved in regulating BMP4 expression in the hypoxic lung,4,27eC29 our in vitro studies suggest that BMP4 secretion could be directly regulated by hypoxia in ECs. Given the critical role of ECs in the regulation of vascular development and remodeling,30 it is reasonable to propose that EC BMP4 could play a role in regulating these pulmonary vascular responses in hypoxia. The mitogenic effects of EC-derived BMP4 on VSMC proliferation are consistent with this hypothesis but are in contrast with earlier studies demonstrating growth inhibitory effects of BMPs on cultured VSMCs.31eC33 However, this is the first study describing the effects of BMPs on cellular proliferation in mouse PASMC cultures, and recent evidence indicates that VSMCs derived from different compartments of the pulmonary vasculature may be growth inhibited or proliferate in response to BMP4.34 This suggests that the cellular origin of VSMCs may play a critical role in defining the type of response to BMP4 in vitro and that discrepancy with previous reports is likely to reflect differences in cell-culture conditions and passage number in our primary cultures.35,36 In addition to these effects, we demonstrate that BMP4 induces migration of cultured VSMCs. Previous studies indicate that increased migration of VSMCs plays an important role in systemic vascular remodeling following injury.37 Furthermore, upregulation of BMP2 expression in injured systemic vasculature has been shown to promote PASMC migration,38 indicating that BMP signaling may play a role in regulating VSMC migration in other vascular beds.
Our in vivo studies show that there is a marked reduction in hypoxia-induced Smad1/5/8 phosphorylation and expression of Id1 in the pulmonary vasculature of Bmp4lacZ/+ mutant mice. As Id1 is a direct target of Smad-dependent BMP signaling,22 this suggests that EC-derived BMP4 could be exerting both autocrine and paracrine effects in the hypoxic pulmonary vasculature. Furthermore, Id1 plays a critical role in promoting cellular proliferation in a variety of cell types and has been shown to mediate BMP-dependent EC migration.22,39 On this basis, we propose that BMP4-dependent activation of the Smad/Id1 axis may be involved in promoting hypoxic vascular remodeling.
Taken together, our findings indicate that hypoxia-induced BMP4 expression by ECs promotes VSMC proliferation and early remodeling events in hypoxic PH. Although further studies are needed to establish the signaling pathways mediating these events, these findings are of importance as chronic hypoxia associated with parenchymal lung disease is the most common underlying cause of PH in humans.1 Hence, selective inhibition of BMP4 may present a useful therapeutic strategy to attenuate aberrant pulmonary vascular remodeling in patients with hypoxia-induced PH.
Acknowledgments
This research was funded through the Vanderbilt University Interdisciplinary Discovery Grant program. We thank John Newman for helpful advice, Brigid Hogan for the Bmp4LacZ mutant mice, Ed Price from the MMPC facility for assistance with RV pressure monitoring, and John Penn for use of hypoxia chambers.
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Pediatrics (H.S.B.), Vanderbilt University School of Medicine, Nashville, Tenn.
Abstract
We show that 1 of the type II bone morphogenetic protein (BMP) receptor ligands, BMP4, is widely expressed in the adult mouse lung and is upregulated in hypoxia-induced pulmonary hypertension (PH). Furthermore, heterozygous null Bmp4lacZ/+ mice are protected from the development of hypoxia-induced PH, vascular smooth muscle cell proliferation, and vascular remodeling. This is associated with a reduction in hypoxia-induced Smad1/5/8 phosphorylation and Id1 expression in the pulmonary vasculature. In addition, pulmonary microvascular endothelial cells secrete BMP4 in response to hypoxia and promote proliferation and migration of vascular smooth muscle cells in a BMP4-dependent fashion. These findings indicate that BMP4 plays a dominant role in regulating BMP signaling in the hypoxic pulmonary vasculature and suggest that endothelium-derived BMP4 plays a direct, paracrine role in promoting smooth muscle proliferation and remodeling in hypoxic PH.
