Date of Award

1-1-2016

Embargo Period

1-1-2019

Document Type

Thesis - MUSC Only

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Satish Nadig

Second Advisor

Carl Atkinson

Third Advisor

Laura Kasman

Fourth Advisor

Omar Moussa

Fifth Advisor

Azizul Haque

Abstract

Purpose: Graft endothelium is the first point of contact with the recipient, and as such it is, arguably, the primary target of the destructive alloimmune response. Events in pre and postoperative phase, such as ischemia reperfusion injury (IRI) and cytokine stimulation, activate and damage the grafted organ, leading to acute rejection and/or allograft vasculopathy (AV), the main manifestation of cardiac chronic dysfunction. Here we investigate the role of the mammalian target of rapamycin (mTOR), rapamycin, on endothelial cell (EC) cytokine production, adhesion molecule expression, co-stimulatory function, and on long-term transplant outcomes using rodent models. Methods: Mouse Aortic Endothelial Cells (MAECs) and Mouse Cardiac Endothelial Cells (MCECs) were exposed to two insults to recapitulate IRI. ECs were either exposed to 6h of cold storage in University of Wisconsin (UW) solution or UW/rapamycin followed by reperfusion, or alternatively cells were exposed to IFNg and TNFa for 6, 24, 48, or 72h with or without rapamycin. Flowcytometry and ELISA techniques were performed to assess the impact of rapamycin on EC adhesion and costimulatory/inhibitory molecules, as well as cytokine production. Transwells and automated cell counter were used for T cell-endothelial cell transmigration experiment. In-vivo, BALB/c aortic grafts were treated with either UW alone or rapamycin- augmented UW solution for 6h in cold hypoxia before transplantation into C57BL/6 recipients for 7 or 28 days. Immune cells infiltrates (neutrophils and T cells) were assessed 7 days posttransplant, using immunohistochemistry (IHC). AV was analyzed 28 days 5 posttransplant, by measuring the percentage of intimal expansion. Results: Rapamycin significantly reduced mouse IL-8 (KC) release by cytokine- activated MAEC and MCEC after both 6 and 24h treatment. Treatment of ECs for 6h had no effect on adhesion and costimulation profiles. However, after 24h treatment, rapamycin upregulated PD-L1 expression by MAEC, and downregulated MCEC expression of VCAM-1. Allogeneic T cell transmigration through the microvascular endothelium was also inhibited by 24h rapamycin treatment. In-vivo, rapamycin relatively reduced neutrophils and T cells recruitment to aortic grafts’ adventitia after 7 days transplants, and AV was significantly reduced by rapamycin therapy 28 days post transplant. Conclusion: Taken together, these results suggest that rapamycin pre-treatment may improve early and the late transplant outcomes. However, further in- vivo studies are required to investigate the impact of rapamycin pre-treatment on the early alloimmune response.

Rights

All rights reserved. Copyright is held by the author.

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