Publication Date

2015

Document Type

Thesis

Committee Members

Norma Adragna (Committee Member), Ji Chen Bihl (Committee Member), Yanfang Chen (Advisor)

Degree Name

Master of Science (MS)

Abstract

Extracellular vesicles are membrane derived vesicles exists in all organs. The two major functioning extracellular vesicles are exosomes (EXMs) and microvesicles (MVs). EXMs released from cell membranes via exocytosis with size between 30-150 nm in diameter and MVs directly budded from plasma membrane and size between 100-1000 nm. Ceramide is one of the most important factor in lipid mechanism for EXM release and also associated with MV release. Ceramide generated from breakdown of sphingomyelin by neutral sphingomyelinase (nSMase). In this study, we used an nSMase inhibitor (GW4869) and an analogue of ceramide (C6-ceramide) to modulate the release of MVs and EXMs in human brain microvascular endothelial cells (ECs), and to test the effects of these modulations on EC proliferation, microRNA126 (miR-126) expression and MV uptake ability and the interaction between EC derived MVs/EXMs (EC-MVs/EXMs) with target cell, human brain smooth muscle cells (SMCs). Endothelial cells were treated with GW4869 in 10 µM and C6-ceramide in 10 µM for 24 h. MVs were isolated from by centrifuge and EXMs were isolated by centrifuge at 20,000 x g for 2 h and EXM were isolated by ultracentrifuge at 120,000 for 2 h with 100 nm filtration. MVs and EXMs size and concentration were analyzed by Nanoparticle Tracking Analysis (NTA) technique. Cell proliferation was detected by MTT. The levels of miRNA126 expression in ECs and EC-MVS/EXMs were detected by qRT-PCR. MV uptake measured by fluorescent microscope. Migration measured by wound healing method. GW4869 significantly decreased EXM release and C6-ceramide significantly increased both MV and EXM release in ECs. ECs treated with C6-ceramide and GW4869 had different miR-126 expression and MV uptake ability. Modulated EC-MVs/EXMs had no effect on normal SMC proliferation and migration, but EC-EXMs under both release regulation decreased angiotensin II (Ang II) induced SMC migration independent of miR-126 level in EXMs. Our data demonstrate that modulation of MV/EXM via C6-ceramide and GW4869 could affects EC function and it`s derived MVs/EXMs function, which may imply in vascular physiology and pathology.

Page Count

52

Department or Program

Department of Pharmacology and Toxicology

Year Degree Awarded

2015

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