Endothelial monocyte activating polypeptide II in children and adolescents with type 1 diabetes mellitus: Relation to micro-vascular complications

Cytokine xxx (2015) xxx–xxx

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Endothelial monocyte activating polypeptide II in children and adolescents with type 1 diabetes mellitus: Relation to micro-vascular complications Amira A.M. Adly a,⇑, Eman A. Ismail b, Lamis M. Tawfik b, Fatma S.E. Ebeid a, Asmaa A.S. Hassan a a b

Pediatric Department, Faculty of Medicine, Ain Shams University, Egypt Clinical Pathology Department, Faculty of Medicine, Ain Shams University, Egypt

a r t i c l e

i n f o

Article history: Received 30 January 2015 Received in revised form 12 April 2015 Accepted 8 June 2015 Available online xxxx Keywords: EMAP II Type 1 diabetes Micro-vascular complications

a b s t r a c t Objectives: Endothelial monocyte-activating polypeptide II (EMAP II) is a multifunctional polypeptide with proinflammatory and antiangiogenic activity. Hyperglycemia and dyslipidemia appears to be significant factors contributing to increased EMAP-II levels. We determined serum EMAP II in children and adolescents with type 1 diabetes as a potential marker for micro-vascular complications and assessed its relation to inflammation and glycemic control. Methods: Eighty children and adolescents with type 1 diabetes were divided into 2 groups according to the presence of micro-vascular complications and compared with 40 healthy controls. High-sensitivity C-reactive protein (hs-CRP), hemoglobin A1c (HbA1c) and EMAP II levels were assessed. Results: Serum EMAP II levels were significantly increased in patients with micro-vascular complications (1539 ± 321.5 pg/mL) and those without complications (843.6 ± 212.6 pg/mL) compared with healthy controls (153.3 ± 28.3 pg/mL; p < 0.001). EMAP II was increased in patients with microalbuminuria than normoalbuminuric group (p < 0.001). Significant positive correlations were found between EMAP II levels and body mass index, fasting blood glucose, HbA1c, serum creatinine, triglycerides, total cholesterol, urinary albumin creatinine ratio (UACR) and hs-CRP (p < 0.05). A cutoff value of EMAP II at 1075 pg/mL could differentiate diabetic patients with and without micro-vascular complications with a sensitivity of 93% and specificity of 82%. Conclusions: We suggest that EMAP II is elevated in type 1 diabetic patients, particularly those with micro-vascular complications. EMAP II levels are related to inflammation, glycemic control, albuminuria level of patients and the risk of micro-vascular complications. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Type 1 diabetes mellitus is the most common endocrine–metabolic disorder of childhood and adolescence, with important consequences for physical and emotional development [1]. Type-1 diabetes constituted nearly 90–100% of all children with diabetes [2]. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of different organs, especially the eyes, kidneys, nerves, heart, and blood vessels [3]. Diabetes complications represent a huge burden for patients and health services. The fight against each single complication has led to significant improvements in diabetes care, especially for microvascular complications, yet macroangiopathy remains a major source of morbidity and mortality. A common approach ⇑ Corresponding author at: 6 A ElSheshini Street, Shoubra, Soudia Buildings, Cairo, Egypt. Mobile: +20 01005245837. E-mail address: [email protected] (A.A.M. Adly).

for the prevention and treatment of diabetes complications relies on the understanding of their complex pathophysiology [4]. Endothelial dysfunction has received increasing attention as a potential contributor to the pathogenesis of vascular disease in diabetes mellitus [5]. In diabetes, angiogenesis is disturbed, contributing to proliferative retinopathy, nephropathy, neuropathy, atherosclerosis, and impaired wound healing [6–9]. Hyperglycemia, hypertension, dyslipidemia, smoking, adiposity, inflammation and oxidative stress may promote vascular complications [6], and some effects of these stresses may be mediated by disturbances in the levels of or balance of pro- and anti-angiogenic factors [10]. Hyperglycemia is the metabolic hallmark of diabetes and leads to wide spread cellular damage. Endothelial cells, which poorly regulate intracellular glucose, may be particularly vulnerable to hyperglycemia [11–13]. Endothelial dysfunction is expressed in increased interactions with leukocytes, smooth muscle growth, vasoconstriction, impaired coagulation, vascular inflammation,

http://dx.doi.org/10.1016/j.cyto.2015.06.006 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Adly AAM et al. Endothelial monocyte activating polypeptide II in children and adolescents with type 1 diabetes mellitus: Relation to micro-vascular complications. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.06.006

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thrombosis, and atherosclerosis [14,15]. Studies suggest that metabolic abnormalities of diabetes alter the angiogenic process [11– 13]. Endothelial monocyte-activating polypeptide II (EMAP II) has been implicated in endothelial dysfunction and disturbed angiogenesis [16]. EMAP II was identified as an endothelial response mediator secreted by a highly tumor necrosis factor (TNF)-sensitive tumor line, the methylcholanthrene A (Meth A) fibrosarcoma [17,18]. EMAP II, which is synthesized as a 34 kDa precursor molecule (proEMAP) and enzymatically cleaved to produce a biologically active 22 kDa mature polypeptide [16], is a multifunctional polypeptide secreted by various cell types (monocytes, macrophages and tumor cells). In vitro, it induces procoagulant activity, increased expression of E- and P-selectins and TNF receptor-1, blocks adhesion of endothelial cells to fibronectin, as well as matrix assembly by binding to a5b1 integrin [19]. EMAP II is also chemotactic for monocytes and neutrophils. In vivo, it induces an acute inflammatory reaction and tumor regression [20]. Moreover, evidence suggests that EMAP II can induce apoptosis in cultured endothelial cells and inhibits proliferation, vascularization and neovessel formation [21]. EMAP II has been shown to suppress tumor growth by its potent anti-angiogenic properties [22] and it reduces vascular endothelial growth factor (VEGF) expression, which itself facilitates tumor growth through induction of angiogenesis [23]. The exact mechanism of increased EMAP II in diabetes is not known. Hyperglycemia and dyslipidemia appears to be significant factors contributing to increased EMAP-II levels [24,25]. Despite such data, EMAP II role in diabetes remains to be fully elucidated and its relation to vascular complications has not been explored. Therefore, the aim of this study was to determine its levels in children and adolescents with type 1 diabetes mellitus as a potential marker for micro-vascular complications, and assess its relation to the clinicopathological characteristics of patients and glycemic control. 2. Materials and methods This cross sectional study was conducted on 80 children and adolescents with type 1 diabetes attending the Pediatric Diabetes Clinic, Pediatric Hospital, Ain Shams University. Patients were defined according to the criteria of American Diabetes Association [26]. They included 28 males and 52 females with a male-to-female ratio of 1:9.1 and a mean age of 13.1 ± 3.3 years. Another group of 40 age- and sex-matched healthy volunteers were enrolled as controls. An informed consent was obtained from each patient or control subject or their legal guardians before enrollment into the study. The study was approved by the local ethical committee of Ain Shams University. Exclusion criteria were the presence of any clinical evidence of infection, history of allergies, rheumatoid arthritis, recent trauma, surgery, liver dysfunction, connective tissue disease, or other autoimmune disorders and any other conditions that could influence CRP. Samples with CRP > 10 mg/L were excluded. Patients were divided into two groups according to the presence of micro-vascular complications: Group 1 (patients with complications): included 40 patients with type 1 diabetes having micro-vascular complications. Patients included in this group had either one or more of these inclusion criteria; nephropathy, retinopathy or neuropathy. The presence of micro-vascular complications was investigated starting from diagnosis of diabetes and further verified at time of the study by clinical examination and investigations. Group 2 (patients without complications): included 40 children with type 1 diabetes who lacked the previously mentioned

