Original article 703
The investigation of relationship between joint findings and serum angiogenic and inflammatory factor levels in severe hemophilia A patients ¨ zekc, O ¨ zlem Tu¨fekc¸ia, Tuba H. Karapınara, Nihal Karadas¸b, Gu¨lcihan O Berna Atabayd, Meral Tu¨rkerd, Faize Yu¨ksele, Deniz Y. Karapınarb, ¨ rena Canan Verginc, Gu¨lersu ˙Irkena and Hale O Despite the use of primary prophylactic Factor VIII replacement in severe hemophilia A patients, bleeding into joints cannot be prevented completely and early diagnosis and treatment of the joint bleedings are important for prevention of permanent joint damage. Recent studies have shown that neoangiogenesis plays important role in development of synovitis after recurrent joint bleedings. This study aimed to investigate the relationship between joint findings and levels of serum angiogenic and inflammatory factors in severe hemophilia A patients.The patient groups consisted of 10 severe hemophilia A patients with acute joint bleeding and 25 severe hemophilia A patients without acute joint bleeding. They were all inhibitor negative. The control group consisted of 22 healthy male children. Complete blood cell count analysis, C-reactive protein (CRP), serum ferritin, lactic acid, and ELISA-based detection of vascular endothelial growth factor (VEGF), intercellular adhesion molecule-1, thrombomodulin, macrophage migration inhibitory factor (MIF), and endostatin were performed from peripheral blood of patient and the control groups. CRP and MIF levels were detected significantly higher in hemophilia patients with acute joint bleeding than patients without acute joint bleeding. There
Introduction Spontaneous intra-articular hemorrhages may occur in 90% of the patients with severe hemophilia A [1]. Recurrent bleeding episodes result in disabilities by inducing persistent damage in these joints. Hemophilic arthropathy decreases the quality of life by restricting the physical activity. In spite of regular factor VIII (FVIII) concentrate replacements that are initiated in early ages, intra-articular hemorrhages (especially subclinical hemorrhages) have not yet been prevented completely and permanent joint damages continue to occur [2–4]. It is not yet possible to determine the onset of intra-articular hemorrhage with clinical, laboratory, and radiologic imaging methods. Although only advanced findings of hemophilic arthropathy can be detected with conventional radiograms, MRI methods enable detection of hemarthrosis findings in earlier phases [5–8]. The pathogenesis of hemophilic arthropathy is not well defined. Recent studies demonstrated that neoangiogenesis plays a major role in development of synovitis secondary to recurrent intra-articular hemorrhages in 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
was a positive correlation between serum thrombomodulin, VEGF, and MIF levels. In this study, we demonstrated that serum CRP and MIF levels increases in acute bleeding period regardless of the presence of previous joint damage in children with severe hemophilia. CRP elevation may be a useful and rapid marker for acute bleeding in these patients. Blood Coagul Fibrinolysis 25:703–708 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Blood Coagulation and Fibrinolysis 2014, 25:703–708 Keywords: angiogenesis, children, hemarthrosis, hemophilia, inflammatory factors a
Department of Pediatric Hematology, Dokuz Eylu¨l University Faculty of Medicine, Department of Pediatric Hematology-Oncology, Ege University Faculty of Medicine, cDepartment of Pediatric Hematology-Oncology, Dr Behc¸et Uz Training and Research Hospital, dDepartment of Pediatric Hematology-Oncology, Tepecik Training and Research Hospital and eDokuz Eylu¨l University Faculty of Medicine, Hematology Laboratory, Izmir, Turkey b
¨ ren, Dokuz Eylu¨l University, Faculty of Correspondence to Professor Dr Hale O Medicine, Department of Pediatric Hematology, 35340 Balcova, Izmir, Turkey Tel: +90 532 6669050; fax: +90 232 4126005; e-mail: [email protected] Received 14 January 2014 Revised 24 March 2014 Accepted 24 March 2014
patients with hemophilia [9–12]. It is shown that histologic abnormalities and neogenesis that occur within the synovia are similar to those in other joint diseases [4]. In this study, relation between joint findings and serum levels of angiogenic and inflammatory factors was investigated whether any of the angiogenic and inflammatory factors may be a marker for early phase of joint bleeding. Rapid diagnosis and an appropriate early therapeutic approach may decrease expected morbidity in these patients.
Materials and methods In this study, which was performed between 01 March 2012 and 01 March 2013, 10 patients with severe hemophilia A (FVIII level 0.5). When hemoglobin, platelet, leukocyte, and monocyte counts were compared between the children with severe hemophilia and the control group, there was no statistically significant difference between the groups (P ¼ 0.07, P ¼ 0.47, P ¼ 0.40, P ¼ 0.18, respectively). Laboratory findings of all patients are shown in Table 1. There was early joint damage in three patients with acute intra-articular hemorrhage and seven patients with acute intra-articular hemorrhage had advanced joint damage. There was early joint damage in three patients without acute intra-articular hemorrhage and six patients without acute intra-articular hemorrhage had advanced joint damage. There was no joint damage in 16 patients. As two of the 10 hemophilic patients who had acute joint hemorrhage did not come to first-month followup, the control values of these two patients could not be obtained. CRP, serum ferritin, and lactic acid levels (Table 1) were found to be higher in hemophilic patients with acute joint bleeding compared with the control group (P < 0.05). Serum lactic acid levels were significantly higher in hemophilia patients who had no bleeding (P < 0.05), but these values were within normal range. CRP levels were found to be more than twice the upper limit of normal in hemophilic patients with acute joint hemorrhage. Serum VEGF, MIF, thrombomodulin, endostatin,
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Angiogenic and inflammatory factors in hemophilia A Karapınar et al. 705
Table 1
Laboratory results of the patients in the study and the control groups Patients with acute intra-articuler hemorrhage
Leukocyte (cells/ml) Monocyte (cells/ml) Hemoglobin (g/dl) Platelet count (cells/ml) CRP (mg/dl) Ferritin (ng/ml) Lactic acid (mmol/l)
Patients without acute intra-articular hemorrhage
Control group
Advanced joint damage (n ¼ 7)
Early joint damage (n ¼ 3)
Advanced joint damage (n ¼ 6)
Early joint damage (n ¼ 3)
No damage (n ¼ 16)
(n ¼ 22)
8577 505 13.4 240000 16.9 48.54 1.11
6233 500 12.1 266000 6.2 47.73 1.27
8516 451 13.9 239000 4.0 86.85 1.33
7500 500 11.9 304000 4.9 92.35 1.16
7753 599 12.7 253000 4.1 51.67 1.59
6572 500 13.1 267000 1.1 26.95 1.07
CRP, C-reactive protein.
and ICAM-1 levels were found to be similar to the control group (Table 2), and there was not any statistically significant difference between the groups (P ¼ 0.95, P ¼ 0.28, P ¼ 0.89, P ¼ 0.83 and P ¼ 0.83, respectively). There was not any significant difference among leukocyte count, hemoglobin levels, serum ferritin, lactic acid, VEGF, thrombomodulin, and endostatin (Table 3). When the laboratory results were compared in patients with early joint damage (n ¼ 3) and with advanced joint damage (n ¼ 7), among the patients who had acute joint hemorrhage, no significant difference was found. When the laboratory results were compared in patients with advanced joint damage (n ¼ 6) and with early joint damage (n ¼ 3), among the patients who had no acute joint hemorrhage, no significant difference was found. However, as the number of the cases in these compared groups were very few, we excluded these results from the evaluation. When the laboratory results were compared in all hemophilic patients with and without acute joint hemorrhage, those who had advanced joint damage (n ¼ 13) had significantly higher hemoglobin levels than those with early joint damage (n ¼ 6) (P < 0.05). There was no significant difference among leukocyte count, serum ferritin and lactic acid, VEGF, thrombomodulin, and endostatin levels (P ¼ 0.32, P ¼ 0.63, P ¼ 0.96, P ¼ 0.89, P ¼ 0.52, P ¼ 0.10, and P ¼ 0.57, respectively). Among all the hemophilic patients, when the laboratory results were compared between those with joint damage (n ¼ 19) and those without (n ¼ 16), CRP was significantly high in the patients with joint damage (P < 0.05) and Table 2
serum lactic acid level was significantly high in those without joint disease (P < 0.05). There was a positive correlation among the patients with early joint damage in terms of thrombomodulin, VEGF, ICAM-1, and lactic acid levels; however, these results were not statistically significant (P ¼ 0.77, P ¼ 0.77, P ¼ 0.77, respectively). No correlation was found between serum VEGF and ICAM-1 and lactic acid (P ¼ 0.15, P ¼ 0.42, P ¼ 0.54, respectively). There was a positive correlation between thrombomodulin levels and MIF in patients with advanced joint damage and a negative correlation was noted between MIF and endostatin levels in these patients; however these results were not statistically significant (P ¼ 0.70, P ¼ 0.33, respectively).
Discussion In spite of initiating regular F VIII concentrate replacements at early ages in patients with severe hemophilia, subclinic and clinically overt intra-articular hemorrhages currently cannot be prevented completely and permanent joint damages continue to occur [2–4]. It is not yet possible to determine the onset of intra-articular hemorrhage with clinical, laboratory, and radiologic imaging methods. This can be an important reason for the increase in joint damage. In our study, an investigation of the relation between plasma angiogenic and inflammatory factor levels in patients with severe hemophilia A and an examination of changes in these factor levels during joint hemorrhage were performed. Leukocytes play an important role in inflammation because of their antimicrobial, secretory, and phagocytic
Results of ELISA tests Patients with acute intra-articular hemorrhage
VEGF (pg/ml) Thrombomodulin (ng/ml) MIF (pg/ml) ICAM-1(pg/ml) Endostatin (ng/ml)
Patients without acute intra-articular hemorrhage
Control group
Advanced joint damage (n ¼ 7)
Early joint damage (n ¼ 3)
Advanced joint damage (n ¼ 6)
Early joint damage (n ¼ 3)
No damage (n ¼ 16)
(n ¼ 22)
113.208 0.42 75952 366.125 27.791
114.681 1.18 34204 360.228 26.215
70.615 0.45 40542 352.061 29.811
71.024 0.8 27104 380.809 24.683
148.089 0.97 19471 377.01 25.049
111.436 0.72 17491 374.289 27.332
ICAM-1, intercellular adhesion molecule-1; MIF, macrophage migration inhibitory factor; VEGF, vascular endothelial growth factor.
