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Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis
BBRC Biochemical and Biophysical Research Communications 307 (2003) 765–770 www.elsevier.com/locate/ybbrc
Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis Toshio Tsutsui,a Hiroko Koide,a Hiroko Fukahori,a Katsuhiro Isoda,a Shinji Higashiyama,a Isamu Maeda,a Fumi Tashiro,b Eiji Yamato,b Jun-Ichi Miyazaki,b Junji Yodoi,c Masaya Kawase,a and Kiyohito Yagia,* a
c
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan b Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan Department of Biological Responses, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan Received 12 June 2003
Abstract A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liverspecific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Thioredoxin; Adenovirus; Hepatocytes; Bioartificial liver; Apoptosis; Necrosis; Gene transfection
A bioartificial liver support system (BAL) using liver cells has been studied for the treatment of patients with fulminant hepatic failure or as a transitional treatment prior to liver transplantation [1]. As the first-generation BAL, clinical trials in the USA have been carried out using porcine hepatocytes cultured on collagen-coated dextran microcarriers and packed in hollow-fiber-type bioreactors [2]. With the aim of developing a high-performance BAL, our previous work was concerned with improving the scaffolds for hepatocyte cultivation. We showed that chitosan and a polyamidoamine dendrimer are effective in the preparation of scaffolds for hepatocytes [3,4]. We also showed that chemical modifications of these polymers are useful for improving the cell
* Corresponding author. Fax: +81-6-6879-8195. E-mail address: [email protected] (K. Yagi).
0006-291X/03/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-291X(03)01253-1
attachment and the maintenance of cell viability and functions [5,6]. The development of a second-generation BAL will require improvement of the cell itself. In a previous study, the gene encoding glutamine synthetase was introduced into hepatoma cells, which were then packed in a bioreactor. These recombinant HepG2 cells improved ammonia removal when applied to pigs with hepatic failure [7]. Tzanakakis et al. [8] transfected hepatocyte spheroids with the cytochrome P450 2B1 gene. In this study, we used the unique strategy of not only enhancing one liver function but of maintaining all liver functions by introducing into hepatocytes a gene that confers resistance to various kinds of stress. We chose the thioredoxin (TRX) gene as a promising candidate for conferring this protection on primary cultured hepatocytes. TRX is a lowmolecular-weight redox protein (12 kDa) found in both prokaryotic and eukaryotic cells [9]. Cysteine residues at
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the conserved, Cys–Gly–Pro–Cys–Lys active site of TRX undergo reversible oxidation–reduction catalyzed by the NADPH-dependent flavoprotein, thioredoxin reductase. TRX was originally identified as a reducing cofactor for ribonucleotide reductase. Recently, the protein has been shown to be involved in cellular protective mechanisms against various kinds of stress, such as ischemia/reperfusion, X-ray irradiation, and inflammatory cytokines [10]. In this study, we constructed an adenovirus vector using the Cre-loxP recombination system [11] and used it to deliver the human TRX gene into primary cultured rat hepatocytes. We demonstrated that the introduced gene confers protection against oxidative stress, apoptosis, and necrosis on hepatocytes. Materials and methods Media. The basal medium used consisted of 100 U/ml penicillin G, 100 lg/ml streptomycin, 50 ng/ml amphotericin B, and 100 ng/ml aprotinin (Nacalai Tesque, Kyoto, Japan) in William’s medium E (WE, ICN Biochemicals, Costa Mesa, CA, USA). Medium A consisted of 10% fetal bovine serum (FBS, ICN Biochemicals) in basal medium. Medium B consisted of 1 nM insulin (Sigma Chemicals, St. Louis, MO, USA) and 1 nM dexamethasone (Nacalai Tesque, Kyoto, Japan) in Medium A. Culture conditions. Hepatocytes were isolated from male Sprague– Dawley rats weighing 150–200 g by perfusing the liver with collagenase (from Clostridium histolyticum Type IV; Sigma Chemicals, St. Louis, MO, USA) according to the method of Seglen [12]. The animals were housed in an air-conditioned room at 22 1 °C prior to the experiment. The experiments were conducted according to the ethical guidelines of the Graduate School of Pharmaceutical Sciences, Osaka University. Cells were seeded at a density of 1 105 cells/cm2 onto 12well polystyrene culture plates (Nippon Becton Dickinson, Tokyo, Japan). After a 6-h cultivation in Medium B, the cells were cultivated in Medium A. The medium was changed every 24 h. Medium A containing 50 lM NADPH was used for the experiments on the effect of recombinant adenoviruses. Construction of recombinant adenoviral vectors. We constructed an E1- and E3-deleted recombinant adenovirus vector using the pALC3 cosmid, which was generated by removing the E3 region of the adenoviral genome from the pALC cosmid, which already lacks E1 [11] (Fig. 1). The TRX expression cassette, shown in Fig. 2, was flanked by SwaI sites and included the CAG promoter [13], human TRX cDNA, and the rabbit b-globin polyA signal. The expression cassette was inserted into the unique SwaI site of pALC3, to make pALC3-TRX. The recombinant adenovirus vector expressing human TRX (Adv-TRX) was produced by