Generation of a Recombinant Heme Oxygenase, Delivered by Cell-Penetrating Peptides, to Protect Transplanted Steatotic Livers from Reperfusion Injury

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Objective: To assay the ability of a cell-penetrating heme oxygenase-1 (HO-1) protein to protect organs from ischemia-reperfusion injury in models of steatotic liver transplantation. Methods: Plasmid constructs expressing cell-penetrating enhanced green fluorescent protein (CPP-EGFP) and HO-1 (CPP-HO-1) were generated by polymerase chain reaction amplification and ligation into a bacterial protein expression system. CPP-HO-1 and CPP-EGFP proteins were isolated from bacterial cultures and purified on nickel agarose columns. HO1-CPP functionality was assayed using spectrophometric quantification of bilirubin production by CPP-HO-1 treated HEK293T cells supplemented with the necessary co-factors. Cell penetration, in vitro, was assayed after treatment of various cell lines with CPP-tagged proteins in tissue culture and visualisation of target proteins by immunohistochemistry followed by fluorescence microscopy. Cellular internalisation of protein in rat livers was performed by ex vivo portal vein perfusion of isolated organs with 2mg of CPP-HO-1 or CPP-EGFP in histidine-tryptophan-ketoglutarate solution. Controls included mock treated organs and organs perfused with proteins lacking a CPP. To measure IRI we assayed for damage-associated molecular patterns (DAMPs) associated with oxidative stress such as mitochondrial DNA (mtDNA) by quantitative PCR. Results: Purified CPP-HO-1 is functional in a biochemical assay of bilirubin production and is able to penetrate cells in vitro. Histological analysis showed cell penetration of CPP-fused proteins in cells surrounding the hepatic portal vein after ex vivo perfusion, but not control proteins. mtDNA was detected in assays for DAMPs in models for IRI and has potential to be used as a marker for protection from IRI. Conclusions: We have successfully generated a CPP-HO-1 protein which is fully functional and is able to penetrate cells in vitro and ex vivo. Further work will determine if 1) CPP-HO-1 can provide cellular protection in an in vitro model of IRI 2) whether treatment of steatotic livers with CPP-HO-1 in a rat model of transplantation can protect these organs from IRI.