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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 18, 2003 as doi:10.1096/fj.03-0229fje. |
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* Immunobiology Research Center, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA;
The Dumont-UCLA Transplant Center, David Geffen School of Medicine at UCLA, Los Angeles, California, USA;
Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA; and
Instituto Gulbenkian de Ciência, Oeiras, Portugal
3Correspondence: Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Avenue, Boston, MA 02215, USA. E-mail: msoares{at}caregroup.harvard.edu
SPECIFIC AIMS
By releasing Fe2+ from heme, heme oxygenase-1 (HO-1) induces the expression of the Fe2+-sequestering protein ferritin, which acts as an antioxidant by limiting the ability of Fe2+ to participate in the generation of free radicals through the Fenton reaction. The aim of this study was to test the hypothesis that expression of the ferritin heavy chain (H-ferritin) gene may have cytoprotective effects that could be used in a therapeutic manner such as to suppress ischemia reperfusion injury (IRI) of transplanted organs.
PRINCIPAL FINDINGS
The main findings of this manuscript are that overexpression of H-ferritin protects rat livers from IRI and prevents hepatocellular damage upon transplantation into syngeneic recipients. The protective effect of H-ferritin is associated with its ability to inhibit endothelial cell (EC) and hepatocyte apoptosis in vivo and in vitro.
1. H-ferritin protects EC from undergoing apoptosis
Control EC (pcDNA3-transfected) showed 60–70% apoptotic cells after exposure to tumor necrosis factor 
(TNF-) in the presence of the transcription inhibitor Actinomycin D (Act.D; Fig. 1
A). Expression of pcDNA3/H-ferritin suppressed TNF--mediated EC apoptosis (10% apoptotic EC; Fig. 1A
). This protective effect was observed in the EC line 2F-2B as well in primary BAEC (Fig. 1A
). The antiapoptotic effect of H-ferritin was also observed with other proapoptotic stimuli such as etoposide or serum deprivation (Fig. 1B
). This effect was dose-dependent, showing protection from 1 ng to 100 ng of the pcDNA3/H-ferritin expression vector per 3 x 105 cells. A level of pcDNA3/H-ferritin over 100 ng was toxic, as evidenced by an increasing number of apoptotic cells in TNF-/Act.D-stimulated cells as well as in control cells (H-ferritin plus Act.D only; Fig. 1C
).
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2. Recombinant adenovirus-mediated H-ferritin expression protects livers from ex vivo reperfusion injury
Rat livers exposed to prolonged cold ischemia [University of Wisconsin (UW) solution, 4°C, 24 h] showed severe signs of injury once reperfused ex vivo with whole syngeneic blood. Injury was evidenced by the relative low increase in portal blood flow (from 0.63±0.076 ml/min/g at time 0 to 1.13±0.23 at ml/min/g at 120 min) and bile production (from 0.00338±0.0078 ml/g at time 0 to 0.025±0.01 ml/g at 120 min; data not shown; see full paper online), as well as by a significant increase in alanine aminotransferase (ALT) release (from 7.2±4.9 IU/l at time 0 to 173±71 IU/l at 120 min) following reperfusion. Similar results were obtained in ß-galactosidase recombinant adenovirus-transduced livers in terms of portal blood flow (from 0.626±0.079 ml/min/g at time 0 to 1.035±0.105 ml/min/g at 120 min), bile production (from 0.0005±0.0002 ml/g at time 0 to 0.03±0.0085 ml/g at 120 min), and ALT release (from 12.5±10.5 IU/l at time 0 to 148±92 IU/l at 120 min). Unlike untreated or ß-galactosidase-transduced groups, H-ferritin-transduced livers showed significantly (P<0.01) greater increases in portal blood flow (from 0.62±0.099 ml/min/g at time 0 to 1.372±0.133 ml/min/g at 120 min) and bile production (from 0.00621±0.0029 ml/g at time 0 to 0.043±0.0088 at 120 min; data not shown; see full paper online) following reperfusion. Further, ALT release in the H-ferritin-transduced livers remained at relatively low levels (from 9.3±4.5 IU/l at time 0 to 68.6±14.8 IU/l at 120 min). At 2 h of reperfusion, myeloperoxidase activity, a marker of neutrophil-mediated oxidative stress injury, was significantly inhibited (P<0.05) in H-ferritin-transduced livers (0.736±0.58 units/g) as compared with untreated (1.35±0.227 units/g) or ß-galactosidase-transduced (3.12±0.9 units/g) livers. These results support the notion that overexpression of H-ferritin protects livers from IRI, despite prolonged periods of cold ischemia. Livers transduced with the H-ferritin gene also had a significantly better preservation of their histological detail as compared with nontransduced (P<0.05) or ß-galactosidase (P<0.05)-transduced livers, as assessed by standard Suzuki’s pathological scoring (data not shown).
3. Recombinant adenovirus-mediated H-ferritin expression prevents IRI following orthotopic liver transplantation
Rat livers exposed to prolonged cold ischemia (UW solution, 4°C, 24 h) showed severe signs of hepatocellular damage following transplantation into syngeneic recipients. This was evidenced by high aspartate aminotransferase (AST) serum levels (3928±1455 IU/L), 24 h post-transplant. Similar results were obtained in recipients transplanted with ß-galactosidase recombinant adenovirus-transduced livers (4887±500 IU/L). In marked contrast, in animals bearing liver transplants transduced with the H-ferritin recombinant adenovirus, AST release (1368±550.8 IU/L) was significantly inhibited as compared with ß-galactosidase/untreated groups (P<0.05). That H-ferritin-transduced livers were protected from IRI is supported by the demonstration that up to 90% of H-ferritin-transduced livers survived for >14 days when transplanted into syngeneic SD recipients. In marked contrast, only 40–50% of nontransduced or ß-galactosidase-transduced livers survived >14 days when transplanted into syngeneic recipients (Fig. 2
). The relative number of cells undergoing apoptosis in H-ferritin-transduced livers transplanted into syngeneic recipients was significantly reduced as compared with nontransduced or ß-galactosidase-transduced livers transplanted under the same conditions (data not shown; see full paper online). This finding is consistent with the hypothesis that the antiapoptotic effect of H-ferritin may contribute to its overall cytoprotective function in transplanted livers (Fig. 3
).
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CONCLUSIONS AND SIGNIFICANCE
Our present findings unveil the previously unrecognized antiapoptotic function of H-ferritin and suggest that expression of this gene can be used in a therapeutic manner. By its ability to suppress liver IRI, overexpression of the H-ferritin gene may result in the safer use of liver transplants despite prolonged periods of cold ischemia.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0229fje; doi: 10.1096/fj.03-0229fje 
2 Contributed equally to this study.
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