Release Of Endothelial Microparticles Research Articles

Endothelial Microparticles: Markers of Inflammatory Response After Sutureless Valve Implantation.

Systemic inflammatory response syndrome (SIRS) is related to increased circulating endothelial microparticles (EMP). The aim of this study was to compare the plasma concentration of EMP between patients undergoing aortic valve replacement with conventional bioprosthesis implantation and Perceval™ S (LivaNova) and to evaluate its impact on the inflammatory response in the short-term follow-up. This is a randomized clinical trial with 24 patients submitted to isolated aortic valve replacement divided into two groups: Perceval™ S (Group P) and conventional bioprostheses (Group C). Incidence of severe SIRS (three or more criteria) in the first 48 hours postoperatively, EMP release profile, interleukins (IL) 6 and 8, C-reactive protein, and procalcitonin were analyzed preand postoperatively at 24 hours and three months. There were 24 patients (12 in each group), mean age was 69.92±5.17 years, 83.33% were female, the incidence of severe SIRS was 66.7% and 50% in groups C and P, respectively (P=0.68), and EMP showed a significant increase in the 24-hour postoperative period (P≤0.001) and subsequent decrease in the three-month postoperative period (P≤0.001), returning to baseline levels. For IL-6 and IL-8, there was a greater increase in group C at 24 hours postoperatively (P=.0.02 and P<0.001). The incidence of severe SIRS was similar in both groups, with significantly higher levels of IL-6 and IL-8, at the 24-hour postoperative period, in group C, however with higher levels of EMP in group P, and subsequent return to baseline levels at the three-month postoperative period in both groups.

Endothelial microparticles: A mechanosensitive regulator of vascular homeostasis and injury under shear stress.

Hemodynamic shear stress (SS), a frictional force generated by blood flow, regulates vascular homeostasis. High and steady SS maintains physiological function of endothelial cells while low and disturbed SS promotes disturbance of vascular homeostasis and the development of atherosclerosis. Endothelial microparticle (EMP), a vesicular structure shed from endothelial cells, has emerged as a surrogate biomarker of endothelial injury and dysfunction. EMP release is triggered by disturbed SS in addition to multiple inflammatory cytokines. This review systematically summarizes the impact of SS on EMPs and the role of EMPs under SS in modulating vascular homeostasis and injury, including endothelial survival, vasodilation, inflammatory response, vascular permeability, and coagulation system.

Angiogenic content of microparticles in patients with diabetes and coronary artery disease predicts networks of endothelial dysfunction

BackgroundElevated endothelial microparticles (EMPs) levels are surrogate markers of vascular dysfunction. We analyzed EMPs with apoptotic characteristics and assessed the angiogenic contents of microparticles in the blood of patients with type 2 diabetes (T2D) according to the presence of coronary artery disease (CAD).MethodsA total of 80 participants were recruited and equally classified as (1) healthy without T2D, (2) T2D without cardiovascular complications, (3) T2D and chronic coronary artery disease (CAD), and (4) T2D and acute coronary syndrome (ACS). MPs were isolated from the peripheral circulation, and EMPs were characterized using flow cytometry of CD42 and CD31. CD62E was used to determine EMPs’ apoptotic/activation state. MPs content was extracted and profiled using an angiogenesis array.ResultsLevels of CD42- CD31 + EMPs were significantly increased in T2D with ACS (257.5 ± 35.58) when compared to healthy subjects (105.7 ± 12.96, p < 0.01). There was no significant difference when comparing T2D with and without chronic CAD. The ratio of CD42-CD62 +/CD42-CD31 + EMPs was reduced in all T2D patients, with further reduction in ACS when compared to chronic CAD, reflecting a release by apoptotic endothelial cells. The angiogenic content of the full population of MPs was analyzed. It revealed a significant differential expression of 5 factors in patients with ACS and diabetes, including TGF-β1, PD-ECGF, platelet factor 4, serpin E1, and thrombospondin 1. Ingenuity Pathway Analysis revealed that those five differentially expressed molecules, mainly TGF-β1, inhibit key pathways involved in normal endothelial function. Further comparison of the three diabetes groups to healthy controls and diabetes without cardiovascular disease to diabetes with CAD identified networks that inhibit normal endothelial cell function. Interestingly, DDP-IV was the only differentially expressed protein between chronic CAD and ACS in patients with diabetes.ConclusionOur data showed that the release of apoptosis-induced EMPs is increased in diabetes, irrespective of CAD, ACS patients having the highest levels. The protein contents of MPs interact in networks that indicate vascular dysfunction.

Abstract 11165: Stimulated Release of Apoptosis- Induced Endothelial Microparticles in Patients with Diabetes and Coronary Artery Disease

Introduction: High endothelial microparticles (EMPs) levels could be used as a marker of vascular dysfunction. Hypothesis: The release of EMPs with apoptotic characteristics in the blood of patients with diabetes is different according to the presence of CAD, and further distinct between chronic CAD and ACS. Methods: A total of 80 participants were recruited and equally classified as: 1) healthy participants without diabetes, 2) healthy diabetics without micro or macro-vascular complications, 3) participants with T2D and chronic CAD, and 4) participants with T2D and ACS. MPs were isolated using ExoQuick-LP TM and analysed using flow cytometry of CD42 and CD31. CD62E was used to determine the apoptotic/activation state of the EMPs. MP content was extracted and profiled using angiogenesis array (R&amp;D systems). Results: Mean age of participants was 56 ±10. Levels of CD42- CD31+ EMPs were significantly increased in diabetics with acute ACS (257.5±35.58) when compared to healthy subjects (105.7±12.96, p&lt;0.01). There was no significant difference when comparing diabetics, with and without chronic CAD. The ratio of CD42-CD62+/ CD42-CD31+ EMPs was reduced in all diabetic groups, with further reduction in ACS when compared to chronic CAD, reflecting a release by apoptotic endothelial cells. The angiogenic content of the full population of MPs was analysed and revealed a significant differential expression of 5 factors in patients with ACS and diabetes: TGF-β1, PD-ECGF, platelet factor 4, serpin E1 and thrombospondin 1 (Figure 1). Ingenuity Pathway Analysis revealed of those 5 differentially expressed molecules, mainly TGF-β1, causes an inhibition of key pathways involved in normal endothelial function. Conclusions: Our data showed that the release of apoptosis-induced EMPs is increased in diabetes, irrespective of the presence of CAD, ACS patients having the highest levels. The protein contents of MPs interact in networks that are indicative of vascular dysfunction.

Effects of structured home-based exercise training on circulating endothelial progenitor cells and endothelial function in patients with intermittent claudication.

Exercise training improves walking capacity in patients with intermittent claudication (IC). Endothelial progenitor cells (EPCs), endothelial microparticles (EMPs), and endothelial dysfunction could play a role in this process. We measured EPCs and EMPs in a group of 60 patients with IC, and in a control group of 20 individuals without IC, before a treadmill test and 2, 24, and 48 hours after the test. Thirty patients with IC were randomly assigned to perform a 12-week home-based exercise training program. The EPC count, flow-mediated dilation (FMD) of the brachial artery, pain-free walking time (PFWT), and maximum walking time (MWT) were measured at the baseline and after the exercise training program. In patients with IC, EMPs significantly increased 2 hours after the treadmill test, whereas EPCs significantly increased after 24 hours. Among the subjects assigned to complete the training program, we observed a significant increase in the number of EPCs after 12 weeks, as well as an improvement in FMD, PFWT, and MWT. A significant correlation between the variation of EPCs, FMD, and MWT was found. The increase of EPCs and FMD were independent determinants of the walking capacity improvement, without significant interaction. Our results suggest that EPCs mobilization contributes to the improvement of walking capacity in patients with IC undergoing structured physical training. A number of different, partly independent, mechanisms are involved in this process, and our results highlight the potential role of EMPs release and endothelial function improvement. ClinicalTrials.gov Identifier: NCT04302571.

Role of endothelial-microparticles and the tissue factor pathway in ginsenoside Rb1-mediated prevention of umbilical vein endothelial cell injury.

Hepatic veno-occlusive disease (VOD) is a life-threatening complication of hematopoietic stem cell transplantation, which urgently requires effective prevention and treatment. Endothelial damage is recognized as the first event in patients with hepatic VOD. However, the mechanism by which endothelial injury induces thrombosis in hepatic VOD is still not clear. In the present study, monocrotaline (MCT) was used to induce endothelial cell injury in EA.hy926 cells to imitate in vitro hepatic VOD. MCT significantly increased apoptosis in EA.hy926 endothelial cells and the secretion of endothelial microparticles (EMPs) which can be used to reflect the level of endothelial injury. Additionally, MCT significantly enhanced the expression of soluble tissue factor (TF) and EMP-bound TF protein, suggesting that EMPs may participate in the development of hepatic VOD by regulating coagulation. Ginsenoside Rb1, a major constituent and effective ingredient of Panax ginseng, was found to significantly decrease MCT-induced endothelial injury and release of EMPs. Moreover, Ginsenoside Rb1 decreased soluble TF released by EA.hy926 cells and EMP-bound TF protein induced by MCT. These data suggest that ginsenoside Rb1 may serve as a potent prophylactic and/or as a treatment of hepatic VOD by protecting endothelial cells and preventing microthrombosis induced by endothelial injury.

PAK4 suppresses TNF-induced release of endothelial microparticles in HUVECs cells.

Tumor necrosis factor-α (TNF) is a pro-inflammatory cytokine upregulated in many inflammatory diseases, and a potent inducer of endothelial cell-derived microparticle (EMP) formation. In this study, we identified the protein kinase PAK4 as a key regulator of the TNF-induced EMP release from human umbilical vein endothelial cells (HUVECs). TNF induces dose- and time-dependent EMP release and downregulation of PAK4 and upstream cdc42 in HUVECs. PAK4 suppression or inhibition of its kinase activity increases TNF-induced EMP release and apoptosis in HUVECs, while PAK4 overexpression reduces EMP release and apoptosis in TNF-stimulated cells. Collectively, these data indicate that PAK4 suppresses TNF-induced EMP generation occurring during apoptosis, and suggest that modulation of PAK4 activity may represent a novel approach to suppress the TNF-induced EMP levels in pro-inflammatory disorders and other pathological conditions.

Sirolimus and propranolol inhibit endothelial proliferation while detergent sclerosants induce endothelial activation, microparticle release and apoptosis in vitro.

To investigate the effects of detergent sclerosants, sodium tetradecyl sulphate and polidocanol, on endothelial cell activation and microparticle release and the effects of detergent sclerosants, sirolimus and propranolol, on apoptosis in vitro. Cultured human umbilical vein endothelial cells and murine haemangioendothelioma (EOMA) cell lines were incubated with different concentrations of sodium tetradecyl sulphate and polidocanol, as well as sirolimus and propranolol. Endothelial activation was assessed using flow cytometry for CD62e (E-Selectin), CD54 (ICAM-1), CD105 (endoglin), CD144 (VE-Cadherin), CD146 (MCAM) and the release of endothelial microparticles. Cell proliferation was assessed using [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and carboxyfluorescein succinimidyl ester assays. Apoptosis was assessed using flow cytometry for lactadherin/propidium iodide staining and for Caspase-3 expression. Sublytic concentrations of sodium tetradecyl sulphate and polidocanol (0.075%-0.3%) increased the expression of the activation markers CD62e and CD54. The expression of CD105 decreased in sclerosant treated cultured human umbilical vein endothelial cells. Both sodium tetradecyl sulphate and polidocanol induced the release of endothelial microparticles. All agents inhibited cell proliferation. Sodium tetradecyl sulphate and polidocanol-induced apoptosis as evidenced by increased phosphatidylserine exposure and caspase-3 expression, whereas sirolimus and propranolol increased caspase-3 expression only. Sublytic concentrations of detergent sclerosants induce endothelial activation and the release of endothelial microparticles. All agents were anti-proliferative in EOMA cell lines, with sodium tetradecyl sulphate and polidocanol inducing cellular apoptosis.

P4145Endothelial NLRP3-Inflammasome impairs vascular function via microparticles

Abstract Background In recent years inflammation has emerged to the centre of attention of cardiovascular research. One of its key figures is the NLRP3-inflammasome a multimeric protein complex that stimulates inflammatory responses in atherogenesis through proinflammatory cytokines like caspases IL-1β and -18. It is activated by danger signals such as cholesterol crystals, oxidized LDL, ATP or uric acids. Microparticles (MP) are extracellular vesicles that are released by activated or apoptotic cells. They are known as a vector for the intercellular transfer of biological information. The body of evidence indicates that endothelial microparticles contribute to the development and complications in atherosclerosis. With this study we sought to elucidate the effects microparticles, that are discharged by inflammasome activated endothelial cells, exert on arterial vascular cells. Methods and results RTPCR experiments showed that activation of human coronary artery endothelial cells (HCAEC) with LPS and Nigericin leads to NLRP3-inflammasome-specific upregulation of NLRP3 and IL1β. Analysis of the supernatant of aforementioned cells via westernblot revealed release of cleaved caspase-1 while donorcells undergo pyroptosis. FACS and electronmicroscopy experiments revealed time dependent release of endothelial microparticles (EMP) by inflammasome activated HCAEC, while western blot demonstrated that EMP enclose active caspase-1. Fluorescence microscopic imaging illustrated time dependent incorporation of EMP by HCAEC. Stimulation of HCAEC with EMP revealed detrimental biological effects on recipient cells as viability assay and scratch assay showed decreased viability and proliferation/ migration, cytotoxity assay showed increased cytotoxity and RT-PCR experiments showed increased expression of NALP3, IL-1b, VCAM and ICAM. The fact that treatment of recipient cells with the NLRP3-Inhibitor isoliquiritigenin (ILG), heat-inactivation of EMP and rupturing the EMP-membrane by freezing is able to diminish harmful effects EMP exert on recipient cells shown by viability assay, scratch assay and microscopic imaging underlines detrimental effects being exerted by EMP-encapsuled inflamasome-components. Conclusion Our findings verify that MP released from inflammasome-activated endothelial cells are incorporated by vascular cells which in turn sustain a reduction of cell viability, migration and proliferation. EMP effectuate activation of the NLRP3-inflammasme in their target cells. The cytotoxic effects of EMP are suppressed by inhibitors of the NLRP3-inflammasome and affection of EMP-membrane. Our results emphasize the immunological role of endothelial cells and indicate that inflammasome activation is transferable through microparticle-associated communication. This in turn facilitates cell death and possibly initiates a vicious cycle of inflammation suggesting a role in the advancement of atherosclerosis. Acknowledgement/Funding Else Kröner Scholarship

The CD105:CD106 microparticle ratio is CD106 dominant in polycystic ovary syndrome compared to type 2 diabetes and healthy subjects

BackgroundA retrospective analysis was carried out from patients and controls during the past 5 years from a series of studies investigating endothelial microparticles (MP).MethodsIn total, 319 samples from 207 individuals were included in this analysis, from patients with type 2 diabetes (T2D, n = 105), women with polycystic ovary syndrome (PCOS, n = 145) and healthy volunteers (n = 69). All data were generated via the same flow cytometry protocol with the same antibody clones. Endothelial markers CD105 (Endoglin) and CD106 (Vascular cell adhesion molecule-1) were used to enumerate MP in venous blood.ResultsThe ratio of CD105MP:CD106MP was significantly different between groups (F = 63.43, p < 0.0001). Women with PCOS were found to have a median CD105MP:CD106MP ratio of 0.40 (IQR 0.24–0.57), suggesting approximately two CD106MP were found per CD105MP. The T2D group showed a median ratio of 2.32 (1.51–3.69) whereas in healthy volunteers the ratio was 2.21 (1.63–3.55). Serum intercellular adhesion molecule-1 was also shown to be significantly increased in PCOS when compared with control or T2D groups (F = 14.5, p < 0.001).ConclusionThese data suggest that women with PCOS have an altered endothelial MP release in favour of CD106. Thus a potential activated endothelial state exists in women with PCOS with a shift towards a predominantly CD106MP profile.

