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Characterization of Microparticle Generation from Blood and Endothelial Cells during Inflammatory Stimulation
Author(s) -
Chatterjee Victor,
Meegan Jamie,
Overstreet Jonathan,
Coleman Danielle,
Szekeres Charles,
Yuan Sarah
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.574.1
Subject(s) - lipopolysaccharide , chemistry , umbilical vein , flow cytometry , cytokine , tumor necrosis factor alpha , endothelial stem cell , platelet , microbiology and biotechnology , stimulation , immunology , thrombin , biology , endocrinology , biochemistry , in vitro
Microparticles (MPs) are small membrane vesicles released by activated cells in response to physical or biological stimulation. They mediate cell‐cell communication by transferring a cargo of cell surface receptors, mRNAs, and microRNAs to target cells. Recent studies have shown an altered MP production in the circulation during inflammation or sepsis. However, cell‐specific mechanisms of MP generation and their functional impact remain poorly understood. In this study, we measured MP generation from platelets, red blood cells (RBC) and leucocytes in human whole blood, as well as in cultured human umbilical vein endothelial cells (HUVEC), during stimulation by different inflammatory mediators, including tumor necrosis factor alpha (TNFα), interferon gamma (INF γ), granulocyte‐macrophage colony stimulating factor (GM‐CSF), bacterial lipopolysaccharide (LPS), thrombin, complement C5a and nitric oxide (via the NO donors DEA NONOate and SNAP). MPs isolated from blood or cell culture conditioned media were analyzed by flow cytometry using cell specific markers and Annexin V, a marker for phosphaditylserine (PS) abundant in MPs. Size‐based gating of MP was performed using a mixture of standardized beads of sub‐micron size and absolute MP numbers calculated based on reference beads of known concentration. The results showed that platelet‐derived MPs were significantly increased by C5a, thrombin, cytokines and LPS. Neutrophil‐derived MPs were increased by LPS, cytokines, GM‐CSF, C5a, and NO donors. RBC‐derived MPs were mainly increased with NO donors. In cultured endothelial cells, TNFα caused a significantly increased, whereas NO decreased production of MPs that were stained positive for ICAM‐1, VCAM‐1, PECAM‐1 and E‐selectin. Interestingly, endothelial cells were able to internalize leukocyte‐derived MPs, evidenced by confocal microscopic confirmation of their intracellular location. Furthermore, neutrophils responded to endothelial‐derived MPs by forming neutrophil extracellular traps. These findings suggest a cell‐specific production and crosstalk of microparticles with blood cell and endothelium origins during stimulation with different inflammatory mediators. Support or Funding Information NIH HL070752, HL126646 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .