Calpain inhibition prevents sinusoidal endothelial cell apoptosis in the cold ischemic rat liver.
BACKGROUND
Cold preservation of the liver followed by reperfusion results in sinusoidal endothelial cell (SEC) apoptosis. Calpain-like activity is dramatically increased during reperfusion and inhibition of calpains results in lower graft injury and longer survival. Recently, calpains have been implicated in inducing apoptosis. Our aim was to determine the effect of calpain inhibition on SEC apoptosis.


METHODS
Livers were stored in the University of Wisconsin solution for 24 hr (survival conditions) and 40 hr (nonsurvival conditions) and ex vivo reperfused for 1 hr at 37 degrees C. Calpain-like activity was inhibited in some experiments using an i.p. injection of a selective inhibitor 2 hr before explantation. Apoptosis was quantified using the terminal deoxynucleotidyl trans. ferase-mediated dUTP nick end-labeling assay. Cross-inhibition by the inhibitor was determined for caspases 1 and 3.


RESULTS
Apoptosis of exclusively the SEC was a key feature of reperfusion injury after both storage periods in University of Wisconsin solution after 1 hr normothermic reperfusion. Inhibition of calpain activity with Cbz-Val-Phe methyl ester resulted in a 50% reduction of apoptotic SEC in the 40-hr preserved liver, and an almost complete abrogation of SEC apoptosis after 24 hr preservation. Only minimal cross-inhibition of caspases was determined at high concentrations in vitro by the calpain inhibitor.


CONCLUSIONS
Apoptosis of exclusively SEC is a key feature of reperfusion injury partially mediated through calpain-dependent processes. Calpain inhibition reduces the number of apoptotic SEC. Based on these data and our previous work, calpain inhibition may prove to be useful in clinical transplantation.
Sindram
D
D
Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710, USA.
Kohli
V
V
Madden
J F
JF
Clavien
P A
PA
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002154:Calpain / Q000037:antagonists & inhibitors*; D015925:Cryopreservation; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004727:Endothelium / Q000166:cytology; D008099:Liver / Q000098:blood supply / Q000166:cytology*; D009926:Organ Preservation; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000517:prevention & control*
D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; C095174:N-benzyloxycarbonyl-valyl-phenylalanine methyl ester; D002154:Calpain