Blood Cells, Molecules and Diseases 53 (2014) 189–193
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Growth differentiation factor-15 in young sickle cell disease patients: Relation to hemolysis, iron overload and vascular complications Azza Abdel Gawad Tantawy a,⁎, Amira Abdel Moneam Adly a, Eman Abdel Rahman Ismail b, Yasser Wagih Darwish b, Marwa Ali Zedan a a b
Pediatrics Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt Clinical Pathology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt
a r t i c l e
i n f o
Article history: Submitted 3 July 2014 Revised 5 July 2014 Accepted 5 July 2014 Available online 25 July 2014 (Communicated by M. Narla, DSc, 05 July 2014) Keywords: Growth differentiation factor-15 Sickle cell disease Ineffective erythropoiesis Iron overload
a b s t r a c t Background: High expression of growth differentiation factor-15 (GDF-15) contributes to pathological iron overload in thalassemia. Sickle cell syndromes are characterized by increased levels of erythropoiesis, although the primary defect involves the destruction of mature erythrocytes. Aim: To determine serum GDF-15 in 35 children and adolescents with sickle cell disease (SCD) compared to 35 healthy controls and assess its relation to markers of hemolysis, iron overload and vascular complications. Methods: GDF-15 was measured and correlated to genotype, frequency of sickling crises, hydroxyurea therapy and serum ferritin. Results: GDF-15 levels were increased in SCD patients whether sickle cell anemia or sickle β° thalassemia compared with controls (p b 0.001) with no significant difference between patients' groups. GDF-15 was significantly higher in patients who had serum ferritin ≥2500 μg/L, previous cerebral stroke, and splenectomy. GDF-15 was not significantly related to frequency of sickling crises, pulmonary hypertension, or hydroxyurea therapy. On regression analysis, transfusion index, lactate dehydrogenase and serum ferritin were independently related to GDF-15. Conclusion: Increased GDF-15 in SCD reflects the importance of ineffective erythropoiesis in the pathophysiology and severity of anemia in SCD. GDF-15 levels are related to hemolysis and iron overload and may provide utility for identifying patients at increased risk of thrombotic events. © 2014 Elsevier Inc. All rights reserved.
Introduction Sickle cell disease (SCD) is a multisystem disease associated with episodes of acute illness and progressive organ damage, and one of the most common monogenic disorders worldwide, affecting an estimated 30 million people. SCD represents a major public health problem because of its associated morbidity and mortality [1,2]. SCD is a hereditary hemoglobinopathy characterized by microvascular vaso-occlusion with erythrocytes containing polymerized sickle (S) hemoglobin, erythrocyte hemolysis, vasculopathy, and both acute and chronic multiorgan injury [3]. Increased expression of adhesion molecules on erythrocytes and endothelial cells, interactions with leukocytes, increased levels of circulating inflammatory cytokines, enhanced microvascular thrombosis, and endothelial damage are all thought to contribute to obstruction of the arterioles by sickled erythrocytes. Vascular dysfunction is the end result, due to complex and multifactorial interactions that ultimately manifest as the clinical phenotypes of SCD [4].
The measurement of growth differentiation factor-15 (GDF-15), a member of the transforming growth factor-β superfamily, is thought to predict ineffective or apoptotic erythropoiesis [5]. GDF-15 levels were found to be significantly elevated in patients with β-thalassemia major [6] and intermedia [7]. Furthermore, sera from those patients suppressed expression of the iron regulatory protein hepcidin, with a subsequent reversal of suppression when GDF-15 was depleted [6]. Like thalassemia syndromes, sickle cell syndromes are characterized by increased levels of erythropoiesis, although the primary defect in sickle cell involves destruction of mature erythrocytes. In severe cases of sickle cell disease, some ineffective erythropoiesis may be found [8]. However, data on GDF-15 levels among patients with SCD are lacking and its relation to disease severity has not been explored. Therefore, the aim of this study was to assess the levels of GDF-15 in SCD patients and its relation to markers of hemolysis and iron overload, and to evaluate its role as potential markers for vascular complications. Materials and methods
⁎ Corresponding author at: 22 Ahmed Amin Street, St. Fatima Square, Heliopolis, Cairo, Egypt. Fax: +20 2 22400507. E-mail address: [email protected] (A.A.G. Tantawy).
http://dx.doi.org/10.1016/j.bcmd.2014.07.003 1079-9796/© 2014 Elsevier Inc. All rights reserved.
This cross sectional study included 35 patients with SCD (23 males and 12 females) recruited from the regular attendants of the Pediatric
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Hematology Clinic, Pediatric Hospital, Ain Shams University. Thirty five age- and sex-matched healthy subjects were enrolled as a control group (19 males and 16 females). The median age of SCD patients was 12 years (range: 3.2–18 years) while that of controls was 10.9 (range: 3–18 years). An informed consent was obtained from the guardian of each patient or control before participation. The procedures applied in this study were approved by the Ethical Committee of Human Experimentation of Ain Shams University, and are in accordance with the Helsinki Declaration of 1975. All included patients were subjected to detailed medical history and thorough clinical examination with special emphasis on disease duration, anthropometric measures, evidence of renal, hepatic or cardiac disease, frequency of sickling crisis, and history of splenectomy (for HbSβ-thalassemia patients). The transfusion received was calculated as the transfusion index: volume of transfused packed red cells in mL per kg body weight per year (expressed as the mean value of the last three years). SCD patients included 22 patients with sickle cell anemia (SCA) and 13 patients with sickle β° thalassemia and all were at steady state at the time of sample collection. Twenty-seven patients received hydroxyurea (Bristol-Meyers-Squibb, NY, USA) as an oral daily dose ranging from 15 to 25 mg/kg/day while deferoxamine therapy (Desferal®, DFO; Novartis Pharma AG, Basel, Switzerland) was given subcutaneously in a dose that ranged from 30 to 40 mg/kg/day 5 days/week.
extremities, back, abdomen, chest, or head that lasted at least 2 hours, led to a clinic visit, and could not be explained except by SCD [10] while steady state was defined as a period without pain or painful crisis for at least 4 weeks [11]. The frequency of sickling crisis in the previous year was divided into mild (defined as 2 or less episodes requiring medical visits) or severe (defined as 3 or more episodes requiring medical visits) [12]. A TRV ≥ 2.5 m/s was used as a proxy for patients at risk for pulmonary hypertension [13,14]. Statistical analysis The analysis of data was done using Statistical Program for Social Science version 15 (SPSS Inc., Chicago, IL, USA). Quantitative variables were described in the form of mean and standard deviation or median and interquartile range (IQR; 75th and 25th percentiles). Qualitative variables were described as number and percent. In order to compare parametric quantitative variables between two groups, Student t-test was applied. For comparison of non-parametric quantitative variables between two groups, Mann–Whitney test was used. Qualitative variables were compared using Chi-square (Χ2) test or Fischer's exact test when frequencies were below five. Correlation studies were done using Pearson's correlation coefficient. Multi-regression linear analysis was employed to determine the relation between GDF-15 and clinical and laboratory variables. A p value b 0.05 was considered significant or p value b0.001 was considered highly significant in all analyses.
Sample collection Results Peripheral blood samples were collected on potassium-ethylene diamine tetra-acetic acid (K2-EDTA) (1.2 mg/mL) for complete blood count (CBC) and hemoglobin analysis. For chemical analysis and enzyme linked immunosorbent assay (ELISA), clotted samples were obtained and serum was separated by centrifugation for 15 min at 1000 ×g then stored at −20 °C till subsequent use in ELISA. Laboratory and radiological assessment Laboratory investigations included CBC using Sysmex XT-1800i (Sysmex, Japan), examination of Leishman-stained smears for differential white blood cell (WBC) count, hemoglobin analysis by high performance liquid chromatography (HPLC) using D-10 (BioRad, Marnes La Coquette, France), liver and kidney function tests, markers of hemolysis (lactate dehydrogenase [LDH] and indirect bilirubin) and high sensitivity C-reactive protein (hs-CRP) as well as serum ferritin on Cobas Integra 800 (Roche Diagnostics, Mannheim, Germany). Patients with any clinical evidence of infection or CRP N 10 mg/L were excluded. Serum ferritin level was measured at the start of the study with calculation of the mean value of the last year prior to the study in order to know the ferritin trend. The determination of serum levels of GDF-15 was done by ELISA using Quantikine Human GDF-15 Immunoassay (R&D systems, Minneapolis, Minnesota). All studied patients were clinically asymptomatic for pulmonary hypertension and cardiovascular abnormalities. Screening for pulmonary hypertension was performed by the non-invasive Doppler echocardiography with different modalities using Vivid E9 (GE Healthcare, Norway) to evaluate pulmonary artery pressure and tricuspid regurgitant jet velocity (TRV). Diagnostic criteria The definition of SCD was based on complete blood picture, reticulocyte count, markers of hemolysis as well as hemoglobin analysis using HPLC and confirmed by genotyping based on the identification of β-globin gene mutations by PCR implications of the DNA and subsequent reverse-hybridization to immobilized allele-specific biotinylated oligonucleotide probes covering the most common Mediterranean mutations [9]. A painful crisis was defined as the occurrence of pain in the
In this study, 31.4% of SCD patients had pulmonary hypertension risk and 25.7% had history of cerebral stroke. In addition, 31.4% of patients were splenectomized and 77.1% were on hydroxyurea therapy (Table 1). The median (IQR) of GDF-15 level in SCD patients was 2750 (1200) pg/mL compared to 90 (120) pg/mL in controls (p b 0.001). Patients with SCA or sickle β° thalassemia had higher GDF-15 levels than controls (p b 0.001; Fig. 1) while no significant difference between both patients' groups (p = 0.338; Table 2). GDF-15 levels were significantly higher in patients who had serum ferritin ≥2500 μg/L (p b 0.001), previous cerebral stroke (p = 0.022), and splenectomy (p = 0.018) (Fig. 2). GDF-15 was not significantly related to age, sex, severity of Table 1 Clinical and laboratory data of the studied sickle cell disease patients. Variable
SCD (n = 35)
Age (years), median (IQR) Males, n (%) Weight SDS, median (IQR) Height SDS, median (IQR) Transfusion index (mL/kg/year), mean ± SD Splenectomized, n (%) Viral hepatitis, n (%) History of cerebral stroke, n (%) Pulmonary hypertension, n (%) History of sickling crisis (≥3 attacks/year), n (%) Positive hydroxyurea therapy, n (%) WBC count (×109/L), mean ± SD Hemoglobin (g/dL), mean ± SD HbS (%), mean ± SD HbF (%), mean ± SD Lactate dehydrogenase (IU/L), mean ± SD Indirect bilirubin (mg/dL), mean ± SD Serum ferritin (μg/L), median (IQR) Ferritin b2500, n (%) Ferritin ≥2500, n (%) GDF-15 (pg/mL), median (IQR) Range
12.1 (8.7) 23 (65.7) −0.5 (2.6) −0.95 (1.93) 200.5 ± 143 11 (31.4) 6 (17.1) 9 (25.7) 11 (31.4) 13 (37.1) 27 (77.1) 12.9 ± 4.4 8.7 ± 1 58.6 ± 22.3 8.8 ± 5.7 1152.1 ± 455.5 1.35 ± 0.54 2890 (3520) 16 (45.7) 19 (54.3) 2750 (1200) 465–4500
SCD; sickle cell disease; SDS: standard deviation score; IQR: inter-quartile range; WBC: white blood cell; Hb: hemoglobin; GDF-15: growth differentiation factor-15; IQR: interquartile range.