Key Words: bone morphogenetic proteins endothelial cells hypoxic pulmonary hypertension signaling pathways Smad vascular remodeling vascular smooth muscle cell proliferation
Introduction
Chronic hypoxia is the most common underlying cause of secondary pulmonary hypertension (PH) in humans.1 This is associated with the development of fixed defects in the pulmonary vasculature, including medial wall thickening and muscularization of the peripheral vasculature, both mimicked by a rodent model of hypoxic PH in rats and mice.2,3 Numerous studies using these experimental models provide evidence of an imbalance in the secretion of vasoactive agents and mitogens in the pulmonary vasculature, leading to structural changes in the pulmonary vasculature.4eC6
Genetic studies in patients with familial primary pulmonary hypertension (FPPH) have identified mutations in 1 of the 3 known type II bone morphogenetic protein receptors (BMP-RII), BMPR2.7eC9 BMP-RII is a member of the transforming growth factor type family of receptors that acts downstream of the BMP family of ligands.10,11 BMP ligands interact with 2 classes of transmembrane receptors, termed type I receptors, (ALK2, 3, and 6), and the type II receptors (BMP-RII and Act-RIIA and -IIB). Ligand binding induces type IeCreceptor phosphorylation by type II receptors, leading to activation of downstream signaling including the classical Smad1/5/8 and the alternative p38 and extracellular signaleCregulated kinase, mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase C pathways.10,11 Evidence of a role for this signaling pathway in hypoxic PH was shown in 2 recent in vivo studies. By manipulating BMP-RII signaling through 2 different approaches, these studies demonstrated that BMP-RII can promote opposite effects on the pulmonary vasculature under different conditions.12,13
To explore the mechanisms mediating these effects, we looked at the regulation BMP ligands and downstream signaling in a mouse model of hypoxia-induced PH. One of these ligands, BMP4, is widely expressed in the adult mouse lung and is upregulated by hypoxia in vivo. These changes are of functional significance as heterozygous null Bmp4 mutant mice show a reduction in hypoxic pulmonary vascular smooth muscle cell (VSMC) proliferation and remodeling, leading to a significant delay in the development of PH following exposure to hypoxia. These findings suggest that BMP4 plays an important role in promoting hypoxic pulmonary vascular remodeling, suggesting a more complex role for BMP signaling in the development of PH.
Materials and Methods
Quantification and Localization of BMP-Signaling Components
To assess Bmp4 expression in the Bmp4lacZ/+ mouse, a modified -galactosidase assay on trachea-perfused lungs was performed, as described.14 Immunohistochemistry was performed using citrate antigen retrieval, biotinylated secondary antibodies with Vectastain Elite ABC amplification (Vector), and developed with diaminobenzidine (Sigma) or 3-amino-9-ethylcarbazole reagents (Zymed). Primary antibodies used for both Western blotting and immunohistochemistry included rabbit polyclonal antibodies for phospho-Smad1/5/8 (Upstate, catalogue No. 06702; Cell Signaling, catalogue No. 9511) and Id1 (Santa Cruz, catalogue No. SC488), monoclonal anti-BMP4 antibody (Santa Cruz, catalogue No. SC12721), and a -actin monoclonal antibody (Sigma, catalogue No. A2228) for normalization of all Westerns. Quantitative RT-PCR (Q-RT-PCR) analysis of lung RNA was performed using a Light Cycler (Roche) and primers crossing exons for Bmp2, 4, 5, 6 and 7 and -actin (Table I in the online data supplement available at http://circres.ahajournals.org). Values are expressed as fold-change in the ratio of Bmp2, 4, 5, 6, or 7/-actin mRNA copies.
For analysis of protein expression in endothelial cells (ECs), primary mouse pulmonary microvascular ECs (mPMVECs) were recovered from wild-type and Bmp4lacZ/+ mutant mice, as described,15 and cultured up to P4 in EC medium (EGM-2; Clonetics). Temperature-sensitive, conditionally immortalized mPMVECs were isolated in the same manner from H-2Kb-tsA58 SV40 large T Ag transgenic mice16 and were characterized by Matrigel-based capillary formation and expression of endothelial-specific markers15 (online supplemental data section No. 1 and online Figure I). Following expansion, cells were maintained at 37°C in the absence of interferon- for 5 days, before serum starving and incubating in 1% oxygen. BMP4 protein was detected by Western blot in cell lysates and conditioned media after pull-down using heparin Sepharose beads. Additionally, other proteins were detected with rabbit polyclonal antibodies to phospho-Smad1/5/8, Id1, and hypoxia-inducible factor (HIF)-1 (Novus Biologicals, catalogue No. NB100449) and normalized to -actin.
Experimental PH
The Bmp4 mutant mice (Bmp4lacZ/+), bred on an ICR background, were a gift from Brigid Hogan (Duke University, Durham, NC).17 Eight to ten-week old heterozygous and wild-type littermates were exposed to 10% normobaric oxygen for 4 days, 3, and 5 weeks. Right ventricular systolic pressure (RVSP), heart rates, RV weights, and lung-tissue collection were performed as described in online supplemental data section No. 2. Experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee.
Analysis of medial wall thickening was performed as described in online supplemental data section No. 3, using a blind-coded analysis. For peripheral muscularization, sections were stained by double immunofluorescence using mouse antieC-SM actin (Sigma, catalogue No. A5228) and rabbit antieCvon Willebrand Factor (Dako, catalogue No. A0082). Peripheral vessels were defined as those small vessels distal to the terminal, muscularized bronchioles, and classified as fully muscularized, partially muscularized, or nonmuscularized.
Analysis of Cell Proliferation and Migration
Isolation of mouse pulmonary artery smooth muscle cells (mPASMCs) was performed as described previously.18 More than 95% of these cells expressed -SM actin and -SM actin by immunofluorescence and were used between P1 to P9. mPASMC proliferation was analyzed by 3H-thymidine incorporation performed in quadruplicate. After 48-hour serum starvation, media were treated with either BMP ligands, a 1:2 dilution of 24-hour 1% oxygen, or normoxic conditionally immortalized mPMVEC-conditioned media with or without recombinant Noggin (Alpha Diagnostics) or neutralizing anti-BMP4 monoclonal antibody (R&D systems, catalogue No. MAB757), and cells were cultured for an additional 24 hours at 1% oxygen while labeling with 3H-thymidine. Cell-migration assays were performed using 8 eol/L polycarbonate Transwell filter inserts (Costar) precoated with growth-factor reduced Matrigel (BD Bioscience). mPASMCs were washed and plated in serum-free media into the upper chamber, and the lower chamber was filled with serum-free media treated with the respective BMP ligands or conditionally immortalized mPMVEC-conditioned media, as described above. Plates were placed in 1% oxygen for 5 hours, fixed, and stained overnight with 2% crystal violet. After washing in PBS, cells in the upper chamber were removed using cotton buds and remaining cells counted in 5 randomly selected fields at x200 magnification for each Transwell, repeated in triplicate.
In vivo cell proliferation was determined by double immunofluorescence using rabbit polyclonal antieCproliferating cell nuclear antigen (PCNA) (Santa Cruz, catalogue No. SC7907), and monoclonal antieC-SM actin and nuclei were counterstained with 4',6-diamidino-2-phenylindole. Proliferation index was determined by counting the proportion of PCNA and actin-positive cells in 40 small/medium-sized arterioles accompanying muscularized airways.
Statistical Analysis
For universal data analysis, the Student t test was applied for testing the unadjusted mean difference between the study groups. For multivariate data analysis, the general linear model (2-way ANOVA) with adjusted least-squares means was used for testing the adjusted mean difference between study groups. All tests of significance were 2-sided, and differences were considered statistically significant when the probability value was P<0.05. All data were expressed as means±SEM.
Results
BMP4 Is Selectively Upregulated in the Lungs of Hypoxic Mice
To evaluate the regulation of BMP ligands during hypoxic PH, we obtained lung-tissue samples from wild-type mice after 3 weeks of exposure to hypoxia. Bmp2, 4, 5, 6 and 7 mRNA levels were quantified using real-time RT-PCR. We saw a 2- to 3-fold increase in Bmp2 and Bmp4 mRNAs in the hypoxic mice compared with normoxic controls but no significant changes in mRNA levels for any of the other Bmp ligands (Figure 1A).
As BMP4 plays a vital role in lung branching and morphogenesis,19,20 we focused on the analysis of BMP4 in hypoxic PH. Bmp4 mRNA levels rose steadily at 4 days, peaked at 3 weeks, and returned to normoxic levels after 5 weeks of exposure to 10% oxygen (Figure 1B). Analysis of BMP4 protein expression confirmed that these changes in Bmp4 mRNA were associated with a significant increase in both mature secreted and preprotein forms of BMP4 in whole-lung lysates following exposure to hypoxia for 4 days (Figure 1C, lanes 1 through 12, and 1D). We used the Bmp4lacZ/+-reporter knock-in mice17 to localize Bmp4-dependent LacZ expression in the adult mouse lung. Using this mouse, we showed that LacZ is widely expressed in EC, VSMC, and epithelial compartments of the mouse lung (Figure 1E), with prominent expression in ECs seen both in muscularized and nonmuscularized vessels throughout the lung (Figure 1F and 1G).
Impaired BMP4 Signaling in Hypoxic Bmp4lacZ/+ Mutant Mice
We used heterozygous null Bmp4lacZ/+ mice to examine the functional role of BMP4 in hypoxic PH. Heterozygous null Bmp4lacZ/+ mutant mice exhibit minor renal, craniofacial, and skeletal phenotypes on different backgrounds,21 but we observed no evidence of structural defects in the lung or vasculature on the ICR background used for these studies. Q-RT-PCR in lung samples from Bmp4lacZ/+ mutant mice showed a 50% decrease in the basal expression of Bmp4 mRNA compared with wild-type mice, with no increase in Bmp4 expression following exposure to hypoxia at any of the time points tested (Figure 1B). Analysis of BMP4 protein confirmed that there was a loss of hypoxia-induced BMP4 expression in Bmp4LacZ/+ mutant mice, although basal expression was unchanged compared with wild-type mice (Figure 1C, lanes 13 through 24, and 1D).
We next examined the effects of hypoxia on the activation of two downstream components of the BMP-signaling pathway, the BMP-receptoreCactivated Smad proteins, Smad1, 5, and 8,10,11 and Id1, an inhibitory transcriptional cofactor that mediates a range of Smad-dependent responses in different cell types.22 Activation of Smad1/5/8 was detected using phospho-specific antibodies that recognize a specific C-terminal eCSSPVSP motif conserved in all 3 BMP-activated Smads but cannot be used to distinguish between phosphorylated forms of Smad1, 5, or 8. Western blots of lung lysates from wild-type mice exposed to hypoxia for 4 days demonstrated increased expression of Id1 associated with a minor, nonsignificant change in phospho-Smad1/5/8 (Figure 2A, lanes 1 through 12, and 2C and 2D), both of which were lost in the Bmp4lacZ/+ mutant mice (Figure 2A, lanes through 24, and 2B and 2C). Immunohistochemical analysis demonstrated a parallel increase in nuclear expression of phospho-Smad1/5/8 in ECs, VSMCs, and in bronchial and alveolar epithelial cells of hypoxic wild-type mice but was absent in hypoxic Bmp4lacZ/+ mutants (Figure 2D through 2G). Phospho-Smad1/5/8 staining was blocked by incubation with a specific phospho-peptide, and similar results were obtained using phospho-Smad1/5/8 antibodies from another commercial source (Cell Signaling; data not shown). Id1 was also induced in the pulmonary vasculature of hypoxic wild-type but not Bmp4lacZ/+ mutant mice (Figure 2H through 2K). Id1 was prominently expressed in the VSMCs of the hypoxic pulmonary vasculature. Specificity of this antibody was conformed in an earlier study showing absent staining in tumor vasculature from Id1-null mutant mice.23 Loss of hypoxia-induced vascular phospho-Smad1/5/8 and Id1 expression occurred despite the fact that Bmp2 mRNA was more highly upregulated in the lungs of hypoxic Bmp4lacZ/+ mutant than wild-type mice (Figure 2L).
Impaired Functional Responses to Chronic Hypoxia in Bmp4lacZ/+ Mice
To explore the functional significance of these changes in BMP4 expression, we induced PH by placing wild-type and Bmp4lacZ/+ mutant mice in hypoxic chambers for 4 days, 3 weeks, and 5 weeks. Whereas we saw very little to no change in RV pressure and hypertrophy after 4 days of hypoxia, there was a marked increase in RVSP and RV hypertrophy in wild-type mice after 3 weeks of hypoxia, both of which were significantly attenuated in Bmp4lacZ/+ mice (Figure 3A and 3B). Multivariate statistical analysis indicated that neither sex nor weight of the mice had significant confounding effects on these differences (online Table II). In addition, although there was a significant increase in hematocrit in hypoxic mice (normoxia versus hypoxia, P<0.001), there was no difference between genotypes (wild type versus Bmp4 mutant, P=0.4896). After 5 weeks of hypoxia, there was a consistent fall in RVSP in wild-type mice compared with 3 weeks. The explanation for this is unclear, but it may reflect adaptive changes associated with prolonged hypoxic exposure on this genetic background. The attenuation of hypoxia-dependent increase in RVSP at 5 weeks in the Bmp4lacZ/+ mice was less pronounced than after 3 weeks, and although the increase in RV weight was still attenuated in Bmp4lacZ/+ mutant mice, this protection was not statistically significant.
Pulmonary Vascular Remodeling
We analyzed the effects of the Bmp4 mutation on two parameters of vascular remodeling, medial wall thickening of muscular arteries, and muscularization of small, peripheral vessels.2 Whereas there was a significant increase in arterial wall thickness in wild-type mice after 3 weeks at 10% oxygen, these changes were absent in mice Bmp4LacZ/+ mutant mice (Figure 4A through 4G). In addition, there was a marked increase in the proportion of peripheral muscularized vessels in wild-type mice exposed to hypoxia, which was significantly reduced in the heterozygous Bmp4 mutants after 3 and 5 weeks of exposure to hypoxia, although the effect was less pronounced at 5 weeks (Figure 5A through 5E). Similar changes were observed by immunohistochemical analysis using horse radish peroxidaseeCconjugated antibodies to detect -SM actin expression in the lung periphery (Figure 5F through 5I).
Hypoxia-Induced VSMC Proliferation
To explore this further, we examined effects of the Bmp4 mutation on hypoxia-induced VSMC apoptosis and proliferation. As previous studies have shown that proliferative effects of hypoxia occur early in the development of PH,2,3 we examined VSMC proliferation and apoptosis after 4 days of hypoxia. Although we were unable to detect any effect of hypoxia on cellular apoptosis in wild-type or heterozygous Bmp4 mutant mice by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (data not shown), we did observe an increase in the proportion of PCNA-positive pulmonary VSMCs in hypoxic wild-type mice, which was reduced in the Bmp4lacZ/+ mutants (Figure 6A through 6J).
Hypoxia-Induced BMP4 Expression in Pulmonary Microvascular Endothelial Cells
We used conditionally immortalized mouse PMVECs to evaluate the potential origin and mechanisms regulating BMP4 expression in the hypoxic lung. When grown under hypoxic conditions, these cells secrete BMP4 into the cell-culture supernatant (Figure 7A, conditioned media). Similar results were obtained using primary wild-type mPMVECs (Figure 7B, lanes 1 through 4). This response was lost in mPMVECs derived from Bmp4lacZ/+ mutant mice (Figure 7B, lanes 5 through 8), indicating that loss of hypoxia-induced BMP4 expression in Bmp4lacZ/+ mutant mice occurs cell autonomously. In addition, analysis of BMP-dependent Smad/Id1 signaling in hypoxic conditionally immortalized mPMVECs demonstrated parallel changes in phospho-Smad1/5/8, Id1, and HIF-1 expression (Figure 7A, cell lysates).
Paracrine Signaling by BMP4 Mediates EC-VSMC Interactions
To determine whether hypoxia-induced BMP4 expression by ECs could induce paracrine effects on VSMCs, we isolated primary mPASMCs and cultured them in the presence of conditioned media from hypoxic conditionally immortalized mPMVECs. Two parameters of VSMC activation, cellular proliferation and migration, were analyzed under hypoxic conditions. In addition, to determine whether these effects were BMP dependent, experiments were performed both with and without the addition of saturating concentrations of the BMP antagonist Noggin, as previously described,23,24 or a commercially available BMP4-specific neutralizing antibody. Incubation of mPASMCs with conditioned media from conditionally immortalized mPMVECs exposed to 1% oxygen or normoxia for 24 hours demonstrated increased mPASMC proliferation and migration compared with baseline, and both responses were inhibited by addition of Noggin or the neutralizing BMP4 antibody inhibited these responses, indicating that they are BMP4 dependent (Figure 8A and 8B). Furthermore, BMP4 induced a dose-dependent increase in thymidine incorporation and migration that was only observed in early passage, cultured mPASMCs (Figure 8C through 8E). In contrast to BMP4, BMP2, which was also upregulated in the hypoxic lung, had opposite effects on cellular proliferation, but it promoted mPASMC migration (Figure 8D and 8E).
Discussion
These studies demonstrate that Bmp4-dependent LacZ expression is widely distributed in the mouse lung and that BMP4 mRNA and protein are upregulated during the early phases of hypoxic PH. This response was lost in the Bmp4 heterozygous null mutant mice, indicating that these mice provide a useful model to study the functional effects of hypoxia-induced BMP4 expression. Furthermore, whereas we showed that the related BMP family member Bmp2 is also upregulated in the hypoxic lung, decreased BMP4 expression in the Bmp4lacZ/+ mutants was associated with a dramatic decrease in phospho-Smad1/5/8 and Id1 expression in the hypoxic pulmonary vasculature. Therefore, we postulate that BMP4 plays a dominant role in regulating hypoxia-induced BMP signaling in the lung. In support of this, a reduction in hypoxic BMP4 expression in Bmp4 mutant mice attenuated pulmonary VSMC proliferation and delayed vascular remodeling associated with chronic hypoxia. This delay in pulmonary vascular responses to hypoxia correlates with the kinetics of Bmp4 expression during the progression of this disease. At stages where protection from the development of PH is greatest, we see the largest difference in Bmp4 mRNA levels between wild-type and Bmp4lacZ/+ mutant mice. Furthermore, these changes in VSMC proliferation after 4 days of hypoxia are not associated with physiological changes in RV pressure, suggesting that the dominant effect of the Bmp4lacZ/+ mutation is on vascular remodeling rather than hypoxic vasoconstriction. This indicates that BMP4 plays a critical role in regulating the early phases of hypoxic pulmonary vascular remodeling.
