- Email: [email protected]
Role of vascular endothelial growth factor and monocyte chemoattractant protein-1 in Behçet's disease
Indian Journal of Rheumatology 2011 December Volume 6, Number 4; pp. 168–172
Original Article
Role of vascular endothelial growth factor and monocyte chemoattractant protein-1 in Behçet’s disease Soha ElDessouki Ibrahim1, Heba Fawzi Elshishtawy2, Amir HelmySamy3, Zeinab Ali Galal4
ABSTRACT Objectives: To study the role of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) in Behçet’s disease and their relation to clinical manifestations particularly vascular manifestations and disease activity. Methods: Forty Behçet’s disease patients (32 males, 8 females), diagnosed according to the criteria proposed by the International Study Group for Behçet’s disease were assessed clinically and serum VEGF and MCP-1 were measured by enzyme-linked immunosorbent assay. Results: Serum VEGF and MCP-1 levels were detected in patients and control groups with highly significant increase in Behçet’s group. A highly significant increase in the level of serum VEGF and MCP-1 was found in patients with vascular manifestations compared to those without vascular complications. A significant increase in the level of VEGF and MCP-1 was found in patients with active disease compared to inactive. Conclusion: Our findings suggest that serum VEGF and MCP-1 levels are elevated in active Behçet’s patients and in patients with vascular complications. So, we conclude that VEGF and MCP-1 play a crucial role in the pathogenesis of vascular complications of Behçet’s disease. Therefore, the serum levels of these cytokines serve to predict a possible acute thrombosis in Behçet’s disease. Keywords: Beçhet, VEGF, MCP-1
INTRODUCTION Behçet’s disease is a refractory multisystem disorder of recurrent acute inflammation characterised by major symptoms as genital ulcers, aphthous ulcers, and uveitis.¹ It is a systemic vasculitis that can affect joints, blood vessels, the central nervous system and the gastrointestinal tract.2 The aetiology of Behçet’s disease is presumed to be multifactorial, implicating immune system dysregulation, cellular and humoural immune defects, genetic pre-disposition, and endothelial cell dysfunction.3 The recruitment of macrophages and monocytes to the sites of inflammation suggests that chemokines might be involved in the development of Behçet’s disease.4 Different sized vessel lesions such as small, large venous or arterial lesion can be involved. Venous lesions are characteristic manifestation of the disease. The most common 1
patterns of vascular involvement are deep or superficial venous thrombosis of the extremities.5 Angiogenesis is defined by the growth of new capillary from pre-existing vasculature. However, the most potent stimulant of angiogenesis is tissue inflammation or hypoxia. Vascular endothelial growth factor (VEGF) is a potent angiogenic cytokine that stimulates functional changes in endothelial cells including increased vascular permeability to fluids and plasma proteins, endothelial migration, Von Willibrand factor release and enhanced procoagulant activity.6 Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine released by lymphocytes, monocytes, mast cells and eosinophils during inflammation.7 It is a strong chemoattractant protein for monocytes and binds to the monocytes via MCP-1 cromokine receptor-2 (CCR-2) receptor. Monocyte chemoattractant protein-1 actively participates in the process of arteriogenesis in which pre-existing collateral
Lecturer, 2Assistant Professor, Department of Rheumatology, 3Lecturer, Department of Internal Medicine, 4Lecturer, Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt. Correspondence: Dr. Soha ElDessouki Ibrahim, email: [email protected]
doi: 10.1016/S0973-3698(11)60202-1
Role of VEGF and MCP-1 in Behçet’s disease
arterioles turn into functional collateral arteries and as well as in organisation and resolution of venous thrombi.8 In view of the above functions of VEGF and MCP-1, we considered these cytokines as targets for investigations in Behçet’s disease. Different studies were done on the role of VEGF and MCP-1 in Behçet’s disease.9 Bozoglu et al. detected increased levels of VEGF and MCP-1 in Behçet’s disease thrombosis suggesting the possible role of those angiogenic cytokines in the pathogenesis of the disease.10 Therefore, it is interesting to study the role of these cytokines in the Egyptian population. The aim of this study was to determine the serum levels of VEGF and MCP-1 in Behçet’s disease to assess their role in the pathogenesis of the disease. The relation between these cytokines and clinical manifestations particularly vascular manifestations and disease activity were also studied.
Original Article
169
of venous thrombosis in the last 15 days prior to examination were considered to be acute venous thrombosis. 5. Assessment of disease activity was done at the time of blood sampling. Patients with at least two of the following were considered to have the active disease: aphthous stomatitis, genital ulceration, pathergy test positivity, skin lesions, anterior iridocyclitis or panuveitis or posterior vasculitis, arthritis, vascular lesions, pulmonary, and central nervous system manifestations.9
Measurement of vascular endothelial growth factor serum levels The blood samples were collected from all study groups. Serum VEGF levels were measured once by enzyme-linked immunosorbent assay (ELISA), using the commercial VEGF-A biolisa kits (Diaclone Research, France).
MATERIALS AND METHODS Forty Behçet’s disease patients were included as the study group. The diagnosis of Behçet’s disease was confirmed according to the criteria proposed by the International Study Group for Behçet’s disease.11 Patients were recruited from the outpatient clinic of Rheumatology and Rehabilitation and Internal Medicine Department of Ain Shams University. Informed consent was obtained from all participants to be included in the study. The patients group were 32 males (80%) and 8 females (20%). Their age ranged from 28 to 52 years with a mean of 40.35 ± 7.34 years. Forty age and sex matched healthy subjects were included as a control group. They were 31 males (77.5%) and 9 females (22.5%). Their age ranged from 28 to 51 years with a mean of 37.3 ± 7.06 years. Exclusion criteria for study and control groups included hyperlipidaemia, diabetes, hypertension, renal impairment, hepatic affection, malignancy, and thyroid diseases. The study and control groups were subjected to: 1. Full history taking. 2. Thorough clinical examination. 3. Laboratory investigations included erythrocyte sedimentation rate (ESR) by Westergren method and complete blood picture by coulter method T660. 4. Radiological investigation. Duplex imaging studies were performed with a Logiq-500 MD (GE Yokogawa Medical scanner equipped with 6–9 MHz wideband linear array transducer with colour flow mapping capability at the Radio Diagnosis Unit, Ain Shams University Hospital. All vessels were examined in the transverse and longitudinal views. Patients who developed symptoms
Measurement of monocyte chemoattractant protein-1 serum levels Monocyte chemoattractant protein-1 levels were measured once in the serum of all Behçet’s disease patients and controls by the use of human MCP-1 ELISA kit (Biosource Europe SA).
Statistical analysis Analysis of data was done by IBM computer using SPSS (statistical programme for social science version 12). Variables were expressed as mean ± standard deviation (SD). Student’s t test was used to compare the two quantitative variables with each other. Pearson correlation co-efficient (r) was used to test the correlation between the two quantitative variables. In all tests, if P value was < 0.01, it is considered as highly significant, if P value was < 0.05, it is considered significant and if P value was > 0.05, it is considered not significant.
RESULTS The disease duration for Behçet’s patients ranged from 1 to 9 years with a mean of 4.55 ± 2.5 years. The ESR level of the patients group ranged from 21 to 56 mm/hr with a mean of 37.1 ± 11.38 mm/hr. Twenty patients were in the active stage of the disease (50%), and the remaining were
170
Indian Journal of Rheumatology 2011 December; Vol. 6, No. 4
inactive (50%). Recurrent oral ulcers were detected in 32 patients (90%), genital ulcers in 12 patients (30%), skin lesions in 20 (50%), positive pathergy test in 16 (40%), articular symptoms in 14 (35%), ocular lesions in 18 (45%) in the form of uveitis in 16 patients (40%) and retinal vasculitis in 2 patient (5%), central nervous system (CNS) in 2 (5%) and vascular lesions in 12 (30%) in the form of venous thrombosis in 10 patients (25%) and arterial thrombosis in 2 patients (5%). Patients with venous thrombosis were further classified into acute venous thrombosis (2 patients, 5%) and chronic venous thrombosis (8 patients, 20%) (Figure1). Vascular endothelial growth factor serum level was detectable in all patients with Behçet’s disease and controls, with a statistically significant difference in the serum levels of VEGF in Behçet’s patients compared to the controls
Figure 1 Duplex ultrasound for acute deep venous thrombosis in left popliteal vein.
Ibrahim et al.
as P < 0.01 (mean ± SD: 249 ± 87.126 for the patients vs. mean ± SD: 56.6 ± 5.84 for the control groups). There was a statistically significant difference in the levels of VEGF in patients with vascular manifestations and ocular affection compared to those without as P < 0.01 (Table 1). There was a statistically significant difference in the serum levels of VEGF among patients with genital ulcers, skin manifestations, articular symptoms and positive pathergy test in comparison to patients without these manifestations as P < 0.05. With regard to disease activity, there was a statistically significant difference between active and inactive patients P < 0.05 (Table 1). Correlation between serum levels of VEGF and different clinical and laboratory parameters showed that there was a statistically significant positive correlation between serum levels of VEGF and ESR (r = 0.582, P < 0.05). Monocyte chemoattractant protein-1 was detected in all Behçet’s patients and controls with a statistically significant difference in the serum levels of MCP-1 in patients compared to control groups as P < 0.01 (mean ± SD: 130.5 ± 40.9 for the patients vs. mean ± SD: 51.73 ± 7.49 for the control groups). There was a statistically significant difference in the serum levels of MCP-1 in patients with vascular lesions in comparison to those without vascular manifestations (P < 0.01). There was a statistically significant difference in the serum levels of MCP-1 in Behçet’s patients with ocular lesions, skin manifestations and genital ulcers compared to patients without (P < 0.05). Regarding the serum levels of MCP-1, a statistically significant difference was present between active patients compared to inactive (P < 0.05) (Table 2). A statistically significant positive correlation was found between serum levels of VEGF and MCP-1 (r = 0.835, P < 0.01).
Table 1 Comparison of serum levels of vascular endothelial growth factor between active patients with vascular manifestations and those without Variables Vascular manifestations Disease activity
No. of patients with
Mean ± SD of VEGF levels in patients with
No. of patients without
Mean ± SD of VEGF levels in patients without
t value
P value
12 20
347.5 ± 51.5 299.5 ± 80.6
28 20
206.78 ± 60.6 198.5 ± 62.09
4.951 3.138
< 0.01 < 0.05
SD = standard deviation; VEGF = vascular endothelial growth factor.
Table 2 Comparison of serum levels of monocyte chemoattractant protein-1 between active patients with vascular manifestations and those without Variables Vascular manifestations Disease activity
No. of patients with
Mean ± SD of MCP-1 levels in patients with
No. of patients without
Mean ± SD of MCP-1 levels in patients without
t value
P value
12 20
181.66 ± 14.7 153 ± 41.3
28 20
107.5 ± 26.5 106.5 ± 28.1
6.379 2.94
< 0.01 < 0.05
SD = standard deviation; MCP-1 = monocyte chemoattractant protein-1.
Role of VEGF and MCP-1 in Behçet’s disease
DISCUSSION We found in our study a significant increase in serum VEGF levels in Behçet’s disease compared to controls. Our results came in agreement with other authors who reported the same findings in their studies.12,13 Endothelial dysfunction is thought to play a central role in the development of Behçet’s disease. It is one of the major factors responsible for increased frequency of thrombogenesis in Behçet’s disease.3 Systemic vascular manifestations include deep vein and arterial thrombosis, superficial thrombophlebitis and arterial aneurysm formation.14 Approximately 32–35% of all Behçet’s patients develop thrombotic lesions mostly in the venous system.15 In our study, we conclude significant increase in the serum levels of VEGF and MCP-1 in active Behçet’s patients with vascular complications. Vascular endothelial growth factor has been shown to be a very potent stimulant for angiogenesis and has been implicated in the pathology of autoimmune and inflammatory disease.16 Vascular endothelial growth factor is a cytokine involved in inflammation with a powerful effect on endothelial cells. Numerous cytokines, including interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)-α were implicated in the regulation of VEGF expression.17 A number of studies demonstrated increased levels of these cytokines in the serum of Behçet’s patients which may explain the increased VEGF levels found in those patients.18 As regards vascular manifestations, there was a statistically significant increase in VEGF levels in patients with vascular manifestations compared to those without. This came in accordance with the study of Bozoglu et al. who found that Behçet’s patients with acute or chronic thrombosis had significantly higher VEGF levels than the patients with mucocutaneous involvement.10 Moreover, Shaker et al. correlated the levels of VEGF with the presence of vascular manifestations.13 Vascular endothelial growth factor has a direct effect on endothelial cells and it is produced by cells participating in the pathophysiology of Behçet’s syndrome, such as neutrophils, macrophages and endothelial cells and can alter vessel permeability.12 Megakaryocytes and platelets contain VEGF in their cytoplasm and it is secreted by aggregating platelets. Together with the fact that platelets aggregation occurs predominantly at sites of endothelial injury, these data explain the elevated VEGF levels in patients with thrombosis.19 Vascular endothelial growth factor production upregulates nitric oxide (NO) synthase expression in endothelial cells and increases endothelial release of NO. Nitric oxide synthase could damage host cells and tissues either directly and/or following reaction with free radicals. So, it plays a critical role in the development of thrombotic events in Behçet’s disease.20
Original Article
171
In the current study, we found a significant increase in serum VEGF levels in patients with ocular manifestations compared to those without ocular affection. These results came in agreement with Shaker et al. and Ozdamar et al. who demonstrated high levels of VEGF with ocular involvement in Behçet’s disease.13,21 Moreover, Kamoun et al. reported a positive association between VEGF bp I/D gene polymorphism and ocular involvement in Behçet’s disease.22 In our study, we found a statistically significant increase in VEGF levels in active Behçet’s patients compared to inactive group. These results came in agreement with the results of Cekmen et al. who deduce the same comparison.12 Shaker et al. found higher VEGF levels in active than inactive Behçet’s disease but the results were not statistically significant.13 The difference between our results and those of Shaker et al. may be due to the difference in methods of assessing disease activity.13 In other study, both clinical and laboratory findings (ESR, neutrophil count) were used to classify the patients as having active or inactive disease. However in our work, we assessed the disease activity clinically only. A significant positive correlation between VEGF levels in Behçet’s disease and marker of inflammation (ESR) had been reported in our study, but the results did not agree with the observation of Erdem et al. who found no correlation between VEGF levels and ESR in Behçet’s disease.9 Monocyte chemoattractant protein-1 is a potent chemokine released by lymphocytes, monocytes, mast cells and eosinophils during inflammation.7 The MCP-1 plays an important role in the recruitment of leucocytes to the site of inflammation. Our results showed significant difference in serum MCP-1 levels in Behçet’s disease compared to healthy controls. These results came in agreement with the study of Cho et al. who found increased MCP-1 concentrations in serum of Behçet’s patients.23 Bozoglu et al. supported these results as they found significantly raised serum MCP-1 levels compared with control group.10 In our study, we found significant increase in MCP-1 levels in Behçet’s disease with vascular manifestations. The results of Bozoglu et al. supported the same findings with significantly higher concentrations of MCP-1 in patients with acute thrombosis than chronic.10 Monocyte chemoattractant protein-1 is known to contribute to the organisation and resolution of venous thrombi by attracting monocytes.8 Endothelial cells are the major source of endogenous MCP-1 and these cells have been shown to express messenger ribonucleic acid for MCP-1 after stimulation by pro inflammatory cytokines including IL-1, TNF-α. Furthermore VEGF has been shown to induce the expression of MCP-1 which may act in concert with MCP-1 in the process of recanalisation and angiogenesis in the study done by Yamada et al.24
172
Indian Journal of Rheumatology 2011 December; Vol. 6, No. 4
These findings came in agreement with our results as we found significant positive correlation between serum VEGF levels and MCP-1 levels. However, Bozoglu et al.10 found no correlation between the serum levels of VEGF and MCP-1. We found significant increase in MCP-1 levels in active patients compared to inactive. However, Bozoglu et al. found no correlation between MCP-1 and ESR. The difference in their results may be due to the difference in methods of assessing disease activity.10 From this study, we can conclude that VEGF and MCP-1 play a crucial role in the pathogenesis of vascular complications of Behçet’s disease. So, these cytokines serve to predict a possible acute thrombosis in Behçet’s disease.