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Fig. 1. GDF-15 and serum ferritin levels in relation to genotype of the studied sickle cell disease patients.
sickling crisis, pulmonary hypertension, or hydroxyurea therapy (Table 2). Patients with sickle β° thalassemia had elevated serum ferritin levels (median [IQR], 2534 [1000–4069) μg/L versus 3125 [2500–6310] μg/L; p = 0.04) than those with SCA (Fig. 1). GDF-15 levels were positively correlated with transfusion index (r = 0.874, p b 0.001), LDH (r = 0.773, p b 0.001), indirect bilirubin (r = 0.432, p = 0.009) and serum ferritin (r = 0.790, p b 0.001), while negatively correlated to hemoglobin (r = − 0.621, p b 0.001) and HbF% (r = −0.837, p b 0.001) (Fig. 3). GDF-15 was not correlated with age (r = 0.135, p = 0.441). Multiple regression analysis revealed that transfusion index (p = 0.001), LDH (p = 0.042) and serum ferritin (p = 0.006) were independently related to GDF-15 levels in SCD patients (r2 = 0.929, p b 0.001). Discussion GDF-15 has been identified in different contexts as a hypoxiainducible gene product and as a molecule involved in hepcidin regulation. Table 2 GDF-15 levels in relation to clinical characteristics of sickle cell disease patients. Variable Sex Male Female Genotype SCA (Homozygous SS) Sickle β° thalassemia Splenectomy Positive Negative Viral hepatitis Positive Negative History of cerebral stroke Positive Negative Pulmonary hypertension Positive Negative History of frequent sickling crisis Positive Negative Serum ferritin (μg/L) Ferritin ≥2500 Ferritin b2500 Hydroxyurea therapy Positive Negative
GDF-15 (pg/mL) Median (range)
p value
GDF-15 expression at the high levels achieved in the setting of ineffective erythropoiesis contributes to pathological iron overloading through a mechanism of incomplete hepcidin suppression [15]. We assessed the level of GDF-15 in young patients with SCD and investigated its role as a potential marker for disease activity. In this study, GDF-15 was significantly elevated in SCD patients, even in patients with SCA, compared with healthy controls denoting the presence of increased erythropoiesis. To our knowledge, no previous studies analyzed GDF-15 levels in SCD; however, increased serum GDF-15 levels in patients with other erythroid disorders have been reported [5]. GDF-15 could constitute a marker for ineffective erythropoiesis in the differential diagnosis of cases of unexplained anemia [6]. A significant increase in GDF-15 has been found in thalassemia major and transfusion-dependent thalassemia intermedia [5,7]. GDF-15 mRNA was absent in normal bone marrow precursors and in peripheral blood cells [5]. Some ineffective erythropoiesis has been shown in SCD [8]. It has been proposed that ineffective erythropoiesis causes iron overloading by not permitting the appropriate rise in hepcidin expression to occur once the erythroid demand is met [16]. Conceivably, while generating systemic iron overload, ineffective erythropoiesis and associated iron fluxes might generate an iron-deficiency signal in a relevant molecular
0.614 2700 (570–4500) 2950 (465–4500) 0.338 2700 (465–4200) 2800 (600–4500) 0.018 3250 (1200–4500) 2600 (465–4200) 0.101 3225 (2250–4500) 2700 (465–4500) 0.022 3450 (660–4500) 2700 (465–3800) 0.414 2800 (660–4500) 2700 (465–4500) 0.434 3000 (660–4500) 2800 (465–4200) b0.001 3250 (2350–4500) 1950 (465–2800) 0.596 2750 (465–4500) 2950 (600–4500)
SCA: sickle cell anemia; GDF-15: growth differentiation factor-15.
Fig. 2. GDF-15 in relation to splenectomy and serum ferritin among sickle cell disease patients.
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Fig. 3. Positive correlation between GDF-15 and lactate dehydrogenase and serum ferritin in sickle cell disease patients.
or cellular context and consequent stimulation of GDF-15 expression in a particular erythroid compartment. Therefore, further dissection of GDF-15 regulation by iron and oxygen in erythroid progenitors should be of interest and may provide mechanistic explanations to elevated GDF-15 levels in thalassemia patients [17]. This could also explain elevated GDF-15 levels in our SCD patients. Hemolysis appears to predispose SCD patients to a vasculopathy that may manifest as one or more of several complications: pulmonary hypertension, leg ulcers, priapism, and possibly stroke [18]. The chronic hemolysis in SCD may be also complicated by arterial or venous thrombosis. Several pathogenic factors contribute to this complication, including a chronic hypercoagulable state [19], increased erythrocyte aggregation and endothelial adhesion of erythrocytes in microvessels. It is often a combination of these factors that leads to thromboembolic events [20]. We found increased GDF-15 levels among patients with previous manifest or silent stroke. This is because GDF-15 is a stressresponsive cytokine that is upregulated under pathologic conditions involving various stimuli such as tissue hypoxia, inflammation, or enhanced oxidative stress that occur in thrombosis [21]. Moreover, ineffective erythropoiesis results in the secondary release into the circulation of damaged red blood cells with thrombogenic potential that reflect in a high rate of thromboembolic events [22,23]. Since GDF-15 has been regarded as a marker of ineffective erythropoiesis, our findings also confirm the substantial role of ineffective erythropoiesis in the pathophysiology and clinical severity of SCD. Ineffective erythropoiesis, and the subsequent chronic anemia and hypoxia, lead to hepcidin suppression, increased iron absorption from the gut, and increased release of recycled iron from the reticuloendothelial system. This results in the depletion of macrophage iron, relatively low levels of serum ferritin, and preferential portal and hepatocyte iron storage [7]. This in turn leads to considerable hepatic iron overload (suggested by a positive correlation between GDF-15 and serum ferritin in this study and the significant increase of GDF-15 among patients with serum ferritin ≥ 2500 μg/L) and releases into the circulation of toxic iron species like nontransferrin-bound iron, which can lead to targetorgan damage [24,25]. GDF-15 was strongly expressed in alveolar macrophages which might indicate a role of this protein in innate immunity [26]. Immunostaining experiments clearly demonstrated strong expression of GDF-15 in the vascular compartment of patients with pulmonary
