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A Link between Mitophagy and Apoptosis in Endothelial Cells: Exosomal Delivery of Mfn‐2 siRNA
Author(s) -
Familtseva Anastasia,
Chaturvedi Pankaj,
Kalani Anuradha,
Tyagi Neetu,
Tyagi Suresh
Publication year - 2015
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.29.1_supplement.974.13
Subject(s) - microvesicles , microbiology and biotechnology , exosome , small interfering rna , apoptosis , mitochondrial fission , flow cytometry , biology , mitochondrion , chemistry , cell culture , transfection , microrna , biochemistry , genetics , gene
Exosomes are lipid‐bilayer vesicles (50‐100 nm) derived from many cell types including endothelial cells that are a promising delivery vehicle for small interfering ribonucleic acid (siRNA). Exosomes have the ability to fuse with cell membranes, avoid phagocytosis and be less immunogenic compared to other carriers. We have previously described the beneficial effects of mitochondrial fusion regulated by mitofusin‐2 (Mfn‐2) and the detrimental effects of excessive mitochondrial fission controlled by dynamin related protein (Drp‐1) on endothelial cell loss in vivo. We hypothesize that in vitro delivery of Mfn‐2 siRNA encapsulated with exosomes decreases Mfn‐2 protein expression in mice aortic endothelial cells (MAEC), triggering excessive mitochondrial fragmentation, activation of mitochondrial apoptotic cascade with Bax, Caspase 9 and Caspase 3 up regulation contributing to endothelial cell loss. In our current study, we isolated and purified exosomes from cultured mice aortic endothelial cell medium (MAEC exosomes). The round shape morphology and exosome size were defined by transmission electron microscopy. Western blotting and Flow Cytometry further validated the expression of three exosome markers: CD63, Flotillin‐1 and tumor susceptibility gene (Tsg101). Immunohistochemistry using PKH67 labeling showed the presence of MAEC exosomes in MAEC. In conclusion, our data suggests that Mfn‐2 siRNA encapsulated with MAEC exosomes silenced Mfn‐2 protein expression in MAEC that triggers excessive mitochondrial fragmentation, activation of mitochondrial apoptotic cascade with Bax, Caspase 9 and Caspase 3 up regulation inducing endothelial cell apoptosis.