title: Delivery of Soluble Heme Oxygenase 1 Cell-Penetrating Peptide into Liver Cells in in vitro and ex vivo Models of Cold Ischemia.
abstract: OBJECTIVE
  Liver transplantation is the treatment of choice in patients with end-stage liver disease. During liver transplantation, ischemia-reperfusion injury (IRI) occurs, which is an inevitable consequence of the transplantation process. To reduce the extent of cellular injury, one of the proteins that have been extensively investigated is heme oxygenase 1 (HO-1), which plays an important role in protecting the organs against IRI. The aim of this study was to introduce an active and functional HO-1 protein conjugated to a cell-penetrating peptide (CPP) in vitro and ex vivo into liver cells in hypothermic and anoxic conditions and to assert its cytoprotective effects.
  
  
  METHODS
  We generated an enzymatically active soluble (s)HO-1-CPP recombinant protein. The ability of the sHO-1-CPP protein to penetrate McA-RH7777, Clone 9, and Hep G2 cells, primary hepatocytes, and Kupffer and human umbilical vein endothelial cells in vitro, as well as its ability to penetrate a whole liver ex vivo under hypothermic and anoxic conditions, was assessed. An in vitro hypoxia-reoxygenation (HR) model was used to determine the cytoprotective effect of the sHO-1-CPP protein.
  
  
  RESULTS
  We showed that our recombinant protein sHO-1-CPP can cross cell membranes into rodent and human liver cells in vitro, and the results were further validated ex vivo, where rodent livers were perfused with an organ preservation solution supplemented with sHO-1-CPP under anoxic and hypothermic conditions. Immunohistochemistry revealed an intracellular localization of sHO-1-CPP in zones 1-3 of the perfused livers. The CPP did not exert any significant toxicity on the cells. Treating cells with sHO-1-CPP showed significant cytoprotection in the in vitro HR model.
  
  
  CONCLUSIONS
  Our findings show that the recombinant protein sHO-1-CPP can be successfully delivered to cells of a whole organ in an ex vivo hypothermic and anoxic perfusion model and that it provides cytoprotection to hepatocytes in an in vitro HR model. These results hold great potential for future repair and protection of donor organs. Future experiments are planned to confirm these data in in vivo models of IRI.
authors.affiliation: Atlantic Centre for Transplantation Research, Dalhousie University, Halifax, NS, Canada.
authors.forename: Ananda Baskaran
authors.initials: AB
authors.lastname: Venkatachalam
authors.forename: Scott Michael
authors.initials: SM
authors.lastname: Livingstone
authors.forename: Qianni
authors.initials: Q
authors.lastname: Hu
authors.forename: Ayush
authors.initials: A
authors.lastname: Ray
authors.forename: Caroline
authors.initials: C
authors.lastname: Wood
authors.forename: Sanem
authors.initials: S
authors.lastname: Cimen
authors.forename: Ian Patrick Joseph
authors.initials: IP
authors.lastname: Alwayn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D057846:Cell-Penetrating Peptides; D050377:Cold Ischemia; D019610:Cytoprotection; D016503:Drug Delivery Systems; D004353:Drug Evaluation, Preclinical; D051547:Heme Oxygenase-1 / Q000008:administration & dosage*; D056945:Hep G2 Cells; D006801:Humans; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology*; D016031:Liver Transplantation; D008969:Molecular Sequence Data; D010477:Perfusion; D017208:Rats, Wistar; D011994:Recombinant Proteins; D015427:Reperfusion Injury / Q000517:prevention & control*
chemical_list: D057846:Cell-Penetrating Peptides; D011994:Recombinant Proteins; D051547:Heme Oxygenase-1