Lucia M. Mauro

Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis.

BACKGROUND Endothelial cells (EC) shed endothelial microparticles (EMP) in activation and apoptosis. OBJECTIVES We compared the antigenic expression of EMP species released during activation as compared to apoptosis, in three cell lines. METHODS EC from renal and brain microvascular (MiVEC) and coronary macrovascular (MaVEC) origin were incubated with TNF-alpha to induce activation, or deprived of growth factors to induce apoptosis. Antigens expressed on EMP and EC were assayed flow cytometrically and included constitutive markers (CD31, CD51/61, CD105), inducible markers (CD54, CD62E and CD106), and annexin V binding. RESULTS It was found that in apoptosis, constitutive markers in EMP were markedly increased (CD31>CD105), with a concomitant decrease in expression in EC. Annexin V EC surface binding and annexin V+ EMP were more sharply increased in apoptosis than in activation. In contrast, in activation, inducible markers in EMP were markedly increased in both EMP and EC (CD62E>CD54>CD106). Coronary MaVEC released significantly less EMP than MiVEC. CONCLUSION EC release qualitatively and quantitatively distinct EMP during activation compared to apoptosis. Analysis of EMP phenotypic signatures may provide clinically useful information on the status of the endothelium.

Effects of Severe Hypertension on Endothelial and Platelet Microparticles

Abstract—The molecular mechanisms by which extreme blood pressure elevation leads to vascular injury are not defined. To explore the hypothesis that activation of endothelium and platelets as manifested by increased concentrations of circulating endothelial microparticles and platelet microparticles could play a role in this target organ injury, we conducted a cross-sectional study of these markers in 3 groups: (1) untreated patients referred specifically for treatment of severe uncontrolled hypertension; (2) untreated patients with established mild hypertension; and (3) normotensive volunteer subjects. By ANOVA, endothelial (P =0.002) and platelet (P =0.01) microparticles were greatest in the severely hypertensive group. There was a significant correlation between both of these markers and blood pressure, even in the setting of multiple risk factors. Our results suggest that these markers may be useful and specific for pressure-induced endothelial and platelet activation in hypertension. Furthermore, because of the combined effects of endothelial and platelet microparticles on coagulation, leukocytes, and endothelium, it is possible that they may play a pathogenic role in mediating target organ injury in severe hypertension.

High levels of circulating endothelial microparticles in patients with acute coronary syndromes.

BACKGROUND Endothelial injury plays a critical role in coronary artery disease (CAD), but the assessment of this injury has been problematical. Recently, it has been shown in vitro that endothelial cells (ECs) release endothelial microparticles (EMPs) on activation or apoptosis and that an assay of EMPs can provide useful information on EC status in patients with thrombotic disorders. This study is aimed at assessing possible correlations between EMPs, which are markers of endothelial injury, and clinical subgroups of patients with CAD. METHODS A prospective, case-controlled study was conducted on 84 patients with CAD and 42 control subjects to investigate EMP profiles. Included were 64 patients with acute coronary syndromes ([ACS], 38 with myocardial infarction [MI] and 26 with unstable angina [UA]) and 20 patients with stable angina (SA). EMPs in platelet-poor plasma were measured flow cytometrically with combinations of fluorescent antibodies (anti-CD31, -51, -42), allowing distinction of EMPs from platelet microparticles (PMPs). Clinical subgroups of patients were correlated with EMP and PMP levels in blood. RESULTS Two species of EMPs (CD31+ and CD51+) were evaluated. Both were significantly higher in patients with CAD than in control subjects. CD31+ EMP was higher in ACS than SA. Among patients with first MI, CD31+ EMP was higher in patients with MI than in patients with UA and was significantly higher than in patients with recurring MI. CD51+ EMP did not discriminate ACS from SA. A simultaneous assay of PMP showed correlation between EMPs and PMPs. However, PMPs did not discriminate patients with SA from control subjects. CONCLUSIONS EMP assay appears promising for assessing EC injury in CAD.

