Joaquin J. Jimenez

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.

Measuring circulating cell-derived microparticles

Cell-derived microparticles (MPs) are receiving increasing attention in recent years, both as a diagnostic aid and investigative tool [1–4]. Because they carry markers of the parent cell, including those induced by activation or apoptosis, endothelial MPs (EMPs) can provide valuable information on the status of the parent cell, obtainable in no other way. In addition, there is a growing belief that MPs can function as important diffusible vectors of specific adhesins and cytokines promoting cellular interactions and signal transmission [2]. ThusMP analysis constitutes a new avenue for investigation of pathologies in various diseases. Although still considered investigational [1–4], recent results from several laboratories suggest that MP analysis may be poised to enter the mainstream of clinical testing. However, a major impediment to that end is the wide variety ofmethodologies used by different laboratories in this field, few of which can be directly compared to the others, and results from which are sometimes inconsistent or conflicting. As a first step in addressing that problem, the Editor has organized this Forum article, consisting of a brief description of the preferred methods and rationality from each of six active laboratories in the field, including our own [5–10]. Table 1 lists some key features of the six methodological approaches. It is seen that major differences exist in the preparation of the MP samples (such as centrifugation), whether or not they are first sedimented and resuspended, means of generic MP detection (4 of 6 use annexin V), and cell lineage-specific antigenic markers. These differences probably account for some of the different findings among the groups.

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.

Endothelial microparticles as markers of endothelial dysfunction.

Endothelial microparticles (EMP) are small vesicles released from disturbed endothelial cells (EC). Owing to the central importance of EC injury in thrombotic and inflammatory conditions, assay of EMP as a marker of EC disturbance has come under intensive development by several laboratories. The review begins with established markers of EC injury, commonly soluble markers such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, von Willebrand factor (vWF), etc., pointing out that many of these are in fact mixtures of true soluble molecules with membrane-bound forms, for example, EMP. Assays of EMP from different labs are reviewed and standardization of assay is recommended. EMP are heterogeneous: those released in activation vs. apoptosis are distinctive in phenotypic markers and procoagulant properties. Application of EMP phenotype analysis can distinguish EC state of activation from apoptosis. Some EMP carry functional vWF with properties different from soluble vWF. Certain EMP bind to and activate monocytes; EMP-monocyte conjugates were found to be a marker of inflammatory disease such as multiple sclerosis (MS), and to enhance transendothelial migration of leukocytes in vitro. Clinical studies have revealed elevated plasma levels of EMP in lupus anticoagulant (LA), multiple sclerosis (MS), thrombotic thrombocytopenic purpura (TTP), coronary artery disease (CAD), hypertension, preeclampsia, and diabetes. Further refinement of EMP assay could open new windows for evaluating and monitoring endothelial injury in thrombotic and inflammatory disorders.

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.

Levels of endothelial and platelet microparticles and their interactions with leukocytes negatively correlate with organ dysfunction and predict mortality in severe sepsis.

Objective:Mortality in sepsis is believed to be associated with exaggerated inflammatory responses, but recent evidence suggests that poor outcome is associated with reduced inflammation. To test this hypothesis, we measured several inflammatory markers to determine whether any of them or any combinations are associated with mortality or organ dysfunction. Design:Clinical study. Setting:School of medicine. Patients:Thirty-five patients with severe sepsis. Interventions:Markers of endothelial, platelet, and leukocyte activation were measured on days 1, 2, and 3 after enrollment. The markers were a) endothelial microparticles (EMPs) and their conjugates with monocytes (EMP/MONO); b) platelet microparticles (PMPs) and platelet activation marker CD62P; c) platelet-leukocyte conjugates (PLT/LEU) and leukocyte activation marker CD11b; and d) intracellular nitric oxide in leukocytes. Measurements and Main Results:The 28-day mortality rate was 51% (18 of 35). Significant differences between survivors and nonsurvivors on day 1 were found in PLT/LEU (p = .001), CD11b (p = 0.02), and EMP/MONO (p = .02) groups. Using logistic regression to assess if these markers predict mortality on day 1, we found that PLT/LEU had the best predictive value among the markers used (area under receiver operating characteristics curve = 0.82). All markers of cell activation and inflammation were significantly higher among survivors on days 2 and 3, except nitric oxide, which was lower. This marker showed significant negative correlation with the Sequential Organ Failure Assessment score throughout the study. Conclusions:Our data support the hypothesis that early increased, not decreased, inflammatory response as measured by our markers is associated with improved survival rate. A high negative correlation was found between some of these markers and Sequential Organ Failure Assessment score.

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.