Endothelial Microparticles in Uremia: Biomarkers and Potential Therapeutic Targets.

Endothelial microparticles (EMPs) are vesicles derived from cell membranes, which contain outsourced phosphatidylserine and express adhesion molecules, such as cadherin, intercellular cell adhesion molecule-1 (ICAM-1), E-selectin, and integrins. EMPs are expressed under physiological conditions and continue circulating in the plasma. However, in pathologic conditions their levels increase, and they assume a pro-inflammatory and pro-coagulant role via interactions with monocytes; these effects are related to the development of atherosclerosis. Chronic kidney dysfunction (CKD) characterizes this dysfunctional scenario through the accumulation of uremic solutes in the circulating plasma, whose toxicity is related to the development of cardiovascular diseases. Therefore, this review aims to discuss the formation of EMPs and their biological effects in the uremic environment. Data from previous research demonstrate that uremic toxins are closely associated with the activation of inflammatory biomarkers, cardiovascular dysfunction processes, and the release of EMPs. The impact of a decrease in circulating EMPs in clinical studies has not yet been evaluated. Thus, whether MPs are biochemical markers and/or therapeutic targets has yet to be established.

Microparticles in systemic sclerosis: Potential pro-inflammatory mediators and pulmonary hypertension biomarkers.

Endothelial microparticles (EMP) are submicron vesicles released from endothelial cells. We aimed to determine the utility of EMP as biomarkers of pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc) patients and the pathogenic role of microparticles (MP) in vascular inflammation. Levels of EMP (CD144+, CD31+, CD62E+ and CD143+) were compared between three groups (10 SSc patients with PAH, 10 SSc patients without pulmonary hypertension (no-PH) and 10 healthy age- and sex-matched controls). Human pulmonary artery endothelial cells (HPAEC) were exposed in vitro to MP obtained from SSc patients or healthy controls, and levels of cytokines and inflammatory adhesion molecules were compared. CD144+ EMP were significantly higher in the SSc-PAH group compared to either the SSc-no PH or healthy controls (diagnostic accuracy 80%, P = 0.02). Compared to controls, SSc patients had higher CD31+/CD62E+ ratios, indicating larger contributions of apoptosis to EMP release (P = 0.04). Patients with limited SSc had significantly higher levels of CD143+ EMP compared to those with diffuse subtype (P = 0.008). When HPAEC were exposed to MP from SSc patients, there was a significant increase in inflammatory cytokines and adhesion molecules. Interestingly, exposure to healthy control MP caused a reduction in inflammatory markers. EMP (particularly CD144+) are promising biomarkers of PAH in SSc but require further study. MP isolated from SSc patients induced an increase in endothelial cell inflammation and may be an important pathogenic factor in SSc.

Natural Killer Cells Exhibit a Peculiar Phenotypic Profile in Systemic Sclerosis and Are Potent Inducers of Endothelial Microparticles Release.

The pathophysiology of systemic sclerosis (SSc) involves early endothelial and immune activation, both preceding the onset of fibrosis. We previously identified soluble fractalkine and circulating endothelial microparticles (EMPs) as biomarkers of endothelial inflammatory activation in SSc. Fractalkine plays a dual role as a membrane-bound adhesion molecule expressed in inflamed endothelial cells (ECs) and as a chemokine involved in the recruitment, transmigration, and cytotoxic activation of immune cells that express CX3CR1, the receptor of fractalkine, namely CD8 and γδ T cells and natural killer (NK) cells. We aimed to quantify circulating cytotoxic immune cells and their expression of CX3CR1. We further investigated the expression profile of NK cells chemokine receptors and activation markers and the potential of NK cells to induce EC activation in SSc. We performed a monocentric study (NCT 02636127) enrolling 15 SSc patients [15 females, median age of 55 years (39–63), 11 limited cutaneous form and 4 diffuse] and 15 healthy controls. Serum fractalkine levels were significantly increased in SSc patients. Circulating CD8 T cells numbers were decreased in SSc patients with no difference in their CX3CR1 expression. Circulating γδ T cells and NK cells numbers were preserved. CX3CR1 expression in CD8 and γδ T cells did not differ between SSc patients and controls. The percentage and level of CX3CR1 expression in NK cells were significantly lowered in SSc patients. Percentages of CXCR4, NKG2D, CD69-expressing NK cells, and their expression levels were decreased in NK cells. Conversely, CD16 level expression and percentages of CD16+ NK cells were preserved. The exposure of human microvascular dermic EC line (HMVEC-d) to peripheral blood mononuclear cells resulted in similar NK cells degranulation activity in SSc patients and controls. We further showed that NK cells purified from the blood of SSc patients induced enhanced release of EMPs than NK cells from controls. This study evidenced a peculiar NK cells phenotype in SSc characterized by decreased chemokine and activation receptors expression, that might reflect NK cells involvement in the pathogenic process. It also highlighted the role of NK cells as a potent mechanism inducing endothelial activation through enhanced EMPs release.

