Number Of Endothelial Microparticles Research Articles

Relationship of Endothelial Microparticles to Obesity and Cardiovascular Disease Risk in Children and Adolescents.

Background Microparticles and endothelial microparticles (EMPs) are implicated in accelerating cardiovascular disease (CVD); however, data in pediatrics are limited. We examined the relationship of microparticles and EMPs with adiposity and subclinical CVD risk measures in a pediatric population to determine their potential as biomarkers of CVD risk. Methods and Results A cross-sectional study of youth (n=280; ages 8-20 years) with a range of body mass index categories was used. Microparticles, EMPs, and activated EMPs were measured by flow cytometry. %Body fat and %visceral adipose tissue were measured by dual X-ray absorptiometry. Measures of arterial stiffness and vascular wall structure were obtained. Linear regression (with log-transformed outcomes) and logistic regression were used to evaluate associations and all results were exponentiated. Youth with overweight/obesity and severe obesity had 2.50 (95% CI, 1.56-4.01) and 3.42 (95% CI, 2.15-5.43) times the geometric means of the total number of microparticles, respectively, compared with those with normal weight. Youth with overweight/obesity and severe obesity had 1.97 (95% CI, 1.09-3.55) and 2.34 (95% CI, 1.31-4.19) times the geometric means of the total number of EMPs, respectively, compared with those with normal weight. There were positive associations between the levels of both microparticles and EMPs with higher adiposity measures and poor CVD risk measures. Youth with higher adiposity showed 1.84 times the odds of having high levels of activated EMPs (%) (odds ratio, 1.84; 95% CI, 1.08-3.14) compared with those with normal weight. Conclusions Levels of microparticles, EMPs, and activated EMPs were positively associated with adiposity and poor subclinical CVD risk in a pediatric population.

Gly14]-Humanin Ameliorates High Glucose-Induced Apoptosis by Inhibiting the Expression of MicroRNA-155 in Endothelial Microparticles.

BackgroundHumanin, a newly emerging endogenously expressed cytoprotective peptide, has been shown to have anti-apoptotic properties effects by protecting neuronal cells injury. Endothelial microparticles (EMPs) are considered as vital mediators in intercellular communication. EMPs may regulate various physiological and pathological processes by transferring mRNAs and microRNAs (miRNAs) to recipient cells.MethodsEMPs were isolated from human umbilical vein endothelial cells (HUVECs) by ultracentrifugation. EMPs were characterized by transmission electron microscopy and nanoparticle tracking analyses. Observation of EMPs uptake into HUVECs and the number of EMPs were realized by confocal microscopy. The expression of miR-155 was examined using real-time PCR. Cell apoptosis was examined by flow cytometry assay.ResultsWe found that high glucose (HG) increased the number of EMPs and upregulated the expression of miR-155 contained within EMPs, which was mitigated by HNG pretreatment. miR-155 overexpression in EMPs reversed the effects of HNG pretreatment and increased apoptosis of target cells. Effects of HNG pretreatment on HG-treated endothelial cells (ECs) were mitigated after miR-155 mimic transfection into HUVECs while were augmented after miR-155 inhibitor transfection into HUVECs.ConclusionHNG inhibited HG-induced apoptosis of ECs and the effect of HNG may be mediated by inhibiting the transfer of EMPs miR-155 from HG-induced HUVECs to normal cells. This study provides a new direction for biological products related to humanin to treat vascular complications associated with all forms of diabetes mellitus.

Circulating Endothelial Microparticles and Aortic Stiffness in Patients with Type 2 Diabetes Mellitus.

Background and objectives: Diabetes mellitus represents a metabolic disorder the incidence of which has been on the increase in recent years. The well-known long-term complications of this disease encompass a wide spectrum of renal, neurological and cardiovascular conditions. The aim of the study was to investigate the serum concentration of endothelial microparticles (EMPs) as well as selected noninvasive parameters of the ascending aorta stiffness calculated with echocardiography. Materials and Methods: 58 patients were enrolled in this study—38 subjects diagnosed with type 2 diabetes mellitus (T2DM) and 20 healthy controls. The analyzed populations did not differ significantly with respect to age, renal function, systolic and diastolic blood pressure. Results: The patients with T2DM and concomitant hypertension presented higher levels of EMPs in comparison with diabetic normotensive subjects. Among patients with T2DM and hypertension, aortic stiffness assessed with the elasticity index (Ep) was higher and the aortic compliance index (D) lower than in the diabetic normotensive group. No correlation between the amount of EMPs and lipid profile, C-reactive protein (CRP) level and glycemia, was observed in the studied group. There was, however, a statistically significant positive correlation between the creatinine level and amount of EMPs, while the negative relationship was documented for EMPs level and the estimated glomerular filtration rate (eGFR). Conclusions: Considering the elevated number of EMPs in diabetic patients with hypertension as well as the positive correlation between EMPs and serum creatinine level, EMPs assessment could be useful in identifying patients who are at high risk of organ damage due to diabetes mellitus.

Increase in circulating ACE-positive endothelial microparticles during acute lung injury.

Circulating endothelial microparticles (EMPs) are considered to be markers of endothelial injury, and lung microvascular endothelial cells express higher levels of angiotensin-converting enzyme (ACE). The aim of this study is to examine whether the number of ACE+ microvascular EMPs could be a prognostic marker for the development of acute respiratory distress syndrome (ARDS) in septic patients.The numbers of EMPs and ACE+ EMPs in the culture supernatant from human microvascular endothelial cells, as well as in the blood of mouse lung injury models and septic patients (n=82), were examined using flow cytometry.ACE+ EMPs in the culture supernatant from pulmonary microvascular endothelial cells increased after exposure to an inflammatory stimulus. In the mouse lung injury models, the circulating ACE+ EMPs and ACE+ EMP/EMP ratio were higher than in the controls (p<0.001). The ACE+ EMP/EMP ratio was correlated with the wet/dry lung ratio (rs=0.775, p<0.001). The circulating ACE+ EMPs and ACE+ EMP/EMP ratio on admission were significantly increased in septic patients who developed ARDS compared with septic patients who did not (p<0.001).Therefore, circulating ACE+ EMPs may be a prognostic marker for the development of ARDS in the septic patients.

Endothelial Microparticles and Vascular Endothelial Growth Factor in Patients With Head and Neck Cancer Undergoing Radiotherapy or Radiochemotherapy.

Endothelial microparticles (EMPs) released from activated or apoptotic endothelial cells may play a role in coagulation and thrombus formation. However, there is insufficient evidence regarding the impact of EMPs on angiogenesis in patients with cancer. Sixteen patients with head and neck cancer (HNC) undergoing radiotherapy/radiochemotherapy (RT/RCT) and 10 healthy controls were studied. Serum EMPs were counted by flow cytometry, and vascular endothelial growth factor (VEGF) was measured by enzyme-linked immunosorbent assay (ELISA). The mean EMP level was significantly higher in patients with HNC before RT/RCT (1,601±1,479 EMP/μl) compared to the control group (782±698 EMP/μl). The number of EMPs was not notably increased after RT/RCT (1,629±769 EMP/μl). There was no significant correlation between the plasma EMP number and concentration of VEGF before (r=0.131; p=0.625), 1 day after (r=-0.042, p=0.874), nor 3 months after RT/RCT (r=0.454, p=0.076). Released EMPs may not influence promotion of neovascularization in patients with HNC.

Changes of microparticle levels before and after daunorubicin-based chemotherapy in patients with acute leukemia

Objective To observe the serum levels of endothelial microparticles (EMP) and tissue factor-bearing microparticles (TF+ MP) in patients with acute leukemia before and after daunorubicin-based chemotherapy. Methods From July 2012 to February 2013, 15 patients with newly diagnosed acute leukemia in Peking Union Medical College Hospital received DA (daunorubicin + cytarabine) regimen or VDCLP (vincristine + daunorubicin + cyclophosphamide + L-asparaginase + prednisone) regimen chemotherapy. There were 8 males and 7 females, and the median age of patients was 44 years old. Eleven patients were acute myeloid leukemia (M0 1 case, M1 1 case, M2 9 cases), and 4 were acute lymphocytic leukemia. The peripheral blood samples were taken before induction chemotherapy and after 3 days of daunorubicin. Levels of EMP and TF+ MP were assessed using flow cytometry. Results The serum EMP and TF+ MP levels were significantly higher after 3-day daunorubicin infusions than those before induction chemotherapy (28.94/μl vs. 10.74/μl, P = 0.001; 64.24/μl vs. 43.80/μl, P = 0.02). Conclusion Daunorubicin-based chemotherapy may cause increased numbers of EMP and TF+ MP in patients with acute leukemia. Key words: Leukemia, acute; Antineoplastic combined chemotherapy protocols; Daunorubicin; Endothelial particles; Tissue factor-bearing microparticles

Systemic lupus erythematosus

: To evaluate blood-borne endothelial microparticles (EMPs) in women with SLE and correlated these to disease activity as defined by the SLEDAI-2K score. The study takes cross-sectional design. A total of 90 age-matched women were recruited including: G1 (healthy volunteers, n = 30), G2 (women with SLE and low disease activity (SLEDAI-2K score ≤4; n = 30) and G3 (women with SLE and moderate/high disease activity (SLEDAI-2K score >4; n = 30). Blood was collected in 3.2% sodium citrate. Subsequently, the microparticles were purified by ultracentrifugation and labeled with anti-CD51/61 and anti-Annexin-V antibodies. Quantification and phenotyping were performed using flow cytometry. The number of EMPs was significantly higher in SLE patients compared with controls (P = 0.0178). When SLE patients were stratified according to disease activity, the number of EMPs was significantly increased in women with moderate-to-high disease activity compared with controls (P = 0.0074). We observed a correlation between the number of EMPs and age (r = -0.34; P = 0.0123) and between the number of EMPs and SLEDAI-2K score (r = 0.30; P = 0.04). Our results suggest that the SLE causes increased EMPs release, especially in patients with SLEDAI-2K score greater than 4. Although measurement of the EMPs could be useful in distinguishing patients with SLE from health controls, they have limited value in differentiating between SLE subtypes.

AGE/RAGE-Induced EMP Release via the NOX-Derived ROS Pathway.

Objective Diabetes is associated with accelerated formation of advanced glycation end products (AGEs) that are extensively found in circulating endothelial microparticles (EMPs). This study aimed to investigate whether AGEs have a direct effect on EMP formation and the possible underlying mechanism. Methods In vitro, cultured human umbilical vein endothelial cells (HUVECs) were incubated with AGEs (200 and 400 μg/ml) for 24 hours with or without pretreatment with anti-RAGE antibody, NOX inhibitor, or ROS scavenger. The number of CD31-positive EMPs was assessed by flow cytometry. Results The number of EMPs was significantly increased in HUVECs stimulated by AGEs in a dose-dependent manner. In addition, receptors for AGEs (RAGE), NAD(P)H oxidase (NOX), and reactive oxygen species (ROS) were increased by AGEs as compared to the control group. These changes could be reversed when HUVECs were pretreated with anti-RAGE antibody. Moreover, inhibition of NOX as well as antioxidant treatment reduced the release of EMPs induced by AGEs. Conclusion Our study suggested that AGEs increased EMP generation, which was mediated by RAGE signaling through NOX-derived ROS.

Increased serum levels of fractalkine and mobilisation of CD34+CD45\u2212 endothelial progenitor cells in systemic sclerosis

BackgroundThe disruption of endothelial homeostasis is a major determinant in the pathogenesis of systemic sclerosis (SSc) and is reflected by soluble and cellular markers of activation, injury and repair. We aimed to provide a combined assessment of endothelial markers to delineate specific profiles associated with SSc disease and its severity.MethodsWe conducted an observational, single-centre study comprising 45 patients with SSc and 41 healthy control subjects. Flow cytometry was used to quantify circulating endothelial microparticles (EMPs) and CD34+ progenitor cell subsets. Colony-forming unit-endothelial cells (CFU-ECs) were counted by culture assay. Circulating endothelial cells were enumerated using anti-CD146-based immunomagnetic separation. Blood levels of endothelin-1, vascular endothelial growth factor (VEGF) and soluble fractalkine (s-Fractalkine) were evaluated by enzyme-linked immunosorbent assay. Disease-associated markers were identified using univariate, correlation and multivariate analyses.ResultsEnhanced numbers of EMPs, CFU-ECs and non-haematopoietic CD34+CD45− endothelial progenitor cells (EPCs) were observed in patients with SSc. Patients with SSc also displayed higher serum levels of VEGF, endothelin-1 and s-Fractalkine. s-Fractalkine levels positively correlated with CD34+CD45− EPC numbers. EMPs, s-Fractalkine and endothelin-1 were independent factors associated with SSc. Patients with high CD34+CD45− EPC numbers had lower forced vital capacity values. Elevated s-Fractalkine levels were associated with disease severity, a higher frequency of pulmonary fibrosis and altered carbon monoxide diffusion.ConclusionsThis study identifies the mobilisation of CD34+CD45− EPCs and high levels of s-Fractalkine as specific features of SSc-associated vascular activation and disease severity. This signature may provide novel insights linking endothelial inflammation and defective repair processes in the pathogenesis of SSc.