hypertension. GDF-15 staining was observed in pulmonary vessels of all sizes, beginning from the microvasculature up to large pulmonary vessels and particularly in the intima of pulmonary arteries [27]. Hypoxia is a potent stimulator of GDF-15 expression in pulmonary endothelial cells. Furthermore, stress might lead to the induction of GDF-15 expression in the pulmonary vasculature [28]. However, GDF-15 was not different between our SCD patients with or without pulmonary hypertension may be because the pathology of pulmonary hypertension in SCD is multi-factorial and differs from that in thalassemia. Therefore, further studies are needed to confirm the association between GDF-15 levels and erythropoietic drive in SCD. In the present work, 31.4% of SCD patients were splenectomized. Although the prominent feature of SCD is autosplenectomy, a possible explanation to patients who underwent splenectomy is the presence of sickle thalassemia with hypersplenism [29]. Splenectomized patients had significantly higher levels of GDF-15 denoting the important role of the spleen in endothelial dysfunction and vascular complications in SCD. Similarly, Musallam et al. [7] evaluated GDF-15 levels in 55 patients with thalassemia intermedia as a marker of ineffective erythropoiesis in several anemias. GDF-15 levels were significantly higher in splenectomized compared to non-splenectomized patients. Splenic reticuloendothelial cells serve a critical function in the removal of cells that express phosphatidylserine on their surface (such as apoptotic and ageing cells) including senescent and damaged erythrocytes [22]. Following surgical or autosplenectomy, the rate of intravascular hemolysis increases and senescent and abnormal erythrocytes in the circulation trigger platelet activation, promoting pulmonary micro-thrombosis and red cell adhesion to the endothelium with subsequent erythrocyte fragmentation during hemolysis and generation of large amount of GDF-15 [8]. In SCD, GDF-15 was significantly increased in patients with serum ferritin N2500 μg/L suggesting a relation between them and iron overload. Iron may contribute directly to hemolysis, endothelial damage and vasculopathy. Iron-derived reactive oxygen species are implicated in the pathogenesis of several vascular disorders including atherosclerosis, microangiopathic hemolytic anemia, vasculitis, and reperfusion injury [30]. The relationship between iron overload and the severity of ineffective erythropoiesis seems to be bidirectional. Iron overload may aggravate ineffective erythropoiesis and the secondary release into the circulation of damaged red blood cells with thrombogenic potential.
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Therefore, managing iron overload with iron chelators or more novel therapeutics could improve the efficiency of erythropoiesis and the survival of the resulting reticulocytes and erythrocytes [31]. In this study, GDF-15 was positively correlated to transfusion index, markers of hemolysis and serum ferritin while negatively correlated to hemoglobin and HbF. This further shows that its increase may be a result of tissue ischemia and hypoxia. Similarly, Musallam et al. [7] found that GDF-15 levels were significantly correlated with anemia, markers of iron overload, and a pre-defined clinical severity score in thalassemia intermedia patients. Tanno et al. [6] reported that individuals with beta-thalassemia syndromes had elevated GDF-15 serum levels that were positively correlated with the levels of soluble transferrin receptor, erythropoietin and ferritin. These results suggest that GDF15 overexpression arising from an expanded erythroid compartment contributes to iron overload in thalassemia syndromes by inhibiting hepcidin expression [6] and subsequently contributes to tissue iron overloading in those patients [32]. Recently, the iron and erythropoiesis-controlled GDF-15 has been shown to inhibit the expression of hepcidin in beta-thalassemia patients, thereby increasing iron absorption despite iron overload [32]. Moreover, in congenital dyserythropoietic anemia which is a rare group of red blood cell disorders characterized by ineffective erythropoiesis and increased iron absorption, GDF-15 was correlated significantly with several erythropoietic and iron parameters including hepcidin-25, ferritin, and hepcidin-25/ferritin ratios. These results suggest that those patients express very high levels of serum GDF-15 which contributes to the inappropriate suppression of hepcidin [8]. A more complete understanding of GDF-15 signal transduction and functions in health and disease are topics for future research [32]. In conclusion, increased GDF-15 in SCD reflects the importance of ineffective erythropoiesis in the pathophysiology and clinical severity of anemia in SCD. GDF-15 levels are significantly related to markers of hemolysis and iron overload and may provide utility for identifying SCD patients who are at increased risk of thrombotic events. Limitations of the study The relatively small number of patients included is an important limiting factor. However, the results obtained suggest the importance of further larger studies to verify the practical utility of GDF-15 measurement in SCD and its potential to predict the clinical severity and disease outcome. It is also worth to investigate GDF-15 levels in SCD patients less than one year of age as sickling is expected to be less severe in this age group [33]. References [1] A. Inati, S. Koussa, A. Taher, S. Perrine, Sickle cell disease: new insights into pathophysiology and treatment, Pediatr. Ann. 37 (2008) 311–321. [2] D.C. Rees, T.H. Williams, M.T. Gladwin, Sickle-cell disease, Lancet 376 (2010) 2018–2031. [3] K.C. Wood, L.L. Hsu, M.T. Gladwin, Sickle cell disease vasculopathy: a state of nitric oxide resistance, Free Radic. Biol. Med. 44 (2008) 1506–1528. [4] S. Moncada, A. Higgs, The L-arginine-nitric oxide pathway, N. Engl. J. Med. 329 (2012) 2002–2012. [5] T. Tanno, P. Noel, J.L. Miller, P.A. Oneal, S.H. Goh, Growth differentiation factor 15 in erythroid health and disease, Curr. Opin. Hematol. 17 (2010) 184–190. [6] T. Tanno, N.V. Bhanu, P.A. Oneal, S.H. Goh, P. Staker, Y.T. Lee, J.W. Moroney, C.H. Reed, N.L. Luban, R.H. Wang, T.E. Eling, R. Childs, T. Ganz, S.F. Leitman, High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin, Nat. Med. 13 (2007) 1096–1101. [7] K.M. Musallam, A.T. Taher, L. Duca, C. Cesaretti, R. Halawi, M. Cappellini, Levels of growth differentiation factor-15 are high and correlate with clinical severity in transfusion-independent patients with β thalassemia intermedia, Blood Cells Mol. Dis. 47 (2011) 232–234.
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Growth differentiation factor-15 in young sickle cell disease patients: Relation to hemolysis, iron overload and vascular complications Azza Abdel Gawad Tantawy a,⁎, Amira Abdel Moneam Adly a, Eman Abdel Rahman Ismail b, Yasser Wagih Darwish b, Marwa Ali Zedan a a b
Pediatrics Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt Clinical Pathology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt
a r t i c l e
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Article history: Submitted 3 July 2014 Revised 5 July 2014 Accepted 5 July 2014 Available online 25 July 2014 (Communicated by M. Narla, DSc, 05 July 2014) Keywords: Growth differentiation factor-15 Sickle cell disease Ineffective erythropoiesis Iron overload
a b s t r a c t Background: High expression of growth differentiation factor-15 (GDF-15) contributes to pathological iron overload in thalassemia. Sickle cell syndromes are characterized by increased levels of erythropoiesis, although the primary defect involves the destruction of mature erythrocytes. Aim: To determine serum GDF-15 in 35 children and adolescents with sickle cell disease (SCD) compared to 35 healthy controls and assess its relation to markers of hemolysis, iron overload and vascular complications. Methods: GDF-15 was measured and correlated to genotype, frequency of sickling crises, hydroxyurea therapy and serum ferritin. Results: GDF-15 levels were increased in SCD patients whether sickle cell anemia or sickle β° thalassemia compared with controls (p b 0.001) with no significant difference between patients' groups. GDF-15 was significantly higher in patients who had serum ferritin ≥2500 μg/L, previous cerebral stroke, and splenectomy. GDF-15 was not significantly related to frequency of sickling crises, pulmonary hypertension, or hydroxyurea therapy. On regression analysis, transfusion index, lactate dehydrogenase and serum ferritin were independently related to GDF-15. Conclusion: Increased GDF-15 in SCD reflects the importance of ineffective erythropoiesis in the pathophysiology and severity of anemia in SCD. GDF-15 levels are related to hemolysis and iron overload and may provide utility for identifying patients at increased risk of thrombotic events. © 2014 Elsevier Inc. All rights reserved.
Introduction Sickle cell disease (SCD) is a multisystem disease associated with episodes of acute illness and progressive organ damage, and one of the most common monogenic disorders worldwide, affecting an estimated 30 million people. SCD represents a major public health problem because of its associated morbidity and mortality [1,2]. SCD is a hereditary hemoglobinopathy characterized by microvascular vaso-occlusion with erythrocytes containing polymerized sickle (S) hemoglobin, erythrocyte hemolysis, vasculopathy, and both acute and chronic multiorgan injury [3]. Increased expression of adhesion molecules on erythrocytes and endothelial cells, interactions with leukocytes, increased levels of circulating inflammatory cytokines, enhanced microvascular thrombosis, and endothelial damage are all thought to contribute to obstruction of the arterioles by sickled erythrocytes. Vascular dysfunction is the end result, due to complex and multifactorial interactions that ultimately manifest as the clinical phenotypes of SCD [4].
The measurement of growth differentiation factor-15 (GDF-15), a member of the transforming growth factor-β superfamily, is thought to predict ineffective or apoptotic erythropoiesis [5]. GDF-15 levels were found to be significantly elevated in patients with β-thalassemia major [6] and intermedia [7]. Furthermore, sera from those patients suppressed expression of the iron regulatory protein hepcidin, with a subsequent reversal of suppression when GDF-15 was depleted [6]. Like thalassemia syndromes, sickle cell syndromes are characterized by increased levels of erythropoiesis, although the primary defect in sickle cell involves destruction of mature erythrocytes. In severe cases of sickle cell disease, some ineffective erythropoiesis may be found [8]. However, data on GDF-15 levels among patients with SCD are lacking and its relation to disease severity has not been explored. Therefore, the aim of this study was to assess the levels of GDF-15 in SCD patients and its relation to markers of hemolysis and iron overload, and to evaluate its role as potential markers for vascular complications. Materials and methods
⁎ Corresponding author at: 22 Ahmed Amin Street, St. Fatima Square, Heliopolis, Cairo, Egypt. Fax: +20 2 22400507. E-mail address: [email protected] (A.A.G. Tantawy).
http://dx.doi.org/10.1016/j.bcmd.2014.07.003 1079-9796/© 2014 Elsevier Inc. All rights reserved.
This cross sectional study included 35 patients with SCD (23 males and 12 females) recruited from the regular attendants of the Pediatric
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Hematology Clinic, Pediatric Hospital, Ain Shams University. Thirty five age- and sex-matched healthy subjects were enrolled as a control group (19 males and 16 females). The median age of SCD patients was 12 years (range: 3.2–18 years) while that of controls was 10.9 (range: 3–18 years). An informed consent was obtained from the guardian of each patient or control before participation. The procedures applied in this study were approved by the Ethical Committee of Human Experimentation of Ain Shams University, and are in accordance with the Helsinki Declaration of 1975. All included patients were subjected to detailed medical history and thorough clinical examination with special emphasis on disease duration, anthropometric measures, evidence of renal, hepatic or cardiac disease, frequency of sickling crisis, and history of splenectomy (for HbSβ-thalassemia patients). The transfusion received was calculated as the transfusion index: volume of transfused packed red cells in mL per kg body weight per year (expressed as the mean value of the last three years). SCD patients included 22 patients with sickle cell anemia (SCA) and 13 patients with sickle β° thalassemia and all were at steady state at the time of sample collection. Twenty-seven patients received hydroxyurea (Bristol-Meyers-Squibb, NY, USA) as an oral daily dose ranging from 15 to 25 mg/kg/day while deferoxamine therapy (Desferal®, DFO; Novartis Pharma AG, Basel, Switzerland) was given subcutaneously in a dose that ranged from 30 to 40 mg/kg/day 5 days/week.
extremities, back, abdomen, chest, or head that lasted at least 2 hours, led to a clinic visit, and could not be explained except by SCD [10] while steady state was defined as a period without pain or painful crisis for at least 4 weeks [11]. The frequency of sickling crisis in the previous year was divided into mild (defined as 2 or less episodes requiring medical visits) or severe (defined as 3 or more episodes requiring medical visits) [12]. A TRV ≥ 2.5 m/s was used as a proxy for patients at risk for pulmonary hypertension [13,14]. Statistical analysis The analysis of data was done using Statistical Program for Social Science version 15 (SPSS Inc., Chicago, IL, USA). Quantitative variables were described in the form of mean and standard deviation or median and interquartile range (IQR; 75th and 25th percentiles). Qualitative variables were described as number and percent. In order to compare parametric quantitative variables between two groups, Student t-test was applied. For comparison of non-parametric quantitative variables between two groups, Mann–Whitney test was used. Qualitative variables were compared using Chi-square (Χ2) test or Fischer's exact test when frequencies were below five. Correlation studies were done using Pearson's correlation coefficient. Multi-regression linear analysis was employed to determine the relation between GDF-15 and clinical and laboratory variables. A p value b 0.05 was considered significant or p value b0.001 was considered highly significant in all analyses.
Sample collection Results Peripheral blood samples were collected on potassium-ethylene diamine tetra-acetic acid (K2-EDTA) (1.2 mg/mL) for complete blood count (CBC) and hemoglobin analysis. For chemical analysis and enzyme linked immunosorbent assay (ELISA), clotted samples were obtained and serum was separated by centrifugation for 15 min at 1000 ×g then stored at −20 °C till subsequent use in ELISA. Laboratory and radiological assessment Laboratory investigations included CBC using Sysmex XT-1800i (Sysmex, Japan), examination of Leishman-stained smears for differential white blood cell (WBC) count, hemoglobin analysis by high performance liquid chromatography (HPLC) using D-10 (BioRad, Marnes La Coquette, France), liver and kidney function tests, markers of hemolysis (lactate dehydrogenase [LDH] and indirect bilirubin) and high sensitivity C-reactive protein (hs-CRP) as well as serum ferritin on Cobas Integra 800 (Roche Diagnostics, Mannheim, Germany). Patients with any clinical evidence of infection or CRP N 10 mg/L were excluded. Serum ferritin level was measured at the start of the study with calculation of the mean value of the last year prior to the study in order to know the ferritin trend. The determination of serum levels of GDF-15 was done by ELISA using Quantikine Human GDF-15 Immunoassay (R&D systems, Minneapolis, Minnesota). All studied patients were clinically asymptomatic for pulmonary hypertension and cardiovascular abnormalities. Screening for pulmonary hypertension was performed by the non-invasive Doppler echocardiography with different modalities using Vivid E9 (GE Healthcare, Norway) to evaluate pulmonary artery pressure and tricuspid regurgitant jet velocity (TRV). Diagnostic criteria The definition of SCD was based on complete blood picture, reticulocyte count, markers of hemolysis as well as hemoglobin analysis using HPLC and confirmed by genotyping based on the identification of β-globin gene mutations by PCR implications of the DNA and subsequent reverse-hybridization to immobilized allele-specific biotinylated oligonucleotide probes covering the most common Mediterranean mutations [9]. A painful crisis was defined as the occurrence of pain in the
In this study, 31.4% of SCD patients had pulmonary hypertension risk and 25.7% had history of cerebral stroke. In addition, 31.4% of patients were splenectomized and 77.1% were on hydroxyurea therapy (Table 1). The median (IQR) of GDF-15 level in SCD patients was 2750 (1200) pg/mL compared to 90 (120) pg/mL in controls (p b 0.001). Patients with SCA or sickle β° thalassemia had higher GDF-15 levels than controls (p b 0.001; Fig. 1) while no significant difference between both patients' groups (p = 0.338; Table 2). GDF-15 levels were significantly higher in patients who had serum ferritin ≥2500 μg/L (p b 0.001), previous cerebral stroke (p = 0.022), and splenectomy (p = 0.018) (Fig. 2). GDF-15 was not significantly related to age, sex, severity of Table 1 Clinical and laboratory data of the studied sickle cell disease patients. Variable
SCD (n = 35)
Age (years), median (IQR) Males, n (%) Weight SDS, median (IQR) Height SDS, median (IQR) Transfusion index (mL/kg/year), mean ± SD Splenectomized, n (%) Viral hepatitis, n (%) History of cerebral stroke, n (%) Pulmonary hypertension, n (%) History of sickling crisis (≥3 attacks/year), n (%) Positive hydroxyurea therapy, n (%) WBC count (×109/L), mean ± SD Hemoglobin (g/dL), mean ± SD HbS (%), mean ± SD HbF (%), mean ± SD Lactate dehydrogenase (IU/L), mean ± SD Indirect bilirubin (mg/dL), mean ± SD Serum ferritin (μg/L), median (IQR) Ferritin b2500, n (%) Ferritin ≥2500, n (%) GDF-15 (pg/mL), median (IQR) Range
12.1 (8.7) 23 (65.7) −0.5 (2.6) −0.95 (1.93) 200.5 ± 143 11 (31.4) 6 (17.1) 9 (25.7) 11 (31.4) 13 (37.1) 27 (77.1) 12.9 ± 4.4 8.7 ± 1 58.6 ± 22.3 8.8 ± 5.7 1152.1 ± 455.5 1.35 ± 0.54 2890 (3520) 16 (45.7) 19 (54.3) 2750 (1200) 465–4500
SCD; sickle cell disease; SDS: standard deviation score; IQR: inter-quartile range; WBC: white blood cell; Hb: hemoglobin; GDF-15: growth differentiation factor-15; IQR: interquartile range.
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Fig. 1. GDF-15 and serum ferritin levels in relation to genotype of the studied sickle cell disease patients.
sickling crisis, pulmonary hypertension, or hydroxyurea therapy (Table 2). Patients with sickle β° thalassemia had elevated serum ferritin levels (median [IQR], 2534 [1000–4069) μg/L versus 3125 [2500–6310] μg/L; p = 0.04) than those with SCA (Fig. 1). GDF-15 levels were positively correlated with transfusion index (r = 0.874, p b 0.001), LDH (r = 0.773, p b 0.001), indirect bilirubin (r = 0.432, p = 0.009) and serum ferritin (r = 0.790, p b 0.001), while negatively correlated to hemoglobin (r = − 0.621, p b 0.001) and HbF% (r = −0.837, p b 0.001) (Fig. 3). GDF-15 was not correlated with age (r = 0.135, p = 0.441). Multiple regression analysis revealed that transfusion index (p = 0.001), LDH (p = 0.042) and serum ferritin (p = 0.006) were independently related to GDF-15 levels in SCD patients (r2 = 0.929, p b 0.001). Discussion GDF-15 has been identified in different contexts as a hypoxiainducible gene product and as a molecule involved in hepcidin regulation. Table 2 GDF-15 levels in relation to clinical characteristics of sickle cell disease patients. Variable Sex Male Female Genotype SCA (Homozygous SS) Sickle β° thalassemia Splenectomy Positive Negative Viral hepatitis Positive Negative History of cerebral stroke Positive Negative Pulmonary hypertension Positive Negative History of frequent sickling crisis Positive Negative Serum ferritin (μg/L) Ferritin ≥2500 Ferritin b2500 Hydroxyurea therapy Positive Negative
GDF-15 (pg/mL) Median (range)
p value
GDF-15 expression at the high levels achieved in the setting of ineffective erythropoiesis contributes to pathological iron overloading through a mechanism of incomplete hepcidin suppression [15]. We assessed the level of GDF-15 in young patients with SCD and investigated its role as a potential marker for disease activity. In this study, GDF-15 was significantly elevated in SCD patients, even in patients with SCA, compared with healthy controls denoting the presence of increased erythropoiesis. To our knowledge, no previous studies analyzed GDF-15 levels in SCD; however, increased serum GDF-15 levels in patients with other erythroid disorders have been reported [5]. GDF-15 could constitute a marker for ineffective erythropoiesis in the differential diagnosis of cases of unexplained anemia [6]. A significant increase in GDF-15 has been found in thalassemia major and transfusion-dependent thalassemia intermedia [5,7]. GDF-15 mRNA was absent in normal bone marrow precursors and in peripheral blood cells [5]. Some ineffective erythropoiesis has been shown in SCD [8]. It has been proposed that ineffective erythropoiesis causes iron overloading by not permitting the appropriate rise in hepcidin expression to occur once the erythroid demand is met [16]. Conceivably, while generating systemic iron overload, ineffective erythropoiesis and associated iron fluxes might generate an iron-deficiency signal in a relevant molecular
0.614 2700 (570–4500) 2950 (465–4500) 0.338 2700 (465–4200) 2800 (600–4500) 0.018 3250 (1200–4500) 2600 (465–4200) 0.101 3225 (2250–4500) 2700 (465–4500) 0.022 3450 (660–4500) 2700 (465–3800) 0.414 2800 (660–4500) 2700 (465–4500) 0.434 3000 (660–4500) 2800 (465–4200) b0.001 3250 (2350–4500) 1950 (465–2800) 0.596 2750 (465–4500) 2950 (600–4500)
SCA: sickle cell anemia; GDF-15: growth differentiation factor-15.
Fig. 2. GDF-15 in relation to splenectomy and serum ferritin among sickle cell disease patients.
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Fig. 3. Positive correlation between GDF-15 and lactate dehydrogenase and serum ferritin in sickle cell disease patients.
or cellular context and consequent stimulation of GDF-15 expression in a particular erythroid compartment. Therefore, further dissection of GDF-15 regulation by iron and oxygen in erythroid progenitors should be of interest and may provide mechanistic explanations to elevated GDF-15 levels in thalassemia patients [17]. This could also explain elevated GDF-15 levels in our SCD patients. Hemolysis appears to predispose SCD patients to a vasculopathy that may manifest as one or more of several complications: pulmonary hypertension, leg ulcers, priapism, and possibly stroke [18]. The chronic hemolysis in SCD may be also complicated by arterial or venous thrombosis. Several pathogenic factors contribute to this complication, including a chronic hypercoagulable state [19], increased erythrocyte aggregation and endothelial adhesion of erythrocytes in microvessels. It is often a combination of these factors that leads to thromboembolic events [20]. We found increased GDF-15 levels among patients with previous manifest or silent stroke. This is because GDF-15 is a stressresponsive cytokine that is upregulated under pathologic conditions involving various stimuli such as tissue hypoxia, inflammation, or enhanced oxidative stress that occur in thrombosis [21]. Moreover, ineffective erythropoiesis results in the secondary release into the circulation of damaged red blood cells with thrombogenic potential that reflect in a high rate of thromboembolic events [22,23]. Since GDF-15 has been regarded as a marker of ineffective erythropoiesis, our findings also confirm the substantial role of ineffective erythropoiesis in the pathophysiology and clinical severity of SCD. Ineffective erythropoiesis, and the subsequent chronic anemia and hypoxia, lead to hepcidin suppression, increased iron absorption from the gut, and increased release of recycled iron from the reticuloendothelial system. This results in the depletion of macrophage iron, relatively low levels of serum ferritin, and preferential portal and hepatocyte iron storage [7]. This in turn leads to considerable hepatic iron overload (suggested by a positive correlation between GDF-15 and serum ferritin in this study and the significant increase of GDF-15 among patients with serum ferritin ≥ 2500 μg/L) and releases into the circulation of toxic iron species like nontransferrin-bound iron, which can lead to targetorgan damage [24,25]. GDF-15 was strongly expressed in alveolar macrophages which might indicate a role of this protein in innate immunity [26]. Immunostaining experiments clearly demonstrated strong expression of GDF-15 in the vascular compartment of patients with pulmonary
