Jung-Lye Kim1, Brenda F Reader2, Curtis Dumond3, Yonggyu Lee1, Nahush A Mokadam4, Sylvester M Black5, Bryan A Whitson6. 1. COPPER Laboratory, Ohio State University Wexner Medical Center, Columbus, Ohio; Department of Surgery, Ohio State University Wexner Medical Center, Columbus, Ohio. 2. COPPER Laboratory, Ohio State University Wexner Medical Center, Columbus, Ohio; Comprehensive Transplant Center, Ohio State University Wexner Medical Center, Columbus, Ohio. 3. COPPER Laboratory, Ohio State University Wexner Medical Center, Columbus, Ohio. 4. Department of Surgery, Ohio State University Wexner Medical Center, Columbus, Ohio. 5. COPPER Laboratory, Ohio State University Wexner Medical Center, Columbus, Ohio; Department of Surgery, Ohio State University Wexner Medical Center, Columbus, Ohio; Comprehensive Transplant Center, Ohio State University Wexner Medical Center, Columbus, Ohio. 6. COPPER Laboratory, Ohio State University Wexner Medical Center, Columbus, Ohio; Department of Surgery, Ohio State University Wexner Medical Center, Columbus, Ohio; Comprehensive Transplant Center, Ohio State University Wexner Medical Center, Columbus, Ohio. Electronic address: bryan.whitson@osumc.edu.
Abstract
BACKGROUND: Lung transplant ischemia-reperfusion injury is typified by toxic metabolites and oxygen free radicals leading to worse graft function. Catalase is an enzyme involved in oxidative-stress detoxification. We hypothesize that direct delivery of highly concentrated polyethylene glycol-catalase (PEG-CAT) during normothermic ex vivo lung perfusion (EVLP) significantly reduces ischemia-reperfusion injury. METHODS: To demonstrate protection, primary culture porcine endothelial cells were treated with PEG-CAT (0 to 1250 U/mL) in a model of oxidative stress (400 μM H2o2). In vivo, rat lungs were subjected to 0 hours or 1 hour of warm ischemic injury and 2 hours of EVLP with or without PEG-CAT. Perfusate was collected throughout the perfusion duration and tissue was collected at the end. Tissue and perfusate underwent analysis for markers of apoptosis and a biometric signature of lung health. RESULTS: Uptake of PEG-CAT into primary endothelial cells was demonstrated with Alexa Fluor 488-labeled PEG-CAT. Oxidatively stressed cells pretreated with PEG-CAT had significantly decreased cytotoxicity and caspase 3/7 activity and increased cell viability and cell membrane integrity. In a rat model of warm ischemia with EVLP, PEG-CAT improved allograft viability as measured by indications of cell membrane integrity (lactate dehydrogenase and hyaluronic acid), presence of vasoconstrictive peptides (endothelin-1 and big endothelin-1) released from endothelial cells, and reduced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling). CONCLUSIONS: In vitro and ex vivo, PEG-CAT protects against oxidative stress-induced cytotoxicity, maintains cellular metabolism, and mitigates lung ischemia-reperfusion in an experimental model. Together, these data suggest that PEG-CAT is a potential therapeutic target for donor organs at risk for ischemia-reperfusion injury.
BACKGROUND: Lung transplant ischemia-reperfusion injury is typified by toxic metabolites and oxygen free radicals leading to worse graft function. Catalase is an enzyme involved in oxidative-stress detoxification. We hypothesize that direct delivery of highly concentrated polyethylene glycol-catalase (PEG-CAT) during normothermic ex vivo lung perfusion (EVLP) significantly reduces ischemia-reperfusion injury. METHODS: To demonstrate protection, primary culture porcine endothelial cells were treated with PEG-CAT (0 to 1250 U/mL) in a model of oxidative stress (400 μM H2o2). In vivo, rat lungs were subjected to 0 hours or 1 hour of warm ischemic injury and 2 hours of EVLP with or without PEG-CAT. Perfusate was collected throughout the perfusion duration and tissue was collected at the end. Tissue and perfusate underwent analysis for markers of apoptosis and a biometric signature of lung health. RESULTS: Uptake of PEG-CAT into primary endothelial cells was demonstrated with Alexa Fluor 488-labeled PEG-CAT. Oxidatively stressed cells pretreated with PEG-CAT had significantly decreased cytotoxicity and caspase 3/7 activity and increased cell viability and cell membrane integrity. In a rat model of warm ischemia with EVLP, PEG-CAT improved allograft viability as measured by indications of cell membrane integrity (lactate dehydrogenase and hyaluronic acid), presence of vasoconstrictive peptides (endothelin-1 and big endothelin-1) released from endothelial cells, and reduced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling). CONCLUSIONS: In vitro and ex vivo, PEG-CAT protects against oxidative stress-induced cytotoxicity, maintains cellular metabolism, and mitigates lung ischemia-reperfusion in an experimental model. Together, these data suggest that PEG-CAT is a potential therapeutic target for donor organs at risk for ischemia-reperfusion injury.
Authors: Yong Gyu Lee; Jung-Lye Kim; Andre F Palmer; Brenda F Reader; Jianjie Ma; Sylvester M Black; Bryan A Whitson Journal: J Vis Exp Date: 2021-10-28 Impact factor: 1.424
Authors: Danielle M Peterson; Eliza W Beal; Brenda F Reader; Curtis Dumond; Sylvester M Black; Bryan A Whitson Journal: ASAIO J Date: 2022-01-20 Impact factor: 3.826