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Published ahead of print on January 6, 2006, doi:10.1165/rcmb.2005-0339OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 592-599, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0339OC

Reactive Nitrogen Species Augment Fibroblast-Mediated Collagen Gel Contraction, Mediator Production, and Chemotaxis

Hisatoshi Sugiura, Xiangde Liu, Tetsu Kobayashi, Shinsaku Togo, Ronald F. Ertl, Shin Kawasaki, Koichiro Kamio, Xing Qi Wang, Lijun Mao, Lei Shen, Cory M. Hogaboam and Stephen I. Rennard

University of Nebraska Medical Center, Omaha, Nebraska; and University of Michigan Medical School, Ann Arbor, Michigan

Correspondence and requests for reprints should be addressed to Stephen I. Rennard, M.D., University of Nebraska Medical Center, 985885 Nebraska Medical Center, Omaha, NE 68198-5885. E-mail: srennard{at}unmc.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Reactive nitrogen species (RNS) such as peroxynitrite cause cellular injury and tissue inflammation. Excessive production of nitrotyrosine, which is a footprint of RNS, has been observed in the airways of patients with asthma and chronic obstructive pulmonary disease, disorders characterized by tissue remodeling. The aim of this study was to evaluate whether RNS can affect tissue remodeling through direct effects on fibroblasts, and to determine if these effects depend on production of transforming growth factor-beta (TGF-beta). To accomplish this, human fetal lung fibroblasts (HFL-1) were used to assess fibroblast-mediated contraction of floating gels and chemotaxis toward fibronectin. In addition, the ability of fibroblasts to release TGF-beta1, fibronectin, and vascular endothelial growth factor (VEGF) was assessed by enzyme-linked immunosorbent assay. Authentic peroxynitrite significantly augmented gel contraction (P < 0.01) and chemotaxis (P < 0.01) compared with control in a concentration-dependent manner. Similarly, the peroxynitrite donor 3-morpholynosidenonimine hydrochloride (SIN-1) also augmented gel contraction (P < 0.01). RNS also significantly increased TGF-beta1 (P < 0.01), fibronectin (P < 0.01), and VEGF (P < 0.01) release into the media in both 3D gel and monolayer culture. Anti–TGF-beta antibody reversed RNS-augmented gel contraction (P < 0.01) and mediator production (P < 0.01). Anti–TGF-beta antibody also partially, but significantly, reversed RNS-augmented chemotaxis toward fibronectin (P < 0.01). Finally, peroxynitrite enhanced expression of {alpha}5beta1 integrin, which is a receptor for fibronectin (P < 0.01), and neutralizing anti–TGF-beta antibody suppressed peroxynitrite-augmented {alpha}5beta1 expression (P < 0.01). These results suggest that RNS can affect the tissue repair process by modulating TGF-beta1.

Key Words: peroxynitrite • transforming growth factor-beta • collagen gel contraction • chemotaxis • tissue repair


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Excessive production of nitric oxide (NO) during inflammatory and immune processes leads to the formation of reactive nitrogen species (RNS) including peroxynitrite and nitrogen dioxide (NO2) (1). These RNS are formed from NO and superoxide anion (2) or via the H2O2/ peroxidase-dependent nitrite oxidation pathway (3). In inflammatory conditions in which superoxide anion is generated, NO is rapidly consumed by reacting with superoxide to produce the highly reactive peroxynitrite. Peroxynitrite is an extremely powerful oxidant and is presumed to be largely responsible for many of the adverse effects of excessive generation of NO. Excessive production of RNS causes tissue injury, lipid peroxidation, and nitration of tyrosine residues (1, 4). Consistent with a role for this pathway in disease, excessive production of 3-nitrotyrosine, which is a footprint of RNS production, has been observed in various inflammatory lung diseases, including chronic obstructive pulmonary diseases (COPD) (5, 6), bronchial asthma (5, 7), cystic fibrosis (8), and idiopathic pulmonary fibrosis (IPF) (9).

Inflammatory processes are frequently accompanied by alterations in tissue structure. Such alterations may result from tissue damage due to active proteases or toxic moieties released by inflammatory cells. In addition, mediators released at inflammatory sites are capable of directly altering cell function, leading to tissue repair and remodeling. Production of RNS causes tissue injury, but whether RNS can affect tissue repair and remodeling remains undefined.

Transforming growth factor-beta1 (TGF-beta1) is a key mediator in a variety of physiologic and pathologic processes, including fibroblast repair responses (10). TGF-beta regulates the fibroblast migration, proliferation, differentiation, and production of matrix and soluble factors. In addition, TGF-beta stimulates fibroblast-mediated contraction of extracellular matrix. Through these actions, TGF-beta is believed to be a major regulator of tissue remodeling. Among the cytokines induced by TGF-beta are fibronectin (11) and vascular endothelial growth factor (VEGF) (12). Fibronectin can form a provisional extracellular matrix after injury and is a potent chemoattractant for fibroblasts (13). VEGF is a multifunctional cytokine that stimulates endothelial cell mitogenesis and migration and modulates endothelial permeability (14). Production of VEGF, therefore, could play a role in the neovascularization that characterizes tissue repair after injury.

The present study, therefore, was designed first to determine if RNS could affect tissue remodeling through an effect on fibroblast chemotaxis or contraction of collagen gels. Next we assessed if peroxynitrite increased fibroblast release of TGF-beta1, fibronectin, and VEGF. Finally we determined if the effects of RNS were mediated through the production of active TGF-beta1.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Materials
Native type I collagen (rat tail tendon collagen [RTTC]) was extracted from rat tail tendons by a previously published method (15). Briefly, tendons were excised from rat tails, and the tendon sheath and other connective tissues were removed carefully. Repeated washing with Tris-buffered saline (0.9% NaCl and 10 mM Tris, pH 7.5) was followed by dehydration and sterilization with 50%, 75%, 95%, and pure ethanol. Type I collagen was then extracted in 6 mM hydrochloric acid at 4°C. Collagen concentration was determined by weighing a lyophilized aliquot from each lot of collagen solution. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) routinely demonstrated no detectable proteins other than type I collagen.

Commercially available reagents were obtained as follows: anti–TGF-beta1 antibody (clone: 9,016.2), TGF-beta1, biotinylated anti–TGF-beta1, anti–TGF-beta, neutralizing antibodies which showed < 2% cross-reactivity with human TGF-beta2 and TGF-beta3 and did not cross-react with other growth factors, and anti-immunoglobulin (IgG) were from R&D Systems (Minneapolis, MN); 3-morpholynosidenonimine hydrochloride (SIN-1), collagenase, 3,3',5,5'-tetramethyl benzidine (TMB), monoclonal anti-human fibronectin antibody, polyclonal anti-human fibronectin antibody, and anti-rabbit IgG antibody were from Sigma (St. Louis, MO); Peroxynitrite was purchased from Calbiochem (La Jolla, CA); Dulbecco's Modified Eagle's Medium (DMEM) and fetal calf serum (FCS) were from Invitrogen Life Technologies (Grand Island, NY).

Cell Culture
Human fetal lung fibroblasts (HFL-1) cells were obtained from the American Type Culture Collection (Rockville, MD). Human lung fibroblasts from healthy adults were a gift from Dr. Cory Hogaboam, University of Michigan. Human bronchial fibroblasts from healthy adults were initiated from endobronchial biopsy at the University of Nebraska Medical Center (IRB#422–01). The cells were cultured on tissue culture dishes (Falcon; Becton-Dickinson Labware, Lincoln Park, NJ) with DMEM supplemented with 10% FCS, 100 µg/ml penicillin, 250 µg/ml streptomycin, and 2.5 µg/ml fungizone. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and passaged every 4–5 d at a 1:4 ratio. HFL-1 cells were used between the 14th and 18th passages. To evaluate mediator production in monolayer culture, cells were seeded in 6 well tissue culture plates at a cell density of 1 x 105/ml. At 90% confluence, cells were treated with various concentrations of peroxynitrite in serum-free DMEM (SF-DMEM). The supernatants of monolayer culture were harvested on Day 2 (for TGF-beta1) or Day 3 (for fibronectin and VEGF) and stored at –80°C until later assay.

Collagen Gel Contraction Assay
Collagen gels were prepared as described previously (16). Briefly, RTTC, distilled water and 4x concentrated DMEM were combined so that the final mixture resulted in 0.75 mg/ml collagen, with a physiologic ionic strength of 1x DMEM and a pH 7.4. Cells were trypsinized (trypsin-EDTA: 0.05% trypsin, 0.53 mM EDTA-4Na; Gibco, Grand Island, NY) and suspended in SF-DMEM. Cells were then mixed with the neutralized collagen solution so that the final cell density in the collagen solution was 3 x 105 cells/ ml, and the final concentration of collagen was 0.75 mg/ml. Aliquots (0.5 ml/well) of the mixture of cells in collagen were cast into each well of 24-well tissue culture plates (Falcon) and allowed to gel. After gelation was completed, normally within 20 min at room temperature, the gels were gently released from the 24-well tissue culture plates and transferred into 60-mm tissue culture dishes (three gels in each dish), which contained 5 ml of freshly prepared SF-DMEM with or without various concentrations of peroxynitrite, SIN-1, or hydrogen peroxide. The gels were then incubated at 37°C in a 5% CO2 atmosphere for 3 d. To investigate the effect of anti–TGF-beta antibody on fibroblast-mediated gel contraction, we added TGF-beta antibody (10 µg/ml) to the culture media after gels were released. Anti-human IgG antibody was used as a control. Gel contraction was quantified using an Optomax V image analyzer (Optomax, Burlington, MA) daily. Data were expressed as percentage of the original gel size.

Measurement of TGF-beta1, Fibronectin, and VEGF by Enzyme-Linked Immunosorbent Assay
TGF-beta1, fibronectin, and VEGF in the media of collagen gel culture or monolayer culture were determined by enzyme-linked immonsorbent assay (ELISA). Quantification of TGF-beta1 was performed as follows. Plates were coated with monoclonal anti–TGF-beta1 antibody at 4°C overnight. After being washed three times (5 min each), standards and samples were added and incubated at room temperature for 2 h. To measure TGF-beta1, all samples were assayed both with and without acidification and neutralization to convert the latent form of TGF-beta1 to the active form. To accomplish this, a 500-µl sample was mixed with 100 µl of 1 N HCl and after 10 min at room temperature, neutralized with 100 µl of 1.2 N NaOH/0.5 M HEPES. After being washed, bound antigen was detected after adding biotinylated anti–TGF-beta1 antibody for 1 h at room temperature. After being washed, HRP–streptavidin (1:20,000 dilution) was then added for 1 h. Bound HRP was detected with TMB. The reaction was stopped with 1 M H2SO4, and the product was quantified at 450 nm with a microreader. Fibronectin was assayed with an ELISA that specifically detects human but not bovine fibronectin (17). Quantification of fibronectin was performed as follows. Plates were coated with monoclonal anti-fibronectin antibody at 4°C overnight. After being washed three times, standards and samples were added and incubated at room temperature for 2 h. After being washed, bound antigen was detected after adding polyclonal anti-human fibronectin antibody (1:2,000 dilution) at room temperature for 1 h. After being washed, HRP-conjugated anti-rabbit IgG antibody (1:10,000 dilution) was added at room temperature for 1 h. After being washed, bound HRP was detected with 0.1 mg/ml OPD. The reaction was stopped with 8 M H2SO4, and the product was quantified at 490 nm with a microreader. Quantification of VEGF was performed as follows. Plates were coated with 50 ng/ml monoclonal anti-human VEGF antibody at 4°C overnight. After being washed three times, standards and samples were added and incubated at room temperature for 2 h. After being washed, bound antigen was detected after adding 100 ng/ml biotinylated anti-human VEGF antibody for 1 h at room temperature. After being washed, HRP–streptavidin (1:20,000 dilution) was then added for 1 h. Bound HRP was detected with TMB. The reaction was stopped with 1 M H2SO4, and the product was quantified at 450 nm with a microreader.

Chemotaxis Assay
Cell migration was assessed using the Boyden blindwell chamber (Neuroprobe Inc., Gaithersburg, MD) as previously described (18). Briefly, 26 µl of SF-DMEM containing human fibronectin (20 µg/ml) was placed into the bottom wells. Eight-micrometer pore polycarbonate membranes (Neuroprobe Inc.), which were precoated with 5 µg/ml gelatin in 0.1% acetic acid, were used. Cells grown to 75% confluence were rinsed, refed with SF-DMEM, and treated with various concentrations of peroxynitrite at 37°C in a humidified atmosphere of 5% CO2 for 24 h. To investigate the role of TGF-beta, the peroxynitrite exposure was performed in the presence of anti–TGF-beta neutralizing antibody or a control antibody. Cells were trypsinized and suspended in SF-DMEM at a density of 1 x 106/ml. Fifty microliters of cell suspension treated with various concentrations of peroxynitrite in the presence of neutralizing anti–TGF-beta antibody or control IgG were then added into each top well. Cells were allowed to migrate at 37°C in a 5% CO2 atmosphere for 6 h. Cells that had not migrated were scraped off the upper surface of the membrane, and the membranes were air-dried. Cells were then stained with PROTOCOL (Fisher Scientific, Swedesboro, NJ) and mounted on a glass microscope slide. Chemotaxis was assessed by counting the number of cells in five high-power fields. Wells with SF-DMEM were used as negative controls and those with chemoattractant alone were used as positive controls. To evaluate the role of {alpha}5beta1 integrin on fibroblast chemotaxis toward fibronectin, cells were treated with control or anti-{alpha}5beta1 integrin antibody (5 µg/106 cells) for 30 min. After pretreatment with antibody, chemotaxis assay was performed as described above.

