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2013 Annual Minutes
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6/19/2013 at 8:10:56 PM GMT
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2013 Annual Minutes

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, 2013

The 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




Last edited Wednesday, July 10, 2013
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