Paediatric Obesity and Cardiovascular Dysfunction
Paediatric Obesity and Cardiovascular Dysfunction
Background Childhood obesity has been related to an impaired cardiovascular structure and
 function. Aims of this study will be to evaluate early cardiovascular abnormalities in a
 large population of obese children and adolescents compared with a normal weight counterpart,
 to investigate the potential association with insulin resistance (IR), serum uric acid (sUA),
 metabolic syndrome (MetS), plasmatic markers of inflammation and oxidative stress and
 adipokines, to evaluate changes in cardiovascular dysfunction after 6 and 12 months of a
 behavioral treatment (isocaloric Mediterranean balanced diet plus daily aerobic physical
 activity).

 Subjects and methods This was a single-center case-control study. Eighty obese (OB) subjects
 (6-16 years) and 20 normal weight (NW) matched controls were consecutively recruited. In the
 whole population we will perform an anthropometric and a cardiovascular assessment. OB
 patients will also undergo an OGTT and biochemical evaluations. In the OB group, all these
 evaluations will be performed at baseline and after 6 (T6) and 12 months (T12) of diet plus
 aerobic training.
Background Childhood obesity causes a wide range of severe complications, increasing the risk
 of premature morbidity and mortality and raising public-health concerns. In addition, obese
 children are more prone to become obese adults, with higher risk of cardiovascular diseases
 (CVD). A cluster of CVD risk factors has been identified in children as young as 5 years of
 age. Furthermore, among adolescents and young adults, the presence of CVD risk factors
 correlates with asymptomatic coronary atherosclerosis. Childhood obesity has been related to
 an impaired cardiac structure and function.

 Atherogenesis and arterial wall damage begin during childhood and, there is evolving evidence
 that clinical indicators of atherosclerosis such as carotid artery intima-media thickness
 (CIMT), arterial stiffness, and endothelial function are altered in obese children. In
 addition, little is known on the potential association between early cardiovascular
 alterations and metabolic abnormalities in obese children. Metabolic syndrome (MetS) is a
 cluster of features, which includes dyslipidemia, hypertension, and visceral obesity,
 conferring with a higher risk of CVD and type 2 diabetes. Few studies investigated the
 association of MetS with cardiovascular changes during childhood. Hyperuricemia has been
 recognized as a risk factor for CVD in adults with a negative impact on longevity. However,
 data in pediatric age are still lacking and the association between hyperuricaemia and
 cardiovascular abnormalities in obese children is still unknown. Furthermore, obesity is a
 state of chronic low-level inflammation and increased oxidative stress. Oxidative stress
 plays an important role in the pathogenesis of cardiovascular alterations by either
 triggering or exacerbating the biochemical processes accompanying endothelial dysfunction.

 Moreover, adipose tissue acts as a secretory gland, releasing hormones and adipokines with
 pro- or anti-inflammatory activity. Clinical studies of obese adults have observed an
 association between plasma levels of adipokines and markers of inflammation and/or oxidative
 stress. Among various adipokines, adiponectin seems to play an important role. Indeed, in
 contrast to other adipokines which are up-regulated in obesity, secretion of adiponectin is
 markedly reduced in obese subjects. Second, adiponectin seems to exert mainly positive
 activities on metabolism, vascular tone and inflammatory reaction. Consequently, in contrast
 to other adipokines, which circulate in excess in obese subjects and exert unbeneficial
 effects when chronically elevated, deficiency rather than excess of adiponectin is implicated
 in obesity-associated complications. Finally, serum concentration of adiponectin is very high
 in comparison to other hormones and cytokines, which suggests that apart from binding to
 specific high-affinity receptors, this protein may also have some less specific low affinity
 targets. Adiponectin has been associated with endothelial improvement and vascular protection
 through the activation of an endothelial isoform of nitric oxide (eNOS)-related signalling
 and with anti-inflammatory properties and antiatherogenic effects. Thus, an impaired
 production of adipokines may be a key mechanism linking obesity with inflammation and
 oxidative stress. The understanding of these complex mechanisms and the identification of
 possible early markers of cardiovascular damage are therefore necessary in order to establish
 preventive and therapeutic measures in childhood and to decrease cardiovascular morbidity and
 mortality in adulthood.

