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Cell Growth & Differentiation Vol. 13, 265-273, June 2002
© 2002 American Association for Cancer Research

Adenoviral Delivery of an Antisense RNA Complementary to the 3' Coding Sequence of Transforming Growth Factor-ß1 Inhibits Fibrogenic Activities of Hepatic Stellate Cells1

Monica Arias, Birgit Lahme, Eddy Van de Leur, Axel M. Gressner and Ralf Weiskirchen2

Institute of Clinical Chemistry and Pathobiochemistry, RWTH- University Hospital, D-52074 Aachen, Germany


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Liver fibrosis occurs as a consequence of the transdifferentiationof hepatic stellate cells into myofibroblasts and is associated with an increased expression and activation of transforming growth factor (TGF)-ß1. This pluripotent, profibrogenic cytokine stimulates matrix synthesis and decreases matrix degradation, resulting in fibrosis. Thus, blockade of synthesis or sequestering of mature TGF-ß1 is a primary target for the development of antifibrotic approaches. The purpose of this study was to investigate whether the administration of adenoviruses constitutively expressing an antisense mRNA complementary to the 3' coding sequence of TGF-ß1 is able to suppress the synthesis of TGF-ß1 in culture-activated hepatic stellate cells. We demonstrate that the adenoviral vehicle directs high-level expression of the transgene and proved that the transduced antisense is biologically active by immunoprecipitation, Western blot, quantitative TGF-ß1 ELISA, and cell proliferation assays. Additionally, the biological function of the transgene was confirmed by analysis of differential activity of TGF-ß1-responsive genes using cell ELISA, Northern blotting, and by microarray technology, respectively. Furthermore, we examined the effects of that transgene on the expression of TGF-ß2, TGF-ß3, collagen type {alpha}1(I), latent transforming growth factor binding protein 1, types I and II TGF-ß receptors, and {alpha}-smooth muscle actin. Our results indicate that the administration of antisense mRNA offers a feasible approach to block autocrine TGF-ß1 signaling in hepatic stellate cells and may be useful and applicable in future to the treatment of fibrosis in chronic liver diseases.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Members of the TGF3 -ß family are multifunctional cytokines that signal through types I and II serine/threonine protein kinase receptors (TßR) and Smad proteins (1 , 2) . TGF-ß regulates cell proliferation and differentiation, the metabolism of ECM components, and it plays a pivotal role in the pathogenesis of fibroproliferative disorders (3 , 4) . During liver fibrogenesis, activated HSCs are strongly responsive to TGF-ß-dependent Smad phosphorylation. Once phosphorylated, receptor-associated Smads (Smad2 and Smad3) dissociate from the receptor, bind to Smad4, and then enter the nucleus. Thereby, HSCs transduce TGF-ß1 dependent signals, which result in growth inhibition of these cells, in part mediated by inhibition of cellular kinase activity or by decreasing the level of cyclins (5 , 6) . As a result, Smads can either positively or negatively regulate the transcription of TGF-ß-responsive genes by direct DNA binding or by association with numerous DNA binding proteins (1 , 2) . Although TGF-ß has antiproliferative effects on various cells in culture (7) , it exerts strong fibrogenic potential in culture-activated HSCs. Typically, TGF-ß stimulates the synthesis and accumulation of {alpha}-SMA and collagen in HSCs (8 , 9) , and as a consequence, the cells transform into a MFB-like phenotype (10) . All three isoforms of TGF-ß (ß1, ß2, and ß3) existing in mammals share 70–80% amino acid sequence identity and are equally able to induce {alpha}-SMA mRNA and protein expression in vitro and in vivo (11) . In addition, an increase in TGF-ß expression is observed during HSC activation and transdifferentiation into MFBs, thus indicating that TGF-ß acts as an autocrine positive regulator for the production of ECM. TGF-ß1 expression is also in vivo associated with the accelerated accumulation of ECM during chronic liver diseases of different etiologies (12) . Therefore, blockade of TGF-ß1 synthesis or signaling is a primary target for the development of antifibrotic approaches, and in recent years, scientists have achieved a design of drugs removing this causative agent. Although a definitive antagonistic therapy for TGF-ß1 in the treatment of liver fibrosis has not been developed yet, recent advances in cell biology have opened several ways to approach the inhibition of TGF-ß action. These include the administration of antioxidants (13, 14, 15) , specific inhibitory drugs (16 , 17) , herbal compounds (18 , 19) , neutralizing antibodies (8) , TGF-ß binding proteins (20 , 21) , antagonistic cytokines (22) , and specific oligonucleotides regulating TGF-ß1 expression (23) . Currently, potential gene therapies using dominant-negative or soluble TßRs are also under close investigation. In rats, the pathogenesis of hepatic fibrosis induced by DMN was markedly reduced by administration of adenoviral vectors (Ad) expressing either a truncated (24) or a soluble TßRII receptor composed of a chimeric protein between an entire ectodomain of TßRII and the Fc portion of an immunoglobulin (25) . Impressively, in these experiments a single injection of adenovirus expressing the truncated receptor, given prior to DMN administration, appeared to prevent both hepatic injury and the development of hepatic fibrosis. In a subsequent study, the same adenoviral vector was administered to animals with on-going fibrosis. In rats that received the receptor fusion, the results were similar with lack of progression and possibly some regression of hepatic fibrosis (26) . The antifibrogenic potential of the soluble TßRII was also demonstrated in the established rat bile duct ligation model (27) . Another possible option to inhibit TGF-ß function is to interfere with postreceptor signaling. Overexpression of Smad7, a natural antagonist of TGF-ß signaling, prevents bleomycin-induced pulmonary fibrosis in mice (28) . Although many of the discussed approaches to block TGF-ß are attractive and were shown to be effective in experimental models, their efficacy and safety in human liver fibrosis or other TGF-ß-induced disorders remain unknown.

In this study, we constructed an adenovirus expressing an antisense mRNA complementary to the 3' coding sequence of TGF-ß1 and show that this transgene is a potentially powerful tool for blocking target gene expression. In HSCs, the antisense mRNA is able to suppress the synthesis and the autocrine signaling of TGF-ß1.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Preparation of Ad Expressing Antisense RNA Complementary to the 3' Coding Sequence of TGF-ß1.
In general, cultured HSCs and MFBs are almost refractory to gene transfer by physical methods (29) . To allow effective delivery of transgenes to this hepatic subpopulation, we used an Ad-based expression system. We cloned an adenoviral shuttle vector p{Delta}E1sp1A-CMV-asTGF-ß1 expressing mRNA complementary to TGF-ß1 and directed the construct under transcriptional control of the CMV enhancer/promoter. To direct the proper processing of the 3' end and to stabilize the encoded antisense mRNA, we fused the SV40 polyadenylation signal downstream to the transgene (Fig. 1)Citation . The integration of the transgene into a recombinant Ad was performed through homologous recombination occurring in 293 between common regions of p{Delta}E1sp1A-CMV-asTGF-ß1 and the backbone vector pJM17, resulting in a replication-defective Ad5-derivative expressing an antisense mRNA complementary to the 3' end of TGF-ß1 mRNA (Fig. 2A)Citation . Viral particles were isolated, centrifuged through a buoyant CsCl density gradient, and used for infection. We performed Southern blot analysis to assess integrity of the encoded transgene. Therefore, viral DNA was amplified in 293 cells, digested with EcoRV/HindIII or EcoRV/BglII and was then probed with a cDNA specific for TGF-ß1 (Fig. 2B)Citation . Specific hybridization signals were detectable in Ad5-CMV-as-TGF-ß1-infected cells and were virtually absent in mock or Ad5-CMV-EGFP infected cells. The obtained hybridization signals matched the expected fragments of ~1.6 kbp in size. We then analyzed the expression capacity of the antisense construct by Northern blotting. In human MFBs, Ad5-CMV-as-TGF-ß1 is able to direct high-level expression of an mRNA species with a calculated size of 450–500 nucleotides (Fig. 2C)Citation , which is compatible with the complexity of the delivered asTGF-ß1 mRNA if a poly(A+)-tail of 150–200 nucleotides is estimated. Additionally, the abundance of the expressed antisense mRNA exceeds endogenous mRNA for TGF-ß1 by one to two orders of magnitude, which is a prerequisite to block effectively the expression of the target gene. The expression capacity of the recombinant Ad was also demonstrated in rat HSCs (see below), in a rat cirrhotic fat storing cell line, and in established cell lines (not shown). To test whether the synthesized antisense is able to block synthesis of TGF-ß1, the amount of mature TGF-ß1 was compared in cell lysates from Ad5-CMV-as-TGF-ß1 and mock-infected cells by Western blot analysis and immunoprecipitation (Fig. 2, D and E)Citation . These experiments reveal that the content of the precipitated TGF-ß1 was significantly lower in cells infected with the antisense construct, indicating that the expressed transgene effectively blocks the synthesis of TGF-ß1 in HSCs.



