Date of Award

1-1-2016

Embargo Period

1-1-2019

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Carl Atkinson

Second Advisor

Satish Nadig

Third Advisor

Laura Kasman

Fourth Advisor

Andrew Goodwin

Fifth Advisor

Xue-Zhong Yu

Abstract

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 promoting 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 cardiac and aortic allograft models, Balb/c donor hearts and aortas were stored in UW or UW/ACT1 for 6 hrs prior to transplantation into C57Bl/6 recipients. Grafts were harvested 48 hrs post-transplant, in the cardiac allograft model, to determine cardiac graft ischemia reperfusion as measured by serum cardiac troponin I and histological analyze. For aortic grafts, aortas were harvested at 28 days to determine the impact of graft pre-treatment on the development of chronic rejection. Efficacy was again determined by pathological indices. Grafts, hearts and aortas, were assessed for innate inflammatory cells (neutrophils and macrophages) and T cell infiltration by immunohistochemistry at their specified harvest time points. Results: In-vitro studies demonstrated that UW/ACT1 solution significantly reduced EC injury and inflammation, as measured by TEER, cell viability and ELISA. In-vivo studies showed that ACT1 pretreatment of the donor heart led to a reduction in IRI, as noted by reduced serum troponin and histological analysis. Furthermore, donor aortic graft pretreatment with ACT1 reduced the size and development of chronic rejection as shown by histological matrices. Subsequent analysis of neutrophil, macrophage, and T cell infiltration showed pretreatment significantly reduced graft infiltrates, acutely (48hrs) and chronically (28 days). 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 posttransplant IRI, and the later development of chronic rejection.

Rights

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