In vitro modeling of nonhypoxic cold ischemia–reperfusion simulating lung transplantation

Abstract

Objective Although anoxia/reoxygenation of cultured cells has been employed to model lung ischemia-reperfusion injury (IRI), 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 non-hypoxic IRI to simulate these events. Methods Human umbilical vein endothelial cells (HUVECs) underwent simulated cold ischemia by replacing 37°C culture media with 4°C Perfadex™ solution for 5 hours in 100% O2. Culture dishes were allowed to warm to room temperature for 1 hour (implantation), then Perfadex™ was replaced with 37°C culture media (reperfusion). Results During cold ischemia, HUVEC 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 LDH retention assay. Endothelial cytoskeletal rearrangement could account for early edema due to IRI with reperfusion. MAPK and NF-κB activation occurred with simulated reperfusion despite normoxia. Pro-inflammatory cytokines IL-6 and IL-8 were significantly elevated in media at the end of reperfusion. Conclusions Exposing HUVECs 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 IRI.

Report of the ISHLT Working Group on Primary Lung Graft Dysfunction Part III: Mechanisms: A 2016 Consensus Group Statement of the International Society for Heart and Lung Transplantation

“…Simulated reperfusion resulted in activation of MAPKs and NF-κB and expression of interleukin (IL)-6 and IL-8 by endothelial cells. 20 In addition, in a cell culture model of simulated warm ischemia, inter-endothelial cell gap formation was prevented by a competitive TLR4 antagonist, implicating a direct link between pulmonary edema and TLR signaling. 19 …”

Nitric Oxide Ventilation of Rat Lungs from Non-Heart-Beating Donors Improves Posttransplant Function

“…We developed a model of simulated cold lung IRI with human umbilical vein endothelial cells (HUVECs) and showed that replacing cold media with Perfadex resulted in rapid alteration in actin cytoskeleton associated with gap formation in the endothelial monolayer (22). To determine the impact of exposure to 40 ppm NO on endothelial behavior, human pulmonary microvascular endothelial cells (HMVECs) were grown to confluence in nine P35 dishes.…”

δV1-1 Reduces Pulmonary Ischemia Reperfusion-Induced Lung Injury by Inhibiting Necrosis and Mitochondrial Localization of PKCδ and p53

“…Additionally, in the lung transplant setting, donor lungs are inflated with oxygen and stored in cold preservation solution. Previously, two studies have used cell culture to simulate lung transplantation (7,8). These studies suggested that detailed molecular mechanisms could be explored using such models.…”