Chest ACCP Education Calendar
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Guest Access | Sign In via User Name/Password
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Article Archive
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (17)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sakao, S.
Right arrow Articles by Kuriyama, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sakao, S.
Right arrow Articles by Kuriyama, T.
(Chest. 2003;124:323-327.)
© 2003 American College of Chest Physicians

Vascular Endothelial Growth Factor and the Risk of Smoking-Related COPD*

Seiichiro Sakao, MD; Koichiro Tatsumi, MD; Tomohiro Hashimoto, MD; Hidetoshi Igari, MD; Yuji Shino, MD; Hiroshi Shirasawa, MD and Takayuki Kuriyama, MD, FCCP

* From the Departments of Respirology (Drs. Sakao, Tatsumi, Hashimoto, Igari, and Kuriyama) and Molecular Virology (Drs. Shino and Shirasawa), Graduate School of Medicine, Chiba University, Chiba, Japan.

Correspondence to: Koichiro Tatsumi, MD, Department of Respirology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuou-ku, Chiba 260-8670, Japan; e-mail: tatsumi{at}med.m.chiba-u.ac.jp


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Vascular endothelial growth factor (VEGF) signaling may be required for maintenance of the alveolar structures, and alveolar septal cell apoptosis could contribute to the pathogenesis of COPD presenting emphysematous changes; however, the common mutation at position 936 in the 3' untranslated region of the VEGF gene, a C to T substitution (the C allele was denoted as 1, and the T allele as 2), VEGF936*2, has been reported to be associated with significantly lower VEGF plasma levels. Based on these concepts, we hypothesized that VEGF936*1/2 polymorphism may be linked to the development of COPD.

Design: The differences in VEGF936*1/2 allele frequency were examined in 113 patients with smoking-related COPD and two control groups (101 smoker/ex-smoker control subjects and 102 population control subjects) using the polymerase chain reaction-restriction fragment length polymorphism technique.

Results: VEGF936*1/2 allele frequencies did not differ among the groups: 0.792/0.208 in COPD patients, 0.822/0.178 in smoker/ex-smoker control subjects, and 0.842/0.152 in population control subjects.

Conclusion: The 936 C/T polymorphism of the VEGF gene (including both homozygous and heterozygous) was not associated with the development of COPD (odds ratio, 1.23; 95% confidence interval, 0.760 to 1.995).

Key Words: emphysema • gene polymorphism • vascular endothelial growth factor


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The chronic airflow limitation characteristic of COPD is caused by a mixture of small airway disease (obstructive bronchiolitis) and parenchymal destruction (emphysema).1 It is generally accepted that cigarette smoking is by far the most important risk factor for COPD. Nevertheless, not all smokers acquire clinically significant COPD, which suggests that genetic factors must modify each individual’s risk. It is believed that many genetic factors increase the risk of acquiring COPD. A study2 demonstrated an increased risk of COPD within families with COPD probands. Some of this risk may be due to shared environmental factors, but several studies in diverse populations also suggest a shared genetic risk.3

Alveolar septal cell apoptosis may contribute to the pathogenesis of COPD. Apoptosis occurs in vascular endothelial and/or alveolar epithelial cells in patients with COPD presenting emphysematous changes of the lung, and is associated with reduced expression of vascular endothelial growth factor (VEGF) in the lung.4 5 VEGF receptor signaling is required for maintenance of the alveolar structures, and withdrawal of VEGF leads to endothelial cell apoptosis in vitro6 7 and in vivo.8 The loss of endothelial cells may be caused both by loss of VEGF or a faulty VEGF signaling.5

Based on these concepts, we hypothesized that a reduced ability of vascular endothelial and alveolar epithelial cells to produce VEGF may be linked to the development of COPD and emphysematous changes in the lungs. Cigarette smoking may act to decrease the expression of VEGF and its receptor 2 (KDR/Flk-1). A reduced ability to produce VEGF and/or a decreased expression of VEGF receptor 2 may lead to apoptosis of these cells triggered by direct exposure to inhaled insults, such as tobacco smoking, potentially contributing to the destruction of lung tissue observed in these patients.