transfecting 293 cells with pALC3-TRX and the titer of the virus stock was determined as described previously [11]. Briefly, virus suspensions were serially diluted with medium and added to a 96-well multiplate seeded with 293 cells. After 10 days, the virus titer was calculated by examining the wells for the presence or absence of a cytopathic effect. The control vector Adv-lacZ was constructed using the Escherichia coli lacZ gene instead of the TRX gene. RT-PCR analysis. RNA was extracted from cultured hepatocytes infected with the recombinant adenovirus. TRX gene expression was analyzed using the following primers: forward 50 -TCTGACTGACCGC GTTACTC-30 and reverse 50 -TCATCCACATCTACTTCAAGGA-30 . Albumin gene expression was analyzed using the following primers: forward 50 -TGAACGTTGCCGCTAGGTTT-30 and reverse 50 -CTTCT GGAGTAATCATAAAG-30 . Glutamine synthetase gene expression was analyzed using following primers: forward 50 -ACCTGACAAATG GCCCTAC-30 and reverse 50 -ACCAAAAAATAACCCCCC-30 . bActin gene expression was analyzed using following primers: forward
Fig. 1. Structure of recombinant adenoviruses. pALC3 (A) consisted of a 29-kb adenoviral genome and 7.1-kb cosmid vector. The cosmid backbone was flanked by loxP sites, indicated by filled arrowheads, and included a cos site, kanamycin-resistance gene (Km), bacterial ori, and ampicillin-resistance gene (Ap). Expression cassettes were inserted into the pALC3 cosmid. The cassette contained TRX cDNA (B) and lacZ (C), resulting in Adv-TRX and Adv-lacZ, respectively. 50 -CATCCCCCAAAGTTCTAC-30 and reverse 50 -CCAAAGCCTTC ATACATC-30 . Assays. Adherent cells were treated with trypsin at 37 °C for 5 min and viability was measured with the trypan blue dye exclusion test. The amount of urea was determined according to the method of Ormsby [14]. The detection of dead cells inside hepatocyte spheroids was carried out using the Vybrant apoptosis assay kit #4 (Molecular Probes Inc., Eugene, OR, USA). Flow cytometry. Hepatocytes (3.8 105 cells) were stained with propidium iodide (PI) and flow cytometric profiles were analyzed using a FACSCalibur analyzer and CELLQuest software (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA).
Results Expression of human TRX in rat hepatocytes Isolated rat hepatocytes were plated onto polystyrene plates and infected with the recombinant
T. Tsutsui et al. / Biochemical and Biophysical Research Communications 307 (2003) 765–770
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Protective effect of TRX against cell death
Fig. 2. Expression of the human TRX gene. Hepatocytes were infected with Adv-TRX at a moi of 10 (lane 1), 1 (lane 2), 0.1 (lane 3), and 0 (lane 4, control). After 48 h of culture, the total RNA was isolated and RT-PCR analysis was carried out using primers for TRX (A) and bactin (B). In cells treated similarly, intracellular TRX was analyzed by Western blotting (C). Hepatocyte viability 2 days after the infection was examined (D). Noninfected (moi ¼ 0) and Adv-lacZ-infected (moi ¼ 1) cells were used as controls (open bars). The initial cell density was 1 105 cells/cm2 . An asterisk indicates a value that is significantly different from that of the controls (p < 0:01).
adenovirus at various concentrations. Infection efficiency was examined using the control vector, AdvlacZ, prior to TRX gene transfection. After 24 h of infection at a multiplicity of infection (moi) of 10, 1, and 0.1, about 100%, 80%, and 20% of the hepatocytes expressed lacZ, respectively. The cytotoxic effects of the adenovirus on the viability and function of hepatocytes were examined using the Adv-lacZ control. The infection at moi <10 did not affect viability or urea synthesis activity (data not shown). The expression of the introduced TRX gene was evaluated by RT-PCR and Western blotting using an anti-human TRX monoclonal antibody. As shown in Figs. 2A–C, the TRX gene was expressed in rat hepatocytes in an moi-dependent manner, especially for the amount of TRX protein. Fig. 2D shows the viability of hepatocytes 2 days after infection. When infected with the Adv-TRX at a moi of 1, more than 80% of the initially plated cells were maintained. On the other hand, only 45% of the initial number of uninfected and Adv-lacZ-infected cells were maintained. These results show that human TRX expression improves rat hepatocyte viability, while lacZ expression does not. Thus, we infected hepatocytes with Adv-TRX at a moi of 1 in the succeeding experiments.
We then examined whether the TRX gene conferred oxidative stress resistance on cultured hepatocytes. Transfected and nontransfected cells were treated with 1 mM hydrogen peroxide for 24 h. The number of viable cells was then determined and urea synthesis activity was measured using 2 mM NH4 Cl as a substrate. As shown in Figs. 3A and B, both the viability and urea synthesis activity of the Adv-TRX-infected cells were significantly higher than those of the control. The ratio of apoptotic cells was analyzed using FACS after staining the cells with PI (Fig. 3C). About 80% of the noninfected cells underwent apoptosis after hydrogen peroxide treatment. The ratio of apoptotic cells in the Adv-TRX-infected hepatocytes was only 24%, much less than in the noninfected control. These results indicate that the introduction of the TRX gene confers oxidative stress resistance on hepatocytes. Since the adenovirus vector supports only the transient expression of an inserted gene, we next examined the effect of TRX on cell viability for 2 weeks (Fig. 4A).
