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Plasma levels of soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) in familial Mediterranean fever
Joint Bone Spine 74 (2007) 52e55 http://france.elsevier.com/direct/BONSOI/
Original article
Plasma levels of soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) in familial Mediterranean fever ¨ mer Basxar a, Mehmet Akif O ¨ ztu¨rk b, Seyfettin Ko¨klu¨ c,*, Selime Ayaz d, Osman Yu¨ksel e, O Sedat Kiraz f, Ihsan Ertenli f, Meral C¸algu¨neri f a
Department of Gastroenterology, Tu¨rkiye Yu¨ksek Ihtisas Hospital, Ankara, Turkey Department of Rheumatology, Gazi University School of Medicine, Ankara, Turkey c Department of Gastroenterology, Ankara Education and Research Hospital, Ankara, Turkey d Department of Hematology, Tu¨rkiye Yu¨ksek Ihtisas Hospital, Ankara, Turkey e Department of Gastroenterology, Numune Hospital, Ankara, Turkey f Department of Rheumatology, Hacettepe University School of Medicine, Ankara, Turkey b
Received 30 August 2005; accepted 2 February 2006 Available online 17 April 2006
Abstract Aims: To assess the levels of soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) in patients with familial Mediterranean fever (FMF). Methods: Plasma levels of sVEGFR-1 were investigated in 33 FMF patients in an attack-free period (mean age 30.8 years; males/females 10/23), in 15 patients with acute FMF attack (mean age 32.7 years; males/females 7/8), and 19 healthy controls (mean age 32 years; males/females 11/ 8). Levels of sVEGFR-1 were also compared among patients who were receiving colchicine and those who were not. Results: Plasma sVEGFR-1 levels were 3.49 1.10, 3.53 1.02, and 0.37 0.28 ng/ml for FMF patients in the attack-free period, FMF patients with acute attack, and healthy controls, respectively. Plasma sVEGFR-1 levels were significantly higher in FMF patients with and without acute attack compared to the control group ( p < 0.05). sVEGFR-1 levels were not statistically significant between patients with acute attack and attack-free FMF patients ( p > 0.05). The plasma levels of sVEGFR-1 were also comparable in colchicine treated and untreated patients. Conclusion: Our data suggest that sVEGFR-1 may have a role in the ongoing inflammatory cascade in FMF. Ó 2006 Elsevier Masson SAS. All rights reserved. Keywords: Familial Mediterranean fever; VEGF; sVEGFR-1
1. Introduction Familial Mediterranean fever (FMF) is an autosomal recessive disease, which affects primarily Turks, Armenians, Arabs and Jews [1]. FMF is characterized by self-limiting acute febrile attacks and inflammatory reactions of serosal membranes. Several non-specific immunological abnormalities and elevations in acute-phase reactant levels were observed during FMF attacks [2]. Moreover, wedalong with * Corresponding author. Karargahtepe mahallesi, Kumrulu sokak, No: 18, Kec¸io¨ren, Ankara, Turkey. Tel.: þ90 312 3612568; fax: þ90 312 3124120. E-mail address: [email protected] (S. Ko¨klu¨).
othersdhave previously demonstrated evidence for ongoing subclinical inflammation, even during attack-free periods in FMF patients [3e5]. Vascular endothelial growth factor (VEGF), a member of the platelet-derived growth factor family, is a potent and specific mitogen for endothelial cells [6,7]. VEGF was originally thought to be an endothelial cell-derived, angiogenic, autocrine factor. However, recent reports have shown that various other cell types including macrophages [8,9] and neutrophils [10,11] also express significant amounts of VEGF. VEGF receptors 1 and 2 (VEGFR-1 and VEGFR-2) are the two highaffinity tyrosine kinase receptors for VEGF. VEGFR-1 gene encodes two polypeptides, a soluble VEGF-binding protein
1297-319X/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.jbspin.2006.02.003
¨ . Bas O xar et al. / Joint Bone Spine 74 (2007) 52e55
(sVEGFR-1) and a full-length membrane protein (receptor form of VEGFR-1) [12]. sVEGFR-1 is a naturally occurring VEGF antagonist, and is proved to inhibit the VEGF induced proliferation and migration of endothelial cells in vitro [13e 15]. sVEGFR-1 has a physiological function in pregnancy by regulating the VEGF in a negative manner by downregulating its activity [16]. sVEGFR-1 was detected in serum and plasma samples of normal healthy donors but the biological and physiological significance of it in vivo is not yet clear [13]. Increased VEGF levels in the circulation have been reported in a number of vascular and inflammatory diseases, including systemic vasculitis and inflammatory arthritis [17e22]. Therefore, VEGF seems to be a non-specific marker of diseases in which endothelial cell injury and/or repair occurs. On the other hand, circulating levels of sVEGFR-1 were also increased in inflammatory arthritis [21]. In this study, we assessed sVEGFR-1 levels in patients with FMF. 2. Methods The study groups comprised 33 FMF patients in an attackfree period (mean age 30.8 years (range 16e47); males/ females 10/23), and 15 patients with acute FMF attack (mean age 32.7 years (range 19e50); males/females 7/8). All of the patients had had acute febrile peritonitis attacks. Sixteen patients had experienced arthritis (33.3%), 8 patients had had pleuritis (16.7%), and one patient had had pericarditis (2.1%) before the diagnosis of FMF. One patient was complicated with amyloidosis. The clinical features of the patients are given in Table 1. The diagnosis of FMF was established according to the TelleHashomer criteria [23]. Patients in the acute FMF attack group had signs and symptoms of peritonitis or acute arthritis accompanied by elevations of acute phase reactants (i.e. erythrocyte sedimentation rate, C-reactive protein, fibrinogen). None of the patients was receiving any drug other than colchicine that could influence cytokine levels. Nineteen healthy subjects from the hospital staff (mean age 32 years (range 23e42); males/females 11/8)) were included as a control group. Plasma samples were from venous blood collected between 9.00 and 10.00 a.m. into vacutainer tubes (SST, BD vacutainer). Plasma was removed from red cells, as soon as possible after clotting and separation, and frozen immediately and stored at 80 C until the measuring time. Standard enzyme immunoassays were used for plasma concentration of
Table 1 Clinical features of patients with familial Mediterranean fever (n ¼ 48) Clinical manifestation
Frequency (%)
Abdominal pain Fever Arthritis Pleuritis Pericarditis Amyloidosis
48 48 16 8 1 1
(100) (100) (33.3) (16.7) (2.1) (2.1)
53
sVEGFR-1 (Bendermed System, Austria). The Statistical Package for Social Sciences (SPSS) version 10.0 for Windows was used to analyze the data. Non-parametric KruskaleWallis test was used to analyze the variance among groups. Statistically significant differences obtained from KruskaleWallis analysis were further tested by ManneWhitney U-test for post hoc pairwise comparisons between groups. The correlation analyses were performed by Pearson correlation test. p values below 0.05 were considered as statistically significant. 3. Results Age and sex distributions in different groups were similar. The disease durations were also comparable in patients in attack-free periods and patients with acute FMF attacks (Table 2). The levels of plasma sVEGFR-1 were expressed as mean and standard deviation (SD). Plasma sVEGFR-1 levels were 3.49 1.10, 3.53 1.02, and 0.37 0.28 ng/ml for FMF patients in an attack-free period, FMF patients with acute attack, and healthy controls, respectively (Table 2). Plasma sVEGFR-1 levels were significantly higher in FMF patients with and without acute attack compared to the control group ( p < 0.05). sVEGFR-1 levels were not statistically significant between patients with acute attack and attack-free FMF patients. There was no significant correlation between CRP levels and sVEGFR-1 levels, and between sVEGFR-1 levels and the disease duration ( p > 0.05). In order to eliminate the confounding effect of colchicine on plasma sVEGFR-1 levels, we compared plasma sVEGFR-1 levels in colchicine treated and colchicine untreated FMF patients. Plasma levels of sVEGFR-1 were 3.61 0.96 ng/ml and 3.46 1.11 ng/ml in patients who did not use colchicine (n ¼ 13) and who did use colchicine (n ¼ 35), respectively ( p > 0.05). We further analyzed plasma sVEGFR-1 levels in colchicine treated and colchicine untreated FMF patients in attack-free periods. sVEGFR-1 levels were 3.37 0.47 ng/ml and 3.54 1.29 ng/ml in attack-free FMF patients who did not use colchicine (n ¼ 10) and who did use colchicine (n ¼ 23), respectively ( p > 0.05). We did not compare sVEGFR-1 levels in FMF patients with acute attack according Table 2 Plasma sVEGFR-1 levels of FMF patients and healthy controls
Number of subjects Age, years (mean (minemax)) Males/females Disease duration, years (mean SD) Plasma sVEGFR-1 level, ng/ml (mean SD) a b c
Patients in attack-free period
FMF patients with acute attack
Healthy controls
33 30.8 (16e47)
15 32.7 (19e50)
19 32 (23e42)
10/23 14.6 9.9
7/8 16.4 9.1
11/8 e
3.49 1.10a,b
3.53 1.02c
0.37 0.28
p < 0.05 for patients in attack free period vs. healthy controls. p > 0.05 for patients in attack free period vs. patients with acute attack. p < 0.05 for patients with acute attack vs. healthy controls.
54
O¨. Bas xar et al. / Joint Bone Spine 74 (2007) 52e55
to the colchicine treatment because of the very small number of subjects in the subgroups (three patients with FMF attack were not using colchicine, and 12 patients with FMF attack were using colchicine). There was no significant difference regarding serum VEGFR-1 levels between patients who had arthritis and patients who did not. Comparisons between patients with or without other clinical manifestations were not performed because of limited number of patients in each subgroup. 4. Discussion In this study, we demonstrated that plasma sVEGFR-1 levels increased in patients with FMF with and without acute attacks. Plasma levels of sVEGFR-1 were comparable in patients with acute FMF attack and in attack-free periods. sVEGFR-1 values were independent of colchicine use. Recurrent and self-limiting febrile attacks of polyserositis are characteristic features of FMF. Serum concentrations of acute phase proteins and proinflammatory cytokines are elevated in FMF patients during acute attacks. Furthermore, certain inflammatory cytokines remain elevated even in the attack-free periods of FMF reflecting ongoing subclinical inflammation [5,24]. Systemic inflammation is characterized by the activation of the vascular endothelium. Endothelial cells play key roles in the initiation and perpetuation of the inflammatory response via expressing cell adhesion molecules and secreting proinflammatory cytokines. Endothelial cell damage is expected to occur during the neutrophil migration to adjacent tissue [25]. There is also some data that endothelial cell activation and/or injury can complicate the inflammatory cascade of FMF. The levels of intercellular adhesion molecule 1 (ICAM-1), a cell surface glycoprotein of the immunoglobulin super family expressed in endothelial cells, were increased in both acute attacks and attack-free periods of FMF patients [26]. We recently demonstrated that thrombomodulin, an endothelial injury marker, was elevated in attack-free FMF patients (unpublished observation). Both VEGF and its natural regulator sVEGFR-1 can be synthesized and secreted by vascular endothelial cells. Therefore, our study offers further data regarding the endothelial injury and/or activation to the inflammatory cascade of FMF. However, there was no significant difference regarding sVEGFR-1 levels in FMF patients with acute attack and attack-free FMF patients. Likewise, ICAM-1 levels were comparable in FMF patients with or without acute attack [26]. FMF attacks are characterized by serosal inflammation rich in neutrophil leucocytes (PNLs) [1]. On the other hand, the gene causing FMF, designated MEFV, is primarily expressed in cells of myelocytic lineage [27]. Therefore, neutrophils appear to play the key role in the pathogenesis of FMF. Apart from endothelial cells, neutrophils can also express significant amounts of VEGF [10,11]. PNL-derived VEGF can modulate PNL migration via an autocrine amplification mechanism [28]. VEGF also stimulates the migration of human peripheral monocytes and VEGFR-1 is thought to play a major role in VEGF-dependent functions of monocyte/macrophages [12].
sVEGFR-1 is capable of inhibiting proliferation and migration of endothelial cells induced by VEGF in vitro. Thus sVEGFR1 appears as a natural antagonist for VEGF, and seems to act as an anti-inflammatory mediator in the inflammatory reactions. Taken together, our results suggest that sVEGFR-1 might also have a regulatory role on the ongoing inflammation in FMF via modulating certain neutrophil and monocyte functions. Although VEGFR-1 is present dominantly on endothelial cells, expression of VEGFR-1 was also found on human monocytes and neutrophils [14,27,29]. Therefore, the endothelium, PNLs and monocytes might be the source of VEGFR-1 in FMF patients. In conclusion, our data suggest that sVEGFR-1 is involved in the pathobiology of FMF. The cytokine network of FMF appears broader than we know, and endothelial cells may contribute to the inflammatory cascade of FMF by expressing and secreting certain mediators including VEGFR-1. sVEGFR-1 might also play regulatory roles on certain neutrophil and monocyte functions during the ongoing inflammation in FMF. Levels of both VEGF and sVEGFR-1 levels should be measured concurrently to support this hypothesis. Since levels of sVEGFR-1 are comparable in patients during the acute attacks and in the attack-free periods, the role of this mediator for the initiation of FMF attacks remains to be established. Future studies to determine serum levels of both VEGF and VEGFR-1 together with other endothelial cell markers such as thrombomodulin, and proinflammatory cytokines such as interleukin 6 at different time points during the clinical course of patients will help us to establish the inflammatory cascade in FMF. References [1] Ben-Chetrit E, Levy M. Familial Mediterranean fever. Lancet 1998;351:659e64. [2] Bar-Eli M, Ehrenfeld M, Levi M, Gallily R, Eiakim M. Leukocyte chemotaxis in recurrent polyserositis (familial Mediterranean fever). Am J Med Sci 1981;281:15e8. [3] Anton PA, Targan SR, Vigna SR, Durham M, Schwabe AD, Shanahan F. Enhanced neutrophil chemiluminescence in familial Mediterranean fever. J Clin Immunol 1988;8:148e56. [4] Bar-Eli M, Territo MC, Peters RS, Scwabe AD. A neutrophil lysozyme leak in patients with familial Mediterranean fever. Am J Hematol 1981;1:387e95. [5] Kiraz S, Ertenli I, Arici M, Calguneri M, Haznedaroglu I, Celik I, et al. Effects of colchicine on inflammatory cytokines and selectins in familial Mediterranean fever. Clin Exp Rheumatol 1998;16:721e4. [6] Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999;13:9e22. [7] Veikkola T, Alitalo K. VEGFs, receptors and angiogenesis. Semin Cancer Biol 1999;9:211e20. [8] McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH, Sharkey AM, et al. Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 1996;98:482e9. [9] Xiong M, Elson G, Legarda D, Leibovich SJ. Production of vascular endothelial growth factor by murine macrophages: regulation by hypoxia, lactate, and the inducible nitric oxide synthase pathway. Am J Pathol 1998;153:587e98. [10] Taichman NS, Young S, Cruchley AT, Taylor P, Paleolog E. Human neutrophils secrete vascular endothelial growth factor. J Leukocyte Biol 1997;62:397e400.
¨ . Bas O xar et al. / Joint Bone Spine 74 (2007) 52e55 [11] Webb NJ, Myers CR, Watson CJ, Bottomley MJ, Brenchley PE. Activated human neutrophils express vascular endothelial growth factor (VEGF). Cytokine 1998;10:254e7. [12] Shibuya M. Structure and dual function of vascular endothelial growth factor receptor-1 (Flt-1). Int J Biochem Cell Biol 2001;33:409e20. [13] Barleon B, Reusch P, Totzke F, Herzog C, Keck C, Martiny-Baron G, et al. Soluble VEGFR-1 secreted by endothelial cells and monocytes is present in human serum and plasma from healthy donors. Angiogenesis 2001;4:143e54. [14] Barleon B, Zossani S, Zhou D, Weich HA, Mantovani A, Marme´ D. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood 1996;87:3336e43. [15] Kendall RL, Wang G, Thomas KA. Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR. Biochem Biophys Res Commun 1996;226:324e8. [16] Clark DE, Smith SK, He Y, Day KA, Licence DR, Corps AN, et al. A vascular endothelial growth factor antagonist is produced by the human placenta and released into the maternal circulation. Biol Reprod 1998;59:1540e8. [17] Maeno N, Takei S, Masuda K, Akaike H, Matsuo K, Kitajima I, et al. Increased serum levels of vascular endothelial growth factor in Kawasaki disease. Pediatr Res 1998;44:596e9. [18] Cekmen M, Evereklioglu C, Er H, Inaloz HS, Doganay S, Turkoz Y, et al. Vascular endothelial growth factor levels are increased and associated with disease activity in patients with Behcet’s syndrome. Int J Dermatol 2003;42:870e5. [19] Topaloglu R, Sungur A, Baskin E, Besbas N, Saatci U, Bakkaloglu A. Vascular endothelial growth factor in Henoch-Schonlein purpura. J Rheumatol 2001;28:2269e73. [20] Vignola S, Picco P, Falcini F, Sabatini F, Buoncompagni A, Gattorno M. Serum and synovial fluid concentration of vascular endothelial growth
[21]
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[27]
[28]
[29]
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factor in juvenile idiopathic arthritides. Rheumatology (Oxford) 2002;41:691e6. Ballara S, Taylor PC, Reusch P, Marme D, Feldmann M, Maini RN, et al. Raised serum vascular endothelial growth factor levels are associated with destructive change in inflammatory arthritis. Arthritis Rheum 2001;44:2055e64. Li CG, Reynolds I, Ponting JM, Holt PJ, Hillarby MC, Kumar S. Serum levels of vascular endothelial growth factor (VEGF) are markedly elevated in patients with Wegener’s granulomatosis. Br J Rheumatol 1998;37:1303e6. Livneh A, Langevitz P, Zemer D, Zaks N, Kees S, Lidar T, et al. Criteria for the diagnosis of familial Mediterranean fever. Arthritis Rheum 1997;40:1879e85. Koklu S, Ozturk MA, Balci M, Yuksel O, Ertenli I, Kiraz S. Interferongamma levels in familial Mediterranean fever. Joint Bone Spine 2005;72:38e40. Aird WC. Endothelium as an organ. Crit Care Med 2004;32:271e9. Direskeneli H, Ozdogan H, Korkmaz C, Akoglu T, Yazici H. Serum soluble intercellular adhesion molecule 1 and interleukin 8 levels in familial Mediterranean fever. J Rheumatol 1999;26:1983e6. Centola M, Wood G, Frucht DM, Galon J, Aringer M, Farrell C, et al. The gene for familial Mediterranean fever, MEFV, is expressed in early leukocyte development and is regulated in response to inflammatory mediators. Blood 2000;95:3223e31. Ancelin M, Chollet-Martin S, Herve MA, Legrand C, El Benna J, PerrotApplanat M. Vascular endothelial growth factor VEGF189 induces human neutrophil chemotaxis in extravascular tissue via an autocrine amplification mechanism. Lab Invest 2004;84:502e12. Borg JP, Barleon B, Marme´ D, Birnbaum D. The family of VEGF tyrosine kinase receptors. In: Lonai P, editor. Towards a molecular analysis of mammalian development. Reading, UK: Harwood Academic; 1996.
Original article
Plasma levels of soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) in familial Mediterranean fever ¨ mer Basxar a, Mehmet Akif O ¨ ztu¨rk b, Seyfettin Ko¨klu¨ c,*, Selime Ayaz d, Osman Yu¨ksel e, O Sedat Kiraz f, Ihsan Ertenli f, Meral C¸algu¨neri f a
Department of Gastroenterology, Tu¨rkiye Yu¨ksek Ihtisas Hospital, Ankara, Turkey Department of Rheumatology, Gazi University School of Medicine, Ankara, Turkey c Department of Gastroenterology, Ankara Education and Research Hospital, Ankara, Turkey d Department of Hematology, Tu¨rkiye Yu¨ksek Ihtisas Hospital, Ankara, Turkey e Department of Gastroenterology, Numune Hospital, Ankara, Turkey f Department of Rheumatology, Hacettepe University School of Medicine, Ankara, Turkey b
Received 30 August 2005; accepted 2 February 2006 Available online 17 April 2006
Abstract Aims: To assess the levels of soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) in patients with familial Mediterranean fever (FMF). Methods: Plasma levels of sVEGFR-1 were investigated in 33 FMF patients in an attack-free period (mean age 30.8 years; males/females 10/23), in 15 patients with acute FMF attack (mean age 32.7 years; males/females 7/8), and 19 healthy controls (mean age 32 years; males/females 11/ 8). Levels of sVEGFR-1 were also compared among patients who were receiving colchicine and those who were not. Results: Plasma sVEGFR-1 levels were 3.49 1.10, 3.53 1.02, and 0.37 0.28 ng/ml for FMF patients in the attack-free period, FMF patients with acute attack, and healthy controls, respectively. Plasma sVEGFR-1 levels were significantly higher in FMF patients with and without acute attack compared to the control group ( p < 0.05). sVEGFR-1 levels were not statistically significant between patients with acute attack and attack-free FMF patients ( p > 0.05). The plasma levels of sVEGFR-1 were also comparable in colchicine treated and untreated patients. Conclusion: Our data suggest that sVEGFR-1 may have a role in the ongoing inflammatory cascade in FMF. Ó 2006 Elsevier Masson SAS. All rights reserved. Keywords: Familial Mediterranean fever; VEGF; sVEGFR-1
1. Introduction Familial Mediterranean fever (FMF) is an autosomal recessive disease, which affects primarily Turks, Armenians, Arabs and Jews [1]. FMF is characterized by self-limiting acute febrile attacks and inflammatory reactions of serosal membranes. Several non-specific immunological abnormalities and elevations in acute-phase reactant levels were observed during FMF attacks [2]. Moreover, wedalong with * Corresponding author. Karargahtepe mahallesi, Kumrulu sokak, No: 18, Kec¸io¨ren, Ankara, Turkey. Tel.: þ90 312 3612568; fax: þ90 312 3124120. E-mail address: [email protected] (S. Ko¨klu¨).
othersdhave previously demonstrated evidence for ongoing subclinical inflammation, even during attack-free periods in FMF patients [3e5]. Vascular endothelial growth factor (VEGF), a member of the platelet-derived growth factor family, is a potent and specific mitogen for endothelial cells [6,7]. VEGF was originally thought to be an endothelial cell-derived, angiogenic, autocrine factor. However, recent reports have shown that various other cell types including macrophages [8,9] and neutrophils [10,11] also express significant amounts of VEGF. VEGF receptors 1 and 2 (VEGFR-1 and VEGFR-2) are the two highaffinity tyrosine kinase receptors for VEGF. VEGFR-1 gene encodes two polypeptides, a soluble VEGF-binding protein
1297-319X/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.jbspin.2006.02.003
¨ . Bas O xar et al. / Joint Bone Spine 74 (2007) 52e55
(sVEGFR-1) and a full-length membrane protein (receptor form of VEGFR-1) [12]. sVEGFR-1 is a naturally occurring VEGF antagonist, and is proved to inhibit the VEGF induced proliferation and migration of endothelial cells in vitro [13e 15]. sVEGFR-1 has a physiological function in pregnancy by regulating the VEGF in a negative manner by downregulating its activity [16]. sVEGFR-1 was detected in serum and plasma samples of normal healthy donors but the biological and physiological significance of it in vivo is not yet clear [13]. Increased VEGF levels in the circulation have been reported in a number of vascular and inflammatory diseases, including systemic vasculitis and inflammatory arthritis [17e22]. Therefore, VEGF seems to be a non-specific marker of diseases in which endothelial cell injury and/or repair occurs. On the other hand, circulating levels of sVEGFR-1 were also increased in inflammatory arthritis [21]. In this study, we assessed sVEGFR-1 levels in patients with FMF. 2. Methods The study groups comprised 33 FMF patients in an attackfree period (mean age 30.8 years (range 16e47); males/ females 10/23), and 15 patients with acute FMF attack (mean age 32.7 years (range 19e50); males/females 7/8). All of the patients had had acute febrile peritonitis attacks. Sixteen patients had experienced arthritis (33.3%), 8 patients had had pleuritis (16.7%), and one patient had had pericarditis (2.1%) before the diagnosis of FMF. One patient was complicated with amyloidosis. The clinical features of the patients are given in Table 1. The diagnosis of FMF was established according to the TelleHashomer criteria [23]. Patients in the acute FMF attack group had signs and symptoms of peritonitis or acute arthritis accompanied by elevations of acute phase reactants (i.e. erythrocyte sedimentation rate, C-reactive protein, fibrinogen). None of the patients was receiving any drug other than colchicine that could influence cytokine levels. Nineteen healthy subjects from the hospital staff (mean age 32 years (range 23e42); males/females 11/8)) were included as a control group. Plasma samples were from venous blood collected between 9.00 and 10.00 a.m. into vacutainer tubes (SST, BD vacutainer). Plasma was removed from red cells, as soon as possible after clotting and separation, and frozen immediately and stored at 80 C until the measuring time. Standard enzyme immunoassays were used for plasma concentration of
Table 1 Clinical features of patients with familial Mediterranean fever (n ¼ 48) Clinical manifestation
Frequency (%)
Abdominal pain Fever Arthritis Pleuritis Pericarditis Amyloidosis
48 48 16 8 1 1
(100) (100) (33.3) (16.7) (2.1) (2.1)
53
sVEGFR-1 (Bendermed System, Austria). The Statistical Package for Social Sciences (SPSS) version 10.0 for Windows was used to analyze the data. Non-parametric KruskaleWallis test was used to analyze the variance among groups. Statistically significant differences obtained from KruskaleWallis analysis were further tested by ManneWhitney U-test for post hoc pairwise comparisons between groups. The correlation analyses were performed by Pearson correlation test. p values below 0.05 were considered as statistically significant. 3. Results Age and sex distributions in different groups were similar. The disease durations were also comparable in patients in attack-free periods and patients with acute FMF attacks (Table 2). The levels of plasma sVEGFR-1 were expressed as mean and standard deviation (SD). Plasma sVEGFR-1 levels were 3.49 1.10, 3.53 1.02, and 0.37 0.28 ng/ml for FMF patients in an attack-free period, FMF patients with acute attack, and healthy controls, respectively (Table 2). Plasma sVEGFR-1 levels were significantly higher in FMF patients with and without acute attack compared to the control group ( p < 0.05). sVEGFR-1 levels were not statistically significant between patients with acute attack and attack-free FMF patients. There was no significant correlation between CRP levels and sVEGFR-1 levels, and between sVEGFR-1 levels and the disease duration ( p > 0.05). In order to eliminate the confounding effect of colchicine on plasma sVEGFR-1 levels, we compared plasma sVEGFR-1 levels in colchicine treated and colchicine untreated FMF patients. Plasma levels of sVEGFR-1 were 3.61 0.96 ng/ml and 3.46 1.11 ng/ml in patients who did not use colchicine (n ¼ 13) and who did use colchicine (n ¼ 35), respectively ( p > 0.05). We further analyzed plasma sVEGFR-1 levels in colchicine treated and colchicine untreated FMF patients in attack-free periods. sVEGFR-1 levels were 3.37 0.47 ng/ml and 3.54 1.29 ng/ml in attack-free FMF patients who did not use colchicine (n ¼ 10) and who did use colchicine (n ¼ 23), respectively ( p > 0.05). We did not compare sVEGFR-1 levels in FMF patients with acute attack according Table 2 Plasma sVEGFR-1 levels of FMF patients and healthy controls
Number of subjects Age, years (mean (minemax)) Males/females Disease duration, years (mean SD) Plasma sVEGFR-1 level, ng/ml (mean SD) a b c
Patients in attack-free period
FMF patients with acute attack
Healthy controls
33 30.8 (16e47)
15 32.7 (19e50)
19 32 (23e42)
10/23 14.6 9.9
7/8 16.4 9.1
11/8 e
3.49 1.10a,b
3.53 1.02c
0.37 0.28
p < 0.05 for patients in attack free period vs. healthy controls. p > 0.05 for patients in attack free period vs. patients with acute attack. p < 0.05 for patients with acute attack vs. healthy controls.
54
O¨. Bas xar et al. / Joint Bone Spine 74 (2007) 52e55
to the colchicine treatment because of the very small number of subjects in the subgroups (three patients with FMF attack were not using colchicine, and 12 patients with FMF attack were using colchicine). There was no significant difference regarding serum VEGFR-1 levels between patients who had arthritis and patients who did not. Comparisons between patients with or without other clinical manifestations were not performed because of limited number of patients in each subgroup. 4. Discussion In this study, we demonstrated that plasma sVEGFR-1 levels increased in patients with FMF with and without acute attacks. Plasma levels of sVEGFR-1 were comparable in patients with acute FMF attack and in attack-free periods. sVEGFR-1 values were independent of colchicine use. Recurrent and self-limiting febrile attacks of polyserositis are characteristic features of FMF. Serum concentrations of acute phase proteins and proinflammatory cytokines are elevated in FMF patients during acute attacks. Furthermore, certain inflammatory cytokines remain elevated even in the attack-free periods of FMF reflecting ongoing subclinical inflammation [5,24]. Systemic inflammation is characterized by the activation of the vascular endothelium. Endothelial cells play key roles in the initiation and perpetuation of the inflammatory response via expressing cell adhesion molecules and secreting proinflammatory cytokines. Endothelial cell damage is expected to occur during the neutrophil migration to adjacent tissue [25]. There is also some data that endothelial cell activation and/or injury can complicate the inflammatory cascade of FMF. The levels of intercellular adhesion molecule 1 (ICAM-1), a cell surface glycoprotein of the immunoglobulin super family expressed in endothelial cells, were increased in both acute attacks and attack-free periods of FMF patients [26]. We recently demonstrated that thrombomodulin, an endothelial injury marker, was elevated in attack-free FMF patients (unpublished observation). Both VEGF and its natural regulator sVEGFR-1 can be synthesized and secreted by vascular endothelial cells. Therefore, our study offers further data regarding the endothelial injury and/or activation to the inflammatory cascade of FMF. However, there was no significant difference regarding sVEGFR-1 levels in FMF patients with acute attack and attack-free FMF patients. Likewise, ICAM-1 levels were comparable in FMF patients with or without acute attack [26]. FMF attacks are characterized by serosal inflammation rich in neutrophil leucocytes (PNLs) [1]. On the other hand, the gene causing FMF, designated MEFV, is primarily expressed in cells of myelocytic lineage [27]. Therefore, neutrophils appear to play the key role in the pathogenesis of FMF. Apart from endothelial cells, neutrophils can also express significant amounts of VEGF [10,11]. PNL-derived VEGF can modulate PNL migration via an autocrine amplification mechanism [28]. VEGF also stimulates the migration of human peripheral monocytes and VEGFR-1 is thought to play a major role in VEGF-dependent functions of monocyte/macrophages [12].
sVEGFR-1 is capable of inhibiting proliferation and migration of endothelial cells induced by VEGF in vitro. Thus sVEGFR1 appears as a natural antagonist for VEGF, and seems to act as an anti-inflammatory mediator in the inflammatory reactions. Taken together, our results suggest that sVEGFR-1 might also have a regulatory role on the ongoing inflammation in FMF via modulating certain neutrophil and monocyte functions. Although VEGFR-1 is present dominantly on endothelial cells, expression of VEGFR-1 was also found on human monocytes and neutrophils [14,27,29]. Therefore, the endothelium, PNLs and monocytes might be the source of VEGFR-1 in FMF patients. In conclusion, our data suggest that sVEGFR-1 is involved in the pathobiology of FMF. The cytokine network of FMF appears broader than we know, and endothelial cells may contribute to the inflammatory cascade of FMF by expressing and secreting certain mediators including VEGFR-1. sVEGFR-1 might also play regulatory roles on certain neutrophil and monocyte functions during the ongoing inflammation in FMF. Levels of both VEGF and sVEGFR-1 levels should be measured concurrently to support this hypothesis. Since levels of sVEGFR-1 are comparable in patients during the acute attacks and in the attack-free periods, the role of this mediator for the initiation of FMF attacks remains to be established. Future studies to determine serum levels of both VEGF and VEGFR-1 together with other endothelial cell markers such as thrombomodulin, and proinflammatory cytokines such as interleukin 6 at different time points during the clinical course of patients will help us to establish the inflammatory cascade in FMF. References [1] Ben-Chetrit E, Levy M. Familial Mediterranean fever. Lancet 1998;351:659e64. [2] Bar-Eli M, Ehrenfeld M, Levi M, Gallily R, Eiakim M. Leukocyte chemotaxis in recurrent polyserositis (familial Mediterranean fever). Am J Med Sci 1981;281:15e8. [3] Anton PA, Targan SR, Vigna SR, Durham M, Schwabe AD, Shanahan F. Enhanced neutrophil chemiluminescence in familial Mediterranean fever. J Clin Immunol 1988;8:148e56. [4] Bar-Eli M, Territo MC, Peters RS, Scwabe AD. A neutrophil lysozyme leak in patients with familial Mediterranean fever. Am J Hematol 1981;1:387e95. [5] Kiraz S, Ertenli I, Arici M, Calguneri M, Haznedaroglu I, Celik I, et al. Effects of colchicine on inflammatory cytokines and selectins in familial Mediterranean fever. Clin Exp Rheumatol 1998;16:721e4. [6] Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999;13:9e22. [7] Veikkola T, Alitalo K. VEGFs, receptors and angiogenesis. Semin Cancer Biol 1999;9:211e20. [8] McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH, Sharkey AM, et al. Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 1996;98:482e9. [9] Xiong M, Elson G, Legarda D, Leibovich SJ. Production of vascular endothelial growth factor by murine macrophages: regulation by hypoxia, lactate, and the inducible nitric oxide synthase pathway. Am J Pathol 1998;153:587e98. [10] Taichman NS, Young S, Cruchley AT, Taylor P, Paleolog E. Human neutrophils secrete vascular endothelial growth factor. J Leukocyte Biol 1997;62:397e400.
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