Heart Vessels DOI 10.1007/s00380-015-0686-9
ORIGINAL ARTICLE
Clinical significance of Angiopoietin‑1 in Behcet’s disease patients with vascular involvement Iman H. Bassyouni1 · Mohammed Sharaf1 · Iman E. Wali2 · Hossam M. Mansour3
Received: 1 November 2014 / Accepted: 1 May 2015 © Springer Japan 2015
Abstract Behcet’s disease (BD) is a chronic multisystem inflammatory disorder of unclear etiology. Vascular inflammation, endothelial dysfunction and angiogenesis may be in part responsible for the pathogenesis of BD. Angiopoietin-1 (Ang-1) is a recent angiogenic mediator. The aim of the present study was to assess Ang-1 in the plasma of BD patients as well as to analyze its association with clinical, and laboratory parameters of the disease. The present study included 47 BD patients and 30 age- and gendermatched healthy controls. Demographic, clinical, disease activity and severity were prospectively assessed. Plasma Ang-1 levels were measured using enzyme-linked immunosorbent assay. The plasma level of Ang-1 in BD patients was significantly lower than healthy controls (p = 0.005). Plasma Ang-1 level in patients with vascular affection was significantly lower than those without vascular affection (p = 0.045). Levels of Ang-1 showed a significant positive correlation with steroid dose. Patients who received cyclophosphamide or steroids showed a significant increase in plasma Ang-1 level. This was further confirmed by the results of the multivariate analysis. There was no significant association between plasma Ang-1 levels and other clinical manifestations or disease activity and severity. Plasma Ang-1 levels were diminished in our BD patients especially * Iman H. Bassyouni [email protected]; [email protected] 1
Department of Rheumatology and Rehabilitation, Faculty of Medicine, El‑Kasr El‑Aini Hospital, Cairo University, Cairo 12613, Egypt
2
Medical Microbiology and Immunology, Faculty of Medicine, Cairo University, Giza, Egypt
3
Cardiovascular Department, Faculty of Medicine, Six of October University, 6th of October, Egypt
in patients with vascular involvement. Larger studies with further investigations of the precise role of Ang-1 in the pathogenesis of BD are needed and might lead to novel therapies for the clinical management of BD Keywords Angiogenesis · Angiopoietin · Behcet’s disease · Vasculitis
Introduction Behcet’s disease (BD) is a multisystem vasculitic disorder of unknown etiology, characterized by relapsing episodes of oral aphthous ulcers, genital ulcers, skin lesions and ocular lesions. Vascular involvement is a common complication of BD which worsens the prognosis of BD. The cause of BD is unknown [1]. It is believed to be due to an autoimmune process triggered by an environmental or infectious agent in a genetically predisposed subject. Interaction of primed neutrophils with endothelial cells is considered a crucial event in the pathogenesis of severe endothelial lesions in BD vasculitis [2]. In this regard, the interaction between neutrophils and endothelial cells has been the subject of much study. Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are antagonistic ligands that bind to the extracellular domain of the Tie-2 receptor, which is expressed by endothelial cells [3]. The Ang–Tie ligand–receptor system was identified as the second class of vascular-specific receptor tyrosine kinases (RTKs). Angiopoietins and Tie-2 receptor control angiogenic remodeling in a context-dependent manner [4]. Tie signaling is involved in angiogenic remodeling process during development, including destabilization of existing vessels, endothelial cell migration, tube formation and stabilization of newly formed tubes by mesenchymal cells [5].
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Ang-1 has vasoprotective and anti-inflammatory actions, mediates vessel maturation and maintains vessel integrity by the recruitment of periendothelial cells. Thus, low-level constitutive Tie2 activation by Ang-1 may be required in the adult to maintain the mature quiescent and non-proliferating phenotype of the vascular endothelium. In turn, Ang-2 acts as a vessel-destabilizing cytokine, thereby playing an essential role in vascular remodeling and inflammatory process [4]. In line with these data, abnormality of the Ang–Tie ligand–receptor system has been detected in patients with autoimmune and vascular diseases [6–12]. Recently, a preliminary study reported elevated Ang1 in Korean BD Patients [13]. But with this exception, no information is available on the role of Ang/Tie system in BD patients. The aim of the present study was to assess Ang-1 concentrations as a recent angiogenic mediator in plasma of BD patients and to analyze its association with disease clinical features, laboratory parameters as well as disease activity and severity.
Patients and methods Patients Forty-seven patients with BD, diagnosed according to the International Study Group criteria [14], were recruited (mean age 33.81 ± 9.27 years) and 30 ageand gender-matched healthy control subjects (mean age 32.74 ± 8.73 years) were included in the study. Patients who had other autoimmune disease, malignancy or infection were excluded from the study. The detailed clinical characteristics of the patients were determined by combined medical history and physical examination. For venous and arterial thrombotic events, Doppler ultrasonography, angiography, MRI, CT, and echocardiography were done. Radiologic evaluations, including, CT scans of the lungs and the abdomen, cranial magnetic resonance imaging (MRI) were performed as clinically indicated. The activity and severity of the disease were assessed as previously described [15]. The total clinical activity index score of each patient represents the numerical sum of the manifestations present only at the time of blood sampling. BD clinical severity score was calculated as the sum of 1 point each for mild symptoms (oral aphthosis, genital ulcers, arthralgia and typical skin lesions such as folliculitis, erythema nodosum, and papulopustular lesions), 2 points each for moderate symptoms (anterior uveitis, arthritis, deep vein thrombosis of the legs, and gastrointestinal involvement) and 3 points each for severe disease manifestations (retinal vasculitis, posterior/panuveitis, arterial thrombosis,
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Heart Vessels
bowel perforation and neuro-Behçet’s). Routine laboratory investigations were collected from the patients’ records [15]. The study was approved by our institutional ethics committee and written consent was obtained from all patients. Plasma Angiopoietin‑1 levels determination Peripheral venous blood samples in EDTA containing tubes were obtained from patients and controls. After centrifugation at 1000g for 10 min, the plasma was frozen and stored at −70 °C until assayed on the same day to eliminate dayto-day interassay variability. A commercial ELISA kit (RayBiotech Inc., GA, USA) was used for assaying Ang-1 levels. This assay employed an antibody specific for human Ang-1 coated on a 96-well plate. In brief, each plasma sample was directly transferred to the wells of the ELISA plate and then assayed according to the manufacturer’s instructions. The absorbance was measured at 450 nm in a microtest plate spectrophotometer, and Ang-1 levels were quantified with a calibration curve using human Ang-1 as a standard. Both standards and samples were evaluated in duplicates and the interassay variations were shown to be within the range given by the manufacture. Statistical analysis The statistical package for social sciences (SPSS) version 15 (LEAD Technology Inc., Charlotle, NC, USA) was used to analyze data. Continuous variables were summarized through the median (range) or mean (SD) and categorical variables as frequency (percentage). Nonparametric Mann–Whitney U test compared two independent groups. Spearman’s rank correlation test was used as a measure of association of quantitative variables. Significant differences were calculated using Mann–Whitney U test for continuous variables. For those variables that were significantly influencing plasma Ang-1 levels (p 10 mg/day Cyclophosphamide Azathioprine Colchicine Laboratory investigationsa ESR (mm/h) Hemoglobin (g/dl) Leucocyte (×103/µl) Neutrophils (×103/µl) Platelets (×103/µl)
BD patients (N = 47)
33.81 ± 9.27 7.46 ± 6.51 37/10 47 (100) 42 (89) 20 (42.6) 19 (40.4) 14 (29.8) 34 (72.3) 6 (12.8) 9 (19.1) 30 (63.8) 10 (21.2) 23 (49) 18 (38.3) 18.49 ± 16.87 13.56 ± 1.41 10.45 ± 3.81 56.87 ± 8.71 287.49 ± 86.42
a
Continuous variables: mean ± SD
b
Categorical variables: number (%)
pseudofolliculitis- and erythema nodosum-like lesions (42 %) were the most common clinical features reported by this group of patients. Previous neurological involvement was detected in 6 (13 %) of these patients which manifested as headache, aseptic meningitis, facial paralysis, intracranial hypertension, and seizures. Vascular events were reported in 9 patients, deep venous thrombosis was notified by 7 of patients, 5 with superficial thrombophlebitis as well, arterial thrombosis was previously detected also in 1 patients and 1 had arterial aneurysms. At the time of blood sampling, the mean BD activity score was 2.65 ± 2.00 (range 0–6), while the total BD severity score was 7.39 ± 2.41 (range 3–13). Plasma Ang‑1 levels and BD clinical and laboratory parameters The median plasma Ang-1 concentrations were reduced in patients with BD compared to healthy controls (78.12; range 24.71–151.66, 84 and 97.74; range
Fig. 1 Plasma Ang-1 levels in: a BD patients and Healthy controls. b BD patients with and without vascular involvement. The lines inside the boxes indicate the median; the outer borders of the boxes indicate 25th and 75th percentiles; the bars extending from the boxes indicate the 10th and 90th percentiles
59.27–140.1 ng/ml, respectively, p = 0.005; Fig. 1a). As BD is a heterogeneous disease, further analysis was performed to compare the levels of plasma Ang-1 in BD patients with the presence and absence of some clinical manifestations and medications used at time of blood sampling (Table 2). We found that patients with vascular involvement had significant reduction of plasma Ang-1 levels than those without (p = 0.045l; Fig. 1b). We did not find significant differences in median plasma Ang-1 levels in relation to any other specific organ involvement (p > 0.05). With respect to medications used, we found significant increase in median plasma Ang-1 levels in patients who received cyclophosphamide than those who did not receive it (p = 0.049). Similarly, Ang-1 levels were higher in patients receiving steroid dose >10 mg/day (p = 0.003). Furthermore, it correlated significantly with steroid dose (r = 0.354, p = 0.018).
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Heart Vessels
Table 2 The differences in median plasma Ang-1 concentration in relation to the presence or absence of different clinical features and the current medications used Clinical parameter/current medications
No (%)
Median Ang-1 levels ng/ml (min–max) Present
Absent
P value
Genital ulcers Pseudofolliculitis/EN Positive Pathergy test Articular involvement Ocular involvement Neurological involvement Vascular involvement Corticosteroids >10 mg/day Cyclophosphamide Azathioprine
42 (89) 20 (42.6) 19 (40.4) 14 (29.8) 34 (72.3) 6 (12.8) 9 (19.1) 30 (63.8) 10 (21.2) 23 (49)
25.64 (24.71–126.64) 68.23 (24.71–151.66) 82.53 (24.95–140.72) 69.62 (24.71–137.93) 79.10 (24.71–151.66) 77.65 (24.71–151.66) 55.54 (24.95–104.54) 86.49 (25.65–151.66) 100.40 (41.12–151.66) 69.62 (24.71–137.93)
78.12 (39.38–151.66) 84.98 (24.95–140.72) 69.62 (24.71–151.66) 91.09 (24.95–151.66) 74.63 (24.95–134.21) 95.22 (69.63–134.21) 82.53 (24.71–151.66) 55.54 (24.71–134.21) 69.62 (24.71–137.93) 85.16 (25.65–151.66)
0.15 0.32 0.58 0.14 0.48 0.147 0.045* 0.003* 0.049* 0.33
Colchicine
18 (38.3)
78.12 (24.95–151.66)
72.24 (24.71–140.72)
0.53
Data are median Ang-1 levels (min–max); Mann–Whitney U test was used to compare variables EN Erythema nodosum * Significant (p 0.05; Table 3). Since patients with BD usually do not present with only one symptom of the disease, same treatment or one risk factor, therefore, we decided to perform a multivariate analysis to estimate the independent association of each covariate with plasma Ang-1 as dependent variable against the variables that yielded p values 10 mg (β = 0.340, p = 0.013), and cyclophosphamide (β = 0.291, p = 0.30). Vascular involvement was found to be a negative predictor of Ang-1 levels (β = −0.274, p = 0.044).
Discussion Vascular inflammation, endothelial dysfunction and neovascularization may be in part responsible for the pathogenesis of BD [1, 2]. One of the systems involved in angiogenesis is Ang–Tie receptor system, it is a novel regulator involved in the maintenance of vascular integrity
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and vascular remodeling and is related to the survival and migration of endothelial cells [5]. We had found a significant reduction of the plasma concentration of Ang-1 in BD patients compared with controls especially in patients with vascular involvement. This implies that Ang-1 might have a significant role in BD [4]. Ang-1 is considered an endothelial survival factor and vasculoprotective agent. Therefore, Ang-1 insufficiency might lead to loss of endothelial integrity, vessel dilatation, increased permeability and promotes formation of perivascular inflammatory infiltrates [16] as observed in BD. On the other hand, Ang-2 is an angiostatic factor to Ang-1, its binding to Tie 2 results in rapid destabilization of the endothelium, suggesting that Ang-2 functions as an autocrine negative regulator of the quiescent resting endothelium [17]. In accordance to our results, Michalska-Jakubus et al. have demonstrated significantly decreased Ang-1 levels in patients with systemic sclerosis (SSc). This reduction in Ang-1 levels has been attributed to the development of SSc-related vasculopathy through promoting activation and apoptosis of ECs and destabilization of blood vessels [18]. Likewise, it is obvious that molecules that favor the stability of new blood vessels, such as Ang-1, are under expressed in inflammatory bowel disease patients, while, destabilizing molecules such as Ang-2 are upregulated in those patients [9]. Similarly, circulating Ang-2, but not Ang-1, has been elevated and closely correlates with disease activity and circulating endothelial cells numbers in anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitis with renal involvement and in systemic lupus erythematosus (SLE) patients [6, 7]. To the best of our knowledge, no reports concerning Ang-1 in BD have
Heart Vessels Table 3 Correlation between plasma Ang-1 levels with disease activity, severity and laboratory features of BD patients Parameter
Fig. 2 Scatter Plot demonstrating correlation of plasma Ang-1 Abs levels with BD disease activity (a), and Severity (b) Scores
been published so far except the study of Choe et al., in BD Korean population [13]. They have found increased levels of plasma Ang-1 in their studied BD patients compared to the healthy controls. The variability in the results regarding Ang-1 in BD might be attributed to the multi-systemic involvement characterizing this disease. We had reported in our study a significant association of Ang-1 with vascular involvement (present in 19 % of our patients). On the other hand, in Choe et al. study, only 2 BD patients had vascular involvement (3.4 % of their patients). Their Ang-1 levels were lower in patients with vascular involvement than those without although it was statistically insignificant probably due to the low number of patients (216.2 ± 15.8 vs 286.8 ± 102.1, p = 0.318). In accordance to our results, Ang-1 has been found to be diminished in several rheumatic and vascular diseases [6–9, 18–20]. The expression of angiogenic factors including Ang-1 has been insufficient in bone marrow of critical limb ischemia patients [12].
Plasma Ang-1 (ng/ml) r
P value
Hb (g/dl) WBCs (103/μL) Neutrophils (%) Lymphocytes (%) Platelets (103/μL) ESR (mm/h) ALT (U/L) AST (U/L) Creatinine (mg/dl) BD disease activity
0.07 0.2 −0.1 0.1 0.08 −0.07 0.04 −0.05 0.1 0.264
0.40 0.08 0.32 0.51 0.52 0.67 0.82 0.72 0.51 0.08
BD disease severity
0.155
0.297
Studies of animal models have confirmed the Ang-1 role within the vasculature. Acute administration of Ang1 significantly reduces vascular inflammation in mice [21]. On the other hand, Ang-2 has been found to trigger an inflammatory response by activating the endothelium and inducing vascular permeability [22]. Decreased activity of the Tie-2 pathway has contributed to right ventricular load in experimental hypoxia-induced pulmonary hypertension, and this effect can be antagonized by Ang-1 administration [23]. It is, therefore, tempting to speculate that Ang-1 deficiency might contribute to the development vascular affection in BD. Regarding disease activity, the results of the present study suggested that plasma Ang-1 levels do not reflect clinical or serologic disease activity. These results were in accordance with those obtained by Choe et al. [13]. These equivocal findings regarding the association between angiogenic factors and activity may be affected by the administration of therapeutic drugs with anti-inflammatory properties, but it is not understood whether these drugs have antiangiogenic potential. The management of a patient with BD is complex and oftentimes requires a multidisciplinary approach. Colchicine is used for mucocutaneous manifestations, non-steroidal anti-inflammatory drugs for joint manifestations, anticoagulation for vascular thrombosis, and cytotoxic drugs for major system involvement [1, 2]. In 2008, The European League Against Rheumatism (EULAR) guidelines have suggested that cyclophosphamide be used preferentially for life-threatening manifestations of BD, including vascular disease and nervous system involvement [24]. The use of cyclophosphamide and steroids in addition to anticoagulation was recommended by several authors for the management of BD arterial and venous thrombosis to prevent their recurrence [25, 26].
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Herein, we had detected a significant positive correlation of Ang-1 levels with the steroid dose and cyclophosphamide. In line with this, Kim et al. have demonstrated that dexamethazone increases the levels of Ang-1 in mouse bone marrow and the quiescence of hematopoietic stem cells [27]. Furthermore, a significant increase in mean plasma Ang-1 levels in patients who received cyclophosphamide was reported. In accordance, Zhen et al. have observed an increase in plasma Ang-1 levels following administration of cyclophosphamide in Neuroblastoma model with improvement of function of the vessels [28]. In line with this, serum Ang-2 levels have been found to decrease significantly after IV cyclophosphamide therapy compared with their initial levels in systemic sclerosis patients [29]. Ang-1 has received significant attention as a potential therapeutic molecule in diabetic retinopathy, allograft vasculopathy, sepsis/endotoxemia, malignancy and diabetesinduced complications [30]. Takahashi and coworkers have reported Ang-1 as a possible candidate proangiogenic factor to be used in the treatment of coronary artery disease [31]. Investigations into the role of Ang-1 may ultimately lead to the development of a potent therapeutic molecule for treating patients with vascular diseases [32]. This study had some limitations. First, this study was not aimed at elucidating pathophysiological pathways of Ang-1 regulation in BD, but was rather designed to evaluate its possible clinical significance. Second, because of its cross-sectional nature; we were unable to show Ang-1 level changes with respect to clinical course and medical treatment. In future prospective large-scale, longitudinal study, we will investigate Ang-1 levels according to relapse, additional patterns of organ involvement, and treatment regimens. This might lead to novel therapies for the clinical management of BD Conflict of interest There are no conflicts of interest related to the manuscript.
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Heart Vessels and Angiopoietin-1 exert opposing effects on cell junctions by regulating the Rho GEF Syx. J Cell Biol 199(7):1103–1115 6. Kümpers P, Hellpap J, David S, Horn R, Leitolf H, Haller H, Haubitz M (2009) Circulating angiopoietin-2 is a marker and potential mediator of endothelial cell detachment in ANCA-associated vasculitis with renal involvement. Nephrol Dial Transplant 24(6):1845–1850 7. Kümpers P, David S, Haubitz M, Hellpap J, Horn R, Bröcker V, Schiffer M, Haller H, Witte T (2009) The Tie2 receptor antagonist angiopoietin 2 facilitates vascular inflammation in systemic lupus erythematosus. Ann Rheum Dis 68(10):1638–1643 8. Karapinar H, Esen O, Emiroglu Y, Akcakoyun M, Pala S, Kargin R, Izgi A, Kirma C, Esen AM (2011) Serum levels of angiopoietin-1 in patients with pulmonary hypertension due to mitral stenosis. Heart Vessels 26(5):536–541 9. Oikonomou KA, Kapsoritakis AN, Kapsoritaki AI, Manolakis AC, Tiaka EK, Tsiopoulos FD, Tsiompanidis IA, Potamianos SP (2011) Angiogenin, angiopoietin-1, angiopoietin-2, and endostatin serum levels in inflammatory bowel disease. Inflamm Bowel Dis 17(4):963–970 10. Zhang BH, Guo CX, Wang HX, Lu LQ, Wang YJ, Zhang LK, Du FH, Zeng XJ (2014) Cardioprotective effects of adipokine apelin on myocardial infarction. Heart Vessels 29(5):679–689 11. Takahashi Y, Satoh M, Tabuchi T, Nakamura M (2012) Prospective, randomized, single-blind comparison of effects of 6 months’ treatment with atorvastatin versus pravastatin on leptin and angiogenic factors in patients with coronary artery disease. Heart Vessels 27(4):337–343 12. Oda M, Toba K, Kato K, Ozawa T, Yanagawa T, Ikarashi N, Takayama T, Suzuki T, Hanawa H, Masuko M, Kobayashi H, Aizawa Y (2012) Hypocellularity and insufficient expression of angiogenic factors in implanted autologous bone marrow in patients with chronic critical limb ischemia. Heart Vessels 27(1):38–45 13. Choe JY, Park SH, Kim SK (2010) Serum angiopoietin-1 level is increased in patients with Behcet’s disease. Bone Spine 77(4):340–344 14. International Study Group for Behcet’s disease (1990) Criteria for diagnosis of Behcet’s disease. Lancet 335:1078–1080 15. Harzallah O, Kerkeni A, Baati T, Mahjoub S (2008) Oxidative stress: correlation with Behçet’s disease duration, activity and severity. Eur J Intern Med 19(7):541–547 16. Hato T, Tabata M, Oike Y (2008) The role of angiopoietin-like proteins in angiogenesis and metabolism. Trends Cardiovasc Med 18(1):6–14 17. Pfaff D, Fiedler Y, Augustin H (2006) Emerging roles of the angiopoietin–Tie and the ephrin–Eph systems as regulators of cell trafficking. J Leukoc Biol 80:719–726 18. Michalska-Jakubus M, Kowal-Bielecka O, Chodorowska G, Bielecki M, Krasowska D (2011) Angiopoietins-1 and -2 are differentially expressed in the sera of patients with systemic sclerosis: high angiopoietin-2 levels are associated with greater severity and higher activity of the disease. Rheumatology (Oxford) 50(4):746–755 19. Lee JS, Song SH, Kim JM, Shin IS, Kim KL, Suh YL, Kim HZ, Koh GY, Byun J, Jeon ES, Suh W, Kim DK (2008) Angiopoietin-1 prevents hypertension and target organ damage through its interaction with endothelial Tie2 receptor. Cardiovasc Res 78:572–580 20. Lee KW, Lip GY, Blann AD (2004) Plasma angiopoietin-1, angiopoietin-2, angiopoietin receptor tie-2, and vascular endothelial growth factor levels in acute coronary syndromes. Circulation 110(16):2355–2360 21. Thurston G, Rudge JS, Ioffe E, Zhou H, Ross L, Croll SD, Glazer N, Holash J, McDonald DM, Yancopoulos GD (2000)
