Endothelial dysfunction and complement activation are independently associated with disease duration in patients with systemic vasculitis.

OBJECTIVES: Systemic vasculitis is a heterogenous group of autoimmune diseases characterized by enhanced cardiovascular mortality. Endothelial dysfunction is associated with accelerated vascular damage, representing a core pathophysiologic mechanism contributing to excess CV risk. Recent studies have also shown that complement activation holds significant role in the pathogenesis of Anti-Neutrophilic Cytoplasmic Autoantibody (ANCA) -associated vasculitis (AAV). Given the potential crosstalk between the endothelium and complement, we aimed to assess, for the first time simultaneously, easily accessible biomarkers of endothelial dysfunction and complement activation in SV. METHODS: We measured circulating endothelial microvesicles (EMVs) and soluble complement components representative of alternative, classical and terminal activation (C5b-9, C1q, Bb fragments, respectively) in a meticulously selected group of patients with systemic vasculitis, but without cardiovascular disease. Individuals free from systemic diseases, who were matched with patients for cardiovascular risk factors(hypertension, diabetes, smoking, dyslipidemia), comprised the control group. RESULTS: We studied 60 individuals (30 in each group). Patients with systemic vasculitis had elevated EMVs, higher levels of C5b-9 [536.4(463.4) vs 1200.94457.3), p = 0.003] and C1q [136.2(146.5 vs 204.2(232.9), p = 0.0129], compared to controls [232.0 (243.5) vs 139.3(52.1), p < 0.001]. In multivariate analysis both EMVs and C5b-9 were independently associated with disease duration (p = 0.005 and p = 0.004 respectively), yet not with disease activity. CONCLUSION: Patients with systemic vasculitis exhibit impaired endothelial function and complement activation, both assessed by easily accessible biomarkers, even in the absence of cardiovascular disease manifestations. EMVs and soluble complement components such as C5b-9 and C1q could be used as early biomarkers of endothelial dysfunction and complement activation, respectively, in clinical practice during the course of SV, yet their predictive value in terms of future cardiovascular disease warrants further verification in appropriately designed studies.

Ponatinib Induces a Procoagulant Phenotype in Human Coronary Endothelial Cells via Inducing Apoptosis.

BCR-ABL tyrosine kinase inhibitors (TKIs) are effective drugs in the treatment of patients with chronic myeloid leukemia. However, based on clinical studies, ponatinib was associated with the development of thrombotic complications. Since endothelial cells (ECs) regulate blood coagulation, their abnormal phenotype may play a role in the development of thrombotic events. We here aimed to investigate the effect of ponatinib on the procoagulant activity of cultured endothelial cells in vitro. Human coronary artery endothelial cells (HCAECs) were incubated with 50, 150, and 1000 nM of ponatinib. Subsequently, phosphatidylserine (PS) exposure and endothelial microvesicles (EMVs) were measured by flow cytometry. In addition, EC- and EMV-dependent thrombin generation was analyzed. To investigate pro-apoptotic effects of ponatinib, the level of Bax and Bcl-xL proteins were studied using Western blot and F3, THBD, and VCAM1 mRNAs were quantified by qPCR. Therapeutic concentrations of ponatinib significantly increased PS expression on ECs and the amount of EMVs which significantly shortened the time parameters of thrombin generation. In addition, these changes were associated with an increased ratio of Bax and Bcl-xL proteins in the presence of the decreased THBD mRNA level. Overall, ponatinib enhances the procoagulant activity of ECs via inducing apoptosis, which may contribute to thrombotic events.

Endothelial-derived extracellular vesicles associated with electronic cigarette use impair cerebral microvascular cell function.

