F. Dignat-George

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

Summary.u2002 Background:u2002Despite 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:u2002To 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:u2002A prospective monocentric study was performed on 144 patients undergoing PCI. PR was evaluated by VASP phosphorylation analysis 24u2003h 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:u2002Of 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, Pu2003<u20030.01). 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:u2002VASP 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.u2002 Background:u2002Chronic 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:u2002To 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:u2002Circulating 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:u2002CD144+ 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.u2002 Background:u2002Platelet 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:u2002The 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:u2002Using a blend of size‐calibrated fluorescent beads (0.5 and 0.9u2003μm) 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:u2002This calibrated‐bead strategy allowed full long‐term control of the FCM‐based microparticle protocol and reproducible PMP counts over time [coefficient of variation (CV)u2003<u200310%]. Optimal settings were easily transferred from one instrument to another, using Megamix as a stable template. Similar PMP counts (CVu2003<u200312%) were obtained using the three instruments. With such a standardized FCM protocol, PMP values were established in healthy subjects (nu2003=u200360) with significantly higher levels in women than in men [median (1st quartile to 3rd quartile): 1775u2003μL−1 (1014–3039u2003μL−1) vs. 656u2003μL−1 (407–962u2003μL−1)]. Conclusions:u2002The present strategy provides a new option for PMP count standardization and thus opens the way for multicenter studies.

Isolation and enumeration of circulating endothelial cells by immunomagnetic isolation: proposal of a definition and a consensus protocol

Summary.u2002 Background:u2002Circulating endothelial cells (CECs) have been identified as markers of vascular damage in a variety of disorders, such as myocardial infarction, vasculitis, and transplantation. CD146‐driven immunomagnetic isolation has gained widespread use, but the technique is hampered by the lack of a definition of CECs and the absence of a consensus for their enumeration. Aim:u2002To evaluate several variables influencing immunomagnetic isolation of CECs, formulate a definition for CECs and propose a consensus protocol for their enumeration. Methods:u2002We devised a protocol based on CD146‐driven immunomagnetic isolation and a subsequent confirmatory step with Ulex‐Europaeus‐Lectin‐1 staining. In a multi‐center effort, we evaluated the preanalytical and analytical phases of this protocol. We evaluated the effects of storage, anticoagulation and density centrifugation, and compiled previous experience with this technique. Results:u2002Our protocol permitted unequivocal identification of CECs with acceptable reproducibility. There was an effect of storage time in that median cell numbers declined to only 87.5% of their baseline values during 24u2003h of storage at 4u2003°C. Recovery was lower with citrate than with ethylene‐diamine tetra‐acetic acid after 4u2003h of storage; density centrifugation was also associated with lower recovery. We provide a comprehensive list of technical recommendations and potential pitfalls. Finally, based on our experience with this protocol and a recent consensus workshop, we formulated a working definition for CECs. Conclusion:u2002Our work represents an important step toward consensus regarding the CECs. Our recommendations represent the experience of three major centers and should now be scrutinized by others in the field.

Endothelial injury induced by coronary angioplasty triggers mobilization of endothelial progenitor cells in patients with stable coronary artery disease1

Despite the wider use of drug-eluting stents, the rate of complications following angioplasty, mainly stent thrombosis and in-stent restenosis, is still limiting [1]. These complications are the consequences of endothelial injury and may depend on the efficiency of the healing process for the re-establishment of the endothelial monolayer. This balance between damage and repair is therefore of critical importance in determining angioplasty outcomes [2]. Circulating endothelial cells (CEC) can be used to monitor both functional and physical endothelial integrity [3]. They are non-hematopoietic cells, originated from sloughing of the injured vessel wall. These cells are defined by the expression of endothelial markers, the absence of leukocyte and immaturitymarkers, and the lack of proliferative potential in vitro [3]. CEC are found in very low concentration in healthy subjects [4] but increased numbers are described in various cardiovascular clinical settings with significant diagnosis and prognostic value [5–9]. Repair of an injured endothelium has been recently shown to involve circulating endothelial progenitor cells (EPC). These cells are defined as bonemarrowderived circulating cells with an immature endothelial phenotype, a high clonogenic potential in vitro and a specific capacity to contribute to angiogenesis in vivo [10,11]. The baseline number of circulating EPC depends on numerous factors among which are coronary artery disease severity, cardiovascular risk factors, and aging [12,13]. The main triggers of EPC mobilization from bone marrow to peripheral blood are ischemia and vascular trauma [14–16]. Although experimental models of vascular injury have suggested that exogenous EPC could play a critical role in the restoration of the endothelial lining [17–19], whether endothelial injury induced by coronary angioplasty is a sufficient stimulus to trigger EPC mobilization in coronary artery disease (CAD) patients is still unknown. We investigated the effect of coronary angioplasty on EPC levels in patients with CADand determine the relationship with the endothelial damage assessed by CEC counts. Patients with stable angina with need for scheduled angioplasty on a native coronary were enrolled after an informed consent was obtained. Exclusion criteria were acute coronary syndromes (ACS) within the two previous weeks, elevated preprocedural troponin Ic (cTnI), concurrent illness that could affect progenitor cell count. Coronary angioplasty and sirolimuscoated stent implantation were performed according to institutional standards, using a balloon inflation time of 30 s at 20 atmospheres. Blood samples were obtained before angioplasty (pre-precutaneous coronary intervention [PCI]), immediately after the end of the procedure (post-PCI), then 6, 24, 48 h and 7 days after PCI. CEC were counted using CD146-based immunomagnetic separation as previously described [4]. Circulating hematopoietic progenitor cells (HPC) were analyzed using flow cytometry (Epics XL Cytometer; Beckman Coulter, Villepinte, France) after staining of whole blood with fluorescein isothiocyanate (FITC)-labeled monoclonal mouse antihuman CD45 antibody, phycoerythrin (PE)-labeled monoclonal mouse antihuman CD34 antibody and 7AAD (Stemkit ; Beckman Coulter). Absolute numbers of HPC lL were determined using calibration beads according to manufacturer’s instructions. The number of circulating EPC was determined using a colony-forming assay according to method ofHill et al. [12]. Results were expressed in number of CFU-EC per 10 cells. Correspondence: Françoise Dignat-George, INSERM UMR 608, UFR de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille cedex 5, France. Tel.: + 33 4 91 83 56 00; fax: + 33 4 91 83 56 02; e-mail: dignat@ pharmacie.univ-mrs.fr

CD146-based immunomagnetic enrichment followed by multiparameter flow cytometry: a new approach to counting circulating endothelial cells

Summary.u2002 Background: Circulating endothelial cells (CECs) have emerged as non‐invasive biomarkers of vascular dysfunction. The most widely used method for their detection is CD146‐based immunomagnetic separation (IMS). Although this approach has provided consensus values in both normal and pathologic situations, it remains tedious and requires a trained operator. Objectives: Our objective was to evaluate a new hybrid assay for CEC measurement using a combination of pre‐enrichment of CD146+ circulating cells and multiparametric flow cytometry measurement (FCM). Patients and methods: CECs were determined in peripheral blood from 20 healthy volunteers, 12 patients undergoing coronary angioplasty, and 30 renal transplant recipients, and blood spiked with cultured endothelial cells. CD146+ cells were isolated using CD146‐coated magnetic nanoparticles and labeled using CD45–fluorescein isothiocyanate and CD146–PE or isotype control antibody and propidium iodide before FCM. The same samples were also processed using CD146‐based immunomagnetic separation as the reference method. Results: The hybrid assay detected CECs, identified as CD45dim/CD146bright/propidium iodide+, with high size‐related scatter characteristics, and clearly discriminated these from CD45bright/CD146dim activated T lymphocytes. The method demonstrated both high recovery efficiency and good reproducibility. Both IMS and the hybrid assay similarly identified increased CEC levels in patients undergoing coronary angioplasty and renal transplantation, when compared to healthy controls. In patients, CEC values from these two methods were of the same order of magnitude and highly correlated. Bland–Altman analysis revealed poor statistical agreement between methods, flerrofluid–FCM providing higher values than IMS. Conclusion: This new hybrid FCM assay constitutes an accurate alternative to visual counting of CECs following CD146‐based IMS.

Kidney transplantation decreases the level and procoagulant activity of circulating microparticles.

Microparticles (MP) are important players in cardiovascular disorders. Renal transplantation significantly improves the survival of hemodialyzed patients, in part because cardiovascular disease (CVD) progression is lessened. We hypothesized that the beneficial effect of renal transplantation on cardiovascular outcome might involve decreased levels of circulating MP. We evaluated the kinetics of MP subpopulations and their procoagulant activity (MP‐PCA) in 52 patients before and 3, 6, 9 and 12 months after graft with reference to 50 healthy controls and we evaluated the impact of cardiovascular complications. During the follow‐up, the increased levels of MP observed before graft were significantly decreased and reached normal values with different kinetics according to their cellular origin whereas MP‐PCA remained significantly higher than in controls. From multivariate analysis, the levels of MP were negatively correlated with renal function. At 12 months, the decrease in MP and MP‐PCA was more pronounced in patients without history of CVD than those with. In conclusion, we demonstrated that renal graft is associated with decreased levels of MP levels and MP‐PCA, even more pronounced so in patients without history of CVD. Therefore, we suggest that MP lowering could be involved in the vascular dysfunction improvements reported after transplantation.

Tissue factor up-regulation in proinflammatory conditions confers thrombin generation capacity to endothelial colony-forming cells without influencing non-coagulant properties in vitro

Summary.u2002 Background:u2002Endothelial progenitor cells (EPC) are good candidates for cell‐based therapy in cardiovascular diseases. However, concerns have been raised about the potential risks of EPC‐based cell therapy, in terms of thrombogenicity particularly in inflammatory conditions, currently observed in such patients. Tissue factor (TF) can trigger coagulation and may support thrombogenicity. TF is also a key receptor in angiogenesis. Objective:u2002The present study was designed to (i) evaluate the capacity of resting and tumour necrosis factor‐alpha (TNF)‐α‐stimulated late‐outgrowth endothelial colony‐forming cells (ECFCs) to express TF and (ii) investigate the effect of TF/FVII(a) interaction on procoagulant and non‐procoagulant activities of ECFCs in vitro. Methods: ECFCs from cord blood (cb) and adult peripheral blood (ab) were analyzed for TF expression and activity using reverse transcription‐polymerase chain reaction (RT‐PCR), flow cytometry, Western blot and a thrombin generation assay. Non‐procoagulant properties of TF‐expressing ECFCs were investigated in vitro using wound‐healing, cell proliferation, tube formation and spheroid‐based assays. Results:u2002ECFCs expressed TF in response to TNF‐α. The up‐regulation of TF conferred to ECFCs a FVII(a)‐dependent thrombin generation activity. Compared with cb‐ECFC, ab‐ECFCs can display a higher level of constitutive TF expression and activity, with a notable heterogeneity among donors. TF/FVIIa interaction did not modify non‐procoagulant properties of TNF‐α stimulated cb‐ECFCs in vitro. Conclusions:u2002Proinflammatory conditions up‐regulate TF expression in ECFCs. This expression confers to ECFCs a strong thrombin generation capacity without influencing their non‐coagulant properties. Our results suggest that EPC‐based cell therapy may be associated with prothrombotic risk which could be limited by inhibiting TF without affecting the proangiogenic capacity of the cells.