Am J Physiol Lung Cell Mol Physiol 290: L897–L908, 2006.
First published December 16, 2005; doi:10.1152/ajplung.00116.2005.
Cigarette smoke disrupts VEGF165-VEGFR-2 receptor signaling complex in
rat lungs and patients with COPD: morphological impact of
VEGFR-2 inhibition
John A. Marwick,1* Christopher S. Stevenson,1 June Giddings,1
William MacNee,2 Keith Butler,1 Irfan Rahman,3* and Paul A. Kirkham1*
1
Novartis Institute for Biomedical Research, Horsham, and 2Respiratory Medicine Unit, Medical Research Council Centre
for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom; and 3Department of Environmental
Medicine, Lung Biology and Disease Program, University of Rochester Medical Center, Rochester, New York
Submitted 14 March 2005; accepted in final form 16 November 2005
kinase insert domain receptor; neuropilin-1; vascular endothelial
growth factor receptor-2; chronic obstructive pulmonary disease
mitogen for vascular endothelial cells
and is integral for endothelial cell migration, proliferation, and
survival (6, 8, 34). VEGF is expressed as several splice
variants, arising from alternate splicing of a single gene (51).
VEGF165 is the most highly expressed isoform in the human
lung, where it is produced mainly by epithelial cells, acting as
VEGF IS A HIGHLY SPECIFIC
* J. A. Marwick, I. Rahman, and P. A. Kirkham contributed equally to this
paper.
Address for reprint requests and other correspondence: I. Rahman, Dept. of
Environmental Medicine, Univ. of Rochester Medical Center, Box 850, 601
Elmwood Ave., Rochester, NY 14642 (e-mail: irfan_rahman@urmc.
rochester.edu).
http://www.ajplung.org
a paracrine mediator (51). As one of the most potent angiogenic factors known, it is fundamental in the development and
maintenance of the vasculature. Deletion of even one allele
disrupts cardiovascular development, leading to embryonic
death (5), and deletion or inhibition of VEGF in specific tissues
results in significant reduction in capillary density with tissue
cell apoptosis (8).
Maintenance of the microvasculature in the lung is critical
for gas exchange and the integrity of the alveolar structure.
Emphysema, one of the major components of chronic obstructive pulmonary disease (COPD), is defined as enlargement of
the distal air spaces as a result of destruction of the alveolar
walls. Cigarette smoke is one of the main etiologic factors in
the development of emphysema and evokes significant alterations in the pulmonary vasculature including decreased capillary diameter and density and a decrease in precapillary
arterioles in both animal models and chronic COPD patients
(56, 60). Cigarette smoke exposure also inhibits angiogenesis
of pulmonary artery cells in vitro (49). However, the molecular
mechanism of inhibition of angiogenesis was not studied.
VEGF stimulates angiogenesis through VEGF receptor
(VEGFR)-2, also known as the kinase insert domain receptor
(KDR), which is almost exclusively expressed on endothelial
cells (6, 9, 15, 34). Inhibition of VEGFR-2 during a critical
period of lung growth in infant rats results in decreased
alveolarization and arterial density (21). Moreover, chronic
inhibition of VEGFR-2 leads to enlargement of the air spaces,
alveolar septal cell death, and the “pruning of the arterial tree,”
which is also a feature of emphysema (23). Recent studies have
shown that lung-targeted ablation of the VEGF gene leads to an
emphysema phenotype in mice (50). Furthermore, human emphysematous lungs display significantly reduced VEGF and
VEGFR-2 expression and increased apoptosis of endothelial
cells compared with healthy lungs (22, 28, 40). This highlights
the concept of VEGF-VEGFR-2 signaling in endothelial cell
survival and the maintenance of the alveolar structures in the
pathogenesis of emphysema and underscores the importance of
the vasculature in what is traditionally thought of as an airway
disease. However, the specific role of VEGFR-2 signaling in
the involvement of COPD is not known.
VEGF165-VEGFR-2 signaling is highly regulated, involving
the VEGFR-2 coreceptor neuropilins (NRPs) and the heparin
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Marwick, John A., Christopher S. Stevenson, June Giddings,
William MacNee, Keith Butler, Irfan Rahman, and Paul A.
Kirkham. Cigarette smoke disrupts VEGF165-VEGFR-2 receptor
signaling complex in rat lungs and patients with COPD: morphological impact of VEGFR-2 inhibition. Am J Physiol Lung Cell Mol
Physiol 290: L897–L908, 2006. First published December 16, 2005;
doi:10.1152/ajplung.00116.2005.—VEGF is fundamental in the development and maintenance of the vasculature. VEGF165 signaling
through VEGF receptor (VEGFR)-2/kinase insert domain receptor
(KDR) is a highly regulated process involving the formation of a
tertiary complex with glypican (GYP)-1 and neuropilin (NRP)-1. Both
VEGF and VEGFR-2 expression are reduced in emphysematous
lungs; however, the mechanism of regulation of VEGF165 signaling
through the VEGFR-2 complex in response to cigarette smoke exposure in vivo, and in smokers with and without chronic obstructive
pulmonary disease (COPD), is still unknown. We hypothesized that
cigarette smoke exposure disrupts the VEGF165-VEGFR-2 complex, a
potential mechanism in the pathogenesis of emphysema. We show
that cigarette smoke exposure reduces NRP-1 and GYP-1 as well as
VEGF and VEGFR-2 levels in rat lungs and that VEGF, VEGFR-2,
GYP-1, and NRP-1 expression in the lungs of both smokers and
patients with COPD are also reduced compared with nonsmokers.
Moreover, our data suggest that specific inhibition of VEGFR-2 alone
with NVP-AAD777 would appear not to result in emphysema in the
adult rat lung. As both VEGF165 and VEGFR-2 expression are
reduced in emphysematous lungs, decreased GYP-1 and NRP-1 expression may yet further disrupt VEGF165-VEGFR-2 signaling.
Whether or not this by itself is critical for inducing endothelial cell
apoptosis and decreased vascularization of the lung seen in emphysema patients is still unclear at present. However, targeted therapies to
restore VEGF165-VEGFR-2 complex may promote endothelial cell
survival and help to ameliorate emphysema.
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CIGARETTE SMOKE DISRUPTS VEGF165-VEGFR-2 COMPLEX
MATERIALS AND METHODS
Animal cigarette smoke exposure. Male Sprague-Dawley rats
(323 ⫾ 2.5 g; Charles River, Margate, UK) were divided into six
exposure groups: 1) 3 day sham exposed (n ⫽ 6), 2) 3 day smoke
exposed (n ⫽ 6), 3) 8 wk sham exposed (n ⫽ 6), 4) 8 wk smoke
exposed (n ⫽ 6), 5) 6 mo sham exposed (n ⫽ 6), and 6) 6 mo smoke
exposed (n ⫽ 6). The rats were exposed to whole body cigarette
AJP-Lung Cell Mol Physiol • VOL
smoke generated from 2R4F research cigarettes (University of Kentucky, Louisville, KY) in 7-liter smoking chambers at four cigarettes
per day, Monday to Friday. The total particulate matter (TPM) was
27.1 ⫾ 0.8 mg/cigarette. To ensure a consistent exposure across
exposed animals, cotinine levels were measured. Plasma cotinine
levels were 2.66 ⫾ 0.12 ␮M 1 h after exposure (cotinine was not
detectable in the plasma from air-exposed animals) and 0.51 ⫾ 0.07
␮M after 24 h. There was no progressive increase in cotinine levels
over 1 wk of exposures. Carboxyhemoglobin levels were measured
immediately after the animals were removed from the chambers. A
peak level of 42 ⫾ 4.0 ␮M was reached after the fourth cigarette,
which quickly decreased after exposure was stopped. Sham-exposed
animals were exposed to medical-grade oxygen under the same
conditions as smoke-exposed animals. Rats were killed 2 h after last
exposure by intraperitoneal injection of 200 mg of pentobarbital
sodium. Studies were conducted with a license issued under the
Animals (Scientific Procedures) Act 1986 by the Home Office (UK
government) and were further subject to local ethical reviews as
required by the Act.
Smokers and patients with COPD. Peripheral lung tissue was
obtained from subjects undergoing lung resection for peripheral bronchial carcinomas. After histological examination the lung tissue was
snap frozen and stored at ⫺80°C until further use. Lung tissue was
obtained from six COPD patients [1 man and 5 women; 65.8 ⫾ 2.5
(SE) yr]. All patients were current smokers (Table 1). Lung tissue was
also obtained from six smokers (3 men and 3 women; 64.7 ⫾ 2.4 yr)
with no clinical or spirometric evidence of COPD (Table 1). Finally,
lung tissue was obtained from six nonsmokers (4 men and 2 women;
62.8 ⫾ 2.9 yr) with little or no smoking history, no clinical evidence
of COPD, and normal lung function. A summary of the data on lung
function tests of these patients is presented in Table 1. All subjects
showed ⬍13% reversibility of the forced expiratory volume in 1 s
after 400 ␮g of inhaled salbutamol. Exclusion criteria included 1)
diffuse pulmonary inflammation associated with lung fibrosis, 2)
absence of tumor-free or pneumonia-free lung tissue specimens, and
3) obstruction of central bronchi due to the tumor. Preoperatively,
Table 1. Characteristics of subjects and patients
Subject
Smoking,
pack䡠yr
FEV1
(Expiratory)
FEV1 (volume
predicted)
FEV1/FVC,
%
Nonsmokers (62.8 ⫾ 2.9 yr)
1
2
3
4
5
6
0
0.027
0
0
0
0
1.24
1.3
2.45
1.56
4.6
2.26
68.9
68.4
111.4
78.0
75.4
107.6
95.38
76.47
83.90
81.68
79.37
77.93
86.8
71.9
81.8
102.4
100
1156
76.66
85.19
71.05
74.04
77.19
73.24
Smokers (64.7 ⫾ 2.4 yr)
7
8
9
10
11
12
112.5
51.75
30
33.75
111.1
30.75
13
14
15
16
17
18
88
31.8
27
45
28
103
2.43
2.3
2.7
1.74
2.2
2.08
COPD (65.8 ⫾ 2.5 yr)
1.8
1.06
1.75
1.9
1.35
1.5
52.9
66.3
76.1
52.8
38.6
45.5
48.65
52.48
62.31
52.02
41.54
34.88
Subjects 1– 6 were nonsmokers, subjects 7–12 were smokers, and subjects
13–18 were chronic obstructive pulmonary disease (COPD) patients who were
current smokers. FEV1, forced expiratory volume in 1 s; FVC, forced vital
capacity.
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sulfate proteoglycan (HSPG) glypican (GYP)-1 in the formation of a tertiary signaling complex (6, 8, 34). NRPs are 130to 140-kDa cell surface glycoproteins that mediate neuronal
guidance and angiogenesis (13). NRP-1 is expressed by neuronal, endothelial, and tumor cells and is essential for the
normal development of both the nervous and cardiovascular
systems (25, 44, 47). An isoform-specific receptor for VEGF,
NRP-1 selectively binds VEGF165, serving as a coreceptor for
VEGFR-2 on endothelial cells, where VEGF165 bridges
VEGFR-2 and NRP-1, thereby enhancing VEGF165 binding to
VEGFR-2 and subsequent bioactivity (24, 43, 45, 47). Crosslinking and quantitative binding studies demonstrate that the
binding of VEGF165 to VEGFR-2 and the chemotaxis to a
VEGF165 gradient is enhanced 4- to 6-fold and 2.5-fold higher,
respectively, in endothelial cells coexpressing NRP-1 compared with cells expressing VEGFR-2 alone (12, 47).
Heparin and heparin sulfate are thought to play a role in the
regulation of angiogenesis. VEGF165 binding to VEGFR-2 is
heparin dependent; concentrations ranging from 0.1 to 1 ␮g/ml
enhance the binding of VEGF165 to VEGFR-2 eightfold (11).
This binding is completely inhibited after the digestion of
endothelial cells with heparinase but can be restored by the
addition of exogenous heparin (16). Heparin sulfate also enhances the binding of VEGF165 to NRP-1 (12, 16, 47). Around
95% of the heparin sulfate of mammalian cell surfaces, basement membranes, and the ECM arises from HSPGs. ECMassociated HSPGs function as extracellular storage deposits or
reservoirs for the heparin-binding VEGF isoforms, acting both
to protect bound ligands from enzymatic degradation and to
compartmentalize VEGF, immobilizing it and thereby preventing unregulated levels resulting in pulmonary edema in the
lung (14, 18, 37, 46).
GYP-1 is a glycosylphosphatidylinositol-anchored cell surface HSPG expressed on vascular endothelial cells and is
implicated in cell adhesion and migration and modulation of
growth factor activities (4, 59). It binds VEGF165 with high
affinity by its heparin sulfate chains. This binding is specific
and saturable and enhances the interaction of VEGF165 with its
signaling receptors (7). GYP-1 has also been shown to restore
both the biological activity and the binding of oxidized
VEGF165 to VEGFR-2; however, the mechanism remains unknown (42).
In this study, we hypothesized that VEGFR-2 and VEGF
expression is decreased by cigarette smoke exposure in rat
lungs and that this reduction is associated with the disruption of
the VEGF165-VEGFR-2 receptor signaling complex. We show
for the first time that cigarette smoke exposure results in
decreased lung expression of the VEGFR-2 coreceptor NRP-1
and the VEGF165 chaperone GYP-1. Moreover, we show that
VEGF165, VEGFR-2, GYP-1, and NRP-1 expression are also
decreased in the lungs of human smokers and COPD patients.
Furthermore, specific inhibition of VEGFR-2 alone does not
result in emphysema-like changes in rat lungs.
CIGARETTE SMOKE DISRUPTS VEGF165-VEGFR-2 COMPLEX
AJP-Lung Cell Mol Physiol • VOL
the large airway in two pieces of the left lobe were counted (i.e., 4
fields, total area ⬃6.5 mm2). When there was a difference of ⬎5
cells/mm2 between the average counts of two and four fields, an extra
field was counted in piece 3 (total area of ⬃8.3 mm2).
Enzyme-linked immunobsorbent assay. ELISA was performed in
total lung homogenate, using rat and human match-paired antibodies
from R&D Systems according to the manufacturer’s instructions; 50
␮g of whole cell lysate protein was made up to 100 ␮l in PBS with
complete cocktail inhibitors (Roche). Plates were read at 570 nm
(Dynex MRXTC, Dynex Technologies, Ashford, UK).
Inhibition of VEGFR-2 in rat lungs. Male Sprague-Dawley rats
(323 ⫾ 2.5 g; Charles River) were divided into four groups, 1) sham
exposed ⫹ vehicle, 2) cigarette smoke exposed ⫹ vehicle, 3) sham
exposed ⫹ VEGFR-2 inhibitor, and 4) cigarette smoke exposed ⫹
VEGFR-2 inhibitor, each group consisting of four animals, and all
treatments were given for 3 wk. Cigarette smoke exposure was
performed as described above. The VEGFR-2 inhibitor NVPAAD777 (31) was dissolved in PEG 400 as vehicle and administered
at 20 mg/kg subcutaneously three times per week. Sham-exposed
animals were given vehicle alone in the same manner. Rats were
killed 2 h after last cigarette smoke/sham exposure by intraperitoneal
injection of 200 mg of pentobarbital sodium. The right lung was tied
off, removed, snap frozen in liquid nitrogen, and then stored at ⫺80°C
until required for assessment of the effectiveness of VEGFR-2 inhibition in vivo by NVP-AAD777 as follows. Frozen lung tissue was
homogenized in whole cell lysis buffer, and the resultant whole cell
lysate was subjected to Western blotting for both phospho-VEGFR-2
(Tyr1175) [New England Biolabs (UK) catalog no. 2478] and total
VEGFR-2 as described above. All samples were assessed under equal
protein loading conditions, and levels of phosphorylated VEGFR-2
detected were normalized against levels of total VEGFR-2. The
remaining left lung was inflated, paraffin embedded, cut, and mounted
onto slides for morphometric analysis. Dewaxing was performed as
described above, and the sections were stained with hematoxylin and
eosin. Morphometric analysis was performed as described by Hind
and Maden (17). Images were acquired with a ⫻10 objective from
four sections per animal, giving at least five nonoverlapping fields/
section. This resulted in 22–34 nonoverlapping fields per animal, from
which the mean linear intercept was determined with a % coefficient
of variation of between 6 and 12%. For vasculature counting, blood
vessels within peripheral lung tissue were counted within a 1-mm2
grid from four nonadjacent fields per section from each animal, using
a ⫻10 objective. The data from all the animals in each treatment
group were averaged and displayed as the mean ⫾ SE vasculature
count per square millimeter.