 Subjects and methods This study is a single-centre longitudinal study. Subjects were
 recruited at Division of Pediatrics, Department of Health Sciences, University of Piedmont
 Orientale, Novara (Italy). The study protocol was in accordance with the ethical guidelines
 of the Declaration of Helsinki and has been approved by the local Ethical Committee. Informed
 written consent was obtained from all subjects and their parents before study. The
 investigators consecutively enrolled 80 Caucasian obese (OB) children and adolescents, aged 6
 to 16 years, and 20 normal weight, age and sex matched controls (NW). NW patients were
 evaluated only at baseline while OB subjects will be evaluated at baseline and after 6 (T6)
 and 12 months (T12) of an isocaloric Mediterranean balanced diet plus aerobic training.

 Assessment in both groups (OB and NW)

 Echocardiographic assessment Transthoracic echocardiogram using a Vivid 7 Pro ultrasound
 scanner (General Electric Healthcare, USA) will be performed by a sonographer and the images
 will be reviewed by an expert pediatric cardiologist, blinded to patients' clinical data.
 Measurements of left ventricle (LV end-diastolic diameter, LVEDD; LV end-systolic diameter,
 LVESD; interventricular septum at end diastole, IVSD; LV posterior wall at end diastole,
 LVPWD) and left atrium diameter (LAD) will be obtained according to established standards.
 The maximum LA volume will be calculated from apical 4- and 2-chamber zoomed views of the LA.
 LV end-diastolic and end-systolic volumes and the LV ejection fraction at rest will be
 computed from 2- and 4-chamber views, using a modified Simpson's biplane method. LV mass
 (LVM) will be derived from the Devereux formula and indexed to body surface area (left
 ventricular mass index [LVMI]). Relative wall thickness (RWT) will be calculated as the ratio
 (LVPWD x 2)/LVEDD. Using pulsed wave Doppler, mitral inflow velocities, peak early diastolic
 velocity (E), peak late diastolic velocity (A), E/A ratio, will be measured.

 Vascular assessment Vascular measurements will be performed with a high-resolution
 ultrasonography (Esaote MyLab25TM Gold, Esaote, Italy) using a 8-megaHertz (mHz) linear
 transducer and a 5 mHz convex transducer for the abdominal aorta, by an expert sonographer
 and images will be then reviewed offline by an expert vascular surgeon blinded to patients
 clinical status. Ultrasonography of the right and left carotid arteries will be performed in
 the supine position with the head turned 45° away from the side being imaged. CIMT will be
 defined as the mean distance from the leading edge of the lumen-intima interface to the
 leading edge of the media-adventitia interface of the far wall, approximately 10 mm distal to
 the common carotid artery. CIMT will be calculated by the average of three measurements
 performed at 0.2 mm intervals.

 The abdominal aortic diameter will be measured at maximum systolic expansion (Ds) and minimum
 diastolic expansion (Dd) at the mid-point between renal arteries origin and iliac carrefour.
 Aortic strain (S) will be calculated using the formula (S = (Ds-Dd)/Dd). Pressure strain
 elastic modulus (Ep) will be calculated from S using the formula (Ep=(Ps-Pd)/S; Ps= aortic
 systolic pressure; Pd= aortic diastolic pressure). Pressure strain normalized by diastolic
 pressure (Ep*), will be calculated using the formula (Ep* = Ep/Pd). While S is the mean
 strain of the aortic wall, Ep and Ep* are the mean stiffness of the aorta. To measure
 brachial artery flow-mediated dilation (FMD), a pneumatic cuff will be placed on the right
 forearm, 2 cm above the antecubital fossa and inflated to a suprasystolic level (300 mmHg)
 for 5 minutes. A continuous Doppler velocity assessment will be obtained simultaneously, and
 data will be collected using the lowest insonation angle (between 30° and 60°). Brachial
 artery diameters, peak systolic velocity (PSV) and end diastolic velocity (EDV) will be
 measured immediately after and 2 minutes after the cuff release and then compared to basal
 values taken immediately before the inflation. The maximum diameter recorded following
 reactive hyperemia will be reported as a percentage change of resting diameter (FMD = peak
 diameter - baseline diameter/baseline diameter).

 Anthropometric variables Height will be measured to the nearest 0.1 cm using a Harpenden
 stadiometer, and body weight to the nearest 0.1 kg using a manual weighing scale. Body mass
 index (BMI) will be calculated as body weight divided by squared height (kg/m2). Waist
 circumference (WC) will be measured at the high point of the iliac crest around the abdomen
 and was recorded to the nearest 0.1 cm. Hip circumference will be measured over the widest
 part of the gluteal region. Pubertal stages will be determined by physical examination, using
 the criteria of Marshall and Tanner. Systolic (SBP) and diastolic (DBP) blood pressure will
 be measured three times at 2-minute intervals using a standard mercury sphygmomanometer with
 an appropriate cuff size. Mean values will be used for the analysis.

 Assessment only in the OB group

 Biochemical variables After a 12-h overnight fast, blood samples will be taken for
 measurement of: glucose (mg/dL), insulin (μUI/mL), total cholesterol (mg/dL), high density
 lipoprotein-cholesterol (HDL-c, mg/dL), triglycerides (mg/dL), sUA (mg/dL), using
 standardized methods in the Hospital's Laboratory. Low density lipoprotein-cholesterol
 (LDL-c) will be calculated by the Friedwald formula. sUA (mg/dL) will be measured by Fossati
 method reaction using uricase with a Trinder-like endpoint.

 OB subjects will also undergo an oral glucose tolerance test (1.75 g of glucose solution per
 kg, maximum 75 g) and samples will be drawn for the determination of glucose and insulin
 every 30 min. Insulin-resistance at fasting will be calculated using the formula of
 homeostasis model assessment (HOMA)-IR. Insulin sensitivity at fasting and during OGTT will
 be calculated as the formula of the Quantitative Insulin-Sensitivity Check Index (QUICKI) and
 Matsuda index (ISI).

 Determination of interleukins (IL), tumor necrosis factor (TNF)α, plasminogen activator
 inhibitor-1 (PAI1), adiponectin and plasmatic markers of oxidative stress IL-8, IL-10, IL-6,
 TNFα, PAI-1, adiponectin, 3-nitrotyrosine, malondialdehyde (MDA), reactive oxygen species
 (ROS) generation, myeloperoxidase (MPO), reduced glutathione (GSH) and superoxide dismutase
 (SOD) will be measured using specific kits. NO will be quantified from blood samples by using
 the Griess reagent.

 Mitochondria morphology and function Mitochondria will be isolated from monocytes.
 Ultrastructural analyses of mitochondria (through transmission electron microscope ZEISS 109)
 will be performed to assess morphologic mitochondrial changes (mitochondrial swelling,
 decrease in matrix density, possible difference in the sub-plasmalemmal and intrafibrillar
 sub-fraction of mitochondria, fission-fusion dynamic mitochondrial propriety, mitophagy).
 Moreover, mitochondria will be used for in vitro assays of mitochondrial oxygen consumption,
 complex I activity (NAD+/NADH), transmembrane potential and mitochondrial dynamic proteins
 expression (fusion and fission ratio through mitofusin 1 and 2 Western blot analysis).

 Time course of measurements in the OB group All the evaluations previously described will be
 performed at baseline and after 6 (T6) and 12 months (T12) of an isocaloric Mediterranean
 balanced diet plus aerobic training.

 Nutritional analysis and interventions A well-trained and experienced clinical paediatric
 endocrinologist will assess food consumption in all subjects and will administer an
 isocaloric Mediterranean balanced diet in OB children. To assess food consumption, foods will
 be divided according to the classic basic food groups by the Italian Institute of Research on
 Food and Nutrition. Food frequencies questionnaires, validated for a wide range of ages, will
 be also completed by parents. The nutritional counselling will be performed at baseline and
 after 6 and 12 months, according to Italian LARN (Livelli di Assunzione di Riferimento di
 Nutrienti) Guidelines and the Italian food pyramid.

 Moreover, obese subjects will undergo an exercise training regimen. Exercise will be
 conducted daily and will consist of 60 minutes of aerobic physical activity. Parents will
 record every day, on a specific questionnaire, the training performed.
Inclusion Criteria:

 -  children and adolescents (6-16 years);

 -  obese (OB) and normal weight (NW, control group) according to the International
 Obesity Task Force (IOTF) criteria;

 -  both genders;

 -  diet naïve.

 Exclusion Criteria:

 -  specific causes of endocrine or genetic obesity;

 -  type 1 or type 2 diabetes;

 -  previous heart, respiratory, liver and kidney diseases,

 -  current or past use of hormonal or interfering therapies (lipid-lowering,
 hypoglycemic, or antihypertensive treatments).