Cristina Zanoni

Oxidative Stress Is Associated With Arterial Dysfunction and Enhanced Intima-Media Thickness in Children With Hypercholesterolemia: The Potential Role of Nicotinamide-Adenine Dinucleotide Phosphate Oxidase

BACKGROUND. Endothelial dysfunction and intima-media thickness are precocious manifestations of hypercholesterolemia, but the mechanism is unclear. OBJECTIVE. The aim of the study was to analyze the interplay among endothelial dysfunction, intima-media thickness, and oxidative stress in children with hypercholesterolemia. METHODS. We performed a cross-sectional study comparing flow-mediated dilation, intima-media thickness, lipid profile, urinary isoprostanes as markers of oxidative stress, and platelet expression of gp91phox, the catalytic unit of nicotinamide-adenine dinucleotide phosphate oxidase, in a population of 50 children with hypercholesterolemia (mean age ± SD: 10.0 ± 3.7 years) and 50 children without hypercholesterolemia (mean age: 9.2 ± 3.5 years). Four children with hereditary deficiency of gp91phox were studied also. RESULTS. Children with hypercholesterolemia had reduced flow-mediated dilation (mean ± SD: 6.2 ± 2.4 vs 9.2 ± 2.5%) and enhanced intima-media thickness (0.45 ± 0.07 vs 0.40 ± 0.06 mm), urinary isoprostanes (86.9 ± 51.6 vs 45.9 ± 25.6 pg/mg creatinine), and gp91phox platelet expression (4.4 ± 3.8 vs 2.0 ± 1.7 mean fluorescence) compared with control subjects. At bivariate analysis, flow-mediated dilation was correlated with low-density lipoprotein cholesterol, intima-media thickness, urinary isoprostanes, and platelet gp91phox. Stepwise multiple linear regression analysis showed that, in children with hypercholesterolemia, flow-mediated dilation and intima-media thickness were significantly associated with low-density lipoprotein cholesterol and urinary isoprostanes; also, gp91phox platelet expression was an independent predictor of urinary isoprostanes. Children with gp91phox hereditary deficiency showed downregulation of platelet gp91phox and reduced urinary excretion of isoprostanes. CONCLUSIONS. The study suggests that gp91phox-mediated oxidative stress may have a pathogenic role in the anatomic and functional changes of the arterial wall occurring in children with premature atherosclerosis.

Early Myocardial Deformation Changes in Hypercholesterolemic and Obese Children and Adolescents: A 2D and 3D Speckle Tracking Echocardiography Study

AbstractDyslipidemia and obesity are considered strong risk factors for premature atherosclerotic cardiovascular disease and increased morbidity and mortality and may have a negative impact on myocardial function.Our purpose was to assess the presence of early myocardial deformation abnormalities in dyslipidemic children free from other cardiovascular risk factors, using 2-dimensional speckle tracking echocardiography (2DSTE) and 3-dimensional speckle tracking echocardiography (3DSTE).We studied 80 consecutive nonselected patients (6–18 years of age) with hypercholesterolemia (low-density lipoprotein [LDL] cholesterol levels >95th percentile for age and sex). Forty of them had normal weight and 40 were obese (body mass index >95th percentile for age and sex). Forty healthy age-matched children were selected as controls. Left ventricular (LV) global longitudinal, circumferential, and radial strains were calculated by 2DSTE and 3DSTE. Global area strain (GAS) was calculated by 3DSTE as percentage of variation in surface area defined by the longitudinal and circumferential strain vectors. Right ventricular (RV) global and free-wall longitudinal strain and LV and RV diastolic strain rate parameters were obtained. Data analysis was performed offline.LV global longitudinal strain and GAS were lower in normal-weight and obese dyslipidemic children compared with normal controls and reduced in obese patients compared with normal-weight dyslipidemic children. LV early diastolic strain rate was lower compared with normals. RV global and free-wall longitudinal strain was significantly reduced in obese patients when compared with the control group. A significant inverse correlation was found between LV strain, LDL cholesterol levels, and body mass index.2DSTE and 3DSTE show LV longitudinal strain and GAS changes in dyslipidemic children and adolescents free from other cardiovascular risk factors or structural cardiac abnormalities. Obesity causes an additive adverse effect on LV strain parameters and RV strain impairment.

Circulating miR-33a and miR-33b are up-regulated in familial hypercholesterolaemia in paediatric age

Hypercholesterolaemia is one of the major causes of CVD (cardiovascular disease). It is associated with enhanced oxidative stress, leading to increased lipid peroxidation which in turn determines endothelial dysfunction and susceptibility to coronary vasoconstriction and atherosclerosis. Different miRNAs are involved in the pathogenesis of CVD and play an important role in inflammatory process control, therefore, together with atherogenic factors, they can stimulate atherosclerotic degeneration of the vessel walls of arteries. miR-33a and miR-33b play a pivotal role in a variety of biological processes including cholesterol homoeostasis, HDL (high-density lipoprotein)-cholesterol formation, fatty acid oxidation and insulin signalling. Our study aimed to determine whether circulating miR-33a and miR-33b expression was altered in familial hypercholesterolaemic children. Total RNA was extracted from plasma, and miR-33a and miR-33b were measured by quantitative real-time PCR. We found that miR-33a and miR-33b were significantly up-regulated in the plasma of 28 hypercholesterolaemic children compared with 25 healthy subjects (4.49±0.27-fold increase, P<0.001, and 3.21±0.39-fold increase, P<0.05 respectively), and for both miRNAs, a positive correlation with total cholesterol, LDL (low-density lipoprotein)-cholesterol, LDL-cholesterol/HDL-cholesterol ratio, apolipoprotein B, CRP (C-reactive protein) and glycaemia was found. OLS (ordinary least squares) regression analysis revealed that miR-33a was significantly affected by the presence of FH (familial hypercholesterolaemia), glycaemia and CRP (P<0.001, P<0.05 and P<0.05 respectively). The same analysis showed that miR-33b was significantly related to FH and CRP (P<0.05 and P<0.05 respectively). Although it is only explorative, the present study could be the first to point to the use of miR-33a and miR-33b as early biomarkers for cholesterol levels in childhood, once validated in independent larger cohorts.

Low dose chromium-polynicotinate or policosanol is effective in hypercholesterolemic children only in combination with glucomannan.

OBJECTIVE A low-fat, fiber-rich diet is the first step in the management for hypercholesterolemic children. Glucomannan (GM) is a natural fiber that has been demonstrated to lower total and LDL-cholesterol. The use of high-dose chromium-polynicotinate (CP) and policosanol (PC) has also shown cholesterol-lowering benefits. We aimed at investigating the effects of low-dose CP or PC and their GM combination in hypercholesterolemic children. METHODS A double-blind trial was conducted in 120 children (60 M, 60 F, 9 ± 4 years, median 9.6 years, range: 3-16 years) randomly assigned to 5 neutraceutical and 1 placebo (only resistant starch) 8-week treatment groups. Fasting blood glucose (FBG), total cholesterol (CholT), triglycerides (TG), HDL and LDL cholesterol were considered. RESULTS GM combination of low-dose CP or PC reduced CholT and LDL without changing HDL, TG and FBG. The highest post-treatment changes were seen after GM combination with CP (CholT 85 ± 3% and LDL 85 ± 5%, of pretreatment) which was significantly (p < 0.01) less than with low-dose CP or PC and starch. When GM was associated with starch, there was no lipid lowering effect, which was an unexpected finding as compared to previous data with GM and no starch. No adverse effects were reported. CONCLUSION This is the first report to show the cholesterol-lowering efficacy of GM combined treatment with low-dose CP or PC. Further studies are needed to investigate the best combinations and doses of nutraceutics to be added to the standard GM treatment. The potential negative association of GM and nutraceutics with starch is clearly shown.

Arterial blood pressure and serum lipids in a population of children and adolescents from Southern Italy: The Calabrian Sierras Community Study (CSCS)

BACKGROUND Lipid standards in Italy are lacking in children and adolescents whereas those for blood pressure (BP) were derived from US surveys. METHODS In a 14-town community in Southern Italy 1657 (64%) of 2594 children aged 6-14 years were enrolled and anthropometric, BP, lipid and glucose serum levels were obtained. RESULTS Average systolic BP was 101 ± 11 (60-150) mm Hg and cholesterol (CholT) level was 156 ± 28 (57-264) mg/dl. There were positive (p<0.00001) age-trends for systolic BP and body mass index (BMI) in both genders whereas age-trends for CholT and heart rate were negative (p<0.00001). A negative age-trend in both genders was also seen for non-HDL cholesterol (p<0.03). Based on 95% percentile gender and age distributions, there were 177 (10.68%) hypertensive (HT) and 82 (4.94%) hypercholesterolemic (HC) children or adolescents. Univariately, HT had higher (p<0.00001) height, weight, BMI, arm circumference, hips, waist, diastolic BP and waist/height, whereas HC had higher LDL-, HDL and non-HDL-cholesterol and triglycerides (p<0.01). Systolic BP was predicted (r(2)=0.2810, p=0.00001) by age (t=2.319, p<0.0205), male gender (t=3.179, p<0.0015), glucose (t=2.357, p<0.0186), height (t=2.473, p<0.0135), arm circumference (t=3.313, p<0.0009) and heart rate (t=4.161, p<0.00001). CholT was related inversely (r(2)=0.1399, p=0.00001) to height (t=-3.928, p<0.0001), weight (t=-3.922, p<0.0001) and waist/height (t=-4.797, p<0.00001) and directly to BMI (t=3.064, p<0.0022), waist (t=5.149, p<0.0000), triglycerides (t=11.332, p<0.00001) and female gender (t=-2.041, p<0.0414). CONCLUSION In these Southern Italian children and adolescents systolic BP and CholT are related with anthropometric and other variables, not confined to height. BP is lower than previously reported.

Metabolic syndrome among children and adolescents from Southern Italy: contribution from the Calabrian Sierras Community Study (CSCS).

Among 1657 children and adolescents aged 6 to 14 years (787, 47% girls and 870, 53% boys) from primary and secondary schools in a 14-town Southern Italian community, HDL cholesterol (54 ± 15 mg/dl), triglycerides (61 ± 29 mg/dl), blood glucose (78 ± 10 mg/dl), systolic (101 ± 11 mm Hg) and diastolic (62 ± 10 mm Hg) blood pressures, waist circumference (WC) (66 ± 10 cm) and WC/height (0.46 ± 0.006) and triglycerides/HDL cholesterol (1.31 ± 0.99) ratios were measured. The distributions were similar in both genders. Age did not affect triglycerides/HDL cholesterol ratio, whereas there was a slightly positive correlation (p<0.00001) between WC/height and triglycerides/HDL cholesterol ratios. We present individual gender and age specific percentile distributions (as Supplementary materials). Using percentile cut-offs (≤ 10th for HDL cholesterol and ≥ 90th for the other components), there were 183 (11%) children or adolescents with low HDL cholesterol, 162 (9.77%) with high triglycerides, 178 (10.74%) with high blood glucose, 178 (10.74%) with high WC, 244 (20.76%) with high systolic or diastolic BP and 126 (7.6%) with high systolic and diastolic BP. Abnormally high BP was seen in 470 (28.36%) children or adolescents. Using abnormal percentile values of 3 of 5 of its components, metabolic syndrome (MS) was diagnosed in 70 (4.2%) subjects, similarly in both genders. To assess out-of-limit distributions of all 5 individual MS components in children and adolescents gender- and age-distributions derived from local epidemiological data should be used: these distributions are presented and they might now be used both for comparative and applicative purposes at least in Southern Europe.

Epigenetics and cardiovascular risk in childhood.

Cardiovascular disease (CVD) can arise at the early stages of development and growth. Genetic and environmental factors may interact resulting in epigenetic modifications with abnormal phenotypic expression of genetic information without any change in the nucleotide sequence of DNA. Maternal dietary imbalance, inadequate to meet the nutritional needs of the fetus can lead to intrauterine growth retardation, decreased gestational age, low birth weight, excessive post-natal growth and metabolic alterations, with subsequent appearance of CVD risk factors. Fetal exposure to high cholesterol, diabetes and maternal obesity is associated with increased risk and progression of atherosclerosis. Maternal smoking during pregnancy and exposure to various environmental pollutants induce epigenetic alterations of gene expression relevant to the onset or progression of CVD. In children with hypercholesterolemia and/or obesity, oxidative stress activates platelets and monocytes, which release proinflammatory and proatherogenic substances, inducing endothelial dysfunction, decreased Doppler flow-mediated dilation and increased carotid intima-media thickness. Primary prevention of atherosclerosis should be implemented early. It is necessary to identify, through screening, high-risk apparently healthy children and take care of them enforcing healthy lifestyle (mainly consisting of Mediterranean diet and physical activity), prescribing nutraceuticals and eventual medications, if required by a high-risk profile. The key issue is the restoration of endothelial function in the reversible stage of atherosclerosis. Epigenetics may provide new markers for an early identification of children at risk and thereby develop innovative therapies and specific nutritional interventions in critical times.

Early increase of NOX2-derived oxidative stress in children: relationship with age.

Background:Studies conducted in the prepubertal period showed that biomarkers of oxidative stress decreased with increasing age in normocholesterolemic children (NC), and, conversely, they are persistently high in hypercholesterolemic children (HC). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is the most important cellular source of reactive oxygen species. No data have been reported concerning the behavior of age-related oxidative stress generated by NOX2, the catalytic subunit of NADPH oxidase, in children.Methods:We performed a cross-sectional study comparing oxidized low-density lipoprotein (ox-LDL), as a marker of oxidative stress, and NOX2 activity, as assessed by blood levels of soluble NOX2–derived peptide (sNOX2-dp), in a population of 250 children, including 125 NC and 125 HC.Results:HC had higher sNOX2-dp (25.8 ± 16.1 pg/ml) and ox-LDL (23.6 ± 14.4 U/l) levels as compared with NC (15.4 ± 10.1 and 11.6 ± 6.4 pg/ml, respectively; P < 0.001). Multiple linear regression analysis showed that ox-LDL and total cholesterol were the only independent predictive variables associated with sNOX2-dp. In NC, ox-LDL and sNOX2-dp significantly decreased from the first to the second quintile of age. In HC, ox-LDL and sNOX2-dp levels did not show significant differences among quintiles of age.Conclusion:This study suggests that NOX2 contributes to generating ox-LDL during the early phase of life in NC and to the persistent high oxidative stress in the prepubertal period of HC.

Mediterranean diet and physical activity impact on metabolic syndrome among children and adolescents from Southern Italy: Contribution from the Calabrian Sierras Community Study (CSCS)

OBJECTIVES The aim of this study was to assess the impact of Mediterranean diet (MD) and physical activity on metabolic syndrome (MS) prevalence in children and adolescents. MATERIAL AND METHODS This cross-sectional study was carried out in 863 boys and 780 girls, 6-14years old, from primary and secondary schools in a 14-town Southern Italian community. We modified the KIDMED questionnaire to adapt it to the local reality. RESULTS Poor adherence to MD was seen in 18.4% of children and adolescents, while 81.6% had a medium-high compliance and the prevalence of MS was 6.6% and 3.7% respectively (OR: 1.8; 95% C.I.: 1.06-3.11; p=0.013). When participants had less physical activity, MS was more frequent as compared to those more active (5.3% versus 2.3%; OR: 2.3; 95% C.I.: 1.3-4.3; p=0.0068) which had a parallel counterpart when comparing those accustomed to seeing television for less or more than 5h per day and MS prevalence was 12.3% versus 3.8% (OR: 3.38; 95% C.I.: 1.66-6.86, p=0.0008), respectively. Finally, there was a specific abnormality in triglyceride levels, both in girls and boys, when participants were classified according to bad lifestyles, based on the combined evaluation of scarce adherence to MD and less extracurricular physical activity, accompanied by a 7-fold increased prevalence of MS as compared to those with the best lifestyle (11.0% versus 1.6%; p=0.025). CONCLUSIONS There is an apparent importance of healthier lifestyle habits including physical activity and adherence to the MD also among children and adolescents.

Circulating miR-200c is up-regulated in paediatric patients with familial hypercholesterolaemia and correlates with miR-33a/b levels: implication of a ZEB1-dependent mechanism

Hypercholesterolaemia provokes reactive oxygen species (ROS) increase and is a major risk factor for cardiovascular disease (CVD) development. We previously showed that circulating miR-33a/b expression levels were up-regulated in children with familial hypercholesterolaemia (FH). miR-33a/b control cholesterol homoeostasis and recently miR-33b has been demonstrated to directly target the transcription factor zinc finger E-box-binding homeobox 1 (ZEB1). The latter acts in a negative feedback loop with the miR-200 family. Our previous studies showed that the ROS-dependent miR-200c up-regulation induces endothelial dysfunction and provokes a ZEB1-dependent apoptosis and senescence. In the present study, we aimed to verify whether circulating miR-200c was induced in FH children, and whether a correlation existed with miR-33a/b Total RNA was extracted from plasma of 28 FH children and 25 age-matched healthy subjects (HS) and miR-200c levels were measured. We found that miR-200c was up-regulated in FH compared with HS (4.00 ± 0.48-fold increase, P<0.05) and exhibited a positive correlation with miR-33a/b. miR-200c did not correlate with plasma lipids, but correlated with C-reactive protein (CRP) plasma levels and glycaemia (GLI). Ordinary least squares (OLS) regression analysis revealed that miR-200c was significantly affected by GLI and by miR-33a (P<0.01; P<0.001 respectively). Moreover, we found that miR-33 overexpression, in different cell lines, decreased ZEB1 expression and up-regulated both the intracellular and the extracellular miR-200c expression levels. In conclusion, circulating miR-200c is up-regulated in FH, probably due to oxidative stress and inflammation and via a miR-33a/b-ZEB1-dependent mechanism. The present study could provide the first evidence to point to the use of miR-33a/b and miR-200c, as early biomarkers of CVD, in paediatric FH.