Protein assay. Protein concentrations were determined with a
bicinchoninic acid protein assay kit according to the manufacturer’s
instructions (Bio-Rad).
Statistical analysis. All data are expressed as means ⫾ SE. Statistical analysis of significance was performed with Minitab software.
The data were normally distributed, and values obtained in the
different groups of rats were compared with one-way ANOVA, using
Tukey’s post hoc test. Statistical significance was accepted at P ⬍
0.05.
RESULTS
VEGF expression levels in rat smoking model lungs. To
investigate the expression of VEGF in rat lungs exposed to
cigarette smoke, RT-PCR for VEGF mRNA expression and
ELISA for protein expression were performed. VEGF mRNA
expression in the lungs remained unchanged after 3 days of
cigarette smoke exposure compared with sham-exposed animals; however, it was significantly decreased after both 8-wk
and 6-mo smoke exposure compared with sham-exposed animals (Fig. 1A). VEGF protein expression was significantly
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none of the patients had clinical evidence of an upper respiratory tract
infection. No antibiotics had been taken in the 4 wk before the
operation, and no glucocorticoids had been taken during the 3 mo
before the operation. Ethical consent was obtained from the local
regional ethics committee, and informed consent was obtained from
all subjects.
Whole cell lung tissue homogenate. Lung tissue (0.1 g) was
homogenized in 1 ml of ice-cold lysis buffer containing 1% NP-40,
0.1% SDS, 0.01 M deoxycholic acid, and a complete protease cocktail
inhibitor tablet with EDTA (Roche UK). The homogenate was kept
on ice for 45 min and then centrifuged at 13,000 rpm for 25 min at
4°C, and the supernatant was aliquoted and stored at ⫺80°C until
further use.
Western blotting. Whole cell lysate (30 ␮g protein) was subject to
electrophoresis on Tris-glycine gels and transferred to Proban BA 85
nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany)
in Tris-glycine buffer containing 20% methanol. Western blots were
visualized with enhanced chemiluminescence fluid (ECF; Amersham). Blots were stripped with a Chemicon Re-Blot Plus Western
recycling kit (Chemicon International, Temecula, CA), blocked, and
reprobed. The antibodies used were anti-Flk-1 (VEGFR-2; Santa
Cruz), anti-NRP1 (Oncogene), anti-GYP-1 (Santa Cruz), and antiGAPDH (Abcam, Cambridge, UK).
Immunoprecipitation. Whole cell lung homogenate (200 ␮g of
protein) in a final volume of 100 ␮l was incubated for 1 h with
anti-VEGFR-2 (Santa Cruz) and then immunoprecipitated with protein A agarose beads (Calbiochem) at 4°C overnight with constant
agitation. Samples were then centrifuged, and the beads were washed.
Sample loading buffer was added to the immunoprecipitated
VEGFR-2 agarose bead suspension, boiled, and run with SDS-PAGE
as described previously (31). Blots were probed with anti-phosphotyrosine (Upstate) and then stripped and reprobed with anti-VEGFR-2
(Santa Cruz) as a loading control.
RNA isolation. Lung tissue (0.1 g) was homogenized in 1 ml of
TRIzol (Invitrogen Life Technologies, Paisley, UK) and left at room
temperature for 15 min. RNA was extracted according to the manufacturer’s instructions. The RNA was then aliquoted and stored at
⫺80°C until further use. RNA was quantified by a spectrophotometer
at 260 nm. Protein contamination was checked at 280 nm, and a ratio
of ⬍1.6 was accepted.
Reverse transcriptase-PCR. RNA (2␮g) was reverse transcribed
into cDNA with oligo(dT) Moloney murine leukemia virus-reverse
transcriptase in a 20-␮l final volume. RT-PCR was then performed
with 5 ␮g of cDNA (primers in Table 1) and 1⫻ PCR buffer (in mM:
50 KCl, 10 Tris 䡠 HCl pH 9.0, and 1.5 MgCl2 with 0.1% Triton X-100),
400 ␮M 2-deoxynucleotide 5⬘-triphosphate, 1 mM MgCl2, 5⬘ and 3⬘
primers at 50 pM, and 2 U of Taq DNA polymerase (Promega).
RT-PCR amplification was carried out for rat VEGFR-2 (29), rat
VEGF (36), and rat NRP-1 (32), and rat GAPDH (52) as a loading
control, using previously described RT-PCR parameters.
RT-PCR reactions were carried out with a thermocycler, and the
PCR products were electrophoresed on a 1.5% agarose gel containing
ethidium bromide. The bands were visualized and quantified by
densitometry with a UV Grab-IT software package.
Immunohistochemistry. Briefly, lung sections were dewaxed in
xylene and rehydrated, and endogenous peroxidase inhibited with
0.5% hydrogen peroxide in methanol for 10 min. A controlled digestion was carried out in warmed trypsin solution (ICN Pharmaceuticals,
Basingstoke, UK). Slides were incubated in anti-active caspase-3
(Abcam) overnight at 4°C. Immunodetection was preformed with
biotinylated rabbit anti-mouse Ig reagent (DakoCytomation, Ely, UK),
streptavidin-biotin complex reagent (Dako), and 3,3⬘-diaminobenzidine (Sigma). The nuclei were counterstained with Cole’s hematoxylin solution. For rat lungs, tonsil acted as an internal positive control.
For negative controls, the primary antibody was omitted from one
section of each of the samples. Cells were identified by both positive
staining and standard morphological criteria. Two fields to the right of
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CIGARETTE SMOKE DISRUPTS VEGF165-VEGFR-2 COMPLEX
decreased after 3-day, but not 8-wk, cigarette smoke exposure
compared with sham-exposed animals. However, a significant
reduction in VEGF protein expression was found after 6-mo
cigarette smoke exposure (Fig. 1B).
VEGF expression levels in human smoker and COPD patient lungs. To confirm the findings from rat lungs in human
tissues, we assessed VEGF protein expression by ELISA in
smokers and patients with COPD. We observed significantly
reduced VEGF protein expression in both smoker and COPD
lung tissue compared with nonsmoker lung tissue (Fig. 2).
VEGFR-2 expression levels in rat smoking model lungs. We
also examined the effects of cigarette smoke on VEGFR-2
expression in rat lungs by assessing KDR protein levels in the
lungs by Western blot analysis and mRNA expression by
RT-PCR. Two bands for VEGFR-2 protein expression were
AJP-Lung Cell Mol Physiol • VOL
Fig. 2. Cigarette smoke decreases VEGF protein expression human smoker
and chronic obstructive pulmonary disease (COPD) lungs. VEGF protein is
significantly decreased in both human smoker and COPD lungs compared with
healthy nonsmoker lungs, as assessed by ELISA (n ⫽ 6). Values are means ⫾
SE of VEGF expression. ***P ⬍ 0.001 compared with healthy nonsmoker
lungs.
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Fig. 1. Cigarette smoke decreases VEGF expression in rat smoking model. A:
VEGF protein is significantly decreased after both 3-day and 6-mo but not
8-wk cigarette smoke exposure in rat lungs compared with sham-exposed
animals, as assessed by ELISA (n ⫽ 6). B: VEGF mRNA expression is
significantly decreased after both 8-wk and 6-mo but not 3-day cigarette smoke
exposure in rat lungs compared with sham-exposed animals, as assessed by
RT-PCR (n ⫽ 6). Values are means ⫾ SE of VEGF expression. ***P ⬍ 0.001
compared with sham-exposed animals.
observed in the rat lung, a semiglycosylated immature 195-kDa
protein and the fully glycosylated cell surface-expressed 235kDa protein. A decrease in the immature 195-kDa protein at 3
days, 8 wk, and 6 mo of cigarette smoke exposure compared
with sham-exposed animals was observed (Fig. 3A). However,
no significant changes were seen in the mature 235-kDa protein
at either 3 days or 8 wk of exposure, although there was a
decreasing trend at 8 wk of exposure (Fig. 3B). There was,
however, a significant decrease in the mature 235-kDa protein
after 6 mo of exposure compared with sham-exposed animals
(Fig. 3B). We observed no change in VEGFR-2 mRNA expression after 3 days of exposure in the lungs; however, there
was a significant decrease in both 8-wk and 6-mo cigarette
smoke-exposed compared with sham-exposed lungs (Fig. 3C).
VEGFR-2 expression levels in human smoker and COPD
patient lungs. We examined whether cigarette smoke-mediated
decrease in VEGFR-2 expression is also mirrored in human
lung tissue. We assessed VEGFR-2 protein expression by
Western blot analysis in lungs of smokers and patients with
COPD. We observed no significant difference in the levels of
VEGFR-2 protein expression between smoker and nonsmoker
lungs. However, there was a significant decrease in VEGFR-2
expression levels between COPD patient and nonsmoker lungs
(Fig. 4).
VEGFR-2 phosphorylation levels in rat smoking model,
smoker, and COPD patient lungs. To assess whether the
observed reduction of VEGFR-2 expression induced by cigarette smoke exposure was paralleled by reduced VEGFR-2
activation, we studied VEGFR-2 phosphorylation by immunoprecipitation and Western blot. Phosphorylated VEGFR-2 was
normalized against native VEGFR-2. We observed no change
in the phosphorylation status of the VEGFR-2 receptor in rat
lungs after 3 days of smoke exposure compared with shamexposed animals. However, a significant decrease in phosphorylated VEGFR-2 levels after 8-wk and 6-mo cigarette smoke
exposure was seen compared with sham-exposed animals (Fig.
5A). This is in agreement with the previous observation in
which the mature VEGFR-2 receptor expression was reduced
at both 8 wk and 6 mo but not at 3 days of cigarette smoke
exposure (21). There was also a small, although significant,
reduction in VEGFR-2 phosphorylation in smokers and a more
distinct reduction in COPD patients (Fig. 5B).
CIGARETTE SMOKE DISRUPTS VEGF165-VEGFR-2 COMPLEX
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Fig. 3. Cigarette smoke decreases VEGF receptor (VEGFR)-2 expression
in rat smoking model. A: immature, semiglycosylated VEGFR-2/kinase
insert domain receptor (KDR) protein is significantly decreased after
3-day, 8-wk, and 6-mo cigarette smoke exposure in rat lungs compared
with sham-exposed animals, as assessed by immunoblotting (n ⫽ 6). B:
mature, fully glycosylated cell-surface expressed VEGFR-2 protein is
significantly decreased after 6-mo cigarette smoke exposure and shows a
decreasing trend at both 3 days and 8 wk of exposure in rat lungs
compared with sham-exposed animals, as assessed by immunoblotting
(n ⫽ 6). C: VEGFR-2 mRNA is significantly decreased after both 8-wk
and 6-mo but not 3-day cigarette smoke exposure in rat lungs compared
with sham-exposed animals, as assessed by RT-PCR (n ⫽ 6). Values are
means ⫾ SE of VEGFR-2 expression. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍
0.001 compared with sham-exposed animals.
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CIGARETTE SMOKE DISRUPTS VEGF165-VEGFR-2 COMPLEX
NRP-1 expression levels in smoker and COPD patient lungs.