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706 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 7
activities [18]. We did not find any difference in terms of leukocyte and monocyte counts between hemophilic patients with and without joint damage and healthy control group in our study. There was no significant difference in patients who had joint hemorrhage. This can be because of the scarcity of the patients with hemorrhage or local bleeding not causing a systemic response. Exposure of the cells to hypoxia in the wound induces angiogenesis. It was shown that both local hypoxia and decrease in glucose occur because of diminishing of vascular perfusion; therefore, cells transform into an anaerobic metabolic pathway, production of lactate increases and extracellular pH is decreased and this new environment results in the secretion of cytokine/ enzyme from the macrophages. The release of angiogenic molecules by macrophages recruited to the wound is central to the formation of neoangiogenesis [19–21]. It is known that increase in the serum lactic acid level indicates tissue hypoxia. We did not notice any significant increase in lactic acid levels during acute joint hemorrhage in patients with hemophilia. Literature suggest that in patients with sepsis, myocardial infarction, and trauma, serum lactate levels rise and this can be used for followup [22–24]. When studying whether acute joint hemorrhage in hemophilic patients resulted in systemic hyperlactatemia, we did not find any significant increase. Blood normally does not exist in articular space. Synovial macrophages and condrocytes absorb the iron released by erythrocytes when intra-articular hemorrhage occurs. If bleeding episodes become more frequent, synovia regeneration becomes inadequate and iron accumulates [25–27]. When all hemophilic patients with or without acute joint bleeding were compared with the healthy control group, we found that serum ferritin levels were significantly higher in hemophilic patients. When a comparison was done between hemophilic patients with acute joint bleeding and the healthy control group, as well as between hemophilic patients and healthy controls, serum ferritin levels were higher compared with healthy controls. When the first-month laboratory findings of eight patients during and after the bleeding were compared, we did not find any significant difference between serum ferritin levels. It was shown that as an acute-phase reactant, ferritin was increased in patients with rheumatoid arthritis [28,29]. In inadequately treated hemophilic mice, it was observed that when hemorrhage recurred during recovery, in spite of more machrophage migration, iron staining persisted and iron was accumulated in deeper tissues. Angiogenesis rate was similar in hemophilic and healthy mice; however, the number of newly formed vessels was higher in hemophilic mice. It was suggested that proinflammatory and proangiogenic effects of tissue iron could result in this [10]. Our finding regarding higher levels of serum ferritin during acute bleeding compared with the healthy controls can be
related to the fact that ferritin is an acute phase reactant. However, in periods when no acute bleeding occurs, as shown in mice models, the presence of ferritin at higher levels compared with the healthy controls could be because of the accumulation of iron in deeper tissues as a result of recurrent bleedings. However, as the increased ferritin levels were within normal range, this is not considered as an important marker in the hemophilic patients. VEGF, one of the proangiogenic molecules released in response to hypoxia, is a basic signaling molecule for angiogenesis [11,30,31]. In this study, we did not find any difference in VEGF levels between all hemophilic patients with and without joint bleeding and the healthy control group. When the first-month laboratory findings of eight patients during and after the bleeding were compared, we did not find any significant difference between serum VEGF levels. In 2011, Acharya et al. [11] demonstrated that both serum and synovial VEGF levels were increased in hemophilic patients with joint hemorrhage. In the mentioned study which consisted of 14 patients, it was shown that both serum and synovial proangiogenic macrophage/monocytes (VEGFþ/CD68þ and VEGFR1þ/CD11bþ) levels were increased in patients’ hemophilic arthropathy. In the same study, a four-fold increase in both synovial and peripheral blood VEGF-A, stromal derivated factor-1, and matrix metalloprotease-9 was demonstrated in hemophilic arthropathy and it was shown by performing cell culture studies, by using human anti-VEGF antibody (bevacizumab) and by demonstrating the halt of synovial cell proliferation that VEGF inhibition could be an alternative in future treatment. It may be possible that we could not show an increase in VEGF levels because of scarcity of the patients. MIF is an important cytokine that plays a role in regulating inflammatory and immune response. In this study, we did not find any difference in MIF levels between all hemophilic patients with and without joint bleeding and the healthy control group. We found that serum MIF levels significantly increased during bleeding when the laboratory findings were compared at the time of and at the first month after the bleeding in patients with acute intra-articular hemorrhage. In recent studies, it was shown that MIF plays a role in several diseases such as hemorrhagic fever, rheumatoid arthritis, autoimmune diseases, and atherosclerosis [32–34]. Thrombomodulin is a membrane glycoprotein that is placed on the endothelial cell surface and acts as a thrombin receptor. It is basically found in vascular endothelial cells in humans [35]. In this study, we did not find any difference in thrombomodulin levels between all hemophilic patients with and without joint bleeding and the healthy control group. We found that serum thrombomodulin levels increased during bleeding when
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Angiogenic and inflammatory factors in hemophilia A Karapınar et al. 707
Table 3
Laboratory results of patients with acute joint bleeding During bleeding
Leukocyte (cells/ml) Hemoglobin (g/dl) CRP (mg/dl) Ferritin (ng/ml) Lactic acid (mmol/l) VEGF (pg/ml) Thrombomodulin (ng/ml) MIF (pg/ml) ICAM-1 (pg/ml) Endostatin (ng/ml)
One month after bleeding
Mean
Median
Mean
Median
P values
7937 13.1 16.3 50.33 1.13 115.689 0.91 74801 359.496 28.076
6300 12.9 7.3 39.94 1.11 111.883 0.68 36336 362.242 25.471
6850 13.2 3.1 39.11 1.18 144.542 0.57 23692 399.759 26.874
6900 13.6 1.9 38.01 1.16 139.127 0.46 12967 404.929 27.255
0.49 0.64 0.02 0.06 0.35 0.48 0.09 0.02 0.01 0.88
CRP, C-reactive protein; ICAM-1, intercellular adhesion molecule-1; MIF, macrophage migration inhibitory factor; VEGF, vascular endothelial growth factor.
the laboratory findings were compared at the time of and at the first month after the bleeding in patients with acute intra-articular hemorrhage, but this difference was insignificant. It was recently detected that thrombomodulin domain 2 and domain 3 has an angiogenic-promoting function. It was shown that TMD23, that has a recombinant thrombomodulin fragment, stimulates endothelial migration, proliferation, and tube formation. It was demonstrated that TMD23 leads to endothelial cell invasion and migration in early phases of angiogenesis by inducing release of matrix metalloproteins and plasminogen activators from the endothelial cells [36]. When a wound forms, monocytes move from circulation into tissue via LFA-1 cytokines on the monocyte surface and ICAM-1 cytokines on the endothelial cell surface [19]. In this study, we did not find any difference in ICAM-1 levels between all hemophilic patients with and without joint bleeding and healthy control group. It was shown that ICAM-1 plays an important role in inflammatory pathway and serum ICAM-1 levels increase in the early phases of osteoarthritis and rheumatoid arthritis [37–39]. We found that MIF and CRP serum levels increase during acute intra-articular hemorrhage. It was very recently shown that MIF increases both ICAM-1 and thrombomodulin release [33]. Since CRP is an acutephase reactant, a rise in CRP during acute hemorrhage is expected. Our study also suggested that CRP was higher in patients with joint damage. In childhood, the increase in CRP levels may be superior than clinical symptoms to demonstrate early acute joint bleeding. In the literature, it is stated that there is a significant relation between high ICAM-1 and high CRP [39,40]. There is a balance between proangiogenic agents and antiangiogenic agents during the process of angiogenesis. Angiostatin, thrombospondin, endostatin, and fibrinogen E fragment are main antiangiogenic agents [19,41]. In this study, we did not find any difference in endostatin levels between all hemophilic patients with and without joint bleeding and healthy control group. When the laboratory findings of eight patients during and 1 month
after the bleeding were compared, we did not find any significant difference between serum endostatin levels. It has been shown that neoangiogenesis is increased in tumor tissues and at the same time, endostatin release is increased [41–43]. There was a positive correlation among the patients with early joint damage in terms of thrombomodulin, VEGF, ICAM-1, and lactic acid levels; however, this result was not statistically significant. These results can be because of low number of the cases in this group. We did not find any correlation between serum VEGF and ICAM-1 and lactic acid levels. There was a positive correlation between thrombomodulin levels and MIF in patients with advanced joint damage and a negative correlation was noted between MIF and endostatin levels in these patients; however, these results were not statistically significant. Although statistically insignificant, we believe that the positive correlation between thrombomodulin levels and VEGF and MIF is important. This correlation between proangiogenic molecules during inflammation was also shown in different patient groups [11,33]. Despite the advantages of MRI over conventional X-ray, because of its rather difficult accessibility, its requirement of sedations for the children and its high cost, biological markers are needed to indicate hemophilic arthropathy in early phases [5,11]. Marked increase in CRP during acute hemorrhage suggested that CRP could be a good indicator. In childhood, the increase in CRP levels may be superior to clinical symptoms to demonstrate early acute joint bleeding. Besides, MIF levels rise in case of hemorrhage and it support the presence of hemorrhage regardless of severity of the joint damage. We think that levels of these factors can give information during early phase in acute bleedings in patients with severe hemophilia A. Therefore, we expect that antiangiogenic or anti-inflammatory treatments can prevent recurrent hemorrhages and following arthropathy.
Acknowledgements This study was supported by Dokuz Eylu¨l University Research Fund.
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708 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 7
Conflicts of interest
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None of the authors express a conflict of interest. 23
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The investigation of relationship between joint findings and serum angiogenic and inflammatory factor levels in severe hemophilia A patients ¨ zekc, O ¨ zlem Tu¨fekc¸ia, Tuba H. Karapınara, Nihal Karadas¸b, Gu¨lcihan O Berna Atabayd, Meral Tu¨rkerd, Faize Yu¨ksele, Deniz Y. Karapınarb, ¨ rena Canan Verginc, Gu¨lersu ˙Irkena and Hale O Despite the use of primary prophylactic Factor VIII replacement in severe hemophilia A patients, bleeding into joints cannot be prevented completely and early diagnosis and treatment of the joint bleedings are important for prevention of permanent joint damage. Recent studies have shown that neoangiogenesis plays important role in development of synovitis after recurrent joint bleedings. This study aimed to investigate the relationship between joint findings and levels of serum angiogenic and inflammatory factors in severe hemophilia A patients.The patient groups consisted of 10 severe hemophilia A patients with acute joint bleeding and 25 severe hemophilia A patients without acute joint bleeding. They were all inhibitor negative. The control group consisted of 22 healthy male children. Complete blood cell count analysis, C-reactive protein (CRP), serum ferritin, lactic acid, and ELISA-based detection of vascular endothelial growth factor (VEGF), intercellular adhesion molecule-1, thrombomodulin, macrophage migration inhibitory factor (MIF), and endostatin were performed from peripheral blood of patient and the control groups. CRP and MIF levels were detected significantly higher in hemophilia patients with acute joint bleeding than patients without acute joint bleeding. There
Introduction Spontaneous intra-articular hemorrhages may occur in 90% of the patients with severe hemophilia A [1]. Recurrent bleeding episodes result in disabilities by inducing persistent damage in these joints. Hemophilic arthropathy decreases the quality of life by restricting the physical activity. In spite of regular factor VIII (FVIII) concentrate replacements that are initiated in early ages, intra-articular hemorrhages (especially subclinical hemorrhages) have not yet been prevented completely and permanent joint damages continue to occur [2–4]. It is not yet possible to determine the onset of intra-articular hemorrhage with clinical, laboratory, and radiologic imaging methods. Although only advanced findings of hemophilic arthropathy can be detected with conventional radiograms, MRI methods enable detection of hemarthrosis findings in earlier phases [5–8]. The pathogenesis of hemophilic arthropathy is not well defined. Recent studies demonstrated that neoangiogenesis plays a major role in development of synovitis secondary to recurrent intra-articular hemorrhages in 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
was a positive correlation between serum thrombomodulin, VEGF, and MIF levels. In this study, we demonstrated that serum CRP and MIF levels increases in acute bleeding period regardless of the presence of previous joint damage in children with severe hemophilia. CRP elevation may be a useful and rapid marker for acute bleeding in these patients. Blood Coagul Fibrinolysis 25:703–708 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Blood Coagulation and Fibrinolysis 2014, 25:703–708 Keywords: angiogenesis, children, hemarthrosis, hemophilia, inflammatory factors a
Department of Pediatric Hematology, Dokuz Eylu¨l University Faculty of Medicine, Department of Pediatric Hematology-Oncology, Ege University Faculty of Medicine, cDepartment of Pediatric Hematology-Oncology, Dr Behc¸et Uz Training and Research Hospital, dDepartment of Pediatric Hematology-Oncology, Tepecik Training and Research Hospital and eDokuz Eylu¨l University Faculty of Medicine, Hematology Laboratory, Izmir, Turkey b
¨ ren, Dokuz Eylu¨l University, Faculty of Correspondence to Professor Dr Hale O Medicine, Department of Pediatric Hematology, 35340 Balcova, Izmir, Turkey Tel: +90 532 6669050; fax: +90 232 4126005; e-mail: [email protected] Received 14 January 2014 Revised 24 March 2014 Accepted 24 March 2014
patients with hemophilia [9–12]. It is shown that histologic abnormalities and neogenesis that occur within the synovia are similar to those in other joint diseases [4]. In this study, relation between joint findings and serum levels of angiogenic and inflammatory factors was investigated whether any of the angiogenic and inflammatory factors may be a marker for early phase of joint bleeding. Rapid diagnosis and an appropriate early therapeutic approach may decrease expected morbidity in these patients.
Materials and methods In this study, which was performed between 01 March 2012 and 01 March 2013, 10 patients with severe hemophilia A (FVIII level 0.5). When hemoglobin, platelet, leukocyte, and monocyte counts were compared between the children with severe hemophilia and the control group, there was no statistically significant difference between the groups (P ¼ 0.07, P ¼ 0.47, P ¼ 0.40, P ¼ 0.18, respectively). Laboratory findings of all patients are shown in Table 1. There was early joint damage in three patients with acute intra-articular hemorrhage and seven patients with acute intra-articular hemorrhage had advanced joint damage. There was early joint damage in three patients without acute intra-articular hemorrhage and six patients without acute intra-articular hemorrhage had advanced joint damage. There was no joint damage in 16 patients. As two of the 10 hemophilic patients who had acute joint hemorrhage did not come to first-month followup, the control values of these two patients could not be obtained. CRP, serum ferritin, and lactic acid levels (Table 1) were found to be higher in hemophilic patients with acute joint bleeding compared with the control group (P < 0.05). Serum lactic acid levels were significantly higher in hemophilia patients who had no bleeding (P < 0.05), but these values were within normal range. CRP levels were found to be more than twice the upper limit of normal in hemophilic patients with acute joint hemorrhage. Serum VEGF, MIF, thrombomodulin, endostatin,
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Angiogenic and inflammatory factors in hemophilia A Karapınar et al. 705
Table 1
Laboratory results of the patients in the study and the control groups Patients with acute intra-articuler hemorrhage
Leukocyte (cells/ml) Monocyte (cells/ml) Hemoglobin (g/dl) Platelet count (cells/ml) CRP (mg/dl) Ferritin (ng/ml) Lactic acid (mmol/l)
Patients without acute intra-articular hemorrhage
Control group
Advanced joint damage (n ¼ 7)
Early joint damage (n ¼ 3)
Advanced joint damage (n ¼ 6)
Early joint damage (n ¼ 3)
No damage (n ¼ 16)
(n ¼ 22)
8577 505 13.4 240000 16.9 48.54 1.11
6233 500 12.1 266000 6.2 47.73 1.27
8516 451 13.9 239000 4.0 86.85 1.33
7500 500 11.9 304000 4.9 92.35 1.16
7753 599 12.7 253000 4.1 51.67 1.59
6572 500 13.1 267000 1.1 26.95 1.07
CRP, C-reactive protein.
and ICAM-1 levels were found to be similar to the control group (Table 2), and there was not any statistically significant difference between the groups (P ¼ 0.95, P ¼ 0.28, P ¼ 0.89, P ¼ 0.83 and P ¼ 0.83, respectively). There was not any significant difference among leukocyte count, hemoglobin levels, serum ferritin, lactic acid, VEGF, thrombomodulin, and endostatin (Table 3). When the laboratory results were compared in patients with early joint damage (n ¼ 3) and with advanced joint damage (n ¼ 7), among the patients who had acute joint hemorrhage, no significant difference was found. When the laboratory results were compared in patients with advanced joint damage (n ¼ 6) and with early joint damage (n ¼ 3), among the patients who had no acute joint hemorrhage, no significant difference was found. However, as the number of the cases in these compared groups were very few, we excluded these results from the evaluation. When the laboratory results were compared in all hemophilic patients with and without acute joint hemorrhage, those who had advanced joint damage (n ¼ 13) had significantly higher hemoglobin levels than those with early joint damage (n ¼ 6) (P < 0.05). There was no significant difference among leukocyte count, serum ferritin and lactic acid, VEGF, thrombomodulin, and endostatin levels (P ¼ 0.32, P ¼ 0.63, P ¼ 0.96, P ¼ 0.89, P ¼ 0.52, P ¼ 0.10, and P ¼ 0.57, respectively). Among all the hemophilic patients, when the laboratory results were compared between those with joint damage (n ¼ 19) and those without (n ¼ 16), CRP was significantly high in the patients with joint damage (P < 0.05) and Table 2
serum lactic acid level was significantly high in those without joint disease (P < 0.05). There was a positive correlation among the patients with early joint damage in terms of thrombomodulin, VEGF, ICAM-1, and lactic acid levels; however, these results were not statistically significant (P ¼ 0.77, P ¼ 0.77, P ¼ 0.77, respectively). No correlation was found between serum VEGF and ICAM-1 and lactic acid (P ¼ 0.15, P ¼ 0.42, P ¼ 0.54, respectively). There was a positive correlation between thrombomodulin levels and MIF in patients with advanced joint damage and a negative correlation was noted between MIF and endostatin levels in these patients; however these results were not statistically significant (P ¼ 0.70, P ¼ 0.33, respectively).
Discussion In spite of initiating regular F VIII concentrate replacements at early ages in patients with severe hemophilia, subclinic and clinically overt intra-articular hemorrhages currently cannot be prevented completely and permanent joint damages continue to occur [2–4]. It is not yet possible to determine the onset of intra-articular hemorrhage with clinical, laboratory, and radiologic imaging methods. This can be an important reason for the increase in joint damage. In our study, an investigation of the relation between plasma angiogenic and inflammatory factor levels in patients with severe hemophilia A and an examination of changes in these factor levels during joint hemorrhage were performed. Leukocytes play an important role in inflammation because of their antimicrobial, secretory, and phagocytic
Results of ELISA tests Patients with acute intra-articular hemorrhage
VEGF (pg/ml) Thrombomodulin (ng/ml) MIF (pg/ml) ICAM-1(pg/ml) Endostatin (ng/ml)
Patients without acute intra-articular hemorrhage
Control group
Advanced joint damage (n ¼ 7)
Early joint damage (n ¼ 3)
Advanced joint damage (n ¼ 6)
Early joint damage (n ¼ 3)
No damage (n ¼ 16)
(n ¼ 22)
113.208 0.42 75952 366.125 27.791
114.681 1.18 34204 360.228 26.215
70.615 0.45 40542 352.061 29.811
71.024 0.8 27104 380.809 24.683
148.089 0.97 19471 377.01 25.049
111.436 0.72 17491 374.289 27.332
ICAM-1, intercellular adhesion molecule-1; MIF, macrophage migration inhibitory factor; VEGF, vascular endothelial growth factor.
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706 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 7
activities [18]. We did not find any difference in terms of leukocyte and monocyte counts between hemophilic patients with and without joint damage and healthy control group in our study. There was no significant difference in patients who had joint hemorrhage. This can be because of the scarcity of the patients with hemorrhage or local bleeding not causing a systemic response. Exposure of the cells to hypoxia in the wound induces angiogenesis. It was shown that both local hypoxia and decrease in glucose occur because of diminishing of vascular perfusion; therefore, cells transform into an anaerobic metabolic pathway, production of lactate increases and extracellular pH is decreased and this new environment results in the secretion of cytokine/ enzyme from the macrophages. The release of angiogenic molecules by macrophages recruited to the wound is central to the formation of neoangiogenesis [19–21]. It is known that increase in the serum lactic acid level indicates tissue hypoxia. We did not notice any significant increase in lactic acid levels during acute joint hemorrhage in patients with hemophilia. Literature suggest that in patients with sepsis, myocardial infarction, and trauma, serum lactate levels rise and this can be used for followup [22–24]. When studying whether acute joint hemorrhage in hemophilic patients resulted in systemic hyperlactatemia, we did not find any significant increase. Blood normally does not exist in articular space. Synovial macrophages and condrocytes absorb the iron released by erythrocytes when intra-articular hemorrhage occurs. If bleeding episodes become more frequent, synovia regeneration becomes inadequate and iron accumulates [25–27]. When all hemophilic patients with or without acute joint bleeding were compared with the healthy control group, we found that serum ferritin levels were significantly higher in hemophilic patients. When a comparison was done between hemophilic patients with acute joint bleeding and the healthy control group, as well as between hemophilic patients and healthy controls, serum ferritin levels were higher compared with healthy controls. When the first-month laboratory findings of eight patients during and after the bleeding were compared, we did not find any significant difference between serum ferritin levels. It was shown that as an acute-phase reactant, ferritin was increased in patients with rheumatoid arthritis [28,29]. In inadequately treated hemophilic mice, it was observed that when hemorrhage recurred during recovery, in spite of more machrophage migration, iron staining persisted and iron was accumulated in deeper tissues. Angiogenesis rate was similar in hemophilic and healthy mice; however, the number of newly formed vessels was higher in hemophilic mice. It was suggested that proinflammatory and proangiogenic effects of tissue iron could result in this [10]. Our finding regarding higher levels of serum ferritin during acute bleeding compared with the healthy controls can be
related to the fact that ferritin is an acute phase reactant. However, in periods when no acute bleeding occurs, as shown in mice models, the presence of ferritin at higher levels compared with the healthy controls could be because of the accumulation of iron in deeper tissues as a result of recurrent bleedings. However, as the increased ferritin levels were within normal range, this is not considered as an important marker in the hemophilic patients. VEGF, one of the proangiogenic molecules released in response to hypoxia, is a basic signaling molecule for angiogenesis [11,30,31]. In this study, we did not find any difference in VEGF levels between all hemophilic patients with and without joint bleeding and the healthy control group. When the first-month laboratory findings of eight patients during and after the bleeding were compared, we did not find any significant difference between serum VEGF levels. In 2011, Acharya et al. [11] demonstrated that both serum and synovial VEGF levels were increased in hemophilic patients with joint hemorrhage. In the mentioned study which consisted of 14 patients, it was shown that both serum and synovial proangiogenic macrophage/monocytes (VEGFþ/CD68þ and VEGFR1þ/CD11bþ) levels were increased in patients’ hemophilic arthropathy. In the same study, a four-fold increase in both synovial and peripheral blood VEGF-A, stromal derivated factor-1, and matrix metalloprotease-9 was demonstrated in hemophilic arthropathy and it was shown by performing cell culture studies, by using human anti-VEGF antibody (bevacizumab) and by demonstrating the halt of synovial cell proliferation that VEGF inhibition could be an alternative in future treatment. It may be possible that we could not show an increase in VEGF levels because of scarcity of the patients. MIF is an important cytokine that plays a role in regulating inflammatory and immune response. In this study, we did not find any difference in MIF levels between all hemophilic patients with and without joint bleeding and the healthy control group. We found that serum MIF levels significantly increased during bleeding when the laboratory findings were compared at the time of and at the first month after the bleeding in patients with acute intra-articular hemorrhage. In recent studies, it was shown that MIF plays a role in several diseases such as hemorrhagic fever, rheumatoid arthritis, autoimmune diseases, and atherosclerosis [32–34]. Thrombomodulin is a membrane glycoprotein that is placed on the endothelial cell surface and acts as a thrombin receptor. It is basically found in vascular endothelial cells in humans [35]. In this study, we did not find any difference in thrombomodulin levels between all hemophilic patients with and without joint bleeding and the healthy control group. We found that serum thrombomodulin levels increased during bleeding when
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Angiogenic and inflammatory factors in hemophilia A Karapınar et al. 707
Table 3
Laboratory results of patients with acute joint bleeding During bleeding
Leukocyte (cells/ml) Hemoglobin (g/dl) CRP (mg/dl) Ferritin (ng/ml) Lactic acid (mmol/l) VEGF (pg/ml) Thrombomodulin (ng/ml) MIF (pg/ml) ICAM-1 (pg/ml) Endostatin (ng/ml)
One month after bleeding
Mean
Median
Mean
Median
P values
7937 13.1 16.3 50.33 1.13 115.689 0.91 74801 359.496 28.076
6300 12.9 7.3 39.94 1.11 111.883 0.68 36336 362.242 25.471
6850 13.2 3.1 39.11 1.18 144.542 0.57 23692 399.759 26.874
6900 13.6 1.9 38.01 1.16 139.127 0.46 12967 404.929 27.255
0.49 0.64 0.02 0.06 0.35 0.48 0.09 0.02 0.01 0.88
CRP, C-reactive protein; ICAM-1, intercellular adhesion molecule-1; MIF, macrophage migration inhibitory factor; VEGF, vascular endothelial growth factor.