Elevated plasma endothelial microparticles in multiple sclerosis

Objective: To assess endothelial dysfunction in patients with MS and to investigate whether plasma from patients with MS induces endothelial cell dysfunction in vitro. Background: Endothelial cell dysfunction may contribute to the pathogenesis of MS. Elevations of soluble adhesion molecules intracellular adhesion molecule, vascular cell adhesion molecule, and platelet-endothelial cell adhesion molecule-1 (CD31) have been reported as markers of blood–brain barrier (BBB) damage in MS, but direct assay of endothelium has been difficult. Endothelial cells release microparticles <∼1.5 μm (EMP) during activation or apoptosis. The authors developed a flow cytometric assay of EMP and studied EMP as markers of endothelial damage in MS. Methods: Platelet-poor plasma (PPP) from 50 patients with MS (30 in exacerbation and 20 in remission) and 48 controls were labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD31 and anti-CD51 (vitronectin receptor) antibodies, and two classes of EMP (CD31+ and CD51+) were assayed by flow cytometry. For in vitro studies, patients’ plasma was added to the microvascular endothelial cell (MVEC) culture and release of CD31+ and CD51+ EMP were measured in the supernatant. Results: Plasma from patients in exacerbation had 2.85-fold elevation of CD31+ EMP as compared with healthy controls, returning to near control value during remission. The CD31+ EMP concentration showed a positive association with gadolinium enhancement in patients with MS. In contrast, CD51+ EMP remained elevated in both exacerbation and remission. This suggests that CD31+ EMP is a marker of acute injury, whereas CD51+ EMP reflects chronic injury of endothelium. MS plasma induced release of both CD31+ and CD51+ EMP from MVEC culture in vitro. Conclusion: Endothelial dysfunction is evident during exacerbation of MS, evidenced by shedding of EMP expressing PECAM-1 (CD31). The in vitro data indicate contribution of one or more plasma factors in endothelial dysfunction of MS.

Elevated endothelial microparticles in thrombotic thrombocytopenic purpura : findings from brain and renal microvascular cell culture and patients with active disease

Endothelial injury is believed to be a key initiating event in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), leading to platelet activation and formation of platelet‐rich thrombi in microvasculature. However, the nature of endothelial injury in TTP is poorly defined and clinical assays to rapidly and reliably monitor endothelial damage are not readily available. Using flow cytometry, we measured endothelial microparticles (EMPs) generated from cultured renal and brain microvascular endothelial cells (MVECs) during activation and apoptosis, and evaluated the effect of TTP plasma on them. EMPs were measured using positivity for monoclonal antibodies (mAbs) CD31 and CD51, and their procoagulant activity was assessed using a Russell viper venom assay. Both cell lines generated procoagulant EMPs when cultured with inducers of activation (tumour necrosis factor alpha; TNF‐α) or apoptosis (mitomycin C). TTP plasma induced a five‐ to sixfold increase of EMP generation and a two‐ to threefold increase of procoagulant activity in cultured brain and renal MVECs. TTP plasma induced a threefold and 13‐fold increase of intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) expression, respectively, on renal MVECs. Procoagulant activity tended to parallel EMP numbers. The effect of TTP plasma on cell viability was similar to that of TNF‐α, implying that it induced activation rather than apoptosis. Control plasma and idiopathic thrombocytopenic purpura (ITP) plasma had little effect. In the clinical study, EMP assay of blood from acute TTP patients showed levels markedly elevated compared with normal controls, but values returned to normal in remission. In conclusion, TTP plasma activated and induced injury to MVECs in culture, judged by production of EMP and expression of activation markers. Released procoagulant EMP may play a role in the pathogenesis of TTP. Assay of EMP may be a useful marker of disease activity and endothelial injury in TTP and possibly other thrombotic disorders.

Postprandial hypertriglyceridemia increases circulating levels of endothelial cell microparticles.

Background—This study evaluated a possible relationship between levels of endothelial microparticles (EMPs), known to be a sensitive indicator of endothelial disturbance, and changes in postprandial lipid levels in healthy volunteers after a low- or high-fat meal. Methods and Results—Eighteen healthy subjects without known cardiovascular risk factors were evaluated. Lipid and EMP levels were measured before and 1 and 3 hours after a single low- or high-fat isocaloric meal. The low-fat meal had no significant postprandial effect on EMPs or lipids compared with fasting levels. In contrast, a single high-fat meal significantly increased EMP levels after 1 and 3 hours, from 389±54 (thousands per milliliter) when fasting to 541±139 (P=0.0002) and 677±159 (P<0.0001), respectively, and correlated with a postprandial elevation in serum triglycerides. Conclusions—A single high-fat meal led to a significant elevation of plasma EMP levels in healthy, normolipidemic subjects and correlated with a postprandial elevation of serum triglycerides. EMPs may be an indirect marker of endothelial dysfunction or injury induced by postprandial triglyceride-rich lipoproteins.

Endothelial microparticles released in thrombotic thrombocytopenic purpura express von Willebrand factor and markers of endothelial activation.

Summary.  It has been suggested that endothelial apoptosis is a primary lesion in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). We tested this hypothesis by examining the phenotypic signatures of endothelial microparticles (EMP) in TTP patients. In addition, the effect of TTP plasma on microvascular endothelial cells (MVEC) in culture was further delineated. EMP released by endothelial cells (EC) express markers of the parent EC; EMP released in activation carry predominantly CD54 and CD62E, while those in apoptosis CD31 and CD105. We investigated EMP release in vitro and in TTP patients. Following incubation of MVEC with TTP plasma, EMP and EC were analysed by flow cytometry for the expression of CD31, CD51, CD54, CD62E, CD105, CD106 and von Willebrand factor (VWF) antigen. EMP were also analysed in 12 TTP patients. In both EC and EMP, CD62E and CD54 expression were increased 3‐ to 10‐fold and 8‐ to 10‐fold respectively. However, CD31 and CD105 were reduced 40–60% in EC but increased twofold in EMP. VWF expression was found in 55 ± 15% of CD62E+ EMP. Markers of apoptosis were negative. In TTP patients, CD62E+ and CD31+/CD42b− EMP were markedly elevated, and preceded and correlated well with a rise in platelet counts and a fall in lactate dehydrogenase. CD62E+ EMP (60 ± 20%) co‐expressed VWF and CD62E. The ratio of CD31+/42b− to CD62E+ EMP exhibited a pattern consistent with activation. In conclusion, our studies indicate endothelial activation in TTP. EMP that co‐express VWF and CD62E could play a role in the pathogenesis of TTP.

ENDOTHELIAL MICROPARTICLES (EMP) BIND AND ACTIVATE MONOCYTES: ELEVATED EMP- MONOCYTE CONJUGATES IN MULTIPLE SCLEROSIS

Elevated plasma endothelial microparticles (EMP) have been documented in MS during exacerbation. However, the role of EMP in pathogenesis of MS remains unclear. We investigated the formation of EMP-monocyte conjugates (EMP-MoC) and their potential role in transendothelial migration of inflammatory cells in MS. EMP-MoC were assayed in 30 MS patients in exacerbation, 20 in remission and in 35 controls. EMP-leukocyte conjugation was investigated flowcytometrically by employing alpha-CD54 or alpha-CD62E for EMP, and alpha-CD45 for leukocytes. EMP-MoC were characterized by identifying adhesion molecules involved and their effect on monocyte function. In vivo (clinical): EMP-MoC were markedly elevated in exacerbation vs. remission and controls, correlating with presence of GD+ MRI lesions. Free CD54+ EMP were not elevated but free CD62E+ EMP were. In vitro: EMP bound preferentially to monocytes, less to neutrophils, but little to lymphocytes. Bound EMP activated monocytes: CD11b expression increased 50% and migration through cerebral endothelial cell layer increased 2.6-fold. Blockade of CD54 reduced binding by 80%. Most CD54+ EMP bound to monocytes, leaving little free EMP, while CD62+ EMP were found both free and bound. These results demonstrated that phenotypic subsets of EMP interacted differently with monocytes. Based on our observations, EMP may enhance inflammation and increase transendothelial migration of monocytes in MS by binding to and activating monocytes through CD54. EMP-MoC were markedly increased in MS patients in exacerbation compared to remission and may serve as a sensitive marker of MS disease activity.

Endothelial microparticles induce formation of platelet aggregates via a von Willebrand factor/ristocetin dependent pathway, rendering them resistant to dissociation.

Summary.  Endothelial microparticles (EMP) released from activated or apoptotic endothelial cells (EC) are emerging as useful markers for detection of EC dysfunction. Our recent observation that EMP carry von Willebrand factor (vWf) led us to investigate their interaction with platelets. EMP were incubated with normal washed platelets in the presence or absence of ristocetin, then platelet aggregates were measured by flow cytometry. In the absence of ristocetin, negligible EMP conjugated with platelets (< 5%) but in the presence of ristocetin (1 mg mL−1), EMP induced up to 95% of platelets to aggregate. EMP‐platelet interaction was 80% blocked by anti‐CD42b, or by 0.1 μm filtration to remove EMP. Platelet aggregates induced by normal plasma or high molecular weight vWf (Humate‐P) dissociated 50% within 15–25 min following 1 : 20 dilution. In contrast, aggregates formed with EMP persisted two‐ to threefold longer with the same treatment, indicating greater stability. A similar degree of prolongation of dissociation was observed using plasma from thrombotic thrombocytopenic purpura (TTP) patients compared with normal plasma. Addition of EMP to plasma from severe von Willebrand disease restored his ristocetin‐induced platelet aggregation. Multimer analysis of vWf on EMP showed unusually large vWf (ULvWf). In summary, EMP carries ULvWf multimers, promote platelet aggregates, and increase the stability of the aggregates thus formed.

Elevated endothelial microparticle—monocyte complexes induced by multiple sclerosis plasma and the inhibitory effects of interferon-β1b on release of endothelial microparticles, formation and transendothelial migration of monocyte-endothelial microparticle complexes

Monocyte migration through the disrupted cerebral endothelial cell (EC) junctions plays an essential role in formation of multiple sclerosis (MS) demyelinating lesions. During pathogenesis of MS, activated ECs release endothelial microparticles (EMP), which possibly facilitate transendothelial migration (TEMIG) of monocytes. To assess functional roles of EMP in MS, specifically, their (i) interaction with monocytes, (ii) effect on monocyte TEMIG in an in vitro model of the brain microvascular endothelial cells (BMVEC), (iii) phenotypic profiles of EMP elicited by MS plasma and (iv) the effects of IFN-b1b on release of EMP and on TEMIG of monocytes (mono) and monocytes:EMP complexes (mono:EMP) through the BMVEC. The effect of IFN-b1b on the release of EMP and the TEMIG of mono and mono:EMP was assessed by preincubating BMVEC cultures of IFN-b1b prior to addition of plasma. Three EMP phenotypes, CD54, CD62E and CD31 were assayed. Plasma specimens from 20 patients with relapsing—remitting MS (11 in exacerbation, MS-E, and 9 in remission, ME-R) and 10 healthy controls were studied. Incubation of BMVEC with MS-E plasma yielded elevated levels of EMPCD54, EMP62E and EMPCD31 relative to MS-R and control plasmas. MS-E but not MS-R or control plasma also augmented TEMIG of monocytes, respectively. Mono:EMP complexes further augmented TEMIG relative to mono alone, but only in the presence of MS-E plasma; there was no significant effect with MS-R or control plasmas. The presence of IFN-b1b inhibited TEMIG of mono and mono:EMP by 20% and 30%, respectively. MS-E but not MS-R plasma elicited release of activation-derived EMP and enhanced TEMIG of mono and mono:EMP. IFN-b1b inhibited TEMIG and release of EMP, suggesting a role of EMP and a novel therapeutic mechanism for IFN-β1b in MS.