Superoxide-induced apoptosis of activated rat hepatic stellate cells Chinnasamy Thirunavukkarasu, Simon Watkins, Stephen A.K. Harvey, Chandrashekhar R. Gandhi  Journal of Hepatology  Volume 41, Issue 4, Pages 567-575 (October 2004) DOI: 10.1016/j.jhep.2004.06.023 Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Apoptosis of aHSC in the fibrotic liver. Liver of control rat (phenobarbital treatment alone) (A and C) shows normal architecture, with α-SMA-positive cells restricted to portal vein and hepatic artery walls (A) and desmin-positive cells scattered in the sinusoidal areas (C). No TUNEL positive cells stained for α-SMA or desmin are seen in A and C. Abundant α-SMA-(B) and desmin-positive (D) cells are seen in the fibrous areas after CCl4 treatment, a significant number of which being TUNEL-positive (arrows). Original magnification ×200. Images of greater magnification (×1000) exhibiting double staining for α-SMA+TUNEL and desmin+TUNEL are shown in the insets. Journal of Hepatology 2004 41, 567-575DOI: (10.1016/j.jhep.2004.06.023) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Extracellular superoxide dismutase (EC-SOD) and heme oxygenase-1 expression in the cirrhotic liver. A. A representative gel showing expression of EC-SOD mRNA in control (CT) and cirrhotic (CR) liver. PCR was carried out by denaturation (30s at 96°C), annealing (30s at 60°C) and extension (30s at 72°C), and the final extension at 72°C for 10min [17]. The same amount of cDNA was used to determine β-actin mRNA expression for normalization. The numbers of PCR cycles employed (30 for β-actin and 50 for EC-SOD) were in the linear range of the reaction for each product. The bar graph shows the ratio of EC-SOD mRNA vs β-actin mRNA±SD from 3 independent experiments. B. Immunohistochemical localization of heme oxygenase-1 in control (A) and cirrhotic (B) livers is shown. Note that no heme oxygenase-1 is seen in the fibrous regions of the cirrhotic liver. Magnification ×200. Journal of Hepatology 2004 41, 567-575DOI: (10.1016/j.jhep.2004.06.023) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Effect of SO on DNA synthesis and cell viability. Cells were incubated with 1mM hypoxanthine and indicated concentrations of xanthine oxidase for 24h. (A) For determination of DNA synthesis, the cells were incubated in serum-free medium containing 1μCi/ml [3H]thymidine for 4h at 37°C, washed with ice-cold PBS, treated with ice-cold 5% TCA (10min), and washed once with TCA followed by 95% ethanol. After digestion with 5% (w/v) SDS, radioactivity was determined. Results are expressed as CPM/μg DNA. (B) Cell viability was determined by the MTT assay as described in Section 2. All values are averages of triplicate determinations ±SD from representative experiments performed at least 4 times. *P<0.05; **P<0.01; #P<0.001 vs 0mU/ml xanthine oxidase. Journal of Hepatology 2004 41, 567-575DOI: (10.1016/j.jhep.2004.06.023) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Flow cytometric analysis of aHSCs after SO treatment. (A, B) Cell cycle analysis was performed using flow cytometry of cells stained with propidium iodide following incubation with 1mM hypoxanthine in the absence or presence of 2.0mU/ml xanthine oxidase for 24h. The cell cycle phases were analyzed by diploid staining profiles and ModFit software program. Forward and side light scatter characteristics were used to exclude cell debris (size and granularity) from the analysis. The percentage of cell aggregates (doublets) and other background were also eliminated using peak vs integral gating of cells. Peaks ‘C’, ‘D’, ‘E’ and ‘F’ represent cells in subG0, G0/G1, S and G2/M phases respectively. The effect of various concentrations of xanthine oxidase on the cell cycle is shown in Table 1. (C, D) The cells were stained with annexin-Vcy3 and 7-AAD following 24h incubation with 1mM hypoxanthine±2mU/ml xanthine oxidase (XO). Flow cytometry was performed as described in Section 2. Normal cells (annexin-Vcy3- and 7-AAD-negative, E3); cells in early apoptosis (annexin-Vcy3-positive and 7-AAD-negative, E4); and cells in late apoptosis/necrosis (annexin-Vcy3- and 7-AAD-positive, E2). For each measurement, 10,000 events were counted. Journal of Hepatology 2004 41, 567-575DOI: (10.1016/j.jhep.2004.06.023) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Time-course of cell death during SO treatment. The cells were stained with annexin-Vcy3 and 7-ADD at indicated time points during incubation with 1mM hypoxanthine and 2mU/ml xanthine oxidase. Flow cytometry was performed as described in Section 2. Other details are given in the legend to Fig. 4. Values are averages±SD from representative experiments performed at least 4 times. Journal of Hepatology 2004 41, 567-575DOI: (10.1016/j.jhep.2004.06.023) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Determination of apoptosis by TUNEL assay. TUNEL assay was performed on the aHSCs treated with 1mM hypoxanthine and 0 (A) or 2mU/ml (B) xanthine oxidase for 24h as described in Section 2. A representative experiment performed in triplicates at least 3 times shows apoptotic nuclei (green). Journal of Hepatology 2004 41, 567-575DOI: (10.1016/j.jhep.2004.06.023) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Effect of SO on DNA laddering and DNA fragmentation. Cells were treated with 1mM hypoxanthine±2mU/ml xanthine oxidase for 24h. DNA was extracted and separated by electrophoresis (A) or the DNA fragmentation assay (B) was performed as described in Section 2. Values are averages±SD from representative experiments performed at least 4 times. Journal of Hepatology 2004 41, 567-575DOI: (10.1016/j.jhep.2004.06.023) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 8 Superoxide-induced release of cytochrome c. Cells were treated with 1mM hypoxanthine and 2mU/ml xanthine oxidase for indicated time points. Western analysis for cytochrome c released into the cytosol was performed as described in Section 2. Journal of Hepatology 2004 41, 567-575DOI: (10.1016/j.jhep.2004.06.023) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 9 Effect of SO on caspases, Bcl-2 family of proteins and PARP. Cells were treated with 1mM hypoxanthine and 2mU/ml xanthine oxidase for indicated time points. Western analysis for various proteins was performed as described in Section 2. Journal of Hepatology 2004 41, 567-575DOI: (10.1016/j.jhep.2004.06.023) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 10 Caspase-3 like activity in SO-treated cells. The cells were treated for the indicated time periods with 1mM hypoxanthine and 0 or 2mU/ml xanthine oxidase. Caspase-3 activity was measured by using caspase fluorescent assay kit (BD Biosciences Clontech). *P<0.01 vs control; **P< 0.001 vs control. Journal of Hepatology 2004 41, 567-575DOI: (10.1016/j.jhep.2004.06.023) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 11 Effect of SO on the nuclear translocation of NFκB. Cells were treated with 1 mM hypoxanthine and 0 or 2mU/ml xanthine oxidase for indicated time points. Western analysis for p65 subunit of NFκB was performed in the nuclear and cytosolic extracts as described in Section 2. Journal of Hepatology 2004 41, 567-575DOI: (10.1016/j.jhep.2004.06.023) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions