Impact of Mitochondrial Permeability on Endothelial Cell Immunogenicity in Transplantation

ABSTRACTBackgroundMicrovascular endothelial cells (ECs) are central to an allograft's immunogenicity. Cold ischemia and reperfusion injury associated with static cold storage and warm reperfusion activates ECs and increases the immunogenicity of the allograft. Following reperfusion, mitochondrial permeability transition pore (mPTP) opening contributes to mitochondrial dysfunction in the allograft, which correlates to alloimmune rejection. Current understanding of this relationship, however, centers on the whole allograft instead of ECs. This study aimed to elucidate the relationship between EC mPTP opening and their immuno-phenotype.MethodsMitochondrial metabolic fitness and glycolysis in ECs were assessed in parallel with metabolic gene microarray postreperfusion. NIM811 was used to inhibit mPTP opening to rescue mitochondrial fitness. The immunogenicity of NIM811-treated ECs was determined via levels of EC's pro-inflammatory cytokines and allogeneic CD8+ T cell co-cultures. Finally, EC surface expression of adhesion, co-stimulatory, co-inhibitory, MHC-I molecules, and MHC-I machinery protein levels were characterized.ResultsGenes for glycolysis, tricarboxylic acid cycle, fatty acid synthesis, gluconeogenesis were upregulated at 6 hours postreperfusion but either normalized or downregulated at 24 hours postreperfusion. As mitochondrial fitness was reduced, glycolysis increased during the first 6 hours postreperfusion. EC treatment with NIM811 during the early postreperfusion period rescued mitochondrial fitness and reduced EC immunogenicity by decreasing CCL2, KC release, and VCAM-1, MHC-I, TAP1 expression.ConclusionsStatic cold storage and warm reperfusion leads to a reduction in mitochondrial fitness in microvascular ECs due to mPTP opening. Further, mPTP opening promotes increased EC immunogenicity that can be prevented by NIM811 treatment. Background Microvascular endothelial cells (ECs) are central to an allograft's immunogenicity. Cold ischemia and reperfusion injury associated with static cold storage and warm reperfusion activates ECs and increases the immunogenicity of the allograft. Following reperfusion, mitochondrial permeability transition pore (mPTP) opening contributes to mitochondrial dysfunction in the allograft, which correlates to alloimmune rejection. Current understanding of this relationship, however, centers on the whole allograft instead of ECs. This study aimed to elucidate the relationship between EC mPTP opening and their immuno-phenotype. Methods Mitochondrial metabolic fitness and glycolysis in ECs were assessed in parallel with metabolic gene microarray postreperfusion. NIM811 was used to inhibit mPTP opening to rescue mitochondrial fitness. The immunogenicity of NIM811-treated ECs was determined via levels of EC's pro-inflammatory cytokines and allogeneic CD8+ T cell co-cultures. Finally, EC surface expression of adhesion, co-stimulatory, co-inhibitory, MHC-I molecules, and MHC-I machinery protein levels were characterized. Results Genes for glycolysis, tricarboxylic acid cycle, fatty acid synthesis, gluconeogenesis were upregulated at 6 hours postreperfusion but either normalized or downregulated at 24 hours postreperfusion. As mitochondrial fitness was reduced, glycolysis increased during the first 6 hours postreperfusion. EC treatment with NIM811 during the early postreperfusion period rescued mitochondrial fitness and reduced EC immunogenicity by decreasing CCL2, KC release, and VCAM-1, MHC-I, TAP1 expression. Conclusions Static cold storage and warm reperfusion leads to a reduction in mitochondrial fitness in microvascular ECs due to mPTP opening. Further, mPTP opening promotes increased EC immunogenicity that can be prevented by NIM811 treatment. Correspondence information: Satish N. Nadig, MD PhD, FACS, 96 Jonathan Lucas Street, MSC 613, CSB 409, Charleston, SC 29425. nadigsn@musc.edu Authorship statement: D.T. participated in research design, performance of the research, data analysis, and manuscript preparation. C.A., S.N. participated in research design, data analysis, and manuscript preparation. J.K. participated in research design and data analysis. S.M. participated in research design. S.E. participated in performance of the research Disclosure: The authors declare no conflicts of interest. Funding: NIH/NIBIB K08 EB019495-01A1 NIH/NHLBI Predoctoral Fellowship T32 HL007260 NIH/MSTP Predoctoral Fellowship T32 GM008716 NIH/NCATS KL2 TR001452 & UL1 TR001450 Patterson Barclay Memorial Foundation Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.

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