Platelet and not erythrocyte microparticles are procoagulant in transfused thalassaemia major patients.

The level of circulating platelet-, erythrocyte-, leucocyte- and endothelial-derived microparticles detected by high-sensitivity flow cytometry was investigated in 37 β-thalassaemia major patients receiving a regular transfusion regimen. The phospholipid procoagulant potential of the circulating microparticles and the microparticle-dependent tissue factor activity were evaluated. A high level of circulating erythrocyte- and platelet-microparticles was found. In contrast, the number of endothelial microparticles was within the normal range. Platelet microparticles were significantly higher in splenectomized than in non-splenectomized patients, independent of platelet count (P<0·001). Multivariate analysis indicated that phospholipid-dependent procoagulant activity was influenced by both splenectomy (P=0·001) and platelet microparticle level (P<0·001). Erythrocyte microparticles were not related to splenectomy, appear to be devoid of proper procoagulant activity and no relationship between their production and haemolysis, dyserythropoiesis or oxidative stress markers could be established. Intra-microparticle labelling with anti-HbF antibodies showed that they originate only partially (median of 28%) from thalassaemic erythropoiesis. In conclusion, when β-thalassaemia major patients are intensively transfused, the procoagulant activity associated with thalassaemic erythrocyte microparticles is probably diluted by transfusions. In contrast, platelet microparticles, being both more elevated and more procoagulant, especially after splenectomy, may contribute to the residual thrombotic risk reported in splenectomized multi-transfused β-thalassaemia major patients.

Changes in endothelial microparticles and endothelial progenitor cells in obese patients in response to surgical stress.

Obese patients undergoing surgery are at increased risk of intraoperative and postoperative cardiovascular complications. The present study was designed to study the changes in endothelial microparticles, endothelial progenitor cells, and adipokines in obese patients in response to limb ischemia during knee surgery. This prospective study included seventy-four patients who underwent total knee arthroplasty. Patients were stratified in tertiles according to their body mass index. Flow cytometry was used for quantification and characterization of endothelial microparticles, endothelial progenitor cells, and adipokines. ELISA (enzyme-linked immunosorbent assay) was used to measure the adiponectin level. The number of endothelial microparticles was greater in obese compared with nonobese patients. The number of endothelial microparticles increased further immediately after surgery in all tertiles. Three days after surgery, endothelial microparticles returned to the basal preoperative level except in the most obese patients. The percentage of endothelial progenitor cells was lower in obese patients. Concentrations of adipokines increased after surgery, but the increase was more accentuated in obese patients. Obese patients present with a high number of endothelial microparticles, a low number of endothelial progenitor cells, and high levels of adipokines, with further increases in adipokines after surgery, suggesting an inflammatory condition that worsens after surgery and may affect endothelial repair.

CD51+ endothelial microparticles as a biomarker of endothelial dysfunction in obese patients with hypertension

Endothelial dysfunction plays a crucial role in the development of hypertension. Furthermore, obesity is associated with hypertension and impaired endothelial function [1, 2]. Recent studies have showed that the number of endothelial microparticles (EMPs) is high in patients with obesity and hypertension [3, 4], and EMPs are involved in the pathophysiology of endothelial function in vivo and in vitro [5]. Therefore, EMPs may contribute to hypertension related to obesity; however, EMPs have many phenotypes that exert different functions in various diseases. Currently, the link between obesity and hypertension caused by endothelial dysfunction is unclear. To our knowledge, no study has thus far investigated the specific phenotype to monitor endothelial function in obese patients with hypertension. The aim of the present study was to investigate whether a specific EMP phenotype can be used to monitor endothelial function in obese patients with hypertension and whether this phenotype is characteristic in obese patients with hypertension. We recruited 29 hypertension patients (19 non-obese and 10 obese), and 15 healthy control subjects. Hypertension was diagnosed as systolic/diastolic blood pressure C140/90 mmHg. Obesity was diagnosed according to the proposed body mass index (BMI) criteria of China’s Ministry of Health as follows: normal: 18.5–23.9, overweight: 24–27.9, and obese C28 kg/m. All patients with a history of inflammatory disease, chronic renal failure requiring dialysis, hepatic or hematologic disorders, or autoimmune or malignant diseases were excluded. All patients were not on statins. Healthy subjects were included if they had no known history of medical illness and a normal blood pressure (\140/90 mm Hg), BMI (18.5–23.9 kg/m), and physical examination. The consent procedure was approved by the Ethics Committee at Institute of Microcirculation Peking Union Medical College & Chinese Academy of Medical Sciences. For microparticles isolation and measurements, blood samples were drawn by venipuncture into blue-top vacutainer tubes containing sodium citrate. Within 1 h of blood sampling, whole blood (3 ml per sample) was centrifuged at 1,500 g for 10 min to prepare platelet-rich plasma and further centrifuged at 13,000 g for 10 min to obtain plateletpoor plasma. Samples were stored at -20 C for 1 week and at -80 C thereafter until analysis. One thawing of stock plasma did not affect microparticle (MP) levels. For the EMPs assay, platelet-poor plasma (50 ll) was incubated with the following fluorescent monoclonal antibodies (4 ll each): phycoerythrin (PE)-labeled anti-CD31 (560983, BD Biosciences), fluorescein isothiocyanatelabeled (FITC) anti-CD51/CD61 (integrin alpha v beta3, 110519, eBioscience), and PE-labeled anti-CD144 (VECadherin12-1449, eBioscience). The samples were incubated at room temperature for 20 min, diluted with 1 ml of phosphate-buffered saline buffer, and analyzed using flow cytometer (Accuri C6, Accuri cytometers). For a precise delineation of CD31positive EMPs (as opposed to platelet-derived CD31positive MPs), CD42b-negative MPs were analyzed in platelet-poor plasma. An isotype antibody was used as a negative control in all measurements. EMPs were defined S.-S. Hu H.-G. Zhang (&) Q.-J. Zhang R.-J. Xiu Institute of Microcirculation, Peking Union Medical College & Chinese Academy of Medical Sciences, Key Laboratory of Microcirculation, National Health and Family Planning Commission, Beijing, China e-mail: zhanghg1966126@imc.pumc.edu.cn

Endothelial Progenitor Cells and Endothelial Microparticles Are Independent Predictors of Endothelial Function

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. 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. 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. 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.

Cyclosporine Induces Endothelial Cell Release of Complement-Activating Microparticles

Defective control of the alternative pathway of complement is an important risk factor for several renal diseases, including atypical hemolytic uremic syndrome. Infections, drugs, pregnancy, and hemodynamic insults can trigger episodes of atypical hemolytic uremic syndrome in susceptible patients. Although the mechanisms linking these clinical events with disease flares are unknown, recent work has revealed that each of these clinical conditions causes cells to release microparticles. We hypothesized that microparticles released from injured endothelial cells promote intrarenal complement activation. Calcineurin inhibitors cause vascular and renal injury and can trigger hemolytic uremic syndrome. Here, we show that endothelial cells exposed to cyclosporine in vitro and in vivo release microparticles that activate the alternative pathway of complement. Cyclosporine-induced microparticles caused injury to bystander endothelial cells and are associated with complement-mediated injury of the kidneys and vasculature in cyclosporine-treated mice. Cyclosporine-induced microparticles did not bind factor H, an alternative pathway regulatory protein present in plasma, explaining their complement-activating phenotype. Finally, we found that in renal transplant patients, the number of endothelial microparticles in plasma increases 2 weeks after starting tacrolimus, and treatment with tacrolimus associated with increased C3 deposition on endothelial microparticles in the plasma of some patients. These results suggest that injury-associated release of endothelial microparticles is an important mechanism by which systemic insults trigger intravascular complement activation and complement-dependent renal diseases.

Endothelium Dependent Vasomotion and In Vitro Markers of Endothelial Repair in Patients with Severe Sepsis: An Observational Study

BackgroundOutcome in sepsis is mainly defined by the degree of organ failure, for which endothelial dysfunction at the macro- and microvascular level is an important determinant. In this study we evaluated endothelial function in patients with severe sepsis using cellular endothelial markers and in vivo assessment of reactive hyperaemia.Materials and MethodsPatients with severe sepsis (n = 30) and 15 age- and gender- matched healthy volunteers were included in this study. Using flow cytometry, CD34+/KDR+ endothelial progenitor cells (EPC), CD31+ T-cells, and CD31+/CD42b- endothelial microparticles (EMP) were enumerated. Migratory capacity of cultured circulating angiogenic cells (CAC) was assessed in vitro. Endothelial function was determined using peripheral arterial tonometry at the fingertip.ResultsIn patients with severe sepsis, a lower number of EPC, CD31+ T-cells and a decreased migratory capacity of CAC coincided with a blunted reactive hyperaemia response compared to healthy subjects. The number of EMP, on the other hand, did not differ. The presence of organ failure at admission (SOFA score) was inversely related with the number of CD31+ T-cells. Furthermore, the number of EPC at admission was decreased in patients with progressive organ failure within the first week.ConclusionIn patients with severe sepsis, in vivo measured endothelial dysfunction coincides with lower numbers and reduced function of circulating cells implicated in endothelial repair. Our results suggest that cellular markers of endothelial repair might be valuable in the assessment and evolution of organ dysfunction.

Increased Circulating Apoptotic CD31+/CD42b- and Activated CD62E+ Endothelial Micro Particles in Coronary Artery Disease

Objective: To examine the phenotypes of endothelial microparticles and platelet microparticles in order to identify a sensitive endothelial microparticle biomarker for monitoring endothelial dysfunction in patients with coronary artery disease, and to examine the mechanisms underlying endothelial dysfunction. Methods: Thirty-three coronary artery disease patients and 53 healthy subjects were recruited. The absolute numbers of endothelial microparticles and platelet microparticles were measured by flow cytometry, and the clinical characteristics of patients and healthy subjects were recorded. Spearman’s correlation analysis and multiple linear regression analysis were performed to analyse the correlation between endothelial microparticles and platelet microparticles, and between microparticles and clinical data. Results: There was a significant difference in the number of CD31+/CD42b- endothelial microparticles and CD62E+ endotheial microparticles between patients with coronary artery diease and healthy subjects (p<0.05). Conclusion: Both endothelial apoptosis and activation play an important role in coronary artery disease, although endothelial apoptosis plays the major role. Endothelial microparticles may be used to monitor endothelial dyfunction in patients with coronary artery disease.

Proteomic analysis of TNF-α-activated endothelial cells and endothelial microparticles

Endothelial microparticles (EMPs) are small vesicles released from endothelial cells (ECs) and found circulating in the blood. EMPs are formed by a plasma membrane surrounding a small amount of cytosol and contain a subset of cellular proteins. As the number of EMPs in the blood increases with certain diseases, they may be an attractive biomarker for clinical diagnosis. Proteomic analysis of EMPs has been previously performed by mass spectrometry. However, the proteomic information of the ECs that secrete EMPs is lacking. This study introduces an invitro model of activated ECs we created for proteomic analyses and reports the changes of the protein content in the ECs and EMPs using proteomic methods. Thus, this study provides valuable information for the analysis of the highly dynamic secretion process of EMPs. There is a direct correlation between the proteins that form EMPs and tumor necrosis factor-α (TNF-α)-activated ECs. The endothelial proteins transferred by EMPs may play important roles in the interaction between EMPs and the target cells, which may lead to endothelial dysfunction.

Dietary flavanols maintain endothelial homeostasis

Dietary intervention studies showed that flavanols can improve endothelial function. We investigated how dietary flavanols affect the balance of endothelial homeostatic regeneration and repair processes. We show in a randomized controlled clinical study that 1 month ingestion of a high flavanol drink (750 mg total flavanols) increased endothelial function (flow‐mediated vasodilation [FMD]) and decreased systolic blood pressure in patients with coronary artery disease. This increase correlated with an increase in the number of circulating angiogenic cells (CACs; CD34+/KDR+ and CD133+/KDR+). Furthermore, flavanols led to a decrease in the numbers of endothelial microparticles (CD31+/CD41‐; CD144+), markers of endothelial injury, as measured by flow cytometry. This was inversely correlated with FMD. Platelet microparticles (CD41+) remained unaffected. In vitro, low nanomolar concentrations of (−)‐epicatechin and flavanol‐rich plasma increased the migratory capacity of CACs and endothelial cells, closure of an endothelial scratch injury, and capillary network formation. Flavanol metabolites increased eNOS activity and led to vasodilation of aortic rings. In mice, epicatechin feeding over one week improved vascular function, angiogenesis (hindlimb ischemia), and aortic GSH levels. Our data lend evidence to the concept that dietary flavanols may help maintain endothelial homeostasis.

