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Am J Physiol Lung Cell Mol Physiol 294: L98-L109, 2008. First published November 16, 2007; doi:10.1152/ajplung.00034.2007
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Increased susceptibility to hypoxic pulmonary hypertension in Bmpr2 mutant mice is associated with endothelial dysfunction in the pulmonary vasculature

David B. Frank,1 Jonathan Lowery,1 Lynda Anderson,2 Monique Brink,2 Jeff Reese,3 and Mark de Caestecker1,2

Departments of 1Cell and Developmental Biology, 2Medicine, and 3Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee

Submitted 22 January 2007 ; accepted in final form 12 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Patients with familial pulmonary arterial hypertension inherit heterozygous mutations of the type 2 bone morphogenetic protein (BMP) receptor BMPR2. To explore the cellular mechanisms of this disease, we evaluated the pulmonary vascular responses to chronic hypoxia in mice carrying heterozygous hypomorphic Bmpr2 mutations (Bmpr2{Delta}Ex2/+). These mice develop more severe pulmonary hypertension after prolonged exposure to hypoxia without an associated increase in pulmonary vascular remodeling or proliferation compared with wild-type mice. This is associated with defective endothelial-dependent vasodilatation and enhanced vasoconstriction in isolated intrapulmonary artery preparations. In addition, there is a selective decrease in hypoxia-induced, BMP-dependent, endothelial nitric oxide synthase expression and Smad signaling in the intact lungs and in cultured pulmonary microvascular endothelial cells from Bmpr2{Delta}Ex2/+ mutant mice. These findings indicate that the pulmonary endothelium is a target of abnormal BMP signaling in Bmpr2{Delta}Ex2/+ mutant mice and suggest that endothelial dysfunction contributes to their increased susceptibility to hypoxic pulmonary hypertension.

bone morphogenetic protein receptors; endothelial dysfunction; pulmonary hypertension; endothelial nitric oxide synthase

GENETIC STUDIES HAVE SHOWN that patients with familial pulmonary arterial hypertension (FPAH) inherit heterozygous mutations in the type 2 bone morphogenetic protein (BMP) receptor gene BMPR2 (8, 17). The majority of BMPR2 mutations encode premature termination codons or amino acid substitutions in critical functional domains of the mature protein, suggesting that these give rise to receptors with complete or partial loss of function (20). Although this implies that decreased function of a single BMPR2 allele predisposes these individuals to clinical disease, the underlying mechanisms and cellular effectors of this abnormal response remain to be established.

BMPR2 is a member of the transforming growth factor-β (TGF-β) family of receptors (6), which act downstream of a large family of ligands, which includes BMP2, BMP4, and BMP7 (all of which are expressed in the lung; Refs. 13, 24). These ligands interact with two classes of transmembrane receptors, termed type 1 receptors, which include ALK2, 3, and 6, and type 2 receptors, which include BMPR2, ActR2A, and ActR2B. Activation of these receptors leads to phosphorylation of a subset of intracellular signaling proteins known as the receptor-activated Smads, resulting in their nuclear translocation and transactivation of target genes. Of the receptor-activated Smad proteins, Smad1, Smad5, and Smad8 are preferentially phosphorylated by BMP receptor complexes. In addition, there is evidence that these receptors regulate a variety of Smad-independent pathways, including the p38 and ERK MAPK signaling pathways. These act in a combinatorial manner with Smads to specify distinct cellular responses under different conditions (6). BMPR2 is expressed in endothelial cells and, to a lesser extent, in vascular smooth muscle and adventitial cells within the intact pulmonary vasculature (1, 23, 24, 29). Limited evidence also indicates that there may be a loss of BMP-activated Smad phosphorylation in smooth muscle and endothelial cells within the vasculature of a patient with FPAH (36). However, it is unclear whether this is a consistent finding, as these in vivo studies were based on the analysis of a single FPAH patient.

Functional studies using isolated pulmonary vascular endothelial vs. smooth muscle cells have also failed to provide definitive information regarding the cellular targets and functional responses that are defective in the pulmonary vasculature of patients with FPAH. BMP ligands have antiproliferative and proapototic effects on human vascular smooth muscle cells derived from the main pulmonary artery (22, 30, 36, 38), and these responses are abrogated (22, 36, 38), or even reversed (30), in cells derived from patients with sporadic and familial forms of pulmonary arterial hypertension (with and without identified BMPR2 mutations). However, BMPs have also been shown to promote proliferation in vascular smooth muscle cells derived from more distal pulmonary arteries (36) and inhibit apoptosis in pulmonary artery endothelial cells (31). On this basis it is unlikely that simple loss of BMP-dependent smooth muscle and/or endothelial cell growth suppression accounts for the enhanced pulmonary vascular cells proliferation and remodeling seen in patients with FPAH.

An alternative approach to address this question has been to study the pulmonary vasculature of mice with germ line mutations at the Bmpr2 locus. Studies in mice carrying heterozygous null Bmpr2 mutations have shown that there is a mild basal increase in pulmonary vascular resistance (2) and increased susceptibility to pulmonary hypertension (PH) in response to inflammatory stress or a combination of chronic hypoxia and serotonin treatment (19, 26). The latter study (19) also demonstrated an associated increase in serotonin-induced vasoconstriction in isolated pulmonary arteries from these mice. These studies suggest that the dominant effect of decreased BMPR2 signaling in Bmpr2{Delta}Ex2/+ mutant mice is to enhance vascular reactivity rather than to promote increased pulmonary vascular remodeling. However, it is unclear whether alterations in pulmonary vasoreactivity result from defects in vascular smooth muscle vs. endothelial cell function in the intact vasculature.

We have addressed these questions by evaluating BMP-signaling defects and endothelial cell function in the pulmonary vasculature of mice carrying partially inactivating heterozygous mutations at the Bmpr2 locus. We show that these mice develop more severe PH after prolonged exposure to hypoxia and that this is associated with defective endothelial-dependent vasodilatation along with a selective decrease in BMP-dependent Smad signaling and endothelial nitric oxide synthase (eNOS) expression in the endothelial compartment of the pulmonary vasculature. These findings indicate that endothelial cells are a target of defective BMP signaling in mice carrying heterozygous inactivating germ line mutations at the Bmpr2 locus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies and reagents. Affinity purified rabbit anti-BMPR-II antibodies were generated by immunizing rabbits with the keyhole limpet hemocyanin conjugated peptide DTTPLSPPHSFNRDE (DTT). Western blots were performed using rabbit anti-phosphoS463/pS465 Smad1 (Cell Signaling, also cross-reacts with pSmad5 and 8 and is referred to as anti-pSmad1/5/8 throughout the text), rabbit anti-phospho-p38 MAPK (Cell Signaling), mouse monoclonal anti-phospho-ERK1/2 MAPK (Clone E4, Santa Cruz), rabbit anti-eNOS (Santa Cruz), rabbit anti-phospho-eNOS (Ser1177; Cell Signaling), rabbit anti-inducible NOS (iNOS; BD Transduction Labs), rabbit anti-soluble guanylate cyclase β1 subunit (Cayman Chemical), and mouse anti-β-actin antibodies (Clone AC74, Sigma). Immunohistochemical studies were performed using rabbit anti-BMPR-II (DTT), anti-von Willebrand factor (vWF; Dako), anti-PCNA (Santa Cruz), anti-pSmad1/5/8 (Cell Signaling), anti-eNOS antibodies (Santa Cruz), mouse anti-{alpha}-SM actin (1A4, Sigma), and goat anti-BMPR-II antibodies (Santa Cruz).

Immunohistochemistry. Immunohistochemistry was performed using citrate antigen retrieval and either horseradish peroxidase conjugated anti-rabbit secondary antibodies (with anti-BMPR-II DTT primary) or biotinylated secondary antibodies with Vectastain Elite ABC amplification (Vector) for all of the other primaries. Indirect immunofluorescence was performed using the respective mouse or rabbit FITC or rhodamine conjugated secondary antibodies (Vector). Peptide blocking studies were performed by incubating the primary antibody (anti-BMPR-II DTT) with ~100-fold molar excess of the immunizing peptide DTT in blocking solution for 1 h before incubation with the tissue section. Sections were heated in citrate antigen retrieval solution (Biogenex) before incubation with primary antibodies. vWF immunostaining was performed after antigen retrieval using trypsin (Zymed).

Mouse line. Mice carrying a heterozygous partially inactivating (hypomorphic) mutation at the Bmpr2 locus (Bmpr2{Delta}Ex2/+) bred on a Balb/cJx129SvJ background were a gift from K. Lyons (7). Genotyping was performed by PCR on tail DNA preparations. Primers and conditions have been established in the Lyons' laboratory and include use of the following primers to detect wild-type (1-kb product) and Bmpr2{Delta}Ex2/ mutant alleles (700-bp product): 1) common forward primer: CCA TGC TCT TTT GAA GAT GG; 2) wild-type reverse primer: GTC CCC TTT TGA TTT CTC CCA; and 3) mutant reverse primer: GGC CGC TTT TCT GGA TTC ATC (detailed conditions for the PCR genotyping provided via personal communication, L. Dornbach).

Experimental PH. Eight- to ten-week-old heterozygous mutant and wild-type littermates were placed into hypoxic chambers maintained in normobaric hypoxia (10% O2 and 90% N2) for the indicated time periods (4 days, 3 wk, and 5 wk). Controls were maintained in room air under the same conditions. Closed chest measurements of right ventricular (RV) systolic pressure were obtained in lightly anaesthetized (ketamine/xylazine) mice by inserting a transdiaphragmatically pressure gauge needle into the right ventricle via an upper abdominal incision. RV pressure tracings were obtained over ~15- to 30-s periods, and results were only documented when pulse rates exceed 300 beats/min. The chest cavity was opened, and blood samples were collected by cardiac puncture for measurement of hematocrit. The left lung was clamped, excised, and snap frozen in liquid nitrogen for subsequent RNA and protein analysis; then the trachea was cannulated; and the right lung was inflated with 10% formalin at 23 cm of water. The heart was then excised, and the free RV wall was removed from the left ventricle and septum. After heart sections were dried overnight at 55°C, RV mass was expressed as the ratio of the RV dry weight divided by the left ventricle plus the septum. All experiments were approved by the Institutional Animal Care and Use Committee at Vanderbilt University. Pulmonary vascular remodeling and proliferation indices were evaluated using a blind-coded analysis, as described previously (13). Dual-color immunofluorescence using rabbit anti-vWF and mouse {alpha}-SM actin antibodies was used to determine the proportion of peripherally muscularized vessels. Rabbit anti-PCNA and mouse anti-{alpha}SM actin antibodies were used to identify PCNA-positive proliferating cells, as described previously (13). Proliferating endothelial and vascular smooth muscle cells were identified as internal to and within the {alpha}-SM actin domain, respectively. As identification of PCNA-positive vs. -negative cells can be subjective, samples were always evaluated by an observer (D. Frank) blind to the genotype and treatment group.

Intrapulmonary arterial pressure myography. Intact intrapulmonary arterial segments were carefully trimmed free from surrounding tissue of the left lung by tracing the main pulmonary artery from the hilum of whole lung blocks under a dissecting microscope (SMZ 1500, Nikon; see Supplemental Fig. 1; the online version of this article contains supplemental data). Vessel isolation was performed under ice-cold modified Krebs buffer: 118 mM NaCl, 25 mM NaHCO3, 4.7 mM KCl, 0.9 mM MgSO4-7(H2O), 2.5 mM CaCl2-2(H2O), 1 mM KH2PO4, and 11.1 mM glucose equilibrated with 5% CO2 balanced to room air to maintain a constant pH of 7.35. Isolated vascular segments were transferred to a custom arteriography chamber (University of Vermont, Instrument and Model Facility, Burlington, VT) with two micrometer-controlled positioning arms holding glass micropipettes in a 5-ml perfusion bath. All studies were performed using invariant, second branch, second and third generation pulmonary arteries with resting external diameters of 75–150 µM (see Supplemental Fig. 1). These were mounted on glass micropipettes at proximal and distal ends and secured using single filaments of 10–0 braided nylon suture. Side branches from the mounted vessel were individually tied off. The arteriography chamber was transferred to the stage of an inverted microscope (Motic AE-21, Richmond, British Columbia, CA) equipped with a video camera and computer-based monitoring system. The vessel image was captured and processed for continuous measurement of the lumen diameter using video dimension analysis software (IonOptix, Milton, MA). The perfusion chamber was initially connected to a 1-liter nonrecirculating reservoir bubbled with the same gas mixture for superfusion of cannulated, pressurized vessels. A second 100-ml recirculating reservoir was used for all drug studies. Krebs buffer prewarmed to 37°C was circulated through the vessel bath at a rate of 6 ml/min. An elevated column of Krebs buffer maintained vessel pressure. During a 30- to 40-min period of equilibration, the vessel was pressurized in 5-mmHg increments until a distending pressure of 15 mmHg was achieved. Inflation pressure was continuously recorded and adjusted by changing column height. The artery was discarded if the pressures were not maintained or there was other evidence of vessel leak. After a 10- to 15-min equilibration period with stable diameter at working pressure, vessel constriction was induced by exposure to Krebs buffer containing 50 mM KCl (repeated 2 times). Preparations that failed to constrict to KCl were excluded from further study. After the vessels were mounted and pressurized to 15 mmHg to optimize vasoconstrictor responses, final distended vessel external diameters ranged from 250–350 µM. Preliminary studies confirmed the expected vasoconstrictor responses to KCl and the thromboxane A2 analog U46619 [GenBank] and endothelial-dependent vasodilator responses to ACh in normal intrapulmonary arteries (IPAs; see Supplemental Fig. 2). Subsequent studies were performed using norepinephrine (NE) as a vasoconstrictor agent, as this produced more consistent and reversible vasoconstriction than U46619. [GenBank] Studies could be performed up to 24 h after removal of the lung, but vascular reactivity decreased markedly if isolated vessels were left unperfused for >2 h. All studies were therefore started within 90 min of vessel isolation. After washout and another 30-min equilibration period at baseline diameter, vessel responses were determined to various reagents (all obtained from Sigma) added to the recirculating water bath. These included 1) vasoconstriction: KCl and NE; 2) endothelial-dependent vasodilatation after submaximal constriction with NE using ACh and the receptor-independent endothelial activator A23187 [GenBank] ; and 3) endothelial-independent vasodilatation evaluated using sodium nitroprusside (SNP). Using this approach, we were able to generate vasoconstriction dose-response curves using KCL and NE (and the thromboxane A2 mimetic U46619 [GenBank] ). However, as previously reported in myography studies using small (150 µM) mouse coronary and mesenteric artery preparations (4, 25), we were unable to generate complete vasodilator dose response curves as the IPAs did not maintain stable tone for prolonged periods. By using single doses of ACh (1 µM), A23187 [GenBank] (1 µM), or SNP (10 µM), we were able to clearly distinguish ACh, A23187 [GenBank] , and SNP-induced vasodilatation from spontaneous decreases in constrictor tone (see Supplemental Fig. 2).

Endothelial cell culture and signaling studies. Primary mouse pulmonary microvascular endothelial cells (PMECs) were recovered from wild-type and heterozygous null Bmpr2{Delta}Ex2/+ mutant mice, as described previously (13). These were cultured up to passage 4 in endothelial cell medium (EGM-2, Clonetics). Temperature sensitive, conditionally immortalized PMECs (cPMECs) were isolated in the same manner from the H-2Kb-tsA58 SV40 large T Ag transgenic mice (Immortomouse; Ref. 34), provided to us by Ambra Pozzi in the Nephrology Division at Vanderbilt University. After expansion, cells were maintained at 37°C in the absence of {gamma}IFN for at least 3 days, before serum starving in DMEM with 0.1% BSA overnight before treatment with BMP ligands or placement in incubators controlling oxygen levels at 1% (PROOX 110, Biospherix) for 24 h, as indicated. BMP2, 4, and 7 were used at concentrations of 10 ng/ml for 24 h. Noggin was added to the media at 200 ng/ml for 24 h. Cells were then lysed, protein content was estimated by Bradford assay, and protein expression was determined by Western blot.

Statistical analysis. Statistical analysis was performed by two-tailed t-test for pair-wise comparisons or one-way ANOVA for comparison between multiple groups using Bonferroni correction for post-hoc, pair-wise between group comparisons for sample sizes of ≥5 per group. Smaller sample sizes (≤4 per group) were compared using the nonparametric Kruskal-Wallis ANOVA test with Siegel (Bonferroni) correction for post-hoc, pair-wise contrasts. The minimal level of significance was set at P < 0.05. Statistical analyses were performed using Analyse-It (General, version 1.73) for Microsoft Excel.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Increased hypoxic PH without remodeling in Bmpr2{Delta}Ex2/+ mutant mice. We evaluated the susceptibility of mice carrying heterozygous mutations at the Bmpr2 locus (Bmpr2{Delta}Ex2/+) to hypoxia-induced PH. This mutation is distinct from the Bmpr2 null mutation described in previous PH papers (2, 19, 26), as it gives rise to a hypomorphic mutant allele (7). Mean RV systolic pressure measurements and RV weights in Bmpr2{Delta}Ex2/+ mutant and wild-type littermates mice were indistinguishable at baseline and after 3 wk of hypoxia. However, both measurements were significantly increased in Bmpr2{Delta}Ex2/+ mutant mice after 5 wk at 10% oxygen (Fig. 1, A and B). There was a significant increase in hematocrit after 3- and 5-wk exposure to hypoxia in both groups of mice but no significant difference between wild-type and Bmpr2{Delta}Ex2/+ mutant mice (Fig. 1C). To determine whether increased PH is associated with enhanced pulmonary vascular remodeling, we evaluated the degree of muscularization of peripheral vessels. There was a significant increase in peripheral muscularization in wild-type and Bmpr2{Delta}Ex2/+ mutant mice after 5 wk of hypoxia but no significant difference between genotypes (Fig. 2A). As previous studies (13) have shown that proliferative effects of hypoxia occur early in the course of hypoxic PH, we examined endothelial and vascular smooth muscle cell proliferation after 4 days of hypoxia. There was a significant increase in proliferation of both cell types within the pulmonary vasculature of wild-type and Bmpr2{Delta}Ex2/+ mutant mice after exposure to hypoxia, but no significant differences in these parameters were observed between genotypes (Fig. 2, B and C).


Figure 1
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  		Fig. 1. Hemodynamic and hematocrit measurements in wild-type (WT) and Bmpr2{Delta}Ex2/+ mutant mice exposed to hypoxia. A: closed chest right ventricular systolic pressure (RVSP). B: RV hypertrophy (RV/LV+S ratios). C: hematocrit values in normoxic mice and mice exposed to 10% oxygen for 3 and 5 wk. Data are means ± SE [wt: normoxia (n = 10), 3-wk hypoxia (n = 11), and 5-wk hypoxia (n = 10); Bmpr2{Delta}Ex2/+ mice: normoxia (n = 13), 3-wk hypoxia (n = 11), and 5-wk hypoxia (n = 11)]. One-way ANOVA with Bonferroni correction for between group comparisons: *P < 0.05 vs. WT normoxia; **P < 0.05 vs. WT hypoxia (at the same time point).

 

Figure 2
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  		Fig. 2. Bmpr2{Delta}Ex2/+ does not affect hypoxic pulmonary vascular remodeling. A: muscularization of peripheral vessels. Mice were exposed to 5-wk hypoxia, and lungs were stained by 2-color immunofluorescence with anti-von Willebrand factor (endothelial cells) and {alpha}-SM actin (vascular smooth muscle cell) antibodies. Vessels classified as nonmuscular (N), partially muscular (P), and fully muscularized (M). Values are means ± SE [WT mice: normoxia (n = 5) and 5-wk hypoxia (n = 5); Bmpr2{Delta}Ex2/+: normoxia (n = 5) and 5-wk hypoxia (n = 6)]. One-way ANOVA with Bonferroni correction for between group comparisons: *P < 0.05 vs. WT normoxia. B and C: hypoxia-induced endothelial cell (EC; B) and vascular smooth muscle cell (VSMC; C) proliferation in the pulmonary vasculature of WT and Bmpr2{Delta}Ex2/+ mice. Mice were exposed to 10% oxygen for 4 days, and lungs were stained using anti-PCNA (proliferating cells) and {alpha}-SM actin antibodies. Proliferating ECs and VSMCs were identified as internal to and within the {alpha}-SM actin domains, respectively, and proliferation index was defined as the proportion of each cells type staining with PCNA antibody [WT mice: normoxia (n = 4) and 4- day hypoxia (n = 6); Bmpr2{Delta}Ex2/+ mice: normoxia (n = 3) and 4-day hypoxia (n = 6)]. Kruskal-Wallis ANOVA: *P < 0.05 vs. WT normoxia.

 
Loss of hypoxia-induced Smad signaling in Bmpr2{Delta}E2/+ mutant pulmonary endothelium. To evaluate defects in BMP signaling that might be mediating these effects, we determined the expression of the activated, phosphorylated forms of Smad1/5/8, p38 MAPK, and ERK MAPK in whole lung lysates of mice exposed to hypoxia. Expression of phospho-Smad1/5/8 was significantly increased in wild-type mice exposed to hypoxia for 4 days (as previously reported in Ref. 13), while phospho-p38 MAPK expression was decreased (Fig. 3). Both responses were reduced in hypoxic Bmpr2{Delta}Ex2/+ mutant mouse lungs. There were no significant changes in phospho-ERK1/2 expression after 4 days of hypoxia in either wild-type or Bmpr2{Delta}Ex2/+ mutant mouse lungs. We also evaluated expression in whole lung lysates from mice exposed to hypoxia for 5 wk (Fig. 4). Phospho-Smad1/5/8 was increased with 5 wk of hypoxia in wild-type mice. Like the 4-day time point, this response was absent in Bmpr2{Delta}Ex2/+ mutant mouse lungs, although changes failed to reach statistical significance. In contrast to the 4-day time point, however, there was no change in phospho-p38 MAPK expression with prolonged hypoxia in either wild-type or Bmpr2{Delta}Ex2/+ mutant mouse lungs.


Figure 3
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  		Fig. 3. Regulation of BMP-activated Smad1/5/8 and MAPK phosphorylation in Bmpr2{Delta}Ex2/+ mutant mouse lungs after 4-day hypoxia. A: Western blot demonstrating phospho-Smad1/5/8, p38 MAPK, and ERK MAPK expression in lung lysates from WT and Bmpr2{Delta}Ex2/+ mutant mice after 4-day hypoxia. The phospho-ERK Western blot illustrates similar changes in expression of upper p44 ERK1 and lower p42 ERK2 bands. B: quantification of the Western blot band densities corrected for β-actin loading. Values are means ± SE of phospho-S1/5/8 and phospho-p42 ERK2 MAPK/β-actin band density ratios. Kruskal-Wallis ANOVA: *P < 0.05 vs. normoxic control.

 

Figure 4
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  		Fig. 4. Regulation of BMP-activated Smad1/5/8 and p38 MAPK phosphorylation in Bmpr2{Delta}Ex2/+ mutant mouse lungs after 5-wk hypoxia. A: Western blot demonstrating phospho-Smad1/5/8 and p38 MAPK expression in lung lysates from WT and Bmpr2{Delta}Ex2/+ mutant mice after 5-wk hypoxia. B: quantification of the Western blot band densities corrected for β-actin loading. Values are means ± SE of the indicated protein/β-actin band density ratios. Kruskal-Wallis ANOVA: P < 0.05, no significant differences between groups.

 
BMPR2 is dominantly expressed in pulmonary endothelium. To identify the cellular targets of abnormal BMP signaling in hypoxic Bmpr2{Delta}Ex2/+ mutant mouse lungs, we first evaluated the cellular localization of BMPR2 in the pulmonary vasculature of wild-type mice. To this end, we generated and characterized an affinity purified rabbit polyclonal antibody raised against the peptide DTT. This corresponds to a conserved sequence within the juxtamembrane extracellular region of the human and mouse BMPR2 proteins that is not found in any of the other TGF-β family receptors. Immunohistochemical analysis of normal mouse lung demonstrated prominent staining of airway epithelia and endothelium (Fig. 5, A and C). A similar staining pattern was seen using a commercially available goat anti-BMPR2 antibody previously used to characterize BMPR2 expression in rat lungs (23, 29), although there was increased background staining using this antibody (Fig. 5B). There was weak, scattered staining of vascular smooth muscle cells, but this was never as prominent as that seen in the endothelium. Specificity of the DTT antibody staining was confirmed by preincubation with immunizing peptide (Fig. 5D). These findings suggest that pulmonary endothelial cells are likely to be particularly susceptible to changes in BMPR2 expression in Bmpr2{Delta}Ex2/+ mutant mice.


Figure 5
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  		Fig. 5. BMPR2 and phospho-Smad1/5/8 expression in pulmonary endothelium. A–D: BMPR2 is expressed in endothelium and bronchial epithelium. Lung tissue sections from WT mice stained using DTTPLSPPHSFNRDE (DTT) anti-BMPR2 antibody (A, C, and D) and a commercially available goat polyclonal BMPR2 antibody (B). D: specific staining is blocked by preincubation of anti-BMPR2 DTT primary antibody with the immunizing peptide. E–H: selective loss of endothelial phospho-Smad1/5/8 expression in hypoxic Bmpr2{Delta}Ex2/+ mutant mouse lungs. Immunohistochemical staining for phospho-Smad1/5/8 in WT (E and F) and Bmpr2{Delta}Ex2/+ mouse lungs (G and H) exposed to normoxia (E and G) or 4-day hypoxia (F and H). Vessels (*) and airways ({dagger}) are shown (x400 magnification).

 
To explore this further, we evaluated phospho-Smad1/5/8 localization in the pulmonary vasculature of wild-type and Bmpr2{Delta}Ex2/+ mutant mice by immunohistochemistry after 4 days exposure to hypoxia. As previously reported (13), most of the hypoxia-induced Smad1/5/8 phosphorylation was detected within the endothelium in wild-type mice. This was markedly reduced in hypoxic Bmpr2{Delta}E2/+ mutant lungs (Fig. 5, E–H). In contrast, patchy expression of phospho-Smad1/5/8 in pulmonary vascular smooth muscle and adventitial cells and high levels of expression in bronchial epithelium were seen under normoxic and hypoxic conditions in both wild-type and Bmpr2{Delta}E2/+ mutant lungs.

Decreased pulmonary endothelial eNOS expression in Bmpr2{Delta}E2/+ mutant mice. To determine the effect of this defect in endothelial BMP signaling on endothelial cell function, we evaluated the regulation of eNOS in Bmpr2{Delta}Ex2/+ mutant mouse lungs. Pulmonary eNOS was upregulated by hypoxia in wild-type mice, but this response was abrogated in Bmpr2{Delta}Ex2/+ mutants (Fig. 6, A and B). This was associated with a corresponding increase in iNOS expression in hypoxic Bmpr2{Delta}Ex2/+ mutant lungs. We were unable to detect changes in neuronal NOS expression between groups (data not shown). Immunohistochemical staining confirmed that hypoxia-induced eNOS expression is restricted to the endothelium in wild-type mice and decreased in Bmpr2{Delta}Ex2/+ mutant lungs (Fig. 6, C–F).


Figure 6
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  		Fig. 6. Decreased endothelial nitric oxide synthase (eNOS) and increased inducible NOS (iNOS) expression in hypoxic Bmpr2{Delta}E2/+ mouse lungs. A: Western blot showing eNOS and iNOS expression in lung lysates from WT and Bmpr2{Delta}E2/+ mutant mice exposed to 5-wk hypoxia. B: quantification of Western blot for eNOS and iNOS band densities corrected for β-actin loading. Values are means ± SE of eNOS/β-actin band density ratios. Kruskal-Wallis ANOVA: *P < 0.05 vs. WT normoxia. C–F: immunohistochemical staining for eNOS in WT (C and D) and Bmpr2{Delta}E2/+ mouse lungs (E and F) exposed to normoxia (C and E) or hypoxia for 5 wk (D and I). Vessels (*) and airways ({dagger}) are shown (x400 magnification).

 
Attenuation of hypoxia-induced eNOS expression and BMP signaling in primary pulmonary microvascular endothelial cells from Bmpr2{Delta}Ex2/+ mutant mice. To further evaluate the regulation of eNOS expression by BMPs in pulmonary endothelium, we used cPMECs. Unlike primary PMECs, which are difficult to isolate and can only be maintained for two to three passages in culture, these cells can be expanded over numerous passages and have properties of differentiated endothelial cells when maintained under nonpermissive conditions (13). This enables us to perform replicate analyses with relative ease. Previous studies from our laboratory have shown that hypoxia induces BMP4 secretion and autocrine BMP-signaling in both primary and conditionally immortalized PMECs. This can be inhibited by incubating cells with the pan-specific BMP inhibitor Noggin (13). Exposure of these cells to 1% oxygen for 24 h increased eNOS and phospho-Smad1/5/8 expression (Fig. 7, A and B). Both responses were inhibited by treatment with Noggin. These changes did not reach statistical significance but demonstrate parallel changes in eNOS and phospho-Smad1/5/8 expression, suggesting both are regulated by the same BMP-dependent pathways. Based on these observations, we went onto evaluate BMP responses directly in cPMECs. Treatment with exogenous BMP2 and 4 (but not BMP7) significantly upregulated eNOS, phospho-Smad1/5/8, and phospho-p38 MAPK expression (Fig. 7, C and D), indicating that all three responses are regulated by BMP2/4 signaling in cPMECs. To determine whether these responses are defective in pulmonary endothelial cells from Bmpr2{Delta}Ex2/+ mutant mice, we performed more limited studies using primary PMEC cultures from wild-type and Bmpr2{Delta}Ex2/+ mutant mice. Consistent with our in vivo observations, exposure of wild-type PMECs to hypoxia for 24 h induced a selective increase in eNOS and phospho-Smad1/5/8 expression, but this response was reduced in PMECs isolated from Bmpr2{Delta}Ex2/+ mutant mice (Fig. 8). Taken together these findings indicate that eNOS expression is regulated by hypoxia-induced BMP signaling in pulmonary endothelial cells and that this response is defective in pulmonary endothelial cells from Bmpr2{Delta}Ex2/+ mutant mice.


Figure 7
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  		Fig. 7. eNOS expression is regulated by hypoxia-dependent BMP signaling in pulmonary microvascular endothelial cells. A and B: hypoxia induces eNOS expression and Smad1/5/8 phosphorylation in conditionally immortalized PMECs (cPMECs) derived from H-2Kb-tsA58 SV40 large T Ag transgenic mice (wild type with respect to Bmpr2). cPMECs were cultured under normoxia or 1% oxygen for 24 h and treated with or without 200 ng/ml Noggin to inhibit autocrine BMP signaling, as indicated. A: eNOS, phospho-Smad1/5/8, and β-actin expression detected by Western blot. B: Quantification of band densities corrected for β-actin loading from 3 independent experiments. C and D: BMP ligands induce eNOS expression and induce phosphorylation of Smad1/5/8 and p38 MAPK in cPMECs. cPMECs were treated with or without 10 ng/ml BMP2, 4, or 7 for 24 h. C: eNOS, phospho-Smad1/5/8, phospho-p38 MAPK, and β-actin expression detected by Western blot. D: quantification of band densities corrected for β-actin loading from 4 independent experiments. Values are means ± SE of protein/β-actin band density ratios. Kruskal- Wallis ANOVA: *P < 0.05 vs. normoxic or untreated controls.

 

Figure 8
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  		Fig. 8. Hypoxia-induced upregulation eNOS and phospho-Smad1/5/8 is attenuated in cultured endothelial cells from Bmpr2{Delta}Ex2/+ mouse lungs. A: primary PMECs derived from WT and Bmpr2{Delta}Ex2/+ mouse lungs were cultured under normoxia or 1% oxygen for 24 h. Representative Western blots show eNOS, phospho-Smad1/5/8, phospho-p38 MAPK, and phospho-ERK MAPK and β-actin expression in the cell lysates. The experiment was performed twice using separate PMEC isolates with similar results. B: quantification of the Western blot corrected for β-actin loading. Values are expressed as protein/β-actin band density ratios. Mut, mutant mice.

 
Abnormal pulmonary arterial tone in Bmpr2{Delta}Ex2/+ mutant mice. To determine whether these changes in pulmonary vascular eNOS expression are of functional importance, we developed a pressure myography system to evaluate vasoconstrictor and dilatation responses in mouse IPA preparations from wild-type and Bmpr2{Delta}Ex2/+ mutant mice (see MATERIALS AND METHODS). Vasoconstriction was induced using KCL and NE, while endothelial-dependent vasodilatation of submaximally constricted vessels was determined in wild-type and Bmpr2{Delta}Ex2/+ mutant IPAs using two different endothelial activators, the calcium ionophore A23187 [GenBank] , which induces receptor-independent endothelial activation, and the muscarinic receptor agonist ACh (15). IPAs from Bmpr2{Delta}Ex2/+ mutant mice showed a significant increase in NE (but not KCl)-induced vasoconstriction compared with wild-type mice (Fig. 9, A and B). In addition, endothelial-dependent vasodilatation in response to ACh and A23187 [GenBank] was absent in Bmpr2{Delta}Ex2/+ mutant IPAs. This was also associated with a partial reduction in endothelial-independent vasodilatation in response to SNP (Fig. 9C). These findings indicate that Bmpr2{Delta}Ex2/+ mutant mice have increased pulmonary arterial vasoconstrictor responses associated with loss of endothelium-dependent vasorelaxation.


Figure 9
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  		Fig. 9. Abnormal pulmonary vasoconstriction and EC-dependent vasodilatation in Bmpr2{Delta}Ex2/+ mutant mouse intrapulmonary artery (IPA) preparations. IPA preparations were isolated and mounted in pressure myography chambers, and changes in vessel diameter were evaluated in response to defined vasoactive agents. A and B: vasoconstrictor responses to KCl (A) and norepinephrine (NE; B) at the indicated concentrations. ({lozenge}WT and {blacklozenge}Bmpr2{Delta}Ex2/+ IPAs, 3 vessels/group). Kruskal-Wallis ANOVA: *P < 0.05 vs. WT at the same concentration of NE. C: cumulative vasodilatation in vessels preconstricted with 10–6 M NE after treatment with 10–6 M ACh, 10–6 M A23187, and 10–5 M SNP. Kruskal-Wallis ANOVA: *P < 0.05 vs. WT.

 
As these myography studies were performed on IPA preparations from normoxic mice, we went onto evaluate the expression of two surrogate markers of eNOS activity in lung tissues from wild-type and Bmpr2{Delta}Ex2/+ mutant mouse lungs under normoxic conditions. There was a reduction in relative level of phospho-eNOS (S1177) (which is required for maximal activation of its enzymatic activity; Ref. 5), associated with a marked and significant decrease in expression of soluble guanylate cyclase (sGC), which is expressed in vascular smooth muscle cells and upregulated by NO signaling in vivo (18; Fig. 10). These findings suggest that there may be a defect in eNOS activity in Bmpr2{Delta}Ex2/+ mutant mice. Furthermore, as sGC is the major receptor mediating NO-dependent vasorelaxation in vascular smooth muscle cells (14), this could also explain the deficiency in endothelial-independent vasorelaxation in Bmpr2{Delta}Ex2/+ IPAs.


Figure 10
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  		Fig. 10. Decreased expression of phospho-eNOS (S1177) and soluble guanylate cyclase (sGC) in normoxic Bmpr2{Delta}E2/+ mouse lungs. A: Western blot showing phospho-eNOS (S1177) (P-eNOS), total eNOS (T-eNOS), and soluble guanylate cyclase (sGC) expression in lung lysates from WT and Bmpr2{Delta}E2/+ mutant mice. B: quantification of Western blot for phospho-eNOS (S1177) in relation to total eNOS band densities, and sGC band density corrected for β-actin loading. Values are means ± SE of band density ratios. Two-tailed t-test: *P < 0.05 vs. WT mice.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
These studies demonstrate that mice carrying a heterozygous hypomorphic mutation at the Bmpr2 locus (Bmpr2{Delta}Ex2/+) develop more severe PH without increased remodeling after prolonged (5 wk) exposure to hypoxia. This is associated with a reduction in hypoxia-induced BMP-dependent Smad signaling and eNOS expression in the pulmonary endothelium and defective endothelium-dependent vasorelaxation of IPA preparations. In addition, we have shown in mouse lungs [along with others in rats (23, 29) and humans (1)] that BMPR2 is predominantly expressed within the endothelial compartment of the pulmonary vasculature. This would explain why partial loss of BMPR2 expression in mice carrying heterozygous mutations at the Bmpr2 locus would have a dominant effect on BMP signaling in the endothelium. Furthermore, a small proportion of patients with FPAH have mutations in other TGF-β superfamily receptors, Alk1 and Endoglin (3, 33), both of which are expressed exclusively in endothelial cells and play a role in regulating BMP-dependent Smad signaling in the endothelium (6). These findings indicate endothelial cells are likely to be a target of abnormal BMP signaling in of Bmpr2{Delta}Ex2/+ mutant mice.

Our experiments were performed using mice carrying the Bmpr2{Delta}Ex2/+ mutation. This germ line mutation is distinct from the Bmpr2 null mutation described in previous PH reports, as it gives rise to a truncated hypomorphic allele (7). Functional evaluation of Bmpr2{Delta}Ex2/+ mutant mice may therefore more closely resemble phenotypic characteristics of FPAH patients carrying partially inactivating rather than null mutations at the BMPR2 locus. Despite this, studies in mice carrying the heterozygous null Bmpr2 mutation show that these animals also develop more severe PH after exposure to a combination of hypoxia and continuous serotonin infusion (19) or exposure to inflammatory stress (26). Unlike our data, however, these studies did not demonstrate increased susceptibility of Bmpr2 mutant mice to PH after exposure to hypoxia alone (2, 19). This discrepancy is likely due to the more prolonged hypoxia exposure times used in our studies, as we were also unable to see differences in RV pressures between wild-type and Bmpr2{Delta}Ex2/+ mutant mice after 3 wk of hypoxia (the maximal period of hypoxic exposure used in previous studies; see Refs. 2, 19). As in our studies (2), heterozygous null Bmpr2 mutants have been shown, if anything, to have impaired pulmonary vascular remodeling in response to chronic hypoxia.

We show that there is a reduction in endothelial expression of eNOS in hypoxic Bmpr2{Delta}E2/+ mutant mouse lungs and further that eNOS expression is upregulated by hypoxia-induced BMP signaling in cultured pulmonary microvascular endothelial cells. Previous studies have shown that eNOS is upregulated by chronic hypoxia in mouse and rat lungs (10, 35) and that eNOS null mice show increased susceptibility to hypoxic PH (9, 28). Like Bmpr2{Delta}E2/+ mutant mice in these studies, this is not associated with a marked increased in pulmonary vascular remodeling. Our observation that iNOS is upregulated in hypoxic Bmpr2{Delta}Ex2/+ mutant mouse lungs is also consistent with earlier studies (18) showing similar changes in chronically hypoxic eNOS null mice. While iNOS is not expressed in endothelial compartment of hypoxic pulmonary vasculature (35), there is evidence that iNOS null mutant mice have mild PH when raised at Denver altitude (11), suggesting that iNOS may play a role in regulating pulmonary vascular responses to chronic hypoxia. On this basis, this increase in iNOS expression may represent an attempt to compensate for the reduction in NOS expression seen both in chronically hypoxic eNOS null and in Bmpr2{Delta}Ex2/+ mutant lungs. These findings suggest that the deficiency in hypoxia-induced endothelial eNOS expression in Bmpr2{Delta}Ex2/+ mutant mice is likely to be of functional importance and that it may play a role in exacerbating PH after prolonged exposure to hypoxia. Furthermore, there is evidence that there may be a reduction in eNOS expression in the nonplexiform pulmonary endothelium of patients with PAH (16, 21), suggesting that our observations in Bmpr2{Delta}EX2/+ mutant mice are likely to be of importance in human disease.

Earlier studies (19) demonstrated enhanced pulmonary vasoconstrictor responses to exogenous serotonin in isolated pulmonary artery preparations from heterozygous null Bmpr2 (Bmpr2+/–) mutant mice. These findings have been used to support the hypothesis that increased pulmonary vascular smooth muscle contractility accounts for their increased susceptibility to PH. In order explore this further, we developed a novel approach to study pulmonary vascular responses in IPA preparations. Functional analysis of these smaller vessels is likely to more closely reflect functional changes in pulmonary resistance vessels than the analysis of extrapulmonary conduit vessels. These studies confirmed that Bmpr2{Delta}EX2/+ IPAs have increased vasoconstrictor responses to NE. These observations contrast with the earlier studies (19) as these did not detect differences in contractile responses between wild-type and Bmpr2+/– mutant mice to phenylephrine (19). Like NE, phenylephrine induces vasoconstriction through activation of the {alpha}-adrenergic receptors in vascular smooth muscle cells. This discrepancy is likely to reflect differences in vascular responses between conduit artery preparations used in the earlier studies (external diameters of ~350–450 µM), and the smaller IPA preparations used in our studies (resting external diameters of 75–150 µM, increasing to 250–350 µM after inflation). Regardless, results from both of these studies indicate that there is increased pulmonary vascular smooth muscle contractility in mice carrying heterozygous mutations at the Bmpr2 locus that contributes to their increased susceptibility to PH. The mechanisms by which these mutations give rise to defects in vascular smooth muscle cell function remain to be established.

Our analysis of BMP-signaling defects and alterations in eNOS expression also suggests that Bmpr2{Delta}EX2/+ mutant mice are likely to have abnormalities in endothelial cell function. Consistent with this, we demonstrated that there is a marked deficiency in endothelial-dependent vasorelaxation (induced both by ACh and the calcium ionophore A23187 [GenBank] ) of preconstricted IPAs from Bmpr2{Delta}EX2/+ mutant mice. There is also a reduction in endothelial-independent vasorelaxation in response to SNP in Bmpr2{Delta}EX2/+ mutant IPAs, but unlike the ACh and A23187 [GenBank] responses, this response is partially preserved (28% vasorelaxation to SNP vs. 1.7% with ACh and 0 with A23187 [GenBank] ). On this basis, while there is a reduction in the level of endothelium-independent vasorelaxation, failure to detect any vasorelaxation in response to ACh and A23187 [GenBank] indicates that there is also significant endothelial cell dysfunction. These findings are consistent with loss of endothelial eNOS expression and/or activity in Bmpr2{Delta}EX2/+ IPAs, as previous studies have shown complete loss of endothelium-dependent vasorelaxation in eNOS null pulmonary arteries (27). However, our studies were performed on IPA preparations from normoxic lungs. As eNOS activity can be regulated independently of expression levels (12), this raised the question as to whether there might also be a defect in eNOS activity in Bmpr2{Delta}Ex2/+ mutant IPA preparations. To explore this possibility, we evaluated expression of two surrogate markers of eNOS activity, phospho-eNOS (S1177) and sGC. These studies demonstrate that there is a reduction in the expression of the activating phospho-eNOS (S1177) and of the NO target sGC in normoxic Bmpr2{Delta}Ex2/+ mutant mouse lungs. This suggests that there may also be a decrease in eNOS activity in Bmpr2{Delta}Ex2/+ mutant mice. This could also account for the defect in endothelial-independent vasorelaxation in response to SNP, as sGC is required to mediate NO-dependent vasorelaxation in vascular smooth muscle cells (14).

Findings from these studies contrast with recent studies (13) from our laboratory demonstrating that mice carrying heterozygous null mutations at the Bmp4 locus (Bmp4LacZ/+) are protected from the development of PH after short-term (3 wk) exposure to hypoxia. This protective effect was lost after 5 wk of hypoxia and was associated with a marked reduction of hypoxic pulmonary vascular remodeling and smooth muscle cell proliferation. These changes were also associated with decreased secretion of BMP4 by hypoxic pulmonary endothelium and are in keeping with the observation that vascular smooth muscle cells derived from the distal pulmonary arteries proliferate in response to exogenous BMP ligands (36). Based on our observations in Bmpr2{Delta}Ex2/+ mutant mice, these findings would suggest that the dominant effects of BMP4 secretion on vascular smooth muscle cells are not mediated by BMPR2 signaling. This is consistent the observation that BMPR2 is dominantly expressed in the endothelial compartment of the pulmonary vasculature. Furthermore, an alternative type 2 BMP receptor, ActR2A, is expressed in pulmonary vascular smooth muscle cells, and recent studies (37) have shown that it is required to mediate BMP-dependent responses in pulmonary vascular smooth muscle cells lacking BMPR2. In addition, while our studies demonstrate that there is a reduction in hypoxia-induced BMP-dependent Smad signaling in the pulmonary endothelium of Bmp4LacZ/+ mutant mice, there is also a quite distinct and selective loss of hypoxia-induced Id1 expression in vascular smooth muscle cells (13). Id1 is an inhibitory transcriptional cofactor that plays an important role in promoting cellular proliferation and de-differentiation in a variety of different cell types (32). It is possible that loss of hypoxia-induced Id1 expression in smooth muscle cells accounts for the reduction in hypoxic pulmonary vascular remodeling in Bmp4LacZ/+ mutant mice. On this basis, it is possible that Id1-dependent smooth muscle cell proliferation results from the activation of non-Smad signaling after engagement of ActR2A by BMP4 in hypoxic pulmonary vascular smooth muscle cells.

Based on these observations, we suggest a new model to explain the role of BMP signaling in regulating the hypoxic PH. According to this model, hypoxia induces BMP secretion by endothelial cells, which promotes smooth muscle proliferation and pulmonary vascular remodeling (increasing pulmonary vascular resistance). At the same time autocrine signaling in endothelial cells promotes endothelial-dependent vasodilatation (decreasing pulmonary vascular resistance). As BMPR2 is dominantly expressed in the endothelial compartment of the pulmonary vasculature, partial loss of BMPR2 expression associated with heterozygous mutations at the Bmpr2 locus has a dominant effect on BMP signaling in the endothelium, leaving the proliferative response to BMP signaling in smooth muscle cells intact. Increased pulmonary vasoconstriction also suggests that this mutation enhances vascular smooth muscle contractility, although the mechanisms by which this occurs remain to be established. These findings have implications for future targeted therapeutic modification of the BMP signaling in the pulmonary vasculature of patients with different forms of PAH.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was funded through the Vanderbilt University Interdisciplinary Discovery Grant Program and a Philip Morris External Research Grant. L. Anderson is recipient of an American Heart Association Post-Doctoral Training Fellowship.


   ACKNOWLEDGMENTS
 
We thank G. Shi for assistance with Western blot analyses and S. Boyle for critical review of the manuscript. In addition, we thank R. Mernaugh for assistance with design of the anti-BMPR2 antibody, T. Takahashi for providing aliquots of the iNOS and neuronal NOS antibodies used in these studies, E. Price from the Mouse Metabolic Core facility for assistance with RV pressure monitoring, and S. Poole for assistance with IPA myography studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. de Caestecker, Division of Nephrology, Vanderbilt Univ. School of Medicine, S3223 Medical Center North, 1161 21st St. South, Nashville, TN 37232-2372 (e-mail: mark.de.caestecker{at}vanderbilt.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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