Up-regulation of Bcl-2 through hyperbaric pressure transfection of TGF-beta1 ameliorates ischemia-reperfusion injury in rat cardiac allografts.
BACKGROUND
Oxidative stress after ischemia-reperfusion of cardiac allografts leads to activation of cardiomyocytes and production of cytokines. Bcl-2, an inhibitor of the apoptotic pathway, also has strong antioxidant properties. Ischemia-reperfusion injury after transplantation leads to decreased bcl-2 and increased tumor necrosis factor (TNF)-alpha levels. Transforming growth factor (TGF)-beta1 is known to attenuate ischemia-reperfusion injury and inhibits apoptosis of myofibroblasts. We hypothesize that TGF-beta1, prevents bcl-2 cleavage and increased TNF-alpha production.


METHODS
Rat PVG donor hearts were heterotopically transplanted into ACI recipients. Donor hearts were procured and assigned to groups: (1) intracoronary TGF-beta1 (200 ng/ml) perfusion and pressure at 78 psi for 45 minutes (n = 4); (2) intracoronary TGF-beta1 perfusion and incubation for 45 minutes without pressure (n = 4), (3) saline perfusion and incubation for 45 minutes without pressure (n = 4). Hearts were procured 4 hours after transplantation and analyzed by reverse transcriptase-polymerase chain reaction for bcl-2 mRNA expression, ELISA for TNF-alpha, and for myeloperoxidase activity (MPO).


RESULTS
Bcl-2 decreased in untreated animals (bcl-2:G3PDH ratio = 0.85 +/- 0.73 vs 1.16 +/- 0.11, not significant [NS]), whereas TNF-alpha increased to 669.99 +/- 127.09 vs 276.84 +/- 73.65 pg/mg total protein in controls (p < 0.003). In TGF-beta(1) pressure-treated hearts, bcl-2 was up-regulated (2.49 +/- 0.6 vs 1.16 +/- 0.11, controls, p < 0.005), whereas TNF-alpha was unchanged (396.1 +/- 100.38 vs 276.84 +/- 73.65 pg/mg, NS). Hearts treated with TGF-beta1 and pressure showed significant up-regulation of bcl-2 compared with hearts treated with TGF-beta1 without pressure (2.49 +/- 0.6 vs 1.17 +/- 0.6, p < 0.02). MPO showed no differences.


CONCLUSIONS
Bcl-2 is down-regulated and TNF-alpha up-regulated in this model of ischemia-reperfusion injury. Furthermore, TGF-beta1 is linked to this process and ameliorates reperfusion injury by up-regulating bcl-2 and inhibiting TNF-alpha. Therapeutic overexpression of myocardial TGF-beta1 may be clinically useful to control ischemia-reperfusion injury associated with cardiac transplantation.
Grünenfelder
Jürg
J
Department of Cardiothoracic Surgery, Falk Cardiovascular Research Building, Stanford University School of Medicine, Stanford, California, USA. jurg.grunenfelder@chi.usz.ch
Miniati
Douglas N
DN
Murata
Seiichiro
S
Falk
Volkmar
V
Hoyt
E Grant
EG
Robbins
Robert C
RC
D000818:Animals; D002140:California; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D019254:Genes, bcl-2 / Q000235:genetics* / Q000502:physiology*; D016027:Heart Transplantation / Q000009:adverse effects*; D006931:Hyperbaric Oxygenation; D008297:Male; D016328:NF-kappa B / Q000378:metabolism; D018384:Oxidative Stress / Q000235:genetics; D009195:Peroxidase / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000188:drug therapy* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000627:therapeutic use*; D053773:Transforming Growth Factor beta1; D014184:Transplantation, Homologous / Q000009:adverse effects; D016896:Treatment Outcome; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
D016328:NF-kappa B; D012333:RNA, Messenger; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D009195:Peroxidase