Overexpression of human Bcl-2 in syngeneic rat donor lungs preserves posttransplant function and reduces intragraft caspase activity and interleukin-1β production

David T Cooke, E. Grant Hoyt, Robert C. Robbins

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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)-1β, 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-1β production in syngeneic rat donor lung grafts. Methods. First, 1.0 × 109 plaque-forming units of AdvBcl-2 in phosphate-buffered saline (PBS), AdvNull empty vector in PBS, or PBS alone was administered intratracheally to ACI (RT1a) 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 PaO2 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 PaO2 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-1β 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.

Original languageEnglish (US)
Pages (from-to)762-767
Number of pages6
JournalTransplantation
Volume79
Issue number7
DOIs
StatePublished - Apr 15 2005
Externally publishedYes

Fingerprint

Caspases
Interleukin-1
Lung
Transplants
Phosphates
Isografts
Lung Transplantation
Caspase 9
Pulmonary Veins
Reperfusion Injury
Caspase 3
Reperfusion
Inbred ACI Rats
Enzyme-Linked Immunosorbent Assay
Cold Ischemia
bcl-2 Genes
Pressure
Proteins
Cytochromes c
Transgenes

Keywords

  • Apoptosis
  • Bcl-2
  • Ischemia-reperfusion
  • Lung transplantation
  • Rat

ASJC Scopus subject areas

  • Transplantation
  • Immunology

Cite this

Overexpression of human Bcl-2 in syngeneic rat donor lungs preserves posttransplant function and reduces intragraft caspase activity and interleukin-1β production. / Cooke, David T; Hoyt, E. Grant; Robbins, Robert C.

In: Transplantation, Vol. 79, No. 7, 15.04.2005, p. 762-767.

Research output: Contribution to journalArticle

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abstract = "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)-1β, 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-1β production in syngeneic rat donor lung grafts. Methods. First, 1.0 × 109 plaque-forming units of AdvBcl-2 in phosphate-buffered saline (PBS), AdvNull empty vector in PBS, or PBS alone was administered intratracheally to ACI (RT1a) 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 PaO2 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 PaO2 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-1β 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.",
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AB - 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)-1β, 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-1β production in syngeneic rat donor lung grafts. Methods. First, 1.0 × 109 plaque-forming units of AdvBcl-2 in phosphate-buffered saline (PBS), AdvNull empty vector in PBS, or PBS alone was administered intratracheally to ACI (RT1a) 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 PaO2 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 PaO2 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-1β 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.

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