title: Upregulation of Bcl-2 through caspase-3 inhibition ameliorates ischemia/reperfusion injury in rat cardiac allografts.
abstract: BACKGROUND
  Oxidative stress after ischemia/reperfusion of cardiac allografts leads to cytokine production. Bcl-2, an inhibitor of apoptosis, also has strong antioxidant properties. Caspase-3 is known to cleave bcl-2. This study tests the hypothesis that bcl-2 is downregulated while tumor necrosis factor-alpha (TNF-alpha) levels increase after cardiac transplantation. Furthermore, the use of caspase-3 inhibition was investigated as a strategy for preserving myocardial bcl-2 and mitochondrial cytochrome c after transplantation.
  
  
  RESULTS
  PVG-to-ACI rat heterotopic cardiac transplantations were performed in 4 groups designed with 30 minutes' ischemia and 4 or 8 hours of reperfusion (n=4 per group). Treatment consisted of DEVD-CHO 500 microgram IP per animal to donor and recipient 2 hours before transplantation and 250 microgram IC into allograft. Controls were treated with saline. Grafts were analyzed by reverse transcription-polymerase chain reaction for bcl-2 mRNA, by ELISA for TNF-alpha, for myeloperoxidase activity, and by Western blot for cytochrome c. In untreated groups, bcl-2 mRNA decreased significantly over time, whereas TNF-alpha increased significantly at 4 hours (P=0.003) and returned to baseline after 8 hours' reperfusion (P=NS compared with normal hearts). Treatment with caspase-3 inhibitor showed significant upregulation of bcl-2 mRNA expression after 4 and 8 hours of reperfusion (P<0.001 versus control), with a concomitant decrease in TNF-alpha to baseline levels. Myeloperoxidase activity in all groups was no different from that of normal hearts. Mitochondrial cytochrome c release increased in both control and treatment groups.
  
  
  CONCLUSIONS
  Bcl-2 is actively downregulated and TNF-alpha is upregulated in this model of cardiac allograft ischemia/reperfusion. Furthermore, the caspase-3 pathway is linked to this process, and blockade of caspase-3 can ameliorate reperfusion injury by upregulating bcl-2 and inhibiting TNF-alpha without affecting cytochrome c release.
authors.affiliation: Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: J
authors.initials: J
authors.lastname: Grünenfelder
authors.forename: D N
authors.initials: DN
authors.lastname: Miniati
authors.forename: S
authors.initials: S
authors.lastname: Murata
authors.forename: V
authors.initials: V
authors.lastname: Falk
authors.forename: E G
authors.initials: EG
authors.lastname: Hoyt
authors.forename: M
authors.initials: M
authors.lastname: Kown
authors.forename: M L
authors.initials: ML
authors.lastname: Koransky
authors.forename: R C
authors.initials: RC
authors.lastname: Robbins
mesh_terms: D000818:Animals; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016027:Heart Transplantation / Q000009:adverse effects; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D009206:Myocardium / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D018384:Oxidative Stress; D009195:Peroxidase / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D015427:Reperfusion Injury / Q000209:etiology / Q000378:metabolism / Q000517:prevention & control*; D014184:Transplantation, Homologous; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C108637:aspartyl-glutamyl-valyl-aspartal; D009195:Peroxidase; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases