José Sampol

In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant.

Microparticles (MPs) resulting from vesiculation of platelets and other blood cells have been extensively documented in vitro and have been found in increased numbers in several vascular diseases, but little is known about MPs of endothelial origin. The aim of this study was to analyze morphological, immunological, and functional characteristics of MPs derived from human umbilical vein endothelial cells (HUVECs) stimulated by TNF, and to investigate whether these MPs are detectable in healthy individuals and in patients with a prothrombotic coagulation abnormality. Electron microscopy evidenced bleb formation on the membrane of TNF-stimulated HUVECs, leading to increased numbers of MPs released in the supernatant. These endothelial microparticles (EMPs) expressed the same antigenic determinants as the corresponding cell surface, both in resting and activated conditions. MPs derived from TNF-stimulated cells induced coagulation in vitro, via a tissue factor/factor VII-dependent pathway. The expression of E-selectin, ICAM-1, alphavbeta3, and PECAM-1 suggests that MPs have an adhesion potential in addition to their procoagulant activity. In patients, labeling with alphavbeta3 was selected to discriminate EMPs from those of other origins. We provide evidence that endothelial-derived MPs are detectable in normal human blood and are increased in patients with a coagulation abnormality characterized by the presence of lupus anticoagulant. Thus, MPs can be induced by TNF in vitro, and may participate in vivo in the dissemination of proadhesive and procoagulant activities in thrombotic disorders.

Circulating endothelial cells Biomarker of vascular disease

Recent research has recognised new populations of non-hematopoietic cells in the blood. One of these, circulating endothelial cells (CECs), often defined by the expression of membrane glycoprotein CD146, are rarely found in the blood in health, but raised numbers are present in a wide variety of human conditions, including inflammatory, immune, infectious, neoplastic and cardiovascular disease, and seem likely to be evidence of profound vascular insult. An additional population are endothelial progenitor cells, defined by the co-expression of endothelial and immaturity cell surface molecules and also by the ability to form colonies in vitro. Although increased numbers of CECs correlate with other markers of vascular disease, questions remain regarding the precise definition, cell biology and origin of CECs. For example, they may be damaged, necrotic or apopototic, or alive, and could possess procoagulant and/or proinflammatory properties. However, since these cells seem to be representative of in situ endothelium, their phenotype may provide useful information. Indeed, whatever their phenotype, there is growing evidence that CECs may well be a novel biomarker, the measurement of which will have utility in various clinical settings related to vascular injury. Despite this promise, progress is impeded by the diversity of methodologies used to detect these cells. Accordingly, results are sometimes inconclusive and even conflicting. Nevertheless, increased CECs predict adverse cardiovascular events in acute coronary syndromes, suggesting they may move from being simply a research index to having a role in the clinic. The objective of the present communication is to condense existing data on CECs, briefly compare them with progenitor cells, and summarise possible mechanism(s) by which they may contribute to vascular pathology.

Vasodilator-stimulated phosphoprotein phosphorylation analysis prior to percutaneous coronary intervention for exclusion of postprocedural major adverse cardiovascular events.

Summary.  Background: Despite dual antiplatelet therapy, the rate of major adverse cardiovascular events (MACE) after percutaneous coronary angioplasty remains high. Studies have shown interindividual variations in response to clopidogrel. Furthermore, there is an apparent link between clinical outcomes and clopidogrel resistance. Objectives: To investigate the value of platelet reactivity index (PRI), assessed by vasodilator‐stimulated phosphoprotein (VASP) phosphorylation analysis, for predicting MACE after percutaneous coronary intervention (PCI) with stent implantation.Methods: A prospective monocentric study was performed on 144 patients undergoing PCI. PR was evaluated by VASP phosphorylation analysis 24 h after they received a 300‐mg loading dose of clopidogrel. MACE were recorded during a 6‐month follow‐up. Patients were divided into quintiles according to PRI, as assessed by VASP analysis. The receiver operating characteristic (ROC) curve served to determine the optimal cut‐off value of VASP analysis to detect MACE.Results: Of the 144 patients, 34% had stable angina pectoris, 40% silent ischemia, and 26% low‐risk non‐ST‐segment elevation acute coronary syndrome. During the follow‐up, 21 MACE were observed. Patients in quintile 1 of VASP analysis had a significantly lower risk of MACE as compared with those among the four higher quintiles (0 vs. 21, P < 0.01). ROC curve analysis of VASP showed an optimal cut‐off value of 50% PR to exclude MACE. The negative predictive value of the test was 100%.Conclusions: VASP phosphorylation analysis can evaluate the individual response to clopidogrel loading dose prior to PCI and predict postprocedural MACE.

Elevation of circulating endothelial microparticles in patients with chronic renal failure

Summary.  Background: Chronic renal failure patients are at high risk of cardiovascular events and display endothelial dysfunction, a critical element in the pathogenesis of atherosclerosis. Upon activation, the endothelium sheds microparticles, considered as markers of endothelial dysfunction that also behave as vectors of bioactive molecules. Aim: To measure plasma levels of endothelial microparticles (EMPs) in chronic renal failure patients (CRF), either undialyzed or hemodialyzed (HD), and to investigate the ability of uremic toxins to induce EMP release in vitro. Methods: Circulating EMPs were numerated by flow cytometry, after staining of platelet‐free plasma with phycoerythrin (PE)‐conjugated anti‐CD144 (CD144+ EMP) or anti‐CD146 (CD146+ EMP) monoclonal antibodies. Platelet MP (CD41+ PMP), leukocyte MP (CD45+ leukocyte microparticles (LMP)), and annexin‐V+ MPs were also counted. In parallel, MPs were counted in supernatant of human umbilical vein endothelial cells incubated with uremic toxins [oxalate, indoxyl sulfate, p‐cresol, and homocysteine (Hcy)], at concentrations found in patients. Results and conclusions: CD144+ EMP and CD146+ EMP levels were significantly higher in CRF and HD patients than in healthy subjects. Furthermore, annexin‐V+ MPs were elevated in both groups of uremic patients, and CD41+ PMP and CD45+ LMP were increased in CRF and HD patients, respectively. In vitro, p‐cresol and indoxyl sulfate significantly increased both CD146+ and annexin‐V+ EMP release. Increased levels of circulating EMP in CRF and HD patients represent a new marker of endothelial dysfunction in uremia. The ability of p‐cresol and indoxyl sulfate to increase EMP release in vitro suggests that specific uremic factors may be involved in EMP elevation in patients.

Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies?

Summary.  Background: Platelet microparticles (PMPs) have proved useful to identify patients with vascular risk. However, PMP counting, which is currently done by flow cytometry (FCM), needs to be standardized. Objectives: The objectives were (i) to standardize FCM settings for PMP counts on a routine instrument (Cytomics FC500) using size‐calibrated fluorescent beads; (ii) to determine intra‐instrument and interinstrument reproducibility; and (iii) to establish PMP values in healthy subjects. Methods: Using a blend of size‐calibrated fluorescent beads (0.5 and 0.9 μm) in a fixed numerical ratio (Megamix), we gated PMPs in a restricted size window. To test intra‐instrument and inter‐instrument reproducibility, annexin V and CD41 coexpression were used to count PMPs in frozen aliquots of the same platelet‐free plasma (PFP) over 4 months and in PFP from 10 healthy subjects on three independent flow cytometers. Results: This calibrated‐bead strategy allowed full long‐term control of the FCM‐based microparticle protocol and reproducible PMP counts over time [coefficient of variation (CV) < 10%]. Optimal settings were easily transferred from one instrument to another, using Megamix as a stable template. Similar PMP counts (CV < 12%) were obtained using the three instruments. With such a standardized FCM protocol, PMP values were established in healthy subjects (n = 60) with significantly higher levels in women than in men [median (1st quartile to 3rd quartile): 1775 μL−1 (1014–3039 μL−1) vs. 656 μL−1 (407–962 μL−1)]. Conclusions: The present strategy provides a new option for PMP count standardization and thus opens the way for multicenter studies.

Impact of pre‐analytical parameters on the measurement of circulating microparticles: towards standardization of protocol

Summary.  Background:  Microparticles (MP) are small vesicles of 0.1–1 μm, released in response to activation or apoptosis. Over the past decade, they received an increasing interest both as biomarkers and biovectors in coagulation, inflammation and cancer. Clinical studies were conducted to assess their contribution to the identification of patients at cardiovascular risk. However, among the limitation of such studies, pre‐analytical steps remains an important source of variability and artifacts in MP analysis.

Circulating endothelial cells in vascular disorders: new insights into an old concept

Abstract: The endothelial contribution to vascular disorders has been widely documented in experimental models. However, its implication in human pathology is difficult to investigate, owing to the paucity of non‐invasive methods and of specific endothelial markers. The enumeration of circulating endothelial cells (CEC) released in peripheral blood after vascular injury represents a direct exploration of the endothelium. For this purpose, we have produced a monoclonal antibody (S‐Endo 1), which recognizes CD 146, a molecule expressed on all types of human endothelial cells but absent from haemopoietic cells. Using this antibody, we have designed a specific and sensitive immunocapture test, which allowed us to detect high numbers of CEC in thrombotic, infectious or immunological disorders, while CEC were found to be very rare (<3/ml) in normal subjects. This quantitative approach using CEC might prove useful as a marker of vascular wall injury. Their enumeration is of interest in the clinical follow‐up of vascular disorders, in the evaluation of therapeutic effectiveness or in the direct diagnosis of infectious diseases involving intra‐endothelial microbial agents. Furthermore, an immunological and/or functional study of CEC could allow one to assess their procoagulant and proadhesive properties, as well as their viability, opening new perspectives for CEC investigation in vascular pathology.

Pattern formation in drying drops of blood

The drying of a drop of human blood exhibits coupled physical mechanisms, such as Marangoni flow, evaporation and wettability. The final stage of a whole blood drop evaporation reveals regular patterns with a good reproducibility for a healthy person. Other experiments on anaemic and hyperlipidaemic people were performed, and different patterns were revealed. The flow motion inside the blood drop is observed and analysed with the use of a digital camera: the influence of the red blood cells motion is revealed at the drop periphery as well as its consequences on the final stage of drying. The mechanisms which lead to the final pattern of the dried blood drops are presented and explained on the basis of fluid mechanics in conjunction with the principles of haematology. The blood drop evaporation process is evidenced to be driven only by Marangoni flow. The same axisymmetric pattern formation is observed, and can be forecast for different blood drop diameters. The evaporation mass flux can be predicted with a good agreement, assuming only the knowledge of the colloids mass concentration.

Circulating endothelial cells, microparticles and progenitors: key players towards the definition of vascular competence

•  Introduction •  Dynamics between endothelial injury and repair: the response to injury theory ‘revisited’ •  Emerging biomarkers reflecting the dynamics between endothelial injury and repair: from pathophysiology to clinical testing. ‐  Circulating endothelial cells ‐  Endothelial microparticles ‐  Endothelial progenitor cells •  Endothelial lesion versus regeneration: towards the definition of ‘vascular competence’ •  Conclusion

Endothelial-derived microparticles: Biological conveyors at the crossroad of inflammation, thrombosis and angiogenesis

Endothelial microparticles (EMP) are complex vesicular structures that can be shed by activated or apoptotic endothelial cells. EMP are composed of a phospholipid bilayer that exposes transmembrane proteins and receptors and encloses cytosolic components such as enzymes, transcription factors and mRNA derived from their parent cells. Thus, EMP behave as biological conveyors playing a key role in the tuning of vascular homeostasis. This review focuses on the multifaceted roles of EMP, notably in coagulation, inflammation and angiogenesis and also on the mechanisms that trigger their formation. In this context, EMP could compromise vascular homeostasis and then represent key players in the pathogenesis of several inflammatory and thrombotic diseases. Consequently, elucidating their role and their mechanisms of formation will bring new insights into the understanding of endothelial-associated diseases. Moreover, in the future, it can open novel therapeutic perspectives based on the inhibition of EMP release.