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Endothelial Microparticles Correlate with Endothelial Dysfunction in Obese Women

Division of Metabolic Diseases (K.E., M.C., B.S., R.G., L.S., G.G., D.G.), Center of Excellence for Cardiovascular Disease (K.E., D.G.), and Division of Diabetes (L.M.), Second University of Naples, 80138 Naples, Italy

Address all correspondence and requests for reprints to: Katherine Esposito, M.D., Ph.D., Division of Metabolic Diseases, Policlinico Universitario, Piazza L. Miraglia 2, Naples, Italy. E-mail: dario.giugliano{at}unina2.it.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Cell-derived microparticles are supposed to be involved in atherogenesis.

Objective: This study aimed to evaluate circulating microparticles in obese women and their relation with anthropometric measures and endothelial dysfunction.

Design, Setting, and Participants: Forty-one obese [body mass index (BMI) > 30 kg/m²] women and 40 normal weight (BMI < 25 kg/m²) age-matched women were studied. Flow cytometry was used to assess microparticles by quantification of circulating endothelial microparticles (EMP, CD31⁺/CD42b^–) and platelet microparticles (PMP, CD31⁺/CD42b⁺) in peripheral blood; endothelium-dependent flow-mediated vasodilation (FMD) was evaluated in the right brachial artery after reactive hyperemia.

Results: Compared with lean women, obese women presented significantly higher numbers of EMP and PMP, and reduced FMD. BMI did not correlate with either EMP (r = 0.02, P = 0.9) or PMP (r = –0.07, P = 0.645), whereas waist-to-hip ratio (WHR) showed significant correlation with both microparticles (r = 0.699, P < 0.001; r = 0.373, P = 0.016, respectively). Both EMP and PMP counts positively correlated with impairment of FMD in obese women. Multivariate analysis correcting for age, anthropometric indices, lipid parameters, and PMP identified EMP as the only independent predictor for impaired endothelial-dependent vasodilation (P = 0.003).

Conclusions: EMP are elevated in obese women and independently involved in the pathogenesis of endothelial dysfunction. WHR is the anthropometric measure more closely related to EMP and endothelial dysfunction.

	Introduction

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OBESITY HAS BECOME nowadays a common condition and a worldwide public health problem of epidemic proportions. In addition to being an independent risk factor for cardiovascular diseases, obesity also increases the incidence of other risk factors, notably diabetes, dyslipidemia, hypertension, and the prothrombotic state (1). Insulin resistance, endothelial dysfunction, and subclinical inflammation have all been demonstrated in the obese population and may contribute to its raised cardiovascular risk (2).

In recent years, the interest for endothelial cell-derived submicroscopic membranous vesicles, termed microparticles, has substantially increased, not only because of their procoagulant properties, but also because of their putative role in inflammatory processes and their ability to directly affect endothelial functions (3, 4). Moreover, microparticles constitute an important component of the human atherosclerotic plaque (5), and elevated numbers of circulating microparticles have been found in patients with a great variety of diseases with vascular involvement and hypercoagulability (6, 7).

To the best of our knowledge, there are no reported studies that investigated the role of microparticles in human obesity. Therefore, the aims of this study were: 1) to evaluate circulating endothelial and platelet microparticles (PMP) in obese women; 2) to disclose the measure of obesity [body mass index (BMI), waist-to-hip ratio (WHR), and waist or hip circumference] with the strongest relation with circulating microparticles; and 3) to address the relation between circulating microparticles and endothelial-dependent vasodilation.

	Subjects and Methods

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Women were recruited among those attending the outpatient department of the Division of Metabolic Diseases at the Second University of Naples (Naples, Italy). The women were sedentary (less than 1 h per week of physical activity) with no evidence of participation in diet reduction programs and with a stable weight (±1 kg) within the last 6 months, and were asked to complete a personal health and medical history questionnaire, which served as a screening tool.