micro-vascular complications both at the study time and in their previous records. The studied patients were subjected to detailed medical history and thorough clinical examination with special emphasis on age of onset of diabetes, disease duration, insulin therapy and chronic complications (retinopathy, neuropathy, nephropathy, or cardiovascular ischemic events). All patients were on insulin therapy using human insulin with a mean dose of 1.5 ± 0.24 IU/kg/day. Anthropometric measurements were recorded and body mass index (BMI) was calculated. Blood pressure was measured after a 5-min rest in the seated position using mercury sphygmomanometer. If it was greater than 90th percentile for age and sex, the blood pressure was repeated twice for the validity of the reading. Diabetic retinopathy was diagnosed by doing complete ocular examination including visual field testing, slit-lamp biomicroscopy, Volk lens and indirect ophthalmoscopy. Fundus examination was performed through dilated pupils using 90-diopter Volk lens and biomicroscope. The presence and grading of diabetic retinopathy was based on the International clinical diabetic retinopathy and macular edema disease severity scale [27]. The simple rapid bedside neuropathy disability score (NDS) was adopted as a screening tool for diabetic peripheral neuropathy. The NDS was derived from examination of vibration perception (by means of a 128-Hz tuning fork), pin-prick and temperature perceptions in the great toe, and the presence or absence of ankle reflexes. The sensory modalities were scored and a score above two was defined as clinical diabetic peripheral neuropathy [28]. Results were confirmed by nerve conduction velocity to provide accurate diagnosis [29]. 2.1. Laboratory investigations Liver and kidney function tests, fasting lipid profile, high sensitivity C-reactive protein (hs-CRP), as well as mean fasting blood glucose (FBG) levels in the last 6 months prior to the study were measured using Cobas Integra 800 (Roche Diagnostics, Mannheim, Germany). Dyslipidemia was defined if at least one of the following was present; serum total cholesterol P 200 mg/dL, low-density lipoprotein (LDL) cholesterol P 100 mg/dL, high-density lipoprotein cholesterol (HDL) < 40 mg/dL, or serum triglyceride P 150 mg/dL [30]. Further analysis was done after controlling for age and pubertal stage to avoid differences in lipid values [31]. Assessment of mean HbA1c% in the year preceding the study was performed using D-10 (BioRad, France). Urinary albumin excretion (as an indicator of nephropathy) was measured in an early morning urine sample as albumin-to-creatinine ratio by an immuno-nephelometric method. Microalbuminuria and macroalbuminuria were present if urinary albumin excretion in at least 2 out of 3 consecutive urine samples, 2 months apart was 30–299 mg/g creatinine and P300 mg/g creatinine, respectively [32,33]. Potential factors affecting urinary albumin excretion were excluded [34]. Determination of serum levels of EMAP II was done by enzyme linked immunosorbent assay (ELISA) using the assay kit for Aminoacyl tRNA Synthetase Complex Interacting Multifunctional Protein 1 (AIMP1) (Uscn, Life Science Inc., USA) according to the manufacturer’s instructions. 2.2. Sampling Peripheral blood (PB) samples were collected on ethylene diamine tetra-acetic acid (EDTA) (1.2 mg/mL) for analysis of HbA1c. Serum obtained from clotted samples by centrifugation for 15 min at 1000g was used for chemical analysis and stored at 20 °C till subsequent use in ELISA.

Please cite this article in press as: Adly AAM et al. Endothelial monocyte activating polypeptide II in children and adolescents with type 1 diabetes mellitus: Relation to micro-vascular complications. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.06.006

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2.3. Statistical analysis

3.2. EMAP II levels in type 1 diabetes

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, SD and qualitative variables were described as number and percent. In order to compare quantitative parametric variables between the three studied groups, Analysis of Variance (ANOVA) test with post hoc test was used while Student t-test was applied for comparison between 2 groups. Categorical variables were compared using Chi-square (X2) test. Spearman’s rank correlation coefficient and multiple linear regression analysis were employed to assess the relation between EMAP II and clinical as well as laboratory variables. Logistic regression was used to examine the relation between EMAP II and micro-vascular complications after adjustment of other variables; age, gender, disease duration, BMI, blood pressure, FBG, HbA1c, hs-CRP, urinary albumin excretion and fasting lipids. Receiver operating characteristic (ROC) curve was used to determine the best cut-off value of EMAP II to detect micro-vascular complications. The area under the curve (AUC) was calculated for each plot. A p value <0.05 was considered significant in all analyses.

Serum EMAP II levels were significantly elevated in all patients with type 1 diabetes as compared with the control group (1156.8 ± 438.2 and 153.3 ± 28.3, respectively; p < 0.001). EMAP II levels were significantly increased in patients with micro-vascular complications (1539 ± 321.5 pg/mL) compared with those without complications (843.6 ± 212.6 pg/mL) and the healthy control group (153.3 ± 28.3 pg/mL). Similarly, its level was significantly increased among patients without complications compared with healthy controls (p < 0.001; Table 1 and Fig. 1). Moreover, ROC curve analysis revealed that an EMAP II cutoff value of 1075 pg/mL could discriminate between patients with and without micro-vascular complications with a sensitivity of 93%, specificity of 82%, area under the curve (AUC) 0.964 and 95% confidence interval 0.779–1.0; p < 0.001. EMAP II levels were significantly increased in patients having individual complications compared with patients lacking them; microalbuminuria (1569.6 ± 314.5 versus 900.3 ± 278.8; p < 0.001; Fig. 2), peripheral neuropathy (1706.4 ± 351.9 pg/mL versus 1033.5 ± 354 pg/mL; p < 0.001) or retinopathy (1583.3 ± 160.7 pg/mL versus 1134.4 ± 437.1 pg/mL; p < 0.001). Significant positive correlations were found between EMAP II levels and BMI (r = 0.288, p = 0.026), FBG (r = 0.949, p < 0.001), HbA1c (r = 0.859, p < 0.001), triglycerides (r = 0.310, p = 0.031), total cholesterol (r = 0.229, p = 0.005), UACR (r = 0.861, p < 0.001) and hs-CRP (r = 0.937, p < 0.011) (Fig. 3). On the other hand, no correlation was found between EMAP II levels and age, disease duration, blood pressure, serum creatinine or HDL cholesterol. Multiple regression analysis showed that FBG, HbA1c, UACR, and hs-CRP were independently related to EMAP II levels in patients with type 1 diabetes (r2 = 0.954, p < 0.001) (Table 2). Logistic regression showed that EMAP II was a significant independent factor for micro-vascular complications (odds ratio 4.91, 95% CI 2.51–6.93; p < 0.001).

3. Results 3.1. Clinical and laboratory characteristics among the studied groups with type 1 diabetes The most common complication encountered in the studied patients was diabetic nephropathy (38.8%) being in 31 out of 80 patients; all of whom had microalbuminuria while none suffered macroalbuminuria. The next common complication was peripheral neuropathy (18.8%), followed by retinopathy (7.5%). As shown in Table 1, patients with micro-vascular complications were older (p = 0.003) with longer disease duration and higher FBG, HbA1c, urinary albumin creatinine ratio and hs-CRP compared with patients without complications and healthy controls (p < 0.001). Levels of all the studied laboratory variables were significantly higher among patients with or without micro-vascular complications compared with controls (p < 0.05) except for hs-CRP which was similarly distributed between patients without complications and controls (p > 0.05).

4. Discussion The abnormal metabolic state that accompanies diabetes mellitus causes arterial dysfunction. Chronic hyperglycemia and dyslipidemia render arteries susceptible to atherosclerosis. Diabetes

Table 1 Comparison of clinicopathological characteristics among diabetic patients with and without micro-vascular complications and control group. Variables

Controls (n = 40)

All patients (n = 80)

Patients without complications (n = 40)

Patients with complications (n = 40)

Males, n (%) Age (years) BMI (kg/m2) Disease duration (years) Systolic BP (mmHg) Diastolic BP (mmHg) FBG (mg/dL) HbA1C (%) Serum creatinine (mg/dL) Triglycerides (mg/dL) Cholesterol (mg/dl) HDL (mg/dL) UACR (mg/g creatinine) hs-CRP (lg/mL) EMAP II (pg/mL)

20 (50) 12.6 ± 3.7 18.3 ± 4.2 – 100.3 ± 9.3 72.7 ± 6.4 82.4 ± 11.2 4.4 ± 0.8 0.4 ± 0.2 87.4 ± 6.4 122.1 ± 10.3 63 ± 14.4 11.3 ± 4.1 2.2 ± 0.6 153.3 ± 28.3

28 (35) 13.1 ± 3.3 20.4 ± 5.3 7.3 ± 2.9 113.7 ± 10.2 73.3 ± 7.8 165.6 ± 14.4 9.3 ± 2.1 0.6 ± 0.3 160.6 ± 13.8 181.5 ± 13.8 44.7 ± 8.2 91.2 ± 29 3 ± 1.4 1156.8 ± 438.2

16 (40) 12.0 ± 3.0 19.5 ± 4.9 6.0 ± 2.4 109.1 ± 10.4 73.8 ± 8 133.9 ± 12.79 8.1 ± 0.9 0.6 ± 0.3 157.7 ± 10.6 173.9 ± 13.7 45.8 ± 8.1 18.5 ± 3.8 2.3 ± 0.7 843.6 ± 212.6

12 (30) 13.9 ± 3.4 21.5 ± 5.6 7.9 ± 3.5 126.2 ± 10.7 72.8 ± 7.6 189.6 ± 16.9 10.7 ± 2.2 0.7 ± 0.2 164.1 ± 14.6 188.6 ± 14.2 43.5 ± 11 155.5 ± 29.8 4.1 ± 1.2 1539 ± 321.5

P-value Overall

P1

P2

P3

0.169 0.003 0.051 – <0.001 0.341 <0.001 <0.001 0.004 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

– 0.377 0.566 – 0.279 0.253 <0.001 <0.001 0.03 <0.001 <0.001 <0.001 0.003 0.912 <0.001

– 0.005 0.05 – <0.001 0.312 <0.001 <0.001 0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

– 0.003 0.312 0.006 <0.001 0.249 <0.001 <0.001 0.117 0.755 0.345 0.43 <0.001 <0.001 <0.001

BMI: body mass index; BP: blood pressure; FBG: fasting blood sugar; Hb: hemoglobin; HDL: high density lipoprotein; UACR: urinary albumin creatinine ratio; hs-CRP: high sensitivity C-reactive protein; EMAP II: Endothelial monocyte activating polypeptide II. Data were expressed as mean ± SD using ANOVA with Post Hoc test for comparison unless specified as number (%) using Chi-square (X2) test for comparisons. P1: Comparison between control group and patients without complications. P2: Comparison between control group and patients with complications. P3: Comparison between patients with and without complications.

Please cite this article in press as: Adly AAM et al. Endothelial monocyte activating polypeptide II in children and adolescents with type 1 diabetes mellitus: Relation to micro-vascular complications. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.06.006

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Fig. 1. Elevated endothelial monocyte-activating polypeptide II (EMAP II) levels in type 1 diabetic patients with and without micro-vacsular complications compared with healthy controls. Data were expressed as mean ± SD using ANOVA with Post Hoc test for comparison.

Fig. 2. Endothelial monocyte-activating polypeptide II (EMAP II) levels among type 1 diabetic patients in different albuminuric stages and controls. Patients with microalbuminuria had significantly higher EMAP II levels than normoalbuminuric group or healthy controls. Data were expressed as mean ± SD using ANOVA with Post Hoc test for comparison.

Table 2 Multiple regression analysis of the relation of EMAP II to clinical and laboratory variables in patients with type 1 diabetes.

(Constant) FBG (mg/dL) HbA1c (%) UACR (mg/g creatinine) hs-CRP (lg/mL)

Unstandardized coefficients

Standardized coefficients

B

Standard error

Beta

2.785 3.094 87.979 107.824 147.823

67.016 0.502 51.481 41.795 21.133

– 0.509 0.078 0.121 0.456

p Value

r2

0.967 <0.001 0.003 0.013 <0.001

0.954

FBG: fasting blood sugar; Hb: hemoglobin; UACR: urinary albumin creatinine ratio; hs-CRP: high sensitivity C-reactive protein. Dependent variable: endothelial monocyte activating polypeptide II.

alters the function of multiple cell types; including the endothelium, smooth muscle cells, and platelets, indicating the extent of vascular disarray in this disease [35]. Many factors contribute to accelerated macrovascular disease in patients with diabetes [36,37]. Beyond impaired insulin secretion and resistance, inflammation has recently attracted much attention as a contributor to diabetes [38]. EMAP II has been shown to have anti-angiogenic

properties. It also appears to play an important role in neovascularization and endothelial abnormalities [19,39]. The most common microvascular complication observed in the studied patients was diabetic nephropathy (38.8%), followed by peripheral neuropathy and retinopathy. Previous studies reported the incidence of nephropathy in type 1 diabetes to be between 20% and 40% [33,40]. Approximately 30% of patients may develop

