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Am J Physiol Lung Cell Mol Physiol 288: L1162-L1170, 2005. First published February 18, 2005; doi:10.1152/ajplung.00180.2004
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A role for platelet-derived growth factor {beta}-receptor in a newborn rat model of endothelin-mediated pulmonary vascular remodeling

Robert P. Jankov,1,2,3,4 Crystal Kantores,1 Rosetta Belcastro,2 Soojin Yi,1,2 Ross A. Ridsdale,2 Martin Post,2,3,4 and A. Keith Tanswell2,3,4

1Clinical Integrative Biology, Sunnybrook & Women's Research Institute, Toronto; 2Lung Biology Programme, The Hospital for Sick Children Research Institute, Toronto; and Departments of 3Pediatrics and 4Physiology, University of Toronto, Toronto, Ontario, Canada

Submitted 18 May 2004 ; accepted in final form 17 February 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Newborn rats exposed to 60% O2 for 14 days develop endothelin (ET)-1-dependent pulmonary hypertension with vascular remodeling, characterized by increased smooth muscle cell (SMC) proliferation and medial thickening of pulmonary resistance arteries. Using immunohistochemistry and Western blot analyses, we examined the effect of exposure to 60% O2 on expression in the lung of receptors for the platelet-derived growth factors (PDGF), which are implicated in the pathogenesis of arterial smooth muscle hyperplasia. We observed a marked O2-induced upregulation of PDGF-{alpha} and -{beta} receptors (PDGF-{alpha}R and -{beta}R) on arterial smooth muscle. This led us to examine pulmonary vascular PDGF receptor expression in 60% O2-exposed rats given SB-217242, a combined ET receptor antagonist, which we found prevented the O2-induced upregulation of PDGF-{beta}R, but not PDGF-{alpha}R, on arterial smooth muscle. PDGF-BB, a major PDGF-{beta}R ligand, was found to be a potent in vitro inducer of hyperplasia and DNA synthesis in cultured pulmonary artery SMC from infant rats. A critical role for PDGF-{beta}R ligands in arterial SMC proliferation was confirmed in vivo using a truncated soluble PDGF-{beta}R intervention, which attenuated SMC proliferation induced by exposure to 60% O2. Collectively, these data are consistent with a major role for PDGF-{beta}R-mediated SMC proliferation, acting downstream of increased ET-1 in a newborn rat model of 60% O2-induced pulmonary hypertension.

pulmonary oxygen toxicity; vascular smooth muscle; growth factors; soluble receptors

PULMONARY HYPERTENSION IN THE NEWBORN is a common end result of a variety of perinatal and postnatal insults. Regardless of the initial etiology, several pathological events that account for the hallmark manifestation of raised pulmonary arterial pressure are consistently found. These are abnormal vasoconstriction and subsequent structural remodeling of the pulmonary vasculature, which render the pulmonary circulation less responsive to vasodilator therapies (19). Recent data from human infants with bronchopulmonary dysplasia (BPD), a chronic neonatal lung injury that affects preterm infants, have demonstrated structural remodeling of pulmonary resistance arteries as early as 2 wk after birth (30). These data provide a pathological correlate for recent echocardiographic studies demonstrating raised pulmonary arterial resistance in infants with BPD (27, 28), which together indicate that pulmonary hypertension remains a common manifestation of this condition.

Newborn rats exposed to 60% O2 for 14 days, a model for human BPD, develop pulmonary hypertension characterized by right ventricular hypertrophy and remodeling of pulmonary arteries with thickening of the medial layer due to increased smooth muscle mass (18, 20). We have previously reported that expression of endothelin (ET)-1, a potent vasoconstrictor, was greatly increased in the 60% O2-exposed lung (18). This increase in ET-1 was critical to the development of pulmonary hypertension in that treatment with a combined ET receptor antagonist, SB-217242, prevented vascular remodeling (17). ET-1 is also implicated as a mediator of human pulmonary hypertension by observational studies in both newborns (24, 26) and adults (11). Despite a well-established role for ET-1 in the pathogenesis of experimental pulmonary hypertension and a likely role in human pulmonary hypertension, the mechanisms by which ET-1 leads to the development of pulmonary vascular remodeling remain poorly understood, especially in the newborn period.

There is direct experimental evidence for the involvement of platelet-derived growth factors (PDGFs) in the pathogenesis of experimental pulmonary vascular remodeling (3, 10). The PDGFs are dimeric molecules that act on two distinct tyrosine kinase receptors: PDGF {alpha}-receptor (PDGF-{alpha}R) and -{beta}-receptor (PDGF-{beta}R), both of which are abundantly expressed on vascular smooth muscle cells (SMC) (13). The PDGF ligands with putative roles in pulmonary hypertension are PDGF-AA, which binds only the PDGF-{alpha}R, and PDGF-BB, which binds to both PDGF receptor isoforms. Both PDGF-AA (25) and -BB (15) have been reported to be potent mitogens of vascular SMC in vitro. We have previously reported that PDGF-BB expression was increased on the pulmonary vessel walls of 60% O2-exposed newborn rats (8). Total lung expression of PDGF-B, PDGF-{alpha}R, and PDGF-{beta}R mRNA was also found to be increased by exposure to 60% O2 (8). In this study, we quantified and localized the expression of both PDGF receptor subtypes in the lungs of newborn rats exposed to 60% O2 and examined the impact of an ET receptor antagonist on these changes. Together, our findings provide evidence for a major role of the PDGF-{beta}R and its ligand, PDGF-BB, acting downstream of ET-1, in 60% O2-induced pulmonary vascular smooth muscle hyperplasia.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Oxygen exposure chambers and automated controllers (OxyCycler model A84XOV) were purchased from Biospherix (Redfield, NY). Tissue culture plates and flasks were from Corning (Acton, MA). SB-217242 was a generous gift from Dr. Douglas Hay (Glaxo SmithKline, King of Prussia, PA). [3H]thymidine was from ICN Biomedicals (Costa Mesa, CA). Total protein assay kits and Tris-glycine gels were from Bio-Rad (Mississauga, ON, Canada). Polyvinylidene difluoride membranes were from VWR (Mississauga, ON, Canada). 5'-Bromo-2'-deoxyuridine (BrdU) and an in situ BrdU immunostaining kit were from BD Biosciences (Mississauga, ON, Canada). Heat-inactivated FBS, DMEM, antibiotic-antimycotic solution, trypsin, and EDTA were from GIBCO BRL (Burlington, ON, Canada). Recombinant human PDGF-AA and -BB, a mouse monoclonal antibody (MAb) against PDGF-{beta}R, and recombinant human PDGF-{beta}R/Fc chimeric protein were from R&D Systems (Minneapolis, MN). Acids, alcohols, chromatography-grade organic solvents, DMSO, paraformaldehyde, Permount, and Superfrost/Plus microscope slides were from Fisher Scientific (Whitby, ON, Canada). Protease inhibitors were from Roche (Laval, QC, Canada), except for PMSF, which was from Sigma (St. Louis, MO). Rabbit polyclonal antibodies against PDGF-{alpha}R and GAPDH (EC 1.2.1.12 [EC] ) and goat anti-rabbit and anti-mouse IgG-biotin secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse MAb against {alpha}-smooth muscle actin was from Neomarkers (Fremont, CA). Goat anti-rabbit and anti-mouse IgG-peroxidase antibodies were from Cell Signaling Technology (Beverly, MA). Goat anti-rabbit IgG-FITC and anti-mouse IgG-rhodamine were from Calbiochem (San Diego, CA). Avidin-biotin-peroxidase complex immunohistochemistry kits, 4',6'-diamidino-2-phenylindole (DAPI) mounting medium, 3,3'-diaminobenzidine staining kits, and normal goat serum (NGS) were from Vector Laboratories (Burlingame, CA). Fluorescent aqueous mounting medium was from Dako Cytomation (Carpenteria, CA). Tissue-Tek OCT was from Sakura Finetek (Torrance, CA). All other chemicals and reagents were purchased from Sigma.

Institutional review. All procedures involving animals were conducted according to criteria established by the Canadian Council for Animal Care. Approval for the studies was obtained from the Animal Care Committees of the Hospital for Sick Children Research Institute and the Sunnybrook & Women's Research Institute.

Oxygen exposure system and intervention. Pathogen-free pregnant Sprague-Dawley rats (250–275 g) were obtained from Taconic (Germantown, NY). On the anticipated day of delivery, each pregnant dam was placed in an 80 x 60 x 50-cm plastic chamber with 12 h/12 h light-dark cycles and with the temperature maintained at 25 ± 1°C, humidity at ~50%, and CO2 concentration <0.5%. For each exposure period, two dams and their litters were paired and exposed to either 60% O2 or 21% O2 (air controls) for 4, 7, 9, 10, or 14 days. Equal litter sizes (10–12 pups) were maintained to control for nutritional effects, and dams were exchanged daily, between paired chambers, to prevent maternal O2 toxicity. Food and water were available ad libitum. O2 and CO2 concentrations, temperature, and humidity were continuously monitored, recorded, and regulated by computer using customized software (AnaWin2 Run-Time, version 2.2.18; Watlow-Anafaze, St. Louis, MO). Gas delivery was automatically adjusted to maintain an O2 concentration within 0.1% of the set point. O2 sensors were calibrated weekly. At the termination of each exposure period, pups were either killed by ether inhalation or exsanguinated after anesthesia. For intervention studies using SB-217242, a combined ET receptor antagonist, daily intraperitoneal (ip) injections of SB-217242 in 0.9% (wt/vol) NaCl/0.5% (vol/vol) DMSO vehicle (5 mg/kg; 1 mg/ml and 5 µl/g body wt), or vehicle alone, were injected through a 30-gauge needle into the right iliac fossa, as previously described (17). For the intervention using the recombinant human soluble PDGF-{beta}R/Fc chimeric protein, 16 rat pups (4 pups randomly selected from each of 2 air- and 2 60% O2-exposed litters) received either 1x PBS with 0.1% (wt/vol) BSA (vehicle control, 8 pups) or 3 µg/g body wt soluble receptor (PDGF-{beta}R/Fc chimeric protein, 8 pups) in 100 µl of 1x PBS with 0.1% (wt/vol) BSA. Interventions were given once on day 8, and animals were killed after 24 h, on day 9. For labeling of cells undergoing DNA synthesis, each pup received BrdU [3 mg/ml and 15 mg/kg in 0.9% (wt/vol) NaCl] ip 12 h before death.

Immunohistochemistry of paraffin-embedded lung tissue. Four randomly selected animals from each group (2 from each of 2 separate litters) were anesthetized with ip ketamine (80 mg/kg) and xylazine (20 mg/kg). After opening of the thoracic cavity and cannulation of the trachea, the pulmonary veins were divided. The pulmonary circulation was flushed with 1x PBS containing 1 U/ml of heparin to clear the lungs of blood and perfusion fixed with 4% (wt/vol) neutral buffered paraformaldehyde while a constant airway pressure of 10 cmH2O was maintained via a tracheal catheter. Paraffin-embedded tissues were cut into 5-µm sections, mounted on Superfrost/Plus slides, and immunostained by an avidin-biotin-peroxidase method (14). Concentrations of the primary antisera were 1/200 for PDGF-{alpha}R (1 µg/ml) and1/25 for PDGF-{beta}R (10 µg/ml). Specificity of the PDGF receptor antibodies, using specific blocking peptides, was determined as previously described (8). After light counterstaining with hematoxylin, sections were dehydrated, cleared in xylene, and mounted using Permount. For detection of BrdU, immunoperoxidase staining was performed using a biotin-conjugated mouse monoclonal antibody as part of a commercially available in situ detection kit according to the manufacturer's instructions. Images from stained sections were digitally captured using a Leica DC200 camera and Leica DC Viewer software (Leica Microsystems, Wetzlar, Germany).

Quantitation of BrdU-labeled SMC nuclei. For quantitation of BrdU-labeled SMC nuclei, eight BrdU-stained sections were examined from each animal (4 from the right lung and 4 from the left) by an observer blinded to group assignment. Using an eyepiece micrometer, small muscular or partially muscular arteries of 30- to 120-µm external diameter were identified (associated with airways of <250-µm external diameter) where the complete circumference of the vessel was visible. All arteries that fulfilled these criteria (on average, >20/animal) were photographed under oil immersion for counting of nuclei. Medial wall SMC nuclei (identified by their location and characteristic elongated shape) that stained positively for BrdU were counted and expressed as a percentage of the total number of SMC nuclei for each animal. Results are shown as mean values from four animals per group.

Fluorescent immunostaining of frozen lung tissue. Two randomly selected animals from each group (from separate litters) were anesthetized with ip ketamine (80 mg/kg) and xylazine (20 mg/kg). After opening of the thoracic cavity and cannulation of the trachea, the pulmonary veins were divided. The pulmonary circulation was flushed with 1x PBS containing 1 U/ml of heparin to clear the lungs of blood while the lungs were inflated with frozen tissue matrix (Tissue-Tek OCT) diluted 1:3 with 20% (wt/vol) sucrose in PBS. The heart and lungs were then removed en bloc and covered with OCT matrix using a plastic mold placed on dry ice until the tissue and matrix were frozen and stored at –80°C until sectioning. Frozen tissue was cut by cryostat into 10-µm sections and mounted on Superfrost/Plus slides, which were then dried for 20 min at room temperature. Sections were fixed in ice-cold neutral buffered 3% (wt/vol) paraformaldehyde for 5 min and permeabilized with 2% (vol/vol) Triton X in 1% (wt/vol) BSA for 4 min at room temperature. After being washed in 1x PBS, sections were blocked with 1% (wt/vol) BSA/5% (vol/vol) NGS for 1 h followed by overnight incubation at 4°C with primary antisera. Concentrations of the primary antisera were as follows: {alpha}-smooth muscle actin (1/500 = 0.4 µg/ml), PDGF-{alpha}R (1/100 = 2 µg/ml), and PDGF-{beta}R (1/25 = 10 µg/ml). Sections were incubated with FITC- or rhodamine-conjugated secondary antisera diluted to 1:300 with blocking solution at room temperature in the dark for 2 h. After washing, staining of cell nuclei with DAPI, and rinsing in water, sections were mounted in aqueous mounting medium and left to dry overnight at room temperature. Images were digitally captured using a Zeiss LSM 510 META laser scanning microscope and Zeiss LSM Image Browser software (Carl Zeiss, Oberkochen, Germany).

Western blot analyses. After opening of the thoracic cavity and cannulation of the trachea, the pulmonary veins were divided and the pulmonary circulation was flushed of blood via a needle placed in the right ventricle using PBS with 1 U/ml of heparin until the lungs became white. Lungs were flash frozen in liquid N2 before being homogenized in RIPA cell lysis buffer [10 mM NaPO4, 0.3 M NaCl, 0.1% (wt/vol) SDS, 1% (vol/vol) Nonidet P-40, 1% (vol/vol) sodium deoxycholate, and 2 mM EDTA, pH 7.2] with the protease inhibitors leupeptin and aprotinin (2 µg/ml each) and 1 mM PMSF. The homogenate was left on ice for 10 min before centrifugation at 7,000 g for 10 min. Samples were then stored at –80°C until analysis. Sample protein concentrations were measured spectrophotometrically using known concentrations of BSA as standards (7). Lung tissue lysates containing 50 µg of total protein were boiled for 5 min in SDS sample buffer [60 mM Tris·HCl, 10% (wt/vol) SDS, 5% (vol/vol) glycerol, and 2 mM 2-mercaptoethanol, pH 6.8] and fractionated under reducing conditions by SDS polyacrylamide gel electrophoresis for 2 h at 120 V. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes. All membranes were blocked with 5% (wt/vol) nonfat milk in TBST [20 mM Tris base, 137 mM NaCl, pH 7.6, with 0.1% (vol/vol) Tween 20] overnight at 4°C and then incubated with antibodies to either PDGF-{alpha}R (190 kDa, 1/500 = 0.4 µg/ml) or PDGF-{beta}R (170 kDa, 1/250 = 1 µg/ml) for >2 h at room temperature. To control for differences in protein loading, each blot was coincubated with antibody to GAPDH (~30 kDa, 1/500; 0.4 µg/ml), which we have previously determined is not affected by exposure to 60% O2 (18). Blots were then washed in TBST and placed in the appropriate secondary antibody for >1 h at room temperature. The protein bands were imaged using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ) and exposed for 30–120 s on Kodak X-Omat Blue XB-1 film (Eastman Kodak, Rochester, NY). The films were electronically scanned and the band densities quantified using ImageJ software (version 1.30; National Institutes of Health, Bethesda, MD).

Pulmonary artery smooth muscle cell culture. Pulmonary artery smooth muscle cells (PASMC) were isolated by an explant technique using pooled pulmonary arteries from a litter (10–12 pups) of day 14 Sprague-Dawley rats, as previously described (16). All experiments using PASMC from the same litter were performed in quadruplicate using cells from the first passage only. Equal numbers of cells (105/well) were seeded in 48-well plates and grown to semiconfluence with 10% (vol/vol) FBS and then serum starved for 48 h in a gas phase of 21% O2, 5% CO2, and 74% N2. PASMC were incubated with DMEM (control) or with added PDGF-AA (20 ng/ml), PDGF-BB (20 ng/ml), PDGF-{beta}R/Fc chimeric protein (12 µg/ml), or 1% (vol/vol) FBS for 24 h at a gas phase of 21% O2, 5% CO2, and 74% N2.

Measurement of PASMC proliferation. Rates of cellular DNA synthesis, as a proliferation marker, were assessed by determination of [3H]thymidine incorporation into DNA as previously described (12, 29). Cells were incubated for 24 h with 1 µCi/ml [3H]thymidine at 37°C at a gas phase of 21% O2, 5% CO2, and 74% N2. The cells were then washed twice in ice-cold 1x PBS, twice in ice-cold 10% (wt/vol) trichloroacetic acid, and once with 75% (vol/vol) ethanol in ether and left to dry at room temperature. The precipitated material was then solubilized with 0.2 N sodium hydroxide at room temperature for >2 h. An aliquot was diluted in scintillation cocktail containing 1% glacial acetic acid and left in the dark overnight before counting. Results are expressed as a percentage of control (no serum or growth factors) values for disintegrations per minute/well.

Data presentation and analysis. All values are expressed as means ± SE. For in vitro studies, at least one replicate using PASMC obtained from another litter of pups was performed to ensure reproducibility of results. Statistical significance (P < 0.05) was determined using SigmaStat 3.0 software (SPSS, Chicago, IL). Where two groups were compared, the Student's t-test was used; for nonparametric data, the Mann-Whitney rank sum test was used. Where three or more groups were compared, one-way or two-way ANOVA was performed followed by pairwise multiple comparisons using Tukey's test if significant differences were found.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Immunohistochemistry for the candidate smooth muscle growth factor receptors, PDGF-{alpha}R and -{beta}R, from the lungs of rat pups exposed to air or 60% O2 for 14 days is shown in Fig. 1. As seen in Fig. 1B, expression of PDGF-{alpha}R was increased by exposure to 60% O2. Compared with air-exposed animals (Fig. 1A), this increased expression was particularly localized to distal airway epithelium where active secondary septation was apparent and to the vascular wall (Fig. 1B). Expression of PDGF-{beta}R was markedly increased in both the lung parenchyma generally and in the walls of vessels by exposure to 60% O2 (Fig. 1D), compared with air-exposed animals, in which very little expression was evident (Fig. 1C). Laser scanning confocal microscopy was performed on sections of frozen lung tissue from neonatal rats exposed to air or 60% O2 for 10 days. Sections were coimmunostained for the smooth muscle marker, {alpha}-actin, to identify smooth muscle, as opposed to endothelial localization of PDGF receptors. As shown in Fig. 2D for PDGF-{alpha}R, and to an even greater extent for the PDGF-{beta}R (Fig. 2F), upregulation was largely evident on SMC of the vascular wall after exposure to 60% O2, compared with no expression in air-exposed controls (Fig. 2, C and E). No increased expression was evident for either of the PDGF receptors after 4 or 7 days of exposure to 60% O2 (data not shown).



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  		Fig. 1. Representative immunohistochemistry for platelet-derived growth factor {alpha}-receptor (PDGF-{alpha}R; A and B) or platelet-derived growth factor {beta}-receptor (PDGF-{beta}R; C and D) expression in the lungs of rat pups exposed to either air (A and C) or 60% O2 (B and D) for 14 days. Bar lengths = 100 µm. Insets: higher power views of pulmonary arteries enclosed by boxes. Air-exposed pups at 14 days had minimal expression of PDGF-{alpha}R (A) or PDGF-{beta}R (C), either in the parenchyma or the arterial vascular (v) walls. B: expression of PDGF-{alpha}R in 60% O2-exposed pups was increased in both distal air space walls where secondary septation is taking place and in the arterial wall (inset, arrow). D: expression of PDGF-{beta}R in 60% O2-exposed pups was generally increased, especially in the arterial wall (inset, arrow).

 


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  		Fig. 2. Laser scanning confocal microscopy for PDGF-{alpha}R (C and D) or PDGF-{beta}R (E and F) expression (green fluorescence) in frozen lung tissue sections from rat pups exposed to either air or 60% O2 for 10 days and coimmunostained with {alpha}-smooth muscle actin (red fluorescence). The medial walls of pulmonary arteries, where colocalization is found, are shown in yellow (arrows). Bar lengths = 20 µm. A: no fluorescence was seen in an artery (shown by phase-contrast microscopy in B) from a 60% O2-exposed pup in which primary antibodies were omitted. Arteries from air-exposed pups showed no expression of either PDGF-{alpha}R (C) or PDHF-{beta}R (E). In 60% O2-exposed pups, areas of colocalization with {alpha}-smooth muscle actin were seen for PDGF-{alpha}R (D). Colocalization with {alpha}-smooth muscle actin was evident to an even greater extent for PDGF-{beta}R (F).

 
Western blot analyses of PDGF-{alpha}R and PDGF-{beta}R in the total lung were performed after 4, 7, and 14 days (Fig. 3) of exposure to air or 60% O2. Consistent with our findings on immunohistochemistry, immunoreactive PDGF-{alpha}R and PDGF-{beta}R were significantly increased after 14 days of exposure to 60% O2 (P < 0.05 compared with air controls) and not at earlier time points, although there was a nonsignificant trend toward increased expression of the PDGF-{beta}R after 7 days of exposure to 60% O2 (P > 0.05 compared with air controls).



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  		Fig. 3. A: Western blot analyses for total lung expression of PDGF-{alpha}R (190 kDa) and PDGF-{beta}R (170 kDa) expression, normalized to GAPDH (~30 kDa), after exposure to air or 60% O2 for 4, 7, or 14 days. *P < 0.05, by t-test (means ± SE of 3 litters/group), compared with air controls at the same time point. B: images of Western blots for the PDGF receptors and GAPDH after 14 days of exposure to either air or 60% O2.

 
As shown in Fig. 4, treatment with the combined ET receptor antagonist SB-217242 had no effect on the O2-induced upregulation of the PDGF-{alpha}R (Fig. 4D) evident in vehicle-treated animals (Fig. 4B). In contrast, as shown in Fig. 5, SB-217242 completely prevented the 60% O2-induced upregulation of PDGF-{beta}R (Fig. 5D) that was evident in vehicle-treated animals (Fig. 5B).



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  		Fig. 4. Representative immunohistochemistry for PDGF-{alpha}R expression in small pulmonary arteries of rat pups exposed either to air (A and C) or 60% O2 (B and D) for 14 days and receiving daily intraperitoneal injections of either vehicle (A and B) or SB-217242 (C and D). Bar lengths = 50 µm. A: pups exposed to air and treated with vehicle had minimal expression of PDGF-{alpha}R. Increased PDGF-{alpha}R expression caused by exposure to 60% O2 in vehicle-treated animals (B) remained increased in SB-217242-treated animals (D).

 


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  		Fig. 5. Representative immunohistochemistry for PDGF-{beta}R expression in small pulmonary arteries of rat pups exposed either to air (A and C) or 60% O2 (B and D) for 14 days and receiving daily intraperitoneal injections of either vehicle (A and B) or SB-217242 (C and D). Bar lengths = 50 µm. A: pups exposed to air and treated with vehicle had minimal expression of PDGF-{beta}R. Increased PDGF-{beta}R expression caused by exposure to 60% O2 in vehicle-treated animals (B) was completely attenuated by treatment with SB-217242 (D).

 
Effects of PDGF-AA and -BB on proliferation (using DNA synthesis as a marker) and hyperplasia in PASMC from infant rats are shown in Fig. 6. PDGF-BB caused a significant increase in PASMC proliferation (P < 0.05 compared with the control group), although not as great as that induced by serum (P < 0.05 compared with all other groups). This PDGF-BB-induced effect was not amplified by the coaddition of PDGF-AA (P > 0.05 compared with PDGF-BB alone), nor did PDGF-AA alone have any effect (Fig. 6A; P > 0.05 compared with control group). PDGF-BB also led to significant PASMC hyperplasia, similar to that observed for serum alone (Fig. 6B; P < 0.05 compared with control group). Cotreatment with the PDGF-{beta}R/Fc chimeric protein significantly decreased PDGF-BB-induced PASMC proliferation to 76 ± 4.0% of cells treated with PDGF-BB alone (P < 0.05) but had no significant effect on serum-induced proliferation (93 ± 4.7% of cells treated with serum alone; P > 0.05).



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  		Fig. 6. A: [3H]thymidine incorporation into DNA, as a proliferation marker, in cultured pulmonary artery smooth muscle cells from infant rat pups in response to either serum- and growth factor-free conditions (control; open bar), PDGF-AA (20 ng/ml), or PDGF-BB (20 ng/ml) alone, PDGF-AA and PDGF-BB combined (black bars), or 1% FBS (gray bar). *P < 0.05, by 1-way ANOVA (means ± SE of 4–6 wells/group), compared with controls. #P < 0.05, by ANOVA, compared with all other groups. B: cell counts of cultured pulmonary artery smooth muscle cells from infant rat pups following exposure to either serum- and growth factor-free conditions (control; open bar) or to PDGF-AA, PDGF-BB (black bars), or FBS (gray bar) at the same concentrations used in A. *P < 0.05, by 1-way ANOVA (means ± SE of 4–6 wells/group), compared with control and PDGF-AA groups.

 
In keeping with our in vitro findings, a major role for PDGF-{beta}R ligands in 60% O2-mediated medial SMC proliferation was confirmed in vivo (Fig. 7). Exposure to 60% O2 for 9 days increased the number of BrdU-labeled nuclei in the medial walls of muscular and partially muscular respiratory bronchiolar arteries (Fig. 7, B and E; P < 0.05, by two-way ANOVA) compared with air-exposed controls (Fig. 7, A and E). Treatment with the PDGF-{beta}R/Fc chimeric protein significantly attenuated this 60% O2-mediated increase (Fig. 7, D and E; P < 0.05, by 2-way ANOVA, compared with vehicle-treated 60% O2-exposed animals).



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  		Fig. 7. Representative immunohistochemistry (A–D) for 5'-bromo-2'-deoxyuridine (BrdU; brown-stained nuclei) as a marker of DNA synthesis in the pulmonary arterial walls of animals exposed to either air or 60% O2 for 9 days and treated with either 1x PBS with 0.1% BSA (vehicle) or soluble PDGF-{beta}R/Fc chimeric protein in 1x PBS with 0.1% BSA (sPDGF-{beta}R) intraperitoneally 24 h before death. Bar lengths = 50 µm. Small pulmonary resistance arteries from vehicle-treated (A) and sPDGF-{beta}R-treated (C) air-exposed animals show no BrdU-stained smooth muscle cell nuclei (unstained nuclei indicated by arrows). B: artery from a vehicle-treated 60% O2-exposed animal with 3 BrdU-stained smooth muscle cell nuclei (arrows). D: artery from sPDGF-{beta}R-treated 60% O2-exposed animal demonstrating a lack of proliferating smooth muscle cells. E: BrdU-stained smooth muscle cells expressed as a percentage of total medial wall nuclei (means ± SE of 4 animals per group). *P < 0.05, by two-way ANOVA, for vehicle-treated 60% O2-exposed animals compared with all other groups.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Exposure of rats to 60% O2 from birth leads to thickening of the medial vascular smooth muscle layer and right ventricular hypertrophy by day 14 of life (20). We have previously reported that the development of these vascular changes was accompanied by greatly increased expression of ET-1 in the lung (16, 18). Treatment with a combined ET receptor antagonist (SB-217242) prevented right ventricular hypertrophy and medial wall thickening (17), confirming a critical role for ET receptors in this model of pulmonary hypertension. In other work, we have also reported a 60% O2-induced upregulation of PDGF-BB on pulmonary vessels of the newborn rat lung (8), a growth factor with a putative role in pulmonary vascular remodeling (3).

In this study, we have shown that 60% O2-induced vascular remodeling was accompanied by increased SMC proliferation in the medial walls of small (≤120-µm external diameter) pulmonary resistance arteries, consistent with previous reports on hypoxia-induced models of pulmonary hypertension (4, 22). As shown by immunohistochemistry and Western blot analyses, these changes in vascular smooth muscle proliferation and mass were accompanied by upregulation of the PDGF-{alpha}R and PDGF-{beta}R, which are putative receptors mediating SMC growth. We hypothesized that PDGF receptor upregulation may have been causally related to increased ET-1 abundance. We tested this hypothesis by examining PDGF receptor expression in the lungs of SB-217242-treated animals and found that 60% O2-induced upregulation of the PDGF-{beta}R, but not the PDGF-{alpha}R, on vascular smooth muscle, was prevented by this intervention.

We examined in vitro proliferative responses of PASMC cultured from infant rats and found that PDGF-BB, but not PDGF-AA, induced SMC DNA synthesis and hyperplasia, suggesting that these effects occurred through stimulation of the PDGF-{beta}R. An important in vitro role for the PDGF-{beta}R was confirmed by treating PASMC with a recombinant truncated soluble PDGF-{beta}R, which inhibits ligand binding to the native receptor by acting as a decoy. Using this approach, we confirmed the specificity of the soluble PDGF-{beta}R for PDGF ligands by inhibiting PDGF-BB-induced, but not serum-induced, PASMC proliferation. Soluble receptors have been widely used in the past to tease out the role of growth factors and their receptors in biological processes such as organogenesis during fetal development (9) and tumor growth (21). We have previously demonstrated that the Fc portion of this chimeric protein has no independent effect on lung cell DNA synthesis in vivo by using the chimeric soluble receptor for nerve growth factor (NGF-R/Fc), a growth factor receptor that has no apparent effect on lung growth, as a negative control (32). A pharmacological soluble receptor approach to specifically analyze the role of the PDGF-{beta}R in vivo has been previously applied by us for studies of alveologenesis (8, 32) and by others for studies of liver injury (6). In this study, we have again been able to make use of this approach to define a role for ligands binding to the PDGF-{beta}R in 60% O2-mediated vascular SMC proliferation in newborn rats.

Together, our findings in this study are consistent with a role for PDGF-BB as a vascular smooth muscle growth factor, through an autocrine and/or paracrine action on the upregulated SMC PDGF-{beta}R. Activation of the PDGF-{beta}R by PDGF-AA in vivo could not be excluded on the basis of the current findings. However, recent direct evidence from the ovine fetus confirming the importance of dimers containing the PDGF-B chain (3) in the pathogenesis of ductal ligation-induced pulmonary hypertension, further supports our conclusions on the likely role of PDGF-BB. The mechanisms of ET-1-induced SMC PDGF-{beta}R upregulation, which appears to be necessary for abnormal SMC proliferation in O2-exposed animals, remain unknown. Several possible explanations are that it represents a direct effect of ET-1 on PDGF-{beta}R gene expression or a mechanically mediated effect through sustained ET-1-induced vasoconstriction, leading to enhanced strain on SMC and subsequent upregulation and activation of the PDGF-{beta}R (31).

A limitation of our study is that we did not examine other potential mechanisms by which PDGF receptors may contribute to vascular remodeling, such as through the induction of SMC hypertrophy (15), through an antiapoptotic effect (33), or through increased synthesis of extracellular matrix proteins (5), which are all believed to play important roles in vascular remodeling (19). Moreover, our findings in the present study do not rule out the possibility of upregulation of the PDGF-{alpha}R, perhaps through a direct oxidant-induced mechanism, also playing a role in vivo. An important additive or synergistic role for other growth factor receptors that are expressed on vascular smooth muscle, such as the insulin-like growth factor type I receptor and one or more of the fibroblast growth factor receptors (1, 2, 23), is also possible.

In conclusion, the observations reported herein strengthen the argument for a major role of the PDGF-{beta}R and its ligands, particularly PDGF-BB, in the pathogenesis of vascular SMC proliferation in newborn pulmonary hypertension. These data provide a further rationale for the development and examination of interventions targeting the PDGF-{beta}R to either prevent or treat pulmonary vascular remodeling. For the newborn, such an intervention should ideally target vascular SMC and not other cell types, since global inhibition of PDGF-{beta}R function in the developing lung, as we have already shown (8), will have an adverse impact on normal postnatal lung growth.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by group grants from the Canadian Institutes of Health Research (CIHR; M. Post and A. K. Tanswell), a seed grant from the Sunnybrook & Women's Research Institute (R. P. Jankov), and an infrastructure grant from the Canada Foundation for Innovation New Opportunities Fund (R. P. Jankov). R. P. Jankov is a CIHR Strategic Training Fellow in the Canadian Child Health Clinician Scientist Program. M. Post holds a Tier 1 Canada Research Chair from the CIHR. A. K. Tanswell holds the Hospital for Sick Children Women's Auxiliary Chair in Neonatal Medicine. S. Yi was supported by a studentship through the Ontario Student Opportunity Trust Fund and the Hospital for Sick Children Foundation Student Scholarship Program.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. P. Jankov, Dept. of Newborn and Developmental Pediatrics, Sunnybrook & Women's College Health Sciences Center, 76 Grenville St., Toronto, Ontario M5S 1B2, Canada (E-mail: robert.jankov{at}sw.ca)

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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