Endothelial Microparticles Research Articles

Altered lung biology of healthy never smokers following acute inhalation of E-cigarettes

BackgroundLittle is known about health risks associated with electronic cigarette (EC) use although EC are rising in popularity and have been advocated as a means to quit smoking cigarettes.MethodsTen never-smokers, without exposure history to tobacco products or EC, were assessed at baseline with questionnaire, chest X-ray, lung function, plasma levels of endothelial microparticles (EMP), and bronchoscopy to obtain small airway epithelium (SAE) and alveolar macrophages (AM). One week later, subjects inhaled 10 puffs of “Blu” brand EC, waited 30 min, then another 10 puff; n = 7 were randomized to EC with nicotine and n = 3 to EC without nicotine to assess biological responses in healthy, naive individuals.ResultsTwo hr. post-EC exposure, subjects were again assessed as at baseline. No significant changes in clinical parameters were observed. Biological changes were observed compared to baseline, including altered transcriptomes of SAE and AM for all subjects and elevated plasma EMP levels following inhalation of EC with nicotine.ConclusionsThis study provides in vivo human data demonstrating that acute inhalation of EC aerosols dysregulates normal human lung homeostasis in a limited cohort of healthy naïve individuals. These observations have implications to new EC users, nonsmokers exposed to secondhand EC aerosols and cigarette smokers using EC to quit smoking.Trial registrationClinicalTrials.gov NCT01776398 (registered 10/12/12), NCT02188511 (registered 7/2/14).

In the View of Endothelial Microparticles: Novel Perspectives for Diagnostic and Pharmacological Management of Cardiovascular Risk during Diabetes Distress.

Acute or chronic exposure to diabetes-related stressors triggers a specific psychological and behavior stress syndrome called diabetes distress, which underlies depressive symptoms in most diabetic patients. Distressed and/or depressive diabetic adults exhibit higher rates of cardiovascular mortality and morbidity, which have been correlated to macrovascular complications evoked by diabetic behavior stress. Recent experimental findings clearly point out that oxidative stress accounts for the vascular dysfunction initiated by the exposure to life stressors in diabetic conditions. Moreover, oxidative stress has been described as the main autocrine and paracrine mechanism of cardiovascular damage induced by endothelial microparticles (anuclear ectosomal microvesicles released from injured endothelial cells) in diabetic subjects. Such robust relationship between oxidative stress and cardiovascular diseases strongly suggests a critical role for endothelial microparticles as the primer messengers of the redox-dependent vascular dysfunction underlying diabetes distress. Here, we provide novel perspectives opened in the view of endothelial microparticles as promising diagnostic and pharmacotherapeutic biomarkers of cardiovascular risk in distressed diabetic patients.

Exercise Intensity Reduces Circulating Annexin V- CD105 Microparticles in Adults With Prediabetes

PURPOSE: Microparticles (MPs) derived from platelets, leukocytes and endothelial cells are important players in cardiovascular disease (CVD) risk. Exercise is established to reduce CVD risk, but no study has tested the effect of exercise intensity on various subtypes of MP in people with prediabetes. We tested the hypothesis that short-term interval (INT) training would reduce MP subtypes vs. continuous (CONT) exercise. METHODS: Eighteen obese adults (age: 63.8±1.5yrs BMI: 31.0±1.3 kg/m2) were screened for prediabetes using ADA criteria (75g OGTT and/or HbA1c). Subjects were randomized to INT (n=10, 3 min intervals at 90% and 50% HRpeak) or CONT (n=8, 70% HRpeak) training for 12 supervised sessions over 2 wks for 60 min/d. Fitness (VO2peak) and weight (kg) were assessed and arterial stiffness (augmentation index; AI) calculated using total AUC during a 75g OGTT. Total MPs, platelet MPs (CD31+/CD41+), endothelial MPs (CD105, CD31-/ CD41+) and leukocyte MPs (CD45+/CD41-) were analyzed from fresh plasma via imaging flow cytometry pre-/post- intervention. RESULTS: Our interventions had no effect on weight loss but INT exercise increased VO2peak (P=0.04) and reduced fasted AI (trend: P=0.08) compared with CONT training. While our intervention had no effect on platelet or leukocyte MPs, INT exercise decreased Annexin V- endothelial MP CD105 (1.6±0.2 vs. 1.4±0.2 count) compared with CONT training (1.2±0.2 vs. 1.8±0.1 count; P=0.04). Increased VO2peak correlated with decreased Annexin V+ CD105 endothelial MPs (r=-0.60, P=0.01). CONCLUSION: Exercise intensity decreases endothelial derived MPs through possibly a cardiovascular fitness related mechanism, independent of weight loss.

Putative mechanisms Underlying Myocardial infarction onset and Emotions (PUME): a randomised controlled study protocol

IntroductionThe experience of negative emotions (eg, anger, anxiety and sadness) is associated with an increased short-term risk of incident cardiovascular disease (CVD) events, independent of traditional CVD risk factors. Impairment...

Imbalance between endothelial damage and repair capacity in chronic obstructive pulmonary disease.

BackgroundCirculating endothelial microparticles (EMPs) and progenitor cells (PCs) are biological markers of endothelial function and endogenous repair capacity. The study was aimed to investigate whether COPD patients have an imbalance between EMPs to PCs compared to controls and to evaluate the effect of cigarette smoke on these circulating markers.MethodsCirculating EMPs and PCs were determined by flow cytometry in 27 nonsmokers, 20 smokers and 61 COPD patients with moderate to severe airflow obstruction. We compared total EMPs (CD31+CD42b-), apoptotic if they co-expressed Annexin-V+ or activated if they co-expressed CD62E+, circulating PCs (CD34+CD133+CD45+) and the EMPs/PCs ratio between groups.ResultsCOPD patients presented increased levels of total and apoptotic circulating EMPs, and an increased EMPs/PCs ratio, compared with nonsmokers. Women had less circulating PCs than men through all groups and those with COPD showed lower levels of PCs than both control groups. In smokers, circulating EMPs and PCs did not differ from nonsmokers, being the EMPs/PCs ratio in an intermediate position between COPD and nonsmokers.ConclusionsWe conclude that COPD patients present an imbalance between endothelial damage and repair capacity that might explain the frequent concurrence of cardiovascular disorders. Factors related to the disease itself and gender, rather than cigarette smoking, may account for this imbalance.

Increased circulating endothelial microparticles in children with FMF

Objective: Endothelial microparticles (EMPs) are considered as markers of endothelial dysfunction. In this study, we aimed to examine whether there is endothelial dysfunction in children with familial Mediterranean fever (FMF), hypothesizing that endothelial dysfunction would be present especially with acute-phase response in the active period of the disease.Methods: This cross-sectional study included 65 FMF patients (41 attack free, 24 attack period) and 35 healthy controls. Circulating EMPs, serum amyloid A (SAA), and other inflammation markers were measured in all groups. Circulating EMPs were measured using flow cytometry. Study groups were compared for circulating EMP and inflammatory markers. The relationship between EMPs and the activation of the disease was evaluated.Results: The levels of CD144+ and CD146+ EMPs in the FMF attack period group were significantly higher than those of the control group (p < 0.05). The levels of inflammation markers in the attack period group were significantly higher than those of the control and attack-free groups (p < 0.05). In the FMF attack group, the CD144+ and CD146+ EMP were significantly correlated with CRP.Conclusions: Our results suggest that endothelial damage is present especially in the active period of the disease in children with FMF. The endothelial dysfunction becomes an overt parallel with inflammation.

Release of endothelial microparticles in patients with arterial hypertension, hypertensive emergencies and catheter-related injury

Background and aimsCirculating endothelial microparticles (EMPs) are increased in arterial hypertension. The role of physicomechanical factors that may induce EMP release in vivo is still unknown. We studied the relationship of EMPs and physicomechanical factors in stable arterial hypertension and hypertensive emergencies, and investigated the pattern of EMP release after mechanical endothelial injury. MethodsIn a pilot study, 41 subjects (50% hypertensives) were recruited. EMPs were discriminated by flow-cytometry (CD31+/41-, CD62e+, CD144+). Besides blood pressure measurements, pulse-wave-analysis was performed. Flow-mediated dilation (FMD), nitroglycerin-mediated dilation (NMD), and wall-shear-stress (WSS) were measured ultrasonographically in the brachial artery; microvascular perfusion by laser-Doppler (Clinicaltrials.gov: NCT02795377). We studied patients with hypertensive emergencies before and 4 h after BP lowering by urapidil (n = 12) and studied the release of EMPs due to mechanical endothelial injury after coronary angiography (n = 10). ResultsHypertensives exhibited increased EMPs (CD31+/41-, CD144+, CD62e+) as compared to normotensives and EMPs univariately correlated with systolic BP (SBP), augmentation index, and pulse wave velocity and inversely with FMD. CD31+/41--EMPs correlated with diameter and inversely with WSS and NMD. CD62e+ and CD144+-EMPs inversely correlated with microvascular function. During hypertensive emergency, only CD62e+ and CD144+-EMPs were further elevated and FMD was decreased compared to stable hypertensives. Blood pressure lowering decreased CD62e+ and CD144+-EMPs and increased FMD. CD31+/41—EMPs, diameter, and WSS remained unaffected. Similar to hypertensive emergency, catheter-related endothelial injury increased only CD144+ and CD62e+-EMPs. ConclusionsEMP release in hypertension is complex and may involve both physicomechanical endothelial injury and activation (CD144+, CD62e+) and decreased wall shear stress (CD31+/41-).

