*Purpose: A growing body of evidence indicates that mitochondrial permeability transition pore (mPTP) formation plays a crucial role in necroptosis – a programmed form of necrosis. We have recently shown that inhibiting cyclophilin D (CypD), a critical mediator of mPTP, decreases necroptosis and that CypD-deficient heart grafts exhibit prolonged survival. In this study, we extended our findings to a clinically relevant scenario of prolonged cold ischemic organ storage followed by transplantation.

*Methods: To model ischemic insult in vitro, endothelial cells (ECs) were exposed to hypoxia in oxygen-depleted glucose-free medium. To model the reperfusion event, ECs were then transferred to a normoxic incubator in oxygenated glucose-rich medium. Necroptosis was induced using TNFα and a pan-caspase inhibitor and monitored by live cell imaging system and flow cytometry. For in vivo studies, C57BL/6 heart grafts were treated with cold ischemia for 4 hours before transplantation into BALB/c mice. Histopathological grading of ischemia-reperfusion injury (IRI) was done by a pathologist.

*Results: Our data indicate that necroptosis plays a significant role in hypoxia induced EC death. We have also shown that both inhibition and silencing of CypD, a critical component of mPTP, decreased hypoxia induced necroptosis. Interestingly, apoptosis inducing factor (AIF) silencing also decreased hypoxia induced necroptosis. Following IRI, apoptosis inducing factor (AIF), a mitochondrial oxidoreductase, translocates to the nucleus and induces DNA fragmentation. We have found that pre-transplant ischemia aggravates heart transplant rejection. Ischemia-treated grafts had short survival time compared to non-ischemic grafts (n=4, p=0.03). Interestingly, CypD deficiency in donor heart grafts attenuates such graft rejection (n=8, p=0.008).

*Conclusions: Our data indicate that inhibition of caspases during hypoxia decreases apoptosis but increases necroptosis. Our studies indicate that CypD and AIF play significant roles in EC necroptosis following IRI and that AIF may be the downstream effector of necroptosis. Targeting mitochondrial permeability and downstream pathways involved in necroptosis is critical in formulating clinically applicable intervention strategies aimed at reducing IRI induced PCD associated with organ transplantation.

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