the laboratory findings were compared at the time of and at the first month after the bleeding in patients with acute intra-articular hemorrhage, but this difference was insignificant. It was recently detected that thrombomodulin domain 2 and domain 3 has an angiogenic-promoting function. It was shown that TMD23, that has a recombinant thrombomodulin fragment, stimulates endothelial migration, proliferation, and tube formation. It was demonstrated that TMD23 leads to endothelial cell invasion and migration in early phases of angiogenesis by inducing release of matrix metalloproteins and plasminogen activators from the endothelial cells [36]. When a wound forms, monocytes move from circulation into tissue via LFA-1 cytokines on the monocyte surface and ICAM-1 cytokines on the endothelial cell surface [19]. In this study, we did not find any difference in ICAM-1 levels between all hemophilic patients with and without joint bleeding and healthy control group. It was shown that ICAM-1 plays an important role in inflammatory pathway and serum ICAM-1 levels increase in the early phases of osteoarthritis and rheumatoid arthritis [37–39]. We found that MIF and CRP serum levels increase during acute intra-articular hemorrhage. It was very recently shown that MIF increases both ICAM-1 and thrombomodulin release [33]. Since CRP is an acutephase reactant, a rise in CRP during acute hemorrhage is expected. Our study also suggested that CRP was higher in patients with joint damage. In childhood, the increase in CRP levels may be superior than clinical symptoms to demonstrate early acute joint bleeding. In the literature, it is stated that there is a significant relation between high ICAM-1 and high CRP [39,40]. There is a balance between proangiogenic agents and antiangiogenic agents during the process of angiogenesis. Angiostatin, thrombospondin, endostatin, and fibrinogen E fragment are main antiangiogenic agents [19,41]. In this study, we did not find any difference in endostatin levels between all hemophilic patients with and without joint bleeding and healthy control group. When the laboratory findings of eight patients during and 1 month
after the bleeding were compared, we did not find any significant difference between serum endostatin levels. It has been shown that neoangiogenesis is increased in tumor tissues and at the same time, endostatin release is increased [41–43]. There was a positive correlation among the patients with early joint damage in terms of thrombomodulin, VEGF, ICAM-1, and lactic acid levels; however, this result was not statistically significant. These results can be because of low number of the cases in this group. We did not find any correlation between serum VEGF and ICAM-1 and lactic acid levels. There was a positive correlation between thrombomodulin levels and MIF in patients with advanced joint damage and a negative correlation was noted between MIF and endostatin levels in these patients; however, these results were not statistically significant. Although statistically insignificant, we believe that the positive correlation between thrombomodulin levels and VEGF and MIF is important. This correlation between proangiogenic molecules during inflammation was also shown in different patient groups [11,33]. Despite the advantages of MRI over conventional X-ray, because of its rather difficult accessibility, its requirement of sedations for the children and its high cost, biological markers are needed to indicate hemophilic arthropathy in early phases [5,11]. Marked increase in CRP during acute hemorrhage suggested that CRP could be a good indicator. In childhood, the increase in CRP levels may be superior to clinical symptoms to demonstrate early acute joint bleeding. Besides, MIF levels rise in case of hemorrhage and it support the presence of hemorrhage regardless of severity of the joint damage. We think that levels of these factors can give information during early phase in acute bleedings in patients with severe hemophilia A. Therefore, we expect that antiangiogenic or anti-inflammatory treatments can prevent recurrent hemorrhages and following arthropathy.
Acknowledgements This study was supported by Dokuz Eylu¨l University Research Fund.
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708 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 7
Conflicts of interest
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None of the authors express a conflict of interest. 23
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