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Circulation. 2005;112:553-562
Published online before print July 18, 2005, doi: 10.1161/CIRCULATIONAHA.104.492488
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(Circulation. 2005;112:553-562.)
© 2005 American Heart Association, Inc.

Hypertension

Increased Susceptibility to Pulmonary Hypertension in Heterozygous BMPR2-Mutant Mice

Yanli Song, MD, PhD; John E. Jones, MD; Hideyuki Beppu, MD, PhD; John F. Keaney, Jr, MD; Joseph Loscalzo, MD, PhD; Ying-Yi Zhang, PhD

From the Whitaker Cardiovascular Institute and Evans Department of Medicine (Y.S., J.E.J., J.F.K., J.L., Y.-Y.Z.), Boston University School of Medicine, Boston, and the Cardiovascular Research Center (H.B.), Massachusetts General Hospital, Harvard Medical School, Charlestown, Mass. Drs Song, Loscalzo, and Zhang are currently affiliated with the Cardiovascular Research Laboratory; Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass.

Correspondence to Ying-Yi Zhang, PhD, Cardiovascular Research Laboratory, Brigham and Women’s Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 0630, Boston, MA 02115. E-mail yyzhang{at}rics.bwh.harvard.edu

Received July 14, 2004; revision received January 26, 2005; accepted February 4, 2005.


*    Abstract
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 *Abstract
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Background— Bone morphogenetic protein receptor-2 (BMPR2)–heterozygous, mutant (BMPR2+/–) mice have a genetic trait similar to that of certain patients with idiopathic pulmonary arterial hypertension (IPAH). To understand the role of BMPR2 in the development of IPAH, we examined the phenotype of BMPR2+/– mice and their response to inflammatory stress.

Methods and Results— BMPR2+/– mice were found to have the same life span, right ventricular systolic pressure (RVSP), and lung histology as those of wild-type mice under unstressed conditions. However, when treated with recombinant adenovirus expressing 5-lipoxygenase (Ad5LO), BMPR2+/– mice exhibited significantly higher RVSP than wild-type mice. The increase of RVSP occurred in the first 2 weeks after Ad5LO delivery. Modest but significant muscularization of distal pulmonary arterioles appeared in BMPR2+/– mice 4 weeks after Ad5LO treatment. Measurement of urinary metabolites of vasoactive molecules showed that cysteinyl leukotrienes, prostacyclin metabolites, and PGE2 were all increased to a similar degree in both BMPR2+/– and wild-type mice during 5LO transgene expression, whereas urinary endothelin-1 remained undetectable. Urinary thromboxane A2 metabolites, in contrast, were significantly higher in BMPR2+/– than in wild-type mice and paralleled the increase in RVSP. Platelet activation markers, serotonin, and soluble P-selectin showed a trend toward higher concentrations in BMPR2+/– than wild-type mice. Cell culture studies found that BMP treatment reduced interleukin-1ß–stimulated thromboxane A2 production in the pulmonary epithelial cell line A549.

Conclusions— BMPR2+/– mice do not develop pulmonary hypertension spontaneously; however, under inflammatory stress, they are more susceptible to an increase in RVSP, thromboxane A2 production, and vascular remodeling than wild-type mice.


Key Words: inflammation • hypertension, pulmonary • vasoconstriction • bone morphogenetic proteins • thromboxane


*    Introduction
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Bone morphogenetic protein receptor-2 (BMPR2) transduces signals of BMPs, which are a family of peptides originally found to induce ectopic bone formation.1 BMPs were later found to be structurally related to transforming growth factor-ßs, activins/inhibins, and mullerian inhibiting substances2–4 and to play critical roles in embryonic development, tissue morphogenesis, and cell differentiation.5

Recent genetic linkage studies found that heterozygous germline mutations in the BMPR2 gene are associated with idiopathic pulmonary arterial hypertension (IPAH)6–8 (formally denoted as primary pulmonary hypertension9), a disease characterized by a sustained increase of pulmonary artery pressure of unknown cause.10–12 Approximately 55% of cases of the familial form and between 10% and 26% of cases of the sporadic form of IPAH are found to have heterozygous BMPR2 mutations.8,13 Family members with BMPR2 mutations have only a 15% to 20% chance of developing (clinical) pulmonary hypertension.13 The onset of the disease varies widely, both within families and among individuals carrying the same mutation.7,14 It has thus been suggested that additional genetic or environmental factors are required to develop pulmonary hypertension in individuals with a heterozygous BMPR2 mutation. The nature of these additional factors is currently unknown.

Homozygous BMPR2 mutant (BMPR2–/–) mice have been shown to die in utero before mesoderm formation.15 Heterozygous BMPR2 mutant (BMPR2+/) mice, by contrast, are reported to be morphologically normal and fertile.15 Transgenic mice expressing a dominant-negative BMPR2 gene have been recently examined.16 The study showed that postnatal expression of the mutant BMPR2 gene in smooth muscle leads to a significant increase of right ventricular systolic pressure (RVSP) and relatively modest pulmonary vascular remodeling in mice.