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Fig. 1. Schematic diagram of the plasmids used for generation of a recombinant adenovirus expressing antisense TGF-ß1 mRNA. A, the plasmid clone pCMV-asTGF-ß1 was constructed by fusing the PvuII fragment of pET3d-rTGF-ß1 in antisense orientation into the expressing vector pEGFP-C1, which was depleted for the EGFP. The transgene harboring the CMV promoter, the asTGF-ß1-fragment, and the SV40-polyadenylation signal was transferred to the adenoviral shuttle vector p{Delta}E1sp1A. B, the integration of vector sequences from p{Delta}E1sp1A-CMV-asTGF-ß1 into the adenoviral backbone vector pJM17 was performed by in vitro homologous recombination in the human embryo kidney cell line 293, which constitutively expresses the E1 transactivators required for propagation of recombinant adenoviruses. In the schematic diagram, the adenoviral DNA sequences are shown as thick black lines, the bacterial vector sequences as thinner black lines, the sequences encoding TGF-ß1 or EGFP as thick dark gray lines, and the markers for ampicillin (Amp), kanamycin (Kan), and tetracycline (Tet) conferring antibiotic resistance in Escherichia coli as thick gray lines, respectively.

 


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Fig. 2. Adenovirus-mediated gene transfer of asTGF-ß1 mRNA. A, a schematic representation of the strategy used for blockage of TGF-ß1 synthesis. The primary sequence of rat TGF-ß1 consists of 390 amino acids. The signal sequence (amino acids 1–22), the latency-associated peptide (amino acids 23–278), and the COOH-terminal part encoding the mature TGF-ß1 (amino acids 279–390) are shown in black, white, and gray, respectively. The asTGF-ß1 is complementary to the 3' end of the mRNA of TGF-ß1, arresting translation by halting ribosome (RS) after duplex formation. B, Southern blot hybridization of mock- (Lane 1), Ad5-CMV-asTGF-ß1 (Lane 2), and Ad5-CMV-EGFP (Lane 3) infected 293 cells. High molecular weight DNA was analyzed by digestion with EcoRV/HindIII or EcoRV/BglII, followed by 1.2% agarose gel electrophoresis and Southern hybridization using the 32P-labeled 278-bp PvuII fragment of clone pET3d-rTGF-ß1. C, Northern analysis of total RNAs isolated from human MFBs infected with Ad5-CMV-EGFP (Lane 1) or Ad5-CMV-asTGF-ß1 (Lane 2). The blot was subsequently hybridized with the 32P-labeled 278-bp PvuII fragment of pET3d-rTGF-ß1 and a GAPDH-specific cDNA probe. D, Western blot analysis of TGF-ß1 expression in HSC (Lane 2) or in cells infected with Ad5-CMV-asTGF-ß1 (Lane 3), or Ad5-CMV-EGFP (Lane 4). Forty ng of recombinant human TGF-ß1 (Lane 1) with a calculated Mr of 12,794 served as a positive control for rat TGF-ß1 (Mr 12,810). E, immunoprecipitation of TGF-ß1 from total protein cell lysates of HSC (Lane 1), mock infected cells (Lane 2), or cells infected with Ad5-CMV-asTGF-ß1 (Lane 3). TGF-ß1 was resolved by SDS-PAGE under reducing conditions, and labeled TGF-ß1 was visualized by autoradiography for 17 days.

 
Effects of TGF-ß1 Antisense RNA in Culture-activated HSCs.
The spontaneous activation of HSCs on plastic is an established in vitro model system for liver fibrosis, and the use of cells in primary culture serves as a useful tool for the elucidation of the roles of TGF-ß. In our study, we used this model system to assess the molecular and cellular processes after administration of the asTGF-ß1. In culture-activated HSCs, TGF-ß1 is profibrogenic as well as antiproliferative agent in a dose-dependent manner. We used the growth-inhibition assay to evaluate the functionality of the expressed asTGF-ß1 mRNA (Fig. 3A)Citation . In HSCs, the construct significantly increased the incorporation of [3H]thymidine, whereas there was no growth-stimulatory effect in mock-infected cells or in cells infected with an Ad5-CMV-EGFP reporter. We obtained the same results also in a BrdUrd colorimetric assay (not shown). Notably, the proliferative effect of Ad5-CMV-as-TGF-ß1 is independent from other stimulatory triggers. In cells treated with PDGF-BB, which in HSCs is the most potent trigger of proliferation, both proliferative effects are additive, indicating that Ad5-CMV-asTGF-ß1 specifically inhibits the TGF-ß-mediated signal but does not affect signaling by other growth factors. To examine whether infection with Ad5-CMV-asTGF-ß1 could abolish the expression of the fibrosis-associated {alpha}-SMA and LTBP-1, we analyzed the expression of these proteins in a cell ELISA (Fig. 3B)Citation . Compared with mock-infected cells, the levels of both proteins were substantially decreased in cells infected with the antisense construct. Consistent with the ELISA, we independently confirmed the {alpha}-SMA suppression by Western blot analysis (Fig. 3C)Citation . In a contradictory manner, total p38-mitogen-activated protein kinase, which we routinely use as a standard to verify equal gel loading, showed no differential expression in these cells (Fig. 3D)Citation . The suppression of {alpha}-SMA and LTBP-1 was not observed in cells infected with Ad5-CMV-EGFP (not shown). In parallel experiments, we also quantified the amount of various isoforms of TGF-ß in these cells. Compared with mock-infected controls, the amounts of all three isoforms (TGF-ß1, TGF-ß2, and TGF-ß3) were significantly reduced in cells infected with Ad5-CMV-asTGF-ß1 (Fig. 3E)Citation , indicating that the transgene is capable of antagonizing the expression of all three isoforms. Additionally, we found that the protein levels of the types I and II receptors (TßRI and TßRII) for TGF-ß were significantly reduced in these cells (Fig. 3F)Citation . To examine whether the infection with Ad5-CMV-asTGF-ß1 could also abolish the synthesis of type I collagen, a classical marker of fibrosis, we examined the steady-state level of col{alpha}1(I) mRNA at different time intervals after infection. In agreement with the functionality of the transgene, the level of col{alpha}1(I) mRNA was significantly reduced in cells infected with the transgene (Fig. 4Citation , left panel). Again, this reduction was specific for the expressed transgene and was not observed in cells infected with other recombinant Ad (Fig. 4Citation , right panel).



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Fig. 3. Cell proliferation and quantitative determination of {alpha}-SMA, LTBP-1, TGF-ß1, TGF-ß2, TGF-ß3, TßRI, and TßRII. A, proliferation of mock-, Ad5-CMV-asTGF-ß1-, or Ad5-CMV-EGFP-infected HSCs. HSCs were seeded in DMEM containing 10% FCS, and at the second day of culture, the serum was reduced to 5% heat-inactivated FCS and infected with indicated adenoviruses. Twenty-four h later, the medium was changed to 0.5% FCS for 24 h, and then [3H]thymidine (75 kBq/ml) was added. Additionally, the cells were stimulated with human recombinant PDGF-BB or left unstimulated. The mean values of triplicate determinations of a representative experiment are shown; bars, SD. B, quantitative determination by cell ELISA of {alpha}-SMA and LTBP-1. C, immunoblot detection of {alpha}-SMA protein levels in untreated HSC (Lane 1), mock-infected (Lane 2) or Ad5-CMV-asTGF-ß1-infected (Lane 3) HSCs. Protein extracts were isolated three (3d) or five (5d) days after infection. D, immunoblot detection of p38-MAPK in untreated (Lane 1), mock-infected (Lane 2) or Ad5-CMV-asTGF-ß1-infected (Lane 3) HSCs. E and F, quantitative analysis of TGF-ß isoforms (E) and type I and type II receptors (F) as determined by cell ELISA. The concentrations of individual proteins are given as fluorescence units (FU) and refer to the DNA content of control cultures.

 


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Fig. 4. Effects of the asTGF-ß1 mRNA in culture-activated HSCs. Equal amounts (5 µg/lane) of total cellular RNAs were analyzed by Northern blotting. In the left panel, RNA was isolated from untreated (Lane 1), mock-infected (Lane 2), or Ad5-CMV-asTGF-ß1-infected (Lane 3) rat HSCs. In the right panel, RNA was isolated from mock-infected (Lane 1) or Ad5-CMV-EGFP-infected (Lane 2) cells. RNA was isolated two (2d), three (3d), or four (4d) days after infection. The blots were subsequently hybridized with a 32P-labeled col{alpha}1(I)-specific probe and the 278-bp PvuII fragment of clone pET3d-rTGF-ß1. Intensities of the 18S and 28S rRNAs and hybridization with a GAPDH-specific cDNA served as controls to verify equivalent loading.