Heart Vessels Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med 6:460–463 22. Fiedler U, Krissl T, Koidl S, Weiss C, Koblizek T, Deutsch U, Martiny-Baron G, Marmé D, Augustin HG (2003) Angiopoietin-1 and angiopoietin-2 share the same binding domains in the Tie-2 receptor involving the first Ig-like loop and the epidermal growth factor-like repeats. J Biol Chem 278:1721–1727 23. Bae JW (2011) Hypertension, vascular rarefaction and angiopoietin-1. Korean Circ J 41(10):575–577 24. Hatemi G, Silman A, Bang D, Bodaghi B, Chamberlain AM, Gul A, Houman MH, Kötter I, Olivieri I, Salvarani C, Sfikakis PP, Siva A, Stanford MR, Stübiger N, Yurdakul S, Yazici H, Expert Committee EULAR (2008) EULAR recommendations for the management of Behcet disease. Ann Rheum Dis 67(12):1656–1662 25. Ahn JK, Lee YS, Jeon CH, Koh EM, Cha HS (2008) Treatment of venous thrombosis associated with Behcet’s disease: immunosuppressive therapy alone versus immunosuppressive therapy plus anticoagulation. Clin Rheumatol 27(2):201–205 26. Desbois AC, Wechsler B, Resche-Rigon M, Piette JC, Huong Dle T, Amoura Z, Koskas F, Desseaux K, Cacoub P, Saadoun D (2012) Immunosuppressants reduce venous thrombosis relapse in Behcet’s disease. Arthritis Rheum 64(8):2753–2760 27. Kim H, Choi JY, Lee JM, Park YS, Suh H, Song HR, Jo SA, Jo I (2008) Dexamethasone increases angiopoietin-1 and quiescent hematopoietic stem cells: a novel mechanism
of dexamethasone-induced hematoprotection. FEBS Lett 582(23–24):3509–3514 28. Zhen Z, Sun X, He Y, Cai Y, Wang J, Guan Z (2011) The sequence of drug administration influences the antitumor effects of bevacizumab and cyclophosphamide in a neuroblastoma model. Med Oncol 28(Suppl 1):S619–S625 29. Takahashi T, Asano Y, Akamata K, Aozasa N, Taniguchi T, Noda S, Masui Y, Ichimura Y, Toyama T, Tamaki Z, Tada Y, Sugaya M, Kadono T, Sato S (2013) Dynamics of serum angiopoietin-2 levels correlate with efficacy of intravenous pulse cyclophosphamide therapy for interstitial lung disease associated with systemic sclerosis. Mod Rheumatol 23(5):884–890 30. Alfieri A, Watson JJ, Kammerer RA, Tasab M, Progias P, Reeves K, Brown NJ, Brookes ZL (2012) Angiopoietin-1 variant reduces LPS-induced microvascular dysfunction in a murine model of sepsis. Crit Care 16(5):R182 31. Takahashi M, Suzuki E, Kumano S, Oba S, Sato T, Nishimatsu H, Kimura K, Nagano T, Hirata Y (2013) Angiopoietin-1 mediates adipose tissue-derived stem cell-induced inhibition of neointimal formation in rat femoral artery. Circ J 77(6):1574–1584 32. Suh W, Lee JS, Kim KL, Song SH, Koh GY, Kim DK (2011) Angiopoietin-1 gene therapy attenuates hypertension and target organ damage in nitric oxide synthase inhibited spontaneously hypertensive rats. Korean Circ J 41(10):590–595
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ORIGINAL ARTICLE
Clinical significance of Angiopoietin‑1 in Behcet’s disease patients with vascular involvement Iman H. Bassyouni1 · Mohammed Sharaf1 · Iman E. Wali2 · Hossam M. Mansour3
Received: 1 November 2014 / Accepted: 1 May 2015 © Springer Japan 2015
Abstract Behcet’s disease (BD) is a chronic multisystem inflammatory disorder of unclear etiology. Vascular inflammation, endothelial dysfunction and angiogenesis may be in part responsible for the pathogenesis of BD. Angiopoietin-1 (Ang-1) is a recent angiogenic mediator. The aim of the present study was to assess Ang-1 in the plasma of BD patients as well as to analyze its association with clinical, and laboratory parameters of the disease. The present study included 47 BD patients and 30 age- and gendermatched healthy controls. Demographic, clinical, disease activity and severity were prospectively assessed. Plasma Ang-1 levels were measured using enzyme-linked immunosorbent assay. The plasma level of Ang-1 in BD patients was significantly lower than healthy controls (p = 0.005). Plasma Ang-1 level in patients with vascular affection was significantly lower than those without vascular affection (p = 0.045). Levels of Ang-1 showed a significant positive correlation with steroid dose. Patients who received cyclophosphamide or steroids showed a significant increase in plasma Ang-1 level. This was further confirmed by the results of the multivariate analysis. There was no significant association between plasma Ang-1 levels and other clinical manifestations or disease activity and severity. Plasma Ang-1 levels were diminished in our BD patients especially * Iman H. Bassyouni [email protected]; [email protected] 1
Department of Rheumatology and Rehabilitation, Faculty of Medicine, El‑Kasr El‑Aini Hospital, Cairo University, Cairo 12613, Egypt
2
Medical Microbiology and Immunology, Faculty of Medicine, Cairo University, Giza, Egypt
3
Cardiovascular Department, Faculty of Medicine, Six of October University, 6th of October, Egypt
in patients with vascular involvement. Larger studies with further investigations of the precise role of Ang-1 in the pathogenesis of BD are needed and might lead to novel therapies for the clinical management of BD Keywords Angiogenesis · Angiopoietin · Behcet’s disease · Vasculitis
Introduction Behcet’s disease (BD) is a multisystem vasculitic disorder of unknown etiology, characterized by relapsing episodes of oral aphthous ulcers, genital ulcers, skin lesions and ocular lesions. Vascular involvement is a common complication of BD which worsens the prognosis of BD. The cause of BD is unknown [1]. It is believed to be due to an autoimmune process triggered by an environmental or infectious agent in a genetically predisposed subject. Interaction of primed neutrophils with endothelial cells is considered a crucial event in the pathogenesis of severe endothelial lesions in BD vasculitis [2]. In this regard, the interaction between neutrophils and endothelial cells has been the subject of much study. Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are antagonistic ligands that bind to the extracellular domain of the Tie-2 receptor, which is expressed by endothelial cells [3]. The Ang–Tie ligand–receptor system was identified as the second class of vascular-specific receptor tyrosine kinases (RTKs). Angiopoietins and Tie-2 receptor control angiogenic remodeling in a context-dependent manner [4]. Tie signaling is involved in angiogenic remodeling process during development, including destabilization of existing vessels, endothelial cell migration, tube formation and stabilization of newly formed tubes by mesenchymal cells [5].
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Ang-1 has vasoprotective and anti-inflammatory actions, mediates vessel maturation and maintains vessel integrity by the recruitment of periendothelial cells. Thus, low-level constitutive Tie2 activation by Ang-1 may be required in the adult to maintain the mature quiescent and non-proliferating phenotype of the vascular endothelium. In turn, Ang-2 acts as a vessel-destabilizing cytokine, thereby playing an essential role in vascular remodeling and inflammatory process [4]. In line with these data, abnormality of the Ang–Tie ligand–receptor system has been detected in patients with autoimmune and vascular diseases [6–12]. Recently, a preliminary study reported elevated Ang1 in Korean BD Patients [13]. But with this exception, no information is available on the role of Ang/Tie system in BD patients. The aim of the present study was to assess Ang-1 concentrations as a recent angiogenic mediator in plasma of BD patients and to analyze its association with disease clinical features, laboratory parameters as well as disease activity and severity.
Patients and methods Patients Forty-seven patients with BD, diagnosed according to the International Study Group criteria [14], were recruited (mean age 33.81 ± 9.27 years) and 30 ageand gender-matched healthy control subjects (mean age 32.74 ± 8.73 years) were included in the study. Patients who had other autoimmune disease, malignancy or infection were excluded from the study. The detailed clinical characteristics of the patients were determined by combined medical history and physical examination. For venous and arterial thrombotic events, Doppler ultrasonography, angiography, MRI, CT, and echocardiography were done. Radiologic evaluations, including, CT scans of the lungs and the abdomen, cranial magnetic resonance imaging (MRI) were performed as clinically indicated. The activity and severity of the disease were assessed as previously described [15]. The total clinical activity index score of each patient represents the numerical sum of the manifestations present only at the time of blood sampling. BD clinical severity score was calculated as the sum of 1 point each for mild symptoms (oral aphthosis, genital ulcers, arthralgia and typical skin lesions such as folliculitis, erythema nodosum, and papulopustular lesions), 2 points each for moderate symptoms (anterior uveitis, arthritis, deep vein thrombosis of the legs, and gastrointestinal involvement) and 3 points each for severe disease manifestations (retinal vasculitis, posterior/panuveitis, arterial thrombosis,
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Heart Vessels
bowel perforation and neuro-Behçet’s). Routine laboratory investigations were collected from the patients’ records [15]. The study was approved by our institutional ethics committee and written consent was obtained from all patients. Plasma Angiopoietin‑1 levels determination Peripheral venous blood samples in EDTA containing tubes were obtained from patients and controls. After centrifugation at 1000g for 10 min, the plasma was frozen and stored at −70 °C until assayed on the same day to eliminate dayto-day interassay variability. A commercial ELISA kit (RayBiotech Inc., GA, USA) was used for assaying Ang-1 levels. This assay employed an antibody specific for human Ang-1 coated on a 96-well plate. In brief, each plasma sample was directly transferred to the wells of the ELISA plate and then assayed according to the manufacturer’s instructions. The absorbance was measured at 450 nm in a microtest plate spectrophotometer, and Ang-1 levels were quantified with a calibration curve using human Ang-1 as a standard. Both standards and samples were evaluated in duplicates and the interassay variations were shown to be within the range given by the manufacture. Statistical analysis The statistical package for social sciences (SPSS) version 15 (LEAD Technology Inc., Charlotle, NC, USA) was used to analyze data. Continuous variables were summarized through the median (range) or mean (SD) and categorical variables as frequency (percentage). Nonparametric Mann–Whitney U test compared two independent groups. Spearman’s rank correlation test was used as a measure of association of quantitative variables. Significant differences were calculated using Mann–Whitney U test for continuous variables. For those variables that were significantly influencing plasma Ang-1 levels (p 10 mg/day Cyclophosphamide Azathioprine Colchicine Laboratory investigationsa ESR (mm/h) Hemoglobin (g/dl) Leucocyte (×103/µl) Neutrophils (×103/µl) Platelets (×103/µl)
BD patients (N = 47)
33.81 ± 9.27 7.46 ± 6.51 37/10 47 (100) 42 (89) 20 (42.6) 19 (40.4) 14 (29.8) 34 (72.3) 6 (12.8) 9 (19.1) 30 (63.8) 10 (21.2) 23 (49) 18 (38.3) 18.49 ± 16.87 13.56 ± 1.41 10.45 ± 3.81 56.87 ± 8.71 287.49 ± 86.42
a
Continuous variables: mean ± SD
b
Categorical variables: number (%)
pseudofolliculitis- and erythema nodosum-like lesions (42 %) were the most common clinical features reported by this group of patients. Previous neurological involvement was detected in 6 (13 %) of these patients which manifested as headache, aseptic meningitis, facial paralysis, intracranial hypertension, and seizures. Vascular events were reported in 9 patients, deep venous thrombosis was notified by 7 of patients, 5 with superficial thrombophlebitis as well, arterial thrombosis was previously detected also in 1 patients and 1 had arterial aneurysms. At the time of blood sampling, the mean BD activity score was 2.65 ± 2.00 (range 0–6), while the total BD severity score was 7.39 ± 2.41 (range 3–13). Plasma Ang‑1 levels and BD clinical and laboratory parameters The median plasma Ang-1 concentrations were reduced in patients with BD compared to healthy controls (78.12; range 24.71–151.66, 84 and 97.74; range
Fig. 1 Plasma Ang-1 levels in: a BD patients and Healthy controls. b BD patients with and without vascular involvement. The lines inside the boxes indicate the median; the outer borders of the boxes indicate 25th and 75th percentiles; the bars extending from the boxes indicate the 10th and 90th percentiles
59.27–140.1 ng/ml, respectively, p = 0.005; Fig. 1a). As BD is a heterogeneous disease, further analysis was performed to compare the levels of plasma Ang-1 in BD patients with the presence and absence of some clinical manifestations and medications used at time of blood sampling (Table 2). We found that patients with vascular involvement had significant reduction of plasma Ang-1 levels than those without (p = 0.045l; Fig. 1b). We did not find significant differences in median plasma Ang-1 levels in relation to any other specific organ involvement (p > 0.05). With respect to medications used, we found significant increase in median plasma Ang-1 levels in patients who received cyclophosphamide than those who did not receive it (p = 0.049). Similarly, Ang-1 levels were higher in patients receiving steroid dose >10 mg/day (p = 0.003). Furthermore, it correlated significantly with steroid dose (r = 0.354, p = 0.018).
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Table 2 The differences in median plasma Ang-1 concentration in relation to the presence or absence of different clinical features and the current medications used Clinical parameter/current medications
No (%)
Median Ang-1 levels ng/ml (min–max) Present
Absent
P value
Genital ulcers Pseudofolliculitis/EN Positive Pathergy test Articular involvement Ocular involvement Neurological involvement Vascular involvement Corticosteroids >10 mg/day Cyclophosphamide Azathioprine
42 (89) 20 (42.6) 19 (40.4) 14 (29.8) 34 (72.3) 6 (12.8) 9 (19.1) 30 (63.8) 10 (21.2) 23 (49)
25.64 (24.71–126.64) 68.23 (24.71–151.66) 82.53 (24.95–140.72) 69.62 (24.71–137.93) 79.10 (24.71–151.66) 77.65 (24.71–151.66) 55.54 (24.95–104.54) 86.49 (25.65–151.66) 100.40 (41.12–151.66) 69.62 (24.71–137.93)
78.12 (39.38–151.66) 84.98 (24.95–140.72) 69.62 (24.71–151.66) 91.09 (24.95–151.66) 74.63 (24.95–134.21) 95.22 (69.63–134.21) 82.53 (24.71–151.66) 55.54 (24.71–134.21) 69.62 (24.71–137.93) 85.16 (25.65–151.66)
0.15 0.32 0.58 0.14 0.48 0.147 0.045* 0.003* 0.049* 0.33
Colchicine
18 (38.3)
78.12 (24.95–151.66)
72.24 (24.71–140.72)
0.53
Data are median Ang-1 levels (min–max); Mann–Whitney U test was used to compare variables EN Erythema nodosum * Significant (p 0.05; Table 3). Since patients with BD usually do not present with only one symptom of the disease, same treatment or one risk factor, therefore, we decided to perform a multivariate analysis to estimate the independent association of each covariate with plasma Ang-1 as dependent variable against the variables that yielded p values 10 mg (β = 0.340, p = 0.013), and cyclophosphamide (β = 0.291, p = 0.30). Vascular involvement was found to be a negative predictor of Ang-1 levels (β = −0.274, p = 0.044).
Discussion Vascular inflammation, endothelial dysfunction and neovascularization may be in part responsible for the pathogenesis of BD [1, 2]. One of the systems involved in angiogenesis is Ang–Tie receptor system, it is a novel regulator involved in the maintenance of vascular integrity
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and vascular remodeling and is related to the survival and migration of endothelial cells [5]. We had found a significant reduction of the plasma concentration of Ang-1 in BD patients compared with controls especially in patients with vascular involvement. This implies that Ang-1 might have a significant role in BD [4]. Ang-1 is considered an endothelial survival factor and vasculoprotective agent. Therefore, Ang-1 insufficiency might lead to loss of endothelial integrity, vessel dilatation, increased permeability and promotes formation of perivascular inflammatory infiltrates [16] as observed in BD. On the other hand, Ang-2 is an angiostatic factor to Ang-1, its binding to Tie 2 results in rapid destabilization of the endothelium, suggesting that Ang-2 functions as an autocrine negative regulator of the quiescent resting endothelium [17]. In accordance to our results, Michalska-Jakubus et al. have demonstrated significantly decreased Ang-1 levels in patients with systemic sclerosis (SSc). This reduction in Ang-1 levels has been attributed to the development of SSc-related vasculopathy through promoting activation and apoptosis of ECs and destabilization of blood vessels [18]. Likewise, it is obvious that molecules that favor the stability of new blood vessels, such as Ang-1, are under expressed in inflammatory bowel disease patients, while, destabilizing molecules such as Ang-2 are upregulated in those patients [9]. Similarly, circulating Ang-2, but not Ang-1, has been elevated and closely correlates with disease activity and circulating endothelial cells numbers in anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitis with renal involvement and in systemic lupus erythematosus (SLE) patients [6, 7]. To the best of our knowledge, no reports concerning Ang-1 in BD have
Heart Vessels Table 3 Correlation between plasma Ang-1 levels with disease activity, severity and laboratory features of BD patients Parameter
Fig. 2 Scatter Plot demonstrating correlation of plasma Ang-1 Abs levels with BD disease activity (a), and Severity (b) Scores
been published so far except the study of Choe et al., in BD Korean population [13]. They have found increased levels of plasma Ang-1 in their studied BD patients compared to the healthy controls. The variability in the results regarding Ang-1 in BD might be attributed to the multi-systemic involvement characterizing this disease. We had reported in our study a significant association of Ang-1 with vascular involvement (present in 19 % of our patients). On the other hand, in Choe et al. study, only 2 BD patients had vascular involvement (3.4 % of their patients). Their Ang-1 levels were lower in patients with vascular involvement than those without although it was statistically insignificant probably due to the low number of patients (216.2 ± 15.8 vs 286.8 ± 102.1, p = 0.318). In accordance to our results, Ang-1 has been found to be diminished in several rheumatic and vascular diseases [6–9, 18–20]. The expression of angiogenic factors including Ang-1 has been insufficient in bone marrow of critical limb ischemia patients [12].
Plasma Ang-1 (ng/ml) r
P value
Hb (g/dl) WBCs (103/μL) Neutrophils (%) Lymphocytes (%) Platelets (103/μL) ESR (mm/h) ALT (U/L) AST (U/L) Creatinine (mg/dl) BD disease activity
0.07 0.2 −0.1 0.1 0.08 −0.07 0.04 −0.05 0.1 0.264
0.40 0.08 0.32 0.51 0.52 0.67 0.82 0.72 0.51 0.08
BD disease severity
0.155
0.297
Studies of animal models have confirmed the Ang-1 role within the vasculature. Acute administration of Ang1 significantly reduces vascular inflammation in mice [21]. On the other hand, Ang-2 has been found to trigger an inflammatory response by activating the endothelium and inducing vascular permeability [22]. Decreased activity of the Tie-2 pathway has contributed to right ventricular load in experimental hypoxia-induced pulmonary hypertension, and this effect can be antagonized by Ang-1 administration [23]. It is, therefore, tempting to speculate that Ang-1 deficiency might contribute to the development vascular affection in BD. Regarding disease activity, the results of the present study suggested that plasma Ang-1 levels do not reflect clinical or serologic disease activity. These results were in accordance with those obtained by Choe et al. [13]. These equivocal findings regarding the association between angiogenic factors and activity may be affected by the administration of therapeutic drugs with anti-inflammatory properties, but it is not understood whether these drugs have antiangiogenic potential. The management of a patient with BD is complex and oftentimes requires a multidisciplinary approach. Colchicine is used for mucocutaneous manifestations, non-steroidal anti-inflammatory drugs for joint manifestations, anticoagulation for vascular thrombosis, and cytotoxic drugs for major system involvement [1, 2]. In 2008, The European League Against Rheumatism (EULAR) guidelines have suggested that cyclophosphamide be used preferentially for life-threatening manifestations of BD, including vascular disease and nervous system involvement [24]. The use of cyclophosphamide and steroids in addition to anticoagulation was recommended by several authors for the management of BD arterial and venous thrombosis to prevent their recurrence [25, 26].
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Herein, we had detected a significant positive correlation of Ang-1 levels with the steroid dose and cyclophosphamide. In line with this, Kim et al. have demonstrated that dexamethazone increases the levels of Ang-1 in mouse bone marrow and the quiescence of hematopoietic stem cells [27]. Furthermore, a significant increase in mean plasma Ang-1 levels in patients who received cyclophosphamide was reported. In accordance, Zhen et al. have observed an increase in plasma Ang-1 levels following administration of cyclophosphamide in Neuroblastoma model with improvement of function of the vessels [28]. In line with this, serum Ang-2 levels have been found to decrease significantly after IV cyclophosphamide therapy compared with their initial levels in systemic sclerosis patients [29]. Ang-1 has received significant attention as a potential therapeutic molecule in diabetic retinopathy, allograft vasculopathy, sepsis/endotoxemia, malignancy and diabetesinduced complications [30]. Takahashi and coworkers have reported Ang-1 as a possible candidate proangiogenic factor to be used in the treatment of coronary artery disease [31]. Investigations into the role of Ang-1 may ultimately lead to the development of a potent therapeutic molecule for treating patients with vascular diseases [32]. This study had some limitations. First, this study was not aimed at elucidating pathophysiological pathways of Ang-1 regulation in BD, but was rather designed to evaluate its possible clinical significance. Second, because of its cross-sectional nature; we were unable to show Ang-1 level changes with respect to clinical course and medical treatment. In future prospective large-scale, longitudinal study, we will investigate Ang-1 levels according to relapse, additional patterns of organ involvement, and treatment regimens. This might lead to novel therapies for the clinical management of BD Conflict of interest There are no conflicts of interest related to the manuscript.
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