Protection by vascular endothelial growth factor against sinusoidal endothelial damage and apoptosis induced by cold preservation.
BACKGROUND
It is well known that sinusoidal endothelial cell (SEC) damage during cold preservation of liver tissue is closely involved in early graft failure. The objective of this study was to investigate the involvement of apoptosis in the SEC damage induced by cold preservation and to demonstrate the protective effect of vascular endothelial growth factor (VEGF) on SEC injury, including apoptotic changes.


METHODS
Isolated SECs and liver tissue of Wistar rats were cold-preserved in University of Wisconsin (UW) solution, and the protective effect of VEGF was then investigated. Isolated SECs were cultured for 24 hr, and divided into the following 3 groups: Group A, in which the cells were cultured for an additional 27 hr, Group B, in which the cells were cold-preserved in UW solution for 3 hr, and then recultured for 24 hr, and Group C, in which 20 ng/ml of VEGF was added to both the culture medium and the UW solution of cells cultured according to the Group B protocol. Each group of SECs was morphologically examined using the phase contrast microscopic method and the transmission electron microscopic method (TEM), and quantitatively analyzed using the WST-1 assay. Rat livers were cold-preserved in UW solution and divided into the VEGF(+) group and the VEGF(-) group, depending on whether VEGF was added or not. Each group of livers were analyzed by scanning electron microscopic method (SEM) after 24 hr of preservation. The hyaluronic acid uptake rate (HUR) was also determined after 6 hr of preservation. After 24 hr of preservation and 6 hr of reperfusion, tissues were examined by TEM and by the terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labeling (TUNEL) assay.


RESULTS
The phase contrast microscopic method and the WST-1 assay showed a protective effect of VEGF against the injury to isolated SECs during cold preservation and subsequent reculturing. Apoptosis was detected immediately by TEM after isolation of SECs, and the number of apoptotic cells increased with the incubation time. This increase was accelerated after cold preservation. The scanning electron microscopic method and the hyaluronic acid uptake rate showed a protective effect of VEGF against SEC damage in the cold-preserved livers. In the liver tissue, the TEM and the TUNEL assay detected apoptosis of SECs only after cold preservation and subsequent reperfusion. VEGF suppressed the apoptosis of SECs induced by cold preservation in both isolated cells and liver tissue.


CONCLUSIONS
We demonstrated that SEC damage in the cold preservation of liver tissue was caused mainly by apoptosis, which required subsequent reperfusion. Moreover, isolated SECs showed spontaneous occurrence of apoptotic changes during culture, and these changes were accelerated by the preceding cold preservation. This is the first report to demonstrate the apoptotic changes of SECs seen here were inhibited by VEGF.
Moriga
T
T
Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University.
Arii
S
S
Takeda
Y
Y
Furuyama
H
H
Mizumoto
M
M
Mori
A
A
Hanaki
K
K
Nakamura
T
T
Fujioka
M
M
Imamura
M
M
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015925:Cryopreservation; D016228:Endothelial Growth Factors / Q000494:pharmacology*; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D006820:Hyaluronic Acid / Q000493:pharmacokinetics; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology*; D008222:Lymphokines / Q000494:pharmacology*; D008297:Male; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000473:pathology / Q000503:physiopathology / Q000517:prevention & control; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D006820:Hyaluronic Acid