Gene transfer of heat shock protein 70 protects lung grafts from ischemia-reperfusion injury.
BACKGROUND
We recently demonstrated that heat stress induction of heat shock protein 70 (HSP70) in donor animals before harvest decreases posttransplant ischemia-reperfusion injury in preserved rat lung isografts. The purpose of this study was to investigate the feasibility of HSP70 gene transfection into rat lung isografts using an adenoviral vector, and to study the effects of gene expression on subsequent ischemia-reperfusion injury.


METHODS
In preliminary studies to determine the optimal titer, animals were injected with various titers of adenovirus-HSP70 (saline, 5 x 10(9), 1 x 10(10), and 2 x 10(10) plaque forming units [pfu]) and sacrificed 5 days after injection. To determine the optimal exposure time, animals were sacrificed at different times (0, 6, 24, and 72 hours) after intravenous injection of adenovirus-HSP70. In a subsequent series of transplant experiments, donors were allocated to three groups according to transfection strategy. Group 1 (n = 8) donors received 5 x 10(9) pfu adenovirus-HSP70 intravenously, group 2 (n = 7) donors received 5 x 10(9) pfu adenovirus-beta-galactosidase (as a virus control), and group 3 (n = 7) donors received saline and served as a negative control. Twenty-four hours after treatment all grafts were harvested and stored for 18 hours before orthotopic left lung transplantation. Twenty-four hours after implantation animals were sacrificed for assessment. The expression of HSP70 was assessed by Western blot analysis.


RESULTS
In preliminary studies, HSP70 was detectable even at low titers (5 x 10(9) pfu) of adenovirus-HSP70, and was detectable at low levels as early as 6 hours after intravenous administration. Heat shock protein 70 expression was maximal at 24 hours. In transplant experiments, Western blot analysis showed that overexpression of HSP70 occurred in the HSP70-transfected lungs. The mean arterial oxygenation 24 hours after reperfusion in group 1 was superior in comparison with other groups (p < 0.05). Wet to dry weight ratio (p < 0.05) and myeloperoxidase activity (p < 0.05) were also significantly less in group 1 grafts compared with the other groups.


CONCLUSIONS
This study demonstrates that in vivo, donor adenovirus-mediated gene transfer of HSP70 decreases subsequent ischemia-reperfusion injury in rat lung isografts.
Hiratsuka
M
M
Department of Surgery, Washington University School of Medicine, Barnes Jewish Hospital, St. Louis, Missouri 63110, USA.
Mora
B N
BN
Yano
M
M
Mohanakumar
T
T
Patterson
G A
GA
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D005240:Feasibility Studies; D015870:Gene Expression; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D016040:Lung Transplantation; D008297:Male; D009195:Peroxidase / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D015427:Reperfusion Injury / Q000517:prevention & control*; D014162:Transfection; D014185:Transplantation, Isogeneic
D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D009195:Peroxidase