High-Mobility Group Box 1 From Hypoxic Trophoblasts Promotes Endothelial Microparticle Production and Thrombophilia in Preeclampsia.

Thrombophilia is a major complication in preeclampsia, a disease associated with placental hypoxia and trophoblast inflammation. Preeclampsia women are known to have increased circulating microparticles that are procoagulant, but the underlying mechanisms remain unclear. In this study, we sought to understand the mechanism connecting placental hypoxia, circulating microparticles, and thrombophilia. We analyzed protein markers on plasma microparticles from preeclampsia women and found that the increased circulating microparticles were mostly from endothelial cells. In proteomic studies, we identified HMGB1 (high-mobility group box 1), a proinflammatory protein, as a key factor from hypoxic trophoblasts in stimulating microparticle production in human umbilical vein endothelial cells. Immunodepletion or inhibition of HMGB1 in the conditioned medium from hypoxic human trophoblasts abolished the endothelial microparticle-stimulating activity. Conversely, recombinant HMGB1 stimulated microparticle production in cultured human umbilical vein endothelial cells. The microparticles from recombinant HMGB1-stimulated human umbilical vein endothelial cells promoted blood coagulation and neutrophil activation in vitro. Injection of recombinant HMGB1 in pregnant mice increased plasma endothelial microparticles and promoted blood coagulation. In preeclampsia women, elevated placental HMGB1 expression was detected and high levels of plasma HMGB1 correlated with increased plasma endothelial microparticles. Our results indicate that placental hypoxia-induced HMGB1 expression and release from trophoblasts are important mechanism underlying increased circulating endothelial microparticles and thrombophilia in preeclampsia.

Effects of HIV‐1 gp120 and TAT on Endothelial Microparticle Release and Oxidative Stress

Human immunodeficiency virus (HIV)‐1 infection is associated with an increased risk and prevalence of atherosclerotic cardiovascular disease (CVD). A major factor underlying the increased CVD burden with HIV‐1‐infection is endothelial damage and dysfunction. Although the mechanisms underlying the HIV‐1‐related vasculopathy are not fully understood, HIV‐1 associated viral proteins are known to have deleterious effects on the endothelium. Endothelial cell release of microparticles and endothelial cell oxidative stress are key characteristics of a proatherogenic endothelial phenotype. The direct effect of HIV‐1 viral proteins on endothelial microparticle release and oxidative stress, however, is currently unknown. The aim of the present study was to determine the effects of X4 and R5 HIV‐1 gp120 and TAT on endothelial cell microparticle release and oxidative stress. Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated at a density of 2 × 106 cells/condition. HUVECs were treated with media alone or media containing either lavgp120 (X4; 100 ng/mL), Balgp120 (R5; 100 ng/mL) or TAT (500 ng/mL) for 24 h to generate endothelial microparticles (EMPs). Viral protein stimulated EMPs were pelleted by centrifugation and resuspended in culture media. Characterization and concentration of EMPs was determined by flow cytometry using VE‐Cadherin (CD 144‐PE). To assess oxidative stress, intracellular reactive oxygen species (ROS) production was determined. To do so, 2.5 ×104 HUVECs were plated in 96‐well tissue culture plates and allowed to adhere overnight. Cells were then incubated with 2′,7′ –dichlorofluorescin diacetate (DCFDA; 25 uM), washed and stimulated with media in the presence or absence of viral proteins. All measurements were done in triplicate and calculated as percent (%) of control by fluorescence spectroscopy. Treatment of HUVECs with X4 (117±28 MP/μL), R5 (105 ± 24 MP/μL) and TAT (101±36 MP/μL) induced markedly higher (~105%) EMP release compared with untreated (51±13 MP/μL) cells. Additionally, X4 (135 ±11%), R5 (139±13%) and TAT (142±7%) significantly increased (~40%) ROS production compared with untreated cells. Of note, the magnitude of increase in EMP release and ROS production was not significantly different between viral proteins. These results indicate that HIV‐1 gp120 and TAT induce EMP release and increase oxidative stress in endothelial cells. Viral protein augmentation of cellular ROS may be the impetus for EMP release, and requires further study. EMP generation and increased endothelial oxidative burden likely contribute to atherogenic effects of HIV‐1 viral proteins.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TCTAP A-040 Endothelial Microparticles-mediated Transfer of MicroRNA-19b Promotes Atherosclerosis via Activating Perivascular Adipose Tissue Inflammation in ApoE-/- Mice

