M. Yang1,2, Q. DU1, P. R. Varley1, J. Goswami1, R. Wang1, B. Chen1, N. D. Anderson1, D. B. Stolz3, D. A. Geller1 1University Of Pittsburgh,Department OF Surgery,Pittsburgh, PA, USA 2The First Hospital Affiliated To Nanchang University,Department OF Surgery And Department Of Transplantation,Nanchang, JIANGXI, China 3University Of Pittsburgh,Center Of Biology Imaging,Pittsburgh, PA, USA
Introduction: Exosomes play an important role in cell communication, and Rab27a is a GTPase that has been shown to promote exosome secretion. However, the role of Rab27a and exosomes in liver I/R injury is unknown. We hypothesized that exosome secretion regulated by Rab27a promotes liver I/R injury.
Methods: 70% liver I/R injury mouse model was used. Adenoviral Rab27a shRNA (Ad-Rab27a shRNA) was used to knock down hepatic Rab27a expression. Circulating serum exosomes (Exo) in vivo or murine hepatocyte-secreted exosomes in vitro were isolated with ultracentrifugation. Exosome pellets were verified by TEM and by exosome markers CD63, CD81 and HSP70. Serum Exo concentration was analyzed with CD81 exosome ELISA kit. Liver damage during I/R was calculated with ALT and liver HE staining. Hypoxia or normoxia primed hepatocyte-secreted exosomes were injected to mice in vivo during warm I/R with/without Rab27a knockdown.
Results: Hepatic Rab27a protein was induced in vivo during liver I/R in a time-dependent manner with strong induction 6 hr after I/R (Fig. A). Liver I/R induced increase in hepatic Rab27a protein expression was diminished by knockdown of Rab27a in vivo with Ad-Rab27a shRNA (Fig. B, last lane), but not scrambled shRNA. Knockdown of Rab27a resulted in a dramatic reduction in liver damage measured by ALT (Fig. C) and liver necrosis on HE staining (not shown).
During liver warm I/R injury, serum exosomes were increased in a time-dependent manner with maximal serum exosome pellet seen 6-12 hr after I/R (Fig. D). Knockdown of hepatic Rab27 in vivo with Ad-Rab27a shRNA decreased serum exosome concentration (Fig. E). Liver damage in vivo during warm I/R was reduced by Rab27a knockdown (Fig. F, third column), and this protective effect was abrogated by injecting hypoxia primed hepatocyte-secreted exosomes (Fig. F, last column).
Conclusion: Hepatic Rab27a protein expression was markedly increased in vivo during liver warm I/R injury, and this resulted in increased serum exosome concentrations. Knockdown of Rab27a decreased exosome secretion and liver damage. Hypoxia primed exosomes recapitulated liver damage reversed by Rab27a knockdown. These findings increase our understanding of fundamental exosome cell signaling and regulation during liver I/R injury.
