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, Christopher S. Stevenson, June Giddings, William MacNee, Keith Butler, Irfan Rahman, Paul A. Kirkham

Abstract

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.

  • kinase insert domain receptor
  • neuropilin-1
  • vascular endothelial growth factor receptor-2
  • chronic obstructive pulmonary disease

vegf is a highly specific 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 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 sulfate proteoglycan (HSPG) glypican (GYP)-1 in the formation of a tertiary signaling complex (6, 8, 34). NRPs are 130- to 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). Cross-linking 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 μg/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. ECM-associated 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.

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 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 μM 1 h after exposure (cotinine was not detectable in the plasma from air-exposed animals) and 0.51 ± 0.07 μM 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 μM 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 μg 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, 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.

View this table:
Table 1.

Characteristics of subjects and patients

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 μg 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 anti-GAPDH (Abcam, Cambridge, UK).

Immunoprecipitation.

Whole cell lung homogenate (200 μg of protein) in a final volume of 100 μl 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 (2μg) was reverse transcribed into cDNA with oligo(dT) Moloney murine leukemia virus-reverse transcriptase in a 20-μl final volume. RT-PCR was then performed with 5 μg of cDNA (primers in Table 1) and 1× PCR buffer (in mM: 50 KCl, 10 Tris·HCl pH 9.0, and 1.5 MgCl2 with 0.1% Triton X-100), 400 μM 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 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 μg of whole cell lysate protein was made up to 100 μl 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 NVP-AAD777 (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 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).

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.

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).

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.

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 observed in the rat lung, a semiglycosylated immature 195-kDa protein and the fully glycosylated cell surface-expressed 235-kDa 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).

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.

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).

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.

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 sham-exposed 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).

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.

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).

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 sham-exposed 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.

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).

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.

Inhibition of VEGFR-2 in rat lungs.

To examine whether specific inhibition of VEGFR-2 alone results in emphysema-like 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 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, AE). 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 NVP-AAD777 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 phospho-VEGFR-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.

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.

View this table:
Table 2.

Kinase inhibition data for NVP-AAD777 compared with SUG-5416

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 exposure 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. VEGF165-VEGFR-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 more significant, this would suggest that it was a disease-related 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 studies 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. 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.

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 smoke-induced 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 previously 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 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 kinases and VEGFR kinases to a greater extent than VEGFR-2 (Table 2). Nevertheless, it is now thought that both VEGFR-2 and VEGFR-1 (Flt-1) are necessary for the maintenance of the vasculature; therefore, inhibition of both may be required for initiation/development of emphysema, as proposed by Voelkel, Tuder et al. (53, 54, 56, 57).

Although these studies provide a number of interesting observations, they also have a number of limitations. For example, chronic functional inhibition of VEGFR-2 over a number of time points extending beyond 3 wk is required to establish any link between chronic VEGFR-2 inhibition and emphysema. Furthermore, although we have shown reduced levels of these components, these is no direct evidence indicating that this reduction has an impact in functional VEGFR-2 signaling in the rat smoking model, smokers, or COPD patients. Moreover, the mechanism of the reduction in these components reported here remains unknown. Reactive oxygen/nitrogen species and electrophilic components derived by cigarette smoke have been shown to result in direct protein modifications, thereby affecting protein function/expression (39). Further functional studies are required to demonstrate whether oxidative or nitrative stress and electrophilic compounds, reactive aldehydes, or carbonyls have any impact on VEGF/VEGFR-2 posttranslational modifications.

In conclusion, the reduction in both NRP-1 and GYP-1 expression by cigarette smoke is likely to have a detrimental impact on VEGF165 binding and signaling through VEGFR-2. In the context of cigarette smoke-induced emphysema, these effects confound an already altered VEGF165-VEGFR-2 expression and further impair their signaling. This is one of the fundamental events in the vasculature of the lung, leading to the decrease in capillary density and structural changes seen in the alveolar region of the lung in emphysema. However, VEGFR-2 inhibition alone does not result in development of emphysema-like changes in the lung, but nevertheless it represents an important part of a global change in VEGF signaling involving other receptors, such as VEGFR-1. Our study shows for the first time that cigarette smoke results in the decreased expression of the VEGFR-2 coreceptor NRP-1 and the VEGF165 chaperone GYP-1. The reduction in VEGF-VEGFR-2 signaling complex was also seen in human smoker and COPD lungs. Furthermore, in our study, specific inhibition of VEGFR-2 alone would suggest that it did not cause emphysema in adult rat lungs. Nevertheless, targeted therapies to restore the VEGF165-VEGFR-2 complex may ameliorate emphysema, as they might allow the lung vasculature to begin regeneration rather than being held in a suppressed state, unable to repair itself.

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).

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.

Footnotes

  • * J. A. Marwick, I. Rahman, and P. A. Kirkham contributed equally to this paper.

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

REFERENCES

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