Overexpression of human Bcl-2 in syngeneic rat donor lungs preserves posttransplant function and reduces intragraft caspase activity and interleukin-1beta production.
BACKGROUND
A significant cause of primary graft failure in lung transplantation is ischemia-reperfusion (I/R). I/R injury generates proinflammatory cytokines, such as interleukin (IL)-1beta, and activates the caspase-mediated pathways of alveolar epithelial apoptosis. The authors investigated whether gene transfer of the human antiapoptotic protein Bcl-2 by means of intratracheal adenoviral administration would preserve posttransplant lung function and reduce intragraft activated caspase activity and IL-1beta production in syngeneic rat donor lung grafts.


METHODS
First, 1.0 x 10(9) plaque-forming units of AdvBcl-2 in phosphate-buffered saline (PBS), AdvNull empty vector in PBS, or PBS alone was administered intratracheally to ACI (RT1(a)) rats. Then, the left lungs were procured after 24 hr of in vivo incubation and orthotopically transplanted after 1 hr of cold ischemia into syngeneic recipients. After 2 hr of reperfusion, peak inspiratory pressures (PIP) and donor pulmonary vein PaO(2) was measured in all grafts; grafts were then excised and protein extracts were analyzed by enzyme-linked immunosorbent assay (ELISA) and activated caspase-3 and caspase-9 assays.


RESULTS
Human Bcl-2 transgene overexpression in donor lung grafts was demonstrated by ELISA of tissue homogenates. Pretreatment of donor lungs with AdvBcl-2 resulted in reduced PIP and increased lung isograft pulmonary vein PaO(2) compared with AdvNull or PBS-alone treated controls. In addition, AdvBcl-2 pretreatment led to diminished cytochrome c release into cytosolic extracts and reduced intragraft IL-1beta production and inhibited intragraft caspase-3 and caspase-9 activity.


CONCLUSIONS
Adenoviral overexpression of human Bcl-2 protein limits I/R injury in rat lung isografts. These data suggest that the use of Bcl-2 gene transfer to human lung donors may reduce the oxidative stress of pulmonary grafts after transplantation in clinical lung transplantation.
Cooke
David Tom
DT
Department of Cardiothoracic Surgery, Stanford University Medical Center, Stanford, CA 94305, USA. dcooke@partners.org
Hoyt
E Grant
EG
Robbins
Robert C
RC
D000818:Animals; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007375:Interleukin-1 / Q000096:biosynthesis* / Q000378:metabolism; D008168:Lung / Q000201:enzymology / Q000378:metabolism*; D016040:Lung Transplantation; D008297:Male; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014185:Transplantation, Isogeneic
D007375:Interleukin-1; D019253:Proto-Oncogene Proteins c-bcl-2; D045304:Cytochromes c; C505407:CASP3 protein, human; C506433:CASP9 protein, human; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases