Abstract 14569: In vitro Transfer of Genetic Material Using Microvesicles Isolated from Circulating Angiogenic Cells
Microvesicles (MVs) are fragments of cell membranes released into extracellular space by most cell types. They have been shown to mediate intercellular communication, immune suppression and tissue regeneration. Circulatory angiogenic cells (CACs), collected from peripheral blood of patients with documented coronary artery disease (CAD) and age and sex matched controls, were cultured in serum free medium supplemented with 0.25% bovine serum albumin. MVs were pelleted from the cell free medium after 72hrs in culture. RNA was isolated from MVs using TRIZOL reagent (Invitrogen) and analyzed using TaqMan Small RNA assay and TaqMan Gene Expression assay kits (Applied Biosystems). Proteins expressed on the MVs surface were detected by flow cytometry. MVs were incorporated by CACs after 30min incubation at 37°C. An in vitro tube formation assay was performed using serum starved HUVECs and CACs co-cultured on matrigel coated culture dishes. Smooth muscle cells (SMCs) were co-cultured with MVs released from healthy CACs. MVs were electroporated using 125uF and 400V electrical current, in the presence of miR-548a-3p, which has recently been implicated in cell cycle control. MVs were then lysed and assayed for miRNA using qRT-PCR. MVs isolated from patients with CAD demonstrated reduced expression of intracellular adhesion molecule 1 (ICAM-1) and, α4 and β1 integrins on their surface, as well as reduced levels of mRNA for NOS3 and CXCR4 and miRs-126, -92a compared with healthy controls. CACs from CAD patients incubated with MVs derived from control CACs showed increased, levels of miR-150 (p<0.01), NOS3 mRNA (p<0.001) and significantly improved angiogenic potential (p=0.004) when compared with CAD CACs. miRNAs-126 and -150 as well as NOS3 mRNA, not normally present in SMCs, were identified after co-culture with MVs isolated from control CACs A significant difference in the expression of miR-548a-3p was detected between native and electroporated MVs (p=0.001). This work demonstrates that MVs can transfer biological material between same and different cell types, and that content of MVs can be manipulated. We believe that MVs may play an important role in therapeutics as a delivery vehicle for biologics, and have potential utility in both cell and gene therapies.
- © 2012 by American Heart Association, Inc.