In vitro modeling of nonhypoxic cold ischemia–reperfusion simulating lung transplantation
Received 1 December 2008; received in revised form 2 April 2009; accepted 26 May 2009.
Objective
Although anoxia/reoxygenation of cultured cells has been used to model lung ischemia–reperfusion injury, this does not accurately mimic events experienced by lung cells while a lung is retrieved from a donor, stored, and transplanted. We developed an in vitro model of nonhypoxic ischemia–reperfusion injury to simulate these events.
Methods
Human umbilical vein endothelial cells underwent simulated cold ischemia by replacing 37°C culture media with 4°C Perfadex (Vitrolife, Kungsbacka, Sweden) solution for 5 hours in 100% O2. Culture dishes were allowed to warm to room temperature for 1 hour (implantation), and then Perfadex solution was replaced with 37°C culture media (reperfusion).
Results
During cold ischemia, the human umbilical vein endothelial cell filamentous actin cytoskeleton quickly became rearranged, and gaps developed in the previously confluent monolayer occupying 20% of the surface area. Simulated reperfusion resulted in reorganization to a confluent monolayer. Development of gaps was not due to enhanced necrosis based on lactate dehydrogenase retention assay. Endothelial cytoskeletal rearrangement could account for early edema caused by ischemia–reperfusion injury with reperfusion. Mitogen-activated protein kinase and nuclear factor κB activation occurred with simulated reperfusion despite normoxia. Levels of the proinflammatory cytokines interleukin 6 and interleukin 8 were significantly increased in media at the end of reperfusion.
Conclusions
Exposing human umbilical vein endothelial cells to simulated cold ischemia without hypoxia causes reversible cytoskeletal alterations, activation of inflammatory pathways, and elaboration of cytokines. Because this model accurately depicts events occurring during lung transplantation, it will be useful to explore mechanisms regulating lung cell response to this unique form of ischemia–reperfusion injury.
aDivision of Cardiothoracic Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC
bDepartment of Thoracic Surgery, Division of Thoracic Surgery, European Institute of Oncology, Milan, Italy
cCystic Fibrosis/Pulmonary Research and Treatment Center and the Division of Pulmonary and Critical Care Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC
dDepartment of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC
Address for reprints: Thomas M. Egan, MD, MSc, Division of Cardiothoracic Surgery, Department of Surgery, University of North Carolina at Chapel Hill, 3040 Burnett-Womack Bldg, CB #7065, Chapel Hill, NC 27599-7065.
Dr Casiraghi was a visiting research scholar at the UNC–Chapel Hill Division of Cardiothoracic Surgery from San Raffaele Hospital, Milan, Italy.
Supported by a Cystic Fibrosis Foundation grant EGAN02P0 (TME) and National Institute of Health grants R01 HL63159 (TME), RO1 HL080322 (SHR), and PO1 HL45100 (KB).
ABSTRACT