Class VI intermediate filament protein nestin is ... - Wiley Online Library

Class VI Intermediate Filament Protein Nestin Is Induced During Activation of Rat Hepatic Stellate Cells TOSHIRO NIKI,1 MILOS PEKNY,3 KARINE HELLEMANS,1 PIETER DE BLESER,1 KIT VAN DEN BERG,1 FREYA VAEYENS1, ERIK QUARTIER,2 FRANS SCHUIT,2 AND ALBERT GEERTS1

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Hepatic stellate cells are considered to be liver-specific pericytes that play a key role in liver fibrosis. Because these cells express desmin and smooth muscle a-actin, they were assumed to be of myogenic origin. This hypothesis became doubtful when it was reported that stellate cells also express glial fibrillary acidic protein and neural cell adhesion molecule. In the present study, we show that activated stellate cells express nestin, a class VI intermediate filament protein originally identified as a marker for neural stem cells. Expression of nestin was first studied during spontaneous activation of stellate cells in culture. Immunohistochemistry showed that nestin-positive stellate cells already appeared at day 3, and nearly all the cells became positive for nestin at day 6 and 15. The immunoreaction was present in filaments except in dividing cells. The presence of messenger RNA transcript for nestin was shown by reverse transcription polymerase chain reaction and sequencing of amplified complementary DNA. We then compared the presence of nestin with that of other intermediate filament proteins and smooth muscle a-actin. Immunoblotting showed that the relative concentrations of nestin, desmin, and vimentin increased between day 2 and 6 in primary culture. After the initial increase vimentin leveled off, while nestin and desmin showed a tendency to decrease. This pattern was quite different from that of glial fibrillary acidic protein, which kept declining, and smooth muscle a-actin, which increased continuously up to day 13 in culture. We then studied the presence of nestin in normal and CCl4-injured rat liver. In normal liver, minimal immunoreaction for nestin was observed within the liver parenchyma. During induction of fibrosis by carbon tetrachlo-

Abbreviations: aSMA, smooth muscle a-actin; GFAP, glial fibrillary acidic protein; N-CAM, neural cell adhesion molecule; mRNA, messenger RNA; RT-PCR, reverse transcription polymerase chain reaction; TBS, tris-buffered saline; mRNA; messenger RNA. From the 1Laboratory for Cell Biology and Histology and the 2Diabetes Research Center, Faculty of Medicine and Pharmacy, Free University of Brussels (VUB), Belgium; and the 3Department of Medical Biochemistry, University of Gothenburg, Sweden. Received May 20, 1998; accepted October 8, 1998. This work was made possible by FWO-V (Fonds voor Wetenschappelijk Onderzoek Vlaanderen) grants nr. 9.0039.93, 9.0011.95 and G.0044.96, and OZR-VUB (Onderzoeksraad Vrije Universiteit Brussel) grants nr. 193 322 1480 and 196 322 1120. Address reprint requests to: Prof. Dr. Albert Geerts, Laboratory for Cell Biology and Histology, Faculty of Medicine and Pharmacy, Free University of Brussels; Laarbeeklaan 103, 1090 Brussel-Jette, Belgium. Email: [email protected]; fax: 32.2.477.4412. Copyright r 1999 by the American Association for the Study of Liver Diseases. 0270-9139/99/2902-0027$3.00/0

ride, nestin-positive stellate cells appeared at 6 weeks, which was late in comparison with the induction of desmin and smooth muscle a-actin. We conclude that nestin is induced in stellate cells during transition from the quiescent to the activated phenotype; culture activation is a stronger stimulus than in vivo activation by injection of CCl4. Taken together with reports on expression of glial fibrillary acidic protein and neural cell adhesion molecule by stellate cells, new experimental studies on the embryonic origin of these cells are required. (HEPATOLOGY 1999;29:520-527.) Hepatic stellate cells exert specific liver functions: storage of large amounts of retinyl esters, synthesis and breakdown of hepatic extracellular matrix, secretion of a variety of cytokines, and control of the diameter of the sinusoids.1,2 Based on the observation that stellate cells express desmin and smooth muscle a-actin (aSMA),3-6 a myogenic origin of these cells was assumed.3 This hypothesis became doubtful when it was reported that stellate cells also express glial fibrillary acidic protein (GFAP)7-10 and neural cell adhesion molecule (N-CAM).11,12 Nestin is a relatively new member of the intermediate filament family. It was originally defined as a protein in neural stem cells recognized by Rat-1 monoclonal antibody.13,14 Based on sequence homology, nestin was classified as a class VI intermediate filament protein, only distantly related to class III intermediate filament proteins such as desmin, vimentin, and GFAP. In spite of the relatively low sequence homology, nestin forms heteropolymers with type III filament proteins.15 In addition to neural precursor cells, nestin is expressed by immature glial cells,16 reactive astrocytes,17,18 primitive neuroectodermal tumors,16,19 and melanomas.20 Also, immature cardiac21 and skeletal muscle cells15,22,23 express nestin before terminal differentiation. Except for some myoblasts, all these cells have a common neural crest origin. In this article we show the induction of nestin in hepatic stellate cells during their spontaneous activation in culture and in CCl4-induced liver fibrogenesis. Also, we compared relative concentrations of nestin with those of other intermediate filament proteins, such as desmin, vimentin, and GFAP, and to the microfilament protein aSMA. MATERIALS AND METHODS Animals. Adult male Wistar rats were used in all experiments. All rats were fed ad libitum and received humane care in compliance with the institution’s guidelines for the care and use of laboratory animals in research. To induce fibrosis, CCl4 (100 µL/100 g body weight) was administered by intraperitoneal injection twice weekly with equal intervals. CCl4 was mixed 1/1 with pharmaceutical grade

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TABLE 1. Antibodies Used in This Study Dilutions Antibodies

Primary antibodies Anti-nestin (rabbit polyclonal)25 Anti-desmin (mouse monoclonal) clone DE-B-5 Anti-chicken desmin (rabbit polyclonal) A 611 Anti-vimentin (mouse IgM monoclonal) clone VIM13.2 Anti-vimentin (goat polyclonal) V-4630 Anti-porcine GFAP (mouse monoclonal) clone G-A-5 Anti-bovine GFAP (rabbit polyclonal) Z 334 Anti-smooth muscle a-actin (mouse monocl.) clone 1A4 Secondary antibodies Anti-mouse antibody PO-conjugated Anti-mouse IgM antibody PO-conjugated Anti-rabbit antibody PO-conjugated Anti-rabbit antibody PO-conjugated Anti-goat antibody PO-conjugated Anti-mouse antibody ALP-conjugated

Sources

IHC

Western

J. Eriksson* 1:200 1:2,000 Boehringer Dako

1:4 —

— 1:1,600

Sigma Sigma

1:500 — — 1:8,000

Biogenex

1:300

—

—

1:1,600

Dako Sigma

1:500 1:5,000

Amersham Sigma Amersham Jackson Jackson Sigma

1:100 1:1,000 1:250 — — 1:1,000 1:500 — — 1:1,000 1:100 —

Abbreviations: IHC, immunohistochemistry; GFAP, glial fibrillary acidic protein; PO, peroxidase; ALP, alkaline phosphatase. *Dr. J. Eriksson, Turku Center for Biotechnology, University of Turku, Finland.

paraffin oil (Federa, Brussels, Belgium) before injection. Groups of three animals were killed 72 hours after 4, 8, 12, and 16 injections. Immunohistochemistry. Immunohistochemistry was performed on acetone-fixed, 10 µm-thick frozen sections as described previously.10,24 For immunodetection of nestin, a rabbit polyclonal antiserum produced and characterized by Dr. J. Eriksson was used.25 Sources and dilutions of all antibodies were summarized in Table 1. Briefly, after blocking with 2% albumin/phosphate-buffered saline, the sections were reacted overnight with primary antibody at 4°C.

FIG. 1. Distribution of nestin in primary cultures of stellate cells. (A) Stellate cells in culture for 3 days. Nestin is mainly present in filaments concentrated around the nucleus. Acetone fixation removed lipid from the numerous vacuoles in the perinuclear area. Magnification: 2403. (B) Six-day-old stellate cell culture. Nestin is present in intracytoplasmic filaments that extend from the nuclear region to the periphery of the cells. Magnification: 2403. Insert: mitotic stellate cell at 6 days in culture. The pericentral region with the spindle figure is devoid of nestin. The periphery of the cell contains immunoreactive material. Magnification: 1753.

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After rinsing three times for 10 minutes, the sections were incubated with peroxidase-conjugated secondary antibody for 1 hour at room temperature. Antigens were visualized with 3,38diaminobenzidine/ H2O2 enhanced with Ni21 and Co21 ions.24,26 Double staining for nestin/desmin and for nestin/aSMA was performed as follows. Sections were incubated overnight at 4°C with a mixture of primary antibodies to nestin (rabbit polyclonal) and desmin (mouse monoclonal), or with primary antibodies to nestin and aSMA (mouse monoclonal). After rinsing three times with phosphate-buffered saline, species-specific peroxidase-conjugated goat antirabbit (Amersham, Little Chalfont, UK) and alkaline phosphatase-conjugated goat antimouse antibodies (Amersham) were applied simultaneously for 90 minutes at room temperature. After rinsing three times with phosphate-buffered saline, alkaline phosphatase was visualized with use of Fast Red TR/Naphtol AS-MX (Sigma-Aldrich, Bornem, Belgium) for 10 minutes at room temperature. This reaction provided a red reaction product. Subsequently, sections were rinsed three times and peroxidase was visualized as described (vide supra). Isolation and Culture of Hepatic Stellate Cells. Hepatic stellate cells were isolated from rats by collagenase/pronase digestion followed by density gradient centrifugation as described previously.27 After isolation, cells were suspended in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin, and cultured at 37°C in a humidified atmosphere with 5% CO2 and 95% air. At day 2, cell debris and nonadherent cells were removed by washing. The medium was changed every 2 days thereafter, unless otherwise mentioned. Purity of cultures was evaluated by examining the characteristic stellate shape and the presence of lipid-droplets under phase-contrast microscopy. C6 Rat Glioma Cells. Glioma cell lines were reported to contain nestin.19 Cultures of these cells were provided by Prof. Dr. W. Van Driessche, Laboratory of Physiology, Catholic University of Louvain (Leuven, Belgium), and were used as positive control cells. Cells were cultured in the same medium as described for stellate cells. RT-PCR and DNA Sequencing. Total RNA was extracted by the method of Chomczynski and Sacchi.28 RT-PCR was performed using GeneAmp RNA PCR Core Kit by using Amplitaq Gold polymerase (Perkin Elmer, NJ), following the manufacturer’s instructions. Total RNA (0.75 µg) was reverse transcribed in a reaction volume of 20 µL

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A

B

FIG. 2. Western blotting of cultured stellate cells. Cells were harvested at indicated time points. (A) For immunodetection of nestin, 10 µg total protein of each cell lysate was separated by 5% SDS-PAGE. (B) For immunodetection of desmin, vimentin, GFAP, and aSMA, 5 µg total protein of lysates was separated by 10% SDS-PAGE. Following electrophoresis, proteins were transferred to nitrocellulose membranes and probed with antibodies against different cytoskeletal proteins as stated in Materials and Methods. Each of the proteins under investigation showed a different pattern of variation of its intracellular concentration. Nestin peaked at day 6. Thereafter, its relative intracellular concentration decreased. Desmin reached a maximum between day 4 and day 6. Then it declined slowly. Vimentin was strongly upregulated at day 4. Thereafter it leveled off. GFAP was clearly present in very early cultures. It declined with time. aSMA showed up at day 4. It increased strongly with culture time. This figure shows representative images. Expression patterns of desmin, vimentin, GFAP, and aSMA were studied in four different cultures; expression of nestin was examined in three different cultures.

HEPATOLOGY February 1999

by using 1 U/µL murine leukemia virus reverse transcriptase at 42°C. PCR was performed by using a forward primer 58-GGA GGG TTG CGT CGG GGA AG-38 and a reverse primer 58-CTC CTC CTC GTG CGC GGC-38 for 35 cycles with cycle times of 1 minute at 94°C, 1 minute at 61°C, and 1 minute at 72°C. These primers correspond to the published nucleotide sequence of rat nestin complementary DNA positions 129 through 149 and 551 through 568.14 This set of primers gives rise to a PCR product of 439 base pairs. The amplified product was electrophoresed in 1.5% agarose gels, stained with ethidium bromide, and photographed. Specificity of PCR product was confirmed by automatic sequencing by using the dideoxynucleotide method by Sanger et al.29 Fluorescent-dye–labeled extention products were produced in a single tube by using the ABI PRISM TM Dye terminator cycle sequencing reaction kit (Perkin Elmer-Applied Biosystems Division; Foster City, CA). The reaction mixture contained: 50 ng DNA template, 20 pmol/L forward or reverse primer, four different fluorescent-dye–labeled dideoxy terminator nucleotides, deoxynucleotides, and AmpliTaq DNA polymerase fluorescent sequencing (FS). The reaction mixture was subjected to 25 PCR cycles. The obtained DNA fragments were purified by ethanol precipitation, redissolved in Template Suppression Reagent, and loaded on a Genetic Analyzer (Perkin Elmer - Applied Biosystems Division). Western Blot. Cultured stellate cells were lysed in Tris-based buffer (50 mmol/L Tris-HCl, pH 6.8, 1% sodium dodecyl sulfate). The lysate was boiled for 5 minutes. Protein concentration was determined by using Protein assay kit (Pierce, IL). For immunodetection of desmin, vimentin, GFAP, and aSMA, 5 µg total protein of each cell lysate was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis as described by Laemmli30 and electroblotted onto nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). For immunodetection of nestin, 10 µg total protein of each cell lysate was electrophoresed in 5% polyacrylamide gels. The antibodies used for immunodetection were summarized in Table 1. After blocking with 5% skim milk/TBS (Tris-buffered saline)/0.1% Tween 20 for 30 minutes at 37°C, the membrane was reacted with primary antibody for 2 hours at room temperature. After rinsing in TBS/0.1% Tween 20 three times for 10 minutes, the membrane was then reacted with peroxidase-conjugated secondary antibody. After washing with TBS/0.1% Tween 20 four times for 10 minutes, antigens were detected by enhanced chemiluminescence by using SuperSignal Western blotting substrate (Pierce). The results were quantitated by Phosphor-Imaging technology (Molecular Imager, GS 525, Biorad, Hercules, CA).

FIG. 3. Demonstration of nestin mRNA by RT-PCR. (A) Whole liver (WL) and 0.75 µg total RNA of C6 rat glioma cells (RGC) was reverse transcribed and amplified by PCR (35 cycles). We were not able to show nestin mRNA in whole liver; as expected rat glioma cells were positive. (B) RT-PCR reaction on total RNA of stellate cells in culture for 3, 7, and 14 days. We found amplicons with a length between 400 and 500 nucleotides (C) Sequence of the PCR product obtained from stellate cell mRNA was homologous to the originally published sequence.14 Bold nucleotides are primer sequences.


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FIG. 4. Immunohistochemical staining for nestin in situ. (A) Normal rat liver. Nestin is occasionally present in some vascular endothelial cells (arrow) but is absent from the cells that constitute the hepatic parenchyma. Magnification: 2403. (B-E) Rat livers exposed to CCl4 for 6 weeks. (B) Nestin-positive cells (arrows) appear in the vicinity of fibrous septa. Immunoreactive cells display several cytoplasmic processes, which is a typical characteristic of stellate cells. Magnification: 2403. (C) Higher magnification of nestin-positive cell in a liver exposed to CCl4. This immunoreactive cell shows typical intracytoplasmic lipid droplets (arrows). Acetone fixation has removed lipid content of the fat-droplets. Magnification: 4803. (D) Nestin-positive bile duct (B). Magnification: 4803. (E) Negative control section. Primary antibody was replaced by nonimmune rabbit serum. Otherwise, the section was treated as described. Magnification: 1853.

RESULTS

During our studies on intermediate filaments in rat hepatic stellate cells, we observed that these cells in culture contained the class VI intermediate filament protein nestin (Fig. 1). Immunohistochemistry showed that nestin appeared in at least 80% of cells at day 3 (Fig. 1A). By day 6 more than 90% of cells were positive (Fig. 1B). Cells at day 15 were also positive. As expected for an intermediate filament protein, nestin staining exhibited a filamentous pattern in interphase cells. In mitotic cells, immunoreactive nestin was clearly redistributed (Fig. 1B, insert).

Next we investigated by immunoblotting the nestin protein levels in stellate cells at different time points in culture (Fig. 2A). We found that induction of nestin occurred between day 2 and 4, which is in agreement with the data obtained by immunohistochemistry. We observed three bands with molecular weights well above 205 kd. The relative intracellular concentration of nestin peaked around day 6. We then compared the expression pattern of nestin to that of desmin, vimentin, GFAP, and aSMA. In previous studies, it has been shown that expression of desmin, vimentin, and aSMA was

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FIG. 5. Double immunohistochemistry on sections of livers exposed to CCl4 for 6 weeks. (A) Double staining for nestin (dark brown) and desmin (red). Clearly, the two labels overlap in some cells (arrows), indicating that nestin is present in some desmin-positive stellate cells. Magnification: 5203. (B) Double staining for nestin (dark brown) and aSMA (red). Again, the two labels overlap in some cells (arrows), indicating that nestin is present in some activated stellate cells. Magnification: 5203.

upregulated4-6 and that of GFAP downregulated8-10 in activated stellate cells. As shown in Fig. 2B, both desmin and vimentin levels were increased strongly between day 2 and 6 in primary cultures; thereafter, desmin levels decreased whereas vimentin levels remained constant. Relative GFAP and aSMA concentrations showed completely different patterns. GFAP signal was paramount at day 2; its level declined with time in culture. aSMA appeared between day 2 and 4; its level increased continuously up to day 13. The expression of nestin mRNA by cells in culture was examined by RT-PCR. Total RNA was extracted from whole liver, rat glioma cells (positive control), and stellate cells kept in culture for 3, 7, and 14 days. As shown in Fig. 3A, we detected an amplified DNA fragment of the expected size in rat glioma cells but not in whole liver. In cultured stellate cells (Fig. 3B), we found an amplicon of the same size. We characterized the amplicon obtained from stellate cells by DNA sequencing. The obtained sequence (Fig. 3C) was homologous to the previously published rat nestin sequence.14 We then investigated whether nestin was also expressed in activated stellate cells in vivo. Figure 4 shows the distribution of nestin in normal and fibrotic rat liver. In normal liver, we found no staining in the liver parenchyma. Occasionally, we observed nestin in some vascular endothelial cells (Fig. 4A). After 2 and 4 weeks of CCl4 exposure, no significant staining was observed either (data not shown). At 6 and 8 weeks nestin-positive cells appeared in the vicinity of, and inside, the fibrous septa (Fig. 4B). At higher magnification, the nestin-positive cells appeared to contain lipid-droplets, strongly suggesting that these cells were hepatic stellate cells (Fig. 4C). Although the majority of nestin-positive cells was

observed within the lobules and fibrous septa, bile duct epithelial cells and a minority of vascular endothelial cells occasionally exhibited some reaction at 6 or 8 weeks of CCl4 (Fig. 4D). When primary antibody was replaced by nonimmune serum, no staining was observed (Fig. 4E). The nestin-positive cells inside the liver parenchyma were further identified by double immunolabeling for nestin/ desmin and nestin/aSMA. Desmin and aSMA are wellaccepted markers for rat stellate cells. As shown in Fig. 5A, some desmin-positive cells (red reaction product) also contained nestin (dark brown reaction product), indicating that a subpopulation of stellate cells expressed nestin. When sections were double-stained for aSMA and nestin (Fig. 5B), staining for nestin (dark brown reaction product) was partially overlapping with that of aSMA (red reaction product), indicating that a minority of activated stellate cells also expressed nestin. The appearance of nestin-positive stellate cells was a late event during induction of liver fibrosis. Therefore, we compared nestin staining with desmin and aSMA staining in normal rat liver and in livers that were exposed to CCl4 for 2, 4, 6, and 8 weeks. The results were summarized in Fig. 6. In normal liver (Fig. 6A, B, and C), many desmin-positive stellate cells were observed. In contrast, no immunoreactivity for nestin or aSMA was present in the lobules. In early fibrosis (2 to 4 weeks) (Fig. 6D, E, and F), accumulation of desmin-positive stellate cells was observed in the pericentral areas. aSMA-positive cells also appeared at this stage. In spite of the induction of these proteins, no significant staining for nestin was found. At 6 to 8 weeks (Fig. 6G, H, and I), nestin-positive cells started to appear in the vicinity of, and


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FIG. 6. Comparison of the immunocytochemical reaction pattern for nestin (A, D, and G), aSMA (B, E, and H), and desmin (C, F, and I) in normal livers (A, B, and C) and livers exposed to CCl4 for two weeks (D, E, and F) and 6 weeks (G, H, and I). Magnification: 803. Nestin is virtually absent in normal liver. Few cells (arrows) have become nestinpositive following CCl4 exposure for 2 weeks. At 6 weeks, more nestinpostive cells became apparent (arrows). aSMA is only present in vascular smooth muscle cells of normal liver. Following exposure to CCl4 for 2 weeks, a modest number of stellate cells has become aSMApositive. At 6 weeks, their number has increased significantly. Desmin is clearly present in cells scattered throughout the lobule as well as in vascular smooth muscle cells of normal liver. Following CCl4 exposure, many stellate cells show increased staining.

inside, the fibrous septa. At this stage, both desmin and aSMA were strongly induced. DISCUSSION

In this study we have shown that nestin was induced in stellate cells during spontaneous activation in culture and following CCl4-induced liver injury. Nestin is a class VI intermediate filament originally isolated as a marker for neural stem cells.13,14 Subsequently, nestin was found to be expressed by immature glial cells,16 astrocytes in injured central nervous system,17,18 primitive neuroectodermal tumors and gliomas,16,19 melanomas,20 myoblasts,21 and immature cardiac- and skeletal-muscle cells.15,22,23 Stellate cells in

normal rat liver contain GFAP,7-10 an intermediate filament protein characteristic of astrocytes. In addition to this cytoskeletal protein, stellate cells express, on their surface, the cell adhesion molecule N-CAM.11,12 These observations suggest that beyond their morphological similarities, hepatic stellate cells and astrocytes may perform similar biological functions in their respective organ. Consistent with this hypothesis, astrocytes play a central role in reactive gliosis, the tissue repair mechanism of the central nervous system,31 whereas hepatic stellate cells play a key role in tissue repair following liver injury.1,2 Human hepatic stellate cells have a different intermediate filament cytoskeleton than rat cells. Quiescent human cells

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express vimentin, but not desmin.32,33 GFAP is expressed by only a small subpopulation of periportal cells (M. Hautekeete, 1998, unpublished data). Whether or not quiescent human cells express nestin is unknown. Second passage, culture-activated human stellate cells are negative for nestin (A. Geerts, data not shown). The expression of nestin by rat stellate cells raises speculation as to their embryonic origin. It is generally accepted that cephalic neural-crest cells give rise to a variety of cell types, including neurons, glial cells, cartilage, and bone- and smooth-muscle cells.34 The cardiac neural crest, located between the cephalic and trunk crest,35 also gives rise to both neural and connective tissue, including all the musculoconnective tissue in the wall of the large arteries.36 Whether mammalian-trunk neural-crest cells also differentiate into smooth-muscle cells is an open question. A recent study has shown that rodent-trunk neural-crest cells have the potential to differentiate into smooth-muscle cells and that transforming growth factor-b promotes this differentiation process.37 These investigators suggest the possibility that some trunk neural-crest cells also contribute to the development of the smooth-muscle cells of blood vessels. Taken together these data, we conclude that new studies on the embryonic origin of hepatic stellate cells are required. The same argument about possible neural-crest origin is not valid for the nestin-positive bile-duct cells and vascularendothelial cells. These cells have never been found to express GFAP nor N-CAM. The intrahepatic vasculature derives largely from the embryonic umbilical veins whereas intrahepatic bile-duct epithelial cells derive from hepatoblasts (fetal hepatic epithelial cells).38 We have compared protein steady-state levels of nestin with those of desmin, vimentin, GFAP, and aSMA in culturedrat stellate cells. The results show that induction of nestin occurs in parallel with increased presence of desmin and vimentin during early primary culture. After the initial increase, all these proteins appear to level off or decrease slightly. This leveling-off is somewhat unexpected, because upregulation of desmin and vimentin has been thought to be a characteristic of activation of stellate cells.4-6 aSMA increased continuously during the 13 days of observation. This confirms the generally held view that this actin isoform is a good marker for activation of stellate cells. GFAP expression declines during the activation process. This is in keeping with previous observations.8-10 Whereas expression of nestin occurs in early primary culture of rat stellate cells, it takes approximately 6 weeks of chronic CCl4 intoxication before the protein gets expressed in stellate cells in vivo. This observation is in agreement with previous findings that report differences between activation in vitro and in vivo.39,40 The role of the four different intermediate filament proteins expressed by stellate cells is still unknown. Their differential regulation during activation suggests that they exert different functions. The type of intermediate filament protein expressed by a given cell depends on the cell lineage, developmental stage, and sometimes the functional status of the cell. It has been postulated that different intermediate filament proteins have different affinities for intracellular structures or different mechanical properties that make a particular intermediate filament suited for one cell type but not for another.41 In summary, we have shown that nestin expression is


induced in activated rat stellate cells. Our data suggest that nestin can be used as an additional activation marker that is regulated differently from desmin and aSMA. The expression of nestin by stellate cells raises questions regarding their embryonic origin and extends the similarities that stellate cells share with astrocytes. Acknowledgments: We thank Mr. J.-M. Lazou for technical assistance and Mrs. C. Derom for the excellent photographic work. We thank Dr. John Eriksson from Turku Center for Biotechnology, University of Turku and Åbo Akademi University (Finland) for the nestin antibody; Prof. Dr. W. Van Driessche, Laboratory of Physiology, Catholic University of Louvain (Belgium) for the C6 rat glioma cells; and Prof. Dr. M. Pinzani, Universita’di Firenze (Italy), for the cultured human stellate cells. This work has been performed while M. Pekny was a fellow of the Swedish Council for Medical Research. REFERENCES 1. Geerts A, De Bleser P, Hautekeete M, Niki T, Wisse E: Fat-storing (Ito) cell biology. In: Arias I, Boyer J, Fausto N, Jacoby W, Schlachter D, Schafritz D, eds. The liver. Biology and Pathophysiology. New York: Raven press, 1994;819-838. 2. Friedman SL. Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. N Engl J Med 1993;328:1828-1835. 3. Yokoi Y, Namihisa T, Kuroda H, Komatsu I, Miyazaki A, Watanabe S, Usui K. Immunocytochemical detection of desmin in fat-storing cells (Ito cells). HEPATOLOGY 1984;4:709-714. 4. Ogawa K, Suzuki J, Mukai H, Mori M. Sequential changes of extracellular matrix and proliferation of Ito cells with enhanced expression of desmin and actin in focal hepatic injury. Am J Pathol 1986;125:611-619. 5. Ballardini G, Fallani M, Biagini G, Bianchi FB, Pisi E. Desmin and actin in the identification of Ito cells and in monitoring their evolution to myofibroblasts in experimental liver fibrosis. Virchows Arch 1988;56: 45-49. 6. Ramadori G, Veit T, Schwogler S, Dienes HP, Knittel T, Rieder H, Meyer zum Buschenfelde KH. Expression of the gene of the alpha-smooth muscle-actin isoform in rat liver and in rat fat-storing (ITO) cells. Virchows Arch 1990;59:349-357. 7. Gard A, White F, Dutton G. Extra-neural glial fibrillary acidic protein (GFAP) immunoreactivity in perisinusoidal stellate cells of rat liver. J Neuroimmunol 1985;8:359-375. 8. Buniatian G, Gebhardt R, Schrenk D, Hamprecht B. Colocalization of three types of intermediate filament proteins in perisinusoidal stellate cells: Glial fibrillary acidic protein as a new cellular marker. Eur J Cell Biol 1996;70:23-32. 9. Neubauer K, Knittel T, Aurisch S, Fellmer P, Ramadori G. Glial fibrillary acidic protein—a cell type specific marker for Ito cells in vivo and in vitro. J Hepatol 1996;24:719-730. 10. Niki T, de Bleser PJ, Xu G, Van Den Berg K, Wisse E, Geerts A. Comparison of glial fibrillary acidic protein and desmin staining in normal and CCl4-induced fibrotic rat livers. HEPATOLOGY 1996;23:15381545. 11. Nakatani K, Seki S, Kawada N, Kobayashi K, Kaneda K. Expression of neural cell adhesion molecule (N-CAM) in perisinusoidal stellate cells of the human liver. Cell Tissue Res 1996;283:159-165. 12. Knittel T, Aurisch S, Neubauer K, Eichhorst S, Ramadori G. Cell-typespecific expression of neural cell adhesion molecule (N-CAM) in Ito cells of rat liver. Up-regulation during in vitro activation and in hepatic tissue repair. Am J Pathol 1996;149:449-462. 13. Hockfield S, McKay RD. Identification of major cell classes in the developing mammalian nervous system. J Neurosci 1985;5:3310-3328. 14. Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell 1990;60:585-595. 15. Sjoberg G, Jiang WQ, Ringertz NR, Lendahl U, Sejersen T. Colocalization of nestin and vimentin/desmin in skeletal muscle cells demonstrated by three-dimensional fluorescence digital imaging microscopy. Exp Cell Res 1994;214:447-458. 16. Tohyama T, Lee VM, Rorke LB, Marvin M, McKay RD, Trojanowski JQ. Nestin expression in embryonic human neuroepithelium and in human neuroepithelial tumor cells. Lab Invest 1992;66:303-313.

HEPATOLOGY Vol. 29, No. 2, 1999 17. Frisen J, Johansson CB, Torok C, Risling M, Lendahl U. Rapid, widespread, and longlasting induction of nestin contributes to the generation of glial scar tissue after CNS injury. J Cell Biol 1995;131:453464. 18. Clarke SR, Shetty AK, Bradley JL, Turner DA. Reactive astrocytes express the embryonic intermediate neurofilament nestin. Neuroreport 1994;5: 1885-1888. 19. Dahlstrand J, Collins VP, Lendahl U. Expression of the class VI intermediate filament nestin in human central nervous system tumors. Cancer Res 1992;52:5334-5341. 20. Florenes VA, Holm R, Myklebost O, Lendahl U, Fodstad O. Expression of the neuroectodermal intermediate filament nestin in human melanomas. Cancer Res 1994;54:354-356. 21. Kachinsky AM, Dominov JA, Miller JB. Intermediate filaments in cardiac myogenesis: Nestin in the developing mouse heart. J Histochem Cytochem 1995;43:843-847. 22. Sejersen T, Lendahl U. Transient expression of the intermediate filament nestin during skeletal muscle development. J Cell Sci 1993;106:12911300. 23. Kachinsky AM, Dominov JA, Miller JB. Myogenesis and the intermediate filament protein, nestin. Dev Biol 1994;165:216-228. 24. Geerts A, Lazou JM, De Bleser P, Wisse E. Tissue distribution, quantitation and proliferation kinetics of fat-storing cells in carbon tetrachlorideinjured rat liver. HEPATOLOGY 1991;13:1193-1202. 25. Frojdman K, Pelliniemi L, Lendahl U, Virtanen I, Eriksson J. The intermediate filament protein nestin occurs transiently in differentiating testis of rat and mouse. Differentiation 1997;61:243-249. 26. Adams J. Heavy metal intensification of DAB-based HRP reaction product (letter). J Histochem Cytochem 1981;29:775. 27. De Bleser P, Geerts A, Van Eyken P, Vrijsen R, Lazou J-M, Desmet V, Wisse E: Tenascin synthesis in cultured rat liver fat-storing cells. In: Wisse E, Knook D, McCuskey R, eds. Cells of the hepatic sinusoid. Rijswijk: Kupffer Cell Foundation, 1991;218-221. 28. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-159.

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