J Pediatr (Rio J). 2015;91(6):560---566
www.jped.com.br
ORIGINAL ARTICLE
Circulating endothelial progenitor cells in obese children and adolescents夽,夽夽 António Pires a,∗ , Paula Martins a , Artur Paiva b , Ana Margarida Pereira c , ¸a c Margarida Marques d , Eduardo Castela a , Cristina Sena c , Raquel Seic a
Service of Pediatric Cardiology, Hospital Pediátrico de Coimbra, Centro Hospitalar e Universitário de Coimbra (CHUC), Coimbra, Portugal b Instituto Português do Sangue e Transplantac¸ão, Coimbra, Portugal c Laboratório de Fisiologia, Instituto de Imagem Biomédica e Ciências da Vida, Faculdade de Medicina, Universidade de Coimbra, Coimbra, Portugal d Laboratório de Estatística, Faculdade de Medicina, Universidade de Coimbra, Instituto de Imagem Biomédica e Ciências da Vida, Coimbra, Portugal Received 1 October 2014; accepted 13 January 2015 Available online 29 August 2015
KEYWORDS Pediatric obesity; C-reactive protein; Monocyte chemoattractant protein-1; E-selectin; Asymmetric dimethylarginine; Endothelial progenitor cells
Abstract Objective: This study aimed to investigate the relationship between circulating endothelial progenitor cell count and endothelial activation in a pediatric population with obesity. Methods: Observational and transversal study, including 120 children and adolescents with primary obesity of both sexes, aged 6---17 years, who were recruited at this Cardiovascular Risk Clinic. The control group was made up of 41 children and adolescents with normal body mass index. The variables analyzed were: age, gender, body mass index, systolic and diastolic blood pressure, high-sensitivity C-reactive protein, lipid profile, leptin, adiponectin, homeostasis model assessment-insulin resistance, monocyte chemoattractant protein-1, E-selectin, asymmetric dimethylarginine and circulating progenitor endothelial cell count.
夽 Please cite this article as: Pires A, Martins P, Paiva A, Pereira AM, Marques M, Castela E, et al. Circulating endothelial progenitor cells in obese children and adolescents. J Pediatr (Rio J). 2015;91:560---6. 夽夽 Study linked to the Hospital Pediátrico, Centro Hospitalar e Universitário de Coimbra (CHUC); Laboratório de Fisiologia and Laboratório de Estatística, Instituto de Imagem Biomédica e Ciências da Vida, Faculdade de Medicina, Universidade de Coimbra; and to the Instituto ¸ão, Coimbra, Portugal. Português do Sangue e Transplantac ∗ Corresponding author. E-mail:
[email protected] (A. Pires).
http://dx.doi.org/10.1016/j.jped.2015.01.011 0021-7557/© 2015 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.
Circulating endothelial progenitor cells
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Results: Insulin resistance was correlated to asymmetric dimethylarginine ( = 0.340; p = 0.003), which was directly, but weakly correlated to E-selectin ( = 0.252; p = 0.046). High sensitivity C-reactive protein was not found to be correlated to markers of endothelial activation. Systolic blood pressure was directly correlated to body mass index ( = 0.471; p < 0.001) and the homeostasis model assessment-insulin resistance ( = 0.230; p = 0.012), and inversely correlated to adiponectin ( = −0.331; p < 0.001) and high-density lipoprotein cholesterol ( = −0.319; p < 0.001). Circulating endothelial progenitor cell count was directly, but weakly correlated, to body mass index (r = 0.211; p = 0.016), leptin ( = 0.245; p = 0.006), triglyceride levels (r = 0.241; p = 0.031), and E-selectin ( = 0.297; p = 0.004). Conclusion: Circulating endothelial progenitor cell count is elevated in obese children and adolescents with evidence of endothelial activation, suggesting that, during infancy, endothelial repairing mechanisms are present in the context of endothelial activation. © 2015 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.
PALAVRAS-CHAVE Obesidade infantil; Proteína C reativa; Proteína quimiotática de monócitos-1; E-seleticna; Dimetilarginina assimétrica; Células progenitoras endoteliais
Células progenitoras endoteliais circulantes em crianc ¸as e adolescentes obesos Resumo Objetivo: O objetivo deste estudo foi investigar a relac ¸ão entre os números de células progenitoras endoteliais circulantes e a ativac ¸ão endotelial em uma populac ¸ão pediátrica com obesidade. Métodos: Estudo observacional e transversal, incluindo 120 crianc ¸as e adolescentes com obesidade primária de ambos de sexos, com idades entre 6 e 17 anos, recrutados de nossa Clínica de Riscos Cardiovasculares. O grupo de controle contou com 41 crianc ¸as e adolescentes com índice de massa corporal normal. As variáveis analisadas foram: idade, sexo, índice de massa corporal, pressão arterial sistólica e diastólica, proteína C reativa de alta sensibilidade, perfil lipídico, leptina, adiponectina, resistência à insulina para avaliac ¸ão do modelo de homeostase, proteína quimiotática de monócitos-1, E-seleticna, dimetilarginina assimétrica e números de células endoteliais progenitoras circulantes. Resultados: A resistência à insulina foi correlacionada a dimetilarginina assimétrica (p = 0,340; p = 0,003), que foi diretamente correlacionada, porém de forma muita amena à E-seleticna ( = 0,252; p = 0,046). Não constatamos que a proteína C reativa de alta sensibilidade está correlacionada a marcadores de ativac ¸ão endotelial. A pressão arterial sistólica foi diretamente correlacionada ao índice de massa corporal = 0,471; p < 0,001) e à resistência à insulina para avaliac ¸ão do modelo de homeostase ( = 0,230; p = 0,012) e inversamente correlacionada a adiponectina ( = -0,331; p < 0,001) e lipoproteína de alta densidade-colesterol = -0,319; p < 0,001). Os números de células progenitoras endoteliais circulantes foram diretamente correlacionados, porém de forma muito amena ao índice de massa corporal (r = 0,211; p = 0,016), à leptina ( = 0,245; p = 0,006), aos níveis de triglicerídeos (r = 0,241; p = 0,031) e à E-seleticna = 0,297; p = 0,004). Conclusão: Os números de células progenitoras endoteliais circulantes são elevados em crianc ¸as e adolescentes obesos com comprovac ¸ão de ativac ¸ão endotelial, sugerindo que, na infância, os mecanismos de reparac ¸ão endotelial estão presentes no contexto da ativac ¸ão endotelial. © 2015 Sociedade Brasileira de Pediatria. Publicado por Elsevier Editora Ltda. Todos os direitos reservados.
Introduction In obesity, various inflammatory agents, such as C-reactive protein1---3 and leptin,4 disturb the production of nitric oxide via the inhibition of its rate-limiting enzyme, endothelial nitric oxide synthase (eNOS).5,6 Other endogenous substances, such as asymmetric dimethylarginine (ADMA), a competitive antagonist of eNOS, also play a role in further compromising nitric oxide bioavailability. Its production is stimulated by inflammatory agents, such as C-reactive
protein.7 Unlike nitric oxide, it can be easily assayed and used as a surrogate for nitric oxide bioavailability. Damaged endothelium is repaired through the proliferation of adjacent endothelial cells, whose regenerative capacity is limited, and through the migration of progenitor cells, which originate in the bone marrow, known as circulating endothelial progenitor cells (EPCs). They express a variety of cell markers similar to those expressed by vascular endothelium, which display vascular regenerative properties and, as such, participate in new vessel formation.8
562 In patients with cardiovascular risk factors, such as arterial hypertension and insulin resistance, the count and function of EPCs is reduced. In these, the risk of cardiovascular events is increased.9,10 Inflammatory markers also appears to reduce the number of EPCs, implying a possible role in obesity.11,12 The aims of the present study were two fold. Firstly, to demonstrate that endothelial activation is present in childhood obesity; and secondly, that repairing mechanisms, through EPC activation, are not, at this early stage, compromised.
Methods Subjects The authors conducted an observational and transversal analysis, in a cohort of obese children and adolescents, recruited randomly, and followed-up at a Cardiovascular Risk Clinic. All parents gave their informed consent for the children to participate in the study, which had been approved by the local Ethics Committee. The inclusion criteria for the study group were primary obesity (body mass index [BMI] above the 95th percentile for sex and age) in children and adolescents aged 6---17 years, without recent or chronic illnesses. The exclusion criteria included secondary causes of obesity, acute infectious or inflammatory disorders (within a month of sampling), and chronic disorders. The control group included healthy children and adolescents, unrelated to the study group, within the same age range with a normal BMI (percentil 5---85), recruited from the Cardiology Clinic where they were sent for evaluation of murmurs, but proved to have no subjacent cardiac anomalies. All had undergone a 12-h fast prior to the clinical evaluation and blood sampling. The study group comprised 120 and the control group 41 children and adolescents, of both sexes. Based on this sample size, a significance level (p-value) of 0.05, a power of 0.80, and an effect size of 0.52 were obtained. These values were calculated using the G* Power 3.1.5 program. The variables analyzed were: age, gender, body mass index, systolic blood pressure, diastolic blood pressure, total cholesterol, high-density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglycerides, high-sensitivity C-reactive protein (hsCRP), monocyte chemoattractant protein-1 (MCP-1), lipid profile, leptin, adiponectin, homeostasis model assessment-insulin resistance (HOMA-IR), E-selectin, asymmetric dimethylarginine, and circulating EPC count.
Clinical and anthropometric evaluation Weight (in kilograms to the nearest 100 g) was determined using a SECA 220® digital weight scale (Medical Scales and Measuring Systems, Hamburg, Germany) and for standing height (in centimeters to the nearest 0.1 cm) a stadiometer included in the same equipment was used, with the children wearing only undergarments. Body mass index was calculated based on the formula: BMI = (weight/height2 ).13 The World Health Organization (WHO) BMI percentile charts were
Pires A et al. use to define obesity (if BMI >percentil 95; normal weight: BMI between percentil 5---85). The criteria for metabolic syndrome in children and adolescents were defined according to the International Diabetes Federation concensus.14
Blood collection and biochemical analysis Fasting venous blood samples (15 mL) were obtained to estimate the hematological parameters. Blood specimens were collected in vacutainer tubes with or without ethylenediaminetetraacetic acid (EDTA) as needed. Serum and plasma were prepared and then frozen (−80 ◦ C) for storage until analysis. High-sensitivity C-reactive protein was determined by immunonephelometry from serum samples and processed in the BN ProSpec® System (Siemens Healthcare Diagnostics Inc, Munich, Germany) analyzer (undetected if
