Luc Bruyndonckx

Quantification of circulating CD34+/KDR+/CD45dim endothelial progenitor cells: analytical considerations.

The discovery of peripheral circulating cells that contribute to vasculogenesis and endothelial repair was one of the most fascinating breakthroughs in the domain of vascular research during the last two decades. The population of vasculogenic cells however, is heterogeneous and can be analyzed using different approaches including in vitro culture and flow cytometry. Circulating CD34(+)/KDR(+)/CD45(dim) endothelial progenitor cells (EPC) have a great potential as biomarkers in various cardiovascular diseases. With the expanding interest in this field, the development of standardized protocols is critical. In this review we describe in detail the pre-analytical and analytical factors that should be taken into account when quantifying CD34(+)/KDR(+)/CD45(dim) EPC using flow cytometry. Moreover, technical suggestions in order to enhance accuracy and reproducibility of this enumeration are provided.

Endothelial progenitor cells and endothelial microparticles are independent predictors of endothelial function.

OBJECTIVE To examine the degree of microvascular endothelial dysfunction in relation to classical cardiovascular risk factors, arterial stiffness, and numbers of circulating endothelial progenitor cells (EPCs) and endothelial microparticles (EMPs), in obese and normal-weight children. STUDY DESIGN Cross-sectional study with 57 obese (15.2±1.4 years) and 30 normal-weight children (15.4±1.5 years). The principal outcome was microvascular endothelial function measured with peripheral arterial tonometry. Fasting blood samples were taken for biochemical analysis and EMPs (CD31+/CD42b- particles) and EPCs (CD34+/KDR+/CD45dim/- cells) flow cytometry. Characteristics between groups were compared by use of the appropriate independent samples test; a stepwise multiple regression analysis was used to determine independent predictors of microvascular endothelial function. RESULTS Microvascular endothelial function was significantly impaired in obese children and inversely correlated with body mass index Z scores (r=-0.249; P=.021) and systolic blood pressure (r=-0.307; P=.004). The number of EPCs was significantly lower in obese children and correlated with endothelial function (r=0.250; P=.022), and the number of EMPs was significantly greater in obese children and correlated inversely with endothelial function (r=-0.255; P=.021). Multivariate analysis revealed that systolic blood pressure and numbers of circulating EPCs and EMPs are important determinants of endothelial function. CONCLUSION Obese children demonstrate impaired endothelial microvascular function, increased arterial stiffness, fewer EPCs, and more EMPs. Besides systolic blood pressure, EPC and EMP counts independently predict the presence of microvascular endothelial dysfunction.

Methodological considerations and practical recommendations for the application of peripheral arterial tonometry in children and adolescents

Endothelial dysfunction is recognized as the primum movens in the development of atherosclerosis. Its crucial role in both cardiovascular morbidity and mortality has been confirmed. In the past, research was hampered by the invasive character of endothelial function assessment. The development of non-invasive and feasible techniques to measure endothelial function has facilitated and promoted research in various adult and paediatric subpopulations. To avoid user dependence of flow-mediated dilation (FMD), which evaluates nitric oxide dependent vasodilation in large vessels, a semi-automated, method to assess peripheral microvascular function, called peripheral arterial tonometry (Endo-PAT(®)), was recently introduced. The number of studies using this technique in children and adolescents is rapidly increasing, yet there is no consensus with regard to either measuring protocol or data analysis of peripheral arterial tonometry in children and adolescents. Most paediatric studies simply applied measuring and analysing methodology established in adults, a simplification that may not be appropriate. This paper provides a detailed description of endothelial function assessment using the Endo-PAT for researchers and clinicians. We discuss clinical and methodological considerations and point out the differences between children, adolescents and adults. Finally, the main aim of this paper is to provide recommendations for a standardised application of Endo-PAT in children and adolescents, as well as for population-specific data analysis methodology.

Assessment of Endothelial Dysfunction in Childhood Obesity and Clinical Use

The association of obesity with noncommunicable diseases, such as cardiovascular complications and diabetes, is considered a major threat to the management of health care worldwide. Epidemiological findings show that childhood obesity is rapidly rising in Western society, as well as in developing countries. This pandemic is not without consequences and can affect the risk of future cardiovascular disease in these children. Childhood obesity is associated with endothelial dysfunction, the first yet still reversible step towards atherosclerosis. Advanced research techniques have added further insight on how childhood obesity and associated comorbidities lead to endothelial dysfunction. Techniques used to measure endothelial function were further brought to perfection, and novel biomarkers, including endothelial progenitor cells, were discovered. The aim of this paper is to provide a critical overview on both in vivo as well as in vitro markers for endothelial integrity. Additionally, an in-depth description of the mechanisms that disrupt the delicate balance between endothelial damage and repair will be given. Finally, the effects of lifestyle interventions and pharmacotherapy on endothelial dysfunction will be reviewed.

Diet, Exercise, and Endothelial Function in Obese Adolescents

BACKGROUND AND OBJECTIVES: Endothelial dysfunction is the first, although reversible, sign of atherosclerosis and is present in obese adolescents. The primary end point of this study was to investigate the influence of a multicomponent treatment on microvascular function. Additional objectives and end points were a reduced BMI SD score, improvements in body composition, exercise capacity, and cardiovascular risk factors, an increase in endothelial progenitor cells (EPCs), and a decrease in endothelial microparticles (EMPs). METHODS: We used a quasi-randomized study with 2 cohorts of obese adolescents: an intervention group (n = 33; 15.4 ± 1.5 years, 24 girls and 9 boys) treated residentially with supervised diet and exercise and a usual care group (n = 28; 15.1 ± 1.2 years, 22 girls and 6 boys), treated ambulantly. Changes in body mass, body composition, cardiorespiratory fitness, microvascular endothelial function, and circulating EPCs and EMPs were evaluated after 5 months and at the end of the 10-month program. RESULTS: Residential intervention decreased BMI and body fat percentage, whereas it increased exercise capacity (P < .001 after 5 and 10 months). Microvascular endothelial function also improved in the intervention group (P = .04 at 10 months; + 0.59 ± 0.20 compared with + 0.01 ± 0.12 arbitrary units). Furthermore, intervention produced a significant reduction in traditional cardiovascular risk factors, including high-sensitivity C-reactive protein (P = .012 at 10 months). EPCs were increased after 5 months (P = .01), and EMPs decreased after 10 months (P = .004). CONCLUSIONS: A treatment regimen consisting of supervised diet and exercise training was effective in improving multiple adolescent obesity-related end points.

Childhood obesity–related endothelial dysfunction: an update on pathophysiological mechanisms and diagnostic advancements

Childhood obesity jeopardizes a healthy future for our society’s children as it is associated with increased cardiovascular morbidity and mortality later on in life. Endothelial dysfunction, the first step in the development of atherosclerosis, is already present in obese children and may well represent a targetable risk factor. Technological advancements in recent years have facilitated noninvasive measurements of endothelial homeostasis in children. Thereby this topic ultimately starts to get the attention it deserves. In this paper, we aim to summarize the latest insights on endothelial dysfunction in childhood obesity. We discuss methodological advancements in peripheral endothelial function measurement and newly identified diagnostic markers of vascular homeostasis. Finally, future challenges and perspectives are set forth on how to efficiently tackle the catastrophic rise in cardiovascular morbidity and mortality that will be inflicted on obese children if they are not treated optimally.

TransFix® for delayed flow cytometry of endothelial progenitor cells and angiogenic T cells.

Endothelial progenitor cells (EPC) and angiogenic T cells have not been validated for use in studies that involve delayed sample processing and analysis. Here, we report our results for the flow cytometric enumeration of circulating EPC and angiogenic T cells using TransFix®-treated whole blood obtained from adult patients with cardiovascular disease and healthy volunteers. Both cell types promote neovascularization and vascular homeostasis. As such they have been put forward as novel diagnostic markers for endothelial dysfunction and may add prognostic information in patients with cardiovascular disease. Our findings indicate that by the addition of TransFix® cellular antigen stabilizing reagent to whole blood, analyses can be postponed up to 7 days after blood collection. Therefore, this procedure may facilitate laboratory workflow, as well as the organization of multicenter studies, which requires analyses to be conducted in a central core laboratory.

Impact of Lifestyle Intervention on HDL-Induced eNOS Activation and Cholesterol Efflux Capacity in Obese Adolescent

Background. Endothelial dysfunction occurs in obese children and adolescent and is regarded as a key step in the development of atherosclerosis. Important components for the development of endothelial dysfunction are reduced activity of endothelial nitric oxide synthase (eNOS) and an increase in cholesterol deposition in the vessel wall, due to reduced reverse cholesterol transport (RCT) activity. High density lipoprotein (HDL) exhibits antiatherosclerotic properties including modulation of eNOS activity and cholesterol efflux capacity. Lifestyle intervention programs can modify endothelial dysfunction in obese adolescents, but their impact on HDL-mediated eNOS activation and RCT is unknown so far. Methods. Obese adolescents (15 ± 1 years, BMI > 35 kg/m2) where randomized either to an intervention group (IG, n = 8; restricted diet and exercise) or to a usual care group (UC, n = 8). At the beginning and after 10 months of treatment HDL-mediated eNOS phosphorylation and cholesterol efflux capacity were evaluated. Results. Ten months of treatment resulted in a substantial weight loss (−31%), an improvement of endothelial function, and an increase in HDL-mediated eNOS-Ser1177 phosphorylation and RCT. A correlation between change in eNOS-Ser1177 phosphorylation or RCT and change in endothelial function was noted. Conclusion. A structured lifestyle intervention program improves antiatherosclerotic HDL functions, thereby positively influencing endothelial function.

Editorial: Assessing cardiovascular risk – should physicians start measuring neck circumference?

The prevalence of overweight and obesity is rapidly increasing worldwide. In 2015 it was estimated that nearly 108 million children and 604 million adults were obese worldwide. Obesity is clearly associated with an increased risk of cardiovascular diseases, mediated through cardiometabolic risk factors such as abnormal lipid profile, higher blood glucose levels and hypertension, and associated comorbidities, such as obstructive sleep apnoea syndrome (OSAS) and type II diabetes mellitus. It is estimated that obesity-related comorbidities account for 8% of all medical health costs every year. More than half of the obese children will become obese adults, if not treated timely and optimally. The cost of obese children becoming obese adults is immense for society and was recently estimated at E19,000 per person. This poses a serious threat for healthcare financing. Obesity is also associated with increased mortality. No less than 4 million deaths in 2015 could be attributed to high body mass index, and 2.7 million of those 4 million could be attributed to cardiovascular disease. Therefore, early detection of patients with increased cardiovascular risk is of the essence, and easily available and non-invasive screening tools are desirable. Fantin et al. studied 95 overweight and obese subjects, with ages ranging from 20 to 77 years and without evidence of cardiovascular disease. All patients underwent extended clinical investigations and medication was carefully noted. Anthropometric measures were taken, including body mass index and neck circumference, registered with the use of a simple flexible ruler at the level of the thyroid cartilage. Blood pressure and both central and peripheral arterial stiffness was registered by measuring carotid-femoral pulse wave velocity (PWVcf) and carotid-radial pulse wave velocity (PWVcr), respectively. Metabolic risk factors were investigated using fasting blood samples: concentrations of glucose, cholesterol, triacylglycerol, high-density lipoprotein (HDL) and insulin were determined. Subjects with obesity and higher values for neck circumference had more cardiometabolic risk factors than subjects with obesity alone; these subjects had higher mean arterial pressure, higher homeostasis model assessment (HOMA) indices and lower HDL concentrations. In addition, these subjects demonstrated increased central (higher PWVcf) and peripheral (higher PWVcr) arterial stiffness. In a step-wise regression analysis they demonstrated that central stiffness was influenced by age, mean arterial pressure, triacylglycerol levels and waist circumference. In contrast, only age and neck circumference were independent predictors of PWVcr, explaining 24% of its variance. Measuring neck circumference seems a promising screening tool for cardiovascular risk. It fulfils most World Health Organization criteria for a good screening tool. Cardiovascular risk associated with obesity is a serious health problem. There is treatment available, the test is easily available, cheap and non-invasive, yet the question arises as to whether the association between neck circumference and cardiovascular risk is direct. It has been demonstrated that upper body fat distribution affects the development of OSAS. Some studies have suggested neck circumference as the most important anthropometric marker for upper body fat distribution. The present data could therefore be attributed to the effects of OSAS. Indeed, according to the recent European Society of Cardiology (ESC) guidelines on cardiovascular disease prevention, OSAS is an important clinical condition affecting cardiovascular disease risk. This effect is partly mediated through repeated hypoxaemia and increased inflammation worsening the endothelial dysfunction present in obese patients. OSAS in obese