Delivery of Heme Oxygenase-1-Cell Penetrating Peptide (HO-1-CPP) Into Hepatocytes, Kupffer and Islet Cells in In Vitro and Ex Vivo Models of Cold Ischemia
1Surgery, Dalhousie University, Halifax, NS, Canada
2Surgery, Pathology, Microbiology & Immunology, Surgical Lead Multi Organ Transplant Program, QEII Health Sciences Center, Dalhousie University, Halifax, NS, Canada.
Meeting: 2015 American Transplant Congress
Abstract number: B63
Session Information
Session Name: Poster Session B: Cell Transplantation and Cell Therapies
Session Type: Poster Session
Date: Sunday, May 3, 2015
Session Time: 5:30pm-6:30pm
Presentation Time: 5:30pm-6:30pm
Location: Exhibit Hall E
In an attempt to increase the number of donor organs available to transplantation extended criteria donor (ECD) organs are more frequently considered. Unfortunately, ECD organs are more susceptible to cold ischemia. Several methods have been described that may reduce the cellular injury in experimental models of cold ischemia. One of the proteins that has gained a lot of traction is heme-oxygenase-1 (HO-1). Induction of HO-1 expression under conditions of cellular stress has targeted it as a key gene for protection against injury during cold ischemic conditions. The aim of the present study is to introduce active and functional HO-1 protein conjugated to a cell penetrating peptide (CPP) directly in vitro to hepatocytes and pancreatic islets, and ex vivo to hepatocytes, and endothelial and Kupffer cells in hypothermic and anoxic conditions. This novel strategy may also be used for the ex vivo delivery of other protective proteins in hypothermic perfusion systems. Here we show our ability to consistently produce a functional HO-1 protein fused to a CPP. To confirm the ability of our fusion protein to cross cell membranes, we have incubated rodent and human hepatocytes and murine islets in vitro with HO-1-CPP and later stained with anti-HO-1 and anti-His antibodies followed by fluorescent secondary antibodies. Localization of HO-1-CPP was revealed by (red) anti-His staining which shows a pattern consistent with HO-1 localization to the endoplasmic reticulum. Further evidence of the cell permeability of HO-1-CPP has been studied in ex vivo perfusion experiments. The HO-1-CPP perfused livers sections were paraffin embedded, sectioned and stained with an anti-HO-1 antibody which revealed intracellular localization of HO-1-CPP 4-5 cell layers deep surrounding the perfused vessels. Our ability to successfully deliver an active protein conjugated to a CPP to cells of a whole organ in an ex vivo hypothermic and hypoxic perfusion model holds great potential for future repair and protection of organs for transplantation. Future studies to determine the ability of HO-1-CPP to modulate the response to reperfusion injury and whole liver transplantation as well as using this technology to assess other bioactive proteins are planned.
To cite this abstract in AMA style:
Venkatachalam A, Wood C, Hu Q, Alwayn I. Delivery of Heme Oxygenase-1-Cell Penetrating Peptide (HO-1-CPP) Into Hepatocytes, Kupffer and Islet Cells in In Vitro and Ex Vivo Models of Cold Ischemia [abstract]. Am J Transplant. 2015; 15 (suppl 3). https://atcmeetingabstracts.com/abstract/delivery-of-heme-oxygenase-1-cell-penetrating-peptide-ho-1-cpp-into-hepatocytes-kupffer-and-islet-cells-in-in-vitro-and-ex-vivo-models-of-cold-ischemia/. Accessed November 21, 2024.« Back to 2015 American Transplant Congress