The aim of this study was to determine the effect of circulating endothelial cell-derived microvesicles (EMVs) isolated from e-cigarette users on human cerebral microvascular endothelial cells (hCMECs) nitric oxide (NO) and endothelin (ET)-1 production and tissue-type plasminogen activator (t-PA) release. Circulating EMVs (CD144-PE) were isolated (flow cytometry) from 27 young adults (19-25 yr): 10 nonsmokers (6 M/4 F), 10 e-cigarette users (6 M/4 F), and 7 tobacco cigarette smokers (4 M/3 F). hCMECs were cultured and treated with isolated EMVs for 24 h. EMVs from e-cigarette users and cigarette smokers induced significantly higher expression of p-eNOS (Thr495; 28.4 ± 4.6 vs. 29.1 ± 2.8 vs. 22.9 ± 3.8 AU), Big ET-1 (138.8 ± 19.0 vs. 141.7 ± 19.1 vs. 90.3 ± 18.8 AU) and endothelin converting enzyme (107.6 ± 10.1 and 113.5 ± 11.8 vs. 86.5 ± 13.2 AU), and significantly lower expression of p-eNOS (Ser1177; 7.4 ± 1.7 vs. 6.5 ± 0.5 vs. 9.7 ± 1.6 AU) in hCMECs than EMVs from nonsmokers. NO production was significantly lower and ET-1 production was significantly higher in hCMECs treated with EMVs from e-cigarette (5.7 ± 0.8 µmol/L; 33.1 ± 2.9 pg/mL) and cigarette smokers (6.3 ± 0.7 µmol/L; 32.1 ± 3.9 pg/mL) than EMVs from nonsmokers (7.6 ± 1.2 µmol/L; 27.9 ± 3.1 pg/mL). t-PA release in response to thrombin was significantly lower in hCMECs treated with EMVs from e-cigarette users (from 38.8 ± 6.3 to 37.4 ± 8.3 pg/mL) and cigarette smokers (31.5 ± 5.5 to 34.6 ± 8.4 pg/mL) than EMVs from nonsmokers (38.9 ± 4.3 to 48.4 ± 7.9 pg/mL). There were no significant differences in NO, ET-1, or t-PA protein expression or production in hCMECs treated with EMVs from e-cigarette users and smokers. Circulating EMVs associated with e-cigarette use adversely affects brain microvascular endothelial cells and may contribute to reported cerebrovascular dysfunction with e-cigarette use.NEW & NOTEWORTHY In the present study, we determined the effect of circulating endothelial cell-derived microvesicles (EMVs) isolated from e-cigarette users on human cerebral microvascular endothelial cells (hCMECs) nitric oxide (NO) and endothelin (ET)-1 production and tissue-type plasminogen activator (t-PA) release. EMVs from e-cigarette users reduced brain microvascular endothelial cell NO production, enhanced ET-1 production, and impaired endothelial t-PA release. EMVs are a potential mediating factor in the increased risk of stroke associated with e-cigarette use.

Propofol inhibits myocardial injury induced by microvesicles derived from hypoxia-reoxygenated endothelial cells via lncCCT4-2/CCT4 signaling.

BACKGROUND: Ischemia-reperfusion (IR) induces increased release of extracellular vesicles in the heart and exacerbates myocardial IR injury. We have previously shown that propofol attenuates hypoxia/reoxygenation (HR)-induced injury in human umbilical vein endothelial cells (HUVECs) and that microvesicles derived from propofol-treated HUVECs inhibit oxidative stress in endothelial cells. However, the role of microvesicles derived from propofol post-treated HUVECs ((HR + P)-EMVs) in IR-injured cardiomyocytes is unclear. In this study, we aimed to investigate the role of (HR + P)-EMVs in cardiac IR injury compared to microvesicles derived from hypoxic/reoxygenated HUVECs (HR-EMVs) and to elucidate the underlying mechanisms. METHODS: Hypoxia/reoxygenation (HR) models of HUVECs and AC16 cells and a mouse cardiac IR model were established. Microvesicles from HR-injured HUVECs, DMSO post-treated HUVECs and propofol post-treated HUVECs were extracted by ultra-high speed centrifugation, respectively. The above EMVs were co-cultured with HR-injured AC16 cells or injected intracardially into IR mice. Flow cytometry and immunofluorescence were used to determine the levels of oxidative stress and apoptosis in cardiomyocytes. Apoptosis related proteins were detected by Western blot. Echocardiography for cardiac function and Evans blue-TTC staining for myocardial infarct size. Expression of lncCCT4-2 in EMVs and AC16 cells was analysed by whole transcriptome sequencing of EMVs and RT-qPCR. The molecular mechanism of inhibition of myocardial injury by (HR + P)-EMVs was elucidated by lentiviral knockdown of lncCCT4-2, plasmid overexpression or knockdown of CCT4, and actinomycin D assay. RESULTS: In vitro and in vivo experiments confirmed that HR-EMVs exacerbated oxidative stress and apoptosis in IR-injured cardiomyocytes, leading to increased infarct size and worsened cardiac function. Notably, (HR + P)-EMVs induced significantly less oxidative stress and apoptosis in IR-injured cardiomyocytes compared to HR-EMVs. Mechanistically, RNA sequencing of EMVs and RT-qPCR showed that lncCCT4-2 was significantly upregulated in (HR + P)-EMVs and cardiomyocytes co-cultured with (HR + P)-EMVs. Reduction of lncCCT4-2 in (HR + P)-EMVs enhanced oxidative stress and apoptosis in IR-injured cardiomyocytes. Furthermore, the anti-apoptotic activity of lncCCT4-2 from (HR + P)-EMVs was achieved by increasing the stability of CCT4 mRNA and promoting the expression of CCT4 protein in cardiomyocytes. CONCLUSIONS: Our study showed that (HR + P)-EMVs uptake by IR-injured cardiomyocytes upregulated lncCCT4-2 in cardiomyocytes and promoted CCT4 expression, thereby inhibiting HR-EMVs induced oxidative stress and apoptosis.

Differential Expression of Vascular-Related MicroRNA in Circulating Endothelial Microvesicles in Adults With Spinal Cord Injury: A Pilot Study.

