Cytoglobin Overexpression Protects against Damage-Induced Fibrosis

ARTICLE

doi:10.1016/j.ymthe.2005.11.027

Cytoglobin Overexpression Protects against Damage-Induced Fibrosis Ruian Xu,1,2 Phillip M. Harrison,3 Miao Chen,1,2 Linyan Li,1,2 Tung-yu Tsui,4 Peter C. W. Fung,5 Pik-to Cheung,6 Guangji Wang,1,2 Hua Li,1 Yong Diao,1,2 Geoffrey W. Krissansen,7 Sue Xu,1 and Farzin Farzaneh8,* 1 Institute of Molecular Medicine, Huaqiao University, Fujian, China Institute of Molecular Medicine, China Pharmaceutical University, Nanjing, China 3 Department of Liver Studies and Transplantation and 8Department of Haematological and Molecular Medicine, King’s College London, London SE5 9NU, UK 4 Department of Surgery, University of Regensburg Medical Center, 93053 Regensburg, Germany 5 Department of Medicine, and 6Department of Paediatrics, University of Hong Kong, Hong Kong 7 Department of Molecular Medicine, University of Auckland, Auckland 1020, New Zealand 2

*To whom correspondence and reprint requests should be addressed. Fax: +44 20 7733 3877. E-mail: [email protected].

Available online 3 April 2006

Cytoglobin (Cygb), a member of the hexacoordinate globin superfamily (hxHb), is expressed in fibroblasts from a broad range of tissues. The physiological functions of hxHb are still unclear, but biochemical studies reveal that they can scavenge toxic species, such as nitric oxide, peroxynitrite, and hydrogen peroxide. We demonstrate that the overexpression of Cygb in rat hepatic stellate cells, both in vitro and in vivo, protects against oxidative stress, inhibiting their differentiation into a myofibroblast-like phenotype. Accordingly, the overexpression of Cygb reduces extracellular matrix deposition in both toxic and cholestatic models of liver injury. The overexpression of Cygb also promotes recovery from previously initiated damage-induced fibrogenesis. By inhibiting free radical-induced activation of hepatic stellate cells, Cygb plays an important role in controlling tissue fibrosis. Therefore, the normal upregulation of Cygb during tissue injury has a homeostatic effect, inhibiting free radical-induced fibroblast activation and tissue fibrosis. Key Words: cytoglobin, stellate cell activation associated protein, hexacoordinate globin superfamily, hepatic stellate cells, liver damage, fibrosis, cirrhosis

INTRODUCTION Cytoglobin (Cygb) is a member of the newly discovered class of the hexacoordinate globin superfamily (hxHb) that is expressed in animals [1–3], cyanobacteria [4,5], and plants [6,7]. The crystal structure reveals that Cygb [8,9], like other hxHb [10,11], uses a mechanism of regulation of ligand binding that differs fundamentally from traditional pentacoordinate hemeproteins [8,12], such as hemoglobin (Hb) and myoglobin (Mb), although hxHb are still capable of binding ligands with high affinity [3]. The physiological roles of Hb and Mb in oxygen transport and respiration are clearly defined, but the physiological functions of hxHb are much less clear. In mammals, Cygb is expressed in the cytoplasm of fibroblasts and fibroblast-like cells in a broad variety of organs such as liver, heart, gut, kidney, lung, and pancreas [13,14]. Cygb mRNA is upregulated by tissue hypoxia [14,15] and Cygb levels are increased during the activation of fibroblasts in liver [2], pancreas [13], and

MOLECULAR THERAPY Vol. 13, No. 6, June 2006 Copyright C The American Society of Gene Therapy 1525-0016/$30.00

kidney [13]. There is growing evidence linking hxHb to a role in the protection of cells from oxidative stress, as they are efficient scavengers of reactive oxidizing species [16], which are generated at high levels during tissue injury. We therefore hypothesized that Cygb overexpression may protect against damage-induced oxidative stress, thus inhibiting free radical-mediated activation of fibroblasts and induction of tissue fibrosis. We used hepatic stellate cells (HSC) as a model to investigate the function of Cygb during the activation of fibroblasts. Hepatic fibrosis requires sustained inflammatory responses, leading to the activation of stellate cells and their transdifferentiation into a fibrogenic and proliferative myofibroblast cell type (see [17] for review). Recent studies have also demonstrated a critical role for macrophages both in the induction of injury-induced fibrogenesis and in the resolution of fibrosis following the termination of injury [18,19]. In the present study we demonstrate that increased expression of Cygb in

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HSC protects the cells from oxidative stress and suppresses their differentiation to a myofibroblast-like phenotype both in vitro and in vivo. Cygb expression reduced tissue fibrosis in both toxic and cholestatic models of liver injury, when administered before or, remarkably, after the onset of liver injury. These data suggest that upregulation of Cygb during tissue injury is a homeostatic mechanism inhibiting free radical-induced activation of stellate cells and the subsequent tissue fibrosis.

RESULTS

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DISCUSSION

Overexpression of Cygb Protects Hepatic Stellate Cells against Oxidative Stress We transduced primary rat HSC (24 h postisolation and 98% pure by desmin staining) with more than 90% efficiency, using a modified recombinant adeno-associated virus-2 (rAAV-2) [20] encoding full-length rat Cygb (rAAV/Cygb, Fig. 1). Forty-eight hours after transduction of primary rat HSC with rAAV-2 encoding Cygb or enhanced green fluorescent protein (eGFP) (Fig. 2A), we induced oxidative stress by exposure to 50 AM ferric nitrilotriacetate and 20 AM arachidonic acid (Fe/AA). The overexpression of Cygb protected the stellate cells against oxidative stress, as assessed by reduced production of malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4HNE), biomarkers of lipid peroxidation (Fig. 2B). In keeping with these data, Cygb expression significantly increased the total oxyradical scavenging capacity (TOSC) compared to the expression of eGFP (Fig. 2C). Cygb overexpression also prevented oxidative stressinduced differentiation of primary HSC into a myofibroblast-like phenotype, as characterized by the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and

TGF-h1 transcripts (Fig. 2D) and high levels of AP-1 and NF-nB DNA binding activities (Fig. 2E). Consequently, induction of collagen synthesis was reduced by about 50% in the HSC transduced with rAAV/Cygb compared to rAAV/eGFP (Fig. 2F). rAAV2-Mediated Overexpression of Cygb Protects against Chronic Injury-Induced Liver Fibrosis The natural tropism of the rAAV-2 vector for HSC (compare Figs. 1C and 1D), which is due to expression of the AAV-2 coreceptor fibroblast growth factor receptor1 (unpublished data), was utilized to express high levels of Cygb in HSC in vivo. Four weeks after administration of rAAV-2 directly into the portal vein (pv) of male Sprague– Dawley (SD) rats, in situ hybridization demonstrated higher levels of Cygb mRNA in liver lobules of the rAAV/Cygb-infected animals compared to normal controls (Figs. 3A and 3B) or rAAV/eGFP-infected animals (data not shown). Staining of liver sections with antiCygb and anti-desmin antibodies demonstrated the preferential in vivo transduction of HSC (Figs. 3C and 3D). Furthermore, flow-cytometric analysis of freshly dissociated liver cells confirmed a time-dependent rise in Cygb levels in HSC (by 70% on day 7 and by 95% on day 14) but not in the parenchymal liver cells (Fig. 3E). To investigate the function of Cygb expression during fibroblast activation in vivo, we used two models of liver injury and fibrogenesis. We induced hepatic necrosis by biweekly administration of carbon tetrachloride (CCl4; 0.5 ml/kg) or generated cholestasis by bile duct ligation (BDL). Two weeks before toxin administration, or 3 days before BDL, we delivered rAAV vectors, or an equivalent volume of PBS carrier, to the rats in each group by pv injection. Following 8 weeks of CCl4 administration,

FIG. 1. In vitro transduction of primary rat HSC with rAAV. (A) Primary rat HSC, 24 h postisolation, were stained with an anti-desmin antibody to determine purity. (B) The 570-bp cDNA encoding the open reading frame of rat cytoglobin was cloned into the EcoRI and NotI sites of the replication-deficient rAAV-2 vector. The vector also contained the AAV-2 ITRs, a CAG promoter, and the woodchuck hepatitis virus posttranslational regulatory element to facilitate expression. The in vitro infection of isolated rat (C) HSC and (D) hepatocytes with the rAAV/eGFP (at an m.o.i. of 5 104 (C0 and 2.5 105 particles/ml (D)) demonstrates the efficient transduction of HSC, but not hepatocytes, at 60 h.

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FIG. 2. Cytoglobin protects primary rat hepatic stellate cells against oxidative stressinduced activation. (A) HSC, 60 h posttransduction with rAAV (m.o.i. of 5 104 particles/cell) encoding Cygb (left) or eGFP (right), were stained with a Cygb-specific antibody. (B) Oxidative stress was induced in HSC by exposure to 50 AM ferric nitrilotriacetate and 20 AM arachidonic acid (Fe/AA), 48 h after transduction with rAAV encoding Cygb or eGFP. Levels of lipid peroxidation were assessed by production of MDA and 4HNE. (C) The total oxyradical scavenging capacity of HSC was measured following exposure to H2O2, after transduction with rAAV encoding Cygb or eGFP. (D) HSC were treated as in (B) and expression of TGF-h1 and TIMP-1 mRNA transcripts was determined by RT-PCR. (E) HSC were treated as in (B) and electrophoretic gel mobility shift analysis was used to measure DNA binding activities for AP-1 (left) and NF-nB (right). (F) Collagen production by HSC was determined by incorporation of [3H]proline (10 ACi/ml) into collagenase-sensitive protein [26].

histological examination demonstrated extensive fibrosis and micronodule development throughout the liver in rats receiving PBS or rAAV/eGFP. In contrast, rats receiving rAAV/Cygb had a preserved liver architecture and histology similar to the non-liver-injury controls (Fig. 4A). Administration of rAAV/Cygb prevented the development of a fibrogenic phenotype, as it inhibited the characteristic rise in procollagen 1 (PC-1), TGF-h1, and TIMP-1 transcripts (Fig. 4B), as well as a-SMA and TGF-h1 proteins (Fig. 4C). Pretreatment with rAAV/Cygb also protected against hepatic necrosis as evidenced by similar


levels of serum aspartate transaminase (AST) in normal controls (86 F 12) and rAAV/Cygb rats (159 F 21), contrasting with the markedly elevated levels in rAAV/ eGFP animals (1100 F 225; P b 0.001). Twenty-eight days after BDL, rats pretreated with rAAV/eGFP had significant cholestatic liver injury and histological evidence of extensive fibrosis with nodule development, identical to PBS controls (Fig. 4D). All rats that were pretreated with rAAV/eGFP developed ascites and 28% died within 3 weeks. In contrast all rats receiving rAAV/Cygb prior to BDL remained alive and

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FIG. 3. Expression of cytoglobin in HSC transduced with rAAV/Cygb in vivo. Four weeks after 5 10 11 particles of rAAV/Cygb were administered directly into the portal vein of male Sprague–Dawley rats, DIG nonradioactive in situ hybridization with a Cygb-specific cRNA probe was used to detect Cygb transcripts in liver sections from (A) normal and (B) rAAV/Cygb-treated rats. Sections were developed using alkaline phosphatase NBT–BCIP detection. Arrows point to the Cygb transcript-positive cells (B). Liver sections were also stained with anti-Cygb (green) and anti-desmin (red) antibodies. Representative liver sections from (C) normal and (D) rAAV/ Cygb rats are shown. Double-stained cells are indicated by arrows. (E) Animals were sacrificed 1, 7, and 14 days after pv injection of 5 1011 particles of rAAV/Cygb. The desmin-positive HSC were separated from parenchymal liver cells by standard methodology. Intracellular staining for Cygb was performed using rabbit anti-Cygb antibody visualized by goat anti-rabbit antibody labeled with PE and levels were detected and analyzed using FACSCalibur. Less than 10% of the desmin-negative cell population expressed Cygb at any time point.