Western Blotting
After treatment with 10 µM authentic peroxynitrite in the presence of neutralizing TGF-beta antibody or control IgG, cells were washed with 4°C PBS and homogenized in cell lysis buffer (35 mM Tris-HCl, pH 7.4, 0.4 mM EGTA, 10 mM MgCl2, 1 µM phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin and 1 µg/ml leupeptin). Samples were solubilized in SDS-PAGE sample buffer. Equal amounts of protein were loaded and separated by electrophoresis on 12% SDS polyacrylamide gels. After electrophoresis, the separated proteins were transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, CA). Primary antibodies against human {alpha}5 and beta1 integrin subunit (1:1,000 dilution; Chemicon, Temecula, CA) were used for detection. Bound antibodies were visualized using peroxidase-conjugated second antibodies and enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK) with a Typhoon Scanner (Amersham Biosciences).

Statistical Analysis
Data were expressed as means ± SEM. Experiments with multiple comparisons were evaluated by one-way ANOVA followed by Bonferroni's test to adjust for multiple comparisons. An unpaired two-tailed Student's t test was used for single comparisons. Probability values of < 0.05 were considered significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Effect of Peroxynitrite on Fibroblast-Mediated Collagen Gel Contraction
To investigate peroxynitrite modulation of collagen gels, cells cast in collagen gels were floated in media with various concentration of authentic peroxynitrite. Authentic peroxynitrite significantly augmented gel contraction compared with control in a concentration-dependent manner (at 10–5 M, on Day 3, gel size was 46.1 ± 2.9% versus 71.1 ± 0.4% of initial size; P < 0.01, Figure 1A). Higher concentrations of peroxynitrite were toxic (at > 10–4 M, not shown). In contrast, hydrogen peroxide (10–7–10–5 M) had no effect on collagen gel contraction compared with control (at 10–5 M, on Day 3, gel size was 75.8 ± 1.8% versus 71.1 ± 0.2% of initial size; not significant, Figure 1B). The peroxynitrite donor, SIN-1 (10–6–10–5 M) resembled peroxynitrite and significantly augmented gel contraction (at 10–5 M, on Day 3, gel size was 45.8 ± 2.5% versus 69.8 ± 1.3% of initial size; P < 0.01; Figure 2).


Figure 1
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  		Figure 1. Effect of authentic peroxynitrite (A) and hydrogen peroxide (B) on collagen gel contraction by human fetal lung fibroblasts (HFL-1). Fibroblasts were cast into three-dimensional collagen gels and floated in medium containing various concentrations of peroxynitrite. Gel size was measured daily. Vertical axis: gel size (% of initial size); horizontal axis: time. All values are mean ± SEM for three separate experiments, each performed in triplicate. **P < 0.01, compared with the values of control. Open circles, control; triangles, 0.1 µM; squares, 1 µM; filled circles, 10 µM.

 

Figure 2
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  		Figure 2. Effect of the peroxynitrite donor, 3-morpholynosidenonimine hydrochloride (SIN-1) on collagen gel contraction by HFL-1 cells. Fibroblasts were cast into collagen gels and maintained in floating culture in medium containing various concentrations of SIN-1. Gel size was measured daily. Vertical axis: gel size (% of initial size); horizontal axis: time. All values are mean ± SEM for four separate experiments, each performed in triplicate. **P < 0.01, compared with the values of control. Open circles, control; triangles, 0.1 µM; squares, 1 µM; filled circles, 10 µM.

 
Effect of Peroxynitrite on TGF-beta1, Fibronectin, and VEGF Release by HFL-1 Cells
It is known that TGF-beta1 and fibronectin are involved in tissue repair and are able to enhance collagen gel contraction by human lung fibroblasts. To determine whether TGF-beta1 and fibronectin could contribute to the augmentation of collagen gel contraction by peroxynitrite, TGF-beta1 and fibronectin release in the contracting gel and monolayer cultures were evaluated. Authentic peroxynitrite (10–6–10–5 M) significantly increased TGF-beta1 release in both 3D gel (P < 0.01; Figures 3A and 3B, left panels) and monolayer culture (P < 0.01; Figures 3A and 3B, right panels), as well as release of fibronectin in both 3D gel (P < 0.01; Figure 4A), and monolayer culture (P < 0.01; Figure 4B).


Figure 3
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  		Figure 3. Effect of peroxynitrite on TGF-beta1 release. Fibroblasts were maintained under two culture regimes. Fibroblasts were cast into three-dimensional collagen gels and maintained in floating culture in medium in the presence of varying concentrations of peroxynitrite (left). Fibroblasts were cultured until confluent, after which medium was changed to serum-free DMEM in the presence of varying concentrations of peroxynitrite (right). After 2 d, media were harvested and assayed for TGF-beta1 by ELISA. (A) Vertical axis: active form of TGF-beta1 (pg/culture); horizontal axis: peroxynitrite concentration (M). (B) Vertical axis: total TGF-beta1 (ng/culture); horizontal axis: peroxynitrite concentration (M). All values are mean ± SEM for six separate experiments, each performed in duplicate. **P < 0.01; compared with the values of control.

 

Figure 4
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  		Figure 4. Effect of peroxynitrite on fibronectin release in three-dimensional (A) and monolayer culture (B). (A) Fibroblasts were cast into three-dimensional collagen gels and maintained in floating culture in medium in the presence of varying concentrations of peroxynitrite. (B) Fibroblasts were cultured until confluent, after which medium was changed to serum-free DMEM with varying concentrations of peroxynitrite. After 3 d, media were harvested and assayed for fibronectin by ELISA. Vertical axes: fibronectin production (ng/culture); horizontal axes: peroxynitrite concentration (M). All values are mean ± SEM for five separate experiments, each performed in duplicate. **P < 0.01; compared with the values of control.

 
VEGF is believed to contribute to vascular cell proliferation and may be a mediator of tissue repair and remodeling. Therefore, we also assessed the effect of peroxynitrite on VEGF production by HFL-1 cells. As shown in Figure 5, 10–6–10–5 M authentic peroxynitrite significantly augmented VEGF production in both 3D gel (P < 0.01; Figure 5A) and monolayer culture (P < 0.01; Figure 5B). Because fibroblasts may be heterogeneous functionally, we also investigated the effect of peroxynitrite on mediator release from adult alveolar and bronchial fibroblasts. As shown in Table 1, there were quantitative differences in mediator release among the strains. However, peroxynitrite significantly augmented the release of mediators in all fibroblast strains, suggesting that peroxynitrite has a similar effect on release of mediators not only in fetal fibroblasts but in both alveolar and airway adult fibroblasts.


Figure 5
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  		Figure 5. Effect of authentic peroxynitrite on vascular endothelial growth factor (VEGF) release in three-dimensional (A) and monolayer culture (B). (A) Fibroblasts were cast into three-dimensional collagen gels and maintained in floating culture in medium in the presence of varying concentrations of peroxynitrite. (B) Fibroblasts were cultured until confluent, after which medium was changed to serum-free DMEM with and without varying concentrations of peroxynitrite. After 3 d, media were harvested and assayed for VEGF by ELISA. Vertical axes: VEGF production (pg/culture); horizontal axes: peroxynitrite concentration (M). All values are mean ± SEM for five separate experiments, each performed in duplicate. **P < 0.01; compared with the values of control.

 

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  		TABLE 1. PEROXYNITRITE CAN STIMULATE THE RELEASE OF MEDIATORS IN ADULT HUMAN NORMAL LUNG OR BRONCHIAL FIBROBLASTS

 
Effect of Peroxynitrite on HFL-1 Chemotaxis
Because fibroblast migration from neighboring connective tissue into sites of inflammation plays an important role in tissue repair in response to injury, we assessed the effect of peroxynitrite on HFL-1 chemotaxis. Using fibronectin (20 µg/ml) as the chemoattractant, authentic peroxynitrite significantly augmented fibroblast chemotaxis compared with control (at 10–5 M, 173 ± 6.7% of control, P < 0.01; Figure 6).