Fig. 3. Resistance of TRX gene-transfected hepatocytes to oxidative stress. After 48 h of culture, including an initial 24-h infection period, cells were incubated with 1 mM hydrogen peroxide for 24 h. Viable cells were then counted (A) and urea synthesis activity was measured (B). Filled and open bars represent Adv-TRX- and Adv-lacZ-infected cells at moi ¼ 1, respectively. An asterisk indicates a value significantly different from that of the control (p < 0:01). The ratio of apoptotic cells was analyzed by FACS (C). After 48 h of culture, including an initial 24-h infection period, Adv-TRX- (black) and Adv-lacZ- (gray) infected cells at moi ¼ 1 were incubated with 1 mM hydrogen peroxide for 24 h. All cells including detached cells were recovered and stained with PI for FACS analysis.
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Fig. 5. RT-PCR analysis of liver-specific gene expression. Hepatocytes were infected with Adv-TRX at a moi of 10 (lane 1), 1 (lane 2), 0.1 (lane 3), and 0 (lane 4, control). After 3 days of culture, the total RNA was isolated and RT-PCR analysis was carried out using primers for albumin, glutamine synthetase (GS), and b-actin.
Fig. 4. Maintenance of cell viability and function in TRX genetransfected hepatocytes. Adv-TRX- (j) and Adv-lacZ- (d) infected cells were cultured for 2 weeks including an initial 24-h infection period. Cell viability and urea synthesis activity were analyzed after infection at the times indicated. An asterisk indicates a value significantly different from that of the control (p < 0:01).
After 2 weeks of cultivation, more than 50% of the AdvTRX-infected cells were still viable. On the other hand, about 70% and 85% of the initially plated control cells were nonviable at 5 days and 2 weeks of culture,
respectively. TRX gene expression also had a significant effect on the maintenance of urea synthesis activity within 2 weeks of culture (Fig. 4B). Fig. 5 shows the expression of liver functions other than urea synthesis in the Adv-TRX-infected hepatocytes. RT-PCR analyses of the albumin and glutamine synthetase (GS) genes were carried out after 3 days of hepatocyte culture. Albumin and GS gene expressions in the Adv-TRX-infected cells were much higher than those in the control. These findings indicated that the viable hepatocytes transfected with the TRX gene would be expected to maintain many differentiated functions in vitro for an extended period. Protective effect of TRX in hepatocyte spheroids Hepatocytes were plated onto culture dishes coated with poly-L -lysine and cultured with 50 ng/ml EGF and 10 lg/ml insulin to induce the formation of multicellular spheroids. The recombinant adenovirus was added at the start of the culture and removed after 24 h. The AdvTRX- and Adv-lacZ-infected hepatocytes formed
Fig. 6. Confocal laser scanning microscopic analyses of apoptosis and necrosis in hepatocyte spheroids. Hepatocyte spheroids formed after infection with Adv-TRX (A–D) and Adv-lacZ (E–H). (A, E) Phase-contrast microscopic observations; (B–D, F–H) confocal laser scanning microscopic observations. Spheroids were stained with PI (B, F) and YO-PRO-1 (C, G). Merged images of the red- and green-stained cells (D, H).
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spheroids after 2–3 days of culture. Neither recombinant adenovirus affected spheroid formation. The Adv-TRX and Adv-lacZ-infected spheroids were then stained with the green fluorescent YO-PRO-1 dye, which stains both apoptotic and necrotic cells, and PI, which stains necrotic cells (Fig. 6). A number of internal cells were stained with both dyes in the Adv-lacZ-infected control spheroids. The merged images of the two stainings indicated that both necrotic and apoptotic cells were present inside the control spheroid. On the other hand, almost no cells were stained with either dye in the Adv-TRX-infected hepatocyte spheroids. Hence, the introduction of the TRX gene did not affect spheroid formation and conferred resistance against necrosis and apoptosis inside the spheroids.
Discussion With the goal of developing a high-performance BAL system, we have sought to maintain all liver functions in primary cultured hepatocytes by introducing certain genes into them. This effort requires a vector with a high transfer efficiency for mammalian cells and a gene that confers resistance to stresses from the external environment. We selected adenovirus as the vector for this study because it is generally used for nonproliferative cells such as isolated hepatocytes. Moreover, its transfection efficiency is much higher than that of nonviral vectors. The TRX gene was chosen because the translated protein can be expected to scavenge reactive oxygen species that can cause apoptosis and necrosis. We confirmed that the human TRX gene was successfully introduced using the adenovirus vector and expressed at both the transcription and translation levels in rat hepatocytes. The introduced TRX gene was expressed for at least 10 days, by RT-PCR analysis (data not shown). This maintenance of gene expression is considered to be satisfactory, especially for recombinant hepatocytes used for BAL. Hepatocyte spheroids have a well-known morphology [15,16] and have been used for BAL applications [17,18]. We have shown in this study that Adv-TRX infection does not affect spheroid formation. These results indicate that the adenoviral vector used in this study is very suitable for the introduction of genes into hepatocytes in a BAL system. The function of the human TRX expressed in rat hepatocytes was verified by hydrogen peroxide treatment experiments. Hepatocytes that express human TRX showed improved viability and liver-specific functions after treatment with 1 mM hydrogen peroxide for 24 h. FACS analysis of DNA fragmentation showed that the human TRX suppressed apoptosis in hepatocytes. Annexin V binding, which detects early-phase apoptosis, observed by confocal laser scanning microscopy also showed the protective effect of TRX (data not
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shown). Recently, extensive studies on the relationship between TRX and oxidative stress-induced apoptosis have been carried out [19–21]. In the absence of stress, apoptosis-signal-regulating kinase 1 (ASK1) exists as an inactive complex with the reduced form of TRX. Oxidative stress causes the oxidation of TRX, which disrupts the ASK–TRX complex, thereby activating ASK1 to induce apoptosis [22]. TRX overexpression appears to protect the TRX–ASK1 complex from such disruption. Here we showed that the introduction of the human TRX gene could extend the life span of hepatocytes that were conventionally cultured on polystyrene plates. Generally, hepatocytes that are isolated and cultured under artificial conditions suffer from various stresses. In our recent study, primary hepatocytes cultured on polystyrene plates expressed high levels of stress-inducible HSPs 60 and 70 [23]. Overexpressed TRX seems to confer protection against stresses induced in vitro. In addition to our findings in monolayer culture, we showed that the introduction of the TRX gene has a protective effect against cell death in a spheroidal cell system. Since hepatocyte spheroids are three-dimensional, highly packed multicellular aggregates, the internal cells undergo necrosis due to oxygen and nutrient deficiencies. In this study we showed that the introduction of the TRX gene extensively suppresses necrosis in these internal cells. In considering a BAL system, it is very important to maintain liver functions as well as viability, because gene-transfected hepatocytes might transform into the dedifferentiated state. Detoxification, such as ammonia removal, is an important liver-specific function. Although primary-cultured hepatocytes usually lose this function rapidly, the TRX gene-transfected cells maintained urea synthesis activity significantly longer and at a higher level than the control cells throughout a 2-week cultivation period. In addition to urea cycle enzyme activity, we showed that expression of the albumin and GS genes was maintained in the TRX gene-transfected cells. These findings indicated that TRX overexpression in primary culture suppressed dedifferentiation and helped maintain many liver-specific functions. We consider these results to be very important for the construction of a high-performance BAL system. Moreover, the introduction of the TRX gene could be used to maintain the viability and functions of other types of mammalian cells. Hence, this technology could be applied to other types of tissue engineering. Recently, therapeutic effects of TRX have been reported in various animal models. The pancreatic b cellspecific expression of TRX prevents autoimmune and streptozotocin-induced diabetes [24]. TRX overexpression in transgenic mice attenuates both focal ischemic damage [25] and adriamycin-induced cardiotoxicity [26], and protects against influenza virus-induced pneumonia [27]. Similarly, TRX expression in the liver may exert its
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therapeutic effects by conferring resistance to various types of stress and cell death. Since adenovirus is known to accumulate in the liver, Adv-TRX could represent a promising medicine for liver disease.
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[13] H. Niwa, K. Yamamura, J. Miyazaki, Efficient selection for highexpression transfectants with a novel eukaryotic vector, Gene 108 (1991) 193–199. [14] A.A. Ormsby, A direct colorimetric method for the determination of urea in blood and urine, J. Biol. Chem. 146 (1942) 595–597. [15] N. Koide, T. Shinji, T. Tanabe, K. Asano, M. Kawaguchi, K. Sakaguchi, Y. Koide, M. Mori, T. Tsuji, Continued high albumin production by multicellular spheroids of adult rat hepatocytes formed in the presence of liver-derived proteoglycans, Biochem. Biophys. Res. Commun. 161 (1989) 385–391. [16] N. Koide, K. Sakaguchi, Y. Koide, K. Asano, M. Kawaguchi, H. Matsushima, T. Takenami, T. Shinji, M. Mori, T. Tsuji, Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent environments, Exp. Cell. Res. 186 (1990) 227–235. [17] K. Funatsu, H. Ijima, K. Nakazawa, Y. Yamashita, M. Shimada, K. Sugimachi, Hybrid artificial liver using hepatocyte organoid culture, Artif. Organs 25 (2001) 194–200. [18] Y. Sakai, K. Naruse, I. Nagashima, T. Muto, M. Suzuki, A new bioartificial liver using porcine hepatocyte spheroids in high-celldensity suspension perfusion culture: in vitro performance in synthesized culture medium and in 100% human plasma, Cell Transpl. 8 (1999) 531–541. [19] T. Andoh, P.B. Chock, C.C. Chiueh, The roles of thioredoxin in protection against oxidative stress-induced apoptosis in SH-SY5Y cells, J. Biol. Chem. 277 (2002) 9655–9660. [20] K. Tobiume, M. Saitoh, H. Ichijo, Activation of apoptosis signalregulating kinase 1 by the stress-induced activating phosphorylation of pre-formed oligomer, J. Cell Physiol. 191 (2002) 95–104. [21] T. Tanaka, F. Hosoi, Y. Yamaguchi Iwai, H. Nakamura, H. Masutani, S. Ueda, A. Nishiyama, S. Takeda, H. Wada, G. Spyrou, J. Yodoi, Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis, EMBO J. 21 (2002) 1695–1703. [22] M. Saitoh, H. Nishitoh, M. Fujii, K. Takeda, K. Tobiume, Y. Sawada, M. Kawabata, K. Miyazono, H. Ichijo, Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1, EMBO J. 17 (1998) 2596–2606. [23] S. Higashiyama, M. Noda, M. Kawase, K. Yagi, Mixed ligandsmodification of polyamidoamine dendrimers to develop effective scaffold for maintenance of hepatocyte spheroids, J. Biomed. Mater. Res. 64 A (2003) 475–482. [24] M. Hotta, F. Tashiro, H. Ikegami, H. Niwa, T. Ogihara, J. Yodoi, J. Miyazaki, Pancreatic beta cell-specific expression of thioredoxin, an antioxidative and antiapoptotic protein, prevents autoimmune and streptozotocin-induced diabetes, J. Exp. Med. 188 (1998) 1445–1451. [25] Y. Takagi, A. Mitsui, A. Nishiyama, K. Nozaki, H. Sono, Y. Gon, N. Hashimoto, J. Yodoi, Overexpression of thioredoxin in transgenic mice attenuates focal ischemic brain damage, Proc. Natl. Acad. Sci. USA 96 (1999) 4131–4136. [26] K. Shioji, C. Kishimoto, H. Nakamura, H. Masutani, Z. Yuan, S. Oka, J. Yodoi, Overexpression of thioredoxin-1 in transgenic mice attenuates adriamycin-induced cardiotoxicity, Circulation 106 (2002) 1403–1409. [27] H. Nakamura, S. Tamura, I. Watanabe, T. Iwasaki, J. Yodoi, Enhanced resistancy of thioredoxin-transgenic mice against influenza virus-induced pneumonia, Immunol. Lett. 82 (2002) 165–170.
Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis Toshio Tsutsui,a Hiroko Koide,a Hiroko Fukahori,a Katsuhiro Isoda,a Shinji Higashiyama,a Isamu Maeda,a Fumi Tashiro,b Eiji Yamato,b Jun-Ichi Miyazaki,b Junji Yodoi,c Masaya Kawase,a and Kiyohito Yagia,* a
c
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan b Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan Department of Biological Responses, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan Received 12 June 2003
Abstract A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liverspecific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Thioredoxin; Adenovirus; Hepatocytes; Bioartificial liver; Apoptosis; Necrosis; Gene transfection
A bioartificial liver support system (BAL) using liver cells has been studied for the treatment of patients with fulminant hepatic failure or as a transitional treatment prior to liver transplantation [1]. As the first-generation BAL, clinical trials in the USA have been carried out using porcine hepatocytes cultured on collagen-coated dextran microcarriers and packed in hollow-fiber-type bioreactors [2]. With the aim of developing a high-performance BAL, our previous work was concerned with improving the scaffolds for hepatocyte cultivation. We showed that chitosan and a polyamidoamine dendrimer are effective in the preparation of scaffolds for hepatocytes [3,4]. We also showed that chemical modifications of these polymers are useful for improving the cell
* Corresponding author. Fax: +81-6-6879-8195. E-mail address: [email protected] (K. Yagi).
0006-291X/03/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-291X(03)01253-1
attachment and the maintenance of cell viability and functions [5,6]. The development of a second-generation BAL will require improvement of the cell itself. In a previous study, the gene encoding glutamine synthetase was introduced into hepatoma cells, which were then packed in a bioreactor. These recombinant HepG2 cells improved ammonia removal when applied to pigs with hepatic failure [7]. Tzanakakis et al. [8] transfected hepatocyte spheroids with the cytochrome P450 2B1 gene. In this study, we used the unique strategy of not only enhancing one liver function but of maintaining all liver functions by introducing into hepatocytes a gene that confers resistance to various kinds of stress. We chose the thioredoxin (TRX) gene as a promising candidate for conferring this protection on primary cultured hepatocytes. TRX is a lowmolecular-weight redox protein (12 kDa) found in both prokaryotic and eukaryotic cells [9]. Cysteine residues at
