Purpose: Organ procurement, cold storage and ischemia reperfusion injury (IRI) promote inflammation, which induces endothelial cell (EC) activation and dysfunction post transplantation. EC gap junctions (GJs) breakdown as a consequence of these injuries and play a key role in graft injury post transplantation. Here we explore the therapeutic potential of adding a novel gap junction (GJ) stabilizing peptide, ACT1, to UW preservation solution as a therapeutic agent to improve endothelial cell health. ACT1, is a small peptide Cx43 mimetic, which impairs the association of ZO-1 with Cx43 thus promotes GJ integrity.

Methods: Mouse cardiac ECs (MCECs) were exposed to 6 hrs of cold storage in UW or UW/ACT1 solution followed by reperfusion to mimic clinical cold storage and reperfusion. Efficacy was determined by trans-endothelial electrical resistance (TEER), a measure of GJ function, cell viability assays, and ELISAs for pro-inflammatory cytokines. In-vivo, utilizing a cardiac allograft model, Balb/c donor hearts were stored in UW or UW/ACT1 for 6 hrs prior to transplantation into C57Bl/6 recipients. Grafts were harvested 48 hrs post-transplant and cardiac graft injury determined by serum cardiac troponin I and histological analyze. Graft inflammation was assessed by immunohistochemistry specific for neutrophil and macrophages.

Results: In-vitro studies demonstrate that UW/ACT1 solution significantly reduced EC injury and inflammation, as measured by TEER, cell viability and ELISA. In-vivo studies similarly showed that ACT1 pretreatment of the donor organ led to a reduction in IRI, as noted by reduced serum troponin and histological analysis. Subsequent analysis of neutrophil and macrophage infiltration showed pretreatment significantly reduced graft infiltrates.

Conclusion: Taken together these novel findings propose a role for GJ in the pathogenesis of cold storage IRI, and further demonstrate that stabilization of GJ with a novel connexin 43 mimetic, ACT1, significantly inhibits post transplant IRI.

CITATION INFORMATION: Finnegan R, Zhu P, Stephenson S, Patel K, Nadig S, Atkinson C. Cold Storage Stabilization of Gap Junctions Reduces Post Transplant Ischemia Reperfusion Injury. Am J Transplant. 2016;16 (suppl 3).

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