Expression and regulation of vascular endothelial growth factor in human pulmonary epithelial cells

Sandrine Boussat1, Saadia Eddahibi1, André Coste1,2,3,4, Virginie Fataccioli1, Mallaury Gouge1, Bruno Housset1,2,3, Serge Adnot1,2, and Bernard Maitre1,2,3

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 at -80°C until used.

Experimental 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)-alpha , epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor (TGF)-beta , 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-alpha , 10 ng/ml of KGF, 5 ng/ml of TGF-beta , 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-beta 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 beta -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-beta -stimulated HBECs was placed on quiescent HUVECs for 72 h. In some experiments, HUVECs were incubated for 72 h with the supernatant from TGF-beta -stimulated HBECs in the presence of anti-VEGF antibody (Neosystem, Microm, France) or anti-TGF-beta antibody (R&D Systems). Cell counts were determined by harvesting the cells with trypsin-EDTA and counting them in triplicate with a hemocytometer. Thymidine uptake studies were done by labeling HUVECs with 1 µCi/ml of [methyl-3H]thymidine during stimulation with the conditioned medium. After treatment, the medium was removed, and the cells were washed with PBS and incubated for 30 min at 4°C in cold 5% trichloroacetic acid. Incorporated [3H]thymidine was measured with a liquid scintillation counter.

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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- cells, and 870 pg/ml for primary bronchial cells. These values correspond to 120, 34, and 625 pg/µg of total protein, respectively. Protein synthesis inhibition by cycloheximide blocked VEGF secretion into the medium, suggesting that VEGF protein was newly synthesized rather than released from intracellular stores (data not shown). VEGF mRNA expression was examined in the various cell types. Three major transcripts of 495, 627, and 699 bp were detected, corresponding to the three major VEGF isoforms, VEGF121, VEGF165, and VEGF189, respectively (Fig. 1B). Immunocytochemistry studies using a specific antibody against VEGF165 confirmed that VEGF was expressed by the three human lung epithelial cell types studied, with an intracytoplasmic perinuclear localization as shown in HBECs (Fig. 1C). Control staining with a nonrelevant antibody did not show any signal, confirming the specificity of the staining.


View larger version (74K):
[in this window]
[in a new window]
 
Fig. 1.   A: 165-amino acid splice variant of vascular endothelial growth factor (VEGF165) protein levels in the medium of primary bronchial cells [human bronchial epithelial cells (HBECs)], 1HAEo- cells, and A549 cells. Cells were cultured in medium without serum or Ultroser G for 48 h. VEGF165 was assayed in the cell culture medium by an ELISA after 6 (H6), 12 (H12), and 48 (H48) h. B: VEGF mRNA expression by A549, 1HAEo-, and HBECs as assessed by RT-PCR. PCR products were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. Three bands were detected corresponding to the transcripts for VEGF121, VEGF165, and VEGF189 (495, 627, and 699 bp, respectively). beta -Actin was used as a control for mRNA integrity for each sample. C: immunostaining of HBECs with VEGF antibody. After fixation, the cultured cells were incubated with VEGF antibody. Positive staining was perinuclear, suggesting a vesicular pattern.

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-beta 1 for 24 h, VEGF protein levels increased 4.5- to 5-fold; a 2-fold increase was seen with the primary cells.

In contrast to TGF-beta 1, cytokines and growth factors (5 ng/ml of TNF-alpha , 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).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of cytokines on VEGF protein secretion by A549 cells, 1HAEo- cells, and HBECs. TNF-alpha , tumor necrosis factor-alpha ; EGF, epidermal growth factor; KGF, keratinocyte growth factor; bFGF, basic fibroblast growth factor; PDGF, platelet-derive growth factor; TGF-beta , transforming growth factor-beta . Cells were cultured in serum-free medium for 24 h. Growth factors were added directly to the medium. VEGF protein levels were quantified with an ELISA.