Background: Spinal cord injury (SCI) is associated with an increased risk and prevalence of cardiopulmonary and cerebrovascular disease-related morbidity and mortality. The factors that initiate, promote, and accelerate vascular diseases and events in SCI are poorly understood. Clinical interest in circulating endothelial cell-derived microvesicles (EMVs) and their microRNA (miRNA) cargo has intensified due to their involvement in endothelial dysfunction, atherosclerosis, and cerebrovascular events. Objectives: The aim of this study was to determine whether a subset of vascular-related miRNAs is differentially expressed in EMVs isolated from adults with SCI. Methods: We assessed eight adults with tetraplegia (7 male/1 female; age: 46±4 years; time since injury: 26±5 years) and eight uninjured (6 male/2 female; age: 39±3 years). Circulating EMVs were isolated, enumerated, and collected from plasma by flow cytometry. The expression of vascular-related miRNAs in EMVs was assessed by RT-PCR. Results: Circulating EMV levels were significantly higher (~130%) in adults with SCI compared with uninjured adults. The expression profile of miRNAs in EMVs from adults with SCI were significantly different than uninjured adults and were pathologic in nature. Expression of miR-126, miR-132, and miR-Let-7a were lower (~100-150%; p < .05), whereas miR-30a, miR-145, miR-155, and miR-216 were higher (~125-450%; p < .05) in EMVs from adults with SCI. Conclusion: This study is the first examination of EMV miRNA cargo in adults with SCI. The cargo signature of vascular-related miRNAs studied reflects a pathogenic EMV phenotype prone to induce inflammation, atherosclerosis, and vascular dysfunction. EMVs and their miRNA cargo represent a novel biomarker of vascular risk and a potential target for intervention to alleviate vascular-related disease after SCI.

Endothelial-derived extracellular vesicles from obese/hypertensive adults increase factors associated with hypertrophy and fibrosis in cardiomyocytes.

Obesity and hypertension, independently and combined, are associated with increased risk of heart failure and heart failure-related morbidity and mortality. Interest in circulating endothelial cell-derived microvesicles (EMVs) has intensified because of their involvement in the development and progression of endothelial dysfunction, atherosclerosis, and cardiomyopathy. The experimental aim of this study was to determine, in vitro, the effects of EMVs isolated from obese/hypertensive adults on key proteins regulating cardiomyocyte hypertrophy [cardiac troponin T (cTnT), α-actinin, nuclear factor-kB (NF-kB)] and fibrosis [transforming growth factor (TGF)-ß, collagen1-α1], as well as endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production. EMVs (CD144+ microvesicles) were isolated from plasma by flow cytometry in 12 normal weight/normotensive [8 males/4 females; age: 56 ± 5 yr; body mass index (BMI): 23.3 ± 2.0 kg/m2; blood pressure (BP): 117/74 ± 4/5 mmHg] and 12 obese/hypertensive (8 males/4 females; 57 ± 5 yr; 31.7 ± 1.8 kg/m2; 138/83 ± 8/7 mmHg) adults. Human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were cultured and treated with EMVs from either normal weight/normotensive or obese/hypertensive adults for 24 h. Expression of cTnT (64.1 ± 13.9 vs. 29.5 ± 7.8 AU), α-actinin (66.0 ± 14.7 vs. 36.2 ± 10.3 AU), NF-kB (166.3 ± 13.3 vs. 149.5 ± 8.8 AU), phosphorylated-NF-kB (226.1 ± 25.2 vs. 179.1 ± 25.5 AU), and TGF-ß (62.1 ± 13.3 vs. 23.5 ± 8.8 AU) were significantly higher and eNOS activation (16.4 ± 4.3 vs. 24.8 ± 3.7 AU) and nitric oxide production (6.8 ± 1.2 vs. 9.6 ± 1.3 µmol/L) were significantly lower in iPSC-CMs treated with EMVs from obese/hypertensive compared with normal weight/normotensive adults. These data indicate that EMVs from obese/hypertensive adults induce a cardiomyocyte phenotype prone to hypertrophy, fibrosis, and reduced nitric oxide production, central factors associated with heart failure risk and development.NEW & NOTEWORTHY In the present study we determined the effect of endothelial microvesicles (EMVs) isolated from obese/hypertensive adults on mediators of cardiomyocyte hypertrophy [cardiac troponin T (cTnT), α-actinin, nuclear factor-kB (NF-kB)] and fibrosis [transforming growth factor (TGF-ß), collagen1-α1] as well as endothelial nitric oxide synthase (eNOS) expression and NO production. EMVs from obese/hypertensive induced significantly higher expression of hypertrophic (cTnT, α-actinin, NF-kB) and fibrotic (TGF-ß) proteins as well as significantly lower eNOS activation and NO production in cardiomyocytes than EMVs from normal weight/normotensive adults. EMVs are a potential mediating factor in the increased risk of cardiomyopathy and heart failure with obesity/hypertension.

Propofol inhibits myocardial injury induced by microvesicles derived from hypoxiareoxygenated endothelial cells via lncCCT4-2/CCT4 signaling

BACKGROUND: Ischemia-reperfusion (IR) induces increased release of extracellular vesicles in the heart and exacerbates myocardial IR injury. We have previously shown that propofol attenuates hypoxia/reoxygenation (HR)-induced injury in human umbilical vein endothelial cells (HUVECs) and that microvesicles derived from propofol-treated HUVECs inhibit oxidative stress in endothelial cells. However, the role of microvesicles derived from propofol post-treated HUVECs ((HR + P)-EMVs) in IR-injured cardiomyocytes is unclear. In this study, we aimed to investigate the role of (HR + P)-EMVs in cardiac IR injury compared to microvesicles derived from hypoxic/reoxygenated HUVECs (HR-EMVs) and to elucidate the underlying mechanisms. METHODS: Hypoxia/reoxygenation (HR) models of HUVECs and AC16 cells and a mouse cardiac IR model were established. Microvesicles from HR-injured HUVECs, DMSO post-treated HUVECs and propofol post-treated HUVECs were extracted by ultra-high speed centrifugation, respectively. The above EMVs were co-cultured with HR-injured AC16 cells or injected intracardially into IR mice. Flow cytometry and immunofluorescence were used to determine the levels of oxidative stress and apoptosis in cardiomyocytes. Apoptosis related proteins were detected by Western blot. Echocardiography for cardiac function and Evans blue-TTC staining for myocardial infarct size. Expression of lncCCT4-2 in EMVs and AC16 cells was analysed by whole transcriptome sequencing of EMVs and RT-qPCR. The molecular mechanism of inhibition of myocardial injury by (HR + P)-EMVs was elucidated by lentiviral knockdown of lncCCT4-2, plasmid overexpression or knockdown of CCT4, and actinomycin D assay. RESULTS: In vitro and in vivo experiments confirmed that HR-EMVs exacerbated oxidative stress and apoptosis in IR-injured cardiomyocytes, leading to increased infarct size and worsened cardiac function. Notably, (HR + P)-EMVs induced significantly less oxidative stress and apoptosis in IR-injured cardiomyocytes compared to HR-EMVs. Mechanistically, RNA sequencing of EMVs and RT-qPCR showed that lncCCT4-2 was significantly upregulated in (HR + P)-EMVs and cardiomyocytes co-cultured with (HR + P)-EMVs. Reduction of lncCCT4-2 in (HR + P)-EMVs enhanced oxidative stress and apoptosis in IR-injured cardiomyocytes. Furthermore, the anti-apoptotic activity of lncCCT4-2 from (HR + P)-EMVs was achieved by increasing the stability of CCT4 mRNA and promoting the expression of CCT4 protein in cardiomyocytes. CONCLUSIONS: Our study showed that (HR + P)-EMVs uptake by IR-injured cardiomyocytes upregulated lncCCT4-2 in cardiomyocytes and promoted CCT4 expression, thereby inhibiting HR-EMVs induced oxidative stress and apoptosis. Highlights Microvesicles from hypoxic/reoxygenated HUVECs (HR-EMVs) exacerbated oxidative stress and apoptosis in IR-injured cardiomyocytes. Microvesicles from propofol post-treated HUVECs ((HR + P)-EMVs) induced diminished oxidative stress and apoptosis in IR-injured cardiomyocytes compared with microvesicles from hypoxic/reoxygenated HUVECs (HR-EMVs). lncCCT4-2 was significantly highly expressed in (HR + P)-EMVs and cardiomyocytes co-cultured with (HR + P)-EMVs, and reduction of lncCCT4-2 in (HR + P)-EMVs enhanced oxidative stress and apoptosis in IR-injured cardiomyocytes. lncCCT4-2 inhibited HR-EMVs induced oxidative stress and apoptosis in HR-injured AC16 cells by increasing the stability of CCT4 mRNA and promoting the expression of CCT4 protein in AC16 cells.

Effects of Postprandial Lipemia Combined With Disturbed Blood Flow on the Flow-Mediated Dilation, Oxidative Stress, and Endothelial Microvesicles in Healthy Subjects.

Aims: Both postprandial lipemia (PPL) and disturbed blood flow (DBF) induce endothelial dysfunction. However, the interactive effect of these stimuli on endothelial function is currently unknown. In the present study, we tested whether PPL plus DBF causes a greater reduction in flow-mediated dilation (FMD) than PPL and if this response is associated with elevations in oxidative stress and endothelial microvesicles (EMVs). Methods: Eighteen individuals (aged 28 ± 1yrs, 3 females, and BMI 24.43 ± 0.8kg/m2) randomly underwent two experimental sessions: PPL and PPL plus DBF. FMD and venous blood samples were obtained at baseline and 30, 70, and 110 min after stimulation. PPL was induced by fat overload via mozzarella pizza ingestion and DBF by forearm cuff inflation to 75 mm Hg per 30 min. Lipidic profile, oxidative stress (thiobarbituric acid reactive substances, TBARS; ferric reducing/antioxidant power, FRAP; hydrogen peroxide, H2O2) and EMVs were measured in blood samples. Results: Hypertriglyceridemia was observed in both sessions. Retrograde shear rate and oscillatory index responses were significantly higher in the PPL plus DBF compared with PPL. PPL plus DBF evoked a greater reduction in FMD than did PPL and EMVs, NADPH oxidase, and H2O2 similarly increased in both sessions, but TBARS and FRAP did not change. Conclusion: These data indicate that the association of PPL plus DBF additively impairs endothelium-dependent function in 110 min after stimulus in healthy individuals, despite a similar increase in oxidative stress and EMVs. Further studies are needed to understand the mechanisms associated with the induced-endothelial dysfunction by association of PPL and DBF.