NRP-1 has been shown to be important in the affinity of
VEGF165 binding to VEGFR-2. To establish whether cigarette
smoke affects the expression of NRP-1, we assessed NRP-1
mRNA expression by RT-PCR and protein expression by
Western blot. We observed no change in the mRNA expression
of NRP-1 at any time points (data not shown); however, there
was a significant decrease in protein expression at 3 days, 8 wk,
and 6 mo (Fig. 6A). This suggests that cigarette smoke disrupts
one of the components of the VEGFR-2 signaling complex in
rat lungs. Furthermore, to establish whether the expression of
NRP-1 is also altered in human smoker and COPD lungs, we
assessed NRP-1 protein expression by Western blot. We observed a significant reduction in protein expression in both
smoker and COPD lung tissue compared with healthy nonsmokers (Fig. 6B).
GYP-1 expression levels in rat smoking model, smoker, and
COPD patient lungs. GYP-1 has been shown to be important in
the bioavailability of VEGF165 to both VEGFR-2 and NRP-1.
To examine whether cigarette smoke affects the expression of
GYP-1, we assessed protein expression by Western blot. We
observed a significant decrease in the protein expression at 3
days, 8 wk, and 6 mo (Fig. 7A). This suggests that cigarette
smoke disrupts the VEGFR-2 signaling complex in rat lungs.
In addition, to establish whether cigarette smoke affects the
expression of GYP-1 in human smoker and COPD lungs, we
assessed GYP-1 protein expression by Western blot. We observed a significant reduction in protein expression in both
smoker and COPD lung tissue compared with nonsmokers
(Fig. 7B).
Inhibition of VEGFR-2 in rat lungs. To examine whether
specific inhibition of VEGFR-2 alone results in emphysemalike changes in the rat lung, as previously proposed (22, 23),
we repeated this published study with a more selective and
specific VEGFR-2 inhibitor, NVP-AAD777 (Ref. 30; Table 2).
Our results showed that after 3 wk of VEGFR-2 inhibition in
AJP-Lung Cell Mol Physiol • VOL
DISCUSSION
In this study we show that cigarette smoke disrupts components of the VEGF165-VEGFR-2 tertiary signaling complex by
decreasing NRP-1 and GYP-1 expression together with reducing expression of VEGFR-2 and VEGF in rat lungs in vivo.
Furthermore, we confirm these results in the lungs of human
smokers and COPD patients. Moreover, our data suggest that
specific inhibition of VEGFR-2 alone does not result in emphysema in the adult rat lung.
VEGF and VEGFR-2 expression are decreased in emphysematous lungs concomitant with increased endothelial cell apoptosis (22). VEGF levels are also reduced in the bronchoalveolar
lavage fluid of smokers (26). Interestingly, VEGF receptor
inhibition leads to the enlargement of the air spaces, increased
endothelial cell death, and decreased capillary density, characteristic of emphysema (21, 23). Moreover, COPD patients also
display reduced vascularization of the lung including decreased
capillary density (56). This has given rise to a vascular hypothesis of emphysema in which a decrease in the vascularization
of the lung, coupled with the inability to revascularize damaged tissue, may aid the initiation and/or progression of the
condition. This is an intriguing concept, as it looks at emphysema as having an important vascular perspective as well as the
established theories of the pathogenesis of COPD related to
enhanced lung inflammation (2, 31, 39).
We have shown in the present study that cigarette smoke
reduces both VEGFR-2 and VEGF expression and VEGFR-2
activation in rat smoking model lungs similar to that seen in
emphysematous lungs. We also show that VEGF expression is
decreased in the lungs of smokers and COPD patients. As the
COPD patients were current smokers, this suggests that this
reduction was a smoking- rather than a disease-related effect.
VEGFR-2 expression levels were significantly reduced in the
lungs of COPD patients but not smokers, compared with
nonsmokers, implying that this effect was disease related rather
than smoking related. Moreover, we have shown that both in
the rat smoking model and in the lungs of human smokers and
COPD patients there is a decrease in the phosphorylation of the
VEGFR-2 receptor. This indicates that cigarette smoke expo290 • MAY 2006 •
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Fig. 4. Cigarette smoke decreases VEGFR-2 protein expression in human
smoker and COPD lungs. VEGFR-2 protein expression shows a decreasing
trend in smoker lungs and is significantly reduced in COPD lungs compared
with healthy nonsmoker (NS) lungs, as assessed by immunoblotting (n ⫽ 6).
Values are means ⫾ SE of VEGFR-2 expression. **P ⬍ 0.01 compared with
healthy nonsmokers.
vivo by NVP-AAD777, using the same dose regimen as
previously reported with SUG-5416, a nonspecific VEGFR
inhibitor (23), there were no morphological emphysematous
changes in the rat lungs. Moreover, this remained the case
whether the animals had been treated with the VEGFR-2
inhibitor NVP-AAD777 with or without cigarette smoke compared with vehicle control animals (Fig. 8, A–E). There were
also no changes in vascular density in the rat lungs, in any of
the groups, compared with vehicle control animals (Fig. 8F).
To confirm that inhibition of VEGFR-2 signaling by NVPAAD777 in vivo had taken place, lung tissue samples (right
lung) from the same animal used for morphological assessment
(left lung) were assessed by Western blotting for phosphoVEGFR-2 (Tyr1175) normalized against total VEGFR-2 and
compared against vehicle control animals. Figure 8G shows
that NVP-AAD77 did indeed inhibit VEGFR-2 signaling in
vivo. Those animals that had been subjected to treatment with
NVP-AAD777 displayed a significantly lower level of the
activated phosphorylated form of VEGFR-2, by as much as
70%, compared with vehicle-treated animals.
CIGARETTE SMOKE DISRUPTS VEGF165-VEGFR-2 COMPLEX
L903
sure not only decreases VEGFR-2 expression but also inhibits
its activation. However, VEGF165 signaling through VEGFR-2
is not just a simple ligand-receptor interaction. VEGF165VEGFR-2 signaling is further regulated by a VEGF165-specific
VEGFR-2 coreceptor, NRP-1, and heparin sulfate binding in
the form of the HSPG GYP-1. Disruption to the expression of
either of these may affect VEGF165 binding and subsequent
VEGFR-2 activation and signaling.
NRP-1 is an isoform-specific receptor for VEGF, selectively
binding VEGF165 and increasing its binding to VEGFR-2
through formation of a ternary complex with VEGF165 and
VEGFR-2 (25, 44, 45, 47). Our observation of a marked
reduction in NRP-1 receptor expression in the rat lung in
response to cigarette smoke suggests that these other components of the VEGFR-2 tertiary signaling complex are also
affected by cigarette smoke exposure. This reduction in NRP-1
was confirmed in the lungs of COPD patients. This indicates
that this result was not unique to our rat smoking model.
Moreover, as the reduction in the lungs of COPD patients was
AJP-Lung Cell Mol Physiol • VOL
more significant, this would suggest that it was a diseaserelated effect and may play a role in the deterioration of
VEGFR-2 receptor signaling. Furthermore, it was originally
postulated that the action of NRP-1 on endothelial cells was
limited to that of a coreceptor for VEGFR-2. This was because
the cytoplasmic domain of NRP-1 lacks a consensus kinase
domain required for signaling (13, 33). However, it has now
been shown that NRP-1 can in fact mediate endothelial cell
migration but not proliferation through its intracellular COOH
terminus, involving phosphatidylinositol 3-kinase (58). Therefore, this reduction in NRP-1 receptor expression may also
further decrease the mitogenic signaling of endothelial cells in
the rat lung exposed to cigarette smoke, through a decrease of
its own direct mitogenic signaling. As NRP-1 increases VEGF
binding to VEGFR-2 four- to sixfold, the reduction in
VEGF165 and VEGFR-2 expression may be further confounded by a reduction in NRP-1, reducing yet further
VEGF165-VEGFR-2 binding and subsequent VEGFR-2 signaling. However, this requires confirmation through binding stud290 • MAY 2006 •
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Fig. 5. Cigarette smoke decreases VEGFR-2/KDR phosphorylation. A: VEGFR-2 phosphorylation in rat lungs remains unchanged
at both 3 days and 8 wk of cigarette smoke exposure but is
significantly decreased after 6 mo of cigarette smoke exposure in
rat lungs compared with sham-exposed animals, as assessed by
immunoprecipitation and immunoblotting (n ⫽ 6). B: VEGFR-2
phosphorylation is significantly decreased in smokers and further
decreased in COPD patients, compared with nonsmokers (n ⫽ 6).
Phosphorylated VEGFR-2 (p-VEGFR-2) was normalized against
native VEGFR-2. Values are means ⫾ SE of VEGFR-2 phosphorylation. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001.
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CIGARETTE SMOKE DISRUPTS VEGF165-VEGFR-2 COMPLEX
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Fig. 6. Cigarette smoke decreases neuropilin (NRP)-1 expression.
A: NRP-1 protein is significantly decreased after 3-day, 8-wk, and
6-mo cigarette smoke exposure in rat lungs compared with shamexposed animals, as assessed by immunoblotting (n ⫽ 6). B: NRP-1
protein expression is significantly decreased in both human smoker
and COPD lungs compared with healthy nonsmoker lungs, as
assessed by immunoblotting (n ⫽ 6). Values are means ⫾ SE of
NRP-1 expression. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001.
ies of VEGF165 and NRP-1 and subsequent activation of
VEGFR-2.
HSPGs such as GYP-1 are also thought to play an important
role in the regulation of angiogenesis and can regulate the
interaction of several growth factors, including VEGF, with
their respective receptors and, consequently, their biological
activity (1, 10, 19, 20, 27, 41, 48). GYP-1 binds VEGF165 with
high affinity and also enhances the interaction of VEGF165
with VEGFR-2 and NRP-1 (7, 15, 16, 38). Our observation that
cigarette smoke decreases the protein expression of GYP-1 in
the rat smoking model is another important and novel finding.
Again, this observation of a decrease in GYP-1 expression was
confirmed in both smoker and COPD lungs compared with
nonsmoker lungs. This reduction was similar in the lungs of
both smokers and COPD patients, suggesting that it was due to
a smoking-related effect rather than being due to the disease.
AJP-Lung Cell Mol Physiol • VOL
This decreased expression may further reduce the incidence of
the formation of the tertiary VEGFR-2 signaling complex,
thereby further reducing VEGF165-VEGFR-2 signaling. Moreover, a common requirement for all tyrosine kinase receptors is
the formation of a threshold of a number of phosphorylated
cytoplasmic domains to initiate a specific signaling cascade.
This implies that a threshold number of active receptor-ligand
complexes must be present on the cell surface for a suitable
period of time to result in effective signaling. GYP-1 may
therefore function to allow sustained coactivation of VEGF165
with both NRP-1 and VEGFR-2 through increased availability
of VEGF165 to both receptors. Therefore, the observed reduction in both GYP-1 and NRP-1 expression may decrease the
VEGF165 binding affinity to VEGFR-2, resulting in suboptimal
receptor occupancy and thereby limiting the degree of stimulation for VEGFR-2 signaling.
290 • MAY 2006 •
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CIGARETTE SMOKE DISRUPTS VEGF165-VEGFR-2 COMPLEX
VEGF plays an important role in processes such as inflammation and wound repair in which the microenvironments
around these areas are subject to increased levels of reactive
oxygen/nitrogen species (55). As oxidation/nitration of
VEGF165 inactivates and thereby reduces its bioavailability,
this can potentially be restored by GYP-1 (35), although the
exact mechanism is unclear. This may represent a mechanism
to ensure optimal levels of VEGF165-VEGFR-2 interactions,
particularly under conditions in which the concentrations of
VEGF are compromised (55). Therefore, cigarette smokeinduced oxidative stress (increased generation of reactive aldehydes and reactive oxygen/nitrogen species) may result in
the oxidation and subsequent inactivation of VEGF165. Furthermore, the reduced levels of GYP-1 in smoker and COPD
lungs is unable to restore VEGF165, thereby reducing the
bioavailability of VEGF165 to both VEGFR-2 and NRP-1.
VEGFR-2/KDR has been shown to be critical in the alveolarization of the developing lung (56). Indeed, it was previAJP-Lung Cell Mol Physiol • VOL
Table 2. Kinase inhibition data for NVP-AAD777
compared with SUG-5416
Kinase Receptor
VEGFR-1 (Flt-1)
VEGFR-2
(KDR)
VEGFR-4
PDGFR-8
C-KIT
CSF-1R (c-Fms)
Flt-3
EGFR
FGFR-1
CDK-1
SUG-5416 IC50,
nM
KDR
NVP-AAD777 IC50,
KDR
Selectivity
nM
Selectivity
43
0.2
3,000
5.9
220
50
2,220
660 (35)
84
23
⬎10,000
⬎10,000
(42,000)
⬎10,000
1.0
0.2
10.1
3 (0.2)
0.4
0.1
⬎50
510
1.0
⬎10,000
6,350
⬎10,000
⬎20
12.5
⬎20
⬎10,000
⬎20
⬎10,000
⬎20
⬎50 (20)
⬎50
Data are from Refs. 3 and 30. VEGFR, VEGF receptor; KDR, kinase insert
domain receptor; PDGFR, PDGF receptor; CSF-1R, colony-stimulating factor-1 receptor; EGFR, EGF receptor; FGFR, FGF receptor; CDK-1, cyclindependent kinase-1.
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Fig. 7. Cigarette smoke decreases glypican (GYP)-1 expression. A:
GYP-1 protein expression is significantly decreased after 3-day,
8-wk, and 6-mo cigarette smoke exposure in rat lungs compared
with sham-exposed animals, as assessed by immunoblotting (n ⫽
6). B: GYP-1 protein expression is significantly decreased in both
human smoker and COPD lungs compared with healthy nonsmoker
lungs, as assessed by immunoblotting (n ⫽ 6). Values are means ⫾
SE. *P ⬍ 0.05, ***P ⬍ 0.001.
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CIGARETTE SMOKE DISRUPTS VEGF165-VEGFR-2 COMPLEX
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Fig. 8. Specific inhibition of VEGFR-2 after administration of 20 mg/kg NVP-AAD777 subcutaneously 3 times/wk for 3 wk, in the presence or absence of
cigarette smoke exposure, results in no emphysema-like changes in the rat lung. Tissue samples from the same animals were subjected to both morphometric
analysis and Western blotting for “activated” phosphorylated VEGFR-2. Representative histological pictures of sham ⫹ vehicle (A)-, smoke ⫹ vehicle (B)-,
sham ⫹ NVP-AAD777 (C)-, and smoke ⫹ NVP-AAD777 (D)-treated rat lungs are shown. E: no significant difference in the mean Lm (mean linear intercept)
measurements of lung alveolar air spaces between treatment groups (n ⫽ 6). Data are displayed as the mean (bars) and individual Lm assessment (symbols) for
each animal within the treatment group. F: no significant difference in mean lung vascular counts between the treatment groups. Data are presented as the mean ⫾
SE for each treatment group. G: the VEGFR-2 inhibitor NVP-AAD777 gives rise to a 70% reduction in VEGFR-2 phosphorylation (normalized to total
VEGFR-2) compared with vehicle. See MATERIALS AND METHODS for further details. Values are means ⫾ SE; **P ⬍ 0.01.
ously shown that inhibition of VEGF receptors leads to the
development of emphysema-like changes in the lung (22). We
replicated this study with a stable, highly bioavailable, and
more specific VEGFR-2 inhibitor NVP-AAD777 (Refs. 3, 30;
see Table 2). Interestingly, we found no evidence of any
morphological changes to the alveolar region or the vasculature
with NVP-AAD777. This suggests that although VEGFR-2
AJP-Lung Cell Mol Physiol • VOL
inhibition with NVP-AAD777 can disrupt VEGFR-2 signaling,
as observed in Fig. 8G, our study appears to show that it does
not result in emphysema-like changes in rat lungs after 3-wk
administration. This apparent contradiction may be explained
by the specificities of the inhibitors, where specific inhibition
of VEGFR-2 alone, as we have shown, does not result in
emphysema-like changes. Indeed SUG-5416 can inhibit other
290 • MAY 2006 •
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CIGARETTE SMOKE DISRUPTS VEGF165-VEGFR-2 COMPLEX
ACKNOWLEDGMENTS
We thank Dr. Jeanette M. Wood, Novartis Institutes for Biomedical Research, for providing the VEGFR-2 inhibitor NVP-AAD777. We also thank
Professor M. Maden, Kings College London, for help in the morphometry on
the rat lungs.
GRANTS
J. A. Marwick is jointly cosponsored by a Novartis/Medical Research
Council PhD studentship award. I. Rahman was supported by an Environmental Health Sciences Center award (ES01247).
AJP-Lung Cell Mol Physiol • VOL
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