Figure 6
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  		Figure 6. Effect of authentic peroxynitrite on HFL-1 cells chemotaxis. Fibroblasts were prepared in monolayer culture, exposed to peroxynitrite, harvested and assayed for chemotaxis toward fibronectin in the Boyden blindwell chemotaxis assay (see MATERIALS AND METHODS). Vertical axis: chemotaxis (% of control); horizontal axis: peroxynitrite concentration (M). All values are mean ± SEM for three separate experiments, each performed in triplicate. **P < 0.01; compared with the values of control.

 
Effect of Anti–TGF-beta Antibodies on Peroxynitrite Modulation of Collagen Gel Contraction, Mediator Production, and HFL-1 Chemotaxis
It is well known that TGF-beta1 can stimulate collagen gel contraction (19), fibronectin (11) and VEGF production (12), and chemotaxis in fibroblasts. To clarify a mechanistic role of TGF-beta1 on peroxynitrite modulation of collagen gel contraction, mediator production, and HFL-1 cell chemotaxis, we incubated cultures in the presence of anti–TGF-beta antibodies or control IgG. As shown in Figure 7, anti–TGF-beta antibodies alone had no significant effect but blocked the peroxynitrite-augmented collagen gel contraction by 69.7 ± 1.5% (P < 0.01). The addition of anti–TGF-beta antibodies also significantly inhibited the release of fibronectin (4,100 ± 380 versus 9,180 ± 510 ng/culture, P < 0.01; Figure 8A) and VEGF (82.0 ± 1.7 pg/culture versus 121.1 ± 2.5 pg/culture, P < 0.01; Figure 8B) from HFL-1 cells cultured in collagen gels. Finally, anti–TGF-beta antibody also partially but significantly suppressed peroxynitrite-augmented HFL-1 cell chemotaxis toward fibronectin compared with control IgG, (P < 0.01; Figure 9).


Figure 7
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  		Figure 7. Effect of anti–TGF-beta antibody on peroxynitrite-augmented collagen gel contraction by HFL-1 cells. Fibroblasts were cast into three-dimensional collagen gels and cultured in floating medium in the presence of anti–TGF-beta antibody (filled bars) or control IgG (open bars) in the presence of varying concentrations of peroxynitrite. Gel size was measured daily. Vertical axis: gel size expressed as percent of initial size after 72 h; horizontal axis: peroxynitrite concentration (M). All values are mean ± SEM for three separate experiments, each performed in triplicate. **P < 0.01; compared with the values of control. ++P < 0.01; compared with the values of control IgG–treated group. NS = not significant.

 

Figure 8
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  		Figure 8. Effect of anti–TGF-beta antibody on peroxynitrite-augmented fibronectin and VEGF release. Fibroblasts were maintained in three-dimensional collagen gel culture and floated in medium containing either anti–TGF-beta antibody (filled bars) or control IgG (open bars) in the presence of varying concentrations of peroxynitrite. After 3 d, media were harvested and assayed for fibronectin (A) and VEGF (B) by ELISA. Vertical axes: mediator production (ng or pg/culture); horizontal axes: peroxynitrite concentration (M). All values are mean ± SEM for five separate experiments, each performed in duplicate. **P < 0.01; compared with the values of control. ++P < 0.01; compared with the values of control IgG–treated group. NS = not significant.

 

Figure 9
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  		Figure 9. Effect of anti–TGF-beta antibody on peroxynitrite-augmented HFL-1 cell chemotaxis toward fibronectin. Fibroblasts were maintained in monolayer culture and pretreated with varying concentrations of peroxynitrite in the presence and absence of anti–TGF-beta antibody for 24 h. Fibroblasts were then harvested by trypsinization, and chemotaxis was measured in the blindwell Boyden chamber assay using fibronectin as the chemoattractant in the presence of anti–TGF-beta antibody (filled bars) or control IgG (open bars). Vertical axis: chemotaxis (% of control); horizontal axis: peroxynitrite concentration (M). All values are mean ± SEM for three separate experiments, each performed in triplicate. **P < 0.01; compared with the values of control. ++P < 0.01; compared with the values of control IgG–treated group. NS = not significant.

 
Effect of Peroxynitrite on Expression of {alpha}5beta1 Integrin
To evaluate the possible mechanism by which peroxynitrite augmented HFL-1 cell chemotaxis toward fibronectin, we investigated the effect of peroxynitrite on expression of {alpha}5beta1 integrin, which is a receptor for fibronectin. Peroxynitrite significantly enhanced expression of {alpha}5beta1 integrin (at 10–5 M, P < 0.01; Figures 10A and 10B). This effect of peroxynitrite was also partially but significantly suppressed by anti–TGF-beta antibody (P < 0.01; Figures 10A and 10B). To evaluate the role of {alpha}5beta1 integrin on fibroblast chemotaxis toward fibronectin, cells were incubated with anti-{alpha}5beta1 integrin antibody. Anti-{alpha}5beta1 integrin antibody significantly inhibited chemotaxis toward fibronectin in the absence of peroxynitrite (P < 0.01; Figure 10C). The antibody partially but significantly inhibited peroxynitrite-augmented fibroblasts chemotaxis (P < 0.01; Figure 10C), suggesting that {alpha}5beta1 integrin has a role on chemotaxis toward fibronectin under baseline conditions and in response to peroxynitrite augmentation.


Figure 10
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  		Figure 10. Effect of peroxynitrite on expression of {alpha}5beta1 integrin. Cultured cells were treated with 10–5 M peroxynitrite for 24 h in the presence of control IgG or neutralizing anti–TGF-beta antibody. Both {alpha}5 and beta1 integrin expression were analyzed by immunoblotting (A) and quantified by densitometry (B). All values are mean ± SEM for three separate experiments. Cells were treated with 10–6–10–5 M peroxynitrite for 24 h and harvested. Cells were pretreated with control or anti-{alpha}5beta1 integrin antibody for 30 min and assayed for chemotaxis toward fibronectin (C). Vertical axis: chemotaxis (% of control); horizontal axis: peroxynitrite concentration (M). All values are mean ± SEM for six separate experiments, each performed in triplicate. **P < 0.01; compared with the values of control IgG treated group. ++P < 0.01; compared with the values of control IgG and peroxynitrite treated group. In C, open bars represent control IgG, and filled bars represent anti-{alpha}5beta1 antibody.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The present study demonstrates that authentic peroxynitrite, and the peroxynitrite donor SIN-1, augment fibroblast-mediated contraction of three-dimensional collagen gels and fibroblast chemotaxis toward fibronectin. RNS also significantly increased TGF-beta1, fibronectin, and VEGF release into the supernatant of both 3D gel and monolayer cultures. Anti–TGF-beta antibody blocked RNS-augmented gel contraction and mediator production. Similarly, anti–TGF-beta antibody partially but significantly inhibited RNS-augmented chemotaxis toward fibronectin. Peroxynitrite also enhanced expression of {alpha}5beta1 integrin, which is receptor for fibronectin, whereas neutralizing anti–TGF-beta antibody partially but significantly suppressed peroxynitrite-augmented {alpha}5beta1 expression. These results suggest that RNS can affect fibroblast-mediated repair processes and that the mechanism of this effect depends on generation of active TGF-beta1.

Peroxynitrite is one of the RNS and is formed from superoxide anion and NO. Peroxynitrite is a potent oxidant and causes cell injury, lipid peroxidation, and nitration of tyrosine residues of protein. Recently, it has been reported that peroxynitrite can enhance the production of IL-8 (20) and TNF-{alpha} (21) via NF-{kappa}B activation and can activate AP-1 signaling (22). In the present study, we showed that peroxynitrite enhanced TGF-beta release in both 3D collagen gel and monolayer culture. Previous studies have shown that RNS could enhance TGF-beta mRNA expression (23). Our findings extend these results and demonstrate that peroxynitrite leads to the increased release of TGF-beta protein and, importantly, leads to the generation of active TGF-beta that drives fibroblast-mediated repair responses. The possible mechanism by which peroxynitrite enhances TGF-beta production is still unclear. However, peroxynitrite modulates the DNA binding activity of the transcription factor AP-1 in lymphocytes (22) and AP-1 is thought to be involved in TGF-beta gene expression (24).

Fibronectin, a multifunctional glycoprotein involved in tissue remodeling, is a chemoattractant for lung fibroblasts (13) and can be released in increased amounts by fibroblasts (11) and epithelial cells (25, 26) in response to a variety of cytokines. TGF-beta is a strong stimulator of fibronectin release from fibroblasts. In the present study, peroxynitrite enhanced fibronectin production and anti–TGF-beta antibody reversed the peroxynitrite-augmented fibronectin production, suggesting that TGF-beta production is the mechanism by which peroxynitrite augmented fibronectin production.

In the current study, fibronectin release was stimulated by peroxynitrite in both monolayer and three-dimensional collagen gel culture. In both conditions, some fibronectin is retained in the pericellular matrix and some is released into the medium. There are several distinct forms of fibronectin that arise from splice variants and from post-translational modification. It is possible that peroxynitrite modulates not only fibronectin release, but also the relative distribution of the various forms of fibronectin on its extracellular organization.

VEGF is a multifunctional growth factor that increases endothelial permeability, induces endothelial cell growth, is required for survival of endothelial cells, and plays a role in angiogenesis (14). We have recently reported that TGF-beta enhances VEGF production via Smad3, which is phosphorylated by the activated TGF-beta receptor (27). In the present study, peroxynitrite enhanced VEGF production and anti–TGF-beta antibody reversed peroxynitrite-augmented VEGF production, suggesting that peroxynitrite-augmented VEGF production is also mediated through TGF-beta production.

Regulation of fibroblast recruitment in vivo is likely to depend on both chemotactic factors and inhibitors. TGF-beta can augment chemotactic activity toward fibronectin (13). The current study demonstrated that peroxynitrite augmented chemotaxis. To explore the possible mechanisms for augmented chemotaxis, we investigated whether peroxynitrite might change expression of {alpha}5beta1 integrin, which is a fibronectin receptor, since TGF-beta has been reported to stimulate expression of {alpha}5beta1 integrin (28). In the present study, we found that peroxynitrite augmented expression of {alpha}5beta1 integrin. Neutralizing anti–TGF-beta antibody partially but significantly blocked not only chemotaxis toward fibronectin but also expression of {alpha}5beta1 integrin. Furthermore, anti-{alpha}5beta1 integrin antibody significantly inhibited both baseline chemotaxis and peroxynitrite-augmented fibroblasts chemotaxis toward fibronectin. While these results do not exclude a role for other mechanisms, peroxynitrite augmentation of {alpha}5beta1 integrin expression due to TGF-beta may be a mechanism contributing to augmented fibroblast chemotaxis in response to peroxynitrite.

Sato and coworkers showed that reactive nitrogen species reduced fibronectin-induced fibroblast migration in vitro (29). They incubated fibronectin directly with peroxynitrite. Peroxynitrite reduced the chemotactic activity of fibronectin due to nitrosylation of tyrosine residues. The current study differs from that of Sato and colleagues, as we incubated the fibroblasts with peroxynitrite. In vivo, therefore, RNS may have several potentially antagonist effects. Augmented chemotaxis could result from increased integrin expression or fibronectin production. Inhibited chemotaxis could result from fibronectin nitrosylation. The in vivo response should depend on the local balance between these effects.

In the present study, we used peroxynitrite at a range of concentrations from 10–7–10–5 M. We are unaware of any study that has directly measured the concentration of peroxynitrite in vivo under normal or pathologic conditions because of its instability and high reactivity (30). However, a previous article showed that NO derived from the inducible type of NO synthase (iNOS) was detected at 20 µM by using the electron spin resonance method in a viral pneumonia model in mice (31). Moreover, peroxynitrite is formed from NO and superoxide by a reaction that is almost diffusion limited (2), on an equimolar basis (32). Taken together, it is highly likely that peroxynitrite achieves the higher nanomolar to lower micromolar concentrations that are necessary to exert significant physiologic and pathologic effects (33). The concentration of peroxynitrite used in this study is in this range, and is, therefore, close to the currently accepted "physiologic" range.

In summary, our data demonstrate that peroxynitrite augmented fibroblast contraction of three-dimensional collagen gels, chemotaxis, and integrin expression. Peroxynitrite also increased TGF-beta1, fibronectin, and VEGF release. The effects of peroxynitrite were significantly blocked by anti–TGF-beta antibody. These results suggest that peroxynitrite can affect tissue repair process by modulating TGF-beta1 and secondarily through other mediator production.


   Acknowledgments
 
The authors thank the excellent secretarial support of Ms. Lillian Richards.


   Footnotes
 
Originally Published in Press as DOI: 10.1165/rcmb.2005-0339OC on January 6, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 2, 2005

Accepted in final form November 28, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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