It was reported9 that there were three common mutations in the 3' untranslated region (3'-UTR) of the VEGF gene; one of them, a cytosine (C) to thymine (T) substitution in the VEGF gene at position 936 in the 3'-UTR (the C allele was denoted as 1, and the T allele 2), VEGF936*2, was associated with significantly lower VEGF plasma levels in healthy men. Regulating elements lie in the 3'-UTR of the VEGF gene, where binding of hypoxia-induced proteins to messenger RNA occurs, resulting in a significantly increased half-life of messenger RNA.10 11 This polymorphism may affect the binding of hypoxia-induced proteins to the VEGF messenger RNA, decreasing thereby VEGF expression.

In this study, the base composition at 936 of the VEGF gene was determined in patients with smoking-related COPD and two control groups (a smoker control group and a population control group) using the polymerase chain reaction (PCR)-restriction fragment length polymorphism technique. The purpose of the present study was to determine whether VEGF936*1/2 polymorphism was associated with the presence of smoking-related COPD.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
The patients group consisted of 113 patients with smoking-related COPD recruited from the Respiratory Outpatient Department, Chiba University Hospital and affiliated hospitals (Chiba, Japan). Sixty-nine men and 44 women were recruited to the COPD patient group. COPD was diagnosed on the basis of history, physical examination, and spirometric data, following the American Thoracic Society guidelines.12 Pulmonary function tests were performed to determine FVC and FEV1. Chronic airflow obstruction was defined as an FEV1/FVC ratio < 70% and an FEV1 < 80% of predicted values. Subjects were excluded if they had a history of productive cough for 3 months in each of 2 successive years (chronic bronchitis). The percentage ratio of the low attenuation area to the corresponding lung area was evaluated using a visual scoring system for CT findings in 84 patients with COPD. A significant correlation was observed between visual scores and FEV1/FVC (r = 0.63, p < 0.01).13

Two control groups were used in the study. The first control group was a smoker control group (n = 101), which included asymptomatic smokers and ex-smokers matched for sex and age with a smoking history of at least 10 pack-years but without COPD or asthma. The smoker control group consisted of 59 men and 42 women. This control group consisted of subjects who visited the same hospital for a health checkup. They had a normal pulmonary function (FEV1/FVC > 70% and FEV1 > 80% of predicted values). The second control group was a population control group (n = 102) of adult Japanese blood donors, aged 22 to 72 years, from Chiba Prefecture, Japan. The smoking history of the population control subjects was unknown. The study was approved by the Research Ethics Committee, Graduate School of Medicine, Chiba University, Chiba, Japan, and all subjects gave their informed consent in writing.

Detection of VEGF Polymorphism
The base composition at 936 of the VEGF gene was determined with the PCR-restriction fragment length polymorphism technique. Genomic DNA was obtained from blood lymphocytes using the QIAamp DNA Blood Mini Kit (Qiagen; Valencia, CA). The 3' untranslated region of the VEGF gene was amplified, and the PCR conditions were similar to those already described14 : the 5' primer was 5'-AAGGAAGAGGAGACTCTGCGCAGAGC and the 3'-primer was 5'-TAAATGTATGTATGTGGGTGGGTGTGTCTACAGG. PCR conditions were as follows: genomic DNA was amplified using 0.2 µmol/L concentrations of the primers, 100 µmol/L of each deoxynucleotide triphosphate, 10 mmol/L Tris, 1.5 mmol/L MgCl2, 50 mmol/L KCl, and 0.1% Triton X-100 (Sigma-Aldrich Japan; Tokyo, Japan). Cycling was as follows: 94°C for 1 min, 65°C for 1 min, and 72°C for 1 min for 30 cycles, followed by 60°C for 1 min and 72°C for 5 min. The PCR product was ethanol precipitated and digested with NlaIII (New England Biolab; Beverly, MA) and analyzed on a 3% NuSieve agarose gel (FMC BioProducts; Rockland, ME). DNA products were visualized by ethidium bromide staining. The VEGF936*1 allele would not be digested (208 base pair [bp]), while the VEGF936*2 allele would be digested into two fragments (122 bp and 86 bp, respectively).

Statistical Analysis
The difference in allele distribution and allele frequency among the groups was examined for statistical significance by {chi}2 test for independence, and with the Fisher exact test when appropriate. Age, smoking index expressed as pack-years, and pulmonary function parameters were compared using the Mann-Whitney U test; p < 0.05 was considered statistically significant.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Age, smoking history, and pulmonary function data of COPD patients and smoker control subjects are summarized in Table 1 . No significant differences were observed in age or smoking history between the two groups.


View this table:
[in this window]
[in a new window]

  		Table 1.. Age, Smoking, and Pulmonary Function in Patients With COPD and Smoker Control Subjects*

 
The results of the comparison of the base composition at position 936 of the VEGF gene are summarized in Table 2 . The distribution of the genotype was not statistically different between patients and smoker control groups, nor between patients and population control groups. {chi}2 analysis showed that the patients group had no higher VEGF936*2 frequency than either the smoker control or population control group. Presence of the VEGF936*2 allele (including both homozygous and heterozygous subjects) was not associated with an increased risk for COPD (odds ratio, 1.23; 95% confidence interval, 0.760 to 1.995) compared with VEGF936*1 homozygous subjects.


View this table:
[in this window]
[in a new window]

  		Table 2.. Genotype and Allele Frequencies in Patients With COPD, Smoker Control Subjects, and Population Control Subjects*

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We compared the frequency of a polymorphism in the 3'-UTR of the VEGF gene (designated VEGF936*1/2) in Japanese patients with smoking-related COPD and asymptomatic smoker/ex-smoker control subjects matched for age and sex. We found no association of VEGF936*1/2 alleles with the occurrence of COPD, although a C to T polymorphism at position 936 in the 3'-UTR of VEGF gene decreases the expression of VEGF.9 This result suggests that this polymorphism in the VEGF gene, at least in the Japanese population and for the considered polymorphisms, is not a major genetic risk factor for COPD; however, we cannot exclude that other, so far unknown, mutations in the VEGF gene, in association with mutations in other genes, may play a role in the risk of susceptibility to develop COPD.

VEGF is an important regulator of angiogenesis and a specific mitogen for endothelial cells.15 VEGF gene expression is modulated by a variety of effectors, including cytokines, hormones, and hypoxia.16 17 Dysregulated VEGF expression is implicated in the pathogenesis of numerous diseases. Increased VEGF expression resulting in marked VEGF-induced angiogenesis is linked to tumor growth and metastases,18 rheumatoid arthritis,19 and diabetic retinopathy.20 The increase in active VEGF protein from cells exposed to hypoxia is partly because of an increased transcription rate, mediated by binding of the transcription factor to a hypoxia responsive element in the VEGF gene.11

The precise mechanisms by which the VEGF plasma levels are lower in subjects expressing the VEGF 936 T allele are currently unknown. Analysis of potential transcription factor binding sites showed that the 936 C to T mutation led to the loss of a potential binding site for activator protein-4 (AP-4).9 AP-4 is a helix-loop-helix transcription factor that enhances the expression of several genes by binding to specific enhancer sites.21 The potential AP-4 binding site abolished by the 936 CT mutation may be an explanation for the association between this mutation and lower VEGF plasma levels. Another possible explanation of the association between this mutation and lower VEGF plasma levels could be a linkage disequilibrium between this mutation and another yet unknown functional mutation elsewhere in the VEGF gene sequence. Future studies using reporter gene constructs are necessary to determine if the alleles of the VEGF936*2 polymorphism have a direct effect at the gene transcription level and ultimately at the protein production level, in the alveolar epithelial cell lines.

Disappearance of lung tissue in COPD may lead to the progressive loss of capillary endothelial and epithelial cells associated with the loss of the extracellular matrix. The loss of these cells might occur through the process of programmed cell death (apoptosis). COPD is a heterogeneous disease, and several predisposing genetic risk factors may be involved in its pathogenesis. Among them, VEGF polymorphism could be associated with the development of smoking-related COPD. In patients with COPD, there might be the reduced VEGF expression or the impairment of the signal transduction via VEGF receptor 2, either because of a reduction in the number of endothelial cell receptors or due to impairment of the VEGF receptor/tyrosine kinase activity. VEGF signaling may be required for the maintenance of adult lung alveolar structures. Cigarette smoking may act by decreasing the expression of VEGF and its receptor 2, thus resulting in lung septal cell death. Kasahara and coworkers5 reported increased septal cell death in human emphysematous lungs, which was associated with reduced lung expression of VEGF and VEGF receptor 2. In addition, Grazia et al22 reported that there appears to exist a subgroup of patients with emphysema and significantly decreased or undetectable plasma levels of VEGF, and very low or undetectable plasma VEGF may characterize patients with end-stage emphysema.

In this study, we hypothesized that the reduced ability of patients with VEGF 936*2 allele to produce VEGF, when exposed to tobacco smoking or hypoxia, was involved in the apoptotic changes of vascular endothelial and alveolar epithelial cells. Patients with COPD tend to be hypoxemic, and hypoxia is a strong inducer of the VEGF gene. Theoretically, in patients with COPD, a decreased VEGF production might contribute to an increase of alveolar septal cells apoptosis. We found no association between VEGF936*1/2 alleles and the occurrence of COPD. This result, however, may not deny the important role of VEGF in the development of emphysematous changes in patients with COPD. It was reported that VEGF induced the expression of antiapoptotic proteins6 and acted as a survival factor for endothelial cells.23 If the amount of VEGF is reduced below threshold levels, alveolar endothelial cells could indeed die as a consequence of a failing VEGF-dependent endothelial cell maintenance program. Therefore a reduction of VEGF in the lungs of patients with emphysema could be one of the several factors facilitating alveolar septal cell apoptosis. In the future, the correlation of VEGF production, initiated by any stimulus, such as tobacco smoking, with a specific genotype may allow the identification of individuals predisposed to acquire VEGF-mediated COPD. Detailed disease association studies may reveal that other yet unknown functional mutations elsewhere in the VEGF gene sequence are linked to a particular type of patients with COPD.


   Acknowledgements
 
The authors thank Dr. Shuji Hashimoto, Department of Hygiene, Fujita Health University School of Medicine, for his help with statistical analysis. The authors also thank Drs. Yasunori Kasahara, Katsushi Kurosu, Nobuhiro Tanabe, and Yuichi Takiguchi for helpful comments.


   Footnotes
 
Abbreviations: AP-4 = activator protein-4; bp = base pair; PCR = polymerase chain reaction; 3'-UTR = 3' untranslated region; VEGF = vascular endothelial growth factor

This study was supported by a Grant-in-Aid for Scientific Research (C)(14570541) from the Ministry of Education, Science, Sports and Culture, and grants to Respiratory Failure Research Group from the Ministry of Health, Labour and Welfare, Japan.