Endothelial Microparticles (EMP) for the Assessment of Endothelial Function: An In Vitro and In Vivo Study on Possible Interference of Plasma Lipids

BackgroundCirculating endothelial microparticles (EMP) reflect the condition of the endothelium and are of increasing interest in cardiovascular and inflammatory diseases. Recently, increased numbers of EMP following oral fat intake, possibly due to acute endothelial injury, have been reported. On the other hand, the direct interference of lipids with the detection of EMP has been suggested. This study aimed to investigate the effect of lipid-rich solutions, commonly administered in clinical practice, on the detection, both in vitro and in vivo, of EMP.MethodsFor the in vitro assessment, several lipid-rich solutions were added to whole blood of healthy subjects (n = 8) and patients with coronary heart disease (n = 5). EMP (CD31+/CD42b−) were detected in platelet poor plasma by flow cytometry. For the in vivo study, healthy volunteers were evaluated on 3 different study-days: baseline evaluation, following lipid infusion and after a NaCl infusion. EMP quantification, lipid measurements and peripheral arterial tonometry were performed on each day.ResultsBoth in vitro addition and in vivo administration of lipids significantly decreased EMP (from 198.6 to 53.0 and from 272.6 to 90.6/µl PPP, respectively, p = 0.001 and p = 0.012). The EMP number correlated inversely with the concentration of triglycerides, both in vitro and in vivo (r = −0.707 and −0.589, p<0.001 and p = 0.021, respectively). The validity of EMP as a marker of endothelial function is supported by their inverse relationship with the reactive hyperemia index (r = −0.758, p = 0.011). This inverse relation was confounded by the intravenous administration of lipids.ConclusionThe confounding effect of high circulating levels of lipids, commonly found in patients that receive intravenous lipid-based solutions, should be taken into account when flow cytometry is used to quantify EMP.

Abstract 16821: Reductions in Vascular Function Correlate with Age in a Novel Non-Human Primate Model for Assessment of Vascular Health

Background: Non human primates develop cardiovascular disease over decades. Whether the progression of vascular dysfunction with age is like that observed in humans is unclear. We hypothesize that, like humans, monkeys show age-related reductions in flow-mediated dilation (FMD, an index of endothelium-dependent vascular reactivity) and increases in pulse-wave velocity (PWV, an index of aortic stiffness). We also explored a potential relationship between the age-related vascular effects and circulating biomarkers of endothelial health, endothelial microparticles (EMP) and endothelial progenitor cells (EPC). Methods and Results: In M. fascicularis monkeys (N=21, 14 males and 7 females, 6 to 25 years of age), blood was sampled for EPC and EMP that were measured using flow cytometry. Monkeys were then sedated (midazolam + torbugesic) and baseline femoral artery diameter (4mm segment) was measured by ultrasonography. FMD was calculated as the peak %change from baseline diameter following a 5-minute cessation of blood flow below the knee. PWV was calculated using a modified SphygmoCor program and pulse waves from the radial and femoral arteries. FMD and PWV ranged from 6 to 33% and from 3 to 7 m/s, respectively, and multiple samplings showed high reproducibility up to 4 months (inter-assay variation &lt;5%). FMD negatively (r=-0.94, p&lt;0.0001), while PWV positively (r=0.60, p&lt;0.002) correlated with age in these monkeys. Numbers of EPC (CD45- CD31+ CD34+ VEGFR2+) and EMP [CD45- CD42a- CD54(ICAM-1)+, CD45- CD42a- CD105(endoglin)+, and CD45- CD42a- CD144(VE-cadherin)+] were highly variable between individuals. Neither the EPC nor any of the EMPs significantly correlated with the age or age-related changes in FMD or PWV. Significance = p&lt;0.05. Conclusion: For the first time in non-human primates, we established two non-invasive, clinically-relevant assessments of vascular function, FMD and PWV. Like humans, monkeys show age-related reductions in endothelium-dependent vascular reactivity and increases in aortic stiffness. The circulating numbers of EMP and EPC, independent of the chronic, age-related changes in FMD and PWV, may better serve as biomarkers of acute changes in vascular health associated with disease or therapeutic interventions.