hypertension. GDF-15 staining was observed in pulmonary vessels of all sizes, beginning from the microvasculature up to large pulmonary vessels and particularly in the intima of pulmonary arteries [27]. Hypoxia is a potent stimulator of GDF-15 expression in pulmonary endothelial cells. Furthermore, stress might lead to the induction of GDF-15 expression in the pulmonary vasculature [28]. However, GDF-15 was not different between our SCD patients with or without pulmonary hypertension may be because the pathology of pulmonary hypertension in SCD is multi-factorial and differs from that in thalassemia. Therefore, further studies are needed to confirm the association between GDF-15 levels and erythropoietic drive in SCD. In the present work, 31.4% of SCD patients were splenectomized. Although the prominent feature of SCD is autosplenectomy, a possible explanation to patients who underwent splenectomy is the presence of sickle thalassemia with hypersplenism [29]. Splenectomized patients had significantly higher levels of GDF-15 denoting the important role of the spleen in endothelial dysfunction and vascular complications in SCD. Similarly, Musallam et al. [7] evaluated GDF-15 levels in 55 patients with thalassemia intermedia as a marker of ineffective erythropoiesis in several anemias. GDF-15 levels were significantly higher in splenectomized compared to non-splenectomized patients. Splenic reticuloendothelial cells serve a critical function in the removal of cells that express phosphatidylserine on their surface (such as apoptotic and ageing cells) including senescent and damaged erythrocytes [22]. Following surgical or autosplenectomy, the rate of intravascular hemolysis increases and senescent and abnormal erythrocytes in the circulation trigger platelet activation, promoting pulmonary micro-thrombosis and red cell adhesion to the endothelium with subsequent erythrocyte fragmentation during hemolysis and generation of large amount of GDF-15 [8]. In SCD, GDF-15 was significantly increased in patients with serum ferritin N2500 μg/L suggesting a relation between them and iron overload. Iron may contribute directly to hemolysis, endothelial damage and vasculopathy. Iron-derived reactive oxygen species are implicated in the pathogenesis of several vascular disorders including atherosclerosis, microangiopathic hemolytic anemia, vasculitis, and reperfusion injury [30]. The relationship between iron overload and the severity of ineffective erythropoiesis seems to be bidirectional. Iron overload may aggravate ineffective erythropoiesis and the secondary release into the circulation of damaged red blood cells with thrombogenic potential.
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Therefore, managing iron overload with iron chelators or more novel therapeutics could improve the efficiency of erythropoiesis and the survival of the resulting reticulocytes and erythrocytes [31]. In this study, GDF-15 was positively correlated to transfusion index, markers of hemolysis and serum ferritin while negatively correlated to hemoglobin and HbF. This further shows that its increase may be a result of tissue ischemia and hypoxia. Similarly, Musallam et al. [7] found that GDF-15 levels were significantly correlated with anemia, markers of iron overload, and a pre-defined clinical severity score in thalassemia intermedia patients. Tanno et al. [6] reported that individuals with beta-thalassemia syndromes had elevated GDF-15 serum levels that were positively correlated with the levels of soluble transferrin receptor, erythropoietin and ferritin. These results suggest that GDF15 overexpression arising from an expanded erythroid compartment contributes to iron overload in thalassemia syndromes by inhibiting hepcidin expression [6] and subsequently contributes to tissue iron overloading in those patients [32]. Recently, the iron and erythropoiesis-controlled GDF-15 has been shown to inhibit the expression of hepcidin in beta-thalassemia patients, thereby increasing iron absorption despite iron overload [32]. Moreover, in congenital dyserythropoietic anemia which is a rare group of red blood cell disorders characterized by ineffective erythropoiesis and increased iron absorption, GDF-15 was correlated significantly with several erythropoietic and iron parameters including hepcidin-25, ferritin, and hepcidin-25/ferritin ratios. These results suggest that those patients express very high levels of serum GDF-15 which contributes to the inappropriate suppression of hepcidin [8]. A more complete understanding of GDF-15 signal transduction and functions in health and disease are topics for future research [32]. In conclusion, increased GDF-15 in SCD reflects the importance of ineffective erythropoiesis in the pathophysiology and clinical severity of anemia in SCD. GDF-15 levels are significantly related to markers of hemolysis and iron overload and may provide utility for identifying SCD patients who are at increased risk of thrombotic events. Limitations of the study The relatively small number of patients included is an important limiting factor. However, the results obtained suggest the importance of further larger studies to verify the practical utility of GDF-15 measurement in SCD and its potential to predict the clinical severity and disease outcome. It is also worth to investigate GDF-15 levels in SCD patients less than one year of age as sickling is expected to be less severe in this age group [33]. References [1] A. Inati, S. Koussa, A. Taher, S. Perrine, Sickle cell disease: new insights into pathophysiology and treatment, Pediatr. Ann. 37 (2008) 311–321. [2] D.C. Rees, T.H. Williams, M.T. Gladwin, Sickle-cell disease, Lancet 376 (2010) 2018–2031. [3] K.C. Wood, L.L. Hsu, M.T. Gladwin, Sickle cell disease vasculopathy: a state of nitric oxide resistance, Free Radic. Biol. Med. 44 (2008) 1506–1528. [4] S. Moncada, A. Higgs, The L-arginine-nitric oxide pathway, N. Engl. J. Med. 329 (2012) 2002–2012. [5] T. Tanno, P. Noel, J.L. Miller, P.A. Oneal, S.H. Goh, Growth differentiation factor 15 in erythroid health and disease, Curr. Opin. Hematol. 17 (2010) 184–190. [6] T. Tanno, N.V. Bhanu, P.A. Oneal, S.H. Goh, P. Staker, Y.T. Lee, J.W. Moroney, C.H. Reed, N.L. Luban, R.H. Wang, T.E. Eling, R. Childs, T. Ganz, S.F. Leitman, High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin, Nat. Med. 13 (2007) 1096–1101. [7] K.M. Musallam, A.T. Taher, L. Duca, C. Cesaretti, R. Halawi, M. Cappellini, Levels of growth differentiation factor-15 are high and correlate with clinical severity in transfusion-independent patients with β thalassemia intermedia, Blood Cells Mol. Dis. 47 (2011) 232–234.
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