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Desmin distinguishes cultured fat-storing cells from myofibroblasts, smooth muscle cells and fibroblasts in the rat
JournalofHepatology, 1988: 6:267-276
267
Elsevier HEP 00406
Desmin distinguishes cultured fat-storing cells from myofibroblasts, smooth muscle cells and fibroblasts in the rat
Shujiro Takase, Maria A. Leo, Toshihiko Nouchi and Charles S. Lieber Section of Liver Disease and Nutrition, Mount Sinai School of Medicine (CUNY), and Alcohol Research and Treatment Center, Bronx Veterans Administration Medical Center, New York, N Y (U.S.A.)
(Received 12 November 1987) (Accepted 19 January 1988)
Summary To differentiate cultured rat liver myofibroblasts, fat-storing cells, aortic smooth muscle cells and skin fibroblasts from each other, desmin and vimentin stainings were undertaken by indirect immunofluorescence using monoclonal antibodies. In myofibroblasts, the reaction with antibodies to vimentin was positive but that with antibodies to desmin was virtually negative. In primary cultures as well as subsequent passage of fat-storing cells, reactions with antibodies to both desmin and vimentin were positive. In primary culture of smooth muscle cells, both reactions were positive, but in the first passage, smooth muscle cells lost the reactivity with antibodies to desmin. Fibroblasts showed a positive reaction with antibodies to vimentin and a negative one with antibodies to desmin. Thus, immunohistochemistry of intermediate filaments allows for the differentiation between fat-storing cells, which are desmin- and vimentin-positive, and myofibroblasts or fibroblasts, which are desmin-negative but vimentin-positive. Smooth muscle cells are also vimentin-positive and become desmin-negative after the first passage.
Introduction Deposition of collagen in the Disse space and in the pericellular interstitium of the liver is one of the characteristic features of alcoholic liver injury. Myo-
fibroblasts (Mfs) are associated with the deposition of collagen in the perivenular area of the liver in alcohol-fed baboons [1] and in alcoholic patients [2]. Fatstoring cells (FSCs), also called lipocytes or Ito cells and located in the space of Disse, have been con-
Supported, in part, by the Veterans Administration and DHHS grants AA 03508 and AM 32810. Correspondence: Charles S. Lieber, M.D., Alcohol Research and Treatment Center, Veterans Administration Medical Center, 130 West Kingsbridge Road, Bronx, New York 10468. U.S.A. Tel: (212) 933 5666. 0168-8278/88/$03.50 (~) 1988 Elsevier Science Publishers B.V. (Biomedical Division)
268 sidered to have a close correlation with fibrogenesis in the liver [3-6]. It also has been suggested that the progression of hepatic fibrosis is associated with transformation of FSCs to transitional cells characterized by depletion of lipid droplets and a hypertrophy of the rough endoplasmic reticulum [7]. Recently, isolation and culture of Mfs from baboon [8] and rat [9] livers were achieved and the capacity of these cells to synthesize types I, llI and IV collagens and laminin was demonstrated. FSCs also were isolated and cultured from rat liver, and their fibrogenic role was determined [10,11]. However, the character or origin of these cells has not been fully elucidated. In studies of the cytoskeleton, Yokoi et al. [12] reported that FSCs of rat liver contained desmin, and De Leeuw et al. [10] observed actin, tubulin and vimentin in the first passage of cultured FSCs. Also, Schtirch et al. [13] demonstrated that human Mfs of infiltrating ductal mammary carcinomas lack desmin and prekeratin, and contain only vimentin. The present study was undertaken to establish differential features of cultured liver Mrs and FSCs, aortic smooth muscle cells (SMCs) and skin fibroblasts (Fbs) in the rat by using antibodies against the intermediate filaments desmin and vimentin. This differentiation may help to establish the respective roles of these cells in the process of fibrogenesis and in defining the purity of cell culture systems. Such studies have been hampered thus far by the lack of relatively convenient methods of identification in vitro.