These findings were unexpected, as previous studies demonstrated that loss of normal BMP-RII signaling is associated with aberrant pulmonary vascular remodeling in patients with FPPH7eC9 and that interference with BMP-RII signaling in VSMCs gives rise to spontaneous PH in mice.12,13 As BMP-RII is 1 of the type II receptors responsible for mediating BMP4 signaling,10,11 we predicted that a reduction in BMP4 expression would exacerbate rather than attenuate vascular remodeling in hypoxic PH. However, these findings in hypoxic PH may bear no relation to the functional effects of BMPR2 mutations in patients with FPPH, which, by definition, excludes states of chronic hypoxia. Although this is possible, an alternative explanation is that our findings suggest that the dominant effects of BMP4 on the pulmonary vasculature are mediated through BMP-RIIeCindependent signaling pathways. Our observation that Bmp2 mRNA is upregulated in the hypoxic lung and that BMP2 can exert an opposite and growth inhibitory effect on VSMCs suggest that BMP2 could be signaling through BMP-RII and that this may be opposing the effects of BMP4 under normal physiological conditions. Recent studies using human PASMC cultures indicate that alterations in BMP-receptor stoichiometry associated with BMPR2 mutations can give rise to a switch from a growth inhibitory to a mitogenic response to BMP2 in patients with sporadic PPH.25 Additionally, there is recent in vitro evidence indicating that interference with normal type IIeCreceptor usage can lead to a net gain in signaling by certain BMP ligands through the use of other alternative type II receptors.26 These findings suggest that aberrant vascular remodeling could result from alterations in the balance in cellular responses to different BMP ligands through multiple type IIeCreceptor complexes, rather than a global loss of BMP responsiveness in patients with FPPH.
Although multiple mechanisms could be involved in regulating BMP4 expression in the hypoxic lung,4,27eC29 our in vitro studies suggest that BMP4 secretion could be directly regulated by hypoxia in ECs. Given the critical role of ECs in the regulation of vascular development and remodeling,30 it is reasonable to propose that EC BMP4 could play a role in regulating these pulmonary vascular responses in hypoxia. The mitogenic effects of EC-derived BMP4 on VSMC proliferation are consistent with this hypothesis but are in contrast with earlier studies demonstrating growth inhibitory effects of BMPs on cultured VSMCs.31eC33 However, this is the first study describing the effects of BMPs on cellular proliferation in mouse PASMC cultures, and recent evidence indicates that VSMCs derived from different compartments of the pulmonary vasculature may be growth inhibited or proliferate in response to BMP4.34 This suggests that the cellular origin of VSMCs may play a critical role in defining the type of response to BMP4 in vitro and that discrepancy with previous reports is likely to reflect differences in cell-culture conditions and passage number in our primary cultures.35,36 In addition to these effects, we demonstrate that BMP4 induces migration of cultured VSMCs. Previous studies indicate that increased migration of VSMCs plays an important role in systemic vascular remodeling following injury.37 Furthermore, upregulation of BMP2 expression in injured systemic vasculature has been shown to promote PASMC migration,38 indicating that BMP signaling may play a role in regulating VSMC migration in other vascular beds.
Our in vivo studies show that there is a marked reduction in hypoxia-induced Smad1/5/8 phosphorylation and expression of Id1 in the pulmonary vasculature of Bmp4lacZ/+ mutant mice. As Id1 is a direct target of Smad-dependent BMP signaling,22 this suggests that EC-derived BMP4 could be exerting both autocrine and paracrine effects in the hypoxic pulmonary vasculature. Furthermore, Id1 plays a critical role in promoting cellular proliferation in a variety of cell types and has been shown to mediate BMP-dependent EC migration.22,39 On this basis, we propose that BMP4-dependent activation of the Smad/Id1 axis may be involved in promoting hypoxic vascular remodeling.
Taken together, our findings indicate that hypoxia-induced BMP4 expression by ECs promotes VSMC proliferation and early remodeling events in hypoxic PH. Although further studies are needed to establish the signaling pathways mediating these events, these findings are of importance as chronic hypoxia associated with parenchymal lung disease is the most common underlying cause of PH in humans.1 Hence, selective inhibition of BMP4 may present a useful therapeutic strategy to attenuate aberrant pulmonary vascular remodeling in patients with hypoxia-induced PH.
Acknowledgments
This research was funded through the Vanderbilt University Interdisciplinary Discovery Grant program. We thank John Newman for helpful advice, Brigid Hogan for the Bmp4LacZ mutant mice, Ed Price from the MMPC facility for assistance with RV pressure monitoring, and John Penn for use of hypoxia chambers.
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