free of ascites. In addition, although liver histology showed bile duct proliferation and concentric periductal fibrosis, the liver architecture was substantially preserved (Fig. 4D). Administration of rAAV/Cygb modulated the development of the activated HSC phenotype, since it inhibited the increase in TGF-h1 and TIMP-1 transcripts and partially inhibited the rise in both PC-1 mRNA expression (Fig. 4B) and collagen synthesis, as assessed by hydroxyproline content of liver tissue (0.87 F 0.43 mg/g for rAAV/eGFP versus 0.09 F 0.03 mg/g for sham operated and 0.42 F 0.26 mg/g for rAAV/Cygb; P b 0.01). Overexpression of Cygb also significantly reduced the degree of liver dysfunction, as assessed by total bilirubin and AST levels (Table 1). Cygb Overexpression Protects against Progressive Liver Damage To explore the role of Cygb during progressive liver fibrosis, we administered rAAV/Cygb or rAAV/eGFP vectors by pv injection to rats at week 8, during a 12week course of CCl4 injections. We continued CCL4 administration after transduction with rAAV vectors because withdrawal of liver injury alone can lead to

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remodeling of fibrosis [21]. CCl4-control and CCl4-rAAV/ eGFP rats had a progressive deterioration in liver fibrosis, whereas liver histology 4 weeks after rAAV/Cygb exposure showed a marked remodeling of the extracellular matrix leading to reduced fibrosis (Fig. 5A). Hepatic necrosis was also resolved as revealed by reduced serum AST values in the rAAV/Cygb-treated group compared to the rAAV/eGFP-treated animals (1280 F 265 U/L at week 8, falling to 179 F 37 U/L at week 12 and 1603 F 397 U/L at week 8, rising to 2080 F 110 U/L at week 12; P b 0.01). We observed a similar effect when BDL was carried out 12 days prior to pv injection of rAAV vectors. Examination of liver histology a further 12 days later showed markedly less fibrosis in rats receiving rAAV/Cygb, compared to the rAAV/eGFP group (Fig. 5B). Induction of Cygb expression after the onset of cholestatic liver injury reduced the degree of liver dysfunction, as assessed by total bilirubin and AST levels (Table 2), and induced a quiescent phenotype in HSC, as assessed by real-time RTPCR quantification of TGF-h1 and PC-1 transcript levels (Fig. 5C). The above studies show that overexpression of Cygb in HSC protects the cells from oxidative stress and conse-


doi:10.1016/j.ymthe.2005.11.027

quently suppresses their differentiation to a myofibroblast-like phenotype, leading to reduced extracellular matrix production both in vitro and in vivo. Cygb overexpression reduced tissue fibrosis in both toxic and cholestatic models of liver injury, when administered before or, remarkably, after the onset of liver injury. Interestingly, liver injury stimulates endogenous expression of Cygb in HSC [2]. However, the present data suggest that upregulation of Cygb during tissue injury does not contribute to the HSC-induced fibrogenesis; rather it provides a homeostatic mechanism inhibiting free radical-induced activation of HSC and tissue fibrosis. Given the pattern of regulation of hxHb in both plants [22] and animals [14,15], under conditions generating nitric oxide and other reactive oxidizing species, it is tempting to speculate that hxHb share a common scavenging function. Indeed, neuroglobin, a hxHb localized to cerebral neurons [23,24], was found to act as an endogenous neuroprotective factor in focal cerebral ischemia [25]. Finally, the mechanism by which Cygb regulates the HSC phenotype remains to be elucidated. Although Cygb may simply redress the redox balance by scavenging oxidizing species, the possibility remains that Cygb may target intracellular signaling pathways directly or act as a transcriptional regulator. These events may in turn regulate the function of hepatic macrophages, which have recently been shown to play a critical role in the induction of injury-induced fibrogenesis and the resolution of fibrosis following the termination of injury [17–19].

MATERIALS

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ARTICLE

CAGTGT-3V and inserted into the EcoRI/NotI site of the replicationdeficient rAAV-2 vector. The construct also contained the AAV-2 ITRs, a CAG promoter, and the woodchuck hepatitis B virus posttranscriptional regulatory element to facilitate expression (Fig. 1B). Recombinant AAV vectors expressing Cygb and eGFP were heparin column purified. The rAAV viral genome titer was quantified by real-time PCR using TaqMan (Perkin–Elmer Biosystems, CA, USA).

METHODS

Animals. Young adult male SD rats (~150 g) were housed at a constant temperature and supplied with laboratory chow and water ad libitum for 3–4 weeks prior to use. The research protocol was approved by the UHK Animal Ethics Committee. cDNA cloning and generation of recombinant AAV vectors. A 570-bp cDNA encoding rat cytoglobin was obtained by PCR from reversetranscribed SD rat liver RNA, using the Cygb-specific primers 5VATGGAGAAAGTGCCGGGCGAC-3V and 5V-CTATGGCCCTGAAGAGGG-

FIG. 4. Prophylactic overexpression of Cygb inhibits injury-induced activation of HSC in vivo. (A) 5 1011 particles of rAAV encoding either Cygb or eGFP, or an equivalent volume of PBS carrier, were delivered to male SD (n = 10 for each group) rats via pv injection 2 weeks before induction of hepatic necrosis by twice weekly intraperitoneal injection of CCl4 0.5 ml/kg, mixed in an equal volume of olive oil. Following 8 weeks of CCl4 administration, liver sections were stained with MassonTs trichrome and hematoxylin–eosin. Representative liver sections are shown from normal controls (top, left) or CCL4-treated rats receiving PBS (top, right), rAAV/eGFP (bottom, left), or rAAV/Cygb (bottom, right). (B) As in (A) or (D) but RT-PCR was used to detect PC-1, TGF-h1, and TIMP-1 transcripts in mRNA isolated from whole liver. (C) As in (A) but Western blot analysis was used to detect a-SMA and TGF-h1 protein levels in whole liver tissue lysates. (D) rAAV vectors were administered as in (A), 3 days before induction of cholestatic liver injury by BDL. Twenty-eight days after BDL, liver sections were stained with MassonTs trichrome and hematoxylin– eosin. Representative liver sections are shown from normal controls (top, left) or BDL rats receiving PBS (top, right), rAAV/eGFP (bottom, left), or rAAV/Cygb (bottom, right). The number of rats in each group was at least 7.


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TABLE 1: Effect of Cygb overexpression on bilirubin and AST levels after cholestatic liver injury Sham rAAV/Cygb rAAV/eGFP

Bilirubin (Amol/L)

AST (U/L)

2.1 F 0.8 51.6 F 30.1* 99.9 F 24.2a

74.3 F 28.9 407 F 209* 807 F 357b

5 1011 particles of rAAV encoding either Cygb or eGFP were delivered to male SD rats (n = 10 for each group) via pv injection 3 days before induction of cholestatic liver injury by BDL. Bilirubin and AST levels were measured 28 days after BDL. a P b 0.01 compared with sham and P = 0.054 compared with rAAV/Cygb. b P b 0.000001compared with sham and P b 0.05 compared with rAAV/Cygb. * P b 0.01 compared with sham.

Hepatic stellate cell isolation and lipid peroxidation. HSC were isolated by standard methodology. Briefly, livers were perfused with a Ca2+ Mg2+free solution and tissue was dispersed with 0.05% collagenase type I. After incubation for 20 min at 378C, the livers were homogenized through a mesh. The homogenate was centrifuged at 500 rpm for 5 min. Cell pellets were resuspended in 10 ml 13% Nycodenz in DMEM and then centrifuged at 2600 rpm for 20 min. The upper fraction, diluted with PBS, was centrifuged at 1500 rpm for 10 min and the pellet containing the HSC washed twice with PBS. Lipid peroxidation was induced, 48 h after rAAV transduction (m.o.i. 5 104), by adding Fe/AA to the culture medium at final concentrations of 50 and 20 AM, respectively. Lipid peroxides, including MDA and 4-HNE, in the cell lysate and medium were determined using an LPO-586 kit (CalBiochem, CA, USA) at 0 and 18 h after peroxidation.

H2O2 stimuli and analysis of TOSC. HSC were maintained at 378C and 5% CO2/air in DMEM containing 100 U/ml penicillin/streptomycin and 10% fetal bovine serum, in six-well plates. At 60–70% confluence the culture medium was replaced with 500 Al serum-free DMEM containing rAAV/Cygb or rAAV/eGFP vector. After 40 min incubation, 2 ml fresh DMEM was added and the incubation continued for a further 36 h, when H2O2 was added (0.5 mM). After 1 h incubation, the medium was replaced with fresh culture medium. The TOSC assay, performed at 0 and 16 h, is based on the reaction between artificially generated oxyradicals and aketo-g-methiolutyric acid, which is oxidized to ethylene [27]. The specific TOSC value was calculated by dividing the TOSC values by the relative protein concentration (in milligrams). Flow-cytometric analysis of cytoglobin expression. Intracellular staining for Cygb was performed using anti-Cygb and goat anti-rabbit–PE (Santa Cruz Biotechnologies, CA, USA). The freshly isolated HSC and non-HSC from experimental animals were analyzed using FACSCalibur (BD Biosciences, CA, USA). Western blotting. Tissues were minced and homogenized in the protein lysate buffer, as previously described. Protein samples (100 Ag) were resolved on nondenaturing 10% polyacrylamide SDS gels and transferred to nitrocellulose Hybond C membranes (Amersham, England). The membranes were blocked with 5% bovine serum albumin (BSA) and incubated with the primary antibody followed by horseradish peroxidaseconjugated secondary antibodies and developed by enhanced chemiluminescence (Amersham) and exposure to X-ray film. Electrophoretic gel mobility shift assay. Double-strand oligonucleotides (Santa Cruz) corresponding to the consensus DNA binding sequences of AP-1 (5V-AGCATGAGTCAGACACCTCTTGGC-3V) and NF-nB proteins (5V-

FIG. 5. Overexpression of Cygb after the onset of liver injury reduces liver fibrosis and induces a quiescent HSC phenotype. Rats were exposed to CCl4 for 8 weeks or BDL for 12 days and then injected via pv with 5 1011 particles of rAAV encoding either Cygb or eGFP or an equivalent volume of PBS carrier. Animals were sacrificed 4 weeks (CCl4) or 12 days (BDL) after transduction with the indicated AAV vectors. Representative liver sections, stained with MassonTs trichrome and hematoxylin–eosin, are shown for (A) CCl4-treated and (B) BDL rats: controls (left), rAAV/ eGFP (middle), or rAAV/Cygb (right). (C) Real-time RT-PCR was used to measure levels of TGFh-1 and PC1 mRNA in HSC from BDL rats, isolated at the time of sacrifice (1, sham operated; 2, BDL-rAAV/eGFP; 3, BDL-rAAV/Cygb; 4, no-template control). For each group n = 5 for the BDL- and n = 10 for the CCl4treated rats.

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TABLE 2: Effect of Cygb overexpression on bilirubin and AST levels in response to a previous cholestatic liver injury Sham rAAV/Cygb rAAV/eGFP

Bilirubin (Amol/L)

AST (U/L)

2.9 F 1.0 77.3 F 35.0* 130 F 11.3a

74.3 F 28.9 497 F 253** 1113 F 112b

Rats had BDL performed to induce cholestatic liver injury. 12 days later they were injected via pv with 5 1011 particles of rAAV encoding either Cygb or eGFP. Bilirubin and AST levels were measured 12 days later. a P b 0.01 compared with sham and P b 0.05 compared with rAAV/Cygb. b P b 0.01 compared with sham and P = 0.065 compared with rAAV/Cygb. * P b 0.05 compared with sham. ** P = 0.066 compared with sham.

AGTTGAGGGGACTTTCCCAGGC-3V) were labeled with 50 ACi [g-32P]ATP (4000 Ci/mmol; ICN, CA, USA) by T4 polynucleotide kinase (Boehringer Mannheim). The reaction consisted of binding buffer, 300 Ag/ml BSA, 1 Al of poly(dI–dC), 1 Al of labeled probe, 2 Ag of nuclear protein extract, and unlabeled competitor probe. Unlabeled AP-1, NF-nB, and C/EBP (5VTGCAGATTGCGCAATCTGCA-3V) oligonucleotides were used in 50-fold excess. The reaction components, except the labeled probe, were incubated on ice for 5 min. The probe was then added and incubated at 258C for 30 min. The protein–DNA complexes were resolved by electrophoresis on nondenaturing 6% polyacrylamide gels and visualized by autoradiography. Determination of gene expression in HSC. Total RNA was isolated from frozen tissue using RNAzol B (Invitrogen) and mRNA levels were determined by RT-PCR (GeneAmp RNA PCR Core Kit; Perkin–Elmer Life Science), using intron-spanning primer pairs specific for TIMP-1, 5V-CCACAGATATCCGGTTCGCCTACA-3V and 5V-GCACACCCCACAGCCAGCACTAT-3V; PC-1, 5V-TACTACCGGGCCGATGATGC-3V and 5VT C C T T G G G G T T C G G G C T G A T G T A -3 V; T G F- h1 , 5 V- T A T A G C A A CAATTCCTGGCG-3V and 5V-TGCTGTCACAGGAGCAGTG-3V; GAPDH, 5V-CCCTTCATTGACCTCAACTACATGG-3V and 5V-CATGGTGGTGAAGACGCCAG-3V. The cycling parameters were 5 min at 948C, followed by 35 cycles of 1 min at 948C, 1 min at 558C, and 1 min at 728C. After amplification, PCR products were examined by standard gel electrophoresis. In situ hybridization. Dehydrated sections were hybridized overnight at 558C with probe solution in accordance with THE manufacturerTs instructions (Ambion) and developed with 1 BCIP–NBT (Zymed) to the desired intensity. Negative controls were hybridized with a sense cRNA probe. Determination of hydroxylproline content. Hydrolysis was carried out using concentrated hydrochloric acid. Hydroxylproline levels were measured by reversed-phase HPLC with fluorometric detection after acid hydrolysis. Real-time RT-PCR. Reaction mixtures were prepared with a SYBR Green PCR Reaction Mix (Applied Biosystems). The reaction was processed and measured in an ABI Prism 7900HT sequence detection system (Applied Biosystems). The thermal cycles were 958C for 10 min (1 cycle) and 958C for 30 s, 508C for 30 s, and 728C for 30 s (40 cycles). Histochemistry and immunohistochemical staining and analysis. Paraffin-embedded sections were stained with MassonTs trichrome and hematoxylin–eosin. For immunohistochemistry, the livers were soaked in 30% sucrose in PBS and 10-Am sections prepared and thaw-mounted onto slides. Sections were rinsed 3 with PBS containing 0.2% Triton X-100 and placed in 1% H2O2 in methanol for 1 min, then rinsed 3 in PBS, and incubated with 4% horse serum in PBS for 1 h. After further PBS–Tween 20 rinses, sections were incubated with the primary antibody overnight, at room temperature, and then washed with PBS–Tween prior to a 2-h incubation with the secondary antibody. Following a distilled water rinse they were mounted with Vectashield (Vector Laboratories, CA, USA). Immunofluorescent signals were captured using a Leica 4d TCS confocal microscope.


Biochemical analysis. Serum albumin, bilirubin, and AST levels in rat blood were determined at the Queen Mary Hospital, Hong Kong. Collagen synthesis assay. Cells were transferred to 24-well plates 4 h prior to transduction with equal amounts of the different rAAV vectors in serum-free RPMI containing ascorbic acid (50 Ag/ml) and h-aminopropionitrile fumarate (50 Ag/ml) for 28 h. Collagen production was determined by incorporation of [2,3-3H]proline (Sigma; 10 ACi/ml) during a further 18-h incubation. The cell and medium proteins were precipitated with 10% trichloroacetic acid. Collagen production was determined by a collagenase digestion method. Statistical analysis. Data are given as means F SEM. ANOVA was performed to test the significance; P values were considered to be statistically significant when less than 0.05.

ACKNOWLEDGMENTS We thank Professors Katsutoshi Yoshizato, Don McGregor, and Norifumi Kawada and Dr. Masanobu Obara for critical discussion of the manuscript; Professor James Wilson for the AAV plasmids; Mr. R. Tong, Ms. Li H., Ms. Shi J., Miss L. F. Hui, Mr. K. W. Ho, Dr. Y. Q. Wang, Dr. Ma Hong, Mr. J. Ma, L.-Y. Tse, and Miss K. X. Cai for technical assistance; and Dr. S. Tam for the bilirubin and AST assays. RECEIVED FOR PUBLICATION JULY 2, 2005; REVISED OCTOBER 30, 2005; ACCEPTED NOVEMBER 2, 2005.

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