Serum and synovial fluid concentration of vascular endothelial growth factor in juvenile idiopathic arthritides

Paediatric Rheumatology/Series Editor: P. Woo

S. Vignola, P. Picco, F. Falcini1, F. Sabatini2, A. Buoncompagni and M. Gattorno

Second Division of Paediatrics (Rheumatology Unit),
2 Division of Pneumology, ‘G. Gaslini’ Scientific Institute for Children, Genoa and
1 Clinic of Pediatrics, University of Florence, Italy

Abstract

Objective. To evaluate the role of vascular endothelial growth factor (VEGF) in the pathogenesis of local joint inflammation in juvenile idiopathic arthritis (JIA).

Methods. Sera from 50 patients affected with JIA and 10 age-matched healthy controls were tested with a commercial ELISA for VEGF. Corresponding synovial fluid (SF) concentrations of VEGF and p75 soluble tumour necrosis factor receptor (sTNFR) were evaluated in 20 active JIA patients.

Results. Serum concentrations of VEGF were significantly higher in patients with active polyarticular disease than in patients with active and inactive oligoarticular disease and healthy controls. In JIA patients, serum concentrations of VEGF displayed a significant correlation with a number of clinical and laboratory parameters of disease activity. VEGF concentrations in SF were significantly higher than those detected in corresponding sera. Moreover, a clear correlation was found between corresponding SF and serum VEGF concentrations. In SF, VEGF showed a strong positive correlation with p75 sTNFR.

Conclusions. Concentrations of VEGF in SF in patients with JIA are higher than corresponding serum concentrations, suggesting that this pro-angiogenic factor may have a major role in the outgrowth of hyperplastic pannus and tissue damage at the site of tissue inflammation.

KEY WORDS: Juvenile idiopathic arthritis, Vascular endothelial growth factor, Angiogenesis, TNF-{alpha}, Synovial fluid, Synovial membranes.

Angiogenesis plays a pivotal role in a number of physiological (embryonic development, female reproductive cycle, wound healing) and pathological (tumour growth, inflammation) conditions [1]. Several recent investigations indicate that exuberant proliferation of new blood vessels should be considered as a crucial step in pannus formation in chronic inflammatory arthritides [2, 3].

A large number of mediators, such as platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor ß(TGF-ß), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF), have been shown to play a role in the induction of new vessel formation during angiogenesis [4].

VEGF is a potent endothelial-cell-specific mitogen produced locally by activated synovial monocytes/macrophages and fibroblasts as well as by synoviocytes upon stimulation by a number of proinflammatory cytokines [interleukin (IL) 1, tumour necrosis factor {alpha} (TNF-{alpha}), IL-6], other inflammatory mediators, such as prostaglandin E2, and physical factors (hypoxia) [58].

In particular, the close relationship between proinflammatory cytokines and VEGF production has been shown in adult rheumatoid arthritis (RA), in which the spontaneous release of VEGF in vitro by synovial membrane cells may be dramatically impaired after the addition of anti-TNF-{alpha} cA2 monoclonal antibodies to the culture, especially in combination with the IL-1 receptor antagonist [8]. Similarly, the serum concentration VEGF in RA patients displayed a significant decrease after treatment with anti-TNF-{alpha} [8].

In adult RA, VEGF has been shown to play a crucial role in vessel neoformation at the level of hyperplastic synovial pannus and to increase capillary permeability [9]. VEGF has been detected in large amounts both in sera and synovial fluid from RA patients, with a good correlation with disease activity parameters [1012]. Moreover, several investigations reported clear overexpression of VEGF in RA at the level of the synovial lining layer [1214].

No studies investigating the production of VEGF in the inflamed joints of children affected with juvenile idiopathic arthritides (JIA) have been published.

The aims of the present study were (i) to investigate VEGF levels in synovial fluid (SF) and, for comparison, in paired serum samples from JIA patients; and (ii) to identify possible correlation between VEGF levels and disease activity parameters and TNF activity.

Patients and methods

Fifty patients affected with JIA according to the ILAR (International League Against Rheumatism) Durban criteria were studied [15]. Fourteen patients displayed a polyarticular course of the disease (three with systemic onset, four with rheumatoid factor-negative polyarticular onset, seven with oligoarticular onset) and 36 had a persistent oligoarticular course.

A serum sample was collected with permission at each control visit or at the moment of hospitalization for a disease flare-up, and was stored at -80° immediately after centrifugation. We recorded concomitantly several clinical (number of active joints, number of joints with limited range of motion and physician's global assessment of overall disease activity) and laboratory [erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), white blood cell count (WBC), platelet count (PLT), haemoglobin (Hb) serum concentration] indicators of disease activity.