Please cite this article in press as: Adly AAM et al. Endothelial monocyte activating polypeptide II in children and adolescents with type 1 diabetes mellitus: Relation to micro-vascular complications. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.06.006

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Fig. 3. Significant positive correlation between endothelial monocyte-activating polypeptide II (EMAP II) and laboratory variables in patients with type 1 diabetes (n = 80). (A) EMAP II and HbA1C; (B) EMAP II and urinary albumin creatinine ratio (UACR). Spearman’s rank correlation coefficient was used.

diabetic nephropathy within 25 years of diabetes [41]. Alternatively, other studies reported a predominance of either diabetic neuropathy [42,43] or diabetic retinopathy [44]. The variation between studies as regards frequency of microvascular complications in type 1 diabetes depends on several factors including disease duration, glycemic control, mean systolic blood pressure and triglyceride level [45]. In the present study, patients with micro-vascular complications had higher age, longer disease duration, higher systolic BP, FBG, HbA1c, UACR, hs-CRP than patients without complications. The duration of diabetes, hyperglycemia, and hypertension are well-established risk factors that correlated with all chronic diabetic complications [46–48]. In this work, serum EMAP II was significantly elevated in all studied patients with type 1 diabetes compared with the healthy control group. EMAP II levels were significantly increased in patients suffering micro-vascular complications when compared with patients without complications or the control groups. EMAP II was significantly increased in relation to individual complications; nephropathy (microalbuminuria), peripheral neuropathy or retinopathy. Such elevated EMAP II levels were found to influence micro-vascular complications independent of other risk factors, where multiple regression linear analysis showed that FBG, HbA1c, UACR and hs-CRP were independently related to EMAP II levels in our studied patients. Our results were in agreement with Mogylnytska [24] who reported significantly increased serum level of EMAP II in 30 patients with type 1 diabetes (mean age, 20.26 ± 2.11) compared with control subjects. EMAP II mRNA is ubiquitously expressed in a wide range of normal human tissue [49,50], but it is upregulated by hypoxia and apoptosis [51,52]. It is well known that diabetes mellitus impairs physiological angiogenesis. Multiple molecular mechanisms have been proposed. Hyperglycemia induces the generation of reactive oxygen species (ROS) that cause endothelial derangements, including the reduced synthesis and accelerated degradation of endothelial-derived nitric oxide (NO). The bioactivity of NO is critical for angiogenic processes, such as the survival, proliferation and migration of endothelial cells. The impairment in NO bioactivity may also explain in part the reduced expression of a major angiogenic cytokine, vascular endothelial growth factor (VEGF) in hyperglycemic states, as NO and VEGF have a reinforcing and reciprocal

relationship [53–55]. Glucose intolerance also reduces the number and function of bone-marrow derived endothelial progenitor cells, circulating cells which participate in the angiogenic response. In addition to generating ROS, hyperglycemia may impair cytoprotective mechanisms against oxidative stress [56]. Increased expression of a specific microRNA (miRNA) that appears to orchestrate a pathophysiological response in diabetes mellitus may be another genomic mechanism for hyperglycemia-induced impairment of angiogenesis [57]. The existence of chronic inflammation in diabetes is mainly based on the increased plasma concentrations of CRP, fibrinogen, interleukin-6 (IL-6), interleukin-1 (IL-1), and TNFa [58]. Inflammatory cytokines increase vascular permeability, change vasoregulatory responses, increase leukocyte adhesion to endothelium, and facilitate thrombus formation. NF-jB consists of a family of transcription factors, which regulate the inflammatory response of vascular cells, by transcription of various cytokines which causes an increased adhesion of monocytes, neutrophils, and macrophages, resulting in cell damage. On the other hand, NF-jB is also a regulator of genes that control cell proliferation and cell survival and protects against apoptosis, among others by activating the antioxidant enzyme superoxide dismutase [59]. NF-jB is activated by TNFa and IL-1 next to hyperglycemia, advanced glycation end products, angiotensin II, oxidized lipids, and insulin. Once activated, NF-jB translocates from the cytoplasm to the nucleus to activate gene transcription [15]. Thus, inflammation, endothelial dysfunction and disturbed angiogenesis in diabetes could trigger EMAP II release by monocytes and macrophages. In view of EMAP II anti-angiogenic properties that include the inhibition of endothelial cell proliferation, tube formation, adhesion to fibronectin and induction of endothelial cell apoptosis [19,39], it has been reported that increased EMAP II levels in type 1 diabetes could reflect an endothelial dysfunction in those patients [24]. The molecular mechanism of such endothelial dysfunction includes EMAP II mediated hypoxia-inducible factor 1 alpha (HIF-1a) activity through interaction with PSMA7, a component of the proteasome; resulting in the inhibition of angiogenic cord formation in endothelial cells [19]. Moreover, elevated EMAP II in patients with type 1 diabetes could also reflect inflammation. This is accomplished through manipulation of the procoagulant properties of endothelial cells,

Please cite this article in press as: Adly AAM et al. Endothelial monocyte activating polypeptide II in children and adolescents with type 1 diabetes mellitus: Relation to micro-vascular complications. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.06.006

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likely through upregulation of tissue factor, and the chemotactic effects toward monocytes and granulocytes. In addition, an increase in intracellular calcium levels was found to be involved in this cellular activation and proinflammatory response. At the molecular level, EMAP II was found to induce E- and P-selectin expression, release of von Willebrand factor and TNF production by monocytes [60]. As inflammation proceeds, the risk for vascular complications increases [61]. Hence, the antiangiogenic and proinflammatory properties of EMAP II could explain the results of the current study of detecting the highest levels of EMAP II among patients with micro-vascular complications. Microalbuminuria was the most common complication encountered among our studied group, and EMAP II serum levels were increased in patients with type 1 diabetes, even in normoalbuminuric ones. This could imply the significant role of this marker as a potential predictor of diabetic nephropathy in type 1 diabetes even before development of microalbuminuria. As microalbuminuria occurs relatively late in the disease process, evaluation of serum EMAP II might become a useful test in the early stages of diabetes to identify normotensive and normoalbuminuric children and adolescents with type 1 diabetes at increased risk of developing diabetic kidney disease later in life, who might benefit from a timely and appropriate intervention. In our study, ROC curve analysis revealed that a cutoff value of EMAP II at 1075 pg/mL could differentiate patients with and without micro-vascular complications. However, to our knowledge, this is the first study that assessed EMAP II in relation to complications and defined a cutoff value. Therefore, further prospective studies are needed to validate this threshold. 5. Study limitation Limitations of our study include the presence of a relatively small number of patients in subgroups with single complication. Although comparison of all patients with microvascular complications revealed similar results to the analyzed subgroups, yet, further longitudinal analysis including larger number of patients for each individual complication might provide additional information. In conclusion, this study highlights the role of EMAP II as a potential marker for diabetic microangiopathy. Serum EMAP II levels are elevated in children and adolescents with type 1 diabetes, particularly those with microvascular complications. EMAP II is related to inflammation, glycemic control, albuminuria level of patients. However, prospective studies are needed to verify these results and the cutoff at which EMAP II can detect complications should be further validated in another group of patients with type 1 diabetes. Thereafter, evaluation of patients by regular measurement of EMAP II levels would be considered to identify those at high risk of developing micro-vascular complications. Control of hyperglycemia, thus, remains the best way to improve endothelial function and to prevent atherosclerosis. The underlying mechanism of EMAP II generation and increase in children with type I diabetes may be multifactorial and represents an important area for future research. References [1] Alemzadeh R, Wyatt DT. Diabetes mellitus in children. In: Kliegman RM, Behrman RE, Jenson HB, Stanton BF, editors. Nelson textbook of paediatrics. Philadelphia: Saunders; 2007. p. 2404–31. [2] Ramachandran A, Snehalatha C, Krishnaswamy CV. Incidence of IDDM in children in urban population in Southern India. Diabetes Res Clin Pract 1996;34:79–82. [3] American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2012;35:S64–71. [4] Fadini GP, Sartote S, Agostini C, Avogaro A. Significance of endothelial progenitor cells in subjects with diabetes. Diabetes Care 2007;30:1305–13.

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Please cite this article in press as: Adly AAM et al. Endothelial monocyte activating polypeptide II in children and adolescents with type 1 diabetes mellitus: Relation to micro-vascular complications. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.06.006