Received for publication August 20, 2002. Accepted for publication January 15, 2003.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lenfant, C Khaltaev, N eds. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: NHLBI/WHO workshop report. 2001 National Institutes of Health. Bethesda, MD: publication No. 2701
  2. Silverman, EK, Speizer, FE Risk factors for the development of chronic obstructive pulmonary disease. Med Clin North Am 1996;80,501-522[ISI][Medline]
  3. Chen, Y Genetics and pulmonary medicine. 10: Genetic epidemiology of pulmonary function. Thorax 1999;54,818-824[Free Full Text]
  4. Kasahara, Y, Tuder, RM, Taraseviciene-Stewart, L, et al Inhibition of VEGF receptors cause lung cell apoptosis and emphysema. J Clin Invest 2000;106,1311-1319[ISI][Medline]
  5. Kasahara, Y, Tuder, RM, Cool, CD, et al Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema. Am J Respir Crit Care Med 2001;163,737-744[Abstract/Free Full Text]
  6. Gerber, HP, Dixit, V, Ferrara, N Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J Biol Chem 1998;273,13313-13316[Abstract/Free Full Text]
  7. Gerber, HP, McMurtrey, A, Kowalski, J, et al Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway: requirement for Flk-1/KDR activation. J Biol Chem 1998;273,30336-30343[Abstract/Free Full Text]
  8. Alon, T, Hemo, I, Itin, A, et al Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nature Med 1995;1,1024-1028[CrossRef][ISI][Medline]
  9. Renner, W, Kotschan, S, Hoffmann, C, et al A common 936 C/T mutation in the gene for vascular endothelial growth factor is associated with vascular endothelial growth factor plasma levels. J Vasc Res 2000;37,443-448[CrossRef][ISI][Medline]
  10. Scandurro, AB, Beckman, BS Common proteins bind mRNAs encoding erythropoietin, tyrosine hydroxylase, and vascular endothelial growth factor. Biochem Biophys Res Commun 1998;246,436-440[CrossRef][ISI][Medline]
  11. Levy, AP, Levy, NS, Goldberg, MA Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J Biol Chem 1996;271,2746-2753[Abstract/Free Full Text]
  12. American Thoracic Society.. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1995;152,S77-S120[Medline]
  13. Sakao, S, Tatsumi, K, Igari, H, et al Association of tumor necrosis factor-{alpha} gene promoter polymorphism with low attenuation areas on high-resolution CT in patients with COPD. Chest 2002;122,416-420[Abstract/Free Full Text]
  14. Sakao, S, Tatumi, K, Igari, H, et al Association of tumor necrosis factor-{alpha} gene promoter polymorphism with the presence of COPD. Am J Respir Crit Care Med 2001;163,420-422[Abstract/Free Full Text]
  15. Leung, DW, Cachianes, G, Kuang, WJ, et al Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989;246,1306-1309[Abstract/Free Full Text]
  16. Ferrara, N Molecular and biological properties of vascular endothelial growth factor. J Mol Med 1999;77,527-543[CrossRef][ISI][Medline]
  17. Neufeld, G, Cohen, T, Gengrinovitch, S, et al Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999;13,9-22[Abstract/Free Full Text]
  18. Claey, KP, Robinson, GS Regulation of VEGF/VPF expression in tumour cells: consequences for tumour growth and metastasis. Cancer Metastasis Rev 1996;15,165-176[CrossRef][ISI][Medline]
  19. Koch, AE, Harlow, LA, Haines, GK, et al Vascular endothelial growth factor: a cytokine modulating endothelial function in rheumatoid arthritis. J Immunol 1994;152,4149-4156[Abstract]
  20. Miller, JW, Adamis, AP, Aiello, LP Vascular endothelial growth factor in ocular neovascularization and proliferative diabetic retinopathy. Diabetes Metab Rev 1997;13,37-50[CrossRef][ISI][Medline]
  21. Hu, YF, Luscher, B, Admon, A, et al Transcription factor AP-4 contains multiple dimerization domains that regulate dimer specificity. Genes Dev 1990;4,1741-1752[Abstract/Free Full Text]
  22. Grazia, TJ, Wescott, J, Voelkel, NF Plasma vascular endothelial growth factor in end-stage emphysema [abstract]. Am J Respir Crit Care Med 2002;165,A825
  23. Alon, T, Hemo, I, Itin, A, et al Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nature Med 1995;1,1024-1028



This article has been cited by other articles:


Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
S. J. Lee, S. Y. Lee, H.-S. Jeon, S. H. Park, J. S. Jang, G. Y. Lee, J. W. Son, C. H. Kim, W. K. Lee, S. Kam, et al.
Vascular Endothelial Growth Factor Gene Polymorphisms and Risk of Primary Lung Cancer
Cancer Epidemiol. Biomarkers Prev., March 1, 2005; 14(3): 571 - 575.
[Abstract] [Full Text] [PDF]

Home page
 ThoraxHome page
A R L Medford, L J Keen, J L Bidwell, and A B Millar
Vascular endothelial growth factor gene polymorphism and acute respiratory distress syndrome
Thorax, March 1, 2005; 60(3): 244 - 248.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
P. J. Barnes
Mediators of Chronic Obstructive Pulmonary Disease
Pharmacol. Rev., December 1, 2004; 56(4): 515 - 548.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Article Archive
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (17)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sakao, S.
Right arrow Articles by Kuriyama, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sakao, S.
Right arrow Articles by Kuriyama, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS