Proteomic analysis reveals presence of platelet microparticles in endothelial progenitor cell cultures

Abstract

IntroductionNumerous studies have demonstrated that endothelial progenitor cells (EPCs) are present among peripheral blood mononuclear cells (PBMNCs) and represent a subset of circulating bone marrow mononuclear cells (BMCs), which have the capacity to differentiate into endothelial cells in vivo. 1 New concepts of stem cell-based therapies for myocardial regeneration resulted in a rapid translation into a clinical context. [2][3][4] Yet, key questions remain unanswered. Importantly, the nomenclature and the phenotype of EPCs are subject to ongoing controversy and there are currently no specific markers that unambiguously identify these cells. 5,6 Thus, a more comprehensive approach is needed to analyze their antigenic profiles.MPs are small membrane vesicles (0.2-1.0 m) that originate from the plasma membrane and are shed from the cell surface after activation and apoptosis. 7 Importantly, MPs retain membrane antigens specific for the parent cell they originate from. Thus, MPs represent an ideal subproteome to clarify the cellular progeny of EPC cultures and mass spectrometry is the instrument of choice for this kind of research. 8 In this study, we used a proteomic approach to identify membrane proteins present on MPs in EPC cultures. Methods EPC cultureThe study was approved by the ethics review board of J. W. Goethe University and King's College London. Peripheral blood was collected from healthy adult volunteers and informed consent was obtained in accordance with the Declaration of Helsinki. EPC cultures were performed as previously described. 9,10 In brief, PBMNCs from healthy volunteers were isolated by Lymphoprep (1.077 g/mL; Axis-Shield PoCAS) density barrier centrifugation. The low-density fraction (Ͻ 1.077 g/mL) was carefully removed from the interface and washed 3 times with PBS (Dulbecco phosphate-buffered saline; Sigma-Aldrich) containing 2% FBS (fetal bovine serum, filtered and heat inactivated; Gibco, Invitrogen). Immediately after isolation, the cells were counted and 8 ϫ 10 6 cells were plated on fibronectin-coated (10 g/mL fibronectin from human plasma; SigmaAldrich) 12-well plates containing 1 mL endothelial basal medium (EBM; Cambrex Bio Science) supplemented with 20% FBS, EGM SingleQuots (10 g/mL epidermal growth factor, 3 g/mL bovine brain extract, 50 g/mL gentamicine, 50 g/mL amphotericin-B, 1 g/mL hydrocortisone; Cambrex Bio Science) and 10 ng/mL human vascular endothelial growth factor 165 (hVEGF 165; R&D Systems). Before use, the medium was passed through a 0.2-m filter. After 3 days in culture, the nonadherent An Inside Blood analysis of this article appears at the front of this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 11, 2018. by guest www.bloodjournal.org From cells were removed and fresh EBM medium was added. T...

Role of extracellular membrane vesicles in the pathogenesis of various diseases, including cancer, renal diseases, atherosclerosis, and arthritis

“…They reported the presence of endothelial biomarkers such as CD31 and VWF in mononuclear cells containing platelet MPs. 117 Finally, as discussed above, the levels of circulating MPs in the peripheral blood measured by flow cytometry have been used to assess the clinical status of patients with thromboembolic diseases, such as TTP and Trousseau's syndrome. 27,29,66,67 To date, the use of clinical laboratory tests to measure circulating, disease-associated MVs has just begun, but will undoubtedly be more frequently used in future to assess diagnosis and prognosis in various diseases.…”

Synergistic effect of HIF-1α gene therapy and HIF-1-activated bone marrow-derived angiogenic cells in a mouse model of limb ischemia

“…It is thought that BMDACs exert their beneficial effect through the incorporation of endothelial progenitor cells into new or remodeling vessels (5) and/or the production of angiogenic cytokines by myeloid, mesenchymal, and hematopoietic stem cells (6,7). Recent work suggests that many prior studies used inadequate criteria to define endothelial progenitor cells and that most BMDACs are in fact proangiogenic myeloid cells (8).…”

Are We There Yet? A Story About Cardiac Stem Cells

“…Such studies resulted in lab-to-lab, equipment-to-equipment and reagent-to-reagent (e.g. antibodies) differences in the results, which lead to discussions of contaminations by other cell types (Prokopi et al, 2009). However, the therapeutic potential of EPCs appears relatively high.…”