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- cells were simultaneously exposed to TGF-beta 1 (5 ng/ml) and hypoxia (0% O2) (Fig. 4). We also performed experiments to investigate TGF-beta release in the HBEC and 1HAEo- media under hypoxia and found no change compared with normoxia (27 vs. 35 pg/ml under hypoxia in HBECs; 23 vs. 26 pg/ml under hypoxia in 1HAEo- cells). Experiments using neutralizing antibody for TGF-beta confirmed that TGF-beta was not involved in the hypoxic induction of VEGF synthesis by 1HAEo- cells as shown in Fig. 4A.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3.   A: hypoxia (0% O2) increased VEGF protein levels in medium of A549 cells, 1HAEo- cells, and HBECs. Cells were exposed to 24 h of hypoxia. VEGF protein levels were quantified with an ELISA. Data are means ± SD from 5 independent experiments. * Significant difference compared with control cells, P < 0.02. B: dose response of VEGF protein upregulation by hypoxia. VEGF protein levels in medium of A549 and 1HAEo- cells cultured under 0 and 3% O2 were quantified with an ELISA. Data are means ± SD from 5 independent experiments. * Significant difference compared with control cells, P < 0.05.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 4.   Hypoxia (0% O2) increased VEGF protein levels in lung epithelial cells. A: VEGF protein levels in cell medium from 1HAEo- cells cultured in Ultroser G-free medium for 24 h under air (C), hypoxia (Hy), Hy+TGF-beta , Hy in the presence of normal chicken IgG (Hy+chicken IgG), and Hy in the presence of anti-TGF-beta antibody (Hy+anti-TGF-beta ) were quantified with an ELISA. Data are means ± SD from 5 independent experiments. B: analysis of VEGF protein expression by immunoblotting. Cellular proteins from A549 cells and HBECs cultured under indicated conditions were separated by electrophoresis. After transfer to nitrocellulose, they were incubated with VEGF antibody and analyzed as described in MATERIALS AND METHODS. MW, molecular mass.

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-beta 1

To determine whether the amount of VEGF synthesized by the epithelial cells was sufficient to enhance endothelial cell proliferation, we examined the effect of conditioned medium on HUVEC thymidine uptake and cell counts. We first confirmed the growth effect of recombinant VEGF on HUVEC proliferation (Fig. 5A) by incubating HUVECs with FCS or various concentrations of recombinant VEGF or recombinant TGF-beta 1. Under these conditions, exogenous TGF-beta 1 did not increase the growth of HUVECs, whereas both VEGF and FCS increased the number of endothelial cells in a dose-dependent manner. Then we studied the effect of HBEC-conditioned medium on HUVEC proliferation. Thymidine uptake was increased after 24 h (data not shown), and the cell count was increased after 72 h with the HBEC-conditioned medium as shown in Fig. 5B. To confirm the direct effect of VEGF and the absence of effect of TGF-beta 1, we used selective neutralizing antibodies to VEGF and TGF-beta in some experiments. As shown in Fig. 5B, incubation of cells with anti-VEGF antibody partially reversed the effect of HBEC-conditioned medium. In contrast, the addition of anti-TGF-beta antibody did not modify HUVEC proliferation. These data establish that VEGF in the amounts produced by epithelial cells increased endothelial cell proliferation via a mechanism mainly dependent on VEGF and independent from TGF-beta 1.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of TGF-beta , VEGF, and HBEC-conditioned medium (CM) on human umbilical vein endothelial cell (HUVEC) proliferation. At the end of incubation, cells were harvested and then counted with a hemocytometer. A: effects of 10% FCS (SVF), VEGF in indicated concentrations, and 5 ng/ml of TGF-beta 1 on HUVEC proliferation evaluated by cell count determination. B: effects of HBEC CM on endothelial cell proliferation. Effects on HUVEC proliferation were evaluated by cell count determination after 72 h of incubation with DMEM-Ham's F-12 medium (C), CM + normal mouse IgG, CM+anti-VEGF antibody, CM+normal chicken IgG, and CM+anti-TGF-beta antibody. Values are means ± SD of 5 experiments. * Significant difference compared with control cells, P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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-alpha , TGF-beta 1, and interleukin-1beta 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-beta 1 and, to a lesser extent, EGF induced VEGF release in the culture medium. TGF-beta 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-beta 1, indicating that TGF-beta 1 may be indispensable to normal blood vessel development (10). TGF-beta 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-beta 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-beta 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-beta 1, and incubation with a specific neutralizing antibody to TGF-beta did not abolish the increase in VEGF synthesis. Also, TGF-beta 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-beta .

VEGF is constitutively expressed by human bronchial and alveolar epithelial cells and can be further induced by TGF-beta 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-beta 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.


    ACKNOWLEDGEMENTS

This work was supported by a grant from the Lancardis Foundation (Switzerland) and a grant from the Academy of Paris (Legs Poix).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Acarregui, M, Penisten S, Goss K, Ramirez K, and Snyder J. Vascular endothelial growth factor gene expression in human fetal lung in vitro. Am J Respir Cell Mol Biol 20: 14-23, 1999[Abstract/Free Full Text].

2.   Adamson, I, and Young L. Alveolar type II cell growth on a pulmonary endothelial extracellular matrix. Am J Physiol Lung Cell Mol Physiol 270: L1017-L1022, 1996[Abstract/Free Full Text].

3.   Baeza-Squiban, A, Romet S, Moreau A, and Marano F. Progress in outgrowth culture from rabbit tracheal explants: balance between proliferation and maintenance of differentiated state in epithelial cells. In Vitro Cell Dev Biol 27A: 453-460, 1991[ISI].

4.   Bernard, A, Marchandise F, Depelchin S, Lauwerys R, and Sibille Y. Clara cell protein in serum and bronchoalveolar lavage. Eur Respir J 5: 1231-1238, 1992[Abstract].

5.   Brogi, E, Wu T, Namiki A, and Isner J. Indirect angiogenic cytokines upregulate VEGF and bFGF gene expression in vascular smooth muscle cells, whereas hypoxia upregulates VEGF expression only. Circulation 90: 649-652, 1994[Abstract].

6.   Carroll, N, Cooke C, and James A. Bronchial blood vessel dimensions in asthma. Am J Respir Crit Care Med 155: 689-695, 1997[Abstract].

6a.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

7.   Clark, R, and Henson P. The Molecular and Cellular Biology of Wound Repair. New York: Plenum, 1988.

8.   Cohen, T, Nahari D, Cerem L, Neufeld G, and Levi B. Interleukin 6 induces the expression of vascular endothelial growth factor. J Biol Chem 271: 736-741, 1996[Abstract/Free Full Text].

8a.   Coste, A, Brugel L, Maitre B, Bousset S, Papon JF, Wingerstmann L, Peynegre R, and Escudier E. Inflammatory cells as well as epithelial cells in nasal polyps express vascular endothelial growth factor. Eur Respir J 15: 367-372, 2000[Abstract/Free Full Text].

9.   Cozens, A, Yezzi M, Yamaya M, Steiger D, Wagner J, Garber S, Chin L, Simon E, and Gruenert D. A transformed human epithelial cell line that retains tight junctions post crisis. In Vitro Cell Dev Biol 28A: 735-744, 1992.

10.   Dickson, MC, Martin JS, Cousins FM, Kulkarni AB, Karlsson S, and Akhurst RJ. Defective haematopoiesis and vasculogenesis in transforming growth factor-beta 1 knock out mice. Development 121: 1845-1854, 1995[Abstract/Free Full Text].

11.   Ferrara, N, Jakeman K, and Leung D. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev 13: 18-32, 1992[ISI][Medline].

12.   Fong, G, Rossant J, Gertsenstein M, and Breitman M. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 376: 66-70, 1995[ISI][Medline].

13.   Frank, S, Hübner G, Breier G, Longaker M, Greenhalgh D, and Werner S. Regulation of vascular endothelial growth factor expression in cultured keratinocytes. J Biol Chem 270: 12607-12613, 1995[Abstract/Free Full Text].

14.   Heimark, RL, Twardzik DR, and Schwartz SM. Inhibition of endothelial regeneration by type-beta transforming growth factor from platelets. Science 233: 1078-1080, 1986[ISI][Medline].

15.   Houck, K, Ferrara N, Winer J, Cachianes G, Li B, and Leung D. The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol 5: 1806-1814, 1991[Abstract].

16.   Jornot, L, and Junod A. Response of human endothelial cell antioxidant enzymes to hyperoxia. Am J Respir Cell Mol Biol 6: 107-115, 1992[ISI][Medline].

17.   Kaipainen, A, Korhonen J, Pajusalo O, Aprelikova O, Persico M, Terman B, and Alitalo K. The related FLT-4, FLT-1 and KDR receptor tyrosine kinase show distinct expression patterns in human fetal endothelial cells. J Exp Med 178: 2077-2088, 1993[Abstract].

17a.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

17b.   Lassus, P, Ristimäki A, Ylikorkala O, Viinikka L, and Andersson S. Vascular endothelial growth factor in human preterm lung. Am J Respir Crit Care Med 159: 1429-1433, 1999[Abstract/Free Full Text].

18.   Li, J, Perrella M, Tsai J, Yet S, Hsieh C, Yoshizumi M, Patterson C, Endege W, Zhou F, and Lee M. Induction of vascular endothelial growth factor gene expression by interleukin-1 beta in rat aortic smooth muscle cells. J Biol Chem 270: 308-312, 1995[Abstract/Free Full Text].

19.   Li, X, and Wilson J. Increased vascularity of the bronchial mucosa in mild asthma. Am J Respir Crit Care Med 156: 229-233, 1997[Abstract/Free Full Text].

20.   Lieber, M, Smith B, Szakal A, Nelson-Rees W, and Todaro G. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int J Cancer 17: 62-70, 1976[ISI][Medline].

21.   Liu, Y, Cox S, Morita T, and Kourembanas S. Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells. Circ Res 77: 638-643, 1995[Abstract/Free Full Text].

21a.   Lowry, OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurements with the Folin phenol reagent. J Biol Chem 193: 265-275, 1951[Free Full Text].

22.   Maniscalco, W, Watkins R, D'Angio C, and Ryan R. Hyperoxic injury decreases alveolar epithelial cell expression of vascular endothelial growth factor in neonatal rabbit lung. Am J Respir Cell Mol Biol 16: 557-567, 1997[Abstract].

23.   Maniscalco, W, Watkins R, Finkelstein J, and Campbell M. Vascular endothelial growth factor mRNA increases in alveolar epithelial cells during recovery from oxygen injury. Am J Respir Cell Mol Biol 13: 377-386, 1995[Abstract].

24.   Minchenko, A, Bauer T, Salceda S, and Caro J. Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo. Lab Invest 71: 374-379, 1994[ISI][Medline].

25.   Monacci, W, Merrill M, and Olfield E. Expression of vascular permeability factor/vascular endothelial growth factor in normal rat tissues. Am J Physiol Cell Physiol 264: C995-C1002, 1993[Abstract/Free Full Text].

26.   Pertovaara, L, Kaipainen A, Mustonen T, Orpana A, Ferrara N, Saksela O, and Alitalo K. Vascular endothelial growth factor is induced in response to transforming growth factor-beta in fibroblastic and epithelial cells. J Biol Chem 269: 6271-6274, 1994[Abstract/Free Full Text].

27.   Pintavorn, P, and Ballermann BJ. TGF-beta and the endothelium during immune injury. Kidney Int 51: 1401-1412, 1997[ISI][Medline].

28.   Rooman, I, Schuit F, and Bouwens L. Effect of vascular endothelial growth factor on growth and differentiation of pancreatic ductal epithelium. Lab Invest 76: 225-232, 1997[ISI][Medline].

29.   Shifren, J, Doldi N, Ferarra N, Mesiano S, and Jaff R. In the human fetus, vascular endothelial growth factor is expressed in epithelial cells and myocytes, but not vascular endothelium: implications for mode of action. J Clin Endocrinol Metab 79: 316-322, 1994[Abstract].

30.   Shweiki, D, Itin A, Neufeld G, Gitay-Goren H, and Keshet E. Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis. J Clin Invest 91: 2235-2243, 1993[ISI][Medline].

31.   Stavri, G, Zachary I, Baskerville P, Martin J, and Erusalimsky J. Basic fibroblast growth factor upregulates the expression of vascular endothelial growth factor in vascular smooth muscle cells. Synergistic interaction with hypoxia. Circulation 92: 11-14, 1995[Abstract/Free Full Text].

32.   Tuder, R, Flook B, and Voelkel N. Increased gene expression for VEGF and the VEGF receptors KDR/Flk and Flt in lungs exposed to acute or to chronic hypoxia. J Clin Invest 95: 1798-1807, 1995[ISI][Medline].

33.   Yao, P, Buhler J, D'Ortho M, Lebargy F, Delclaux C, Harf A, and Lafuma C. Expression of matrix metalloproteinase gelatinase A and B by cultured epithelial cells from human bronchial explants. J Biol Chem 271: 15580-15589, 1996[Abstract/Free Full Text].

34.   Yoshida, S, Ono M, Shono T, Izumi H, Ishibashi T, Suzuki H, and Kuwano M. Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis. Mol Cell Biol 17: 4015-4023, 1997[Abstract].

35.   Zeng, X, Wert S, Federici R, Peters K, and Whitsett J. VEGF enhances pulmonary vasculogenesis and disrupts lung morphogenesis in vivo. Dev Dyn 211: 215-227, 1998[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 279(2):L371-L378
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society