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Inhibition of Ferroptosis Enables Safe Rewarming of HEK293 Cells following Cooling in University of Wisconsin Cold Storage Solution

The prolonged cooling of cells results in cell death, in which both apoptosis and ferroptosis have been implicated. Preservation solutions such as the University of Wisconsin Cold Storage Solution (UW) encompass approaches addressing both. The use of UW improves survival and thus extends preservatio...

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Autores principales: Gartzke, Lucas P., Hendriks, Koen D. W., Hoogstra-Berends, Femke, Joschko, Christian P., Strandmoe, Anne-Lise, Vogelaar, Pieter C., Krenning, Guido, Henning, Robert H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10342108/
https://www.ncbi.nlm.nih.gov/pubmed/37446116
http://dx.doi.org/10.3390/ijms241310939
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author Gartzke, Lucas P.
Hendriks, Koen D. W.
Hoogstra-Berends, Femke
Joschko, Christian P.
Strandmoe, Anne-Lise
Vogelaar, Pieter C.
Krenning, Guido
Henning, Robert H.
author_facet Gartzke, Lucas P.
Hendriks, Koen D. W.
Hoogstra-Berends, Femke
Joschko, Christian P.
Strandmoe, Anne-Lise
Vogelaar, Pieter C.
Krenning, Guido
Henning, Robert H.
author_sort Gartzke, Lucas P.
collection PubMed
description The prolonged cooling of cells results in cell death, in which both apoptosis and ferroptosis have been implicated. Preservation solutions such as the University of Wisconsin Cold Storage Solution (UW) encompass approaches addressing both. The use of UW improves survival and thus extends preservation limits, yet it remains unclear how exactly organ preservation solutions exert their cold protection. Thus, we explored cooling effects on lipid peroxidation and adenosine triphosphate (ATP) levels and the actions of blockers of apoptosis and ferroptosis, and of compounds enhancing mitochondrial function. Cooling and rewarming experiments were performed in a cellular transplantation model using Human Embryonic Kidney (HEK) 293 cells. Cell viability was assessed by neutral red assay. Lipid peroxidation levels were measured by Western blot against 4-Hydroxy-Nonenal (4HNE) and the determination of Malondialdehyde (MDA). ATP was measured by luciferase assay. Cooling beyond 5 h in Dulbecco’s Modified Eagle Medium (DMEM) induced complete cell death in HEK293, whereas cooling in UW preserved ~60% of the cells, with a gradual decline afterwards. Cooling-induced cell death was not precluded by inhibiting apoptosis. In contrast, the blocking of ferroptosis by Ferrostatin-1 or maintaining of mitochondrial function by the 6-chromanol SUL150 completely inhibited cell death both in DMEM- and UW-cooled cells. Cooling for 24 h in UW followed by rewarming for 15 min induced a ~50% increase in MDA, while concomitantly lowering ATP by >90%. Treatment with SUL150 of cooled and rewarmed HEK293 effectively precluded the increase in MDA and preserved normal ATP in both DMEM- and UW-cooled cells. Likewise, treatment with Ferrostatin-1 blocked the MDA increase and preserved the ATP of rewarmed UW HEK293 cells. Cooling-induced HEK293 cell death from hypothermia and/or rewarming was caused by ferroptosis rather than apoptosis. UW slowed down ferroptosis during hypothermia, but lipid peroxidation and ATP depletion rapidly ensued upon rewarming, ultimately resulting in complete cell death. Treatment throughout UW cooling with small-molecule Ferrostatin-1 or the 6-chromanol SUL150 effectively prevented ferroptosis, maintained ATP, and limited lipid peroxidation in UW-cooled cells. Counteracting ferroptosis during cooling in UW-based preservation solutions may provide a simple method to improve graft survival following cold static cooling.
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spelling pubmed-103421082023-07-14 Inhibition of Ferroptosis Enables Safe Rewarming of HEK293 Cells following Cooling in University of Wisconsin Cold Storage Solution Gartzke, Lucas P. Hendriks, Koen D. W. Hoogstra-Berends, Femke Joschko, Christian P. Strandmoe, Anne-Lise Vogelaar, Pieter C. Krenning, Guido Henning, Robert H. Int J Mol Sci Article The prolonged cooling of cells results in cell death, in which both apoptosis and ferroptosis have been implicated. Preservation solutions such as the University of Wisconsin Cold Storage Solution (UW) encompass approaches addressing both. The use of UW improves survival and thus extends preservation limits, yet it remains unclear how exactly organ preservation solutions exert their cold protection. Thus, we explored cooling effects on lipid peroxidation and adenosine triphosphate (ATP) levels and the actions of blockers of apoptosis and ferroptosis, and of compounds enhancing mitochondrial function. Cooling and rewarming experiments were performed in a cellular transplantation model using Human Embryonic Kidney (HEK) 293 cells. Cell viability was assessed by neutral red assay. Lipid peroxidation levels were measured by Western blot against 4-Hydroxy-Nonenal (4HNE) and the determination of Malondialdehyde (MDA). ATP was measured by luciferase assay. Cooling beyond 5 h in Dulbecco’s Modified Eagle Medium (DMEM) induced complete cell death in HEK293, whereas cooling in UW preserved ~60% of the cells, with a gradual decline afterwards. Cooling-induced cell death was not precluded by inhibiting apoptosis. In contrast, the blocking of ferroptosis by Ferrostatin-1 or maintaining of mitochondrial function by the 6-chromanol SUL150 completely inhibited cell death both in DMEM- and UW-cooled cells. Cooling for 24 h in UW followed by rewarming for 15 min induced a ~50% increase in MDA, while concomitantly lowering ATP by >90%. Treatment with SUL150 of cooled and rewarmed HEK293 effectively precluded the increase in MDA and preserved normal ATP in both DMEM- and UW-cooled cells. Likewise, treatment with Ferrostatin-1 blocked the MDA increase and preserved the ATP of rewarmed UW HEK293 cells. Cooling-induced HEK293 cell death from hypothermia and/or rewarming was caused by ferroptosis rather than apoptosis. UW slowed down ferroptosis during hypothermia, but lipid peroxidation and ATP depletion rapidly ensued upon rewarming, ultimately resulting in complete cell death. Treatment throughout UW cooling with small-molecule Ferrostatin-1 or the 6-chromanol SUL150 effectively prevented ferroptosis, maintained ATP, and limited lipid peroxidation in UW-cooled cells. Counteracting ferroptosis during cooling in UW-based preservation solutions may provide a simple method to improve graft survival following cold static cooling. MDPI 2023-06-30 /pmc/articles/PMC10342108/ /pubmed/37446116 http://dx.doi.org/10.3390/ijms241310939 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Gartzke, Lucas P.
Hendriks, Koen D. W.
Hoogstra-Berends, Femke
Joschko, Christian P.
Strandmoe, Anne-Lise
Vogelaar, Pieter C.
Krenning, Guido
Henning, Robert H.
Inhibition of Ferroptosis Enables Safe Rewarming of HEK293 Cells following Cooling in University of Wisconsin Cold Storage Solution
title Inhibition of Ferroptosis Enables Safe Rewarming of HEK293 Cells following Cooling in University of Wisconsin Cold Storage Solution
title_full Inhibition of Ferroptosis Enables Safe Rewarming of HEK293 Cells following Cooling in University of Wisconsin Cold Storage Solution
title_fullStr Inhibition of Ferroptosis Enables Safe Rewarming of HEK293 Cells following Cooling in University of Wisconsin Cold Storage Solution
title_full_unstemmed Inhibition of Ferroptosis Enables Safe Rewarming of HEK293 Cells following Cooling in University of Wisconsin Cold Storage Solution
title_short Inhibition of Ferroptosis Enables Safe Rewarming of HEK293 Cells following Cooling in University of Wisconsin Cold Storage Solution
title_sort inhibition of ferroptosis enables safe rewarming of hek293 cells following cooling in university of wisconsin cold storage solution
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10342108/
https://www.ncbi.nlm.nih.gov/pubmed/37446116
http://dx.doi.org/10.3390/ijms241310939
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