VEGF gene polymorphisms and susceptibility to rheumatoid arthritis

S. W. Han, G. W. Kim, J. S. Seo, S. J. Kim, K. H. Sa, J. Y. Park, J. Lee1, S. Y. Kim2, J. J. Goronzy3, C. M. Weyand3 and Y. M. Kang

Department of Internal Medicine, 1 Diagnostic Radiology and 2 Orthopedic Surgery, Kyungpook National University School of Medicine, Daegu, Republic of Korea and 3 Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA.

Correspondence to: Y. M. Kang, Kyungpook National University Hospital, Samduk 2-Ga, Junggu, Daegu, Republic of Korea. E-mail: ymkang{at}knu.ac.kr


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. To investigate polymorphisms of the VEGF gene in patients with rheumatoid arthritis (RA), their relationship to clinical features and the radiographic progression of joint disease.

Methods. One hundred and forty patients with RA and 149 healthy unrelated controls were recruited. We examined four polymorphisms of the VEGF gene which are reported to be associated with production of vascular endothelial growth factor (VEGF), using polymerase chain reaction (PCR) restriction fragment length polymorphism assay and amplification refractory mutation system (ARMS) PCR. Haplotypes were predicted by Bayesian algorithm using the Phase program.

Results. All four polymorphisms were in Hardy–Weinberg equilibrium in both patients and controls. The frequency of the 936 T allele, which has been associated with lower production of VEGF, was significantly increased in RA patients compared with controls (22.7 vs 13.4%, P = 0.002). The frequencies of two haplotypes (CGCT and AAGT) which were predicted using the Phase program were significantly increased in RA patients compared with controls [33 vs 14%, odds ratio (OR) 2.636, 95% confidence interval (CI) 1.38–5.04 for CGCT; 17 vs 6%, OR 3.08, 95% CI 1.20–7.92 for AAGT]. The carriers of the susceptible haplotypes in RA patients had a younger age at disease onset but did not show a difference in the progression rate of radiographic joint destruction.

Conclusions. Our data suggest that the VEGF gene may play a role in the development of RA

KEY WORDS: Vascular endothelial growth factor, Rheumatoid arthritis, Polymorphism


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a chronic destructive joint disease characterized by proliferative synovitis and pannus formation. Angiogenesis is an essential process in the proliferative synovitis, occurs in the early stage of RA and is suggested to be crucial for the progression of the arthritic lesion [1–3].

Once the autoimmune process has been initiated in the early stage of RA, inflammation within the joint structure may induce relative hypoxia of synovial tissue which may aggravate tissue injury by release of hydrolytic enzymes, increase in vascular permeability and acceleration of the inflammatory process [4]. Because vascular endothelial growth factor (VEGF), which can be strongly induced by hypoxia, is a potent endothelial cell-specific angiogenic factor, the potential for VEGF production might be crucial in the initiation of RA.

After establishment of persistent inflammation within the joint, an increased number of inflammatory cells, activation of resident and recruited cells and increased intra-articular pressure due to expanded tissue mass and joint effusion may increase metabolic demand but cause perfusion failure [3]. A state of persistent hypoxia within the rheumatoid joint has been confirmed [5], which may further induce VEGF production and angiogenesis.

In RA, serum VEGF concentration is higher than in controls, is correlated with disease activity and inflammation markers such as the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), and is associated with destructive change [6–8]. VEGF levels in rheumatoid synovial fluid are higher than in osteoarthritis [6]. VEGF messenger ribonucleic acid (mRNA) and protein expression have been localized to the lining layer and endothelial cells in rheumatoid synovial tissue [9, 10]. New vessels are abundant within hyperplastic synovial tissue [3]. These phenomena do not necessarily mean that increased VEGF concentration and abundant new vessel formation increase the susceptibility to this autoimmune disorders. Increased angiogenesis might rather be the result of inflammation and ensuing relative hypoxia.

Polymorphisms within the VEGF gene have been associated with production of VEGF protein and reported to be involved in susceptibility to several disorders in which angiogenesis may be critical in disease development [2, 11–15]. Hypoxia-related VEGF expression has been attributed to increase in both transcriptional and post-transcriptional mechanisms [16, 17].

In spite of the importance of angiogenesis in RA, there have been no published studies on the association of VEGF gene polymorphism and RA. In the present study we assessed whether the VEGF gene is associated with disease risk and/or progression of RA. We determined four candidate polymorphisms of the VEGF gene in the Korean population and investigated their associations with RA—clinical features such as seropositivity, age at onset and disease duration and radiographic progression of joint destruction.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study population
On hundred and forty Korean patients with RA diagnosed by the 1987 revised classification criteria of American College of Rheumatology [18] were recruited from Kyungpook National University Hospital between November 2001 and December 2002. A control group of 149 unrelated, healthy Korean individuals who had no known medical problems on a health screening questionnaire were enrolled. All individuals gave informed consent for study participation and the study was approved by the Institutional Review Board of Kyungpook National University Hospital. Clinical and demographic data including age at onset, disease duration and rheumatoid factor (IgM) determined by nephelometric assay were collected from RA patients.

The radiographic severity of joint disease was scored on a postero-anterior and oblique view of both hands using the modified Sharp method [19] by a trained rheumatologist (YMK) and a musculoskeletal radiologist (JL). Observers were unaware of each patient's identity.

RA patients included 28 men and 112 women who were between 19 and 82 years of age (mean 46.5 yr): the mean duration of disease and age at onset were 7.4 yr and 39.1 yr respectively. There were 109 (77.9%) patients who had IgM rheumatoid factor. The control group included 32 men and 117 women who were between 23 and 69 years of age (mean 42.4 yr).

Genotyping of the VEGF gene polymorphisms
Genomic deoxyribonucleic acid (DNA) was isolated from peripheral blood mononuclear cells by a standard extraction method. The nucleotide sequence of four VEGF gene polymorphisms which were in the promoter region at –2578 and –1154, in the 5' untranslated region (UTR) at –634 and in the 3'-UTR at 936, were amplified by polymerase chain reaction (PCR). The PCR primers for –2578C/A, –1154G/A, –634C/G and 936C/T were 5'-GGCCTTAGGACACCATACC-3' (forward) and 5'-CACAGCTTCTCCCCTATCC-3' (reverse); 5'-TCCTGCTCCCTCCTCGCCAATG-3' (forward) and 5'-GGCGGGGACAGGCGAGCCTC-3' (reverse); 5'-CGACGGCTTGGGGAGATTGC-3' (forward) and 5'-GGGCGGTGTCTGTCTGTCTG-3' (reverse); and 5'-AGGGTTTCGGGAACCAGATC-3' (forward) and 5'-CTCGGTGATTTAGCAGCAAG-3' (reverse), respectively. PCR was performed in a final volume of 25 µl, containing 1x Taq polymerase buffer, 0.5 µmol/l of each primer, 0.75 mmol/l of dNTP, 0.5 U of Taq polymerase and 50–100 ng of genomic DNA. After the initial denaturation step at 95°C for 10 min, 30 cycles consisted of: denaturation at 95°C for 45 s, annealing at 62°C for 45 s, extension at 72°C for 30 s, final extension lasting 10 min at 72°C. Genotypes were determined by restriction fragment length polymorphism (RFLP). The restriction enzymes which detect –2578C/A, –1154G/A, –634C/G and 936C/T were BstYI, MnlI, BsmFI and NlaIII respectively. Amplified DNA was digested with 1–3 U of endonucleases for 3 h to overnight at optimal temperatures which were indicated by the manufacturers (New England Biolabs, Beverly, MA, USA), and then electrophoresed on 2% agarose gel. Because the RFLP results of –1154G/A were only discernible on 12% polyacrylamide gel, the genotype was confirmed using the amplification refractory mutation system (ARMS)-PCR method as described previously [20].

Statistical analysis
The differences in genotype distribution and allele frequency amongst the groups were examined for statistical significance by the {chi}2 test for independence and Fisher's exact test when appropriate. Hardy–Weinberg equilibrium for each polymorphism was tested by {chi}2 analysis. For the comparison of mean values, Student's t-test was performed. We analysed the association of VEGF polymorphism with age at onset and disease duration using analysis of variance (ANOVA). Haplotypes were determined based on a Bayesian algorithm using the Phase program [21] (available at http://www.stat.washington.edu/stephens/phase.html). The progression of the radiographic severity of RA, measured by the modified Sharp method, was expressed as a regression line of total score over disease duration in each group with a different allele. The statistical significance of each regression line was determined using Spearman correlation coefficient analysis. To compare the rate of radiographic deterioration according to the genotype, differences in the slopes of the regression lines were tested using an interaction between a dummy and time variable based on a multiple linear regression model. All analyses were conducted using SPSS, v10.0 (SPSS, Chicago, IL, USA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The genotype frequencies of the four polymorphisms of the VEGF gene did not show a significant deviation from the Hardy–Weinberg expectation. The distributions of genotypes and alleles are shown in Table 1. Genotype and allele frequencies of the –2578, –1154 and –634 polymorphisms were not significantly different between RA and controls. For the 936C/T polymorphism which is located on the long 3'-UTR of the VEGF gene, genotype distribution and allele frequencies were significantly different between RA and controls (P = 0.010 and P = 0.002 respectively). In the dominant genetic analysis (CC = 0, CT + TT = 1), carriers of the T allele were significantly more frequent in RA compared with controls (40.7 vs 24.2%, P = 0.004).


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TABLE 1. VEGF genotype distributions (%) and allele frequencies in patients with RA and controls

 
In patients with RA, positivity for rheumatoid factor (RF), disease duration and age at onset were compared according to the genotype of each polymorphism (Table 2). These clinical features were not significantly different between genotypes of all four polymorphisms. The number of patients who had a disease duration longer than 12 yr was 21 (25.3%) in CC, which is significantly higher than 6 (10.5%) in CT + TT for the 936 polymorphism (P = 0.029). For the –2578, –1154 and –634 polymorphisms there were no significant differences between genotypes in the number of patients with a disease duration of longer than 12 yr.


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TABLE 2. Clinical features according to VEGF genotypes in patients with RA

 
Haplotype frequencies of four VEGF biallelic polymorphisms were determined by a Bayesian algorithm using Phase software (Table 3). Phase has been estimated to correctly reconstruct the haplotypes of ≥80% of the tested data sets [21] and interpretation of statistically reconstructed haplotypes should be cautious. Of the 16 possible haplotypes, nine were estimated to be present. The frequencies of haplotypes CGCT and AAGT were significantly higher in RA (P = 0.003 and 0.018 respectively), and CGGC was more frequent in controls, although it was not statistically significant (P = 0.051).


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TABLE 3. Distribution of haplotypes at the –2578, –1154, –634 and 936 polymorphisms of the VEGF gene, predicted by Bayesian algorithm

 
We then investigated the distribution of carriers of the susceptible haplotypes (CGCT or AAGT) in patients with RA and controls (Table 4). The frequency of carriers of one or more of the susceptible haplotypes was significantly higher in RA (P<0.001). Among the carriers of the T allele at position 936, carriers of wild type–wild type or polymorphic type–polymorphic type at position –1154 and –634 were susceptible to RA but carriers of wild type–polymorphic type were not.


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TABLE 4. Frequency of carriers of the predicted susceptible haplotypes (CGCT or AAGT) of the VEGF gene

 
In RA patients, RF positivity was no different between carriers of the susceptible haplotypes and non-susceptible haplotypes (Table 5). The mean disease duration was similar in both groups but the number of patients with a disease duration longer than 12 yr was lower in the carriers of the susceptible haplotype (5/46 vs 22/94) although it was not significant (P = 0.077). In the susceptible haplotype carriers, RA tended to begin at a younger age (mean 36.2 vs 40.8 yr, P = 0.064) and the proportion of patients with age of onset at or less than 43 yr was significantly higher (P = 0.045).


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TABLE 5. Analysis of RF, age at onset and disease duration in patients with RA according to the presence of susceptible haplotypes

 
In established chronic RA, joint destruction may progress even with apparently effective medical treatment. We determined the severity of joint destruction using the modified Sharp score plotted against disease duration to evaluate the progression rate of joint damage. For each genotype of the four susceptible polymorphisms, there were highly significant positive correlations between the modified Sharp score and disease duration (data not shown). The slopes of the linear regression lines were not significantly different between genotypes for respective polymorphism (data not shown).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present data demonstrate that the 936 T allele of the VEGF gene is associated with increased susceptibility to RA. In the analysis of predicted haplotypes, carriers of the susceptible haplotypes (CGCT and AAGT at –2578, –1154, –634 and 936) for RA had a marginally higher proportion of patients with an age at onset younger than 43 yr.

All four selected single-nucleotide polymorphisms (SNPs) on the VEGF gene have been reported to be associated with VEGF synthesis. The two polymorphisms at position –2578 and –1154 are located within the promoter region and have been shown to be associated with VEGF production from stimulated peripheral mononuclear cells (PBMC) [12]. The –634 CC genotype has been associated with a higher serum VEGF concentration in a normal population [14], and with higher VEGF production from lipopolysaccharide (LPS) stimulated PBMC compared with the CG and GG genotypes. The transcriptional regulation of VEGF under hypoxia may be a common mechanism which has been linked to 5'-flanking elements of the VEGF gene [22, 23]. Our data shows that the polymorphisms at positions –2578, –1154 and –634 within the 5'-flanking region of the VEGF gene were not associated with the development of RA.

Carriers of the 936 T allele have been shown to have lower VEGF plasma levels than non-carriers [11, 24]. Because of the possible transcriptional regulation linked to the 3'-UTR of the VEGF gene, especially at position 936, we searched for potential transcription factor binding sites using MatInspector online access (http://www.genomatix.de/cgi-bin/matinspector_prof/) [25]. The 936 C allele is one of the core sequences for the potential binding of Papillomavirus regulator E2 and the C to T change at position 936 results in the loss of the core binding sequence for this transcription factor. Although Papillomavirus regulator E2 protein is known to interact with several transcriptional coactivators and modulate transcription in human cells [26–28], there have been no data on its role in the transcriptional regulation of the VEGF gene.

In addition to transcriptional regulation, post-transcriptional mechanisms have been suggested to be involved in VEGF expression [29]. In contrast to the 5'-UTR length which is remarkably consistent in organisms ranging from plants to humans, the 3'-UTR shows the evolutionary expansion which suggests the potential for 3'-UTR-based translational regulation in humans [30]. The 3'-UTR-binding proteins may interact with the 5'-UTR-binding proteins forming a circular structure of mRNA which can regulate the translation of mRNA. The predicted proteins which have the potential to bind the site encompassing position 936 could interact with the 5'-UTR-binding proteins. The predicted susceptible haplotypes described in the present study, involving the 936 T allele and polymorphisms at the 5'-flanking region of the VEGF gene may be at least partially explained by this potential mechanism. The secondary structure of mRNA may also affect the expression of VEGF by modulating stability, but the predicted secondary structure determined at the GeneBee website (http://www.genbee.msu.su) did not show any change with either C or T allele at the 936 polymorphism. Measurement of VEGF production from the carriers of susceptible and non-susceptible haplotypes among the 936 T allele carriers may provide further information on the interaction between 936 and other polymorphisms at the 5'-flanking region of the VEGF gene.

Among these four functionally significant SNPs, only the 936 polymorphism was significantly associated with RA in our study. In tumours which need angiogenesis as a critical element for their rapid growth, the 936 C allele, which is associated with higher VEGF production, has been associated with increased susceptibility [11]. Although new vessel formation in rheumatoid synovitis has been reported to be markedly increased, as is the case in tumours, the role of angiogenesis in the development of RA might be distinct from that in tumour development. In RA, the combination of increased metabolic demands caused by local inflammatory process and reduced local perfusion due to intra-articular pressure load, results in microcirculatory compromise which induces release of hydrolytic enzyme, up-regulates inflammatory process and increases vascular permeability perpetuating tissue injury [3]. As VEGF is a potent stimulator of angiogenesis, the inherent tendency of lower VEGF production in the carriers of the 936 T allele may exacerbate the microcirculatory compromise resulting in the perpetuation of joint inflammation [3]. Among RA patients, joint disease initiated at a younger age in carriers of the susceptible haplotypes may support the propensity for development of chronic persistent inflammation within a hypoxic joint compartment.

When RA patients were divided into two groups according to disease duration, the number of patients with disease duration longer than 12 yr was higher in those with the 936 CC genotype than in CT + TT genotypes. Although it was not statistically significant, there was a similar tendency between the carriers of the susceptible and non-susceptible haplotypes. The reason for this difference is unknown. It is possible that the carriers of the T allele might suffer from complications of RA which may reduce their life expectancy. Alternatively, one may consider a selection bias. However, in the case of the other polymorphisms the frequencies of patients with disease duration longer than 12 yr were very similar between genotypes. Because of the potential to utilize the 936 polymorphism as a prognostic parameter in RA, larger prospective studies are warranted.

The progression of joint destruction in RA was no different between carriers of susceptible and non-susceptible haplotypes in the present study. A previous report suggested that high serum VEGF levels at an early stage of disease are associated with an increase in subsequent damage to joints observed by radiography [8]. In collagen-induced arthritis, the production of VEGF by synovial cells primed in the inflammatory milieu of the joint was higher in mice with severe disease [31]. PBMC from RA patients with severe disease produced a larger amount of VEGF when stimulated by cobalt, which mimics hypoxia, compared with that from patients with minimal disease [32]. After establishment of chronic persistent inflammation, a variety of inflammatory mediators as well as the local hypoxia modulate the production of VEGF within the joint [1, 3]. The genetic factor for regulating VEGF production may be overwhelmed by these local inflammatory mediators during the progression of joint destruction.

The determination of genetic predisposition for the development of RA is challenging because of the number of confounding factors. Because the contribution of HLA-DR polymorphism has been estimated to account for only one third of the genetic component of RA aetiology [33], polymorphisms of non-major histocompatibility complex genes, which are critical in the development and maintenance of the disease, will also contribute to the predisposition to RA [34]. Here we describe a possible role for a polymorphism of the VEGF gene that predisposes to earlier onset of RA. Further studies on the functional relevance of the VEGF polymorphisms in RA will be needed to confirm these observations.


    Acknowledgments
 
We are grateful for the critical help in statistical analysis by Mr Won Ki Lee. This study was supported by a grant of the Korea Health 21 R&D project, Ministry of Health and Welfare, Republic of Korea (01-PJ3-PG6-01GN11-0002).

The authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Koch AE. The role of angiogenesis in rheumatoid arthritis: recent developments. Ann Rheum Dis 2000;59(Suppl 1):i65–71.[Medline]
  2. McCarron SL, Edwards S, Evans PR et al. Influence of cytokine gene polymorphisms on the development of prostate cancer. Cancer Res 2002;62:3369–72.[Abstract/Free Full Text]
  3. Paleolog EM. Angiogenesis in rheumatoid arthritis. Arthritis Res 2002;4(Suppl 3):S81–S90.[CrossRef][Medline]
  4. Rothschild BM, Masi AT. Pathogenesis of rheumatoid arthritis: a vascular hypothesis. Semin Arthritis Rheum 1982;12:11–31.[CrossRef][ISI][Medline]
  5. Lund-Olesen K. Oxygen tension in synovial fluids. Arthritis Rheum 1970;13:769–76.[ISI][Medline]
  6. Lee SS, Joo YS, Kim WU et al. Vascular endothelial growth factor levels in the serum and synovial fluid of patients with rheumatoid arthritis. Clin Exp Rheumatol 2001;19:321–4.[ISI][Medline]
  7. Sone H, Sakauchi M, Takahashi A et al. Elevated levels of vascular endothelial growth factor in the sera of patients with rheumatoid arthritis correlation with disease activity. Life Sci 2001;69:1861–9.[CrossRef][ISI][Medline]
  8. Ballara S, Taylor PC, Reusch P et al. Raised serum vascular endothelial growth factor levels are associated with destructive change in inflammatory arthritis. Arthritis Rheum 2001;44:2055–64.[CrossRef][ISI][Medline]
  9. Fava RA, Olsen NJ, Spencer-Green G et al. Vascular permeability factor/endothelial growth factor (VPF/VEGF): accumulation and expression in human synovial fluids and rheumatoid synovial tissue. J Exp Med 1994;180:341–6.[Abstract]
  10. Koch AE, Harlow LA, Haines GK et al. Vascular endothelial growth factor. A cytokine modulating endothelial function in rheumatoid arthritis. J Immunol 1994;152:4149–56.[Abstract/Free Full Text]
  11. Krippl P, Langsenlehner U, Renner W et al. A common 936 C/T gene polymorphism of vascular endothelial growth factor is associated with decreased breast cancer risk. Int J Cancer 2003;106:468–71.[CrossRef][ISI][Medline]
  12. Shahbazi M, Fryer AA, Pravica V et al. Vascular endothelial growth factor gene polymorphisms are associated with acute renal allograft rejection. J Am Soc Nephrol 2002;13:260–4.[Abstract/Free Full Text]
  13. Lin CC, Wu HC, Tsai FJ, Chen HY, Chen WC. Vascular endothelial growth factor gene-460 C/T polymorphism is a biomarker for prostate cancer. Urology 2003;62:374–7.[CrossRef][ISI][Medline]
  14. Awata T, Inoue K, Kurihara S et al. A common polymorphism in the 5'-untranslated region of the VEGF gene is associated with diabetic retinopathy in type 2 diabetes. Diabetes 2002;51:1635–9.[Abstract/Free Full Text]
  15. Howell WM, Bateman AC, Turner SJ, Collins A, Theaker JM. Influence of vascular endothelial growth factor single nucleotide polymorphisms on tumour development in cutaneous malignant melanoma. Genes Immun 2002;3:229–32.[CrossRef][ISI][Medline]
  16. Ikeda E, Achen MG, Breier G, Risau W. Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells. J Biol Chem 1995;270:19761–6.[Abstract/Free Full Text]
  17. Levy AP, Levy NS, Goldberg MA. Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J Biol Chem 1996;271:2746–53.[Abstract/Free Full Text]
  18. Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24.[ISI][Medline]
  19. Sharp JT, Young DY, Bluhm GB et al. How many joints in the hands and wrists should be included in a score of radiologic abnormalities used to assess rheumatoid arthritis? Arthritis Rheum 1985;28:1326–35.[ISI][Medline]
  20. Perrey C, Turner SJ, Pravica V, Howell WM, Hutchinson IV. ARMS-PCR methodologies to determine IL-10, TNF-alpha, TNF-beta and TGF-beta 1 gene polymorphisms. Transpl Immunol 1999;7:127–8.[CrossRef][ISI][Medline]
  21. Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 2001;68:978–89.[CrossRef][ISI][Medline]
  22. Wang GL, Semenza GL. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J Biol Chem 1993;268:21513–18.[Abstract/Free Full Text]
  23. Liu Y, Cox SR, Morita T, Kourembanas S. Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells. Identification of a 5' enhancer. Circ Res 1995;77:638–43.[Abstract/Free Full Text]
  24. Renner W, Kotschan S, Hoffmann C, Obermayer-Pietsch B, Pilger E. 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;37:443–8.[CrossRef][ISI][Medline]
  25. Quandt K, Frech K, Karas H, Wingender E, Werner T. MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res 1995;23:4878–84.[Abstract]
  26. Lee D, Hwang SG, Kim J, Choe J. Functional interaction between p/CAF and human papillomavirus E2 protein. J Biol Chem 2002;277:6483–9.[Abstract/Free Full Text]
  27. Hadaschik D, Hinterkeuser K, Oldak M, Pfister HJ, Smola-Hess S. The Papillomavirus E2 protein binds to and synergizes with C/EBP factors involved in keratinocyte differentiation. J Virol 2003;77:5253–65.[Abstract/Free Full Text]
  28. Massimi P, Pim D, Bertoli C, Bouvard V, Banks L. Interaction between the HPV-16 E2 transcriptional activator and p53. Oncogene 1999;18:7748–54.[CrossRef][ISI][Medline]
  29. Claffey KP, Shih SC, Mullen A et al. Identification of a human VPF/VEGF 3' untranslated region mediating hypoxia-induced mRNA stability. Mol Biol Cell 1998;9:469–81.[Abstract/Free Full Text]
  30. Mazumder B, Seshadri V, Fox PL. Translational control by the 3'-UTR: the ends specify the means. Trends Biochem Sci 2003;28:91–8.[CrossRef][ISI][Medline]
  31. Miotla J, Maciewicz R, Kendrew J, Feldmann M, Paleolog E. Treatment with soluble VEGF receptor reduces disease severity in murine collagen-induced arthritis. Lab Invest 2000;80:1195–205.[ISI][Medline]
  32. Bottomley MJ, Webb NJ, Watson CJ, Holt PJ, Freemont AJ, Brenchley PE. Peripheral blood mononuclear cells from patients with rheumatoid arthritis spontaneously secrete vascular endothelial growth factor (VEGF): specific up-regulation by tumour necrosis factor-alpha (TNF-alpha) in synovial fluid. Clin Exp Immunol 1999;117:171–6.[CrossRef][ISI][Medline]
  33. Deighton CM, Walker DJ, Griffiths ID, Roberts DF. The contribution of HLA to rheumatoid arthritis. Clin Genet 1989;36:178–82.[ISI][Medline]
  34. Fisher SA, Lanchbury JS, Lewis CM. Meta-analysis of four rheumatoid arthritis genome-wide linkage studies: confirmation of a susceptibility locus on chromosome 16. Arthritis Rheum 2003;48:1200–6.[CrossRef][ISI][Medline]
Submitted 1 March 2004; revised version accepted 19 May 2004.