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
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Abstract |
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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 HardyWeinberg 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) 1.385.04 for CGCT; 17 vs 6%, OR 3.08, 95% CI 1.207.92 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
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Introduction |
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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 [68]. 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, 1115]. 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 RAclinical features such as seropositivity, age at onset and disease duration and radiographic progression of joint destruction.
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Materials and methods |
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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 50100 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 13 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 2 test for independence and Fisher's exact test when appropriate. HardyWeinberg equilibrium for each polymorphism was tested by
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).
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Results |
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Discussion |
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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 [2628], 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.
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Acknowledgments |
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The authors have declared no conflicts of interest.
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References |
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