The following parameters were considered to be reliable indicators of disease activity: (i) presence of active arthritis (swelling or, if swelling was absent, tenderness with limitation of motion) in at least one joint at the time of clinical examination [16], (ii) physician's global estimate of disease activity, ranging from 6 to 10 (measured on a 10 cm visual analogue scale); (iii) ESR in first hour >15 mm (for polyarticular course only) [17].

The criteria of disease inactivity were as follows: (i) no history of arthritis or arthralgias 2 weeks before and absence of any sign of active arthritis (as defined above) at the time of clinical examination; (ii) physician's global estimate of disease activity =0 [18]; (iii) ESR in first hour <15 mm (for polyarticular course only).

Ten age-matched healthy subjects attending our clinic for preoperative evaluation were used as controls after informed consent had been given by their parents. Subjects were excluded if they had a history of inflammatory or infectious disorders in the 4 weeks before the examination, or clinical or laboratory (elevated ESR or CRP) features of inflammation at the time of the study.

A total of 60 sera were tested with commercial enzyme-linked immunosorbent assay (ELISA) kits for VEGF (Amersham, Little Chalfont, UK) and p75 soluble TNF receptor (sTNFR; Medgenix, Fleurus, Belgium) according to the manufacturer's instructions. According to previous experience, p75 sTNFR is the most reliable marker of TNF-{alpha} involvement, particularly at supernatant level [19, 20].

Paired synovial fluid (SF) concentrations of VEGF and p75 sTNFR were evaluated in 20 active JIA patients (four with a polyarticular and 16 with a oligoarticular course) who underwent SF needle aspiration before local therapeutic infiltration with steroids. A previous steroid infiltration into the same joint in the last 6 months was an exclusion criterion.

Serum VEGF and p75 sTNFR concentrations were compared among the four subgroups examined (active polyarticular JIA patients, active oligoarticular JIA patients, JIA patients in clinical remission and healthy controls) using the non-parametric Mann–Whitney U-test. Correlations among all the variables considered were evaluated using the non-parametric Spearman rank test. Concomitant serum and SF determinations were evaluated with the Wilcoxon rank test.

Results

The clinical characteristics of JIA patients at the time of the study are reported in Table 1Go. We evaluated a total of 40 sera (14 from patients with a polyarticular course and 26 patients with an oligoarticular course) corresponding to an active phase of disease activity according to the criteria described above. Sera from 10 patients with oligoarticular course with inactive disease were also studied (Table 1Go).


View this table:
[in this window]
[in a new window]
 
TABLE 1.  Clinical and laboratory characteristics and treatment of JIA patients at the time of the study

 
Serum concentrations of VEGF were significantly higher in active polyarticular patients (median 315.5 pg/ml, range 74–1895 pg/ml) than in active oligoarticular JIA patients (median 118.2 pg/ml, range 47–1081 pg/ml; P=0.01), inactive oligoarticular JIA patients (median 123.0 pg/ml, range 86–265 pg/ml; P=0.02) and healthy controls (median 51 pg/ml, range 44–269 pg/ml; P=0.01) (Fig. 1Go). No statistical differences were noted between active and inactive oligoarticular JIA.



View larger version (15K):
[in this window]
[in a new window]
 
FIG. 1.  Serum concentrations of VEGF in individual subjects with active polyarticular, active oligoarticular and inactive oligoarticular JIA and in normal controls. Serum concentrations were significantly higher in patients with active polyarticular JIA than in patients with active oligoarticular JIA (P=0.01) and inactive oligoarticular JIA (P=0.02) and in healthy controls (P=0.01). Active and inactive oligoarticular JIA did not show a statistically significant difference. Horizontal lines represent median values for each study group.

 
Globally, in JIA patients serum concentrations of VEGF displayed a significant correlation with a number of clinical (number of active joints, r=0.52, P=0.001; global physician index, r=0.55, P=0.001) and laboratory parameters of disease activity (PLT count, r=0.48, P=0.001; ESR, r=0.39, P=0.007; CRP, r=0.34, P=0.03; Hb concentration, r -0.57, P=0.001) (Fig. 2Go). Notably, when the serum concentration of VEGF was evaluated according to the different clinical subsets, similar behaviour was observed for the oligoarticular JIA patients but not for the polyarticular subgroup (not shown).



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 2.  Correlations between serum concentrations of VEGF and Hb and between serum concentration of VEGF and the number of active joints in JIA patients (Spearman's rank correlation test).

 
Paired SF concentrations of VEGF and p75 sTNFR were evaluated in 20 active JIA patients. Four of them had a polyarticular course, whereas 16 had a persistent oligoarticular course.

In active polyarticular and oligoarticular JIA, the concentration of VEGF in SF (median 637.5 pg/ml, range 158–2000 pg/ml) was significantly higher than in corresponding sera (median 111.5 pg/ml, range 47–1081 pg/ml; P=0.001, Wilcoxon rank test). Notably, a clear correlation was found between the corresponding SF and sera VEGF concentrations (P=0.007) (Fig. 3Go).



View larger version (12K):
[in this window]
[in a new window]
 
FIG. 3.  Correlation between VEGF concentrations in concomitant samples of serum (sVEGF) and SF fluid (sfVEGF) in 20 JIA patients.

 
As TNF-{alpha} is thought to play a pivotal role in the induction of VEGF expression, a further aim of the study was to look for possible correlations among serum and SF concentrations of VEGF and reliable markers of TNF-{alpha} activity in JIA. Thus, according to previous experience all the samples were tested concomitantly for p75 sTNFR [19, 20]. Notably, in the SF, VEGF showed a strong positive correlation with p75 sTNFR (r=0.86, P=0.001, Spearman rank test). Conversely, no statistical correlation was found for serum concentrations.

Discussion

Angiogenesis is a physiological process consisting of the formation of new blood vessels from the pre-existing microvascular bed. The fundamental steps in this process are concomitant proteolysis of the extracellular matrix surrounding the blood vessels and the proliferation and migration of endothelial cells [1, 21, 22]. The regulation of angiogenesis is modulated strictly by a delicate balance between angiogenic and angiostatic factors.

A number of angiogenic mediators, including angiogenin, proinflammatory and regulatory cytokines (IL-1, IL-2, IL-8, TNF-{alpha}) and growth factors, such as VEGF, bFGF, TGF-ß, PDGF, EGF, platelet-derived endothelial cell growth factor, insulin-like growth factor 1 and hepatocyte growth factor, have been identified [2, 23, 24]. These mediators act on the endothelial cells that induce the production of proteolytic enzymes (metalloproteinases, plasminogen activator, proteinases) and stimulate the degradation of the basement membrane of the venule [22]. This process may be considered the first step in a process that allows the subsequent migration and proliferation of stimulated endothelial cells and the formation of new vessels [25, 26].

A number of angiostatic factors (angiostatin, endostatin, thrombospondin, placental ribonuclease inhibitor and tissue inhibitors of metalloproteinases) act in balancing the processes of angiogenesis [2, 23, 24]. Angiogenesis is thought to play an important role in a number of physiological processes (embryonic development, the female reproductive cycle, wound healing) and in pathological conditions (tumour growth, inflammation) [1, 21]. In all these situations a disproportion between angiogenic and angiostatic factors has been shown [14].

The RA pannus has been compared to a solid tumour because of its unregulated outgrowth, leading to the invasion and destruction of normal tissues, namely joint cartilage and bones. Angiogenesis supports the extensive vascularization occurring during the proliferation of RA pannus [27]. New vessel growth provides the oxygen and nutrients necessary to supply the high metabolic requirement of synoviocytes and permits the migration and progressive infiltration of other inflammatory cells into the joint [14, 12, 27].

Among the several angiogenic factors detected in RA synovial tissue, VEGF has been shown to be particularly over-expressed [4, 8, 10, 1214, 21, 28]. VEGF is a homodimeric glycosylated protein that is structurally homologous with PDGF. Five isoforms of VEGF, derived by alternative splicing of VEGF mRNA, are known [12, 29].

Initially known as a potent mediator of vascular permeability, VEGF has been shown to act as a selective endothelial cell mitogen, playing an important role both in angiogenesis and vasculogenesis (formation of new blood vessels during embryogenesis) as well as in the chemotaxis of endothelial cells and monocytes [1, 6, 10, 28, 30].

VEGF is highly expressed in both the serum and the SF of patients affected with RA, and shows a good correlation with disease activity parameters [10, 11]. Particularly, Koch et al. [12] have shown that SF concentrations of VEGF are higher in RA patients than in patients with osteoarthritis (OA) and other arthritides. On immunohistochemical staining, clear overexpression of VEGF at the level of the synovial lining layer has been found in RA patients, especially when compared with normal synovial tissue. This has been confirmed in later studies [13, 14]. In a collagen-induced arthritis model in rats, reduction in the severity of arthritis using angiogenesis inhibitors resulted in a significant reduction in the serum level of VEGF [31]. From this evidence, many authors have suggested that VEGF has a pivotal role in the angiogenesis that occurs during pannus formation in RA [314, 25].

So far, little information is available regarding the possible role of VEGF in the juvenile arthritides. In a recent study, serum concentrations of VEGF were evaluated in Japanese patients with JIA [28]. Serum levels of VEGF were significantly higher than in healthy controls and patients with acute infectious diseases. Moreover, a good correlation was found with a number of disease activity parameters, especially in the polyarticular subgroup [28]. In our study, the polyarticular subgroup displayed significantly higher serum concentrations of VEGF than the oligoarticular subgroup and healthy controls. However, a significant correlation with a number of disease activity parameters was found only in the oligoarticular subgroup. This could be related to the relative disproportion in the number of poly- vs oligoarticular patients. In the paper of Maeno et al. [28], the same numerical disproportion was present but favoured the polyarticular subgroup. These data support the need to verify the actual role of possible pathogenic mediators at the site of tissue inflammation.

Therefore, the main goal of our study was to evaluate the production of VEGF at the site of synovial inflammation, which, to our knowledge, has not yet been evaluated in JIA. It is of note that the concentration of VEGF was clearly higher in SF than in corresponding sera, showing a possible major role of this pro-angiogenic factor at the site of tissue inflammation. Notably, the significant correlation found between SF and serum VEGF concentrations may support the hypothesis that this growth factor spills out from SF to serum.

An additional aim of the present study was to look for a correlation between VEGF levels and TNF activity. As mentioned previously, the addition to culture of anti-TNF monoclonal antibodies has been shown to decrease the in vitro production of VEGF by RA synovial membrane cells significantly. Moreover, patients treated with the same antibodies displayed a significant reduction in the serum concentration of VEGF [8]. These data support the hypothesis of a close relationship between TNF activity and angiogenesis in chronic inflammatory arthritides.

In the present study we evaluated the possible correlation between VEGF and p75 sTNFR in sera and SF from patients with JIA. A clear correlation was found between these two parameters at the level of tissue damage. According to Paleolog [32], inhibition of TNF activity could interfere with synovial angiogenesis in JIA also, thus attributing other possible therapeutic effects to anti-TNF treatment.

Acknowledgments

The authors would like to thank Dr V. Pistoia for helpful suggestions and critical reading of this paper.

Notes

Correspondence to: M. Gattorno, Second Division of Paediatrics (Rheumatology Unit), ‘G. Gaslini’ Scientific Institute for Children, Largo G. Gaslini 5, 16147 Genoa, Italy. Back

References

  1. Folkman J, Klagsbrun M. Angiogenic factors. Science1987;335:442–7.
  2. Koch AE. Angiogenesis. Arthritis Rheum1998;41:951–62.[ISI][Medline]
  3. Firestein GS. Starving the synovium: angiogenesis and inflammation in rheumatoid arthritis. J Clin Invest1999;103:3–4.[Free Full Text]
  4. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med1995;1:27–31.[ISI][Medline]
  5. Berse B, Hunt JA, Diegel RJ et al. Hypoxia augments cytokine (transforming growth factor-beta (TGF-ß) and IL-1)-induced vascular endothelial growth factor secretion by human synovial fibroblasts. Clin Exp Immunol1999;115:176–82.[ISI][Medline]
  6. Jackson JR, Minton JAL, Ho ML, Wei N, Winkler JD. Expression of vascular endothelial growth factor in synovial fibroblasts is induced by hypoxia and interleukin 1ß. J Rheumatol1997;24:7:1253–9.[ISI][Medline]
  7. Ben-Av P, Crofford LJ, Wilder RL, Hla T. Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin e and interleukin-1: a potential mechanism for inflammatory angiogenesis. FEBS Lett1995;372:83–7.[ISI][Medline]
  8. Paleolog EM, Young S, Stark AC, McCloskey RV, Feldmann M, Maini RN. Modulation of angiogenic vascular endothelial growth factor by tumor necrosis factor {alpha} and interleukin-1 in rheumatoid arthritis. Arthritis Rheum1998;41:1258–65.[ISI][Medline]
  9. Paleolog EM. Angiogenesis: a critical process in the pathogenesis of RA—a role for VEGF? Br J Rheumatol1996;35:917–9.[ISI][Medline]
  10. Harada Mitsuyama K, Yoshida H, Sakisaka S et al. Vascular endothelial growth factor in patients with rheumatoid arthritis. Scand J Rheumatol1998;27:377–80.[ISI][Medline]
  11. Kikuchi K, Kubo M, Kadono T, Yazawa N, Ihn H, Tamaki K. Serum concentrations of vascular endothelial growth factor in collagen disease. Br J Dermatol1998;139:1049–51.[ISI][Medline]
  12. Koch AE, Harlow LA, Haines GK et al. Vascular endothelial growth factor. A cytokine modulating endothelial function in rheumatoid arthritis. J Immunol1994;152:41–9.
  13. Nagashima M, Yoshino, Ishiwata T, Asano G. Role of vascular endothelial growth factor in angiogenesis of rheumatoid arthritis. J Rheumatol1995;22:1624–30.[ISI][Medline]
  14. Walsh DA, Wade M, Mapp PI, Blake DR. Focally regulated endothelial proliferation and cell death in human synovium. Am J Pathol1998;152:691–702.[Abstract]
  15. Petty RE, Southwood TR, Baum J et al. Revision of the proposed classification criteria for juvenile idiopathic arthritis: Durban, 1997. J Rheumatol1998;25:1991–4.[Medline]
  16. Ravelli A, Viola S, Ruperto N, Corsi B, Ballardini G, Martini A. Correlation between conventional disease activity measures in juvenile chronic arthritis. Ann Rheum Dis1997;56:197–200.[Abstract/Free Full Text]
  17. Giannini EH, Ruperto N, Ravelli A, Lovell DJ, Felson DT, Martini A. Preliminary definition of improvement in juvenile arthritis. Arthritis Rheum1997;40:1202–9.[ISI][Medline]
  18. Ruperto N, Ravelli A, Migliavacca D et al. Responsiveness of clinical measures in children with oligoarticular juvenile chronic arthritis. J Rheumatol1999;26:1827–30.[ISI][Medline]
  19. Cope AP, Aderka D, Doherty M et al. Increased levels of soluble tumor necrosis factor receptors in the sera and synovial fluid of patients with rheumatic diseases. Arthritis Rheum1992;35:1160–9.[ISI][Medline]
  20. Gattorno M, Picco P, Buoncompagni A et al. Serum p55 and p75 tumour necrosis factor receptors as markers of disease activity in juvenile chronic arthritis. Ann Rheum Dis1996;55:243–7.[Abstract]
  21. Polverini PJ. The pathophysiology of angiogenesis. Crit Rev Oral Biol Med1995;6:230–47.[Abstract]
  22. Moses MA. The regulation of neovascularization by matrix metalloproteinases and their inhibitors. Stem Cells1997;15:180–9.[Abstract/Free Full Text]
  23. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell1996;86:353–64.[ISI][Medline]
  24. Antoniades HN, Heofanis G, Neville-Golden J, Kiritsy CP, Lynch SE. Injury induces in vivo expression of platelet-derived growth factor (PDGF) and PDGF receptor mRNAs in skin epithelial cells and PDGF mRNA in connective tissue fibroblasts. Proc Natl Acad Sci USA1991;88:565–9.[Abstract]
  25. Sang QX. Complex role of matrix metalloproteinases in angiogenesis. Cell Res1998;8:171–7.[Medline]
  26. Stetler-Stevenson WG. Matrix metalloproteinases in angiogenesis. A moving target for therapeutic intervention. J Clin Invest1999;103:1237–41.[Free Full Text]
  27. Colville-Nash PR, Scott DL. Angiogenesis and rheumatoid arthritis: pathogenic and therapeutic implications. Ann Rheum Dis1992;51:919–25.[Abstract]
  28. Maeno N, Takei S, Imanaka H et al. Increased circulating vascular endothelial growth factor is correlated with disease activity in polyarticular juvenile rheumatoid arthritis. J Rheumatol1999;26:2244–8.[ISI][Medline]
  29. Thomas KA. Vascular endothelial growth factor, a potent and selective angiogenic agent. J Biol Chem1996;271:603–6.[Free Full Text]
  30. Siemeister G, Schirner M, Reusch P, Barleon B, Marmé D, Martiny-Baron G. An antagonist vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells. Proc Natl Acad Sci USA1998;95:4625–9.[Abstract/Free Full Text]
  31. Connolly DT, Heuvelman DM, Nelson R et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest1989;84:1470–8.[ISI][Medline]
  32. Paleolog E. Target effector role of vascular endothelium in the inflammatory response: insight from the clinical trials of anti-TNF alpha antibody in rheumatoid arthritis. Mol Pathol1997;50:225–33.[Abstract]
Submitted 5 January 2001; Accepted 12 February 2002