REFERENCES 1.
Suzuki KM, Suzuki N. Behçet’s disease. Clin Exp Med 2004; 4: 10–20. 2. Yang P, Fang W, Meng Q, Ren Y, Xing L, Kijlstra A. Clinical features of Chinese patients with Behçet’s disease. Ophthalmology 2008; 115: 312–8, e314. 3. Sakane T, Takeno M, Suzuki N, Inaba G. Behçet’s disease. N Engl J Med 1999; 341: 1284–91. 4. Hou S, Yang P, Du L, Jiang Z, Mao L, Shu QL, et al. Monocyte chemoattractant protein-1-2518 A/G single nucleotide polymorphism in Chinese Han patients with ocular Behçet’s disease. Human Immunol 2010; 71: 79–82. 5. Houman MH, Ben Ghorbeli I, Khiari Ben Salah I, Lamloum M, Ben Ahmed M, Milled M. Deep venous thrombosis in Behçet’s disease. Clin Exp Rheumatol 2001; 19: 548–50. 6. Drouart M, Saas P, Billot M, Cedoz JP, Tiberghien P, Wendling D, et al. High serum vascular endothelial growth factor correlates with disease activity of spondyloarthropathies. Clin Exp Immunol 2003; 132: 158–62. 7. Daly C, Rollins BJ. Monocytes chemoattractant protein-1 (CCL2) in inflammatory disease and adaptive immunity: therapeutic opportunities and controversies. Microcirculation 2003; 10: 247–57. 8. Van Royen N, Piek JJ, Buschmann I, Hoefer I, Voskuli W, Schaper W. Stimulation of arteriogenesis: a new concept for the treatment of arterial occlusive disease. Cardiovasc Res 2001; 49: 543–53. 9. Erdem F, Gundogdu M, Kiki O, Sari RA, Kiziltunc A. Vascular endothelial and basic fibroblast growth factor serum levels in patients with Behçet’s disease. Rheumatol Int 2005; 24: 599–603. 10. Bozoglu E, Dinc A, Erdem H, Pay S, Simsek I, Kocar IH. Vascular endothelial growth factor and monocyte chemoattractant
11. 12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Ibrahim et al.
protein-1 in Behçet’s patients with venous thrombosis. Clin Exp Rheumatol 2005; 23(Suppl 38): 42–8. International Study Group for Behcet’s disease. Criteria for diagnosis of Behçet’s disease. Lancet 1990; 335: 1078–80. Cekmen M, Evereklioglu CE, 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 Behçet’s syndrome. Int J Dermtol 2003; 42: 870–5. Shaker O, Ay El-Deen MA, El Hadidi H, Grace BD, El Sherif H, Abdel Halim A. The role of heat shock protein 60, vascular endothelial growth factor and antiphospholipid antibodies in Behçet’s disease. Clin Lab Invest 2007; 156: 32–7. Kiraz S, Ertenli H, Ozturk MA, Haznedaroglu IC, Celic I, Calguneri M. Pathological haemostasis and prothrombotic state in Behçet’s disease. Thromb Res 2002; 105: 125–35. Ames PR, Stever A, Pap AP, Denman AM. Thrombosis in Behçet’s disease: a retrospective survey from a single UK centre. Rheumatology (Oxford) 2001; 40: 652–5. Roscoe WA, Welsh ME, Carter DE, Karlik SJ. VEGF and angiogenesis in acute and chronic MOG (35–55) peptides induced EAE. J Neuro Immunol 2009; 209: 6–15. Klagsbrun M, D’amore PA. Vascular endothelial growth factor and its receptors. Cytokine Growth Factor Rev 1996; 7: 259–70. Baggiolini M. Chemokines in Behçet’s disease, a field to be explored as a potential basis for therapy. Adv Exp Med Biol 2003; 528: 239–43. Meloney JP, Silliman CC, Ambruso DR, Wang J, Tuder RM, Voelkel NF. In vitro release of vascular endothelial during platelets aggregation. Am J Physiol 1998; 275: H1054–6. Sancak B, Onder M, Oztas MO, Bukan N, Gurer MA. Nitric oxide levels in Behçet’s disease. J Eur Acad Dermatol Venereal 2003; 17: 7–9. Ozdamar Y, Berker N, Bahar G, Soykan E, Bicer T, Ozkan SS, et al. Inflammatory mediators and posterior segment involvement in ocular Behçet disease. Eur J Ophthalmol 2009; 19: 998–1003. Kamoun M, Houman MH, Hamzaoui A, Hamzaoui K. Vascular endothelial growth factor gene polymorphism and serum levels in Behçet’s disease. Tissue Antigens 2008; 72: 581–7. Cho ML, Kim JY, Ko HJ, Kim YH, Kim WV, Cho CS, et al. The MCP-1 promoter-2518 polymorphism in Behçet’s disease: correlation between allele types, MCP-1 production and clinical symptoms among Korean patients. Autoimmunity 2004; 37: 77–80. Yamada M, Kim S, Egashira K, Takeya M, Ikeda T, Mimura O, et al. Molecular mechanism and role of endothelial monocyte chemoattractant protein-1 induction by vascular endothelial growth factor. Arterioscler Thromb Vasc Biol 2003; 23: 1996–2001.
Original Article
Role of vascular endothelial growth factor and monocyte chemoattractant protein-1 in Behçet’s disease Soha ElDessouki Ibrahim1, Heba Fawzi Elshishtawy2, Amir HelmySamy3, Zeinab Ali Galal4
ABSTRACT Objectives: To study the role of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) in Behçet’s disease and their relation to clinical manifestations particularly vascular manifestations and disease activity. Methods: Forty Behçet’s disease patients (32 males, 8 females), diagnosed according to the criteria proposed by the International Study Group for Behçet’s disease were assessed clinically and serum VEGF and MCP-1 were measured by enzyme-linked immunosorbent assay. Results: Serum VEGF and MCP-1 levels were detected in patients and control groups with highly significant increase in Behçet’s group. A highly significant increase in the level of serum VEGF and MCP-1 was found in patients with vascular manifestations compared to those without vascular complications. A significant increase in the level of VEGF and MCP-1 was found in patients with active disease compared to inactive. Conclusion: Our findings suggest that serum VEGF and MCP-1 levels are elevated in active Behçet’s patients and in patients with vascular complications. So, we conclude that VEGF and MCP-1 play a crucial role in the pathogenesis of vascular complications of Behçet’s disease. Therefore, the serum levels of these cytokines serve to predict a possible acute thrombosis in Behçet’s disease. Keywords: Beçhet, VEGF, MCP-1
INTRODUCTION Behçet’s disease is a refractory multisystem disorder of recurrent acute inflammation characterised by major symptoms as genital ulcers, aphthous ulcers, and uveitis.¹ It is a systemic vasculitis that can affect joints, blood vessels, the central nervous system and the gastrointestinal tract.2 The aetiology of Behçet’s disease is presumed to be multifactorial, implicating immune system dysregulation, cellular and humoural immune defects, genetic pre-disposition, and endothelial cell dysfunction.3 The recruitment of macrophages and monocytes to the sites of inflammation suggests that chemokines might be involved in the development of Behçet’s disease.4 Different sized vessel lesions such as small, large venous or arterial lesion can be involved. Venous lesions are characteristic manifestation of the disease. The most common 1
patterns of vascular involvement are deep or superficial venous thrombosis of the extremities.5 Angiogenesis is defined by the growth of new capillary from pre-existing vasculature. However, the most potent stimulant of angiogenesis is tissue inflammation or hypoxia. Vascular endothelial growth factor (VEGF) is a potent angiogenic cytokine that stimulates functional changes in endothelial cells including increased vascular permeability to fluids and plasma proteins, endothelial migration, Von Willibrand factor release and enhanced procoagulant activity.6 Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine released by lymphocytes, monocytes, mast cells and eosinophils during inflammation.7 It is a strong chemoattractant protein for monocytes and binds to the monocytes via MCP-1 cromokine receptor-2 (CCR-2) receptor. Monocyte chemoattractant protein-1 actively participates in the process of arteriogenesis in which pre-existing collateral
Lecturer, 2Assistant Professor, Department of Rheumatology, 3Lecturer, Department of Internal Medicine, 4Lecturer, Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt. Correspondence: Dr. Soha ElDessouki Ibrahim, email: [email protected]
doi: 10.1016/S0973-3698(11)60202-1
Role of VEGF and MCP-1 in Behçet’s disease
arterioles turn into functional collateral arteries and as well as in organisation and resolution of venous thrombi.8 In view of the above functions of VEGF and MCP-1, we considered these cytokines as targets for investigations in Behçet’s disease. Different studies were done on the role of VEGF and MCP-1 in Behçet’s disease.9 Bozoglu et al. detected increased levels of VEGF and MCP-1 in Behçet’s disease thrombosis suggesting the possible role of those angiogenic cytokines in the pathogenesis of the disease.10 Therefore, it is interesting to study the role of these cytokines in the Egyptian population. The aim of this study was to determine the serum levels of VEGF and MCP-1 in Behçet’s disease to assess their role in the pathogenesis of the disease. The relation between these cytokines and clinical manifestations particularly vascular manifestations and disease activity were also studied.
Original Article
169
of venous thrombosis in the last 15 days prior to examination were considered to be acute venous thrombosis. 5. Assessment of disease activity was done at the time of blood sampling. Patients with at least two of the following were considered to have the active disease: aphthous stomatitis, genital ulceration, pathergy test positivity, skin lesions, anterior iridocyclitis or panuveitis or posterior vasculitis, arthritis, vascular lesions, pulmonary, and central nervous system manifestations.9
Measurement of vascular endothelial growth factor serum levels The blood samples were collected from all study groups. Serum VEGF levels were measured once by enzyme-linked immunosorbent assay (ELISA), using the commercial VEGF-A biolisa kits (Diaclone Research, France).
MATERIALS AND METHODS Forty Behçet’s disease patients were included as the study group. The diagnosis of Behçet’s disease was confirmed according to the criteria proposed by the International Study Group for Behçet’s disease.11 Patients were recruited from the outpatient clinic of Rheumatology and Rehabilitation and Internal Medicine Department of Ain Shams University. Informed consent was obtained from all participants to be included in the study. The patients group were 32 males (80%) and 8 females (20%). Their age ranged from 28 to 52 years with a mean of 40.35 ± 7.34 years. Forty age and sex matched healthy subjects were included as a control group. They were 31 males (77.5%) and 9 females (22.5%). Their age ranged from 28 to 51 years with a mean of 37.3 ± 7.06 years. Exclusion criteria for study and control groups included hyperlipidaemia, diabetes, hypertension, renal impairment, hepatic affection, malignancy, and thyroid diseases. The study and control groups were subjected to: 1. Full history taking. 2. Thorough clinical examination. 3. Laboratory investigations included erythrocyte sedimentation rate (ESR) by Westergren method and complete blood picture by coulter method T660. 4. Radiological investigation. Duplex imaging studies were performed with a Logiq-500 MD (GE Yokogawa Medical scanner equipped with 6–9 MHz wideband linear array transducer with colour flow mapping capability at the Radio Diagnosis Unit, Ain Shams University Hospital. All vessels were examined in the transverse and longitudinal views. Patients who developed symptoms
Measurement of monocyte chemoattractant protein-1 serum levels Monocyte chemoattractant protein-1 levels were measured once in the serum of all Behçet’s disease patients and controls by the use of human MCP-1 ELISA kit (Biosource Europe SA).
Statistical analysis Analysis of data was done by IBM computer using SPSS (statistical programme for social science version 12). Variables were expressed as mean ± standard deviation (SD). Student’s t test was used to compare the two quantitative variables with each other. Pearson correlation co-efficient (r) was used to test the correlation between the two quantitative variables. In all tests, if P value was < 0.01, it is considered as highly significant, if P value was < 0.05, it is considered significant and if P value was > 0.05, it is considered not significant.
RESULTS The disease duration for Behçet’s patients ranged from 1 to 9 years with a mean of 4.55 ± 2.5 years. The ESR level of the patients group ranged from 21 to 56 mm/hr with a mean of 37.1 ± 11.38 mm/hr. Twenty patients were in the active stage of the disease (50%), and the remaining were
170
Indian Journal of Rheumatology 2011 December; Vol. 6, No. 4
inactive (50%). Recurrent oral ulcers were detected in 32 patients (90%), genital ulcers in 12 patients (30%), skin lesions in 20 (50%), positive pathergy test in 16 (40%), articular symptoms in 14 (35%), ocular lesions in 18 (45%) in the form of uveitis in 16 patients (40%) and retinal vasculitis in 2 patient (5%), central nervous system (CNS) in 2 (5%) and vascular lesions in 12 (30%) in the form of venous thrombosis in 10 patients (25%) and arterial thrombosis in 2 patients (5%). Patients with venous thrombosis were further classified into acute venous thrombosis (2 patients, 5%) and chronic venous thrombosis (8 patients, 20%) (Figure1). Vascular endothelial growth factor serum level was detectable in all patients with Behçet’s disease and controls, with a statistically significant difference in the serum levels of VEGF in Behçet’s patients compared to the controls
Figure 1 Duplex ultrasound for acute deep venous thrombosis in left popliteal vein.
Ibrahim et al.
as P < 0.01 (mean ± SD: 249 ± 87.126 for the patients vs. mean ± SD: 56.6 ± 5.84 for the control groups). There was a statistically significant difference in the levels of VEGF in patients with vascular manifestations and ocular affection compared to those without as P < 0.01 (Table 1). There was a statistically significant difference in the serum levels of VEGF among patients with genital ulcers, skin manifestations, articular symptoms and positive pathergy test in comparison to patients without these manifestations as P < 0.05. With regard to disease activity, there was a statistically significant difference between active and inactive patients P < 0.05 (Table 1). Correlation between serum levels of VEGF and different clinical and laboratory parameters showed that there was a statistically significant positive correlation between serum levels of VEGF and ESR (r = 0.582, P < 0.05). Monocyte chemoattractant protein-1 was detected in all Behçet’s patients and controls with a statistically significant difference in the serum levels of MCP-1 in patients compared to control groups as P < 0.01 (mean ± SD: 130.5 ± 40.9 for the patients vs. mean ± SD: 51.73 ± 7.49 for the control groups). There was a statistically significant difference in the serum levels of MCP-1 in patients with vascular lesions in comparison to those without vascular manifestations (P < 0.01). There was a statistically significant difference in the serum levels of MCP-1 in Behçet’s patients with ocular lesions, skin manifestations and genital ulcers compared to patients without (P < 0.05). Regarding the serum levels of MCP-1, a statistically significant difference was present between active patients compared to inactive (P < 0.05) (Table 2). A statistically significant positive correlation was found between serum levels of VEGF and MCP-1 (r = 0.835, P < 0.01).
Table 1 Comparison of serum levels of vascular endothelial growth factor between active patients with vascular manifestations and those without Variables Vascular manifestations Disease activity
No. of patients with
Mean ± SD of VEGF levels in patients with
No. of patients without
Mean ± SD of VEGF levels in patients without
t value
P value
12 20
347.5 ± 51.5 299.5 ± 80.6
28 20
206.78 ± 60.6 198.5 ± 62.09
4.951 3.138
< 0.01 < 0.05
SD = standard deviation; VEGF = vascular endothelial growth factor.
Table 2 Comparison of serum levels of monocyte chemoattractant protein-1 between active patients with vascular manifestations and those without Variables Vascular manifestations Disease activity
No. of patients with
Mean ± SD of MCP-1 levels in patients with
No. of patients without
Mean ± SD of MCP-1 levels in patients without
t value
P value
12 20
181.66 ± 14.7 153 ± 41.3
28 20
107.5 ± 26.5 106.5 ± 28.1
6.379 2.94
< 0.01 < 0.05
SD = standard deviation; MCP-1 = monocyte chemoattractant protein-1.
Role of VEGF and MCP-1 in Behçet’s disease
DISCUSSION We found in our study a significant increase in serum VEGF levels in Behçet’s disease compared to controls. Our results came in agreement with other authors who reported the same findings in their studies.12,13 Endothelial dysfunction is thought to play a central role in the development of Behçet’s disease. It is one of the major factors responsible for increased frequency of thrombogenesis in Behçet’s disease.3 Systemic vascular manifestations include deep vein and arterial thrombosis, superficial thrombophlebitis and arterial aneurysm formation.14 Approximately 32–35% of all Behçet’s patients develop thrombotic lesions mostly in the venous system.15 In our study, we conclude significant increase in the serum levels of VEGF and MCP-1 in active Behçet’s patients with vascular complications. Vascular endothelial growth factor has been shown to be a very potent stimulant for angiogenesis and has been implicated in the pathology of autoimmune and inflammatory disease.16 Vascular endothelial growth factor is a cytokine involved in inflammation with a powerful effect on endothelial cells. Numerous cytokines, including interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)-α were implicated in the regulation of VEGF expression.17 A number of studies demonstrated increased levels of these cytokines in the serum of Behçet’s patients which may explain the increased VEGF levels found in those patients.18 As regards vascular manifestations, there was a statistically significant increase in VEGF levels in patients with vascular manifestations compared to those without. This came in accordance with the study of Bozoglu et al. who found that Behçet’s patients with acute or chronic thrombosis had significantly higher VEGF levels than the patients with mucocutaneous involvement.10 Moreover, Shaker et al. correlated the levels of VEGF with the presence of vascular manifestations.13 Vascular endothelial growth factor has a direct effect on endothelial cells and it is produced by cells participating in the pathophysiology of Behçet’s syndrome, such as neutrophils, macrophages and endothelial cells and can alter vessel permeability.12 Megakaryocytes and platelets contain VEGF in their cytoplasm and it is secreted by aggregating platelets. Together with the fact that platelets aggregation occurs predominantly at sites of endothelial injury, these data explain the elevated VEGF levels in patients with thrombosis.19 Vascular endothelial growth factor production upregulates nitric oxide (NO) synthase expression in endothelial cells and increases endothelial release of NO. Nitric oxide synthase could damage host cells and tissues either directly and/or following reaction with free radicals. So, it plays a critical role in the development of thrombotic events in Behçet’s disease.20
Original Article
171
In the current study, we found a significant increase in serum VEGF levels in patients with ocular manifestations compared to those without ocular affection. These results came in agreement with Shaker et al. and Ozdamar et al. who demonstrated high levels of VEGF with ocular involvement in Behçet’s disease.13,21 Moreover, Kamoun et al. reported a positive association between VEGF bp I/D gene polymorphism and ocular involvement in Behçet’s disease.22 In our study, we found a statistically significant increase in VEGF levels in active Behçet’s patients compared to inactive group. These results came in agreement with the results of Cekmen et al. who deduce the same comparison.12 Shaker et al. found higher VEGF levels in active than inactive Behçet’s disease but the results were not statistically significant.13 The difference between our results and those of Shaker et al. may be due to the difference in methods of assessing disease activity.13 In other study, both clinical and laboratory findings (ESR, neutrophil count) were used to classify the patients as having active or inactive disease. However in our work, we assessed the disease activity clinically only. A significant positive correlation between VEGF levels in Behçet’s disease and marker of inflammation (ESR) had been reported in our study, but the results did not agree with the observation of Erdem et al. who found no correlation between VEGF levels and ESR in Behçet’s disease.9 Monocyte chemoattractant protein-1 is a potent chemokine released by lymphocytes, monocytes, mast cells and eosinophils during inflammation.7 The MCP-1 plays an important role in the recruitment of leucocytes to the site of inflammation. Our results showed significant difference in serum MCP-1 levels in Behçet’s disease compared to healthy controls. These results came in agreement with the study of Cho et al. who found increased MCP-1 concentrations in serum of Behçet’s patients.23 Bozoglu et al. supported these results as they found significantly raised serum MCP-1 levels compared with control group.10 In our study, we found significant increase in MCP-1 levels in Behçet’s disease with vascular manifestations. The results of Bozoglu et al. supported the same findings with significantly higher concentrations of MCP-1 in patients with acute thrombosis than chronic.10 Monocyte chemoattractant protein-1 is known to contribute to the organisation and resolution of venous thrombi by attracting monocytes.8 Endothelial cells are the major source of endogenous MCP-1 and these cells have been shown to express messenger ribonucleic acid for MCP-1 after stimulation by pro inflammatory cytokines including IL-1, TNF-α. Furthermore VEGF has been shown to induce the expression of MCP-1 which may act in concert with MCP-1 in the process of recanalisation and angiogenesis in the study done by Yamada et al.24
172
Indian Journal of Rheumatology 2011 December; Vol. 6, No. 4
These findings came in agreement with our results as we found significant positive correlation between serum VEGF levels and MCP-1 levels. However, Bozoglu et al.10 found no correlation between the serum levels of VEGF and MCP-1. We found significant increase in MCP-1 levels in active patients compared to inactive. However, Bozoglu et al. found no correlation between MCP-1 and ESR. The difference in their results may be due to the difference in methods of assessing disease activity.10 From this study, we can conclude that VEGF and MCP-1 play a crucial role in the pathogenesis of vascular complications of Behçet’s disease. So, these cytokines serve to predict a possible acute thrombosis in Behçet’s disease.
REFERENCES 1.
Suzuki KM, Suzuki N. Behçet’s disease. Clin Exp Med 2004; 4: 10–20. 2. Yang P, Fang W, Meng Q, Ren Y, Xing L, Kijlstra A. Clinical features of Chinese patients with Behçet’s disease. Ophthalmology 2008; 115: 312–8, e314. 3. Sakane T, Takeno M, Suzuki N, Inaba G. Behçet’s disease. N Engl J Med 1999; 341: 1284–91. 4. Hou S, Yang P, Du L, Jiang Z, Mao L, Shu QL, et al. Monocyte chemoattractant protein-1-2518 A/G single nucleotide polymorphism in Chinese Han patients with ocular Behçet’s disease. Human Immunol 2010; 71: 79–82. 5. Houman MH, Ben Ghorbeli I, Khiari Ben Salah I, Lamloum M, Ben Ahmed M, Milled M. Deep venous thrombosis in Behçet’s disease. Clin Exp Rheumatol 2001; 19: 548–50. 6. Drouart M, Saas P, Billot M, Cedoz JP, Tiberghien P, Wendling D, et al. High serum vascular endothelial growth factor correlates with disease activity of spondyloarthropathies. Clin Exp Immunol 2003; 132: 158–62. 7. Daly C, Rollins BJ. Monocytes chemoattractant protein-1 (CCL2) in inflammatory disease and adaptive immunity: therapeutic opportunities and controversies. Microcirculation 2003; 10: 247–57. 8. Van Royen N, Piek JJ, Buschmann I, Hoefer I, Voskuli W, Schaper W. Stimulation of arteriogenesis: a new concept for the treatment of arterial occlusive disease. Cardiovasc Res 2001; 49: 543–53. 9. Erdem F, Gundogdu M, Kiki O, Sari RA, Kiziltunc A. Vascular endothelial and basic fibroblast growth factor serum levels in patients with Behçet’s disease. Rheumatol Int 2005; 24: 599–603. 10. Bozoglu E, Dinc A, Erdem H, Pay S, Simsek I, Kocar IH. Vascular endothelial growth factor and monocyte chemoattractant
11. 12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Ibrahim et al.
protein-1 in Behçet’s patients with venous thrombosis. Clin Exp Rheumatol 2005; 23(Suppl 38): 42–8. International Study Group for Behcet’s disease. Criteria for diagnosis of Behçet’s disease. Lancet 1990; 335: 1078–80. Cekmen M, Evereklioglu CE, 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 Behçet’s syndrome. Int J Dermtol 2003; 42: 870–5. Shaker O, Ay El-Deen MA, El Hadidi H, Grace BD, El Sherif H, Abdel Halim A. The role of heat shock protein 60, vascular endothelial growth factor and antiphospholipid antibodies in Behçet’s disease. Clin Lab Invest 2007; 156: 32–7. Kiraz S, Ertenli H, Ozturk MA, Haznedaroglu IC, Celic I, Calguneri M. Pathological haemostasis and prothrombotic state in Behçet’s disease. Thromb Res 2002; 105: 125–35. Ames PR, Stever A, Pap AP, Denman AM. Thrombosis in Behçet’s disease: a retrospective survey from a single UK centre. Rheumatology (Oxford) 2001; 40: 652–5. Roscoe WA, Welsh ME, Carter DE, Karlik SJ. VEGF and angiogenesis in acute and chronic MOG (35–55) peptides induced EAE. J Neuro Immunol 2009; 209: 6–15. Klagsbrun M, D’amore PA. Vascular endothelial growth factor and its receptors. Cytokine Growth Factor Rev 1996; 7: 259–70. Baggiolini M. Chemokines in Behçet’s disease, a field to be explored as a potential basis for therapy. Adv Exp Med Biol 2003; 528: 239–43. Meloney JP, Silliman CC, Ambruso DR, Wang J, Tuder RM, Voelkel NF. In vitro release of vascular endothelial during platelets aggregation. Am J Physiol 1998; 275: H1054–6. Sancak B, Onder M, Oztas MO, Bukan N, Gurer MA. Nitric oxide levels in Behçet’s disease. J Eur Acad Dermatol Venereal 2003; 17: 7–9. Ozdamar Y, Berker N, Bahar G, Soykan E, Bicer T, Ozkan SS, et al. Inflammatory mediators and posterior segment involvement in ocular Behçet disease. Eur J Ophthalmol 2009; 19: 998–1003. Kamoun M, Houman MH, Hamzaoui A, Hamzaoui K. Vascular endothelial growth factor gene polymorphism and serum levels in Behçet’s disease. Tissue Antigens 2008; 72: 581–7. Cho ML, Kim JY, Ko HJ, Kim YH, Kim WV, Cho CS, et al. The MCP-1 promoter-2518 polymorphism in Behçet’s disease: correlation between allele types, MCP-1 production and clinical symptoms among Korean patients. Autoimmunity 2004; 37: 77–80. Yamada M, Kim S, Egashira K, Takeya M, Ikeda T, Mimura O, et al. Molecular mechanism and role of endothelial monocyte chemoattractant protein-1 induction by vascular endothelial growth factor. Arterioscler Thromb Vasc Biol 2003; 23: 1996–2001.