Ischaemic tissue released microvesicles induce monocyte reprogramming and increase tissue repair by a tissue factor-dependent mechanism.

AIMS: Despite increasing evidence that monocytes may acquire endothelial features, it remains unclear how monocytes participate in angiogenesis after ischaemic damage. We investigated whether ischaemic cells can release microvesicles (MVs) and promote neovascularization in a model of peripheral artery disease (PAD). METHODS AND RESULTS: To model PAD, we used an in vivo experimental model of hind-limb ischaemia (HLI) in mice. MVs were isolated from the ischaemic muscle and from peripheral blood at different times after unilateral femoral artery ligation. MVs were phenotypically characterized to identify cell origin. HLI in mice induced the release of MVs with a much higher content of tissue factor (TF) than non-HLI control mice both in the MVs isolated from the affected limb muscle area and from blood. MVs were mainly released from endothelial cells (ECs) and induced Mo differentiation to endothelial cell-like (ECL) cells. Differentiation to ECL cells encompassed highly strict hierarchical transcription factor activation, initiated by ETS1 activation. MVs secreted by microvascular ECs over-expressing TF (upTF-EMVs), were injected in the ischaemic hind-limb in parallel with control EMVs (from random siRNA-treated cells) or EMVs released by silenced TF ECs. In animals treated with upTF-EMVs in the ischaemic zone, there was a highly significant increase in functional new vessels formation (seen by magnetic resonance angiography), a concomitant increase in the pool of circulating Ly6Clow Mo expressing vascular EC markers, and a significantly higher number of Mo/macrophages surrounding and integrating the newly formed collaterals. CONCLUSION: Ischaemia-activated ECs release EMVs rich in TF that induce monocyte differentiation into ECL cells and the formation of new vessels in the ischaemic zone. TF by this mechanism of formation of new blood microvessels can contribute to ischaemic tissue repair.

Endothelial Microvesicles Induce Pulmonary Vascular Leakage and Lung Injury During Sepsis.

Sepsis is a prevalent severe syndrome in clinic. Vascular leakage and lung injury are important pathophysiological processes during sepsis, but the mechanism remains obscure. Microvesicles (MVs) play an essential role in many diseases, while whether MVs participate in vascular leakage and lung injury during sepsis is unknown. Using cecal ligation and puncture induced sepsis rats and lipopolysaccharide stimulated vascular endothelial cells (VECs), the role and the underlying mechanism of endothelial microvesicles (EMVs) in pulmonary vascular leakage and lung injury were observed. The role of MVs from sepsis patients was verified. The results showed that the concentration of MVs in blood was significantly increased after sepsis. MVs from sepsis rats and patients induced apparent pulmonary vascular leakage and lung injury, among which EMVs played the dominant role, in which miR-23b was the key inducing factor in vascular leakage. Furthermore, downregulation and upregulation of miR-23b in EMVs showed that miR-23b mainly targeted on ZO-1 to induce vascular leakage. MVs from sepsis patients induced pulmonary vascular leakage and lung injury in normal rats. Application of classic antidepressants amitriptyline reduced the secretion of EMVs, and alleviated vascular leakage and lung injury. The study suggests that EMVs play an important role in pulmonary vascular leakage and lung injury during sepsis by transferring functional miR-23b. Antagonizing the secretion of EMVs and the miR-23b might be a potential target for the treatment of severe sepsis.

Plasma endothelial microvesicles and their carrying miRNA-155 serve as biomarkers for ischemic stroke.

Endothelial microvesicles (EMVs) could reflect the status of endothelial cells (ECs) which are involved in the pathogenesis of ischemic stroke (IS). MiR-155 could regulate EC functions. However, their roles in IS remain unclear. This study aimed to investigate the levels of plasma EMVs and EMVs carrying miRNA-155 (EMVs-miR-155) in IS patients to explore their potential roles as biomarkers. Ninety-three IS patients and 70 controls were recruited in this study. The levels of circulating EMVs and EMVs-miR-155 were detected by fluorescence nanoparticle tracking analysis and quantitative real-time PCR, respectively. The correlations between level of EMVs/EMVs-miR-155 and the onset time, severity, infarct volume, and subtypes of IS were analyzed. The severity and infarct volume were assessed by NIHSS and magnetic resonance imaging, respectively. Multivariate logistic regression analysis was used to investigate the risk factors of IS. The ROC curve and area under ROC curve (AUC) of EMVs and EMVs-miR-155 were determined. The levels of plasma EMVs and EMVs-miR-155 were increased significantly in acute and subacute stages of IS and remained unchanged in chronic stage, and were positively related to the infarct volume and NIHSS scores and were associated with large artery atherosclerosis and cardioembolism subtypes defined by Trial of Org 10 172 in acute stroke treatment (TOAST) classification. Multivariate logistic regression analysis demonstrated that plasma EMVs and EMVs-miR-155 were significant and independent risk factors of IS and their AUC were 0.778 and 0.851, respectively, and increased to 0.892 after combination. Our study suggests that plasma EMVs and EMVs-miR-155 are promising biomarkers for IS. The diagnostic value of EMVs-miR-155 is higher and their combination is the best.

Electronic cigarettes containing nicotine increase endothelial and platelet derived extracellular vesicles in healthy volunteers.

BACKGROUND AND AIMS: E-cigarette use is increasingly common. Whether e-cigarettes are harmful to human health is an intensely debated subject. In order to investigate whether e-cigarettes with and without nicotine cause different vascular responses, we obtained blood samples from healthy young volunteers who performed brief active e-cigarette inhalations. Extracellular vesicles (EVs) of endothelial and platelet origin were measured to determine vascular changes. METHODS: Using a randomized, double-blind, crossover design, 17 healthy occasional smokers inhaled 30 puffs of e-cigarette vapor during 30 min. Blood samples were collected at baseline, as well as at 0, 2, 4 and 6 h post-exposure. EVs from platelets and endothelial cells were measured by flow cytometry. RESULTS: Platelet and endothelial derived EVs were significantly increased with peak levels seen at 4 h following exposure to active inhalation of e-cigarette vapor with nicotine. Moreover, platelet derived EVs, expressing platelet activation marker P-selectin and the inflammation marker, CD40 ligand, were also significantly increased following inhalation of e-cigarette vapor with nicotine. In addition, platelet derived EVs expressing CD40 ligand was increased after inhalation of e-cigarette vapor without nicotine. CONCLUSION: As few as 30 puffs of nicotine-containing e-cigarette vapor caused an increase in levels of circulating EVs of endothelial and platelet origin, which may signify underlying vascular changes. Although e-cigarette vapor without nicotine caused an increase in platelet EVs expressing CD40 ligand, nicotine, as a component in the vapor, seems to have a more compelling effect on extracellular vesicle formation and protein composition.

Changes in circulating endothelial microvesicles in men after myocardial infarction.

PURPOSE: The objective of the study was to determine the differences in the numbers of endothelial microvesicles (EMV) after myocardial infarction (MI) and their association with oxidative stress. MATERIALS AND METHODS: We included 15 post MI patients and 28 healthy controls. Samples were analysed by flow cytometry. We examined four EMV populations: 1) CD144+, CD42a-, CD61-, 2) CD144+, CD42a+, CD61-, 3) CD105+, CD42a-, CD61-and 4) CD31+, CD42a-, CD61-and determined a percentage of CD62e + EMV. Malondialdehyde concentration was determined by ultra-high performance liquid chromatography. RESULTS: The median of EMV counts differed between controls and patients in: CD105+ (10.91 microvesicles/µl vs. 33.68 microvesicles/µl, P = 0.006), CD144+, CD42a+ (312.87 microvesicles/µl vs. 73.29 microvesicles/µl, P < 0.001) and CD31+ (2 microvesicles/µl vs. 1.38 microvesicles/µl, P = 0.021). The median of percentage of CD62e expression differed between controls and patients in: CD105+ (1.35% vs. 14.8%, P < 0.001), CD144+, CD42a+ (56.45% vs. 98.99%, P < 0.001) and CD144+, CD42a- (173.03% vs. 215.56%) EMV. In patients, EMV counts correlated with low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) concentrations: CD105+: R = -0.69, P = 0.004 (LDL-C), R = -0.64, P = 0.01 (TC); CD144+, CD42a-: R = -0.68, P = 0.005 (LDL-C), R = -0.63, P = 0.011 (TC); CD144+: R = -0.54, P = 0.038 (HDL-C) and CD144+, CD42a-, CD62e+: R = 0.78, P = 0.001 (HDL-C). In controls, HDL-C concentration correlated with CD105+ (R = -0.395, P = 0.038) and CD105+, CD62e+ (R = -0.716, P < 0.001) counts. Malondialdehyde concentration correlated with CD144+, CD42a- (P = 0.01, R = 0.48) and CD105+, CD62e+ (P = 0.012, R = 0.47) counts. CONCLUSIONS: Changes in EMV levels after the MI period were observed. Counts of EMV and their CD62e expression correlated with dyslipidaemia and oxidative stress.

Endothelial microvesicles induced by physiological cyclic stretch inhibit ICAM1-Dependent leukocyte adhesion.

Physiological cyclic stretch (CS), caused by artery deformation following blood pressure, plays important roles in the homeostasis of endothelial cells (ECs). Here, we detected the effect of physiological CS on endothelial microvesicles (EMVs) and their roles in leukocyte recruitment to ECs, which is a crucial event in EC inflammation. The results showed compared with the static treatment, pretreatment of 5%-CS-derived EMVs with ECs significantly decreased the adherence level of leukocytes. Comparative proteomic analysis revealed 373 proteins differentially expressed between static-derived and 5%-CS-derived EMVs, in which 314 proteins were uniquely identified in static-derived EMVs, 34 proteins uniquely in 5%-CS-derived EMVs, and 25 proteins showed obvious differences. Based on the proteomic data, Ingenuity Pathways Analysis predicted intercellular adhesion molecule 1 (ICAM1) in EMVs might be the potential molecule involved in EC-leukocyte adhesion. Western blot and flow cytometry analyses confirmed the significant decrease of ICAM1 in 5%-CS-derived EMVs, which subsequently inhibited the phosphorylation of VE-cadherin at Tyr731 in target ECs. Moreover, leukocyte adhesion was obviously decreased after pretreatment with ICAM1 neutralizing antibody. Our present research suggested that physiological stretch changes the components of EMVs, which in turn inhibits leukocyte adhesion. ICAM1 expressed on CS-induced EMVs may play an important role in maintaining EC homeostasis.

Blockade of the kallikrein-kinin system reduces endothelial complement activation in vascular inflammation.

BACKGROUND: The complement and kallikrein-kinin systems (KKS) are activated during vascular inflammation. The aim of this study was to investigate if blockade of the KKS can affect complement activation on the endothelium during inflammation. METHODS: Complement deposition on endothelial microvesicles was assayed in vasculitis patient plasma samples and controls. Plasma was perfused over glomerular endothelial cells and complement deposition assayed by flow cytometry. The effect of the kinin system was assessed using kinin receptor antagonists and C1-inhibitor. The in vivo effect was assessed in kidney sections from mice with nephrotoxic serum-induced glomerulonephritis treated with a kinin receptor antagonist. FINDINGS: Vasculitis patient plasma had significantly more C3- and C9-positive endothelial microvesicles than controls. Perfusion of patient acute-phase plasma samples over glomerular endothelial cells induced the release of significantly more complement-positive microvesicles, in comparison to remission or control plasma. Complement activation on endothelial microvesicles was reduced by kinin B1- and B2-receptor antagonists or by C1-inhibitor (the main inhibitor of the classical pathway and the KKS). Likewise, perfusion of glomerular endothelial cells with C1-inhibitor-depleted plasma induced the release of complement-positive microvesicles, which was significantly reduced by kinin-receptor antagonists or C1-inhibitor. Mice with nephrotoxic serum-induced glomerulonephritis exhibited significantly reduced glomerular C3 deposition when treated with a B1-receptor antagonist. INTERPRETATION: Excessive complement deposition on the endothelium will promote endothelial injury and the release of endothelial microvesicles. This study demonstrates that blockade of the KKS can reduce complement activation and thereby the inflammatory response on the endothelium. FUNDING: Full details are provided in the Acknowledgements/Funding section.

Statins and antiplatelet agents are associated with changes in the circulatory markers of endothelial dysfunction in chronic kidney disease. / El uso de estatinas y antiagregantes se asocia con cambios en los marcadores de disfunción endotelial en la enfermedad renal crónica.

BACKGROUNDS AND PURPOSES: Patients with chronic kidney disease (CKD) have higher risk of developing cardiovascular disease. In CKD patients the mechanisms involved in, endothelial damage and the role of different drugs used on these patients are not completely understood. The aim of this work is to analyze the effect of statins and platelet antiaggregant (PA) on endothelial microvesicles (EMVs) and other markers of endothelial dysfunction. EXPERIMENTAL APPROACH: Cross-sectional study of 41 patients with CKD 3b-4 and 8 healthy volunteers. Circulating levels of EMVs, vascular endothelial growth factor (VEGF), and advance oxidized protein products (AOPPS) were quantified and the correlation with different comorbidity variables and therapeutic strategies were evaluated. RESULTS: EMVs are increased in CKD patients as compared with controls (171.1 vs. 68.3/µl, P<.001). It was observed a negative correlation between age and EMVs. Statins and PA were associated with a reduction in EMVs and VEGF levels, independently of the serum total cholesterol levels (TC). The levels of AOPPS and VEGF were not different in CKD vs. controls. CONCLUSION: CKD is associated with a change in EMVs, VEGF and AOPP levels. The treatment with statins and PA normalizes these values to almost the observed in controls and this effect is independently of the prevailing TC level. These findings explain the existence of the pleiotropic effects of statins and PA which deserve further studies.

Sodium Intake, Circulating Microvesicles and Cardiovascular Outcomes in Type 2 Diabetes.

There is ongoing debate surrounding the complex relationship between dietary sodium intake and cardiovascular morbidity and mortality. The existing literature consists largely of observational studies that have demonstrated positive, negative, U-/J-shaped or unclear associations between sodium intake and cardiovascular outcomes. Our group and others have previously demonstrated an inverse relationship between dietary sodium intake and cardiovascular outcomes in people with type 2 diabetes. Increased activity of the renin-angiotensin-aldosterone system and sympathetic nervous system is postulated to contribute to these paradoxical findings through endothelial dysfunction, a precursor to the development of cardiovascular disease. Microvesicles are submicron (0.1 - 1.0µm) vesicles that form during cellular activation, injury or death with endothelial microvesicles being recognized markers of endothelial dysfunction. They are pathologically elevated in a variety of vascular-related conditions including type 2 diabetes. Lower habitual sodium intake in type 2 diabetes has been associated with higher pro-coagulant platelet microvesicles levels but not with endothelial microvesicles. Research utilizing endothelial microvesicles to evaluate the mechanistic relationship between dietary sodium intake and adverse cardiovascular outcomes in type 2 diabetes remains scarce.

Endothelial microvesicles in hypoxic hypoxia diseases.

Hypoxic hypoxia, including abnormally low partial pressure of inhaled oxygen, external respiratory dysfunction-induced respiratory hypoxia and venous blood flow into the arterial blood, is characterized by decreased arterial oxygen partial pressure, resulting in tissue oxygen deficiency. The specific characteristics include reduced arterial oxygen partial pressure and oxygen content. Hypoxic hypoxia diseases (HHDs) have attracted increased attention due to their high morbidity and mortality and mounting evidence showing that hypoxia-induced oxidative stress, coagulation, inflammation and angiogenesis play extremely important roles in the physiological and pathological processes of HHDs-related vascular endothelial injury. Interestingly, endothelial microvesicles (EMVs), which can be induced by hypoxia, hypoxia-induced oxidative stress, coagulation and inflammation in HHDs, have emerged as key mediators of intercellular communication and cellular functions. EMVs shed from activated or apoptotic endothelial cells (ECs) reflect the degree of ECs damage, and elevated EMVs levels are present in several HHDs, including obstructive sleep apnoea syndrome and chronic obstructive pulmonary disease. Furthermore, EMVs have procoagulant, proinflammatory and angiogenic functions that affect the pathological processes of HHDs. This review summarizes the emerging roles of EMVs in the diagnosis, staging, treatment and clinical prognosis of HHDs.

Endothelial cell-derived microvesicles: potential mediators and biomarkers of pathologic processes.

This review focuses on the formation, composition and function of endothelial microvesicles (MV), often called microparticles (MP). MV release is a controlled event and is considered a hallmark of cellular activation or alteration. MV may affect the function of target cells through surface interaction and receptor activation, cellular fusion and the delivery of intravesicular cargo. Endothelial MV are released as a consequence of endothelial activation during inflammation and have been described to affect hemostasis, various aspects of inflammatory reaction, vessel formation, apoptosis and cell survival, endothelial cell differentiation and function. Recent data suggest the potential use of MV in diagnostics, assessment of severity and prediction of outcomes in inflammatory diseases and their utilization as targets, mediators and vectors in therapy.

Microvesicles Derived from Indoxyl Sulfate Treated Endothelial Cells Induce Endothelial Progenitor Cells Dysfunction.

Cardiovascular disease is a major cause of mortality in chronic kidney disease patients. Indoxyl sulfate (IS) is a typical protein-bound uremic toxin that cannot be effectively cleared by conventional dialysis. Increased IS is associated with the progression of chronic kidney disease and development of cardiovascular disease. After endothelial activation by IS, cells release endothelial microvesicles (EMV) that can induce endothelial dysfunction. We developed an in vitro model of endothelial damage mediated by IS to evaluate the functional effect of EMV on the endothelial repair process developed by endothelial progenitor cells (EPCs). EMV derived from IS-treated endothelial cells were isolated by ultracentrifugation and characterized for miRNAs content. The effects of EMV on healthy EPCs in culture were studied. We observed that IS activates endothelial cells and the generated microvesicles (IsEMV) can modulate the classic endothelial roles of progenitor cells as colony forming units and form new vessels in vitro. Moreover, 23 miRNAs were contained in IsEMV including four (miR-181a-5p, miR-4454, miR-150-5p, and hsa-let-7i-5p) that were upregulated in IsEMV compared with control endothelial microvesicles. Other authors have found that miR-181a-5p, miR-4454, and miR-150-5p are involved in promoting inflammation, apoptosis, and cellular senescence. Interestingly, we observed an increase in NFκB and p53, and a decrease in IκBα in EPCs treated with IsEMV. Our data suggest that IS is capable of inducing endothelial vesiculation with different membrane characteristics, miRNAs and other molecules, which makes maintaining of vascular homeostasis of EPCs not fully functional. These specific characteristics of EMV could be used as novel biomarkers for diagnosis and prognosis of vascular disease.