In the present study, we examined the phenotype of BMPR2+/– mice with respect to (1) their survival rate during prenatal development, (2) their RVSP and lung histology in the basal state, and (3) their responses to inflammatory stress resulting from adenovirus-mediated pulmonary overexpression of 5-lipoxygenase (5LO). Inflammation has been previously suggested to be one of the mechanisms involved in the development of PAH (for reviews, see Voelkel et al,17 Tuder and Voelkel,18 Jefferey and Morrell,19 and Dorfmuller et al20). 5LO catalyzes leukotriene formation and facilitates an inflammatory process.21 Increased 5LO expression or leukotriene production has been demonstrated in patients with PAH and an animal model of PAH.22–24 Thus, using this inflammatory mediator as a stimulus may yield useful information about the role of inflammation in the development of pulmonary hypertension in individuals with BMPR2 haploinsufficiency.


*    Methods
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 *Methods
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Animals
BMPR2-mutant mice were established by Beppu and colleagues15 via targeted gene disruption. Because homozygous BMPR2-mutant mice die in utero, breeding of BMPR2+/– mice was carried out by crossing BMPR2+/– mice with wild-type mice (C57/BL6 mice). Genotyping of the offspring was performed after weaning. The mutated BMPR2 allele in the mice was identified by a polymerase chain reaction with primer pairs 5'-GCTAA AGCGC ATGCT CCAGA CTGCC TTG-3' and 5'-AGGTT GGCCT GGAAC CTGAG GAAAT C-3'.15 The amplification conditions were as follows: 1 cycle of 120 seconds at 95°C; 30 cycles of 30 seconds at 95°C, 30 seconds at 63°C, and 90 seconds at 72°C; and 5 minutes at 72°C.

The mice were maintained at the Boston University Laboratory Animal Science Center under stress-free conditions, ie, unlimited food and water access, regular light cycles (12-hour/12-hour light:dark), no forced exercise, and generally pathogen-free. All animals received humane care. The study was approved by the Institutional Animal Care and Use Committee at Boston University.

Right Heart Catheterization
Mice underwent right heart catheterization at various time points during the study. Before catheterization, mice were anesthetized with 90 mg/kg ketamine and 6 mg/kg xylazine. After dissection to expose the right jugular vein, a 1.4F Millar Mikro-Tip pressure catheter (Millar Instruments) was inserted into the vein and advanced to the RV. The catheter was connected to a transducer unit interfaced with a signal amplifier and recorder (Gould Instrument Systems), and RVSP was recorded.

Histology
Mouse lungs were perfused with saline and inflated with 10% phosphate-buffered formalin at a pressure of 20 cm H2O. After fixation for 20 hour at 4°C, the lung tissue was processed and paraffin-embedded with a Hypercenter XP System and Embedding Center (Shandon Inc) and cut into 5-µm sections. The tissue sections were then heat-dried on slides at 56°C for 1 hour. Deparaffinization and rehydration were carried out by immersing the slides in xylene (2x 5 minutes), 100% ethanol (2x 1 minute), 95% ethanol (2x 1 minute), and deionized water (5 minutes).

For hematoxylin and eosin staining, tissue sections were incubated in Gill-2 hematoxylin (Fisher) for 2 minutes, rinsed with water for 2 minutes, dipped once in acid alcohol (70% ethanol and 1% concentrated HCl), rinsed with water for 1 minute, dipped in 1% NH4OH for 15 seconds, rinsed with water for 1 minute, and dipped twice in 1% eosin Y (Fisher). The stained sections were dehydrated by incubation in 95% ethanol for 2x 30 seconds, 100% ethanol for 2x 30 seconds, and xylene for 2x 2.5 minutes and then mounted with Cytoseal60.

For smooth muscle {alpha}-actin staining, the lung sections were incubated with a mouse monoclonal antibody against smooth muscle cell {alpha}-actin (Sigma, 1:800 dilution in 1% immunohistochemical grade bovine serum albumin [BSA]/phosphate-buffered saline [PBS]) at 4°C overnight. The sections were washed in PBS twice for 5 minutes and incubated with biotinylated goat anti-mouse IgG (Jackson Immunoresearch) at a dilution of 1:500 in 1% BSA/PBS for 30 minutes at room temperature. The sections were then incubated with avidin DH and biotinylated-alkaline phosphatase H (provided in the Vectastain ABC-AP kit, Vector Laboratories) for 30 minutes and then with alkaline phosphatase substrate (provided in Vector Red Substrate kit, Vector Laboratories) for 20 minutes. Two PBS washes were performed between these incubations, and the final washing was performed under running tap water for 5 minutes. The sections were counterstained with Harris modified hematoxylin for 10 seconds, washed with water, dipped in acid alcohol, and rinsed with water.

Delivery of 5LO Transgene
Recombinant replication-deficient adenovirus expressing human 5LO (Ad5LO) was prepared as previously described25 and was delivered to the lungs of mice by intratracheal instillation. In this procedure, mice were anesthetized with ketamine (90 mg/kg)/xylazine (6 mg/kg) and given 1% lidocaine by local injection into the midline neck region. Surgical exposure of the trachea was then performed through a midline incision and subsequent blunt dissection to the level of the trachea. A 29-gauge needle bent at a 60° angle was inserted into the trachea, and 2x108 plaque forming units (PFU) of Ad5LO was slowly instilled into the trachea. After instillation, the neck incision was closed with No. 4 silk surgical suture. Buprenorphine, 0.1 mg/kg sc, was given every 8 to 12 hours after the procedure for 3 doses, and animals were monitored until full recovery from anesthesia before returning them to their regular cages.

RNA Isolation and Northern Blotting
Mouse lungs were perfused with saline and homogenized immediately in 10 mL TRIzol reagent (Invitrogen) followed by chloroform extraction for total RNA. Northern blotting was carried out by electrophoresing 20 µg RNA in 1.2% agarose gels containing 4% formaldehyde. The RNA was transferred onto nitrocellulose membranes and hybridized with an [{alpha}-32P]dCTP-labeled 5LO probe that encompasses nucleotides 45 to 290 of human 5LO cDNA (GenBank accession No. J03600). Hybridization was carried out at 68°C overnight in MiracleHyb solution (Stratagene) containing 10 µg/mL sheared salmon sperm DNA. The membrane was washed with 2x saline sodium citrate (SSC)/0.1% sodium dodecyl sulfate (SDS) at room temperature for 15 minutes and then with 0.1x SSC/0.1% SDS at 68°C for 20 minutes before exposure to X-ray film. The membrane was then washed in 0.1x SSC/0.1% SDS at 100°C twice for 15 minutes to strip the bound 5LO probe and hybridized with a [{alpha}-32P]dCTP-labeled mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe (Ambion) to estimate total RNA loading.

Urine Collection and Measurement of Urinary Creatinine
Urine samples were collected from the mice 2 days before Ad5LO treatment (day –2) and on days 3, 7, 12, and 21 after treatment. During the collection, mice were maintained in metabolic cages with water supplied but food withheld for 16 hours. Urine samples were collected during the 16-hour period, and the mice were returned to their regular cages after collection. The urine samples were centrifuged at 20 000g for 5 minutes, and the supernatants were stored in aliquots at –80°C.

Urinary creatinine concentration was determined by the picric acid assay and creatinine assay reagents from Teco Diagnostics. In this assay, 0.01 mL creatinine standards (1.25, 2.5, 5, and 10 mg/mL) or urine samples (diluted 5- to 15-fold in water) were added to a 96-well plate and mixed with 0.2 mL of creatinine picric acid reagent and creatinine buffer reagent (mixed before assay at 1:1, vol/vol). The plate was incubated at 37°C for 40 minutes and read at 490 nm. The absorbance from this reading was registered as the total absorbance. Acetic acid reagent (0.01 mL) was then added to each well, and the plate incubated at room temperature (22°C to 25°C) for 15 minutes. The plate was read again at 490 nm, and the absorbance from the second reading (nonspecific absorbance) was subtracted from the first reading (total absorbance). The absorbance difference (specific absorbance) was used to construct the standard curve and calculate the sample urinary creatinine concentration.

Urinary Vasoactive Metabolites
The production of cysteinyl leukotrienes, prostacyclin, prostaglandin (PG) E2, endothelin-1, and thromboxane (Tx) A2 in mice was determined by analyzing the urinary concentration of the products or their metabolites by ELISA. For cysteinyl leukotriene measurements, an ELISA kit detecting leukotrienes C4, D4, and E4 from Assay Designs (No. 900-070) was used. Urine samples were diluted a minimum 20-fold for the assay to avoid nonspecific binding. For urinary prostacyclin measurements, an ELISA kit detecting 6-keto-PGF1{alpha} and 2,3-dinor-6-keto-PGF1{alpha}, 2 major metabolites of prostacyclin in urine, was used (Assay Designs No. 901-025). Urine samples were diluted a minimum of 40-fold for these assays. For PGE2 measurements, a high-sensitivity ELISA kit detecting PGE2 from Assay Designs (No. 930-001) was used. Urine samples were diluted a minimum of 20-fold for the assay. For endotheline-1 measurements, an ELISA kit detecting endothelin-1, -2, and -3 was used (No. 583151, Cayman Chemical). Urine samples were diluted a minimum of 10-fold for the assay. For TxA2 measurements, ELISA kits detecting TxB2 (Assay Designs No. 901-002) and 11-dehydro-TxB2 were used (Assay Designs No. 901-092), and urine samples were diluted a minimum of 50-fold and 30-fold for the assays with the TxB2 and 11-dehydro-TxB2 kit, respectively. For urinary serotonin measurement, an ELISA kit from Labor Diagnostika Nord GmbH/Rocky Mountain Diagnostics was used. All samples were analyzed in duplicate and repeated at least twice.

Plasma Soluble P-Selectin Measurement
Plasma soluble P-selectin (sP-selectin) was measured with a mouse sP-selectin Quantikine kit (No. MPS00) from R&D Systems. Blood samples were collected from mice immediately after euthanasia and mixed with EDTA as an anticoagulant. The samples were centrifuged first at 2000g for 20 minutes at room temperature and then at 10 000g for 10 minutes at 2°C to 8°C. Aliquots of the supernatants were kept at –80°C before use.