To be enrolled in the study, women had to have a BMI more than 30 kg/m². Diabetes mellitus (fasting plasma glucose > 126 mg/dl, or plasma glucose > 200 mg/dl 2 h after a 75-g oral glucose challenge), smoking, cardiovascular disease, use of drugs, or alcohol abuse were exclusion criteria. Also excluded were those women with conditions known or suspected to increase levels of microparticles, including acute infections, pregnancy, antiphospholipid syndrome, thrombotic thrombocytopenia purpura, and sepsis. The study was approved by the institutional committee of ethical practice of our institution, and all the study subjects gave informed written consent. Sedentary normal-weight women (BMI < 25 kg/m²) were recruited from the medical and paramedical staff and matched for age with the obese women.

Endothelium-dependent vasodilation

Endothelium-dependent flow-mediated vasodilation (FMD) was evaluated in the right brachial artery as previously described (8). Endothelium-independent vasodilation was assessed after 0.4 mg sublingual nitroglycerin administration.

Blood samplings and flow cytometry

Blood was drawn into citrate Vacutainers at morning time after an overnight fast. Samples were processed within 2 h after being obtained; samples were centrifuged for 10 min at 160 x g to prepare platelet-rich plasma, then centrifuged for 10 min at 1000 x g to prepare platelet-poor plasma. Microparticles were measured by flow cytometry. In brief, 50 µl of platelet-poor plasma was incubated (20 min) with 4 µl of anti-CD31-FITC (PharMingen, San Diego, CA), plus 4 µl anti-CD42b-PE (Becton Dickinson, San Jose, CA), then diluted with 1 ml of PBS and analyzed on BD FACSCalibur cytometer. Microparticles were identified by their characteristic forward and side scatter, and their ability to bind cell-specific monoclonal antibodies. Endothelial microparticles (EMP) were defined as CD31⁺/CD42b^– particles, whereas PMP were defined as CD31⁺/CD42b⁺ particles. The rationale of two-color method (CD31 and CD42b) was that significant CD31 occurs on both EMP and PMP, whereas CD42b is restricted to platelets, allowing discrimination between them. The possibility of leukocyte microparticles (CD31⁺/CD45⁺) was tested but accounted for a negligible percentage of all CD31⁺ microparticles in both normal and obese women. Control isotope IgG1 and IgG2a antibodies were obtained from Becton Dickinson. Events were counted by triggering on the fluorescence signal of phycoerythrin, above background noise, on the y-axis of the dot plot, whereas the green signal of CD31-FITC was on the x-axis (four-decade log scales, x and y). The coefficient of variation of the flow assay is less than 5%. Data were processed using the Macintosh CellQuest software program (Becton Dickinson). Values were reported as counts per microliter).

Laboratory analysis

All laboratory analyses were performed as previously described (8, 9).

Statistical analysis

Data are presented as mean ± SD unless otherwise indicated. Continuous variables were tested for normality distribution with the Kolmogorov-Smirnov test. Values of microparticles were log-transformed to be handled as continuous variables. A two-tailed, unpaired Student t test was used for comparison of means between groups. The one-way ANOVA test with post hoc Bonferroni analysis was used for comparisons of categorical variables. Univariate correlation was performed with the Pearson correlation coefficient. Multiple linear regression analysis was performed to identify independent variables influencing the prediction of microparticles changes in blood. All analyses were performed using SPSS 11.5 for Windows.

	Results

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The baseline clinical and metabolic characteristics of participants in the study are shown in Table 1. Compared with lean women, obese women had greater anthropometric measurements (BMI, WHR, and waist circumference), and C-reactive protein levels. FMD was slightly but significantly lower in obese women, whereas the level of circulating EMP and PMP was significantly greater. Endothelium-independent vasodilation was not different. BMI did not correlate with either EMP (r = 0.02, P = 0.9) or PMP (r = –0.07, P = 0.645), whereas WHR showed significant correlation with both microparticles (r = 0.699, P < 0.001; r = 0.373, P = 0.016, respectively) (Fig. 1). However, neither waist nor hip in isolation correlated with EMP (r = 0.143, r = 0.59; r = –0.276, r = 0.2, respectively) or PMP (r=0.28, P = 0.2; r=-0.15, P = 0.78, respectively). Age, blood pressure, glucose, and lipid [cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol] parameters did not correlate with circulating levels of both EMP and PMP. However, homeostasis model assessment (HOMA) correlated with EMP (r = 0.34, P = 0.04) and FMD (r = –0.29, P = 0.045), whereas C-reactive protein correlated with EMP only (r = 0.29, P = 0.043).

View larger version (25K): [in this window] [in a new window]   FIG. 1. Correlation between WHR (top left) or BMI (bottom left) and the number of circulating EMPs. The number of endothelial (top right) and platelet (bottom right) microparticles negatively correlates with flow-mediated dilation in the brachial artery of obese women. The number of EMPs are log transformed (log base, 10).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Obesity is a major risk factor for cardiovascular disease (1). The results of the present study demonstrate that circulating endothelial and platelet-derived microparticles are higher in obese women as compared with age-matched lean women, and also negatively correlated with endothelium-dependent, but not with endothelium-independent vasodilation. Moreover, WHR was strongly related to microparticles, whereas BMI was not, indicating that the ratio of abdominal to gluteal circumferences is the anthropometric measure more closely related to the cardiovascular risk associated with obesity. As in multivariate analysis, EMP remained the only independent predictor of flow-mediated dilation in the brachial artery; it seems likely that these microparticles may represent a link between abdominal obesity and future atherosclerosis.

To the best of our knowledge this is the first demonstration that circulating microparticles are elevated in women with obesity, particularly those with a high WHR, and without clinical and instrumental evidence of cardiovascular disease. Our findings agree with the results of the INTERHEART study (10), which was a standardized case-control study of acute myocardial infarction with 27,098 participants in 52 countries. In that study, the WHR was the strongest anthropometric predictor of myocardial infarction in men and women across all age and ethnic groups, whereas the association of BMI with myocardial infarction disappeared when adjusted for the other risk factors.

Microparticles are small membrane vesicles that are released from cells upon activation or during apoptosis (6). The majority of in vivo microparticles in blood are derived from platelets, whereas microparticles from erythrocytes, granulocytes, monocytes, lymphocytes, and endothelial cells usually circulate at lower numbers. Microparticles are vesicles smaller than 1.5 µm (11, 12) and can influence the endothelial function; microparticles from patients with acute coronary syndromes directly impaired endothelium-dependent vasodilatation in rat aorta-rings, presumably by inhibition of the nitric oxide-mediated signal transduction (13). Although microparticles are elevated in condition of endothelial cell loss, as a consequence of inflammatory processes and the associated vascular damage, they may play a direct role in atherogenesis, also considering that EMP can directly activate and stimulate cells to produce inflammatory substance mediators such as cytokines (14).

Obesity is a state of chronic oxidative stress and inflammation (15), which may increase free radical formation, which quenches and deactivates nitric oxide, reducing its availability for target cells. It has also been shown that the effect induced by EMP is related to an increase in superoxide anion production (4), which may reduce the availability of nitric oxide. This hypothesis seems consistent with the reduced circulating levels of nitrotyrosine in obese women, considered a good marker for the formation of endogenous peroxynitrite (16) and hence of reduced nitric oxide availability.

Moreover, the raised inflammatory milieu found in obesity could lead to insulin resistance and endothelial dysfunction (17). Regardless of the mechanisms, microparticles may be a cause of endothelial dysfunction but also a consequence of endothelial damage (18, 19, 20), as they are able to induce vascular dysfunction (4) and can be formed and released on cell activation by inflammatory mediators such as cytokines (7).

In conclusion, our findings show that EMP are elevated in women with visceral obesity and independently involved in the pathogenesis of endothelial dysfunction. Whether assessment of circulating endothelial microparticles may be a novel marker for risk stratification, a useful tool for monitoring efficacious antiatherogenic strategies, or a target for novel therapeutic options remains to be defined in future investigations.

	Footnotes

 
The authors have nothing to disclose.

First Published Online July 5, 2006

Abbreviations: BMI, Body mass index; EMP, endothelial microparticle; FMD, flow-mediated vasodilation; HDL, high-density lipoprotein; HOMA, homeostasis model assessment; PMP, platelet microparticle; WHR, waist-to-hip ratio.

Received April 20, 2006.

Accepted June 28, 2006.

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