 
Blocking TGF-ß1 Function in Human MFBs.
The human cytokine expression array from R&D Systems provides researchers with a rapid, semiquantitative tool to identify differentially expressed cytokines and cytokine-related genes. To evaluate the biological effects of the constitutive expressed transgene in human MFBs, we conducted a cytokine expression profile analysis (Fig. 5)Citation . Of the 375 different cloned cDNAs, printed in duplicate as PCR products, ~10 individual clones were differentially expressed after infection with Ad5-CMV-asTGF-ß1 (Fig. 5A)Citation . Inspection of the relevant regions of the microarray revealed that the hybridization signal of the TGF-ß1 target mRNA was higher in the Ad5-CMV-asTGF-ß1-infected cells. This finding was not observed in cells infected with other recombinant Ad (not shown) and is consistent with the fact that under cellular environment, mRNA/mRNA hybrids are resistant to the attack of RNase H, the key enzyme of RNA degradation. Additionally, we found that decorin, which was published previously as a TGF-ß1-responsive gene (30) , was significantly suppressed in cells expressing the antagonistic mRNA (Fig. 5C)Citation . The suppression of the decorin mRNA was also confirmed in Northern blot analysis (Fig. 5D)Citation . Apart from decorin, we found that the expression of MPIF-1, IGFBP4, IGFBP5, IGFBP8, CD34, and PARC was also changed in these cells (not shown). Although we have not examined the significance of this differential gene expression yet, future studies should enlighten this subject.



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Fig. 5. Microarray analysis. A, RNAs from mock (left panel) or Ad5-CMV-asTGF-ß1-infected (right panel) human MFBs were isolated, labeled by reverse transcription, and used as probes in a cytokine expression microarray analysis. After hybridization, the filters were washed and subjected to autoradiography for 16 h. B, comparison of the obtained signals of the TGF-ß superfamily in mock-infected MFBs (upper panel) or in MFBs expressing asTGF-ß1 (lower panel). C, suppression of decorin in cells infected with Ad5-CMV-asTGF-ß1. Compared with mock-infected MFBs (upper panel), cells infected with Ad5-CMV-asTGF-ß1 (lower panel) show a significant reduction of decorin expression. D, verification of decorin suppression by Northern blot analysis. RNAs from mock (Lane 1) or Ad5-CMV-asTGF-ß1 (Lane 2) infected human MFBs were subsequently analyzed for expression of decorin, asTGF-ß1, and GAPDH, respectively.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
There is firm evidence of the role of activated HSCs in hepatic inflammation and fibrogenesis. In activated HSCs, TGF-ß1 is the major cytokine in regulation of the production, degradation, and accumulation of components of the ECMs. Liver fibrogenesis is linked to an up-regulated expression of TGF-ß isoforms, reflecting autocrine effects in cellular activation of HSCs. Concomitant with increased TGF-ß production, HSCs increase the production of collagen, and it is suggested that the unbalanced TGF-ß activity during wound repair induces fibrotic responses, scaring, and parenchymal cell apoptosis. These damaging responses can be inhibited by anti-TGF-ß treatments using neutralizing antibodies or soluble TßRs. In previous studies of DMN-treated rats, the infusion of Ads expressing a soluble TßRII demonstrated that TGF-ß intervention could be therapeutically useful as a means of inhibiting fibrosis and preserving liver function (24) . In other studies, blockade of TGF-ß by in vivo gene transfer of a soluble TßRII in the muscle inhibited corneal opacification, edema, and angiogenesis (31) . However, the fusion of the extracellular domain of TßRII with the Fc portion of an immunoglobulin might create immunogenic epitopes. Therefore, the finding that the direct blockage of TGF-ß1 synthesis is possible by application of the antisense mRNA may greatly increase the safety of future intervention strategies. Comparable with the results obtained with soluble or truncated receptors, our results provide clear evidence that the designed transgene is sufficient to block the expression of TGF-ß and can abolish critical fibrogenic reactions of culture-activated HSCs. In other applications, antisense technology has been used widely for regulating gene expression, and it was demonstrated that single-stranded RNA complementary to the target mRNA can inhibit the translation of endogenous mRNA (32) . Likewise, our therapeutic transgene driven under the control of the CMV promoter is effectively transcribed and might form a duplex in the presumed hybridization area, which in HSCs blocks the synthesis of TGF-ß1. Consequently, we observed: (a) an increase in cell proliferation based on [3H]thymidine and BrdUrd incorporation; (b) the suppression of fibrogenic marker proteins (e.g., {alpha}-SMA, col{alpha}1(I), LTBP-1, TßRI, and TßRII); and (c) alterations in expression of potential TGF-ß1-sensitive genes (decorin and IGFBPs). It is tempting to postulate that this induction of cell proliferation is directly linked to the inhibition of TGF-ß1 synthesis, because cells infected with control viruses did not display substantial induction of cell proliferation. Additionally, Western blot analysis and immunoprecipitation further confirm that the synthesis of TGF-ß1 is indeed significantly inhibited in cells infected with the transgene. The high degree of sequence identity between different isoforms of TGF-ß may contribute to the observed accompanied suppression of TGF-ß2 and TGF-ß3 in these cells. The simultaneous reduction of all three isoforms may partly account for the observed dramatic inhibition of fibrogenic activities in our cell culture model.

Antisense RNA could block expression by a number of mechanisms. Several investigators have argued that the duplex between antisense and sense RNA results in accelerated turnover of the mRNA, leading to a reduction in its abundance in the cell. This mechanism does not appear to account for the observed decrease in TGF-ß1 because the abundance of endogenous mRNA encoding TGF-ß1 estimated from our microarray analysis was higher in cells expressing the transgene. Possibly, the duplex of antisense mRNA/RNA is resistant to the degradation by cellular ribonucleases. Additional experiments are needed to establish or reject the proposed mechanism.

The disruption of the autocrine TGF-ß signaling pathway in culture-activated HSCs has profound effects for ongoing transdifferentiation. An important finding in this study is that the amounts of col{alpha}1(I), {alpha}-SMA, LTBP-1, TßRI, and TßRII, which are increased in the fibrogenic process, decreased after expression of the antisense mRNA, supporting the notion that our construct has indeed antifibrotic potential. Furthermore, the finding that decorin, another TGF-ß1-responsive gene, is markedly suppressed in cells harboring the transgene clearly indicates that our transgene has TGF-ß1-antagonistic capacity. At present, we do not know the relevance of the observed differential gene expression of MPIF-1, IGFBPs, CD34, and PARC in TGF-ß1-depleted cells but in line with our findings previous studies demonstrated that HSCs, in their activated phenotype, constitutively produce IGFBPs and that TGF-ß interferes with their expression and synthesis (33) . Additionally, recent investigations raise the question of whether there is a direct link between the signal cascade induced by TGF-ß1 and IGFBPs, because IGFBPs can stimulate the phosphorylation of receptor-associated Smads (Smad2 and Smad3), potentiate the TGF-ß1-stimulated Smad phosphorylation, and cooperate with exogenous TGF-ß1 in cell growth inhibition (34 , 35) . This will need to be investigated carefully in future studies.

In summary, our results indicate that Ad5-CMV-asTGF-ß1 can block the synthesis of TGF-ß and its downstream targets in activated cultured HSCs. Although the effect of the delivered antisense mRNA was not tested in an experimental in vivo model for liver fibrosis, the present in vitro data provide the basis that the presented transgenic expression vehicle is a possible candidate for use in future gene therapy.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Isolation and Culture of HSCs.
HSCs were isolated from male Sprague Dawley rats by the Pronase-collagenase method with slight modification reported before (36 , 37) . Briefly, livers were subsequently perfused with HBSS (PAA Laboratories, Linz, Austria), 0.35% (w/v) Pronase E in HBSS, and with 0.015% (w/v) collagenase in HBSS. Then the livers were homogenized in HBSS containing 100 µg of DNase, type II (Roche, Mannheim, Germany) per ml and filtered through nylon mesh, centrifuged, and washed in ice-cold HBSS containing 0.25% (w/v) BSA. HSCs were further purified by a single-step density gradient centrifugation with 8.25% (w/v) Nycodenz (Nycomed Pharma AS, Oslo, Norway) as described in detail elsewhere (38 , 39) . Cells were seeded in DMEM (BioWhittaker Europe, Verviers, Belgium) supplemented with FCS (Biochrom KG, Berlin, Germany), L-glutamine (PAA Laboratories), and penicillin/streptomycin (PAA Laboratories). The purity of the cell preparations was assessed by light microscopic appearance and positive immunofluorescence stainings (40) . Human MFBs were isolated as outgrowths from human liver tissues and subcultured for several passages as described previously (41) .