Methods
Arabinogalactan (Stractan), retinol acetate, collagenase type I, protease type XIV, elastase type lII and DNAase were purchased from Sigma Chemical Company, St. Louis, MO, and Percoll density gradient material from Pharmacia Fine Chemicals AB, Uppsala, Sweden. Tissue culture media and fetal bovine serum were obtained from Grand Island Biological Company (Grand Island, NY). We purchased fluorescein isothiocyanate (FITC)-conjugated antibodies from Cappel Laboratories (Westchester, PA), and mouse monoclonal antibodies (IgG class) to des-
s. TAKASE ct al. min and vimentin from Labsystems (Helsinki, Finland). The latter had been lyophilized in diluted ascites fluid containing phosphate-buffered saline (PBS), and 0.1% thiomersal as a preservative. The lyophilized material was reconstituted by adding 0.25 m[ of distilled water.
Animal procedures Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) weighing about 250-300 g were fed Purina Chow diet. For isolation of FSCs, the rats were injected subcutaneously with 60 000 IU of retinol acetate three times a week for 2 weeks. Retinol acetate was dissolved in corn oil and sonicated.
Cell isolation and culture Isolation and culture of Mrs from rat liver was carried out by the method of Leo et al. [9]. Primary cultures were established in Falcon 50-ml plastic flasks (Falcon Labware, Oxnard, CA) with Ham's medium containing 10% fetal bovine serum, 100 units of penicillin, and 100 pg of streptomycin per ml of medium and the subcultures were grown in Dulbecco's modified Eagle's (DME) medium supplemented as noted above. FSCs were isolated and cultured by the method of Friedman et al. [ll]. The liver was first perfused through the portal vein in situ with Ca -'+- and Mg 2+free Hanks" balanced salt solution (HBSS) at 37 °C for 10 rain at a flow rate of 15 ml/min. Then, 0.03% collagenase type I in Leibovitz L-15 medium (200 ml) and 0.1% protease type XIV in the same medium (100 ml) at 37 °C was used. After the liver capsule and big vessels were removed, the liver was cut into small pieces and incubated in 100 ml of medium containing 0.04% protease type XIV and 1 mg of DNAase at 37 °C for 30 min. The pH was kept at about 7.4 with 1 N NaOH. At the end of the incubation period, the cell suspension was filtered through nylon gauze and the filtrate centrifuged at 450 x g for 7 min. The cell pellet was washed twice with medium, resuspended and dispersed through a 20 gauge needle to further dissociate the cells. The non-parenchymal cell suspension was centrifuged on a discontinuous
DESMIN IN LIPOCYTES AND MYOFIBROBLASTS gradient of 6, 8, 12 and 20% Stractan at 20 000 rpm for 30 rain using a swinging bucket rotor (Beckman, SW 41Ti). The FSC fraction was collected from the top interface of the gradient and washed twice with medium. Primary and subcultures were carried out in DME medium containing 20% fetal bovine serum, 100 units of penicillin and 100 pg of streptomycin per ml of medium. Cultures of aortic SMCs and skin Fbs from rats were carried out as follows [14]: The thoracic aorta was dissected and rinsed in HBSS. The adventitia and intima were scraped off with a scalpel and the inner layer of the media was cut into 1 mm 3 pieces. Skin including epidermis and dermis was taken out from the back of the rat under sterile conditions. Biopsied skin was minced into 3 - 4 mm 3 pieces. These tissue pieces were placed in culture chambers with sufficient D M E medium with 10% fetal bovine serum to prevent drying. Explants attached on to the coverslips or the culture flasks within a few days, at which time additional medium was added. After cell migration around the explants was recognized, tissue pieces were removed from culture chambers and cell cultures were maintained in D M E medium with 10% fetal bovine serum. In addition, isolation and culture of SMCs were carried out by the method of collagenase-elastase digestion [15]. A collagenase (type I, 0.4%)-elastase (type Ill, 0.05%) mixture in D M E medium without fetal bovine serum was added to small pieces of aorta. The enzyme digestion was carried out with gentle stirring at 37 °C for 60 rain. The resulting cell suspension was centrifuged at 400 x g for 5 min, and the cell pellet obtained was washed twice with D M E medium containing 10% fetal bovine serum. For subculture, the cultures were washed at confluency in Ca 2+- arid M f + - f r e e HBSS followed by trypsinization for 10 min in Ca 2+- and Mg2+-free HBSS containing trypsin (0.5%) and E D T A - 4 N a (0.2%). Trypsinization was stopped by adding fetal bovine serum and the cell suspension was centrifuged at 450 x g for 4 rain. The supernatant was removed and the cell pellet was resuspended in D M E medium. The culture was carried out at 37 °C in a humidified atmosphere containing 5% CO 2 in air.
