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Microparticle Uptake by Pulmonary Microvascular Endothelial Cells
Author(s) -
Scruggs April K,
Rich Thomas C,
Bauer Natalie N
Publication year - 2017
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.31.1_supplement.689.7
Subject(s) - microbiology and biotechnology , endosome , golgi apparatus , organelle , chemistry , cell type , cell , calcein , confocal microscopy , extracellular , intracellular , biology , biochemistry , membrane
Microparticles (MPs) are important for the exchange of information between cells. MPs are extracellular vesicles that range in size from 0.1 to 1 μm in diameter. They are constitutively released from many cell types and are thought to play a role in vascular homeostasis. Furthermore the number and content of MPs are altered in pathologies involving inflammation and vascular cell activation. MPs carry proteins and RNAs from parent cells and are extracellular packets of information that affect target cell function. However, the mechanisms of targeting and delivery of the message are unknown. Understanding the message delivery system between MPs and target cells is the focus of this project. We first tested whether MPs are internalized by rat pulmonary microvascular endothelial cells (PMVECs) in culture. Cells labeled with Hoechst nuclear stain were incubated with calcein green and PKH26 labeled MPs for four hours. The cells were imaged using an environmental chamber on a confocal microscope (Nikon A1R). An image stack (Z‐stack) was acquired every 15 minutes. MPs were observed within cells and the number of MPs within cells increased over the first 90 minutes of the experiment. Maximum MP‐cell association was observed at 1 hour and few MPs were observed inside cells after 3 hours. These results indicate that MPs are taken up by target PMVECs in a time‐dependent manner. We next examined MP localization with organelles within cells. Organelle markers included monoclonal antibodies against early endosomes (Rab5 and EEA1), lysosomes (LAMP1), cis‐golgi (GRASP65) and trans‐golgi (TGN65), as well as labels for mitochondria, microtubules and cell nuclei. MPs were isolated from cell culture media via serial centrifugation and stained with a lipophilic membrane stain (PKH26). PMVECs were treated with stained MPs for 1, 2, 4 and 6 hours before organelle labeling. At one hour MPs colocalized with early endosomes (EEA1) and a small percentage colocalized with lysosomes (LAMP1). At six hours MPs colocalized with trans‐golgi. No overlap between MP‐staining and cis‐golgi, mitochondria, microtubules, or nuclei was observed at any time point. Taken together, these data indicate that MPs are internalized by target PMVECs. After internalization by target cells, most MPs are trafficked to the trans‐golgi for possible recycling of MP‐contents. Future studies will investigate the mechanisms responsible for internalization, whether payloads of MPs are delivered to specific organelles, and whether MP‐delivered proteins alter target cell function. Support or Funding Information This work was supported by NIH T32HL076125, P01HL066299, S10RR027535, and AHA 11SDG7390037.