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2016 Annual Minutes Locked Topic 0 R. Nieuwland SSC MINUTES VASCULAR BIOLOGY 2016 MONTPELLIER   Extracellular vesicles. Chairs: Chris Gardiner (UK) and Pia Siljander (Finland)   Role of EV-bound polyphosphate in prostate cancer driven thrombosis (Katrin Nickel; Hamburg, Germany). Katrin Nickel described a novel mechanism for cancer-related thrombosis. Prostate cancer cells release prostasomes, heterogeneous extracellular vesicles (30-400 nm) which expose not only tissue factor but also long chain polyphosphates (polyP). PolyP co-localises with factor (F)XIIa and initiates FXII-dependent coagulation in vitro. Mice injected with prostasomes develop lethal pulmonary embolism (PE) within 30 minutes. In contrast, mice deficient in FXI, FXII, or high-molecular-weight kininogen, are protected from lethal prostasome-induced PE. Pre-treatment of wild type mice with the anti-FXIIa 3F7 prior to injection of prostasomes prevents PE but does not increase the bleeding time. Pre-treatment of prostasomes with polyP inhibitors also prevents PE. The plasma from patients with prostate cancer shows increased thrombin generation, which is abolished by the removal of prostasomes. The isolated prostasomes trigger thrombin generation in normal plasma. Katrin concluded that targeting factor XIIa and/or polyphosphate may provide a novel opportunity for safe anticoagulation therapy in patients with cancer.   TF-EV in clinical trials and the need for TF-EV standardisation (Johannes Thaler; Vienna, Austria) Johannes Thaler stated the need for clarification regarding the performance of tissue factor (TF)-extracellular vesicles (EV) assays. He referred to the work of Butenas et al., who showed that in plasma of normal healthy individuals the concentration of functional TF does not exceed 20fM. In clinical trials investigating the role of TF-EV in predicting thrombotic risk in cancer patients, two methods are commonly used: flow cytometry (FCM) and activity assays. The specificity of antibodies used to detect TF by FCM may be assessed by testing unstimulated and lipopolysaccharide-stimulated monocytes. Anti-TF antibodies that bind to unstimulated monocytes or antibodies failing to detect a substantial increase in TF-EV following lipopolysaccharide stimulation are not suitable. Further standardisation of FCM measurement of EVs are necessary, and this work is now being performed the Vascular Biology SSC in collaboration with ISEV, ISAC and METVES (www.evflowcytometry.org). There is better agreement between functional TF-EV assays, but most methods cannot reliably detect TF levels of < 0.5 pg/mL. Consequently, TF-EV is only detectable in specific patient groups e.g. disseminated intravascular coagulation and pancreatic cancer. More studies will be essential to investigate the performance of the various TF-EV assays.   Desirable properties of a TF-EV reference preparation (Chris Gardiner; London, UK) There are many different methods for measuring tissue factor (TF) antigen and activity by extracellular vesicles (EV), and it is clear that these methods are not all measuring the same thing. Chris Gardiner presented data from the laboratory of Nigel Mackman, who demonstrated that detection of TF antigen by either flow cytometry or ELISA do not correlate with TF-coagulant activity. It was concluded from these results that antigenic assays lack sensitivity and specificity. The importance of determining the specificity of assays by using reliable positive controls, e.g. lipopolysaccharide-stimulated monocytes, and negative controls, e.g. an inhibitory antibody against TF or FVIIai were discussed. There are several methodological approaches for measuring TF-EV activity and each  approach has its own advantages and limitations. At present, the only available material to standardize these assays is commercial thromboplastin. Unfortunately, the amount of TF varies between batches, and, as the TF is in soluble form, it cannot be used in most capture assays and cannot be pelleted by centrifugation. Consequently, thromboplastin can only be used to calibrate/standardise the final measurement. Chris proposed to use TF-EV from the human primary pancreatic adenocarcinoma cell line BxPC-3, which constitutively releases EV exposing coagulant TF. Application of reconstituted EV from BxPC-3 in citrate-anticoagulated plasma would allow a “like with like” approach to facilitate standardization of TF-EV activity assays.   Consideration and challenges in generating reference materials –tissue factor extracellular vesicles (Elaine Gray; London, UK) Elaine Gray asked “Why do we need reference materials?” She explained the role of bioassays and the concept of biological standardisation. The use of a common reference standard improves agreement between laboratories. A previous working party on tissue factor (TF) standardization in cancer was initiated 2005 under the Haemostasis and Malignancy Subcommittee of the ISTH. Two TF preparations and several cell lysates were compared and although all showed linearity, assay parallelism was not observed between either TF preparation and the cell lysates. However, the cell lysates showed linearity and parallelism. Ultimately, the SSC did not proceed with the TF standard. The challenges of manufacturing reference standards were described Elaine outlined the questions that the subcommittee would need to address. Pilot studies will be required to determine the purpose of the reference standard and whether the standard will support a single method or multiple methods. The SSC must decide on the type of reference preparation (i.e. SSC or WHO), the methods for which it will be used and who will coordinate large collaborative studies to qualify the suitability. Funding will be required to produce and run a collaborative study and ultimately the production of the reference standard.   Endothelial progenitor cells. Chairs: Anna Randi (UK), Florence Sabatier (France)   The BOEC as a model for organ-specific endothelial assessment – apples versus oranges (M Toshner)   Standardization of measurements of extracellular vesicles. Chair: Rienk Nieuwland (Netherlands) Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicentre collaborative workshop and future directions (Romaric Lacroix, Marseille, France) Romaric presented the final results of a collaborative workshop on platelet microparticle (PMP) standardization by flow cytometry. This workshop evaluated a forward or scatter bead-based strategy adapted to different flow cytometry platform. The study included 44 laboratories and 52 cytometers of 14 different types. Using resolution capability and background noise level as criteria to qualify the instruments, the standardization strategy proved to be compatible with 85% (44/52) instruments. The inter-laboratory variability of PMP counts was 37% and 28% for each sample. No difference was found between instruments using forward or side scattered light as the relative sizing parameter. Romaric concluded that despite remaining limitations, this study demonstrates a potential of bead-based strategies to standardize of MP enumeration. However, additional standardization efforts such as standardization of fluorescence signals, development of sub-micron reference materials and MP-dedicated reagents, are still mandatory to evaluate the clinical relevance of (P)MP at a multicentre level.   Size and refractive index determination by flow cytometry discriminate extracellular vesicles from lipoprotein particles (Leonie de Rond; Amsterdam, Netherlands) Flow cytometry is the most widely used method to study single extracellular vesicles (EV). However, flow cytometry data have arbitrary units, which hampers data interpretation and comparison. Here we present Flow-SR, a label-free method to obtain the diameter (d) and refractive index (RI) from the light scattering signals of a flow cytometer. Mie theory and reference beads were used to obtain the relation between the measured forward and side scattering of a flow cytometer (A50-Micro; Apogee, UK) and d and RI of a particle. We validated this approach by determining d and RI of a mixture with 5 populations of silica and polystyrene beads <500 nm. For each population in the beads mixture, d and RI were determined with measurement errors on the mean <8% and <2%, respectively. We, subsequently, applied  Flow-SR on platelet-depleted supernatant of outdated platelet concentrates containing EV and lipoproteins. Two populations with different RI were clearly discernable. Using fluorescent labeling, we confirmed that the population with RI ≤1.42 contains EV and the population with RI>1.42 contains lipoproteins. Flow-SR is the first method that converts the scattering signals of a commercial flow cytometer to the comparable measurement units of dimension and RI. We have demonstrated that this method is accurate and that it allows label-free discrimination between EV and lipoprotein particles in human plasma. Quantitative light scattering to standardize flow cytometry measurements of extracellular vesicles (Edwin van der Pol; Amsterdam Netherlands) The comparison of flow cytometry results between laboratories remains a challenge. At present, most laboratories select extracellular vesicles (EV) by setting an inclusion gate based on the scatter signal of two polystyrene bead sizes. However, due to the refractive index (RI) difference between EV (RI≈1.4) and polystyrene (RI≈1.6) and the variety of optical configurations among flow cytometers, the current standard leads to poor reproducibility. A well-defined polystyrene beads mixture (www.metves.eu) and an EV reference sample (CD235a-FITC labeled erythrocyte EV) were measured on 46 instruments in 33 laboratories worldwide. Stand-alone software (www.exometry.com) provided the relation between particle diameter, RI, and scatter for the specific optical configuration of each instrument. This relation was used to set EV size gates of 300-600 nm, 600-1200 nm, and 1200-3000 nm. Each site applied the size gates to their own analyses software and independently reported the EV concentration within each size gate. The polystyrene beads mixture contains a population of 400 nm FITC-labeled polystyrene beads, which could not be detected by 34% of the instruments. For comparison, a 400 nm polystyrene bead scatters light more efficiently than a 1 µm EV. Thus, roughly one third of the flow cytometers used by laboratories with expertise in EV detection detect EV or particles >1 µm. Among the instruments capable of detecting 1200-3000 nm EV, the established standard resulted in a CV of 74%, whereas our approach resulted in a CV of 59%. Among the other size gates, no reproducibility was obtained. Final data and results will be submitted to JTH in 2016. Taken together, well-defined beads and Mie theory were used to standardize the size range detected by flow cytometry. Roughly one third of the instruments do not detect EV. Within the 1200-3000 nm size range, an EV size gate by Mie theory (CV=59%) leads to a better reproducibility than a gate on beads (CV=74%). We expect that knowledge on the RI of EV may further improve our approach. Hitherto, standardization of EV <1200 nm is ineffective and requires more flow cytometers with nanoparticle detection sensitivity.   Receptor shedding from platelets in pathological environments. Chair: Elizabeth Gardiner (Australia) Soluble GPVI as a potential biomarker of platelet activation in patients with thermal injury and sepsis (Paul Harrison; Birmingham, UK) Paul measured soluble glycoprotein VI (sGPVI), a platelet activation biomarker, in plasma samples which were collected as part of the Scientific Investigation of the Biological Pathways Following Thermal Injury Study (SIFTI) study. Whilst platelets are traditionally recognized for their central role in haemostasis, numerous studies now highlight how platelets can be potent protagonists in an inflammatory response. Platelets directly recognize, sequester and kill pathogens to activate and recruit leukocytes to sites of infection and inflammation, and to modulate leukocyte behaviour, enhancing their ability to phagocytose and kill pathogens. Work from Paul’s laboratory showed that whilst sGPVI is not increased in the first few days post thermal injury, there are significantly higher sGPVI levels at later time points in patients that developed sepsis compared to non-septic patients. Systemic platelet activation during septic episodes may contribute to the pathogenesis of multi-organ failure. Future work will assess the advantage of stratifying patient groups based on plasma sGPVI levels in samples collected within 4 days post thermal injury to help predict the onset of sepsis and multi-organ failure and aid clinical decisions on possible early therapeutic intervention.   Platelet receptors and coagulopathy (Robert Andrews; Melbourne, Australia) GPVI, which binds collagen, and GPIbα (the ligand-binding subunit of GPIb-IX-V) that binds von Willebrand factor(VWF), form a unique adhesion-signaling complex on the platelet surface that initiates platelet adhesion, spreading, activation and secretion, and integrin αIIbβ3-mediated platelet aggregation (thrombus formation) at high shear rates in flowing blood. Shedding of GPVI generates a ~55-kDa soluble GPVI ectodomain fragment (sGPVI) and a ~10-kDa platelet-associated remnant fragment, which provide a sensitive read-out of receptor engagement, since all GPVI is intact on the surface of resting platelets, unless shedding is induced. Depletion or blockade of platelet GPVI in experimental models results in profoundly decreased arterial thrombosis or stroke, impaired procoagulant activity and thrombin generation ex vivo or in vivo, and protection from tissue factor-induced thromboembolism. Robert showed that FXa induces sGPVI release since shedding could occur in recalcified whole blood or anticoagulated blood treated with Russell Viper Venom (a strong specific activator of FX). Shedding was strongly blocked by the FXa-inhibitors rivaroxaban and enoxaparin, but unaffected by the thrombin inhibitors dabigatran, lepirudin and hirudin. Whether FXa or FXa inhibitors affect ADAM10-mediated processing of other substrates on human platelets and other vascular cells is unknown. Defining the mechanism of FXa-mediated activation of ADAM10/sheddase activity and GPVI shedding may identify new therapeutic targets regulating thrombus growth and stability, determine the relationship between coagulation, platelet activation and ADAM10-mediated substrate processing, and also enable potential clinical consequences of anticoagulation to be recognized.  
by R. Nieuwland
Tuesday, July 12, 2016
2015 Annual Minutes Locked Topic 0 L. Schmeidler Chairman:  Rienk Nieuwland Co-Chairs:  Alexander Brill, Elizabeth E. Gardiner, Chris Gardiner, Anna Randi, Florence Sabatier, Pia R. Siljander Circulating endothelial progenitor cells; chairs Anna Randi (UK) and Florence Sabatier (France) From VSELs to endothelial progenitor cells: What is the “perfect” vasculogenic cell? David Smadja (France) In the setting of cardiovascular disease, the vascular compartment undergoes complex molecular and cellular changes that determine the range of perfusion recovery within the ischemic tissue. Thus, stimulation of vessel growth and/or remodeling has emerged as a new therapeutic option in patients with ischemic diseases. In particular, strategies based on the administration of endothelial progenitor cells (EPCs), or cell population thought to contain these vascular progenitor cells, have been shown to augment neovascularization in experimental models of ischemia and in patients with cardiovascular ischemic diseases. Several methods to obtain a better way to isolate and expand them have been developed. However, cardiovascular risk factors constitute an inhibitory environment and induce an impairment of their vasculogenic cell functions. Moreover, precise origin and identity of EPCs are controversial. Very small embryonic-like stem cells (VSELs) are multi-potent stem cells localized in adult bone marrow (BM) that may be mobilized into peripheral blood (PB) in response to tissue injury. We aimed to quantify VSELs in BM and PB of patients with critical limb ischemia (CLI) and to test their angiogenic potential in vitro as well as their therapeutic capacity in mouse model of CLI. We quantified VSELs in BM and PB from CLI patients compared to healthy controls. Our results suggest that ischemia may trigger VSELs mobilization in this patient population. Sorted BM-VSELs cultured in angiogenic media acquired a mesenchymal phenotype (CD90+, Thy-1 gene positive expression). VSEL-derived cells had a pattern of secretion similar to that of endothelial progenitor cells, as they released low levels of VEGF-A and inflammatory cytokines. Noteworthy, VSELs triggered post-ischemic revascularization in immune-deficient mice (p< 0.05 vs PBS treatment), and acquired an endothelial phenotype either in vitro when cultured in the presence of VEGF-B (Cdh-5 gene positive expression), or in vivo in Matrigel implants (human CD31+ staining in neo-vessels from plug sections). In conclusion, VSELs are a potential new source of therapeutic cells that may give rise to cells of the endothelial lineage in humans. Given the potential usefulness of a cell therapy product, real ontogeny of vascular cells, their thorough characterization as well as a deep understanding of the molecular and cellular mechanisms involved in their pro-angiogenic capacities is of major importance. Blood outgrowth endothelial cells offer novel insights into von Willebrand disease. Richard Starke (UK) Blood out growth endothelial cells (BOEC) are endothelial cells that can be derived from circulating progenitors in blood. BOEC provide access to the normally inaccessible endothelium of patients. We have extensive experience in the isolation, expansion and culture of BOEC from a variety of patients with vascular disorders, including Von Willebrand disease (VWD). VWD describes a deficiency or dysfunction of VWF in blood. Although there is some heterogeneity, BOEC from type 1 VWD patients confirmed the long-held belief that type 1 VWD is due to quantitative defects in the ability of the endothelium to produce VWF. VWF synthesis and release from type 2 VWD patients was mostly normal supporting the belief that this subtype of VWD is mainly due to dysfunctional VWF. Type 3 VWD patients are defined by the absence of plasma VWF and we demonstrated a similar absence of VWF in a type 3 patient’s BOEC. Although a good consistency is seen between repeat isolations from the same donor, some patients with the same genotype show variations in endothelial VWF production consistent with their different plasma VWF levels, suggesting that VWF production by BOEC from these patients is influenced by factors in addition to the mutation.  In conclusion, BOEC can provide novel insight into the cellular biology of VWD and could be useful in the testing of novel treatment approaches on the patients’ endothelium prior to their use in the patient.  Derivation of endothelial colony forming cells from pluripotent stem cells. Nattan Prasain (US) Since vascular endothelial growth factor ligand (VEGF)/receptor 2 (KDR) signaling pathway is critical in the emergence of endothelial cells during development, we have developed a novel pluripotent stem (PS) cell differentiation protocol and found neuropilin-1 (NRP-1), a VEGF co-receptor, as an early marker for identifying the emergence of ECFCs. Compared to other sub-sets of sorted cells, only NRP-1+CD31+ cells exhibited umbilical cord blood (CB) ECFC-like properties and produced a clinically relevant number of cells. We identified NRP-1+CD31+ selected cells that displayed a stable endothelial phenotype exhibiting high clonal proliferative potential, formation of human vessels that inosculated with host vasculature in vivo, and made significant contributions to vascular repair of the ischaemic limb, but lacked teratoma formation potential in vivo. This protocol advances the field by generating highly replicative but stable endothelial cells for use as a potential cell therapy for human clinical disorders. Receptor shedding from platelets in the inflamed vasculature; chairs Elizabeth Gardiner (Australia) and Alexander Brill (UK) Platelets and the inflamed vasculature. Paul Kubes (Canada) Paul gave an elegant presentation showcasing State-of-Art imaging approaches to study the role of platelets in inflammation responses. Whilst platelets are traditionally recognized for their central role in haemostasis, numerous studies now highlight how platelets can be potent immune modulators and effectors. Platelets directly recognize, sequester and kill pathogens to activate and recruit leukocytes to sites of infection and inflammation, and to modulate leukocyte behaviour, enhancing their ability to phagocytose and kill pathogens. Paul highlighted a ‘touch and go’ surveillance property of platelets in liver and then demonstrated first responder roles for platelet chemokines and chemotactic gradients in mice exposed to Staphylococcus aureus where the majority of bacteria were sequestered immediately by hepatic Kupffer cells, resulting in a robust neutrophil infiltration into the liver. A marginal zone proximal to these Kupffer cells and devoid of neutrophils was described as a “necrotactic” zone. This zone displayed severe blood vessel constriction and platelet aggregation. Future work will more fully describe the molecular properties of this zone. Mechanistic insights into platelet receptor shedding. Yotis Senis (UK) Yotis provided an update on signaling properties of platelets from mice deficient in G6b-B. Mice lacking this ITIM-containing receptor exhibit macrothrombocytopenia and aberrant platelet function. The loss of response to collagen was explained by an enhanced ADAMs mediated shedding of the platelet collagen receptor GPVI. Yotis was able to demonstrate enhanced phosphorylation of protein-tyrosine kinase Syk (a key component of platelet activation signaling pathways) in these mice and could rescue significant platelet function by crossing G6b-B deficient mice with mice bearing an arginine to alanine point mutation within Syk which modulates Syk phosphorylation events, possibly reducing the pathway that activates ADAMs-mediated removal of GPVI. Yotis also outlined some opportunities to unify and standardize protocols that measure platelet biomarkers of activation, in particular focusing on the unique platelet/megakaryocyte receptor GPVI.  Glycoprotein Iba shedding and platelet clearance. Renhao Li (US) Renhao described some of the unique metalloproteinase blocking properties of an anti-GPIb antibody that recognizes the ADAM17 cleavage sequence within GPIbα. Clone 5G6, and its monomeric Fab fragment specifically bound purified GPIb-IX complex, human platelets, and transgenic murine platelets expressing human GPIbα, but did not affect platelet activation and aggregation. 5G6 blocked GPIbα shedding induced by calmodulin inhibition  or mitochondrial ageing reagent CCCP with similar potency to broad shedding inhibitor GM6001 in human platelets. 5G6 does not recognize mouse GPIbα or inhibit shedding of other platelet receptors. 5G6 was shown to have utility in increasing the longevity of platelets stored for transfusion in bags, by reducing GPIbα metalloproteolysis observed as part of the platelet storage lesion. This new focus of research is significant as it could potentially extend the life of platelet transfusion bags beyond 4-5 days, and enhance platelet function in people in receipt of platelet transfusion. Microparticles and standardization; chairs Pia Siljander (Finland) and Chris Gardiner (UK) Vesicle size approximation enables inter-laboratory comparison of vesicle measurements by flow cytometry. Rienk Nieuwland (Netherlands) Measuring single extracellular vesicles  by flow cytometry is a challenge. Vesicles scatter light, which is detected by flow cytometry and expressed in arbitrary units. These light scattering signals / arbitrary units are incomparable between instruments, and interpretation is hampered by differences in sensitivity, the unknown refractive index of vesicles, etc. To standardize these measurements, we (1) selected traceable reference materials (www.metves.eu), (2) developed a method to determine the refractive index of single particles < 500 nm diameter in suspension (Nano Letters 2014, E. van der Pol et al.), (3) developed reference materials and software to correct for differences in optical configurations of commercially available flow cytometers (www.exometry.com), (4) distributed reference materials, software and biological samples to 33 laboratories worldwide, (5) determined the vesicle size gate by measuring reference materials, and (6) measured and compared vesicle concentrations. Overall, 34% of included flow cytometers was too insensitive to measure any vesicles in the largest size gate (1,200-3,000 nm). In addition, marked differences in sensitivity are observed between similar instruments (brand, type) in different laboratories. Finally, a comparison of vesicle measurements in the AMC on 4 different instruments results in a CV of 20% for the largest size gate. Taken together, beads and software have been developed to set vesicle size gates in absolute units (nm) on most types of flow cytometers, the CV is improved compared to previous strategies, and 33% of included flow cytometers are too insensitive to detect vesicles. Importantly, further standardization of vesicle measurements will be done also in collaboration with ISEV (www.isev.org) and ISAC (www.isac-net.org). See also www.evflowcytometry.org for additional information. The goal of this collaboration is to develop standards and reference materials, protocols, rules for minimal requirements, education, etc. At present, a follow-up of METVES (www.metves.eu), entitled METVESII, is being written and Letters of Support are needed. Additional information is available at http://msu.euramet.org/health_2015/SRTs/SRT-h03.pdf and please contact Rienk Nieuwland (r.nieuwland@amc.uva.nl) for questions.l   Studies of the microparticle cargo and its functional components. Eric Boilard (Canada) Platelets are anucleated blood element highly potent at generating extracellular vesicles (EV) called microparticles (MPs). Whereas EVs appear as an important means of intercellular communication, the complexity of the platelet MP cargo is starting to be delineated. Studies show that platelet MPs contain functional enzymes and a broad repertoire of nucleic acid (e.g. microRNA). A subset of platelet MPs also bears organelles, such as respiratory-competent mitochondria. Including specific mitochondrial markers for MP assessment thus permits to reveal the diversity of platelet MPs. Furthermore, mitochondria are thought to originate from the endosymbiosis of the Alphaproteobacterium Rickettsia prowazekii during the development of eukaryotic cells. As mitochondria are released concomitantly with MPs from activated platelets, they can trigger highly potent pro-inflammatory signals. The platelet MP cargo is thus complex, and its contribution to physio(patho)logical conditions must be considered. Progress of endothelial microparticle detection and characterization by flow cytometry. Romaric Lacroix (France) Endothelial-derived microparticles (EMPs) are a useful marker of endothelium injury in vascular disorders. However, measuring EMP by flow cytometry remains a challenge, which explains the diversity of (CD) markers used in the literature for identification. Therefore the objective of the presented work was to evaluate the capacity of antibodies used in the literature to detect EMPs. Several clones of antibody were selected for each specificity, and their detection capacity was evaluated on EMPs from primary endothelial cells of different territories both in unstimulated and inflammatory conditions. This work showed that among published markers CD31 (PECAM), CD54 (ICAM1) and CD146 have the best capture efficiency by immune-magnetic separation and detection capacity by flow cytometry to detect EMPs. Interestingly, despite the expression on leukocytes, these markers were not detected on leukocyte-derived MPs in the conditions of the study. It was also demonstrated that combining markers is an interesting approach to improve the signal to noise ratio, the repeatability and lowered the detection limit of the EMP detection. Finally, a strategy was proposed to evaluate and select antibodies for the different subpopulation of MPs whose performances cannot be extrapolated from cellular data.
