Volume 18, Issue 5, Pages 1019-1025 (May 2010) Adoptive Transfer of Ex Vivo HO-1 Modified Bone Marrow–derived Macrophages Prevents Liver Ischemia and Reperfusion Injury  Bibo Ke, Xiu-Da Shen, Feng Gao, Haofeng Ji, Bo Qiao, Yuan Zhai, Douglas G Farmer, Ronald W Busuttil, Jerzy W Kupiec-Weglinski  Molecular Therapy  Volume 18, Issue 5, Pages 1019-1025 (May 2010) DOI: 10.1038/mt.2009.285 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 In vitro and in vivo X-Gal staining. (a,b) The expression of β-gal gene in bone marrow–derived macrophages (BMDMs). Cells were transfected with Ad-β-gal, incubated for 48 hours, and then stained with X-Gal. Note: The expression of β-gal (blue cells) was >90% in BMDMs (b), as compared with control BMDMs (a). (c,d) The expression of β-gal gene in mouse livers after infusion of Ad-β-gal-transfected BMDMs. Note: The expression of β-gal was 40–50% in ischemic livers after transfer of BMDMs transfected with Ad-β-gal (d), as compared with control livers (c). Representative of three experiments; magnification, ×200. Molecular Therapy 2010 18, 1019-1025DOI: (10.1038/mt.2009.285) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Western blot analysis of HO-1, Bcl-2/Bcl-xL protein expression after transfection with Ad-HO-1/Ad-β-gal or HO-1 siRNA/NS siRNA supplemented with CoPP in BMDMs. The expression was probed by using rabbit anti-mouse HO-1, Bcl-2, and Bcl-xL Abs. Lane 1, BMDMs alone; lane 2, BMDMs transfected with HO-1 siRNA plus CoPP (10 µg/ml); lane 3, BMDMs transfected with nonspecific siRNA plus CoPP (10 µg/ml); lane 4, BMDMs transfected with Ad-HO-1; lane 5, BMDMs transfected with Ad-β-gal; lane 6, BMDMs plus CoPP (10 µg/ml). Note: Selectively increased expression of HO-1, Bcl-2/BclxL in Ad-HO-1-treated BMDMs (lane 4), as compared with Ad-β-gal (lane 5). Diminished expression of HO-1, Bcl-2/Bcl-xL in BMDMs treated with HO-1 siRNA plus CoPP (lane 2), compared with nonspecific siRNA plus CoPP (lane 3). Anti-β-actin Ab was used to assure equal protein amounts between samples. Data shown are representative of three separate experiments. Molecular Therapy 2010 18, 1019-1025DOI: (10.1038/mt.2009.285) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Liver tissue evaluation after 90 minutes of warm ischemia followed by 6 hours of reperfusion. (a) Quantitative RT-PCR-assisted measurement of HO-1 mRNA. Note: Increased HO-1 expression after treatment with Ad-HO-1-transfected BMDMs (*P < 0.01), as compared with Ad-β-gal. Decreased HO-1 expression in HO-1 siRNA treated group (**P < 0.005), as compared with nonspecific siRNA and Ad-HO-1 groups. Mean ± SD; n = 3–4 samples/group. (b) Hepatocellular damage, as analyzed by sGOT levels (IU/l). Note: Decreased sGOT levels in mice treated with Ad-HO-1-transfected BMDMs, as compared to Ad-β-gal, HO-1 siRNA, or BMDMs controls (*P < 0.05). Increased sGOT levels after infusion of HO-1 siRNA, as compared with nonspecific control siRNA (*P < 0.05). Mean ± SD; n = 4–6/group. (c) Neutrophil accumulation, as analyzed by MPO enzymatic activity (U/g) in ischemic liver lobes. Note: MPO activity significantly decreased in mice treated with Ad-HO-1-transfected BMDMs, as compared with those given Ad-β-gal or BMDMs (*P < 0.05). HO-1 siRNA increased MPO activity, as compared with nonspecific siRNA (*P < 0.05). Mean ± SD; n = 4–6/group. Molecular Therapy 2010 18, 1019-1025DOI: (10.1038/mt.2009.285) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Histological evaluation of liver tissue after 90 minutes of warm ischemia followed by 6 hours of reperfusion. (a) Representative histological findings. (A) Sham control; (B) treatment with HO-1 siRNA (Suzuki score = 3.5 ± 0.55); (C) treatment with nonspecific scrambled control siRNA (score = 1.8 ± 0.98); (D) treatment with BMDMs only (score = 3.0 ± 0.63); (E) treatment with Ad-HO-1-transfected BMDMs (score = 0.8 ± 0.75); (F) treatment with Ad-β-gal-transfected BMDMs (score = 3.3 ± 0.52); n = 4–6 mice/group; original magnification, ×200. (b) TUNEL-assisted detection of apoptosis. Note: Dense infiltration by apoptotic cells in mouse livers receiving HO-1 siRNA (B), BMDMs (D), and Ad-β-gal-transfected BMDMs (F), as compared with nonspecific siRNA (C; P < 0.0001). Decreased frequency of apoptotic cells in mice given Ad-HO-1-transfected BMDMs (E; P < 0.0005), as compared with HO-1 siRNA or Ad-β-gal groups. The results were scored semiquantitatively by averaging the number of apoptotic cells (mean ± SD)/field at ×200 magnification. A minimum of six fields was evaluated/sample. Representative of 4–6 mice/group. Molecular Therapy 2010 18, 1019-1025DOI: (10.1038/mt.2009.285) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Analysis of liver tissue exposed to 90 minutes of warm ischemia followed by 6 hours of reperfusion. (a) Caspase-3 activity. Note: Treatment with Ad-HO-1-transfected BMDMs but not with Ad-β-gal or BMDMs alone decreased caspase-3 activity (*P < 0.001). Augmented caspase-3 levels after infusion of HO-1 siRNA, as compared with nonspecific control siRNA (*P < 0.001). Mean ± SD; n = 4–6/group. (b) Western blot analysis of HO-1, Bcl-2, Bcl-xl, and caspase-3 gene products. Lane 1, sham; lane 2, BMDMs alone; lane 3, HO-1 siRNA; lane 4, scrambled siRNA; lane 5, Ad-HO-1-transfected BMDMs; lane 6, Ad-β-gal-transfected BMDMs. Note: Selectively increased expression of HO-1, Bcl-2/Bcl-xl, and markedly decreased cleaved caspase-3 levels in mice treated with Ad-HO-1-transfected BMDMs, as compared with HO-1 siRNA or Ad-β-gal. Infusion of HO-1 siRNA decreased HO-1, Bcl-2/Bcl-xl, and increased cleaved caspase-3 expression. Representative of three different experiments. Molecular Therapy 2010 18, 1019-1025DOI: (10.1038/mt.2009.285) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Quantitative RT-PCR-assisted expression of mRNA coding for TNF-α/IL-1β, IFN-γ/E-selectin, and IP-10/MCP-1 in hepatic lobes at 6 hours after 90 minutes of warm ischemia. Note: The expression of TNF-α/IL-1β, IFN-γ/E-selectin, and IP-10/MCP-1 remained depressed in mice treated with Ad-HO-1-transfected BMDMs (P < 0.05), as compared with respective controls. Each column represents the mean ± SD; n = 4–6 samples/group. Molecular Therapy 2010 18, 1019-1025DOI: (10.1038/mt.2009.285) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions