Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Pathology and Laboratory Medicine

Supervisor

Zhang, Zhuxu

2nd Supervisor

Jevnikar, Anthony

Co-Supervisor

Abstract

The clinical relevance of ischemia-reperfusion injury (IRI) in the context of organ transplantation is well-established. IRI is associated with various forms of programmed cell death (PCD), of which necroptosis is particularly significant. Necroptosis is an inflammatory form of PCD that promotes alloimmunity and adversely affects allograft viability and function. We have recently shown the role of cyclophilin D (CypD), a critical mediator of mitochondrial permeability transition pore (mPTP) formation, in necroptosis. In this study, we investigated the downstream mechanism of hypoxia/reoxygenation-induced necroptosis and the effect of CypD inhibition in mitigating IRI-induced allograft injury and the subsequent alloimmune response in a clinically relevant model of cardiac transplantation. Our data indicate that inhibition of caspases during cold hypoxia-reoxygenation injury decreases apoptosis but increases necroptosis and that inhibition of CypD attenuates hypoxia/reoxygenation-induced necroptosis (n=3; p≤0.001). Interestingly, we found that hypoxia/reoxygenation-induced necroptosis involves apoptosis-inducing factor (AIF) translocation to the nucleus and that AIF silencing also attenuates hypoxia/reoxygenation-induced necroptosis (n=3; p≤0.001). Our in vivo studies confirm that CypD deficiency in ischemia-treated donor hearts mitigates IRI and allograft rejection (n=8; p=0.008). Our findings suggest that CypD inhibition following transplantation substantially attenuates necroptosis and mitigates allograft injury and the subsequent alloimmune response. Our data also indicate that AIF may be the downstream effector molecule that executes IRI-induced DNA damage in necroptosis. As such, targeting mitochondrial permeability may be a plausible approach in formulating therapeutic strategies aimed at improving allograft viability and function.

Summary for Lay Audience

Organ transplantation is essential for patients with end-stage organ disease. However, the procedure itself carries its own set of risks. One of the most pertinent risks is damage to the donor organ after its blood supply is cut off. This type of injury is associated with cell death and is called ischemic injury. It may be severe enough to render the donor organ unusable. Despite stringent protocols and guidelines for organ procurement, preservation and transportation, current strategies aimed at reducing ischemic injury are insufficient. Therefore, targeting cell death pathways in ischemic injury may be a better approach. Before such strategies can be developed, a thorough understanding of the cell death pathways in ischemic injury is required. In this study, we investigated a type of cell death called necroptosis. Necroptosis is a severe form of cell death that triggers the recipient's immune response against the donor organ. Hence, inhibiting necroptosis can help prolong donor organ survival following transplantation.

Although necroptosis is involved in ischemic injury, the precise cellular mechanism(s) involved are not yet clear. According to recent research, a specialized cellular structure called mitochondrion that generates the cells' energy may be involved in necroptosis. Our data from this study indicate that mitochondrial components called 'Cyclophilin D' and 'Apoptosis-Inducing Factor' play essential roles in necroptosis. We used pharmacological agents and genetic engineering techniques to inhibit the function of Cyclophilin D and Apoptosis-Inducing Factor in individual cells and found that it protects against ischemic injury. We then extended our findings to an experiment simulating real-life conditions using mice hearts. We found that inhibiting Cyclophilin D and Apoptosis-Inducing Factor provides protection against ischemic injury and helps promote donor heart survival. We hope to extend our findings to clinical studies and believe that our research will make the organ transplantation process better.

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