Background
Ischemia/reperfusion (IR) injury is an unavoidable consequence of tissue transplantation or replantation that often leads to inflammation and cell death. Excessive complement activation following IR induces endothelial cell injury, altering vascular and endothelial barrier function causing tissue dysfunction. To mitigate the IR response, various systemic anti-complement therapies have been tried. Recently, we developed a localized therapy that uses biotinylated fusogenic lipid vesicles (BioFLVs) to first incorporate biotin tethers onto cell membranes, which are then used to bind therapeutic fusion proteins containing streptavidin (SA) resulting in the decoration of cell membranes. The therapy is applied in two steps using solutions delivered intra-arterially.
Materials and methods
Alteration of formulation, concentration and duration of incubation of BioFLVs were conducted to demonstrate the ability of the system to modulate biotin tether incorporation in cultured cells. Using a rat hind limb model, the ability of BioFLVs to decorated endothelium of femoral vessels with FITC-labeled SA for 48 h of reperfusion was demonstrated. The feasibility of a BioFLV-based anti-complement therapy was tested in cultured cells using SA fused with vaccinia virus complement control protein (SA-VCP), a C3 convertase inhibitor. Human ovarian carcinoma (SKOV-3) cells were incubated with BioFLVs first and then with SA-VCP. To activate complement the cells were treated with a SKOV-3-specific antibody (trastuzumab) and incubated in human serum.
Results
Decoration of cells with SA-VCP effectively reduced complement deposition.
Conclusions
We conclude that BioFLV-mediated decoration of cell membranes with anti-complement proteins reduces complement activation and deposition in vitro and has the potential for application against inappropropriate complement activation in vivo.
Ischemia and reperfusion of organs is an unavoidable consequence of transplantation. Inflammatory events associated with reperfusion injury are in part attributed to excessive complement activation. Systemic administration of complement inhibitors reduces reperfusion injury but leaves patients vulnerable to infection. Here, we report a novel therapeutic strategy that decorates cells with an anti-complement peptide. An analog of the C3 convertase inhibitor Compstatin (C) was synthesized with a hexahistidine (His6) tag to create C-His6. To decorate cell membranes with C-His6, fusogenic lipid vesicles (FLVs) were used to incorporate lipids with nickel (Ni2+) tethers into cell membranes, and these could then couple with C-His6. Ni2+ tether levels to display C-His6 were modulated by changing FLV formulation, FLV incubation time and FLV levels. SKOV-3 cells decorated with C-His6 effectively reduced complement deposition in a classical complement activation assay. We conclude that our therapeutic approach appears promising for local ex vivo treatment of transplanted organs to reduce complement-mediated reperfusion injury.
Excessive complement activation following ischemia and reperfusion (IR) induces endothelial cell injury, altering vascular and endothelial barrier function causing tissue dysfunction. To mitigate IR response various systemic anti‐complement therapies have been tried. Recently, we developed a local therapy that uses biotinylated fusogenic lipid vesicles (BioFLVs) to incorporate biotin tethers onto cell membranes, which then bind to therapeutic fusion proteins containing streptavidin (SA) resulting in decoration of cell membranes. Alteration of formulation, concentration and duration of incubation of BioFLVs were conducted to demonstrate the ability of the system to modulate biotin tether incorporation. A rat hind limb model was used to demonstrate the efficacy of this system for 48 h of reperfusion. The feasibility of a BioFLV‐based anti‐complement therapy was tested in human ovarian carcinoma (SKOV3) cells using SA fused with vaccinia virus complement control protein (SA‐VCP), a C3 convertase inhibitor. To activate complement cells were treated with a SKOV‐3‐specific antibody (trastuzumab) and incubated in human serum. Decoration of cells with SA‐VCP effectively reduced complement deposition. We conclude that BioFLV‐mediated decoration of cell membranes with anti‐complement proteins has potential to reduce IR‐mediated complement injury in transplantated or replanted tissues.
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