1 Institut National de la Santé et de la Recherche Médicale Unité U492, 2 Département de Physiologie, 3 Services de Pneumologie et 4 d'Oto-Rhino-Laryngologie, Hôpitaux Henri Mondor et Intercommunal de Créteil, Assistance Publique-Hôpitaux de Paris, Université Paris XII, 94010 Créteil, France
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ABSTRACT |
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Vascular endothelial growth
factor (VEGF) is a potent endothelial cell growth and permeability
factor highly expressed in rodent alveolar epithelium after injury and
repair. To investigate VEGF synthesis in human lung epithelial cells,
we examined VEGF expression by cultured cells under basal conditions
and after cytokine treatment or oxidative stress. Basal VEGF expression was detected in transformed human epithelial cell lines (A549 and
1HAEo) and in primary human bronchial epithelial cells with RT-PCR,
Western blot, and immunocytochemistry. Among the cytokines tested, only
transforming growth factor-
1 increased the levels of excreted
VEGF165 as measured by ELISA. Under hypoxia (0%
O2 for 24 h), the VEGF165 level increased
fivefold, and this effect was O2 concentration dependent.
VEGF concentrations in the medium of all the cell types studied reached
values similar to those found in bronchoalveolar lavage fluids from
normal patients. Endothelial cells (human umbilical vein endothelial
cells) exposed to conditioned medium from primary bronchial epithelial
cell cultures showed an increased growth rate, which was inhibited in
the presence of a specific neutralizing antibody to VEGF. These results
suggest that lung epithelial cells participate in the endothelial
repair and angiogenesis that follow lung injury through the synthesis of VEGF.
angiogenesis; bronchial epithelial cells; lung injury
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INTRODUCTION |
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AFTER ACUTE LUNG INJURY, the main step of the repair process involves the ability of epithelial stem cells to proliferate and heal the loss of substance. This healing process is not restricted to epithelial cell proliferation but also requires other events such as fibroblast proliferation and migration, deposition of extracellular matrix, remodeling, and angiogenesis (7). Endothelial cells are involved in several key processes during wound healing, such as formation of new vessels and restitution of the air-blood barrier in the alveoli (2). In response to angiogenic stimuli, endothelial cells can change their morphology, cause vascular dilatation, degrade the basement membrane, proliferate, migrate, and form new capillaries (7). These angiogenic processes, normally associated with growth and development, may depend in part on chemical and environmental factors including cytokines, hypoxia, and oxyradicals.
Vascular endothelial growth factor (VEGF), one of the most potent angiogenic factors, is a highly conserved dimeric heparin-binding glycoprotein (molecular mass 46 kDa) (11). At least four different VEGF transcripts, resulting from alternative splicing of a single gene, have been described in human cells (15). VEGF121 and VEGF165 are secreted as soluble compounds, whereas VEGF189 and VEGF206 remain cell surface associated or are primarily deposited in the extracellular matrix. VEGF may be a specific endothelial cell growth and permeability factor because selective binding of VEGF to endothelial cells has been demonstrated in vivo and in vitro. Two high-affinity transmembrane tyrosine kinase receptors for VEGF have been described, namely fms-like tyrosine kinase-1 (Flt-1) and kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1) (12). Flk-1 expression seems restricted to endothelial cells, and activation of this receptor may mediate the mitogenic effect of VEGF.
The regulation of angiogenesis and endothelium repair in the lung is probably crucial to satisfactory healing, although it remains poorly understood. Accumulating evidence suggests that VEGF is abundantly expressed in the lung. Previous studies (23, 32) in laboratory animals have demonstrated alveolar epithelial cell expression of VEGF, particularly in response to hypoxia or hyperoxia. High levels of VEGF mRNA and protein have also been detected ex vivo in airway epithelial cells of midtrimester human fetal lungs (1, 17, 29) as well as in a human epithelial cell line (26). However, the exact mechanisms underlying VEGF synthesis by human epithelial cells have not been extensively studied, and it is not known whether VEGF is constitutively expressed in the normal human lung. We designed the present study to explore VEGF expression by cultured human lung epithelial cells under basal conditions and after exposure to cytokines, oxidative stressors, and hypoxia. We also measured VEGF concentrations in bronchoalveolar lavage (BAL) supernatants from patients with normal lungs and sought to determine whether basal VEGF levels in BAL fluids could affect endothelial cell growth.
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MATERIALS AND METHODS |
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Cell Cultures
Cell lines.
A549 human lung adenocarcinoma cells were grown in Earle's
minimal essential medium supplemented with 4 mM glutamine, 50 U penicillin/ml, 50 µg streptomycin/ml, and 10% fetal bovine serum (20). 1HAEo bronchial cells (a gift from D. Gruenert,
University of California, San Francisco, CA) were grown in
Dulbecco's modified Eagle's medium on a collagen G (I + III)
coating; the 1HAEo
line is derived from human tracheal epithelial
cells transfected with mutated SV40T antigen and has been extensively
characterized (9).
Isolation and culture of primary human bronchial epithelial cells. Human bronchial epithelial biopsies were obtained by fiber-optic bronchoscopy for several diseases including bronchopulmonary carcinoma. Biopsies were taken at a distance from the diseased area. All procedures were reviewed and approved by the institutional review board of the Henri Mondor Hospital (Créteil, France). Written informed consent was obtained from each patient. For each specimen, histopathology confirmed that the bronchial mucosa was normal. Human bronchial epithelial cells (HBECs) were cultured according to a variant of the method of Baeza-Squiban et al. (3). Explants were placed on sterile plastic dishes coated with a collagen G matrix (type I and type III collagen) (33). The culture medium was Dulbecco's modified Eagle's medium-Ham's F-12 medium supplemented with 2% Ultroser G, antibiotics (50 U/ml of penicillin, 10 µg/ml of streptomycin sulfate, and 25 µg/ml of amphotericin B), and 4 mM glutamine. Explants were cultured for 2 wk until there was confluence of HBECs. The epithelial nature of all cultured bronchial cells was previously confirmed by positive staining with an antibody to cytokeratin and negative staining for vimentin.
Isolation and culture of endothelial cells. Human umbilical vein endothelial cells (HUVECs) were obtained by collagenase digestion at 37°C for 20 min as previously described (16). The HUVECs were plated onto 0.1% gelatin-coated wells and grown in RPMI medium supplemented with 10% fetal bovine serum, 30 µg/ml of endothelial cell growth supplement, 50 U/ml of penicillin, 10 µg/ml of streptomycin sulfate, and 4 mM glutamine. HUVECs were used between passages 1 and 5. Endothelial cells were characterized based on morphological criteria (phase-contrast microscopy) and staining with an antibody to factor VIII.
Human BAL supernatants were obtained from six patients who underwent fiber-optic bronchoscopy and BAL to assist in the diagnosis of suspected neurological and renal sarcoidosis. BAL fluid was considered normal if spirometry, CT scan, and BAL fluid cytology findings were normal and the patient remained free of lung disease in the absence of treatment. Three 50-ml aliquots of sterile, pyrogen-free 0.9% NaCl were instilled and recovered with gentle suction. BAL fluid was filtered through moistened coarse gauze and centrifuged at 300 g for 10 min immediately after collection and then divided into aliquots and frozen atExperimental Conditions
For the control conditions, cells were cultured in a 5% CO2-95% air atmosphere at 37°C in serum-free medium.Cytokines.
To investigate whether cytokines affect angiogenesis by influencing
VEGF expression, we tested the effects of tumor necrosis factor
(TNF)-, epidermal growth factor (EGF), keratinocyte growth factor
(KGF), transforming growth factor (TGF)-
, basic fibroblast growth
factor (bFGF), and platelet-derived growth factor (PDGF) on VEGF
synthesis in lung epithelial cells. These cytokines were selected
because they have been reported to stimulate VEGF production in various
cell cultures and are present at wound sites during the early phase of
healing. Cells were grown to confluence and incubated for 24 h in
fresh, serum-free medium containing purified growth factors or
cytokines. Final concentrations were 5 ng/ml of TNF-
, 10 ng/ml of
KGF, 5 ng/ml of TGF-
, 10 ng/ml of EGF, 10 ng/ml of PDGF-BB, and 10 ng/ml of bFGF (R&D Systems, Minneapolis, MN).
Hypoxia.
Cells were grown under hypoxia (0 and 3% O2) in
serum-free medium for 24 h. To achieve hypoxia, a preanalyzed gas
mixture (95% N2-5% CO2) was infused into an
air chamber. The inspired fraction of O2 was checked with a
gas analyzer at the end of each experiment. In some hypoxia
experiments, cells were exposed to hypoxia (0% O2) for
24 h in the presence of anti-TGF- antibody (R&D Systems) or
chicken IgG.
Hydrogen peroxide and nitric oxide donors. Hydrogen peroxide (10 mM), sodium nitroprusside (100 µM), and diethylenetriamine (100 µM) were obtained from Sigma. These compounds were incubated with the cells for 24 h. Diethylenetriamine requires neither cellular metabolism nor reductive activation, and its half-life is ~20 h.
Analysis of mRNA Expression by RT-PCR Amplification
Total RNA was extracted from cells with TRIzol Reagent according to an improvement to the single-step RNA isolation method developed by Chomczynski and Sacchi (6a). Total RNA was quantified at a ratio of 260 to 280 nm, and sample integrity was checked by 1.5% agarose gel electrophoresis.Total RNA (2 µg) was converted to cDNAs by exposure to 5 U of
Moloney murine leukemia virus and 0.5 µg of oligo(dT) for 1 h at
37°C. RT-generated cDNAs encoding VEGF and -actin (acting as a
control of RNA integrity and as an internal standard) were amplified
with RT-PCR. Amplification of 5 µl of cDNA was performed with 0.2 nM
sense and antisense VEGF primers and 2.5 U of Taq polymerase. The oligonucleotide primer sequences for VEGF were 5'-CCATGAACTTTCTGCTCTCTTG-3' (sense) and 5'-GGTGAGAGGTCTCCCGA-3' (antisense) (14). 32P-labeled dCTP was added
(0.3 µM for each sample). Samples were amplified for 35 PCR cycles,
each of which involved denaturation for 1 min at 94°C and extension
for 3 min at 72°C. Annealing time was 2 min; annealing temperature
was 55°C for the VEGF transcripts.
A 3-µl aliquot from each PCR was subjected to polyacrylamide gel
electrophoresis (100 V for 1 h). The amplified products were visualized by autoradiography (24-48 h at 80°C). The
authenticity of the amplified products was confirmed by direct
nucleotide sequencing of the amplified RT-PCR products. Primers were
chosen so as to amplify all VEGF splice forms. All RT-PCR studies were
performed at least four times with mRNAs from different cells.
Immunoblotting of VEGF Isoforms
Whole cell lysates were prepared from cultures in lysis buffer containing 10 mM Tris and 1% SDS, pH 7.4. Protein content in the various cells was determined with the method of Lowry (21a) as modified for the DC protein assay (Bio-Rad, Richmond, CA). Samples (50 µg/lane) were subjected to polyacrylamide gel electrophoresis according to the method of Laemmli (17a). The separated peptides were then electrotransferred to nitrocellulose membranes that were incubated overnight with a blocking solution containing Tris-buffered saline (TBS) with 0.05% Tween 20 (TBS-T) and 3% bovine serum albumin (BSA). VEGF proteins were detected by incubating the membrane with a polyclonal anti-human VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Then the blots were washed and incubated with peroxidase-conjugated goat anti-rabbit IgG (DAKO, Glostrup, Denmark). Immune complexes were visualized with the enhanced chemiluminescence detection system (Amersham).ELISA
Cells were grown to confluence in medium supplemented with 10% FCS. The medium was replaced with serum-free medium. The conditioned medium was used to quantitatively measure VEGF. Sandwich enzymetric methods with specific anti-VEGF antibodies (R&D Systems) were used. The supernatant was collected and used for the ELISA according to the manufacturer's guidelines. In brief, 200 µl of cell supernatant were incubated with 50 µl of assay diluent for 2 h at room temperature in a 96-well plate coated with a monoclonal antibody against VEGF165. After three washes, a conjugate consisting of a polyclonal VEGF antibody and horseradish peroxidase was added and incubated for 2 h at room temperature. After addition of a color reagent, absorbance was measured at 450 nm in a Thermo-Max microplate reader. For standardization, serial dilutions of recombinant human VEGF165 were assayed at the same time.Immunohistochemistry Procedures
The cultured cells were washed in PBS and then fixed in methanol in the culture dishes. The samples were preincubated in proteinase K buffer for 4 h, preincubated in TBS with 3% BSA overnight, and then incubated for 1 h with the primary antibody in TBS-T with 1% BSA. The modified alkaline phosphatase-anti-alkaline phosphatase technique was used.Rabbit anti-mouse immunoglobulin antiserum and alkaline phosphatase-anti-alkaline phosphatase complex were purchased from DAKO. Revelation substrate (naphthol AS-TR and Fast Red TR salt; Sigma) was added and incubated protected from light. Samples were counterstained with hematoxylin or light green (2%) and examined under a light microscope.
Proliferation Studies and Thymidine Uptake
Before the experiments, HUVECs were made quiescent by incubation overnight in medium 199 supplemented with 0.5% FCS. The supernatant of TGF-Statistical Analysis
All data are expressed as means ± SD. Between-group comparisons were done with the nonparametric Mann-Whitney U-test. Significance was defined as P < 0.05. ![]() |
RESULTS |
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Constitutive Expression of VEGF in Untreated Epithelial Cells
VEGF165 was detected in the medium of cells in concentrations that increased gradually with culture time (Fig. 1A). Mean VEGF165 concentration in the serum-free medium after 12 h of culture was 342 pg/ml for A549 cells, 514 pg/ml for 1HAEo
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In BAL fluids from human patients (n = 6), ELISA detected VEGF in concentrations ranging from 67 to 289 pg/ml (mean concentration, 141 pg/ml).
Effect of Various Cytokines on VEGF Expression
When confluent cultures of cell lines in serum-free medium were exposed to 5 ng/ml of TGF-In contrast to TGF-1, cytokines and growth factors (5 ng/ml of
TNF-
, 10 ng/ml of KGF, 10 ng/ml of bFGF, and 10 ng/ml of PDGF-BB)
failed to alter VEGF expression. Only EGF (10 ng/ml) tended to slightly
increase VEGF excretion in the cell medium (Fig.
2).
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Induction of VEGF Expression by Hypoxia
After exposure of human lung epithelial cells to hypoxia (0% O2) for 24 h, VEGF protein levels increased three- to sixfold (Fig. 3A) in an O2 concentration-dependent manner (Fig. 3B). Hypoxia induced a fivefold increase in VEGF expression by HBECs. Experiments involving VEGF measurement by ELISA and Western blot failed to show any additional increase when the 1HAEo
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To investigate the specificity of hypoxia-induced VEGF expression by epithelial cells, we studied the effects of two oxidative stressors, namely hydrogen peroxide and nitric oxide donors, both of which have been shown to modulate VEGF expression by various cell lines. Surprisingly, neither stressor modified VEGF synthesis in lung epithelial cells. It should be emphasized that neither sodium nitroprusside nor NONOates modified VEGF synthesis by the cells (data not shown).
Conditioned Medium From HBECs Induced HUVEC Proliferation via a
Mechanism Not Mediated by TGF-1
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DISCUSSION |
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In this study, we found that both primary and transformed lung
epithelial cells from adult humans were significant sources of the
angiogenic growth factor VEGF. Major stimuli of VEGF production in
these cells were hypoxia and TGF-1. VEGF was detected in cell culture media and BAL fluids in concentrations capable of stimulating endothelial cell growth. These data support a key physiological role of
epithelial cells and VEGF in the regulation of endothelial cell growth
and function in the lung.
In animals free of disorders associated with neoangiogenesis, large amounts of VEGF mRNAs are found in several organs, most notably the kidney, adrenal gland, lung, and heart. In the rat lung, VEGF transcripts have been detected by in situ hybridization in the epithelial cells of the lung alveoli (25). VEGF mRNA and protein have been demonstrated in several epithelia, including those in the skin, intestine, choroid, and uterus. Preliminary reports suggest that VEGF may be expressed by human epithelial cells from nasal polyps (8a) and by a transformed human lung epithelial cell line (26). We demonstrated in vitro expression of VEGF by human primary and transformed lung epithelial cells. Primary human bronchial cells produced a far larger amount of VEGF165 than an immortalized human bronchial cell line or a tumorderived alveolar cell line, suggesting that VEGF production was not related to cell cycle disregulation. Both bronchial and alveolar human epithelia produced VEGF.
VEGF expression is known to be regulated by a variety of factors.
Growth factors and cytokines including PDGF-BB, KGF, EGF, TNF-,
TGF-
1, and interleukin-1
have each been reported to induce VEGF
expression in a variety of cultured cells (5,
8, 18, 26, 31,
34). Among the growth factors tested in our cell culture
system, only TGF-
1 and, to a lesser extent, EGF induced VEGF release
in the culture medium. TGF-
1 has been shown to strongly stimulate
VEGF expression and release by a number of nonendothelial cells
(26) and to be widely expressed in airway epithelia,
suggesting that it may play an important role in angiogenesis. Yolk sac
blood vessel development was absent in transgenic mice with null
mutations for TGF-
1, indicating that TGF-
1 may be indispensable
to normal blood vessel development (10). TGF-
1 tended
to inhibit endothelial cell proliferation and migration in
two-dimensional cultures (14) but promoted the formation
of capillary-like structures in three-dimensional gels
(27). The mechanism underlying stimulation of VEGF
expression by TGF-
1 has been partly elucidated and seems to involve
activation of activator protein-1 transcription factors
(27).
VEGF expression is also affected by several oxidant stressors. In
the rat lung, hypoxia has been reported to increase VEGF mRNA
expression primarily by alveolar epithelial cells (32). Hyperoxia, on the contrary, has been associated with a decrease in
rabbit lung VEGF mRNA levels, which was reversed as soon as the animals
returned to air breathing (22). In human lung epithelial cells, hypoxia is the only stressor known to upregulate VEGF
expression. Many cell lines have been shown to express increased
amounts of VEGF when subjected to hypoxia (21,
24, 30), an effect that may involve both
transcriptional and posttranscriptional mechanisms. Hypoxia may induce
VEGF expression via a variety of other mechanisms, for instance, by
inducing other growth factors that, in turn, induce VEGF expression.
However, our data suggest that TGF-1 was not involved in
hypoxia-induced VEGF upregulation in our model; the increase in VEGF
protein was smaller under hypoxia than under normoxia when the cells
were exposed to TGF-
1, and incubation with a specific neutralizing
antibody to TGF-
did not abolish the increase in VEGF synthesis.
Also, TGF-
release in the medium of HBECs was not increased under
hypoxia. Both experiments confirmed that VEGF induction during hypoxia
was not directly related to TGF-
.
VEGF is constitutively expressed by human bronchial and alveolar
epithelial cells and can be further induced by TGF- or hypoxia. It
would be of interest to know whether VEGF is present in vivo in the
airways and alveolar region of the normal human lung. We found that
alveolar fluid from six patients contained VEGF in concentrations
similar to those in the culture supernatants, taking into account the
generally accepted estimate that pooled BAL fluid is diluted 100 times
compared with alveolar fluid (4). The role of VEGF present
in the airways of the normal lung is unknown. An important issue is
whether the amounts of VEGF present are sufficient to affect
endothelial cell proliferation. In the present study, we found that
adding exogenous VEGF in concentrations similar to those measured in
normal alveolar fluid as well as adding conditioned medium of primary
bronchial cells stimulated endothelial cell growth. Although the
conditioned medium may contain cytokine or growth factors able to
enhance or diminish the proliferative response of endothelial cells,
experiments using specific neutralizing antibodies were consistent with
the proposal that VEGF may be the main endothelial growth factor
released by lung epithelial cells. These results suggest an important
role for VEGF on alveolar and bronchial vessels under either
physiological and pathological conditions.
Several experimental studies (1, 17b) have sought to clarify the potential role of VEGF during lung development and repair. Recently, Zeng et al. (35) described the effects of VEGF expression by developing respiratory epithelial cells under the control of the surfactant protein C promoter. Surfactant protein C-VEGF transgenic mice died at birth, and their lungs consisted of large dilated tubules with increased peritubular vascularity. These findings support the hypothesis that VEGF expression by the respiratory epithelium may be critical for pulmonary angiogenesis. More surprisingly, VEGF expression by the respiratory epithelium disrupted branching morphogenesis, an effect that might be either an indirect consequence of VEGF-mediated changes in vascularity or a manifestation of an autocrine role of VEGF in lung epithelial cells. Although the literature contains no studies designed to investigate the cellular specificity of VEGF receptor expression in the lung, data reported by Tuder et al. (32) militate against expression of a VEGF receptor by lung epithelial cells. In our culture model, cell counts were not increased by exogenous VEGF (data not shown), suggesting that this compound is not an autocrine enhancer of lung epithelial cell proliferation. However, VEGF has been described as a mitogen for adult rat pancreatic duct epithelial cells expressing Flk-1 receptors, suggesting that the growth-promoting effect of VEGF may not be confined to endothelial cells (28). Furthermore, VEGF may act on lung epithelial cells as an autocrine factor capable of modifying parameters unrelated to proliferation, such as migration or permeability.
Angiogenesis in the adult lung remains incompletely understood. However, angiogenesis is an integral feature of normal tissue repair, particularly after dermal injury (13). It is well known that bronchial vessels, in contrast to pulmonary vessels, have a remarkable capacity for development and proliferation in various lung diseases. Recent studies (6, 19) have shown that the airways of asthma patients were more "vascularized" than those of control subjects, suggesting that angiogenesis may be a component of the chronic inflammatory response and that the newly formed vessels may provide an increased amount of mediators to the bronchi, resulting in a vicious circle. Regulation of VEGF synthesis in the lung may affect lung injury repair as suggested by a model of delayed skin wound healing used by Frank et al. (13).
This study demonstrated that VEGF gene expression is detectable
in human lung epithelial cells at the alveolar and bronchial levels.
TGF-1-induced and hypoxia-induced VEGF expression by human lung
epithelial cells may promote neovascularization, thereby contributing
to the repair of injuries to the lung endothelium.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the Lancardis Foundation (Switzerland) and a grant from the Academy of Paris (Legs Poix).
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. Maitre, Unité de Pneumologie, CHU H. Mondor, 51 av. du Mal de Lattre de Tassigny, 94010 Créteil, France (E-mail: bernard.maitre{at}hmn.ap-hop-paris.fr).
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. §1734 solely to indicate this fact.
Received 23 July 1999; accepted in final form 15 March 2000.
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