Organ preservation solutions increase endothelial permeability and promote loss of junctional proteins.
OBJECTIVE
To investigate the effects of the organ preservation solutions UW and Plegisol on endothelial permeability; occludin and vascular endothelial (VE)-cadherin content in human umbilical vein endothelial cells (HUVEC); and junctional localization of these proteins after exposure to these solutions.


BACKGROUND
Organ preservation for transplantation is limited by several challenges, including loss of tissue function, tissue injury, and tissue edema. Occludin and VE-cadherin are responsible for maintaining and regulating the endothelial solute barrier. Several studies have noted organ edema and dysfunction with preservation, as well as gaps between endothelial cells suggesting that 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 using FITC-dextran tracer during the reperfusion phase. Occludin and VE-cadherin content at different time points was measured by Western blotting. Treated groups were also examined by immunofluorescence for occludin, VE-cadherin, and F-actin.


RESULTS
Compared with untreated controls, cold preservation for 3 and 6 hours increased endothelial permeability after rewarming, which appears to depend on the duration of cold exposure. Monolayers exposed to 3 hours of cold preservation 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 points. Western blotting demonstrated that occludin content was decreased to a similar extent with all solutions after 3 hours of cold preservation. Six hours of cold preservation in Plegisol reduced the occludin content significantly 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 and discontinuous staining of occludin and VE-cadherin with all cold preservation protocols; changes in F-actin organization were observed at 3 and 6 hours after cold preservation.


CONCLUSIONS
The changes in occludin, VE-cadherin, and F-actin content and organization and increased permeability associated with cold storage demonstrate that alterations of the tight and adherens junctions may underlie organ edema associated with cold organ preservation. These data also suggest that novel strategies to maintain the content and integrity of endothelial junctional proteins may provide an important therapeutic avenue for organ preservation.
Trocha
S D
SD
Department of Surgery, Louisiana State University Medical Center, Shreveport 71130, USA.
Kevil
C G
CG
Mancini
M C
MC
Alexander
J S
JS
D000241:Adenosine / Q000494:pharmacology; D000493:Allopurinol / Q000494:pharmacology; D001639:Bicarbonates / Q000494:pharmacology; D015153:Blotting, Western; D015820:Cadherins / Q000032:analysis / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007365:Intercellular Junctions / Q000648:ultrastructure; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D062793:Occludin; D019188:Organ Preservation Solutions / Q000494:pharmacology*; D010539:Permeability / Q000187:drug effects; D011189:Potassium Chloride / Q000494:pharmacology; D011887:Raffinose / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors
D001639:Bicarbonates; D015820:Cadherins; D007328:Insulin; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D019188:Organ Preservation Solutions; C041711:St. Thomas' Hospital cardioplegic solution; C055206:University of Wisconsin-lactobionate solution; D012965:Sodium Chloride; D000493:Allopurinol; D011189:Potassium Chloride; D005978:Glutathione; D008274:Magnesium; D000241:Adenosine; D002122:Calcium Chloride; D011887:Raffinose