Romaric Lacroix

Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.

During the past decade, extracellular vesicles (EVs), which include apoptotic bodies, microvesicles, and exosomes, have emerged as important players in cell-to-cell communication in normal physiology and pathological conditions. EVs encapsulate and convey various bioactive molecules that are further transmitted to neighboring or more distant cells, where they induce various signaling cascades. The message delivered to the target cells is dependent on EV composition, which, in turn, is determined by the cell of origin and the surrounding microenvironment during EV biogenesis. Among their multifaceted role in the modulation of biological responses, the involvement of EVs in vascular development, growth, and maturation has been widely documented and their potential therapeutic application in regenerative medicine or angiogenesis-related diseases is drawing increasing interest. EVs derived from various cell types have the potential to deliver complex information to endothelial cells and to induce either pro- or antiangiogenic signaling. As dynamic systems, in response to changes in the microenvironment, EVs adapt their cargo composition to fine-tune the process of blood vessel formation. This article reviews the current knowledge on the role of microvesicles and exosomes from various cellular origins in angiogenesis, with a particular emphasis on the underlying mechanisms, and discusses the main challenges and prerequisites for their therapeutic applications.

Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.

PURPOSE: Despite the strong association between malignant disease and thromboembolic disorders, the molecular and cellular basis of this relationship remains uncertain. We evaluated the hypothesis that tumor-derived tissue factor-bearing microparticles in plasma contribute to cancer-associated thrombosis. EXPERIMENTAL DESIGN: We developed impedance-based flow cytometry to detect, quantitate, and size microparticles in platelet-poor plasma. We evaluated the number of tissue factor-bearing microparticles in a cohort of cancer patients of different histologies (N = 96) and conducted a case-control study of 30 cancer patients diagnosed with an acute venous thromboembolic event (VTE) compared with 60 cancer patients of similar age, stage, sex, and diagnosis without known VTE, as well as 22 patients with an idiopathic VTE. RESULTS: Tissue factor-bearing microparticles were detected in patients with advanced malignancy, including two thirds of patients with pancreatic carcinoma. Elevated levels of tissue factor-bearing microparticles were associated VTE in cancer patients (adjusted odds ratio, 3.72; 95% confidence interval, 1.18-11.76; P = 0.01). In cancer patients without VTE, a retrospective analysis revealed a 1-year cumulative incidence of VTE of 34.8% in patients with tissue factor-bearing microparticles versus 0% in those without detectable tissue factor-bearing microparticles (Gray test P = 0.002).The median number of tissue factor-bearing microparticles in the cancer VTE cohort (7.1 x 10(4) microparticles/microL) was significantly greater than both the idiopathic VTE and cancer-no VTE groups (P = 0.002 and P = 0.03, respectively). Pancreatectomy in three patients eliminated or nearly eliminated these microparticles which coexpressed the epithelial tumor antigen, MUC-1. CONCLUSION: We conclude that tumor-derived tissue factor-bearing microparticles are associated with VTE in cancer patients and may be central to the pathogenesis of cancer-associated thrombosis.