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2014 Annual Minutes
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6/19/2014 at 6:22:07 PM GMT
Posts: 4
2014 Annual Minutes
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
  1. platelet specific
  2. readily measurable
  3. demonstrate a specific increase in response to a disease situation
  4. 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; 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.  

Last edited Tuesday, October 14, 2014
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