by L. Schmeidler
Sunday, June 21, 2015
2014 Annual Minutes Locked Topic 0 R. Nieuwland Vascular Biology Chairman: Rienk Nieuwland (the Netherlands)Co-Chairmen: Francoise Dignat-George (France), Elizabeth Gardiner (Australia), Florence Sabatier (France), Pia Siljander (Finland), Anna Randi (UK), Florence Toti (France) Thursday, 26 June (8:45-12:45)  Shedded proteins and receptors (Chair: Elizabeth Gardiner, Australia)    Background and latest developments of metalloproteolytic shedding in vascular biology. Bruce Walcheck, University of Minnesota, USA Walcheck gave an outstanding overview of the A Disintegrin and Metalloproteinase (ADAM) family in the context of vascular biology. He focused on shedding/release of chemokines and Fc receptors from human leukocytes, which have important implications in regulation of the immune response. The Fc receptor CD16 is present on essentially all CD56(dim) peripheral blood natural killer (NK) cells. Upon recognition of antibody-coated cells, CD16 delivers a potent signal to NK cells, which in turn eliminate targets through direct killing and cytokine production. He discussed regulation of CD16 surface expression after NK cell activation. Cytokine activation and target cell stimulation led to marked decreases in CD16 expression. Activation of CD56(dim) NK cells by cross-linking CD16 with antibodies resulted in a loss of CD16 and CD62L, which correlated with increased interferon-γ production. His research group demonstrated that  ADAM17 is expressed by NK cells, and its selective inhibition abrogated CD16 and CD62L shedding, and led to enhanced interferon-γ production, especially when triggering was delivered through CD16. Fc-induced production of cytokines by NK cells exposed to rituximab-coated B cell targets was also enhanced by ADAM17 inhibition. This supports an important role for targeting ADAM17 to prevent CD16 shedding and improve the efficacy of therapeutic antibodies. Over-activation of ADAM17 in NK cells may be detrimental to their effector functions by down-regulating surface expression of CD16 and CD62L.    Platelet receptor metalloproteolytic regulation and impact on platelet age and function. Emma Josefsson, Walter and Eliza Hall Institute, Melbourne, Australia Josefsson gave an excellent update on mechanisms relating to loss of glycoprotein Ib (GPIb), the platelet receptor for von Willebrand Factor (VWF), which is cleaved by ADAM17 during activation. Platelet concentrates for transfusion must be stored at temperatures above 15oC as chilling leads to rapid clearance of platelets in humans after transfusion. She described the mechanism that causes GPIb receptors to cluster on blood platelets at cold temperatures. Hepatic macrophage β2-integrin binding to β-N-acetylglucosamine (β-GlcNAc) residues on the GPIb-α chain leads to rapid clearance of acutely chilled platelets after transfusion. Although capping β-GlcNAc moieties by galactosylation prevents clearance of short-term-cooled platelets, this strategy is ineffective after prolonged refrigeration. Prolonged refrigeration increased the density and concentration of exposed galactose residues on platelets such that hepatocytes, through Ashwell-Morell receptor binding, become increasingly involved in platelet removal. ADAM17 mediates shedding of significant amounts of GPIb-α in transfusion bags of platelets. Macrophages rapidly removed a large fraction of transfused platelets independent of their storage conditions. Survival of transfused platelets could be improved by treating bags with inhibitors of ADAM17. By minimizing ADAM-mediated receptor shedding in platelet transfusion bags or in human blood circulation in thrombocytopenic patients with platelet production defects, platelet survival could be enhanced.    Discussion on clinical aspects of receptor shedding. Chris Ward, Kolling Research Institute and Royal North Shore Hospital, Sydney, Australia Ward gave a timely and important update on the utility of platelet shed proteins as biomarkers of human hematological diseases. Importantly he suggested the criteria that a prospective biomarker should conform to, in order to be useful in clinical diagnostic situations. Specifically, a biomarker should be platelet specific readily measurable demonstrate a specific increase in response to a disease situation levels should change upon commencement of successful therapy or change in disease status.   He then outlined some of the challenges faced by clinicians in making a correct diagnosis of hemolytic uremic syndrome versus thrombotic thrombocytopenic purpura and indicated some encouraging recent data regarding the utility of soluble GPVI as a biomarker of TTP that reflects the increased platelet activation and clearance.    Circulating Endothelial Progenitor Cells (Chair: Anna Randi, UK)    Background and latest developments in isolation of circulating endothelial progenitor cells. Reinhold Medina, Centre for Experimental Medicine, Queen’s University Belfast, UK Dr Medina provided a comprehensive and clear review of the field of Endothelial progenitor cells (EPCs), which suffers from confusion in nomenclature and methods.  The first reports that circulating cells, identified as progenitor for the endothelial lineage, had the capacity to repair and regenerate damaged blood vessels, were met with great enthusiasm. However, these early EPC were identified using assays that overlapped with cells of the hematopoietic lineage. Major disputes and controversy in the field damaged the "credibility” of these cells, with some even doubting their existence. Over the past decade, a consensus view has finally been reached. The term EPC in the literature has been used to identify essentially two types of cells: one from myeloid origin and another from a yet unknown origin.  Both have a role in tissue regeneration by promoting the repair of blood vessels and aiding in the re-perfusion of ischemic areas; however only the latter can be considered a "true” endothelial progenitor or EPC. True EPC can be isolated from whole blood, and expanded in culture to generate colonies with robust clonal proliferative potential, endothelial markers and ability to form de novo vessels in vivo. These cells have been called with various names, most commonly endothelial colony forming cells (ECFC) or blood outgrowth endothelial cells (BOEC). EPCs are very rare, probably accounting for only ~0.01% of circulating cells. Their origin remains uncertain; they are currently thought to derive from hematopoietic stem cells in the bone marrow as well as vascular stem cell niches in the vessel wall. Pre-clinical and clinical investigations evaluating the therapeutic potential of cells labelled as EPCs have produced variable results. This may be, at least in part, due to the use of different populations, mainly including cells of the myeloid lineage.  Circulating "EPC”s have also been proposed as useful cell biomarkers of disease. Dr. Medina recommended that the scientific community should define the nomenclature and characteristics of EPC, and standardize markers and methods across laboratories, to achieve a uniform terminology to identify cells and their various sub-types.    Isolation of blood outgrowth endothelial cells. Anna Randi, National Heart and Lung Institute, Vascular Sciences, Imperial College London, UK Randi focused on the population of "true” endothelial progenitor cells, also called blood outgrowth endothelial cells (BOEC). These cells can be isolated from ~50 ml whole blood; they have been also isolated from cord blood and bone marrow. These cells have been used to investigate EC function in patients; to generate iPS cells; for gene & drug delivery; for tissue engineering. A number of protocols have been published which are similar in the basic steps and in the robustness of the procedure, which is successful in 70-75% of isolations. Randi listed key papers which report BOEC isolation methods: Blood 2004;104 :2752–2760; Nature Protocols 2012; 7:1709–1715; Journal of Visualized experiments 2009; 32: 1524. She then listed key methodological points: Time from blood sample collection to processing; Time of colonies appearance; Number of colonies / ml; Colonies expansion. After showing some examples of isolation and characterization, Prof. Randi pointed out some outstanding method development issues, namely the isolation from frozen samples, crucial to be able to expand patients’ studies, and optimization of cell culture conditions for cell therapy applications.  Conclusions from her presentation were: To isolate BOEC for cell biology studies, current methods are adequate Experience in endothelial cell culture required Standardization for consistency and reproducibility Optimization is required for isolation from Frozen samples Modifications to the standard methods should be introduced for cell therapy applications    Flow cytometry techniques to detect circulating endothelial progenitor cells. Florence Sabatier, Aix Marseille University, Marseille, France Sabatier showed that flow cytometry (FCM) is an alternative to colony forming assays that allows a direct counting of endothelial progenitor cells (EPC) suitable for clinical assessment of vascular repair capacity. However, FMC analysis of EPC remains a technical challenge.  First, the very low level of EPC in blood requires the implementation of various recommendations for rare events detection. Main technical aspects are the need for a pre-enrichment step, the strict exclusion of non-viable cells, debris and non-nucleated cells that may generate false positive events, and adapt strategies for defining positivity of labeling of rare events. Second, due to the absence of specific marker, EPC phenotype is derived from the presence of a combination of antigens that attest for stemness or progenitor state and endothelial engagement, along with markers allowing identification of EPC subtypes. Indeed, the identity of the heterogeneous population of cells covered by the term "EPC” has been clarified recently based on a clear distinction between vasculogenic EPC of non haematopoietic origin (the true EPC subset) and haematopoietic cells with angiogenic activity. EPC have to be detected as viable CD34+/CD45- expressing endothelial antigens such as CD146 or CD31. While such phenotype is clearly identified in cord blood cells, it is near undetectable in peripheral blood from healthy subject. The clinical usefulness of this EPC subset remains to be demonstrated.  By contrast, hematopoietic progenitor cells with angiogenic potential can be detected using recently optimized FCM protocols. These cells are viable, CD34+/CD45dim with forward scatter and side scatter characteristics similar to lymphocyte and expressing KDR. Based on this phenotype, the previous biomarker value assigned to CD34+/KDR+ cells is currently being confirmed in cardiovascular risk patients. Thus, the recent advances in the field of EPC biology now translate in a more consensual terminology and optimized FCM protocols. Detection of hematopoietic progenitors with angiogenic capacity seems a good compromise between sensitivity, specificity and applicability in clinical practice.  Improving technical performance of FCM for rare cells analysis may provide better sensitivity for detection of non-hematopoietic endothelial progenitors.  Altogether, these advances pave the way for improved standardization.    Microparticles (Chair: Françoise Dignat-George, France)      Isolation and characterization of platelet-derived vesicles. Pia Siljander, Hensinki University, Helsinki, Finland The participation of platelets in physiological functions other than haemostasis and thrombosis is also reflected by the formation of platelet-derived extracellular vesicles (EVs) under various conditions and agonists. These EVs are considered to participate in the processes mediated by platelets and therefore these EV should be carefully characterized to better understand platelet function. Siljanders research group has optimized a platelet-derived EV isolation protocol from leukocyte-free platelets, and characterized both thrombin and collagen co-induced EVs and LPS-induced EVs and compared these to Ca2+ ionophore -induced EVs and time-matched controls. In addition to the quantitative differences in EV numbers depending on the activating condition, also qualitative (proteomics) and the quantitative protein content of the EV subpopulations (microparticles and exosomes) were observed under the various conditions. Measured by nanoparticle tracking analysis and transmission electron microscopy, about 90% of the vesicles are < 500 nm and 65-80% were < 250 nm.  In addition, she observed that the protocol of separating microparticles from exosomes by a centrifugation at 20,000 x g for 40 minutes does not yield homogenously sized EV (sub)populations, but rather overlapping vesicle pools, indicating that the platelet-derived EVs are extremely heterogeneous. Due to the activation-dependent differences in the protein/vesicle ratios of the various EVs, it was concluded that protein content of EVs should not be used as a means for sample standardization. Finally, the use of Ca2+ -ionophore as an EV-inducer is discouraged because the resulting EVs greatly differ from those induced by physiologically relevant agonists. Taken together, the presented results promote further optimization of platelet-derived EV isolation and additional characterization and functional studies.    Measurements, applications, and impact of the refractive index of extracellular vesicles. Edwin van der Pol,  Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands Vesicles are often studied by methods that detect light scattering, such as flow cytometry and nanoparticle tracking analysis (NTA). The amount of light scattered by a vesicle depends on its size and refractive index (RI). Consequently, knowledge of the RI is required to determine the vesicle size and provide insight in the smallest detectable vesicles. We have measured the diameter and light scattering of vesicles and beads by tracking their Brownian motion with NTA (NS500, Nanosight Ltd). We analytically described the relation between the diameter, RI, and light scattering of beads using Mie theory to determine the RI of vesicles from cell-free human urine. The mean refractive index of urine vesicles was 1.37, which is in agreement with preliminary results from others. Edwin used the most recent knowledge on the RI of vesicles to discuss recent achievements and insights in the optical detection of vesicles, including Swarm detection, flow cytometry calibration, and the detection limit of NTA and Raman microspectroscopy. Furthermore, he hypothesized that the RI may provide a novel label-free parameter to distinguish vesicles from protein aggregates. This work was funded by the European Metrology Research Programme (EMRP) under the joint research project HLT02 (Metves). The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union.  Standardization (Chair: Rienk Nieuwland, the Netherlands)  Overview of Microparticle standardization. Romaric Lacroix, Aix Marseille Université, Marseille, France Larcoix summarized the key steps achieved by the Scientific Subcommittee on Vascular Biology towards standardizing microparticle (MP) analysis. About pre-analytics, a protocol has been proposed and validated in a multi-center study. Such consensual protocol significantly reduces the variability of platelet and erythrocytes-derived MP measurement. About analytics, a collaborative workshop has also been organized to define the inter-laboratory reproducibility of MP counts using flow cytometry (FCM). The bead-based calibration system proved to be useful to allow instrument qualification and monitoring. However, differential behavior among FCM sub-types still impedes standardization for MP enumeration. Consequently, a modified strategy has been proposed to overcome this issue and has been evaluated in a multi-center study including 61 flow cytometers of 14 different types. The new bead-based strategy is accessible on most commercially available instruments. As a result, no significant variability was observed between instrument types measuring PMPs with different optical systems. Finally, provided that instrument intrinsic behaviors for size-related measurements are taken into account, beads can be used as an efficient standardization tool for MPs. Lacroix also stressed on the need of educational efforts which have to be associated with the MP-related guidelines. These standardization achievements represent an important step towards the use of MPs as biomarker in clinical practice.    Standardization of vesicle detection by flow cytometry through vesicle size approximation. Frank Coumans, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands Coumans presented the basic theory behind the standardization effort based on a Mie model of the scatter to size relationship. Coumans and van der Pol developed a software application to determine the Mie relationship for most commercial flow cytometers based on a measurement of a vial of beads. The draft protocol is finished, and looks well throughout. Samples of selected (METVES; www.metves.eu) reference materials and biological samples have been shipped to the 32 participating sites worldwide (Europe, US, Australia, etc.). Coumans and van der Pol ran an internal test of their protocol in the AMC. For the 4 different flow cytometers, the tested Mie approach functioned better than a bead-based approach similar to the Megamix approach. The lively discussion after the presentation focused on the choice of refractive index, and on the absence of a pan-vesicle marker that would aid triggering on fluorescence.  
by R. Nieuwland
Thursday, June 19, 2014
2013 Annual Minutes Locked Topic 0 F. Dignat-George Vascular Biology Chairman: Francoise Dignat-George (France) Co-Chairs: Elizabeth E. Gardiner (Australia), Nigel Key (USA) , R Nieuwland (Netherlands), Florence Toti-Orfanoudakis (France) June 29, 2013The session was divided into two sections: the Educational session and the business session, This business session was divided into four parts addressing respectively: Part A, "Shedded protein and receptors". Part B "Circulating endothelial cells and progenitors", Part C "Microparticles" and Part D "Standardization studies" The success of this SSC VB was attested by the presence of at least 200 participants during the whole session The Educational session, chaired by Françoise Dignat-George, covered the emerging role of microparticles in cellular therapy and regenerative medicine, the regulation of membrane protein shedding by tetraspanins and the interest of endothelial progenitors in cardiovascular diseases studies with blodd outgrowth endothelial cells. Florence TOTI, France Microparticles : emerging role in cellular therapy and regenerative medecine "In her presentation "Membrane Microparticles: emerging role in cellular therapy and regenerative medicine" Florence Toti reviewed the recent data of the literature concerning microparticles as pathogenic markers of tissue remodeling. She focused on the particular settings of cellular therapy and transplantation, namely ischemia reperfusion and allograft rejection and graft function. She proposed that microparticles are not only indicators of vascular and graft tissue remodeling but contributors to the dynamic exchanges between graft and host tissue. She proposed that the pharmacological control of microparticle release during ischemia could improve engraftment at the early stage and emphasized on the need for a better understanding of the mode of action of immunosuppressive drugs on the long term." Mike Tomlinson, United Kingdom Tetraspanins as new regulators of membrane protein shedding The tetraspanins, are emerging as important regulators of the trafficking, lateral mobility and clustering of specific so-called "partner proteins” with which they interact. Tetraspanins further self-associate to form membrane microdomains that function as platforms for optimal cell adhesion and signal transduction. A major tetraspanin-associated protein is A Disintegrin and Metalloprotease 10 (ADAM10), a ubiquitous transmembrane protein that acts as a "molecular scissor” by shedding the extracellular regions from at least 40 different transmembrane protein targets. The consequences of ADAM10-mediated shedding include the removal of activatory receptors from the cell surface (e.g. platelet GPVI), the removal of adhesion molecules to increase vascular permeability (e.g. vascular endothelial (VE)-cadherin), the release of chemokines to promote leukocyte transmigration during inflammation (e.g. endothelial CX3CL1 and CXCL16), or the initiation of intracellular signalling to direct vascular development (e.g. Notch). ADAM10 is a major component of tetraspanin microdomains, Last year the group of M Tomlinson identified a subgroup of six related ADAM10-interacting tetraspanins termed the TspanC8s. ADAM10 interaction with a TspanC8 promotes its exit from the endoplasmic reticulum and maturation into a mature protease at the cell surface. Indeed, endothelial cells and red blood cells rendered deficient for their major TspanC8 (Tspan14 and Tspan33, respectively), had substantially reduced surface ADAM10 expression. Different TspanC8s could therefore localise ADAM10 to distinct substrates and allow ADAM10 therapeutic targeting in a cell type and/or substrate-specific manner, so minimising the likely severe side-effects of targeting ADAM10 on every cell in the body. Anna. M. Randi, UK Endothelial progenitors in cardiovascular disease: studies with blood outgrowth endothelial cells (BOEC) Over the last 15 years, many claims have been made regarding the origin of Endothelial progenitor cells (EPC) and their potential as therapeutic as well as biomarkers. Unfortunately, because of differences in the methods employed to isolate or quantify EPC, the field has become rather confusing and the value of EPC as research as well as translational tool risks being undermined. One of the most important properties of EPC is that they allow us to investigate endothelial biology in patients. In this presentation, A Randi reviewed the key steps in the identification and characterisation of circulating EPC and highlighted some of the controversial aspects. She then focussed on a type of circulating endothelial progenitor, named blood outgrowth endothelial cells (BOEC), which more than others have been shown to display the characteristics that a "true” EPC should display: clonal proliferative potential, differentiation to the endothelial lineage, ability to form stable blood vessels when implanted into tissues. She showed that BOEC represent a valid tool to investigate endothelial dysfunction in patients with primary endothelial defects, such as von Willebrand Disease (VWD), as well as patients with acquired endothelial dysfunction, such as Chronic Obstructive Pulmonary Disease (COPD). By studying BOEC from VWD patients she identified complex cellular phenotypes in patients previously thought to carry only a synthesis defect (Type 1 VWD). This approach also allowed to validate a novel function for von Willebrand Factor, namely the regulation of angiogenesis. In COPD, BOEC are senescent due to epigenetic modifications which involve the histone deacetylase SIRT1. Ex vivo treatment with drugs such as resveratrol can improve the function of BOEC. A. Randi concluded with future potential applications of BOEC in translational medicine. The Business Session was divided into 4 parts: Part A, "Shedded protein and receptors". Part B "Circulating endothelial cells and progenitors", Part C "Microparticles" and Part D "Standardization studies" PART A: Shedded proteins and receptors, chaired by Elizabeth E. Gardiner Robert Andrews , Australia Measurement of shed vascular proteins in clinical settings Robert Andrews gave an excellent introduction to methodology currently available to measure soluble ectodomains and levels of intact and cleaved receptor fragments in biological samples by flow cytometry, western blot and ELISA. He highlighted the need to develop standard protocols for the preparation of samples for measurement including the collection tubes, the processing steps and the timing of collection and processing. He discussed the mechanisms responsible for the activation-dependent shedding of platelet adhesion receptors and in the case of GPIb and GPVI, linked the regulated shedding of these receptors respectively to platelet ageing and to accelerated platelet clearance in diseases with immunodestruction such as ITP and clinical situations where blood is chronically and transiently exposed to elevated levels of shear stress such as in acute coronary syndrome and in individuals who have Left Ventricular Assist Devices (LVADS). He highlighted variability in healthy donors and indicated the potential of soluble receptor ectodomains to be linked to clinical outcomes – for bleeding or thrombotic events. He recommended analysis in combination with other bio-markers. Giovanni Davi, Italy Shed proteins as biomarkers in ACS G. Davi gave an update on circulating platelet-derived protein from platelet sheddome. He reviewed several shed proteins that have been proposed as biomarkers of acute coronary syndromes (ACS). He reminded the definition of a biomarker, that should be objectively measured, unaltered by pre-analytical artifacts and reproducibly assayed by easily performed methods.Activated platelets shed surface proteins, potentially modifying cell function as well as providing a source of bioactive fragments. Recently, about seventy shed proteins have been identified in the human platelet sheddome. Among them, sP-selectin, sCD40L, sGPV, and sGPVI have been found elevated in patients with stable angina. The shedding of membrane-associated CD40L produces sCD40L that in the Women’s Health Study, in the CAPTURE study, and in the MIRACLE study has been identified as an independent risk factor for recurrent cardiovascular (CV) events in ACS, particularly in combination with troponin-T levels. Reduced levels of sGPVI in ACS, at difference with stable angina, may indicate that this shedded protein binds to sites of vascular injury where collagen is exposed, thus being removed from circulation. Several proteins may be shedded from endothelial cell membrane. In particular, thrombomodulin, E-selectin, ICAM-1, and VCAM-1 have been found elevated within 1-hour of chest pain onset in ACS, and ICAM-1, and VCAM-1 remain elevated for months. High levels of sVCAM-1 on ACS presentation are associated with more in-hospital adverse coronary events and major CV event at 6-months. Soluble CD14, CD163, LR11 have been identified as protein shedded from macrophage surface membrane and found elevated in the ACS setting. Emerging shedded (?) proteins form uncertain cell origin (platelets and/or monocytes/macrophage and/or endothelial sheddome) have been identified in the context of ACS. MRP8/14 predicts CV events in both apparently healthy population and survivors of ACS, and may add prognostic information to that conveyed by standard risk factors and CRP evaluation. ROS-modified compounds induce a prothormbotic phenotype via interaction with platelet CD36, and sCD36 is well related to platelet activation markers in type 2 diabetes mellitus. Highly up-regulated expression of CD36 in circulating monocytes have been demonstrated in ACS patients. Finally, the soluble receptor for advanced glycation end-products (RAGE), an inflammation propagating factor contributing to accelerated atherosclerosis, has been found inversely associated with cardiac troponin in patients with NSTEMI-ACS. Despite increasing data on the relevance of these shed proteins in the ACS setting, the lack of standardized methodological approaches and the complexity of pathophysiology of these proteins renders not easy the use of these shed molecules as biomarkers in clinical settings. Lawrence Brass, USA Mining the platelet sheddome for biomarkers and insight into mechanism Professor Brass discussed data describing proteins released from PMA-treated platelets using mass spectrometry techniques and highlighted the need to treat proteins identified through this process as candidate shed proteins and to specifically analyse and validate individual protein shedding mechanisms. Surface expression, cleavage, and shedding of semaphorin 7A were demonstrated, as was its association with α-granules. Semaphorins are of particular interest because of their role in communication between cells. Cleavage of semaphorin 7A and 12 other proteins was substantially reduced by an inhibitor of ADAM17, a known sheddase. Cleavage of 16 of the proteins, including GPVI, was essentially unaffected. These results define a subset of membrane proteins as sheddome candidates, forming the basis for further studies examining the impact of ectodomain shedding on platelet function. Ectodomain shedding of platelet cell surface proteins may regulate platelet function by modifying adhesive interactions, removing ligand-receptor signaling pairs and liberating soluble fragments that harbour biological activity. PART B: Circulating endothelial cells and progenitors, chaired by Florence Toti-Orfanoudakis David M. Smadja, France Circulating endothelial cells and progenitors in pulmonary hypertension: potential in diagnosis and treatment monitoring The respective abundance of circulating endothelial cells and endothelial progenitor cells may reflect the balance between vascular injury and repair. Pulmonary arterial hypertension (PAH) is a heterogeneous pulmonary vascular disease associated with a strong pulmonary vessel remodelling. The group of D. Smadja aimed to identify non-invasive biomarkers of endothelial turnover that could be used to identify subgroups of PAH, responses to vasodilatator therapy or clinical deterioration. They quantified Circulating endothelial cells (CECs) isolated with CD146-coated beads, circulating CD34+CD133+ progenitor cells and endothelial progenitor cells in patients with congenital heart disease (CHD) associated with PAH, idiopathic PAH, or post thrombotic PAH. He alos présented the follow up of these cells after PAH-specific vasodilator therapy. A proangiogenic effect of treprostinil (a prostacyclin analog) administration targeting endothelial progenitor cells was described that helps to understand the beneficial effect of this drug in kids with refractory PAH. D. Smadja also presented results allowing to propose CECs as a valuable tool to define reversibility of PAH during CHD and as a biomarker that could help optimization of therapeutic strategies. Because of numerous technical challenges to quantify CECs by the reference method, D. Smadja also presented results of CECs detection using the Attune® Acoustic Focusing Cytometer and in particular results of application of this new technique as a biomarker in PAH. Michael Hristov, Germany Flow cytometry detection of circulating endothelial progenitor cells Accumulating evidence intensively advises circulating endothelial progenitor cells (EPCs) as surrogate cellular biomarkers in cardiovascular and cancer disease. However, a general standard on their quantification is still elusive, thus precluding a routine monitoring and comparative interpretation of clinical studies. M. Hristov intended to develop an advanced flow cytometric protocol for proper ex vivo quantification of EPCs in human blood. This protocol employs lyse/no-wash procedure and bead-based determination of absolute cell counts and uses three-color antibody panels at appropriate compensation. Analysis of rare events and low antigen expression in the EPC experiment is strengthening by 1/ sequential gating with strict exclusion of dead cells, as well as by matching high-intensity fluorochromes to low-density markers and 2/ by implementing the fluorescence-minus-one control. This current protocol provides quantitative information under a simple gating logic while using commonly accepted fluorochromes. This assay is therefore highly adapted for routine use. PART C : Microparticles, chaired by Rienk Nieuwland Edwin van der Pol, The Netherlands Determination of the refractive index of vesicles using nanoparticle tracking analysis Microparticles and exosomes are often studied by optical detection methods based on light scattering, such as flow cytometry. Light scattering is a complex process involving the size, shape, and refractive index of these vesicles. Consequently, the refractive index affects the detection limit of optical techniques and is essential to relate the detected light scatter signal to the diameter of vesicles. The refractive index may also provide a new label-free parameter. However, knowledge on the refractive index of vesicles is limited, since no methods are available to determine the refractive index. The group of Van der Pol has applied nanoparticle tracking analysis (NS500, Nanosight Ltd) to detect light scattering from single beads of known size and refractive index. He analytically described the relation between the diameter, refractive index, and light scattering of beads by Mie theory to determine the refractive index of vesicles from cell-free urine and platelet poor plasma. He obtained a median refractive index of urine vesicles of 1.36, with 95% of the vesicles having a refractive index between 1.35 and 1.45. The refractive index distribution of particles from plasma was much broader and had a median refractive index of 1.49. Thus Nanoparticle tracking analysis can be used to asses the refractive index of vesicles. Urine vesicles had a different median refractive index (ñ=1.36) than particles from plasma (ñ=1.49). This group hypothesizes that the relatively high refractive index of plasma particles is due to the presence of lipoproteins (1.45≤n≤1.50). Without considering light scattering theory, the low refractive index of urine vesicles implies that neither polystyrene beads (n=1.61) nor silica beads (n=1.45) are ideal to calibrate optical instruments for vesicle detection. Chris Gardiner, United Kingdom An update on light scattering techniques, especially identification of cellular sources of MPs using fluorescent markers The analysis of extracellular vesicles (EV) presents unique challenges due to their small size, low refractive index, polydispersity, interference from lipoproteins in plasma, and low numbers of antigens. Chris Gardiner recently published a protocol for EV enumeration by Nanoparticle Tracking Analysis using silica microspheres to establish camera settings and calibrate concentration measurements. Specific fluorescent labelling of EV is hampered by low numbers of antigens, steric hindrance, protein-label aggregates and limited availability of bright stable fluorophores. Work of Gardiner’s group, with GFP expressing proteins, membrane specific dyes and DNA has shown that the limitations lie within the labelling techniques rather than detection methodology. Elimination of unbound fluorophore to increase the signal to noise ratio improves sensitivity but there remains a need for suitably conjugated antibodies specific for antigens commonly expressed on EV. Esther N.M. Nolte-‘t Hoen, The Netherlands Intercellular communication by RNA containing extracellular vesicles Immune cells can communicate with each other via regulated secretion of nano-sized membrane vesicles, the majority of vesicles being in the nano-sized range (< 200nm). To unravel the molecular composition of individual vesicles, single particle-based, high-throughput, multi-parameter techniques are needed. This group developed a high resolution flow cytometry-based method that allows simultaneous quantification and characterization of individual extracellular vesicles. This method yields integrated information on the light scattering, buoyant density and the presence of specific proteins on extracellular vesicles. In addition to proteins and lipids, extracellular vesicles contain RNA. Horizontal vesicle-mediated transfer of RNA between cells uniquely allows the dissemination of genetically encoded messages, which may modify the function of target cells. Whereas the presence of miRNAs in these vesicles is frequently reported, they applied deep sequencing for an unbiased screen of small (<70 nt) RNAs in vesicles released during interaction of dendritic cells and T cells. They found that miRNAs formed only a minority of vesicle-enclosed small RNA species. In contrast, several other classes of small noncoding RNAs were highly abundant in extracellular vesicles. The detected small noncoding RNAs overlapped with protein coding regions, repeat sequences, or structural RNAs. Many of these RNAs were enriched relative to cellular RNA, indicating that cells destine specific RNAs for extracellular release. Several of the highly abundant small noncoding transcripts in vesicles have previously been associated to gene regulatory functions. Topical questions in the field are whether all vesicle subsets contain RNA, whether subsets of vesicles contain different types of RNA, and how the RNA content of vesicles changes upon cellular activation. This group recently obtained evidence that subpopulations of vesicles released during dendritic cell-T cell interactions differed in their RNA content. These data present leads for unraveling how immune cells modify the function of other cells via horizontal transfer of specific noncoding RNA species. PART D: Standardization studies, chaired by Nigel Key Rienk Nieuwland, The Netherlands First results of the European Metrology Research Program on traceable measurements of vesicles Rienk Nieuwland presented the advancement of the European Metrology Research Program (EMRP) project METVES (Metrological characterization of microvesicles (MV) from body fluids as non-invasive diagnostic biomarkers) whose goal is "to develop reliable, comparable and quantitative analysis of MV in biological fluids”. A "Questionnaire on particle reference materials and methods” has been prepared and distributed to laboratories working on detection of MV. A total of 20 questionnaires have been returned from 19 laboratories in 7 countries. The results of the questionnaire have been summarized in a "Report on the needs, specification and commercial sources of microvesicle reference materials”, prepared by Anaïs Nicolet and Felix Meli of the Federal Office of Metrology (METAS; Wabern, Switzerland). The report can be downloaded from the METVES website (www.metves.eu), and includes a summary of (i) contributors, (ii) methods comparison, and (iii) requested needs for MV reference materials. Reference materials for size and concentration measurements have been selected and are being measured by 3D metrology atomic force microscopy at METAS. First results have presented showing that not all commercially available reference materials are useful for standardization of measurements on MV. For those willing to participate in future standardization measurements of METVES selected reference materials, please register by providing your name and contact details at www.metves.eu. Romaric Lacroix, France Standardization of pre-analytical variables Romaric Lacroix presented an update about the SSC workshop on standardization of pre-analytical variables in plasma MP determination. The main objective of this collaborative study was to compare inter-laboratory variability of platelet-MP count by flow cytometry or MP-dependent procoagulant activity, using a common pre-analytical protocol versus a non-standardized protocol. 14 laboratories participated. As a main results, the standardized protocol resulted in a reduction of the inter-laboratory variability in MP count by flow cytometry although a significant variability did remain. These data have been published this year as a SSC report in Journal of Thrombosis and Haemostasis (Lacroix et al. J. Thromb. Haemost. 2013). Additional information were reported about the impact of pre-analytics on the different MP subpopulations measured by flow cytometry. Large platelet-MP and erythrocyte-MP are the MP subpopulations showing the highest interlaboratory variability compared to leukocytes and endothelial MP. This variability was reduced when preanalytics was standardized. However, for all MP subpopulations significant variations remained between laboratories stressing the need to identify others sources of variability. Françoise Dignat-George, France Standardization of microparticle enumeration across different flow cytometry platforms : An update Françoise Dignat-George reported the first data about the SSC workshop on the standardization of MP enumeration across different flow cytometry platforms. This study evaluate the inter-instrument reproducibility of platelet-MP counts among different platforms using a new standardization strategy based on different sets of beads experimentally adapted to each FCM platform. 44 laboratories registered accounting for 59 cytometers. Françoise Dignat-George presented the results of the first stage whose goal was to qualify the instruments according to performance in term of scatter resolution and background noise. The main conclusion was that the standardization strategy was compatible with a large proportion of instruments (78%) including instruments of old generation. The next step will be to evaluate the inter-instrument reproducibility of different PMP levels, using common reagents and the standardized FCM protocol. Summary of the ongoing collaborative studies and publications 2012-2013 Ongoing collaborative studies on the standardization of microparticles in ISTH SSC Vascular Biology. 1) Standardization of pre-analytical variables in plasma MP determination. It has involved 14 laboratories. Results of on platelet MP and functional procoagulant tests have been published as SSC report at the beginning of this year. And results on others MP subpopulations have been presented at this meeting. 2) Standardization of MP enumeration across different flow cytometry platforms. This study started this year. 44 laboratories registered accounting for 59 cytometers. Results of the first step related to instrument qualification have been presented and final results are expected for Milwaukee. 3) A third project, the European Metrology Research Program (EMRP) project METVES coordinated by Rienk Nieuwland, aims to develop standard for reliable, comparable and quantitative analysis of extracellular vesicles in biological fluids. Reference materials have been selected with the collaboration of 19 laboratories that answered a questionnaire. These materials are being evaluated by 3D metrology atomic force microscopy. A report on the needs, specification and commercial sources of microvesicle reference materials is available online (www.metves.eu). Publications related to SSC work (2012-2013) - Lacroix R, Judicone C, Poncelet P, Robert S, Arnaud L, Sampol J and Dignat-George F. Impact of pre-analytical parameters on the measurement of circulating microparticles: towards standardization of protocol. J. Thromb. Haemost. 2012; 10:437-46. - Robert S, Lacroix R, Poncelet P, Harhouri K, Bouriche T, Judicone C, Wischhusen J, Arnaud L, Dignat-George F. High-Sensitivity Flow Cytometry Provides Access to Standardized Measurement of Small-Size Microparticles. Arterioscler Thromb Vasc Biol. 2012 ; 32 :1054-8. - Lacroix R., Judicone C., Mooberry M., Boucekine M., Key N.S. and Dignat-George F, on behalf of the ISH SSC WorkshopStandardization of the pre-analytical variables in plasma microparticles determination: results of the ISTH SSC Collaborative Workshop. J. Thromb. Haemost. 2013
by F. Dignat-George
Wednesday, June 19, 2013
2012 Annual Minutes Locked Topic 0 F. Dignat-George Vascular Biology Subcommittee Minutes 29 June 2012 Chairman: Françoise Dignat George (FR) Co-chairmen: Elizabeth Gardiner (Australia), John Griffin (USA), Nigel Key (USA), Peter Newman (USA), Rienk Nieuwland (The Netherlands), Florence Toti-Orfanoudakis (France) This year, the SSC VB was divided into three parts addressing respectively: Part A, "Shed proteins/receptors". Part B "Detection and Characterization of circulating endothelial cells and their progenitors", and Part C "Detection and enumeration of circulating microparticles ." The success of this SSC VB was attested by the presence of an average of 80 to100 participants during the whole session Part A of the session, chaired by Elizabeth Gardiner and Peter Newman, covered the mechanism of protein shedding and their relevance as clinical biomarkers of vascular disease.  Elizabeth Gardiner (Australia) gave a talk on shear-induced release of platelet receptors by metalloproteinases based on previous results showing that ADAM10 is the key platelet surface metalloproteinase activated by coagulation or platelet collagen receptor GPVI engagement by ligand. Absence of GPVI from the surface of platelets either through genetic disposition or presence of anti-platelet autoantibodies causes a marked bleeding defect indicating that functional GPVI is essential for normal haemostasis. Loss of GPVI from the surface of a circulating platelet by shedding leads to changes in platelet responsiveness and reduced ability of platelets to activate. Elizabeth Gardiner examined whether shear force, in the absence of GPVI ligand, was sufficient to induce shedding of GPVI. She examined how chronic exposure to elevated and changing shear stresses differentially regulates metalloproteolysis of GPVI, a key platelet adhesion-signalling receptor. Human citrated PRP or washed platelets were subjected to increasing shear rates in a cone-plate viscometer and levels of intact and cleaved GPVI were examined by western blot and ELISA. Patho-physiological shear rates (3,000-10,000 s-1) induced platelet aggregation and metalloproteinase-dependent appearance of soluble GPVI ectodomain, and GPVI platelet remnant. Shedding of GPVI continued after transient exposure to shear. Blockade of aIIbb3, GPIba or intracellular signalling inhibited shear-induced platelet aggregation but minimally affected shear-induced shedding of GPVI. Shear-induced GPVI shedding also occurred in PRP or washed platelets isolated from a von Willebrand Disease Type 3 patient with no detectable VWF, implying that shear-induced activation of platelet metalloproteinases can occur in the absence of GPVI and GPIba ligands.Finally, Elizabeth Gardiner showed that significantly elevated levels of sGPVI were observed in 10 patients with stable angina pectoris, with well-defined single vessel coronary artery disease.These data suggest that loss of GPVI in platelets exposed to shear could have potential implications for the stability of a forming thrombus at arterial shear rates.  Elaine W. Raines(USA) presented data showing that proteolytic shedding by ADAM 17 functions as a gatekeeper for leukocyte accumulation at inflammatory sites and for resolution of inflammation. Leukocyte recruitment to inflammatory sites involves sequential adhesive and de-adhesive interactions associated with dynamic changes in expression and function of cell surface proteins. Elaine W. Raineshypothesizes that proteolysis of cell surface proteins allows instantaneous uncoupling of adhesive and signaling events, and thus could be a key regulator of the inflammatory response. ADAM17 (also known as tumor necrosis factor-a-converting enzyme or TACE) proteolytically sheds multiple cell surface proteins, but the physiological importance of cleavage of specific substrates is incompletely understood. She presented in vivo analyses of leukocyte trafficking demonstrating that substrate-, context-, and myeloid cell-type-specificity of ADAM17-mediated cleavage of its substrates. For neutrophils, but not monocytes, ADAM17 targets their initial interaction with activated endothelium through cleavage of L-selectin, and thus limits neutrophil rolling and subsequent downstream adhesion events required for their transendothelial emigration into tissues. In contrast to neutrophils, ADAM17 regulates monocyte accumulation within inflammatory sites by apparent proteolytic release of its mitogen, macrophage colony-stimulating factor. Finally, spontaneous exiting of inflammatory macrophages, critical for resolution of inflammation, is controlled by ADAM17 through its cleavage of the integrin aMb2 (Mac-1). In the absence of leukocyte ADAM17, the number of macrophages remaining at the inflammatory site is two-fold elevated relative to wildtype, a cell-instrinsic characteristic. Taken together Elaine W. Rainesconcludes that ADAM17 functions are critical for restriction of neutrophil infiltration, promotion of monocyte proliferation and resolution of the inflammatory response.  Andreas Ludwig (Germany), gave an overview of the pro-inflammatory activities of vascular ADAMS proteins. The metalloproteinases ADAM10 and ADAM17 are responsible for several shedding events leading to increased levels of soluble vascular surface molecules in the course of vascular inflammation. Among the shed proteins there are proinflammatory cytokines, adhesion molecules that are involved in endothelial barrier function or transendothelial transmigration of leukocytes and growth factors that have been implicated in transactivation of vascular cells and vascular remodelling. Inhibition as well as knock-down experiments have demonstrated that both ADAM10 and ADAM17 are critically involved in leukocyte transendothelial migration or detachment and also mediate increased vascular permeability. Andreas Ludwig presented recent data that identity ADAM10 and ADAM17 as sheddases for transmembrane chemokines CX3CL1 and CXCL16, junctional adhesion molecule JAM-A, syndecan-1 and -4 and VE-cadherin in vitro and in vivo. He recently showed that intranasal application of ADAM10/ADAM17 inhibitors reduces the development of acute lung injury induced by endotoxin. Conditional knockout of ADAM10 or ADAM17 in endothelial reduces LPS-induced permeability changes and protects mice from acute lung inflammation (5). Conditional knockout of ADAM17 in smooth muscle cells also protects mice by suppression of growth factor-mediated vascular transactivation leading to reduced production of cytokines and chemokines. All together, these data show that ADAM17 activation in endothelial and smooth muscle cells promotes acute pulmonary inflammation in response to endotoxin by multiple shedding events leading to enhanced vascular permeability, proinflammatory mediator production and leukocyte recruitment.  François Lanza (France) presented an overview of GPV function and its utility as a marker of thrombosis in vascular disorders. Despite its identification more than 30 years ago the real function of GPV is still subject to question. This 82 KDa platelet specific glycoprotein and late marker of thrombopoiesis non-covalently associates with the GPIb-IX complex, a process required for its efficient surface exposure. GPV-deficient mice do not exhibit a Bernard-Soulier phenotype, their platelets normally express GPIb-IX, but they exhibit decreased and increased responses to low concentrations of collagen and thrombin, respectively. These subtle defects could explain the variable anti- or prothrombotic tendencies reported in different models of thrombosis, but have no consequence on haemostatic responses. GPV’s sensitivity to a series of proteases (thrombin, elastase, ADAM10, -17), with the production of 69 to 80 kDa soluble fragments, is conserved in other species, suggesting a role of the released forms in thrombotic and inflammatory situations. These hypotheses could be tested in models expressing GPV mutated at these cleavage sites. Based on GPV expression on platelets coupled to its thrombin sensitivity, François Lanza discussed the utility of GPV as a marker of thrombosis in cardiovascular disorders. Increased levels of soluble GPV were recorded in arterial (myocardial infarction, stroke, and peripheral arterial disease), as well as in venous thrombotic manifestations (deep vein thrombosis, pulmonary embolism). A similar increase in diabetic patients indicated thrombin-independent GPV release mechanisms in certain cardiovascular diseases. GPV has also demonstrated its utility in the quality control of platelet manufacturing processes in transfusion. In summary, despite having demonstrated its utility as a biomarker, GPV has yet to reveal its exact function in fields other than haemostasis.  The Part B of the session, chaired by Francoise Dignat George, covered the detection and measurement of circulating endothelial cells and their progenitors. Gian Paolo Fadini ( Italy ) , presented an overview on the clinical relevance of circulating endothelial progenitors ( EPCs) for assessment of cardiovascular risk . Indeed , a significant proportion of cardiovascular events (CV) occur in subjects categorized into intermediate risk categories. Therefore, discovery of new CV risk biomarkers is important to aid a better CV risk stratification. Endothelial progenitor cells (EPCs) are involved in endothelial repair and angiogenesis. EPC level is reduced in the presence of CV risk factors compared to healthy subject and further decline when CV disease develops and worsens. Gian Paolo Fadini gives a review of the main clinical studies that identify EPCs as a new candidate mechanistic biomarker to assess CV risk. A few longitudinal studies have assessed the ability of EPCs and other progenitor cell phenotypes to predict CV outcomes in different categories of patients. These studies consistently show that a low baseline progenitor cell level is associated with a poor outcome at follow-up. However, a meta-analysis of patients’ level data of such studies has shown that the incremental information provided by progenitor cell level over and beyond traditional risk assessment is limited. Intriguingly, however, EPC level is amenable to pharmacologic modulation by several mechanisms. Therefore, EPC-based intervention may represent a new avenue for the prevention and treatment of CV disease. Whether restoring EPC levels improves long term cardiovascular outcomes will need to be ascertained in future studies. Therefore, methodological standardisation is needed for a better evaluation of their clinical relevance. Hovewer, methodological standardisation is needed for a better evaluation of their clinical relevance  Jamie Case (USA ) presented data on the potential of polychromatic flow cytometryto identify immature and mature circulating endothelial cells . Jamie Case, (USA ) identifies and characterize two distinct populations of bona fide circulating endothelial cells, including the endothelial colony forming cell (ECFC), by electron microscopy, colony assays, immunomagnetic selection and polychromatic flow cytometry (PFC). A population of cells containing both ECFCs and mature circulating endothelial cells (CECs) were determined by varying expressions of CD34, CD31 and CD146, but not AC133and CD45. HE showed that after immunomagnetic separation, these cells failed to form hematopoietic colonies. Clonogenic endothelial colonies with proliferative potential were obtained, thus verifying their identity as ECFCs. The frequency of ECFCs were increased in human umbilical cord blood and were extremely rare in the peripheral blood of healthy adults. In addition, Jamie Case, also detected another mature endothelial cell population in the circulation that was apoptotic. Finally, when comparing this new PFC protocol to a prior method, he determined that the present protocol identifies circulating endothelial cells while the earlier protocol identified extracellular vesicles. Jamie Case concluded that two populations of circulating endothelial cells including the functionally characterized ECFC are now identifiable in human umbilical cord blood and peripheral blood by PFC. Therefore, methodological standardisation is needed for a better evaluation of their clinical relevance  Florence Sabatier ( France ) presented data showing that changes in circulating endothelial cells and their progenitors during percutaneous coronary angioplasty (PCI) reflect endothelial response to injury. Percutaneous coronary intervention (PCI) became the most commonly used revascularization strategy in coronary artery disease patients due to the advents of stents and improvement of anti-platelet therapy. However, the main pitfall of this procedure remains the development of in-stent restenosis (ISR) and the need for subsequent target lesion revascularization (. Disruption of endothelial integrity is recognized as the primary event triggering inflammatory and proliferating signals leading to intimal hyperplasia. This endothelial injury results from mechanical trauma but also inflammatory processes involving platelet activation and interactions with the vessel wall. Animals studies indicate that early regeneration of a functional endothelial layer is critical for preventing pathological remodeling of the arterial wall and may involve bone marrow derived endothelial progenitor cells. Florence Sabatier investigated the usefulness of numeration of circulating endothelial cells (CEC) reflecting endothelial injury and endothelial progenitor cells (EPC) indicators ofrepair capacity. EPC and CEC wereenumerated to monitor the endothelial response to PCI and the prediction of cardiovascular outcomes, in patients with stable CAD undergoing PCI with bare metal stent implantation enrolled in a prospective longitudinal study. CEC were enumerated using the standardize CD146 based immunomagnetic separation assay. Enumeration of circulating CD34+ progenitor cells and CD34+KDR+ EPC was performed using multicolor flow cytometry protocols adapted from the standardized ISHAGE strategy. Measurements were performed before PCI and at 6h and 24 hours after the procedure to analyze dynamic changes of these markers. She showed thatPCI induced transient rise in CEC levels attesting significant endothelial lesion. Changes in CEC levels were more pronounced in patients presenting with high on treatment platelet reactivity (VASP index >50) after a loading dose of 75mg clopidogrel compared to good responder patients (VASP <50%). In multivariate analysis, VASP group, the number of disease vessels and the number of implanted stents independently predicted endothelial injury. PCI also induce the mobilization of CD34+ PC and CD34+KDR+ EPC as indicated by increased levels at H6 and H24 comapred to baseline levels. Interestingly, Changes in CD34+KDR+ cells independently predicted the occurrence and Major acute cardiovascular events at 6 months follow up. These data indicate that efficient inhibition of platelet reactivity in response to clopidogrel therapy exert protective effect on endothelial cells during PCI. In addition, the mobilization of cells with a capacity for endothelial differentiation and repair determines the risk for ISR in CAD patients undergoing PCI and BMS implantation. These data provide new clues of the usefulness of endothelial biomarkers in the identification of patients at risk of poor outcome after PCI and to design and evaluate preventive therapeutics.  The Part C of the session, chaired by Francoise Nigel Key and Florence Toti covered the detection and measurement of circulating cell-derived miroparticles   John. Nolan (San Diego), as actual President of ISAC (Int. Soc. for Advancement of Cytometry) works to encourage merging ISTH expertise in Hemostasis and technological expertise in FCM present among ISAC members. In order to help communication in the field of MP/MV studies, an Interest Development Group (IDG) on microvesicles has been launched that can be reached on the ISAC website . Due to its multi-parametric analysis capability FCM remains the most widely used technology for the evaluation of MP/MV. However, their small size challenges FCM sensitivity limits for both sizing and immunological detection. Among the many technological issues that are to be considered (and will be in the context of ISAC), emphasis is made on 1/ choosing the best triggering approach, with a growing interest towards fluorescence-based triggering,2/ avoiding artefacts generated by coincidence (when more than one particle at a time is present in the laser beam, a phenomenon improperly named as "swarming")by doing dilution experiments by doing dilution experiments, 3/ the relevance of calibrating fluorescence axis in addition to scatter axis for the sake of standardization 4/ developing new reference standards such as for example fluorescent liposomes.  Alain R. Brisson ( France )presented a characterization of microparticles from blood and activated platelets by cryo- electronmicroscopy. He provides a comprehensive structural description of circulating MP/MV found in various body fluids in physiological and pathological situations. In this first study, he focused plasma from healthy donors and on microparticles derived from activated platelets, with the objectives of characterizing the morphology and size distribution of the whole microparticle population using two probes: annexin 5 that bind to phosphatidylserine and an antibody to GP-1b platelet protein He used cryo-Electron Microscopy and specific probes were made of Annexin-5 covalently coupled to gold nanoparticles. Cryo-EM provides the unique advantage to reveal the genuine structure of microparticles in pure PPP (Plasma-Poor-Platelet) samples, in the absence of any treatment, like fixation, staining or drying. Microparticles could be classified into several morphological families, namely isolated microparticles, microparticles made of clusters of vesicles and aggregate-like particles. Plasmatic microparticles range in size from 50 nm to 3 µm. About 75% of them are smaller than 500 nm. Microparticles derived from activated blood platelets present the same morphologies and the same size range. Size histograms were determined for the whole microparticle population and for the sub--population of PS-exposing microparticles. The data presented by Alain R. Brisson provide novel structural information on plasmatic microparticles and open avenues for characterizing circulating microparticles present in various physio-pathological situations.  Jovan Antovic, (Sweden )proposed a strategy to study microparticles in hemophilia . He analyzed samples of patients with hemophilia A and determined microparticles (MP) on a Beckman Coulter Gallios flow cytometer using the 5-color protocol he used: 1/phalloidine as a quality control – MP were defined as phalloidine negative particles < 1µm. 2/lactadherin positive, which binds PS in a Ca-independent manner 3/ CD42a for the determination of platelet MP (PMP), CD45 for leukocyte MP (LMP) and CD144 for endothelial MP (EMP)). He reported a paradoxical decrease of circulating TMP, PMP and EMP after on-demand treatment with FVIII concentrate while TMP and PMP inversely correlated with hemostatic activation and fibrin gel tightness. A plausible explanation may be that MP are incorporated in the hemostatic plug formed after FVIII substitution on the site of injury (injury and bleeding usually precede the concentrate injection in patients treated on-demand). He proposed that MP may provide a negatively charged surface for the activation of coagulation in situ and also promote coagulation via the expression of TF and P-selectin on their surface.  Micah Mooberry ( USA ) presented the effect of different techniques onmicroparticle analysis by flow cytometry. Flow cytometry remains the most commonly used methodology for the detection, enumeration and characterization of microparticles (MPs).Despite this fact, there are inherent limitations with flow cytometry that impede the detection of MPs, most of which are technologically driven.Of these, the intrinsic resolution of the cytometer and the level of background noise have significant impact.To determine the effect of different triggering techniques on these limitations, and thus the sensitivity for MP detection, Micah Mooberry evaluated several newer generation flow cytometers.For each cytometer, 2 sets of biological samples were analyzed using SSC, FSC +/- SSC, and FITC triggers.Biological samples included PFP obtained from whole blood incubated with LPS and the supernatant of washed platelets treated with ionophore, both of which were analyzed for PMPs (AnnexinV+, CD41+).Overall, sensitivity for PMP detection using FSC trigger was roughly equivalent amongst all cytometers on both sets of biological samples and included a portion of the smaller subset of MPs < 0.5 µm.SSC trigger data were more variable across machines, however compared to FSC, it offered a better sensitivity for PMP detection, when performed on a cytometer with high sensitivity SSC PMT, which detected > 2x the number of PMPs compared to FSC trigger. Fluorescence triggering provided the overall highest sensitivity for PMP detection, although the reliability of these results are questionable due to potential problems with specificity and nonspecific fluorescent signal.Micah Mooberryconcluded that the optimal trigger technique may vary with cytometer, based upon the individual strengths of the machine.Fluorescence triggering may also offer the greatest sensitivity for MP detection, but possibly at the expense of specificity.  Philippe Poncelet ( France ) presented forward versus side scatter strategies to standardize microparticle count by flow cytometry. Reliable measurement of cell-derived microparticles (MP) by flow cytometry (FCM)is hampered by their low size range (0.1–1µm). He showed that extending a 1st standardized protocol for MP counts in the limited size range of 0.5 to 1µm bead-eq., expanded this range down-to 0.3µm on Beckman-Coulter (BC) Gallios, thus detecting previously undetectable"small" MP. VB SSC studies showed that this strategy, optimized on BC FCMrs measuring Forward Scatter (FSC) at high solid angle (1-19°), did not totally fit with FCMrs using low angle (1-8°) collection, including Becton-Dickinson (BD) FACS and LSRs. Side Scatter (SSC) was a more robust size-related triggering signal on such FCMrs3but different bead-based reference systems ("Megamix-Plus SSC" rather than "Megamix-Plus FSC") were needed when using this alternative parameter. Philippe Poncelet aimed to provide scatter-based reference levels for threshold and/or gating permitting general standardization of MP counts across platforms. Comparisons of PMP counts were operated on the same plasma samples on FCMrs of each group (FSC- and SSC-optimized). Delineation of small PMP was made in FSC on BC Gallios using 0.3µm and 0.5µm beads; large PMP were found between 0.5µm and 0.9µm beads.The boundaries of the same two subsets were found on the SSC scale of BD FCMrs between 0.17 and 0.22 µm bead-eq (small PMP) and between 0.22 and over 0.5 µm bead-eq (large PMP). Similar counts were obtained on these size-defined PMP subsets using different platforms, demonstrating the ability of this standardization strategy to monitor inter-instrument reproducibility.  On the behalf of Rienk Nieuwland,(The Netherlands ), Edwin van der Pol ( NL ) presented a new project , granted within the "European Metrology Research Programme” of EURAMET., and based on metrological characterization of microvesicles from body fluids as non invasive diagnostic biomarkers In this project, which is collaboration between a university hospital (AMC, Amsterdam) and four European metrological institutes (Netherlands, Germany, Belgium, and Switzerland), Rienk Nieuwland had two major aims:1/ Detailed characterisation of extracellular vesicles (size, distribution, concentration, morphology, (bio) chemical composition) using several techniques in parallel such as SAXS, ASAXS + XRF, AFM, NTA, RPS, etc.2/ Develop standardized methods, protocols & reference materials to enable comparison of MV data between laboratories. With regard to point 2, the aim is to develop MV reference standards which are stable, provide repeatable measurements, and have properties similar to MV (such as size, morphology, refractive index). In his presentation he explained the background and aims(s) of the project, with emphasis on point 2, explaining his approach. As one of the last steps in this project, was inter-clinical laboratory comparison of selected MV reference materials (at least 10 different European clinical laboratories from at least 5 different countries). Via the SSC of the ISTH, he asked for participants in this survey.  Closing remarks Finally, Francoise Dignat George presented the update and perspectives of the ISTH Vascular Biology Working Group on the Measurement of microparticles by flow cytometry. Important questions were raised during previous SSC VB, namely: 1/ the possibility of standardizing MP enumeration by flow cytometry; 2/ the need for new generation flow cytometers or alternative technologies that would allow enumeration and characterization of particles of smaller sizes; 3/ study the impact of pre-analytic parameters on MP determination . Some of these questions have been partially been addressed. During the ISTH SSC that took place in Vienna in 2008, a first collaborative workshop on the standardization of microparticle counts was set up to define the inter-laboratory reproducibility of PMP counts using flow cytometry. Presented in Boston in 2009, the results of this workshop have been published in the Journal of Thrombosis and Haemostasis . : J Thromb Haemost. 2010 Nov;8(11):2571-4 "Standardization of platelet-derived microparticle enumeration by FCM using calibrated beads : results of ISTH SSC. Colll workshop”: R., S. Robert, P. Poncelet, S. Glover, N.S. Key, F. Dignat-George, and all SSC participant. We thank all the investigators who helped to set up this workshop as well as those who actively participated. However some questions remained unsolved because this strategy was not suitable to all types of FCM . Future directions are : to test new syntheticreference standards to enlarge the compatibility to all types of FCM In 2010, the pre-analytical phase was identified as a critical target for future standardization studies, and the Cairo SSC in VB was the opportunity to propose a new collaborative workshop on the impact of pre-analytic variables on MP determination. The first conclusions of this ongoing study have been presented in Kyoto. The results demonstrated that a standardized protocol results in a significant reduction of the inter-laboratory variability in PMP analysis that was more marked for flow cytometry . The future steps are now to evaluate the impact of this improvement of preanalytics on other MP subsets .  In addition, important advances were made during the Kyoto meeting indicating that: 1/ recent technological improvements maintain flow cytometry as a competitive analytical method to measure MP of smaller size; 2/ alternative technologies (NTA, DLS , ISADE, AFM, … ) open new options to enumerate MP of small size.  During Liverpool meeting ,themost critical new questions were1/ Propose homogeneous preparations ofbiological and non biological particlesas standards within the community ( See proposalsofnew subcommittes projects)2/ Propose new reagents (probes, antibodies…)specifically dedicated to MP determination These question points directions for two novels collaborative working parties in the VB SSC that were proposed during Liverpool . New project 1 Name of the project: Collaborative SSC projecton standardization of MP counting using calibrated bead standards and high sensitivity-Flow Cytometric platforms Person responsible: Francoise Dignat-George Methodology : candidate material ( Frozen Platelet Free Plasma with defined levels of MP andcalibratedbeads used as standard ) will be send to participating laboratories to 1/ check the adequacy of their high sensitivity-Flow Cytometric platform 2/ evaluate the sensitivityandreproducibility ofMP counts. Year of starting : 2012( Liverpool ) Annual report : 2013( Amsterdam ) Year ofexpected completion: 2014 ( Milwaukee) New project 2 Name of the project: Collaborative SSC projecton development and distribution of traceable reference materials Person responsible: Rienk Nieuwland Methodology : validate developed protocols using : - traceable reference materials ( synthetic particles and biological particles) - various detection methods available for particle determination (FCM , NTA, DLS , ISADE, AFM, Raman spectroscopy ‘’’’’ ) Year of starting : 2012( Liverpool ) Distribution of reference material : 2014 Expected completion and Publication as SSC report :2016 Instructions and documents to download will be available from the ISTH / SSC website www.isth.org If you are interested to participate , Send an e-mail to: Project 1francoise.dignat-george@univmed.fr before october 2012 Project 2 : r.nieuwland@amc.uva.nl, before october 2012
by F. Dignat-George
Friday, September 14, 2012
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