Organ preservation solutions increase endothelial permeability and promoteloss of junctional proteins

Objective To investigate the effects of the organ preservation solutions UW and Plegi sol on endothelial permeability; occludin and vascular endothelial (VE)-cad herin content in human umbilical vein endothelial cells (HUVEC); and juncti onal localization of these proteins after exposure to these solutions. Summary Background Data Organ preservation for transplantation is limited by several challenges, in cluding loss of tissue function, tissue injury, and tissue edema. Occludin and VE-cadherin are responsible for maintaining and regulating the endothel ial solute barrier. Several studies have noted organ edema and dysfunction with preservation, as well as gaps between endothelial cells sug gesting th at disorganization of junctional proteins (e.g., occludin and VE-cadherin) is responsible for interstitial edema. Methods HUVEC monolayers were treated with 4 degrees C UW and Plegisol for 3 and 6 hours and then reperfused with normal buffer. Permeability was examined usi ng FITC-dextran tracer during the reperfusion phase. Occludin and VE-cadher in content at different time points was measured by Western blotting. Treat ed groups were also examined by immunofluorescence for occludin, VE-cadheri n, and F-actin. Results Compared with untreated controls, cold preservation for 3 and 6 hours incre ased endothelial permeability after rewarming, which appears to depend on t he duration of cold exposure. Monolayers exposed to 3 hours of cold preserv ation did not have increased permeability in the first hour after rewarming but had significantly increased permeability after the first hour and all subsequent time points. Monolayers exposed to 6 hours of cold preservation had increased permeability after the first hour and at all later time point s. Western blotting demonstrated that occludin content was decreased to a s imilar extent with all solutions after 3 hours of cold preservation. Six ho urs of cold preservation in Plegisol reduced the occludin content significa ntly compared with UW and control. VE-cadherin content was unchanged after 3 hours of cold preservation but was dramatically reduced in all groups at 6 hours. Immunofluorescent staining demonstrated junctional gap formation a nd discontinuous staining of occludin and VE-cadherin with all cold preserv ation protocols; changes in F-actin organization were observed at 3 and 6 h ours after cold preservation. Conclusion The changes in occludin, VE-cadherin, and F-actin content and organization and increased permeability associated with cold storage demonstrate that al terations of the tight and adherens junctions may underlie organ edema asso ciated with cold organ preservation. These data also suggest that novel str ategies to maintain the content and integrity of endothelial junctional pro teins may provide an important therapeutic avenue for organ preservation.