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Am J Physiol Lung Cell Mol Physiol 292: L559-L566, 2007. First published October 6, 2006; doi:10.1152/ajplung.00075.2006
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Cyclic stretch induces PlGF expression in bronchial airway epithelial cells via nitric oxide release

Kamal A. Mohammed,1 Najmunnisa Nasreen,1 Robert S. Tepper,2 and Veena B. Antony1

1Division of Pulmonary and Critical Care Medicine, Department of Medicine, Malcom Randall Veterans Affairs Medical Center, University of Florida, Gainesville, Florida; and 2Department of Pediatrics, Indiana University Medical Center, Indianapolis, Indiana

Submitted 3 March 2006 ; accepted in final form 29 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mechanical strain of lung tissue is an important stimulus for the production of growth factors that are critical for lung growth and development. However, excessive mechanical strain, as may occur during mechanical ventilation, may produce an increase in growth factors that may contribute to lung injury. We hypothesized that mechanical strain of primary bronchial airway epithelial cells (BAEpCs) induced the production of placental growth factor (PlGF), a member of the VEGF family. BAEpCs were cultured on a deformable silicoelastic membrane and exposed to different magnitudes of stretch. Stretch induced PlGF and nitric oxide (NO) production that increased with increasing magnitude of stretch. Stretch also induced PlGF and inducible NO synthase (iNOS) gene expression. The stretch-induced PlGF production and NO synthesis were attenuated by PD98059, a specific mitogen-activated protein kinase kinase-1 and -2 inhibitor. Inhibition of NO generation by L-NAME or L-NMMA or scavenging NO by carboxy-PTIO prevented stretch-mediated erk1/2 activation. In addition, in unstretched BAEpCs, exogenous NO enhanced erk1/erk2 activation. Our data suggest that mechanical stretch of BAEpCs induces iNOS expression and induces PlGF release in an erk1/2 activation-dependent manner.

placental growth factor; mitogen-activated protein kinases

MECHANICAL STRAIN OF LUNG tissue is an important stimulus for lung growth, particularly early in life (6, 15). Stretching lung tissue in vivo, as well as in cell culture, increases the production of growth factors in the VEGF family, which are critical for lung growth and development (14). However, overexpression of growth factors in vivo can produce abnormal lung development. In mice, constitutive overexpression of placental growth factor (PlGF), which is a member of the VEGF family, produces enlarged alveolar air spaces (26, 27), similar in appearance to the pathological finding of premature infants that require mechanical ventilation and develop bronchopulmonary dysplasia (BPD) (4). In addition, an elevated level of PlGF in cord blood of premature infants is associated with an increased risk of developing more severe BPD, which includes airways, as well as parenchymal disease (17, 27). PlGF binds with high affinity to VEGF receptor-1 [VEGFR-1; also known as FMS-related tyrosine kinase-1(flt)], which is present on airway epithelium of human fetal and neonatal lungs (9). Expanding the airways during normal breathing and during mechanical ventilation stretches the bronchial epithelium as well as the other components of the airway wall. Because the bronchial epithelium is an important modulator of epithelial-mesenchymal structure and function, straining bronchial epithelial cells may be a source of PlGF. The cellular mechanisms for induction of PlGF by mechanical strain of bronchial epithelial cells have not been defined.

Mitogen-activated protein kinase (MAPK) cascades play a key role in transduction of extracellular signals to cellular responses. Extracellular signal-regulated kinase (erk1/erk2) belongs to MAPK, and erk1/erk2 is activated by various extracellular stimuli through Raf-1 -> MAPK kinase-1 (Mek-1) -> erk1/erk2. This kinase cascade participates in regulation of gene expression by connecting the extracellular signal to intracellular transcriptional elements and regulatory proteins (5, 24). In addition, nitric oxide (NO) has long been implicated as an important signaling molecule in cells exposed to mechanical strain (8). In endothelial cells, physiological levels of mechanical strain induce NO production via erk1/2 MAPK activation (18). Therefore, we hypothesized that, in primary bronchial airway epithelial cells, mechanical stretch would trigger erk1/2 activation and PlGF expression via NO synthesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents and cell cultures. Recombinant PlGF and anti-PlGF antibodies with matching isotype antibodies were purchased from Peprotech. L-NG-monomethyl arginine monoacetate (L-NMMA), N-nitro-L-arginine methyl ester (L-NAME), sodium nitroprusside (SNP), and carboxy-PTIO were purchased from Calbiochem. Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Norcross, Atlanta, GA), bronchial epithelial growth media (BEGM) were purchased from Clonetics. Human PlGF Quantikine ELISA kits and primers for inducible NO synthase (iNOS) and PlGF were purchased from R&D Systems (Minneapolis, MN). Primary human bronchial airway epithelial cells (BAEpCs) were purchased from Cell Applications (San Diego, CA).

BAEpC culture and cell stretch. The cell culture flasks were precoated with BEGM coating media supplemented with 10 µg/ml fibronectin, 30 µg/ml vitrogen, and 10 µg/ml BSA. Human BAEpCs were cultured in BEGM as reported earlier (1). Human BAEpCs were cultured in BEGM according to the supplier's recommendations. The Flexercell 2000 cell stretching system (Flexercell) was used to stretch BAEpCs. Bronchial airway epithelial cells were subcultured on deformable silicoelastic membranes (BioFlex, Flexcell International). The membranes were subjected to variable (5–20%) cyclic stretch with 30 cycles/min for varying time intervals. The application of this negative pressure results in deformation of the membrane and, in turn, the BAEpCs attached on the upper surface of the membrane. Some culture plates were incubated simultaneously in the same incubators but without stretch to serve as controls. The conditioned media were saved at –70°C until estimation of PlGF levels by ELISA. BAEpC viability was checked by Trypan blue dye exclusion after 72 h. The cell viability was >98% in all selected magnitudes of stretch. No significant difference in cell viability was noticed compared with unstretched cultures.

NO estimation. NO production was estimated via assay for nitrite (NO2) and nitrate (NO3). BAEpCs were cultured in silicoelastic plates and subjected to variable magnitudes of stretch, and cell culture supernatant was harvested after a designated time interval. Fifty microliters of supernatants from each sample were assayed for NO production. Experiments were repeated three times with duplicate samples. Briefly, 25 mU of nitrate reductase with 40 µM NADPH in 25 µl of buffer were added to the samples and incubated for 1 h at 25°C. The reaction was stopped by the addition of 25 µl of buffer containing 4 mM beta-ketoglutarate, 100 mM ammonium chloride, and 200 mM glucose-6-phosphate dehydrogenase. After further incubation for 10 min, 150 µl of Griess reagent (0.1% naphthyl ethylene diamine dihydrochloride-1% sulfonic acid in 5% phosphoric acid mixed in equal volumes) were added to each sample at room temperature. After thorough mixing for 5 min, optical density was measured at 540 nm, and the amount of NO3/NO2 was calculated on the basis of standard curves generated by sodium nitrite.

PlGF ELISA. PlGF levels in stretched BAEpC culture supernatants were measured by a "sandwich" enzyme immunoassay (Quantikine ELISA, R&D Systems) as previously described (1). Briefly, the samples were added to 96-well microtiter plates coated with murine monoclonal antibody to PlGF. The unbound protein was washed three times, and an enzyme-linked polyclonal antibody specific to PlGF was added. The plates were again washed three times, and substrate solution was added to the wells. After 30 min of incubation, to each well, stop solution was added. The amount of PlGF was determined by optical density of the samples by comparison with the standards at 450 nm using an ELISA reader. The detection limit of the PlGF kit was 2.0 pg/ml for cell culture supernatants.

Total RNA extraction and quantitative real-time PCR analysis. Total RNA from cultured BAEpCs was purified using the High Pure RNA isolation kit (Roche Applied Science, Indianapolis, IN). The total RNA samples were diluted with RNase-free water to 100 ng/µl, and 10 µl of each sample with 1 µl of 3.5 µM anchored oligo(dT) 23 (Sigma, St. Louis, MO) were denatured at 70°C for 10 min followed by a 2-min incubation on ice. The cDNA strand was generated using avian myeloblastosis virus (AMV) RT and 20 units of RNase inhibitor, and the reaction mix was incubated for 50 min at 42°C followed by an enzyme inactivation step of 15 min at 70°C. The cDNA was stored at –80°C until subsequent analysis. For PCR, the reaction mix contained a total 50 µl that contain 10 µl of cDNA, 25 µl of SYBR Green JumpStart Taq ReadyMix, 0.5 µl of internal reference dye, and 14.5 µl of corresponsive oligonucleotide primers (80 nM final concentration). The following primer sequences were used: PlGF, sense 5'-GAGAGAAGCCAGCCACAGAC-3' and antisense 5'-GTTTCT-CATCCAGGCAGCTC-3'; beta-actin, sense 5'-agagctacgagctg-cctgac-3' and antisense 5'-aaagccatgccaatctcatc-3'. The iNOS primer pair was purchased from R&D Systems. Amplification and detection were performed by the SYBR Green method using the Applied Biosystems 7500 Real Time PCR system with the following profile: 1 cycle at 94°C for 2 min, 40 cycles at 94°C for 15 s, 60°C for 1 min, and 72°C for 1 min. The fluorescence resulted from the incorporation of SYBR Green dye into the double-stranded DNA produced during the PCR reaction, and emission data were quantified using the threshold cycle (Ct) value. Ct readings obtained represent measurements on the log scale. A melting point dissociation curve generated by the instrument was used to confirm that only a single product was present. To validate the specificity of a primer set, RNA (1–3 µg) and the RT negative were analyzed in triplicate to confirm that there was no fluorescence resulting from either genomic DNA contamination or from the RT step. Each PCR run also included triplicate wells of no template control (NTC), where RNase-free water was added to reaction wells. The PCR products were confirmed by electrophoresis on a 2% agarose gel (data not shown).

Data analysis was carried out by ABI sequence detection software using relative quantification. Ct, which is defined as the cycle at which PCR amplification reaches a significant value, is given as the mean value. The relative expression of each mRNA was calculated by the {Delta}Ct method [where {Delta}Ct is the value obtained by subtracting the Ct value of the housekeeping gene (subtract actin mRNA from the Ct value of the target mRNA)]. The amount of the target gene relative to the house keeping gene was expressed as 2–({Delta}{Delta}Ct).

erk1/erk2 MAPK estimation by Western blot analysis. Stretched and unstretched BAEpC pellets were collected using a rubber policeman. All cell pellets were stored at –20°C until use. Cell pellets were resuspended in 60 µl of Laemmli buffer and sonicated for 10 s. Concentrations of proteins were determined by the Bio-Rad Calibrated Imaging Densitometer using Quantity One software. Equal concentrations of protein were loaded onto 12-well precast 7.5% Criterion gels (Bio-Rad Pharmaceuticals, Hercules, CA). Gels were run in 25 mM Tris, 192 mM glycine, and 0.1% SDS at pH 8.3 at 200 V for 45 min. Proteins were then transferred to a polyvinyl difluoride (PVDF; 0.45-µm pore size) membrane using 25 mM Tris, 192 mM glycine, and 20% methanol at pH 8.3 at 200 V for 1 h. PVDF membranes were washed with PBS-T, and Western analysis was performed according to the manufacturer's directions. Anti-erk1/erk2 (1:400) or anti-phosphotyrosine erk1/erk2 (1:200) and anti-mouse IgG horseradish peroxidase (1:1,000)-linked whole antibodies were used as primary and secondary antibodies, respectively. Protein detection was performed using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).

Statistical analysis. The significance of differences between experimental and control groups was tested by ANOVA using Sigma-Stat statistical software. The significance of difference between the two groups was tested by an all pairwise multiple-comparison procedure (Student-Newman-Keuls method), and P value < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cyclic stretch induced PlGF expression in BAEpCs. The release of PlGF was dependent on the magnitude of stretch (Fig. 1A). When BAEpCs were subjected to 5% cyclic stretch, the PlGF production was significantly greater than PlGF production by unstretched BAEpCs (P < 0.001). Increasing the magnitude of the stretch to 10 and 20% resulted in statistically significant increases in PlGF production at each increased level of stretch (P < 0.001). Stretches of 5, 10, and 20% produced 5-, 8-, and 11-fold increases, respectively, in PlGF production compared with unstretched cells. The time course for PlGF production was evaluated with BAEpCs exposed to 20% stretch for time intervals of 6, 24, 48, and 72 h (Fig. 1B). An increase in PlGF was not detected following 6 h of stretch; however, following 24 h, PlGF was significantly increased compared with unstretched cells. There was also a progressive increase in PlGF with increasing time at 48 and 72 h, whereas there was no significant increase in PlGF with time for unstretched BAEpCs.


Figure 1
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  		Fig. 1. Cyclic stretch induced placental growth factor (PlGF) release in bronchial airway epithelial cells. Primary bronchial airway epithelial cells (BAEpCs) were subjected to varying percent stretch for 24 h, and PlGF released into culture media was measured. A: PlGF release vs. percentage of stretch in BAEpCs. Data presented are means ± SE of 3 independent experiments, each evaluated separately. Stretched vs. no stretch: *P < 0.001. Comparison between 2 different magnitudes of stretch: #P < 0.001. B: PlGF release over time in BAEpCs. BAEpCs were subjected to 20% stretch for varying time intervals (6–72 h). Data presented are means ± SE of 3 independent experiments, each evaluated separately. Stretched vs. no stretch: *P < 0.001. Comparison between 2 different times of stretch: #P < 0.001. NS, not significant.

 
Stretch induced NO release in BAEpCs. BAEpCs were subjected to cyclic stretch of 5, 10, and 20%, and NO release was measured. BAEpCs exposed to 5% stretch released significantly higher amounts of NO than unstretched BAEpCs (P < 0.001) (Fig. 2A). Increasing the magnitude of stretch resulted in progressively higher amounts of NO at 10 and 20% (P < 0.001). The time response of NO production was evaluated in BAEpCs exposed to 20% stretch (Fig. 2B). Following 6 h of stretch, there was a significant increase in NO production compared with unstretched BAEpCs (P < 0.001). There was a significantly greater increase in NO production following 12 h of stretch (P < 0.001), and then NO production remained relatively constant at an elevated level compared with unstretched BAEpCs. For unstretched BAEpC, there was no change in NO production over 72 h.


Figure 2
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  		Fig. 2. Cyclic stretch induced nitric oxide (NO) release in bronchial airway epithelial cells. BAEpCs were subjected to varying percent stretch for 24 h, and NO released into culture media was measured. A: NO release under variable percentage of stretch in BAEpCs. Data presented are means ± SE of 3 independent experiments, each evaluated separately. Stretched vs. no stretch: *P < 0.001. Comparison between 2 different magnitudes of stretch: #P < 0.001. B: NO release over time in BAEpCs. BAEpCs were subjected to 20% stretch for varying time intervals (6–72 h). Data presented are means ± SE of 3 independent experiments, each evaluated separately. Comparison of stretch vs. no stretch: *P < 0.001. Comparison between 2 different times of stretch: #P < 0.001. @P < 0.05 compared with stretch at 72 vs. 48 h.

 
Cyclic stretch-induced PlGF release in BAEpCs was NO dependent. To understand the role of NO in stretch-mediated PlGF induction in BAEpC, a NO scavenger (PTIO, 300 µM) was included in some cultures of stretched BAEpCs. PTIO significantly inhibited PlGF release by BAEpCs stretched for 24, 48, and 72 h (Fig. 3A). In addition, some BAEpCs were stretched in the presence of L-NMMA (8 mM), an iNOS inhibitor. Stretch-mediated PlGF production was significantly decreased in the presence of L-NMMA at 24, 48, and 72 h postincubation in BAEpCs (Fig. 3B).


Figure 3
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  		Fig. 3. Effect of NO synthase (NOS) inhibitor (L-NMMA) and NO scavenger (PTIO) on cyclic stretch-induced PlGF release in BAEpCs. BAEpCs were subjected to 20% stretch for varying time intervals (6–72 h). A: BAEpC cultures were pretreated with PTIO (300 µM) 30 min before stretch, as discussed in MATERIALS AND METHODS. B: BAEpC cultures were pretreated with L-NMMA (4 mM) 30 min before stretch, as discussed in MATERIALS AND METHODS. Data presented are means ± SE of 3 independent experiments, each evaluated separately. *P < 0.001 compared with stretched cells.

 
Cyclic stretch of BAEpCs induced erk1/erk2 MAPK activation that was NO dependent. MAPKs relay and integrate signals from a diverse range of stimuli to cells. Cyclic stretch induced erk1/erk2 (p44/42 MAPK) phosphorylation in BAEpCs in a time-dependent manner (Fig. 4). erk1/erk2 phosphorylation was noticed as early as 5 min poststretch, and phosphorylation gradually decreased after 20 min of cyclic stretch. After 60 min of cyclic stretch, the erk1/erk2 phosphorylation dramatically declined in BAEpCs.


Figure 4
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  		Fig. 4. Effect of stretch on erk1/erk2 phosphorylation in BAEpCs. BAEpCs were stretched over time, and erk1/erk2 phosphorylation was measured. This is representative of 3 similar observations noticed in separate experiments. Top: phosphorylated erk1/erk2. Middle: total erk1/erk2. Bottom: beta-actin as an internal control to demonstrate equal loading of samples. Lanes: 0, 5, 10, 20, 40, and 60 min poststretch.

 
To evaluate the role of NO in stretch-mediated erk1/erk2 phosphorylation, BAEpCs were stretched for 10 min in the presence of an iNOS inhibitor (L-NMMA) or L-NAME, a NOS inhibitor, or carboxy-PTIO, a NO scavenger. Carboxy-PTIO at a concentration of <150 µM partially inhibited stretch-induced erk1/erk2 phosphorylation, whereas, at a concentration of >300 µM, it completely blocked stretch-induced phosphorylation of erk1/erk2 in BAEpCs (Fig. 5A). L-NMMA also demonstrated concentration-dependent inhibition of stretch-induced erk1/erk2 phosphorylation in BAEpCs. L-NMMA at <4 mM produced a partial inhibition of stretch-induced erk1/erk2 phosphorylation, whereas at >8 mM, a complete inhibition was obtained in BAEpCs (Fig. 5B). Similar results were noticed with L-NAME. L-NAME at <500 µM demonstrated a partial inhibition of stretch-induced erk1/erk2 phosphorylation, whereas at >1,000 µM, a complete inhibition was achieved in BAEpCs (Fig. 5C).


Figure 5
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  		Fig. 5. Effect of NOS inhibitors and NO scavenger (PTIO) on stretch-induced erk1/erk2 phosphorylation. BAEpCs were stretched for 10 min, and erk1/erk2 phosphorylation was measured. A: BAEpC cultures pretreated with NO scavenger (carboxy-PTIO) for 30 min. B: BAEpC cultures pretreated with inducible NOS (iNOS) inhibitor L-NMMA for 30 min. C: BAEpC cultures pretreated with NOS inhibitor L-NAME for 30 min. Each (A, B, and C) is a representative finding of 3 similar observations. A–C: top, phosphorylated erk1/erk2; middle, total erk1/erk2; bottom, beta-actin as an internal control to demonstrate equal sample loading in gel. Lane c, BAEpCs stretched in presence of serum-free media alone.

 
Exogenous NO activated erk1/erk2 MAPKs in unstretched BAEpCs. We estimated the effect of exogenous NO on erk1/erk2 activation in unstretched BAEpCs using SNP, a NO donor. BAEpCs were treated with varying concentrations of SNP (2.5–20 µmol) for 10 min, and erk1/erk2 phosphorylation was estimated by Western blot analysis. SNP enhanced erk1/erk2 phosphorylation at lower concentrations (5 and 10 µmol) in unstretched BAEpCs (Fig. 6), whereas, at a higher concentration (20 µM), erk1/erk2 phosphorylation declined.


Figure 6
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  		Fig. 6. Effect of exogenous NO on erk1/erk2 phosphorylation in BAEpCs. BAEpCs were exposed to varying concentrations of NO donor sodium nitroprusside (SNP) for 10 min, and erk1/erk2 phosphorylation was measured by Western blot analysis. Top: phosphorylated erk1/erk2. Middle: total erk1/erk2. Bottom: beta-actin as an internal control to demonstrate equal sample loading in gel. Lane c, BAEpC-treated with serum-free media alone. Each (A–C) is a representative finding of 3 similar observations.

 
Inhibition of erk1/erk2 (p44/42 MAPK) activity ameliorated cyclic stretch-induced PlGF expression in BAEpCs. We evaluated whether BAEpCs subjected to cyclic stretch express and release PlGF in an erk1/erk2 activation-dependent manner. Inclusion of PD98059, a specific inhibitor of MAPK kinase-1 (Mek-1) activation, which is an upstream activator of erk1/erk2, significantly blocked the stretch-induced PlGF production in BAEpCs (Fig. 7). At the low concentration (0–2.5 µmol), PD98059 did not block stretch-induced PlGF release in BAEpC; however, a 5-µmol concentration of PD98059 significantly blocked the stretch-induced PlGF release (Fig. 7A). Maximum inhibition of PlGF was noticed with a 10-µmol concentration, while a higher concentration (20 µmol) produced an inhibition similar to that obtained with a 10-µmol concentration, which was used in all other experiments to effectively block erk1/erk2 activation. The role of p38 MAPK in stretch-induced PlGF production was evaluated by incubating BAEpCs with SB203580, a p38 MAPK inhibitor (10 µmol) selected on the basis of previous studies in our laboratory. Inhibition of p38 MAPK by SB203580 had no significant effect on stretch-induced PlGF release in BAEpCs (Fig. 7B).


Figure 7
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  		Fig. 7. Effect of PD98059 and SB203580 on stretch-induced PlGF release in BAEpCs. BAEpCs were stretched (20% cyclic stretch) for 24 h, and the PlGF released in culture media was measured. A: BAEpCs were pretreated with varying concentrations of PD98059 for 30 min and stretched for 24 h. B: some BAEpC cultures were incubated either with PD98059 (10 µM) to inhibit erk1/erk2 activity or with SB203580 (10 µM) to inhibit p38 MAPK activity. Data presented are means ± SE of 3 independent experiments, each evaluated separately. Comparison of stretched cells in serum-free medium (SFM) and stretched cells with inhibitor: *P < 0.001.

 
Inhibition of erk1/erk2 (p44/42 MAPK) activity blocked cyclic stretch-induced NO production in BAEpCs. We evaluated the role of erk1/erk2 on stretched-induced NO release using PD98059. BAEpCs were pretreated with varying concentrations (ranging from 1.25 to 20 µmol) for 30 min and stretched for 10 min. At low concentrations (<2.5 µmol), PD98059 did not block stretch-induced NO release in BAEpCs (Fig. 8A), whereas, at a concentration of 2.5 µmol, it significantly blocked stretch-induced NO release. Maximum inhibition was achieved with 10 µmol; there was no further inhibition with 20 µmol. We also evaluated the role of erk1/erk2 in NO generation over time; some BAEpCs were pretreated with PD98059 (10 µmol) 30 min before initiation of stretch. These cultures were stretched for 6, 12, 24, 48, and 72 h, and NO was measured in cell culture media. Inhibition of erk1/erk2 activation by PD98059 significantly blocked stretch-induced NO release in BAEpCs at all the time points evaluated (Fig. 8B).


Figure 8
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  		Fig. 8. Effect of PD98059 on stretch-induced NO release in BAEpCs. BAEpCs were stretched (20% cyclic stretch) over time, and the NO released in culture media was estimated. Some BAEpC cultures were incubated with PD98059 (10 µM) to inhibit erk1/erk2 activity. Data presented are means ± SE of 3 independent experiments, each evaluated separately. Comparison of stretched cells with and without inhibitor: *P < 0.001.

 
Inhibition of erk1/erk2 activation blocked cyclic stretch-induced iNOS expression in BAEpCs. NO is synthesized by various isoforms of NOS present in airway epithelial cells. To further evaluate the role of erk1/erk2 in NO generation, BAEpCs were stretched in the presence of PD98059, and iNOS mRNA expression was evaluated. Stretch induced a 10-fold increase in iNOS gene expression in BAEpCs (Fig. 9). BAEpC treatment with PD98059 significantly inhibited iNOS mRNA expression.


Figure 9
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  		Fig. 9. Effect of PD98059 on stretch-induced iNOS mRNA expression in BAEpCs. BAEpCs were stretched (20% cyclic stretch) for 24 h, and the iNOS mRNA expression was measured. Some BAEpC cultures were incubated with PD98059 (10 µM) to inhibit erk1/erk2 activity. Data are means ± SE of 3 independent experiments, each evaluated separately. Comparison of stretched and nonstretched cells: *P < 0.001. Comparison of stretched cells with and without PD98059: #P < 0.001.

 
Inhibition of erk1/erk2 activation or scavenging NO blocked cyclic stretch-induced PlGF mRNA expression in BAEpCs. We also evaluated the effects of stretch, erk1/erk2, and NO on transcriptional regulation of PlGF mRNA levels. Stretched BAEpCs expressed a 16-fold increase in PlGF mRNA levels compared with unstretched cells (Fig. 10). Including PD98059 (10 µM) in some cultures before the onset of stretch significantly blocked stretch-induced PlGF mRNA expression in BAEpC; this finding indicates that, during mechanical stretch, erk1/erk2 activation controls PlGF gene expression at the transcriptional level. Including a NO scavenger (PTIO) in some of the cultures during stretch also significantly inhibited stretch-induced PlGF mRNA expression in BAEpC, which indicates that NO modulates PlGF gene expression at the transcriptional level.


Figure 10
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  		Fig. 10. Effect of PD98059 and carboxy-PTIO on stretch-induced PlGF mRNA expression in BAEpCs. BAEpCs were stretched (20% cyclic stretch) for 24 h, and the PlGF mRNA expression was measured. Some BAEpC cultures were incubated either with PD98059 (10 µM) to inhibit erk1/erk2 activity or with carboxy-PTIO (300 µM) to scavenge NO released. Data presented are means ± SE of 3 independent experiments, each evaluated separately. Comparison of stretched cells in SFM: *P < 0.001. Comparison of stretched cells with PD98059 or PTIO vs. SFM: #P < 0.001.

 
We also measured the effect of exogenous NO on PlGF mRNA expression in unstretched BAEpCs 24 h postactivation. SNP, a NO donor, induced PlGF expression in a concentration-dependent manner (Fig. 11). At lower concentrations (2.5 and 5.0 mM), SNP demonstrated a stimulatory effect, whereas at >10 µM, the stimulatory potential significantly (P > 0.001) diminished.


Figure 11
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  		Fig. 11. Effect of exogenous NO donor on PlGF mRNA expression in unstretched BAEpCs. BAEpCs were cultured for 24 h in the presence of the NO donor SNP, and PlGF expression was estimated by quantitative PCR analysis as described in MATERIALS AND METHODS. Data presented are means ± SE of 3 independent experiments, each evaluated separately. Comparison (*P < 0.001): presence vs. absence of NO donor. Comparison: significant increase (#P < 0.001) and significant decrease ($P < 0.001).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our study is the first to demonstrate that cyclic stretch of cultured airway epithelial cells upregulates production of PlGF. In addition, the amount of PlGF produced increases with the magnitude of the mechanical stretch, as well as with the time the cells are exposed to mechanical stretch. We found that the stretch-induced production of PlGF is NO dependent via iNOS and that inhibition of the p44/42 MAPK pathway depresses production of PlGF, iNOS, and NO. We also found that mechanical stretch enhanced PlGF mRNA expression and inhibition of p44/42 MAPK activation, while scavenging NO blocked cyclic stretch-induced PlGF mRNA expression in BAEpCs. This stretched-induced production of PlGF and NO by the airway epithelium may have an important role in modulating the stretch-induced changes in lung structure and function that occur in vivo during lung growth and development, as well as stretch-induced lung injury.

We observed a stepwise increase in the production of PlGF and NO with increasing magnitude of the cyclic stretch from 5 to 20%, without causing cell death. Tidal breathing increases airway diameter and perimeter by ~5%, while a deep inspiration to total lung capacity can increase airway diameter by >50% (21). Therefore, we believe that we evaluated a magnitude of stretch that was within the physiological range. However, these magnitudes of stretch in vitro may not truly reflect the in vivo situation because of possible variance in extracellular matrix components.

Increased PlGF production by the stretched airway epithelial cells was present at 24 h, while the increased levels of NO were present at 6 h. The earlier NO production compared with PlGF production suggested that the increase in PlGF might be NO dependent. Culturing the cells with a NO scavenger (PTIO) markedly inhibited the stretch-induced PlGF protein. NO has previously been implicated as an important molecule in cells stimulated by mechanical strain (7, 8, 19, 30). In addition, recent studies reveal that NO released during mechanical stretch modulated several cellular functions including release of growth factor (3, 10, 23). In unstretched BAEpCs, we noticed that exogenous NO induced PlGF mRNA expression. We also noticed that cyclic stretch upregulated PlGF mRNA expression and scavenging NO masked stretch-induced PlGF mRNA expression. In a similar study, NO induced hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) expression during hypoxia via pericellular diffusion (22). These data indicate that, in BAEpCs during mechanical stretch, the NO generated in cytosol may diffuse into the nucleus and modulate PlGF expression at the transcriptional level and thereby modulate its production. Our study has now demonstrated that mechanical strain induces PlGF production in cultured airway epithelial cells via a NO-dependent pathway. Our data also suggest that the observed NO signaling is upstream of PlGF.

The cyclic stretch-induced PlGF and NO production was attenuated by PD98059, a specific MAPK kinase-1 and -2 inhibitor. In contrast, inhibition of p38 MAPK by SB203580 had no effect on PlGF or NO production. We evaluated whether NO produced by stretch of the airway epithelial cells also induced activation of the p44/42 MAPK pathway, erk1/2 phosphorylation. Blocking NO generation by L-NAME or L-NMMA or scavenging the generated NO with PTIO blocked stretch-induced erk1/erk2 activation, and, in unstretched BAEpCs, exogenous NO activated erk1/erk2. These findings suggest that the NO signaling we observed in primary bronchial epithelial cells occurred upstream of erk1/2.

NO is a highly reactive molecule that acts in various signaling pathways in the lung (11). In addition, NO has long been implicated as an important signaling pathway in cells exposed to mechanical strain (8). Of the three isoforms of NOS (neuronal, endothelial, and inducible), iNOS is transcriptionally regulated and produces sustained NO production in epithelial cells (12, 13, 28). In our cultured BAEpCs, cyclic stretch caused a significant increase in iNOS which was markedly inhibited by blocking the erk1/2 MAPK pathway. These findings indicate that the stretch-induced erk1/2 MAPK activation is upstream of iNOS induction in the airway epithelial cells.

In the airways, NO modulates neurotransmission, smooth muscle relaxation, bacteriostasis, ciliary function, and plasma exudation (17). NOS expression and NO production in the airway epithelium are developmentally regulated (20), and the bronchial epithelium is the primary source of expired NO (2). In ventilated animals, increasing the level of continuous positive airway pressure, which stretches the airways, produces an increase in the level of expired NO (16). In addition, we have recently demonstrated that applying mechanical strain to airways in vivo, as well as in vitro, can reduce airway responsiveness as well as increase the size of the airway (25, 29). Our current finding that mechanical strain of bronchial epithelium upregulates NO and PlGF production suggests that NO and PlGF may contribute to strain-induced modulation, airway structure, and function.

In summary, we have demonstrated that cyclic stretch of cultured airway epithelial cells upregulates production of PlGF, which is NO dependent (Fig. 12). The amount of PlGF produced increases with the magnitude of the mechanical stretch, as well as with the time the cells are exposed to mechanical stretch. In addition, inhibition of the p44/42 MAPK pathway depresses the stretch-induced production of PlGF, iNOS, and NO. Our findings that mechanical strain induced PlGF production in airway epithelial cells may have important implications for normal growth and development of the lung, while overexpression, particularly in a premature lung, may contribute to pathological lung development.


Figure 12
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  		Fig. 12. Schematic presentation of sequence of signaling events during stretch-induced PlGF release in BAEpCs. Sequence of events is indicated by nos. 1–6 as follows. 1, Cyclic stretch causes phosphorylation of erk1/erk2, and, in the absence of stretch, erk1/erk2 do not phosphorylate; 2, erk1/erk2 activation leads to iNOS expression, and inhibition of erk1/erk2 activation blocks iNOS expression; 3, iNOS induces NO release, and inhibition of iNOS blocks NO production; 4, NO induces PlGF mRNA expression; 5, NO also activates/phosphorylates erk1/erk2; 6, NO induces PlGF production, and scavenging or inhibition of NO blocks PlGF production. a,b,cSpeculated responses: erk1/erk2 activation leads to NF-{kappa}B activation (a), which translocates to the nucleus (b) and regulates iNOS expression (c).

 

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This work was supported by National Institutes of Health Grants RO1-AI37454-08, RO1-AI45338-04, and RO1-HL48522.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. A. Mohammed, Division of Pulmonary & Critical Care Medicine, Dept. of Medicine, Univ. of Florida, PO Box 100225, Gainesville, FL 32610-0225 (e-mail: mkamal{at}medicine.ufl.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.


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