A549 Cell Activity Assay
The pulmonary epithelial cell line A549 was obtained from American Type Culture Collection (Manassas, Va) and maintained at 37°C in RPMI 1640 medium containing 10% fetal bovine serum, 100 U penicillin, and 100 µg streptomycin. Treatment of the cells with interleukin (IL)-1ß and/or BMPs was carried out in RPMI 1640 medium containing 1% fetal bovine serum. To determine the TxA2 synthesis, cells were washed with Dulbecco’s phosphate buffered saline (D-PBS) and incubated with 0.01 mmol/L arachidonic acid in D-PBS for 10 minutes at 37°C. The released TxB2 in the assay medium was analyzed by ELISA as described earlier. Total cell protein concentration was determined with DC protein assay reagents from Bio-Rad.

Statistics
Data are presented as the mean±SEM. Statistical analysis was performed by {chi}2 analysis (the Table), Student t test (Figures 4, 10, and 11 DownDown), or 2-way ANOVA (Figures 5 and 7–9DownDownDown) with the SigmaStat program. P<0.05 indicated statistical significance.


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  		Genotypic Ratio of Offspring From BMPR2+/– and BMPR2+/+ Breeding Pairs*



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  		Figure 4. RVSP in mice treated with Ad5LO. Wild-type (white column) and BMPR2+/– (gray column) mice were treated with nothing (No Tx), 2x108 PFU adenovirus expressing green fluorescence protein (GFP) or 2x108 PFU Ad5LO via intratracheal instillation at day 0. Right heart catheterization was performed on mice at day 12 after treatment. Data are presented as mean±SEM; n=6 to 10 per group. *P<0.05 vs no-treatment group in same type of mouse; #P<0.05, BMPR2+/– vs wild-type group with same treatment. Statistical analysis was performed by Student t test.



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  		Figure 10. Plasma sP-selectin and urinary serotonin concentrations. Plasma samples were prepared from wild-type (white column) and BMPR2+/– (gray column) mice with no treatment (No Tx) or treated with 2x108 PFU Ad5LO 7 days previously (day 7). sP-selectin was measured by ELISA (A). Urinary serotonin analysis was performed on same samples as used for measurements depicted in Figure 7. *P<0.05 vs No Tx group (sP-selectin) or day –2 group (serotonin) in same type of mouse. Difference between BMPR2+/– and wild-type mice in both sP-selectin and serotonin measurement did not reach statistical significance. Data are presented as mean±SEM; n=6 to 10 per group. Statistical analysis was performed by Student t test.



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  		Figure 11. Effect of BMPs and IL-1ß on TxB2 production in A549 cells. A549 cells were treated with 0.5 ng/mL IL-1ß and/or 100 ng/mL BMP-2 or BMP-6 for 20 hours. Cells were then washed and incubated with 0.01 mmol/L arachidonic acid in D-PBS for 10 minutes. TxB2 released in assay medium was analyzed by ELISA. *P<0.05 vs untreated cells; #P<0.05 vs cells treated with IL-1ß alone. Data are presented as mean±SEM of 3 measurements, each performed in duplicate. Statistical analysis was performed by Student t test.



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  		Figure 5. Time course of RVSP change in Ad5LO-treated mice. Wild-type (white column) and BMPR2+/– (gray column) mice were treated with 2x108 PFU Ad5LO via intratracheal instillation at day 0. RVSP was measured at days 0, 3, 7, 10, 12, 14, and 21 for each group. *P<0.05 vs day 0 group in same type of mouse; #P<0.05 vs wild-type group at same time point. Data are presented as mean±SEM; n=6 to 10 per group. Statistical analysis was performed by 2-way ANOVA.



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  		Figure 7. Time course of cysteinyl leukotriene production in Ad5LO-treated mice. Wild-type (white column) and BMPR2+/– (gray column) mice were treated with 2x108 PFU Ad5LO via intratracheal instillation at day 0. Urine samples were collected from mice 2 days before (day –2) and 3, 7, 12, and 21 days after treatment. Urinary cysteinyl leukotrienes (CysLT) and creatinine were determined by ELISA and picric acid assay, respectively. *P<0.05 vs day –2 group. Urinary CysLT concentration in either type of mouse was undetectable at days –2 and 21; no statistical difference existed between BMPR2+/– and wild-type mice at any time point. Data are presented as mean±SEM; n=6 to 10 mice per group. Statistical analysis was performed by 2-way ANOVA.



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  		Figure 8. Prostacyclin (PGI2) and PGE2 production in Ad5LO-treated wild-type and BMPR2+/– mice. Analyses were performed on same set of urine samples as used for measurements depicted in Figure 7. PGI2 production (A) in mice was determined by ELISA by measuring urinary 6-keto-PGF1{alpha} and 2,3-dinor-6-keto-PGF1{alpha} concentration. *P<0.05 vs day –2 group. No statistical difference existed between BMPR2+/– and wild-type mice groups at any time point. Data are presented as mean±SEM; n=6 to 10 per group. Statistical analysis was performed by 2-way ANOVA.



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  		Figure 9. TxA2 production in Ad5LO-treated wild-type and BMPR2+/– mice. Analyses were performed on same set of urine samples as used for measurements depicted in Figure 7. Urinary concentrations of TxB2 (A) and 11-dehydro-TxB2 (B) were determined by ELISA. *P<0.05 vs day –2 group; #P<0.05 BMPR2+/– vs wild-type group at same time point. Data are presented as mean±SEM; n=6 to 10 per group. Statistical analysis was performed by 2-way ANOVA.


