Moesin functionality in hypothermic liver preservation injury.
The objective of this study was to determine how expression and functionality of the cytoskeletal linker protein moesin is involved in hepatic hypothermic preservation injury. Mouse livers were cold stored in University of Wisconsin (UW) solution and reperfused on an isolated perfused liver (IPL) device for one hour. Human hepatocytes (HepG2) and human or murine sinusoidal endothelial cells (SECs) were cold stored and rewarmed to induce hypothermic preservation injury. The cells were transfected with: wild type moesin, an siRNA duplex specific for moesin, and the moesin mutants T558D and T558A. Tissue and cell moesin expression and its binding to actin were determined by Western blot. Liver IPL functional outcomes deteriorated proportional to the length of cold storage, which correlated with moesin disassociation from the actin cytoskeleton. Cell viability (LDH and WST-8) in the cell models progressively declined with increasing preservation time, which also correlated with moesin disassociation. Transfection of a moesin containing plasmid or an siRNA duplex specific for moesin into HepG2 cells resulted in increased and decreased moesin expression, respectively. Overexpression of moesin protected while moesin knock-down potentiated preservation injury in the HepG2 cell model. Hepatocytes expressing the T558A (inactive) and T558D (active) moesin binding mutants demonstrated significantly more and less preservation injury, respectively. Cold storage time dependently caused hepatocyte detachment from the matrix and cell death, which was prevented by the T558D active moesin mutation. In conclusion, moesin is causally involved in hypothermic liver cell preservation injury through control of its active binding molecular functionality.
Department of Surgery, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, VA 23298, United States.
Tao
T
Tian
Department of Surgery, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, VA 23298, United States.
Susanne L
SL
Lindell
Department of Surgery, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, VA 23298, United States.
Chris
C
Kowalski
Department of Surgery, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, VA 23298, United States; Department of Emergency Medicine, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, VA 23298, United States; Department of Physiology and Biophysics, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, VA 23298, United States. Electronic address: mjmangino@vcu.edu.
Martin J
MJ
Mangino
D000818:Animals; D002460:Cell Line; D050377:Cold Ischemia; D015925:Cryopreservation / Q000379:methods*; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000473:pathology*; D006801:Humans; D008099:Liver / Q000293:injuries*; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D009926:Organ Preservation; D019188:Organ Preservation Solutions / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering; D015424:Reperfusion
D008840:Microfilament Proteins; D019188:Organ Preservation Solutions; D034741:RNA, Small Interfering; C071058:moesin
Cytoskeletal system; ERM; Liver transplantation; Organ donor; Plasma membrane; Reperfusion injury