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the conserved, Cys–Gly–Pro–Cys–Lys active site of TRX undergo reversible oxidation–reduction catalyzed by the NADPH-dependent flavoprotein, thioredoxin reductase. TRX was originally identified as a reducing cofactor for ribonucleotide reductase. Recently, the protein has been shown to be involved in cellular protective mechanisms against various kinds of stress, such as ischemia/reperfusion, X-ray irradiation, and inflammatory cytokines [10]. In this study, we constructed an adenovirus vector using the Cre-loxP recombination system [11] and used it to deliver the human TRX gene into primary cultured rat hepatocytes. We demonstrated that the introduced gene confers protection against oxidative stress, apoptosis, and necrosis on hepatocytes. Materials and methods Media. The basal medium used consisted of 100 U/ml penicillin G, 100 lg/ml streptomycin, 50 ng/ml amphotericin B, and 100 ng/ml aprotinin (Nacalai Tesque, Kyoto, Japan) in William’s medium E (WE, ICN Biochemicals, Costa Mesa, CA, USA). Medium A consisted of 10% fetal bovine serum (FBS, ICN Biochemicals) in basal medium. Medium B consisted of 1 nM insulin (Sigma Chemicals, St. Louis, MO, USA) and 1 nM dexamethasone (Nacalai Tesque, Kyoto, Japan) in Medium A. Culture conditions. Hepatocytes were isolated from male Sprague– Dawley rats weighing 150–200 g by perfusing the liver with collagenase (from Clostridium histolyticum Type IV; Sigma Chemicals, St. Louis, MO, USA) according to the method of Seglen [12]. The animals were housed in an air-conditioned room at 22 1 °C prior to the experiment. The experiments were conducted according to the ethical guidelines of the Graduate School of Pharmaceutical Sciences, Osaka University. Cells were seeded at a density of 1 105 cells/cm2 onto 12well polystyrene culture plates (Nippon Becton Dickinson, Tokyo, Japan). After a 6-h cultivation in Medium B, the cells were cultivated in Medium A. The medium was changed every 24 h. Medium A containing 50 lM NADPH was used for the experiments on the effect of recombinant adenoviruses. Construction of recombinant adenoviral vectors. We constructed an E1- and E3-deleted recombinant adenovirus vector using the pALC3 cosmid, which was generated by removing the E3 region of the adenoviral genome from the pALC cosmid, which already lacks E1 [11] (Fig. 1). The TRX expression cassette, shown in Fig. 2, was flanked by SwaI sites and included the CAG promoter [13], human TRX cDNA, and the rabbit b-globin polyA signal. The expression cassette was inserted into the unique SwaI site of pALC3, to make pALC3-TRX. The recombinant adenovirus vector expressing human TRX (Adv-TRX) was produced by transfecting 293 cells with pALC3-TRX and the titer of the virus stock was determined as described previously [11]. Briefly, virus suspensions were serially diluted with medium and added to a 96-well multiplate seeded with 293 cells. After 10 days, the virus titer was calculated by examining the wells for the presence or absence of a cytopathic effect. The control vector Adv-lacZ was constructed using the Escherichia coli lacZ gene instead of the TRX gene. RT-PCR analysis. RNA was extracted from cultured hepatocytes infected with the recombinant adenovirus. TRX gene expression was analyzed using the following primers: forward 50 -TCTGACTGACCGC GTTACTC-30 and reverse 50 -TCATCCACATCTACTTCAAGGA-30 . Albumin gene expression was analyzed using the following primers: forward 50 -TGAACGTTGCCGCTAGGTTT-30 and reverse 50 -CTTCT GGAGTAATCATAAAG-30 . Glutamine synthetase gene expression was analyzed using following primers: forward 50 -ACCTGACAAATG GCCCTAC-30 and reverse 50 -ACCAAAAAATAACCCCCC-30 . bActin gene expression was analyzed using following primers: forward
Fig. 1. Structure of recombinant adenoviruses. pALC3 (A) consisted of a 29-kb adenoviral genome and 7.1-kb cosmid vector. The cosmid backbone was flanked by loxP sites, indicated by filled arrowheads, and included a cos site, kanamycin-resistance gene (Km), bacterial ori, and ampicillin-resistance gene (Ap). Expression cassettes were inserted into the pALC3 cosmid. The cassette contained TRX cDNA (B) and lacZ (C), resulting in Adv-TRX and Adv-lacZ, respectively. 50 -CATCCCCCAAAGTTCTAC-30 and reverse 50 -CCAAAGCCTTC ATACATC-30 . Assays. Adherent cells were treated with trypsin at 37 °C for 5 min and viability was measured with the trypan blue dye exclusion test. The amount of urea was determined according to the method of Ormsby [14]. The detection of dead cells inside hepatocyte spheroids was carried out using the Vybrant apoptosis assay kit #4 (Molecular Probes Inc., Eugene, OR, USA). Flow cytometry. Hepatocytes (3.8 105 cells) were stained with propidium iodide (PI) and flow cytometric profiles were analyzed using a FACSCalibur analyzer and CELLQuest software (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA).
Results Expression of human TRX in rat hepatocytes Isolated rat hepatocytes were plated onto polystyrene plates and infected with the recombinant
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Protective effect of TRX against cell death
Fig. 2. Expression of the human TRX gene. Hepatocytes were infected with Adv-TRX at a moi of 10 (lane 1), 1 (lane 2), 0.1 (lane 3), and 0 (lane 4, control). After 48 h of culture, the total RNA was isolated and RT-PCR analysis was carried out using primers for TRX (A) and bactin (B). In cells treated similarly, intracellular TRX was analyzed by Western blotting (C). Hepatocyte viability 2 days after the infection was examined (D). Noninfected (moi ¼ 0) and Adv-lacZ-infected (moi ¼ 1) cells were used as controls (open bars). The initial cell density was 1 105 cells/cm2 . An asterisk indicates a value that is significantly different from that of the controls (p < 0:01).
adenovirus at various concentrations. Infection efficiency was examined using the control vector, AdvlacZ, prior to TRX gene transfection. After 24 h of infection at a multiplicity of infection (moi) of 10, 1, and 0.1, about 100%, 80%, and 20% of the hepatocytes expressed lacZ, respectively. The cytotoxic effects of the adenovirus on the viability and function of hepatocytes were examined using the Adv-lacZ control. The infection at moi <10 did not affect viability or urea synthesis activity (data not shown). The expression of the introduced TRX gene was evaluated by RT-PCR and Western blotting using an anti-human TRX monoclonal antibody. As shown in Figs. 2A–C, the TRX gene was expressed in rat hepatocytes in an moi-dependent manner, especially for the amount of TRX protein. Fig. 2D shows the viability of hepatocytes 2 days after infection. When infected with the Adv-TRX at a moi of 1, more than 80% of the initially plated cells were maintained. On the other hand, only 45% of the initial number of uninfected and Adv-lacZ-infected cells were maintained. These results show that human TRX expression improves rat hepatocyte viability, while lacZ expression does not. Thus, we infected hepatocytes with Adv-TRX at a moi of 1 in the succeeding experiments.
We then examined whether the TRX gene conferred oxidative stress resistance on cultured hepatocytes. Transfected and nontransfected cells were treated with 1 mM hydrogen peroxide for 24 h. The number of viable cells was then determined and urea synthesis activity was measured using 2 mM NH4 Cl as a substrate. As shown in Figs. 3A and B, both the viability and urea synthesis activity of the Adv-TRX-infected cells were significantly higher than those of the control. The ratio of apoptotic cells was analyzed using FACS after staining the cells with PI (Fig. 3C). About 80% of the noninfected cells underwent apoptosis after hydrogen peroxide treatment. The ratio of apoptotic cells in the Adv-TRX-infected hepatocytes was only 24%, much less than in the noninfected control. These results indicate that the introduction of the TRX gene confers oxidative stress resistance on hepatocytes. Since the adenovirus vector supports only the transient expression of an inserted gene, we next examined the effect of TRX on cell viability for 2 weeks (Fig. 4A).
Fig. 3. Resistance of TRX gene-transfected hepatocytes to oxidative stress. After 48 h of culture, including an initial 24-h infection period, cells were incubated with 1 mM hydrogen peroxide for 24 h. Viable cells were then counted (A) and urea synthesis activity was measured (B). Filled and open bars represent Adv-TRX- and Adv-lacZ-infected cells at moi ¼ 1, respectively. An asterisk indicates a value significantly different from that of the control (p < 0:01). The ratio of apoptotic cells was analyzed by FACS (C). After 48 h of culture, including an initial 24-h infection period, Adv-TRX- (black) and Adv-lacZ- (gray) infected cells at moi ¼ 1 were incubated with 1 mM hydrogen peroxide for 24 h. All cells including detached cells were recovered and stained with PI for FACS analysis.
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Fig. 5. RT-PCR analysis of liver-specific gene expression. Hepatocytes were infected with Adv-TRX at a moi of 10 (lane 1), 1 (lane 2), 0.1 (lane 3), and 0 (lane 4, control). After 3 days of culture, the total RNA was isolated and RT-PCR analysis was carried out using primers for albumin, glutamine synthetase (GS), and b-actin.
Fig. 4. Maintenance of cell viability and function in TRX genetransfected hepatocytes. Adv-TRX- (j) and Adv-lacZ- (d) infected cells were cultured for 2 weeks including an initial 24-h infection period. Cell viability and urea synthesis activity were analyzed after infection at the times indicated. An asterisk indicates a value significantly different from that of the control (p < 0:01).
After 2 weeks of cultivation, more than 50% of the AdvTRX-infected cells were still viable. On the other hand, about 70% and 85% of the initially plated control cells were nonviable at 5 days and 2 weeks of culture,
respectively. TRX gene expression also had a significant effect on the maintenance of urea synthesis activity within 2 weeks of culture (Fig. 4B). Fig. 5 shows the expression of liver functions other than urea synthesis in the Adv-TRX-infected hepatocytes. RT-PCR analyses of the albumin and glutamine synthetase (GS) genes were carried out after 3 days of hepatocyte culture. Albumin and GS gene expressions in the Adv-TRX-infected cells were much higher than those in the control. These findings indicated that the viable hepatocytes transfected with the TRX gene would be expected to maintain many differentiated functions in vitro for an extended period. Protective effect of TRX in hepatocyte spheroids Hepatocytes were plated onto culture dishes coated with poly-L -lysine and cultured with 50 ng/ml EGF and 10 lg/ml insulin to induce the formation of multicellular spheroids. The recombinant adenovirus was added at the start of the culture and removed after 24 h. The AdvTRX- and Adv-lacZ-infected hepatocytes formed
Fig. 6. Confocal laser scanning microscopic analyses of apoptosis and necrosis in hepatocyte spheroids. Hepatocyte spheroids formed after infection with Adv-TRX (A–D) and Adv-lacZ (E–H). (A, E) Phase-contrast microscopic observations; (B–D, F–H) confocal laser scanning microscopic observations. Spheroids were stained with PI (B, F) and YO-PRO-1 (C, G). Merged images of the red- and green-stained cells (D, H).
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spheroids after 2–3 days of culture. Neither recombinant adenovirus affected spheroid formation. The Adv-TRX and Adv-lacZ-infected spheroids were then stained with the green fluorescent YO-PRO-1 dye, which stains both apoptotic and necrotic cells, and PI, which stains necrotic cells (Fig. 6). A number of internal cells were stained with both dyes in the Adv-lacZ-infected control spheroids. The merged images of the two stainings indicated that both necrotic and apoptotic cells were present inside the control spheroid. On the other hand, almost no cells were stained with either dye in the Adv-TRX-infected hepatocyte spheroids. Hence, the introduction of the TRX gene did not affect spheroid formation and conferred resistance against necrosis and apoptosis inside the spheroids.
Discussion With the goal of developing a high-performance BAL system, we have sought to maintain all liver functions in primary cultured hepatocytes by introducing certain genes into them. This effort requires a vector with a high transfer efficiency for mammalian cells and a gene that confers resistance to stresses from the external environment. We selected adenovirus as the vector for this study because it is generally used for nonproliferative cells such as isolated hepatocytes. Moreover, its transfection efficiency is much higher than that of nonviral vectors. The TRX gene was chosen because the translated protein can be expected to scavenge reactive oxygen species that can cause apoptosis and necrosis. We confirmed that the human TRX gene was successfully introduced using the adenovirus vector and expressed at both the transcription and translation levels in rat hepatocytes. The introduced TRX gene was expressed for at least 10 days, by RT-PCR analysis (data not shown). This maintenance of gene expression is considered to be satisfactory, especially for recombinant hepatocytes used for BAL. Hepatocyte spheroids have a well-known morphology [15,16] and have been used for BAL applications [17,18]. We have shown in this study that Adv-TRX infection does not affect spheroid formation. These results indicate that the adenoviral vector used in this study is very suitable for the introduction of genes into hepatocytes in a BAL system. The function of the human TRX expressed in rat hepatocytes was verified by hydrogen peroxide treatment experiments. Hepatocytes that express human TRX showed improved viability and liver-specific functions after treatment with 1 mM hydrogen peroxide for 24 h. FACS analysis of DNA fragmentation showed that the human TRX suppressed apoptosis in hepatocytes. Annexin V binding, which detects early-phase apoptosis, observed by confocal laser scanning microscopy also showed the protective effect of TRX (data not
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shown). Recently, extensive studies on the relationship between TRX and oxidative stress-induced apoptosis have been carried out [19–21]. In the absence of stress, apoptosis-signal-regulating kinase 1 (ASK1) exists as an inactive complex with the reduced form of TRX. Oxidative stress causes the oxidation of TRX, which disrupts the ASK–TRX complex, thereby activating ASK1 to induce apoptosis [22]. TRX overexpression appears to protect the TRX–ASK1 complex from such disruption. Here we showed that the introduction of the human TRX gene could extend the life span of hepatocytes that were conventionally cultured on polystyrene plates. Generally, hepatocytes that are isolated and cultured under artificial conditions suffer from various stresses. In our recent study, primary hepatocytes cultured on polystyrene plates expressed high levels of stress-inducible HSPs 60 and 70 [23]. Overexpressed TRX seems to confer protection against stresses induced in vitro. In addition to our findings in monolayer culture, we showed that the introduction of the TRX gene has a protective effect against cell death in a spheroidal cell system. Since hepatocyte spheroids are three-dimensional, highly packed multicellular aggregates, the internal cells undergo necrosis due to oxygen and nutrient deficiencies. In this study we showed that the introduction of the TRX gene extensively suppresses necrosis in these internal cells. In considering a BAL system, it is very important to maintain liver functions as well as viability, because gene-transfected hepatocytes might transform into the dedifferentiated state. Detoxification, such as ammonia removal, is an important liver-specific function. Although primary-cultured hepatocytes usually lose this function rapidly, the TRX gene-transfected cells maintained urea synthesis activity significantly longer and at a higher level than the control cells throughout a 2-week cultivation period. In addition to urea cycle enzyme activity, we showed that expression of the albumin and GS genes was maintained in the TRX gene-transfected cells. These findings indicated that TRX overexpression in primary culture suppressed dedifferentiation and helped maintain many liver-specific functions. We consider these results to be very important for the construction of a high-performance BAL system. Moreover, the introduction of the TRX gene could be used to maintain the viability and functions of other types of mammalian cells. Hence, this technology could be applied to other types of tissue engineering. Recently, therapeutic effects of TRX have been reported in various animal models. The pancreatic b cellspecific expression of TRX prevents autoimmune and streptozotocin-induced diabetes [24]. TRX overexpression in transgenic mice attenuates both focal ischemic damage [25] and adriamycin-induced cardiotoxicity [26], and protects against influenza virus-induced pneumonia [27]. Similarly, TRX expression in the liver may exert its
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therapeutic effects by conferring resistance to various types of stress and cell death. Since adenovirus is known to accumulate in the liver, Adv-TRX could represent a promising medicine for liver disease.
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