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-).

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.

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.

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.

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.

Deferiprone inhibits iron overload-induced tissue factor bearing endothelial microparticle generation by inhibition oxidative stress induced mitochondrial injury, and apoptosis

Iron overload-induced cardiovascular toxicity is one of the most common causes of morbidity and mortality in beta-thalassemia major patients. We have previously shown that iron overload-induced systemic arterial changes characterized by endothelial dysfunction are associated with increased endothelial microparticle (EMP) release. In this study, we further demonstrate how EMP release is associated with iron-induced mitochondrial injury and apoptosis of endothelial cells. Iron increased the production of reactive oxygen species (ROS) and calcium influx into mitochondria [Ca2+]m. Iron also disturbed mitochondrial respiration function and eventually led to loss of mitochondrial membrane potential (ΔΨm). A significant increase in apoptotic cells and EMPs were found under iron treatment. EMPs contained tissue factor (TF), which has potential clinical impact on thromboembolic phenomenon. Then, we investigated the salvaging effect of deferiprone (L1) on endothelial cell damage and EMP release. We found that L1 could inhibit iron-induced ROS generation, and decrease mitochondrial damage with the resultant effect of less endothelial cell apoptosis and EMP release. L1 could protect endothelial cells from iron-induced toxic effects and minimize EMP release, which could be potentially helpful in a subgroup of thalassemia patients who have increased thromboembolic complications.

Oxidized low-density lipoprotein-induced microparticles promote endothelial monocyte adhesion via intercellular adhesion molecule 1.

Oxidized low-density lipoprotein (oxLDL) accumulates early in atherosclerotic lesions and plays an important role in the progressive formation of atherosclerotic plaques. Endothelial derived microparticles (EMPs) form a heterogeneous population of <1-μm particles that shed from endothelial membranes upon activation. While EMPs are shown to be involved in atherosclerotic pathophysiology and progression, there is no report regarding the relationship between oxLDL and EMPs. In this study, we aim to determine the influence of oxLDL on endothelial microparticle release and the subsequent regulation of the endothelial activation. EMPs were collected from the medium of human umbilical vein endothelial cells (HUVECs) treated with oxLDL or PBS as control. We find that oxLDL increases the release of EMPs containing intercellular adhesion molecule 1 (ICAM-1) but not vascular cell adhesion molecule 1 (VCAM-1). Confocal microscopy analysis further demonstrates that these EMPs interact with endothelial cells and increase the expression of ICAM-1 in HUVECs. The fact that injecting oxLDL-induced EMPs via the tail vein of ICR mice augments ICAM-1 expression on aortic endothelial cells confirms our results in vivo. Finally, oxLDL-induced EMPs from HUVECs increase the adhesion of monocytes to endothelial cells as determined by the adhesion assay. Our study suggests that oxLDL may augment the release of EMPs harboring increased levels of ICAM-1 that can be transferred to endothelial cells elsewhere. This leads to increased monocyte recruitment in other regions where oxLDL accumulation was initially more limited. EMPs may therefore serve as the mediator that propagates oxLDL-induced endothelial inflammation.