Microparticles are the major carriers of microRNAs in circulation. Our previous study has shown that microRNA (miR)-19b in endothelial microparticles (EMP) is significantly elevated in patients with unstable angina. However, little is known about the relationship between EMP mediated miR-19b

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.

Association between vitamin D and vascular endothelial injury in hypertensive patients

Objective To evaluate the association between vitamin D (VD) and vascular endothelial injury in hypertensive patients. Methods Ninety nine patients with hypertension and 126 healthy subjects (control group) from the same community in Nanjing were recruited in the study. The serum levels of fasting blood glucose (GLU), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) were measured. Serum 25-hydroxy vitamin D (25-OH-D) and nitric oxide (NO) levels were determined by enzyme-linked immunosorbent assay. The level of plasma endothelial microparticles was measured by flow cytometry. The data were statistically analyzed with SPSS version 19.0. Results The serum 25-(OH)-D[(55.22±11.92)nmol/L vs. (64.17±21.52) nmol/L, t=-3.71], vitamin D binding protein (VDBP)[(257.32±141.31) ng/ml vs. (314.21±124.13) ng/ml, t=-3.21], NO levels [(39.35±13.33) μmol/L vs. (43.42±11.83) μmol/L, t=-2.42]in hypertension group were significantly lower than those in control group. And the circulating endothelial cell particles(4.95±1.78 vs. 2.84±2.07, t=8.05)in hypertension group was significantly higher than that in control group. In hypertensive patients, the circulating endothelial cell particles in VD deficiency group was significantly higher than that in low VD group and normal VD group [(6.42±1.25)% vs. (4.6±1.68)%, (3.15±0.90)%, F=15.08]. And the serum NO level in VD deficiency group was significantly lower than that in other two groups[(36.57±10.52) μmol/L vs. (39.77±13.01) μmol/L, (46.26±25.90) mol/L, F=1.20]. Multiple linear regression analysis showed that low serum 25 (OH) D(B=-0.189, SE=0.033, β=0.509, t=-5.72)and gender (B=-0.682, SE=0.351, β=-0.182, t=-2.054)were the risk factors of vascular endothelial injury. Conclusion The serum vitamin D levels in hypertensive patients is decreased, and vitamin D deficiency may be a risk factor for endothelial injury in hypertension. Key words: Vitamin D; Hypertension; Vascular endothelial cells injury

Microparticles as prognostic biomarkers in dengue virus infection

Promising biomarkers which may help predict the risk of developing severe dengue virus infection (DVI) are lacking and will be helpful. Thus the main aim of this study was to analyze the role of cell-derived microparticles (MP) in DVI. Sixty patients with DVI i.e. 18: dengue with warning signs (DWS); 1: DSS and 41: dengue without warning signs (DWOS); along with 15 controls (other febrile illness) were included in the study. The following MPs were assessed: annexinV, platelet (CD41a), red blood cell (RBC) (CD235a) and activated endothelial (CD62e) MPs. Patients with profound thrombocytopenia without bleeding had statistically elevated platelet MP (PMP) levels when compared to patients with profound thrombocytopenia with bleeding (p < .001). RBC MPs were found to be significantly elevated in the 2nd phase in patient with DWS which was seen earliest on day 4 of infection with a cut off of ≥2200 MPs/μl when compared to patients with DWOS (p < .0001). PMPs may prove to be a promising novel biomarker which helps discriminate patients in need of prophylactic platelet transfusion from those who do not. RBC MPs, on the other hand could be potential biomarkers capable of identifying potentially severe patients who require immediate care. Thus, MPs seem to be a promising important biomarker in many aspects of DVI.

Abstract 171: Endothelial Microparticle Levels and Silent Stroke in the Framingham Heart Study

Introduction: Endothelial microparticles (EMP) are membrane vesicles, with a characteristic diameter of 0.1-1.0μ, shed by endothelial cells during cellular activation or apoptosis. EMP are associated with cardiovascular disease and risk factors, but data are lacking on the relations of EMP with subclinical vascular brain injury. Methods: We studied 487 individuals (mean age 66.9±8.8 years, 55% women) from the Framingham Offspring Cohort, who were free from clinical stroke. CD31+/CD41- EMP were identified and quantified using flow cytometry. Silent cerebral infarcts (SCI) were quantified as parenchymal lesions on brain MRI &gt;3mm in size with characteristics of previous infarcts. Logistic regression models were used to investigate cross-sectional associations of EMP levels with prevalent SCI. We tested for interaction between EMP levels and statin use in their relationship with SCI. Results: SCI was observed in 49 (10%) participants. Figure 1 displays results of the logistic regression analysis. Circulating CD31+/CD41- EMP levels were inversely associated with SCI, adjusting for age, sex, and time interval between blood draw and brain MRI (OR 0.90 [0.82-0.98], p=0.016). Similar results were observed when adjusting for additional vascular risk factors. We observed an interaction between statin use and EMP levels in their association with SCI. There was an inverse association (OR 0.76 [0.64-0.89], p=0.001) among statin users and no association among non-statin users. Conclusions: In our analysis of the relationship of CD31+/CD41- EMP with SCI, in a well-defined community cohort, we observed an inverse association of higher EMP levels, primarily in participants on statins. Our data question whether constitutive or induced release of EMP, associated with a pleiotropic effect of statin medication, might mitigate vascular risk factor exposure and reduce risk of SCI. Further studies are needed to determine the associations of EMP with SCI and clinical stroke.

MicroRNA-145-5p and microRNA-320a encapsulated in endothelial microparticles contribute to the progression of vasculitis in acute Kawasaki Disease

Kawasaki Disease (KD) is an acute inflammatory disease that takes the form of systemic vasculitis. Endothelial microparticles (EMPs) have been recognized as an important transcellular delivery system. We hypothesized whether EMPs are involved in vasculitis in acute KD. Fifty patients with acute KD were enrolled, divided into two subgroups: those with coronary artery lesions (CAL) (n = 5) and those without CAL (NCAL) (n = 45). EMPs were measured using flow cytometry, and microRNA (miR) expression profiling was performed by microRNA array. The percentage of EMPs in acute KD was significantly higher than in controls (P < 0.0001). EMPs in patients with CAL rapidly increased after the initial treatment, and was significantly higher than those in NCAL (P < 0.001). In patients with CAL, we identified 2 specific miRs encapsulated in EMPs, hsa-miR-145-5p and hsa-miR-320a, which are predicted to affect monocyte function using in silico analysis, and were demonstrated to upregulate inflammatory cytokine mRNAs in THP-1 monocytes. In situ hybridization confirmed that hsa-miR-145-5p was preferentially expressed in CAL. EMPs may serve as a sensitive marker for the severity of vasculitis in acute KD. Moreover, these 2 specific miRs encapsulated in EMPs might be involved in inflammatory cytokine regulation and the pathogenesis of vasculitis in acute KD.

Circulating small-sized endothelial microparticles as predictors of clinical outcome after chemotherapy for breast cancer: an exploratory analysis.

Therapeutic exploitation of angiogenesis in breast cancer has been limited by the lack of reliable biomarkers. Circulating small-sized endothelial microparticles (sEMP) are likely to play a significant role as messengers of angiogenesis. Higher levels of EMP have been observed in cancer patients, but their prognostic value in breast cancer is unknown. Our aim was to determine the value of circulating sEMP as a marker of response to chemotherapy in breast cancer. We included patients with breast cancer treated with neoadjuvant or first-line chemotherapy. Baseline and post-treatment circulating sEMP (CD144+) were quantified using a flow cytometer approach specifically designed for analysis of small-sized particles (0.1-0.5 μm). Small-sized EMP response was defined as a post-treatment decrease of sEMP larger than the median decrease of sEMP after chemotherapy. Baseline and post-chemotherapy VEGFA levels were determined with ELISA. Forty-four breast cancer patients were included (19 with metastatic and 25 with locally advanced disease). Median levels of sEMP decreased after chemotherapy (P=0.005). Response to chemotherapy showed a non-significant trend to associate with sEMP response (P=0.056). A sEMP response was observed in 51% of patients and was associated with better overall survival (HR 0.18; 95% CI 0.04-0.87; P=0.02) and progression free survival (HR 0.30; 95% CI 0.09-0.99; P=0.04) in the group of women with metastatic disease. Post-chemotherapy decrease of VEGFA levels was not associated with breast cancer prognosis. Our results did not support sEMP as a marker of response to chemotherapy. However, our exploratory analysis suggests that in patients with metastatic breast cancer, the decrease of sEMP levels after chemotherapy is associated with better overall and disease free survival and might be superior to VEGFA levels as an angiogenesis-related prognostic marker.

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.

Bubbles Induce Endothelial Microparticle Formation via a Calcium-Dependent Pathway Involving Flippase Inactivation and Rho Kinase Activation

Background/Aims: Intravascular bubbles can exert pleiotropic detrimental effects, partly by inducing endothelial microparticles (EMPs) production, which play critical roles in cell communication and vascular inflammation cascades. However, the underlying mechanisms remain unclear. This study aimed to delineate the possible mechanisms involving bubble-induced EMPs formation. Methods: Human umbilical vein endothelial cells (HUVECs) were contacted by bubbles and EMPs level in supernatant were quantified by flow cytometry. Cytoplasmic calcium (Ca²⁺) was measured by the Ca²⁺ binding dyes Fluo-3 AM and flippase activity was assessed by translocation rate of fluorescent phosphatidylserine (PS) analogue NBD-PS. Protein levels of phospho-myosin light chain (MLC, a Rho kinase substrate) and phospho-extracellular signal-regulated kinase 1 or 2 (ERK1/2) were determined by western blotting. The score of actin colocalization was assessed by phalloidin-FITC using an immunofluorescent microscopy. Results: EMPs level markedly increased after bubble stimulus. Cytoplasmic calcium (Ca²⁺) significantly elevated (P< 0.05), flippase activity decreased (P< 0.05), protein levels of phospho- MLC and phospho- ERK1/2 significantly increased (P< 0.05, P < 0.05), and the score of actin colocalization markedly reduced (P< 0.05) in bubble-stimulated HUVECs. All the above changes except the increase in phospho-ERK1/2 can be reversed by Ca²⁺ channel blocker LaCl₃ (P< 0.05). Additionally, MLC phosphorylation was significantly inhibited and actin colocalization markedly increased by Rho kinase inhibitor pretreatment and more importantly, bubble-induced EMPs markedly decreased. Conclusions: These results demonstrate that bubble stimulates EMPs formation by cytoplasmic Ca²⁺ elevation and subsequently activating Rho kinase pathway and cytoskeleton reorganization. Simultaneously, cytoplasmic Ca²⁺ inhibits the flippase activity and subsequently increases phosphatidylserine exposure, which also contributes to EMPs formation.

Changes of Circulating Endothelial Cells and Microparticles after Conventional Chemotherapy in Patients with Extensive Small-Cell Lung Cancer: Relevance to Prognosis and Treatment Response

Objectives: Tumor angiogenesis is controlled by inhibitors and angiogenic factors. However, circulating endothelial cells (CECs) and microparticles (MPs) may be related to tumor angiogenesis in malignant tumors. We therefore investigated the significance of CECs and MPs variation for the prediction of treatment efficacy andprognosis in patients with extensive small-cell lung cancer (SCLC). Materials and methods: Isolation and quantification of CECs from the whole-blood was performed using immune magnetic separation (IMS) technique, while flow cytometry measurement (FCM) was used for enumerating MPs in plasma samples.Results: Twenty-six patients were included in this prospective study. The relative change in CEC and MP counts after treatment, correctly identified treatment response in 22/26 patients (84.6%) and 17/23 patients (73.9%) respectively. The difference in themedian duration of time to progression (TTP) between patient with high relative changes and those with low relative changes in CEC and MP values was not statistically significant (p=0.39 and p=0.2, respectively). By contrast, the increase in CEC and MP levels after chemotherapy tended to be inversely correlated with longer TTP duration (r²=0.111 and r²=0.171, respectively). However, onlym decreased MP values after chemotherapy have the trend to be correlated with longer TTP duration (r²=0.167).Conclusion: The relative change in CEC and MP values after treatment seems to be a useful biomarker for the evaluation of treatment response in patients with extensive SCLC. Moreover, this relative change in both biomarkers might be considered as a prognostic tool in SCLC.

The Relation between circulating endothelial microparticles and carotid intima media thickness in Vitamin B12 deficient adolescents

The Relation between circulating endothelial microparticles and carotid intima media thickness in Vitamin B12 deficient adolescents