*    Results
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 *Results
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BMPR2+/– Mice Breeding
BMPR2+/– mice were bred by crossing BMPR2+/– with wild-type mice (C57/BL6 mice). The genotypes of the offspring were determined after weaning. The Table shows the summary of the numbers of BMPR2+/– and wild-type mice obtained from breeding and the distribution of male and female mice in each population. As demonstrated, the numbers of BMPR2+/– and wild-type mice in the offspring were significantly different, 498 and 618, respectively ({chi}2= 12.9, P<0.001). The ratio of BMPR2+/– to wild-type mice was 81:100, indicating that {approx}20% of BMPR2+/– mice were lost in early development. The majority of the missing BMPR2+/– mice died in utero, although some may have died in the immediate postnatal stage, because the number of pups in each litter was not counted immediately after birth. The female:male distribution in either BMPR2+/– or wild-type offspring was not statistically different. The surviving BMPR2+/– mice were morphologically normal and had no apparent problems in the unstressed state. These mice also had a similar life span ({approx}18 months) to wild-type mice.

Lung Structure and RVSP in BMPR2+/– Mice
To assess lung development in the BMPR2+/– mice, lung tissue sections from BMPR2+/– and wild-type mice were prepared and stained with hematoxylin and eosin. As shown in Figure 1, no apparent gross abnormalities were observed in the sections from either mouse type, although a minor increase in adhesion of leukocytes to the vessel wall was noted in the BMPR2+/– mice (Figure 1B).



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  		Figure 1. Lung histology of wild-type and BMPR2+/– mice. Paraffin-embedded lung tissues from wild-type (A) and BMPR2+/– (B) mouse were cut into 5-µm sections and stained with hematoxylin and eosin. Photomicrographs were taken at x400 magnification.

The pulmonary artery pressure in the BMPR2+/– mice was assessed by measuring RVSP via right heart catheterization. In the absence of pulmonary valve disease, RVSP is equivalent to the pulmonary artery systolic pressure. As shown in Figure 2, the RVSP in BMPR2+/– mice was similar to that of wild-type mice from 2 to 18 months of age, indicating that BMPR2+/– mice did not develop pulmonary hypertension spontaneously under unstressed conditions.



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  		Figure 2. RVSP. Right heart catheterization was performed on wild-type (white column) and BMPR2+/– (gray column) mice at 2, 3, 4.5, 6, 9, 12, or 18 months of age. No statistical differences were found between BMPR2+/– and wild-type mouse groups and of any ages. Data are presented as mean±SEM; n=4 to 8 mice per group.

Expression of 5LO in Lungs of Mice
To examine the responses of BMPR2+/– mice to pulmonary inflammation, recombinant human 5LO was expressed in the lungs of mice via adenovirus-mediated gene transfer. Approximately 2x108 PFU of replication-deficient adenovirus expressing recombinant human 5LO (Ad5LO) was administered to the mice via intratracheal instillation. The time course of transgene expression was examined by Northern blotting. As shown in Figure 3, expression of 5LO peaked at day 7 after Ad5LO delivery and decreased significantly by day 21. No difference in transgene expression was found between wild-type and BMPR2+/– mice.



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  		Figure 3. Northern blotting analysis of time course of 5LO transgene expression. Wild-type mice were treated with 2x108 PFU Ad5LO via intratracheal instillation at day 0. Total RNA was isolated from lung tissue of mice at days 0, 3, 7, 10, 14, and 21. Northern blotting was carried out with [{alpha}-32P]dCTP-labeled human 5LO cDNA probe and probe against mouse GAPDH. Two RNA samples from 2 individual mice were examined at each time point.

RVSP of Mice After Receiving Ad5LO
The RVSP of wild-type and BMPR2+/– mice was examined initially at day 12 after administration of Ad5LO and compared with that of mice that received adenovirus-expressing green fluorescence protein (AdGFP). The choice of time point was based on a previous study, which showed that rats receiving Ad5LO and monocrotaline treatment developed pulmonary hypertension 10 days later.26 As shown in Figure 4, RVSP was significantly increased in Ad5LO-treated BMPR2+/– mice compared with those with no treatment (23±1 versus 12±1 mm Hg, P<0.05). The RVSP response of BMPR2+/– mice was not absolutely specific for exogenous 5LO expression, because the mice that received AdGFP had an elevated RVSP as well, though to a significantly milder degree (Figure 4). Adenovirus-mediated gene transfer is known to cause inflammation. Thus, the increase in RVSP found in BMPR2+/– mice likely reflects a general response to inflammation, which is intensified by 5LO overexpression. Wild-type mice exhibited little increase in RVSP under either Ad5LO or AdGFP treatment, suggesting that BMPR2+/– mice are more sensitive to an inflammation-mediated RVSP increase than are wild-type mice.

To investigate the time course of change of RVSP in the mice, BMPR2+/– or wild-type mice were treated with Ad5LO and examined by right heart catheterization at days 0, 3, 7, 10, 12, 14, or 21 after Ad5LO delivery. As shown in Figure 5, the increase of RVSP in BMPR2+/– mice peaked at day 7 (33±3 mm Hg) and returned to near normal by day 21 (14±2 mm Hg, compared with day 0, 12±1 mm Hg). The RVSP of wild-type mice increased mildly at day 7 (18±2 mm Hg) and became normal at day 12 (13±1 mm Hg). The increase of RVSP in the BMPR2+/– mice was consistent with the time course of transgene expression (shown in Figure 3) and was significantly higher than that of wild-type mice.

To determine whether the change in RVSP was associated with pulmonary vascular remodeling, immunohistochemical staining for smooth muscle {alpha}-actin was carried out in lung tissue sections prepared from wild-type and BMPR2+/– mice after Ad5LO delivery. As shown in Figure 6, lung sections obtained at day 7 after Ad5LO delivery showed no sign of vascular remodeling in either type of mouse, although significant inflammatory cell infiltration was present. Increased muscularization of distal pulmonary arterioles was found in lung sections obtained at day 28 after Ad5LO treatment. Counting the {alpha}-actin–stained distal pulmonary arterioles (vessels that are located distal to terminal bronchioles, adjacent to the alveolar duct, noncollapsed, and with diameters <40 µm) showed that BMPR2+/– mouse lungs had a significantly greater number of muscularized vessels than lungs from wild-type mice (36±3 versus 7±1) (Figure 6G). The muscularized distal arterioles accounted for {approx}10% of total distal arterioles in the BMPR2+/– lungs. The degree of muscularization (thickness of the muscle layer), however, was mild. Because this increase in muscularization occurred after the maximal increase in RVSP, the increased RVSP observed in BMPR2+/– mice in the first 2 weeks after Ad5LO delivery was not due to pulmonary vascular remodeling but rather to enhanced pulmonary vasoconstriction.



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  		Figure 6. Muscularized distal pulmonary vessels in Ad5LO-treated mice. Mice were untreated (No Tx) or treated with 2x108 PFU Ad5LO at day 0, and lung tissues were prepared at days 7 and 28. Lung sections were stained with anti-smooth muscle {alpha}-actin antibody (red) and counterstained with hematoxylin (blue). Number of muscularized pulmonary vessels with diameter <40 µm, located distal to terminal bronchioles and adjacent to alveolar ducts, was counted in 20 consecutive fields (x200) per section. A to E show photomicrographs (x400) of stained lung sections prepared at day 7 (A and B) and day 28 (C–F) from wild-type (A and C) and BMPR2+/– (B and D-F) mice. G shows average number of muscularized distal vessels in lung sections obtained from 3 to 6 mice in each group. *P<0.05 vs No Tx group; #P<0.05 BMPR2+/– vs wild-type group at same time point. Arrows indicate partially (D) and fully (E and F) muscularized distal pulmonary vessels. Bars=50 µm.

Generation of Vasoactive Molecules During 5LO Overexpression
To understand the molecular basis of the enhanced pulmonary vasoconstriction in BMPR2+/– mice during 5LO expression, urine samples were collected from the mice 2 days before (day –2) and 3, 7, 12, and 21 days after Ad5LO delivery. Urinary levels of several vasoactive molecules, including cysteinyl leukotrienes, prostacyclin metabolites, PGE2, TxB2, and endothelin-1, were determined. As shown in Figure 7, the concentration of cysteinyl leukotrienes in urine was undetectable before Ad5LO delivery (day –2) but was markedly increased after 5LO expression. The production of cysteinyl leukotrienes peaked at day 7 ({approx}20 000 pg/mg cysteinyl leukotrienes/urinary creatinine) and diminished by day 21. There was no difference between wild-type and BMPR2+/– mice in cysteinyl leukotriene production.

Urinary levels of prostacyclin metabolites, 6-keto-PGF1{alpha} and 2,3-dinor-6-keto-PGF1{alpha}, were next measured. As shown in Figure 8A, the basal level of urinary prostacyclin metabolites in BMPR2+/– and wild-type mice was similar, {approx}10 000 pg/mg (prostacyclin metabolites/urinary creatinine). The prostacyclin concentration was increased by 3- to 3.5-fold at days 3 and 7 after Ad5LO treatment and returned to the basal level by day 12. The increase was similar in BMPR2+/– and wild-type mice.

Urinary levels of PGE2, a pulmonary vasodilator, in BMPR2+/– and wild-type mice are shown in Figure 8B. The basal level of the compound in the 2 types of mice was 2200 to 4500 pg/mg (PGE2/urinary creatinine). During 5LO overexpression in the mice, the concentration increased to 16 000 to 20 000 pg/mg at days 3 and 7 and returned to basal level by day 12. There was no significant difference in PGE2 production between the wild-type and BMPR2+/– mice. Interestingly, the pattern of PGE2 production in the mice was comparable to that of prostacyclin, which suggests that the increased production of these compounds was due to activation of an upstream enzyme(s) in PG synthesis, such as cyclooxygenase-2.27

To determine whether the increased RVSP in the BMPR2+/– mice was due to overproduction of vasoconstrictive molecules, the generation of endothelin-1 and TxA2 in mice during 5LO expression was examined. Results showed that urinary endothelin-1 was undetectable in these mice either before or after Ad5LO delivery, indicating no marked increase in the production of this vasoactive peptide in these mice.

TxA2 production in the mice was assessed by measuring urinary concentrations of TxB2 and 11-dehydro-TxB2, metabolites of TxA2. As shown in Figure 9A, the basal level of urinary TxB2 in both wild-type and BMPR2+/– mice was {approx}30 000 pg/mg (TxB2/creatinine). During 5LO expression, TxB2 concentration in the BMPR2+/– mice increased to 71 000, 116 000, 91 000, and 59 000 pg/mg at days 3, 7, 12, and 21, respectively. The change in Tx production in wild-type mice, by contrast, was less striking and did not reach statistical significance compared with pretreatment controls, with values of 47 000, 60 000, 39 000, and 24 000 pg/mg on days 3, 7, 12, and 21, respectively. Urinary 11-dehydro-TxB2 in the Ad5LO-treated mice showed a similar pattern of change as TxB2 (Figure 9B). Thus, among the vasoactive molecules examined in this study, a difference between wild-type and BMPR2+/– mice was found only in TxA2 production. The time course of the change in TxB2 level was comparable to that of the RVSP change in the mice.

Platelet Activation
Platelet activation is the major source of Tx production in vivo,28 and urinary Tx metabolites have been used as markers for platelet activation. To determine whether the increased TxA2 production in BMPR2+/– mice under inflammatory stress was due to augmented platelet activation, we examined 2 other platelet activation markers, plasma sP-selectin and urinary serotonin, in the mice. As shown in Figure 10A, untreated wild-type and BMPR2+/– mice had similar plasma sP-selectin levels, 725±26 and 751±99 ng/mL, respectively. Ad5LO treatment increased the plasma sP-selectin concentration to 1326±100 and 1791±187 ng/mL in wild-type and BMPR2+/– mice, respectively. BMPR2+/– mice tended toward a higher sP-selectin level than did wild-type mice, but the difference did not reach statistical significance. A similar trend was found in urinary serotonin concentrations in the 2 types of mice. As shown in Figure 10B, before Ad5LO treatment, the urinary serotonin concentrations in wild-type and BMPR2+/– mice were similar, 73±9 and 73±8 pg/mg urinary creatinine, respectively. Seven days after Ad5LO treatment, urinary serotonin concentration increased to 173±13 and 215±25 pg/mg in wild-type and BMPR2+/– mice, respectively. The difference between wild-type and BMPR2+/– mice did not reach statistical significance. The relatively small difference in sP-selectin and serotonin levels found between wild-type and BMPR2+/– mice after Ad5LO treatment suggested that platelet activation was not the primary source of the increased TxA2 production in the Ad5LO-treated BMPR2+/– mice.

Effect of BMP on TxA2 Production in A549 Cells
Pulmonary epithelial cells are another source of TxA2 production in vivo.28,29 We therefore examined the effect of BMPs on TxA2 production in a human pulmonary epithelial cell line, A549. As shown in Figure 11, untreated A549 cells released a small amount of TxB2 (207±19 pg/mg total cell protein) in the basal state, and IL-1ß stimulated TxB2 production by >10-fold (2822±41 pg/mg). BMP-2 did not affect basal level of TxB2 production (208±11 pg/mg) but reduced IL-1ß–stimulated production by {approx}20% (2215±186 pg/mg). BMP-6 had an effect (2339±41 pg/mg) similar to BMP-2 in the assay. These data indicated that BMP signaling did not directly affect TxA2 production in A549 cells but reduced the stimulating effect of IL-1ß on the synthesis of this prostanoid.


*    Discussion
up arrowTop
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
To understand the relation between BMPR2 haploinsufficiency and pulmonary hypertension, this study examined BMPR2+/– mice and explored 3 issues: (1) whether BMPR2 haploinsufficiency affects the development of the mice; (2) whether haploinsufficiency alone leads to pulmonary hypertension; and (3) whether haploinsufficiency increases the susceptibility to pulmonary hypertension in the setting of inflammation. The results showed that BMPR2 heterozygosity caused {approx}20% fatality in mice during embryonic development but did not lead to pulmonary hypertension under unstressed conditions. The study also showed that BMPR2+/– mice were more sensitive to inflammation-induced pulmonary hypertension than wild-type mice.

BMP signaling plays an important role in embryonic development and is involved in gastrulation, mesoderm formation, neural patterning, skeletal development, and organogenesis (for reviews, see Hogan5 and Zhao30). Homozygous knockout mice deficient in BMP signaling proteins, such as BMP-2, BMP-4, BMPR2, BMPR-IA (Alk-3), ActR-I (Alk-2), Smad1, or Smad5, all die during embryonic development.30 By contrast, animals with heterozygous mutations in these genes are viable and have been reported to be grossly phenotypically normal. In this study, we found that {approx}20% of BMPR2+/– mice died in utero and/or during weaning, which suggests that haploinsufficiency of BMPR2 affects early development. Although we observed no apparent abnormality in the surviving BMPR2+/– mice under unstressed conditions in this study, some of these mice may have had minor developmental defects, and these defects could affect the phenotype of the mice under stressed conditions.

Inflammation has been suggested to be an important mechanism in the development of PAH. Evidence supporting the hypothesis includes the following: (1) Inflammatory cells are found in the vicinity of remodeled pulmonary vessels with plexiform lesion; (2) proinflammatory cytokines and chemokines are increased in patients with PAH; (3) PAH is a common complication of autoimmune diseases involving systemic inflammation, such as scleroderma and systemic lupus erythematosus; and (4) in a rat model of PAH, monocrotaline administration causes pulmonary endothelial injury and inflammation, which is followed by pulmonary vascular remodeling (for reviews, see Voelkel et al,17 Tuder and Voelkel,18 Jeffery and Morrell,19 and Dorfmuller et al20). In the present study, we examined the effect of inflammation on the development of pulmonary hypertension in BMPR2+/– mice. Adenovirus-mediated overexpression of 5LO in the lung was used as the inflammatory stress because 5LO expression is increased in the human disease. The results showed that BMPR2+/– mice responded to the inflammation with an immediate marked increase of RVSP (peaking at day 7) and delayed muscularization of distal pulmonary arterioles (28 days after Ad5LO treatment). The early increase of RVSP is related to enhanced pulmonary vasoconstriction, due, at least in part, to increased TxA2 production in the mice. The later muscularization of distal arterioles could be caused by the initial vasoconstriction, increased release of growth factors by inflammatory cells, and/or endothelial cell activation/injury caused by the inflammation. Further study is required to identify the specific mechanism. The overall degree of the muscularization found in the Ad5LO-treated BMPR2+/– mice is mild. This could be due to the transgene expression, as well as that inflammation is transient. Further studies are required to demonstrate whether increased pulmonary vascular injury by sustained inflammation leads to extensive pulmonary vascular remodeling and persistent pulmonary hypertension in BMPR2+/– mice.

BMPR2+/– mice were found to produce significantly higher amounts of TxA2 than wild-type mice during 5LO overexpression. This observation is consistent with previous reports that TxA2 production is significantly enhanced in patients with PAH.31–33 Mechanistically, however, this finding raises the question of how BMPR2 deficiency leads to increased TxA2 production. No report in the literature has linked BMP signaling to TxA2 production. Because the TxA2 levels in the wild-type and BMPR2+/– mice were the same before Ad5LO delivery, the difference in TxA2 production in these mice resides in the different responses to 5LO overexpression or inflammation. We therefore examined platelet activation in vivo and pulmonary epithelial cell activation in cell culture, because Tx synthase is most abundantly expressed in platelets (2187 ng/mg protein),34 and the lung has the highest content of the enzyme, 765 ng/mg, among solid-organ tissues.34 The cells in lung that express Tx synthase are mainly bronchial epithelial cells and alveolar macrophages in humans and also small pulmonary artery smooth muscle cells in rats.29 Examining plasma sP-selectin and urinary serotonin, 2 platelet activation markers, showed that both of the markers tended to be higher in BMPR2+/– than wild-type mice, but neither difference reached statistical significance. Thus, platelet activation may not be the primary or the only source of the enhanced TxA2 production in BMPR2+/– mice during 5LO expression. The pulmonary epithelial cell line A549 was found to produce a very small amount of TxA2 under basal conditions, but the production of this prostanoid was stimulated markedly by IL-1ß. BMP did not affect the basal level of TxA2 production but reduced IL-1ß stimulation significantly by 20%. These effects suggest that BMP signaling does not regulate TxA2 production directly but interferes with IL-1ß signaling.

IL-1ß signaling activates 2 pathways, one leading to nuclear factor (NF)-{kappa}B activation and the other, mitogen-activated protein kinase, Janus NH2 terminal kinase (JNK), and subsequently, activator protein (AP)-1. A previous study has shown that BMP-7 inhibits IL-1ß–induced JNK and AP-1 activation but does not affect IL-1ß–induced NF-{kappa}B activation in human mesangial cells.35 If a similar selective inhibition occurs in pulmonary epithelial cells, it could explain the partial inhibitory effect of BMPs on the IL-1ß–stimulated TxA2 production in A549 cells. Further studies are required to understand the specific interaction between BMP and IL-1 signaling in these cells.

TxA2 is a potent vasoconstrictor and platelet activator.36,37 It also inhibits voltage-gated potassium channels38 and has synergistic effects with serotonin in causing vascular smooth muscle cell proliferation.39 These effects, when persistently produced by multiple inflammatory insults or stresses, could contribute to pulmonary vascular remodeling and sustained pulmonary hypertension. Understanding the relation between BMPR2 haploinsufficiency and TxA2 production under inflammatory stress could shed light on the mechanism of heterozygous BMPR2 mutation–mediated IPAH.


*    Acknowledgments
 
This study was supported by NIH grants HL58976, HL55993, and HL61795 (to Dr Loscalzo) and grants from the American Heart Association (0256282N) and Pfizer (Atorvastatin Research Award) (to Dr Zhang).


*    Footnotes
 
Guest Editor for this article was Mark A. Creager, MD.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
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 up arrowDiscussion
 *References
 

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