Metabolic Labeling, Immunoprecipitation, and Fluorography of TGF-ß1.
HSCs at the second day of primary culture (58-cm2 Petri dishes) were infected for 1 day with indicated recombinant Ad. Thereafter, cells were washed three times and labeled for 16 h with the PRO-MIX L-[35S] in vitro cell labeling mix (>1000 Ci/mmol; Amersham Pharmacia Biotech, Freiburg, Germany) in a cysteine- and methionine-free DMEM and in the absence of FCS. The crude cell homogenates were prepared as described previously (42) . After centrifugation, the cell-free supernatant was precleared by incubating with a nonimmune rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA), followed by precipitation with Protein G Plus-agarose (Santa Cruz). The resulting final supernatants were then incubated with 10 µg/ml of the polyclonal TGF-ß1 (V) antibody (Santa Cruz) for 16 h. The immunoprecipitates were recovered by addition of one-tenth of Protein G Plus-agarose, washed as described (42) and subjected to a 18% (w/v) SDS-PAGE (Novex, Groningen, the Netherlands) under reducing conditions. The gel was fixed in 25% (v/v) 2-propanol, 10% (v/v) acetic acid, soaked in Amplify (Amersham) for 30 min, dried, and exposed to a BIOMAX MR film (Eastman Kodak, Rochester, NY) for indicated times.

SDS-PAGE and Immunoblotting.
Whole-cell protein extracts were prepared in lysis buffer [50 mM Tris-HCl (pH 6.8), 250 mM NaCl, 2% (v/v) NP40, 2.5 mM EDTA, 0.1% (w/v) SDS, 0.5% (w/v) deoxycholate] with the addition of a protease inhibitor mixture (Roche). Equal amounts of proteins were resolved in NuPAGE 4–12% Bis-Tris gels (Novex) under reducing conditions and electroblotted onto a Protran membrane (Schleicher & Schuell, Dassel, Germany) according to standard procedures. {alpha}-SMA was detected with monoclonal mouse antibody clone asm-1 (Roche) and visualized using a horseradish peroxidase-conjugated antimouse IgG (Santa Cruz) and the supersignal chemiluminescent substrate (Pierce, Rockford, IL). Polyclonal antibody against p38-mitogen-activated protein kinase (Westburg BV, Leusden, the Netherlands) and TGF-ß1 [TGF-ß1(V); Santa Cruz] were detected with antirabbit IgG-horseradish peroxidase (Santa Cruz).

Construction of Recombinant Adenoviruses.
The reporter Ad5-CMV-EGFP, expressing the EGFP directed under the transcriptional control of the CMV promoter, has been described previously (29) . For construction of the adenoviral shuttle vector p{Delta}E1sp1A-CMV-asTGF-ß1, the 278-bp PvuII fragment of pET3d-rTGF-ß1 (43) was cloned in antisense orientation into the expression vector pEGFP-C1 (Clontech, Palo Alto, CA), which was depleted for EGFP by cutting with NheI and BamHI and filled in by the Klenow fragment. The 1518-bp Alw44I/SspI fragment was then subcloned into the blunted EcoRI site of p{Delta}E1sp1A (44) . The integrity of the cloning boundaries was verified by sequencing with primers specific for p{Delta}E1sp1A using the ABI PRISM BigDye termination reaction kit (PE Applied Biosystems, Weiterstadt, Germany). The transgenic region of the p{Delta}E1sp1A-asTGF-ß1 was then inserted into the Ad5-backbone vector pJM17 (45) by homologous recombination as described in detail elsewhere (29) .

Determination of Cell Proliferation.
On the second day after seeding, the serum content was reduced from 10% FCS to 5% heat-inactivated FCS, and cells were infected with indicated recombinant Ad or left untreated. One day later, serum was reduced to 0.5%, and after 24 h, cells were treated with 20 ng/ml recombinant rat PDGF-BB (R&D Systems, Wiesbaden, Germany) for 24 h and were then exposed to 75 kBq/ml [6-3H]thymidine (NEN Life Science Products, Dreieich, Germany) for another 24-h labeling period. Radioactivity incorporated into DNA was measured as described before (40) . Alternatively, the cell proliferation was measured in a commercially available cell proliferation ELISA, BrdUrd colorimetric assay (Roche).

Microarray Analysis.
Human cytokine expression arrays were obtained from R&D Systems, and [{alpha}-32P]dCTP-labeled probes were generated by reverse transcription of 2 µg of total RNA with human cytokine-specific primers (R&D). Each array was prehybridized for 1 h and was then hybridized at 65°C in 20 ml of hybridization mix [5x SSPE, 2% (w/v) SDS, 5x Denhardt’s reagent, and 100 µg/ml sonicated salmon testes DNA]. After hybridization for 16 h, the arrays were subsequently washed for 20 min at 65°C twice in solution I [0.5x SSPE and 1% (w/v) SDS] and then once in solution II [0.1x SSPE and 1% (w/v) SDS]. Autoradiographs were exposed for indicated times to Kodak X-OMAT AR films at -80°C using intensifying screens.

RNA Isolation and Northern Blot Analysis.
Isolation of RNA from rat liver cells was carried out as described previously (37) . An equal amount of purified total RNA was separated by electrophoresis in a 1.2% (w/v) denaturing agarose gel, transferred to Hybond-N membrane (Amersham), and fixed by baking for 2 h at 80°C. Hybridization probes were the 278-bp PvuII fragment of pET3d-rTGF-ß1, the 975-bp EcoRI fragment of pGEM-col{alpha}1(I) (a kind gift of K. Knoch, Institute of Physiological Chemistry, Carl Gustav Carus University, Dresden, Germany), and the ~1.7-kbp EcoRI fragment of pBABE-hdecorin (a kind gift of H. Kresse, Department of Physiological Chemistry and Pathobiochemistry, University of Munster, Germany), respectively. As an internal standard, the blots were rehybridized with a cDNA specific for GAPDH.

Southern Blot Analysis.
High molecular weight genomic DNA was isolated by a standard proteinase K/phenol extraction protocol. Ten-µg portions of DNA were digested and separated on a 1.2% agarose gel, denatured, and transferred to Hybond-N nylon membrane (Amersham). The blots were hybridized with the [{alpha}-32P]dCTP-labeled 278-bp PvuII fragment of vector pET3d-rTGF-ß1 (43) . Hybridization and washing was carried out using standard procedures (46) .

Cell ELISA.
Cells were cultured and infected in black 96-well plates (Nunc, Wiesbaden, Germany). Cultures were fixed in 4% paraformaldehyde in PBS (pH 7.4), permeabilized 2 min on ice with 0.1% Triton X-100 in 0.1% sodium citrate, and analyzed in a cell ELISA assay described before (47) . Antibodies obtained from Santa Cruz were TGF-ß1 (V), TGF-ß2 (V), TGF-ß3 (V), TßRI (V-22), TßRII (L-21), normal rabbit IgG, and normal mouse IgG. Other primary antibodies used were monoclonal mouse anti {alpha}-SMA (clone asm-1; Roche) and polyclonal rabbit antihuman LTBP-1 (provided by Prof. C. H. Heldin, Ludwig Institute for Cancer Research, Uppsala, Sweden). Secondary antibodies conjugates were antimouse IgG-biotin (Sigma, Deisenhofen, Germany) and porcine antirabbit IgG-biotin (Dako, Hamburg, Germany). The streptavidin-alkaline phosphatase and the AttoPhos substrate were obtained from Roche.


    Acknowledgments
 
We are very grateful to Prof. C. H. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden) for sending polyclonal rabbit antihuman LTBP-1, Prof. H. Kresse (Department of Physiological Chemistry and Pathobiochemistry, University of Munster, Germany) for providing cDNA encoding human decorin, and Dr. K. Knoch (Institute of Physiological Chemistry, Carl Gustav Carus University, Dresden, Germany) for sending cDNA specific for rat col{alpha}1(I). Adenoviral expression technology in our laboratory is covered by permission of the Landesumweltamt Nordrhein-Westfalen (Az. 521-K-1.59/99), solely indicating this fact.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Studies were supported by grants from IZKF BIOMAT and from the Start program Aachen (to R. W.). Back

2 To whom requests for reprints should be addressed, at Institute of Clinical Chemistry and Pathobiochemistry, RWTH-University Hospital, D-52074 Aachen, Germany. Phone: 49-(0)241-80-88-683; Fax: 49-(0)241-80-82-512; E-mail: rweiskirchen{at}ukaachen.de Back

3 The abbreviations used are: TGF, transforming growth factor; TßRI (TßRII), TGF-ß receptor type I (II); ECM, extracellular matrix; HSC, hepatic stellate cell; {alpha}-SMA, {alpha}-smooth muscle actin; col{alpha}1(I), collagen type {alpha}1(I); MFB, myofibroblast; DMN, dimethylnitrosamine; Ad, adenoviral vector; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; BrdUrd, bromodeoxyuridine; IGFBP, insulin-like growth factor binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LTBP-1, latent transforming growth factor binding protein 1; PDGF, platelet derived growth factor. Back

Received for publication 2/21/02. Revision received 5/ 6/02. Accepted for publication 5/10/02.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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