269
Morphology For electron microscopy, the cell monolayers were scraped off, fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and post-fixed in 1% OsO4 in 0.1 M cacodylate buffer (4.5% sucrose was added to both fixatives). The cells were dehydrated in graded ethanol and embedded in Epon 812. Thin sections were cut and examined in a Zeiss EM 10-C electron microscope (Carl Zeiss, Inc., Thornwood, NY).
Indirect immunofluorescence For indirect immunofluorescence staining of desmin and vimentin, the cells were grown on coverslips. The cells were washed with HBSS (to remove the culture medium) and with PBS three times. Fixation of cultured cells was carried out with acetone at - 2 0 °C for 5 min. After three washes with PBS the cells were covered with normal rabbit serum for 20 rain in order to reduce non-specific background fluorescence. Then, the cells were incubated with mouse monoclohal antibodies to desmin and vimentin (diluted 1:10 with PBS), at room temperature for 1 h in a humidified chamber, and washed carefully with PBS. The cells were then incubated with FITC-conjugated anti-mouse IgG at room temperature for 1 h, and finally washed with PBS. After mounting with 90% glycerol in PBS (pH 8-9) containing 1% o-phenylenediamine [10], the slides were examined and photographed using a Zeiss fluorescence microscope equipped with epiillumination. Contro.s were carried out by omitting the first antibody and including instead either PBS or a preimmune mouse serum or mouse ascites fluid at the same dilution as used for the monoclonal antibody. Negative results were obtained with these controls.
Results
Cell growth of Mfs was observed within 4 - 5 days after the initiation of the culture, and confluency was reached within 1 month. The Mfs in secondary cultures reached confluency in about 2 weeks. By transmission electron microscopy, Mfs in primary culture
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s. TAKASE et al.
Fig. 1. Electron micrograph of cultured Mfs in primary culture. These cells display abundant microfilaments (mf), dense bodies (db). ~,nd basal lamina-like structures (bl). (Uranyl acetate and lead citrate. ×4160.)
lipid droplets in the second passage and also a welldeveloped rough endoplasmic reticulum (Fig. 3). In cultures of SMCs and Fbs, cell migration around the tissue explants was recognized within 4-5 days; confluency was reached in 10-14 days, and subcultures were carried out each week. Cultures of SMCs obtained by the enzyme digestion method displayed the same growth as noted above. In primary cultures and 1st and 5th passage of Mrs, the reaction with the antibody to desmin was negative or virtually negative (Fig. 4a), but that with antibody to vimentin was positive (Fig. 4b). Passaged myofibroblasts maintained these characteristic features, as described before [8,9]. By contrast, in FSCs, reactions with antibodies to both desmin and vimentin were positive in primary cultures as well as subsequent passages (Fig. 5a and b). Numerous fibrils of desmin or vimentin were clearly recognized in the cytoplasm, and the same reactive pattern with each antibody was observed (Fig. 5c and d). In primary cultures of SMCs, both reactions were positive (Fig. 6a and b), but in the first passage, SMCs lost reactivity to desmin antibody (Fig. 6c) and maintained that to vimentin antibody (Fig. 6d). One morphologic feature of these SMCs was the abundance of microfilaments and lysosomes, as previously reported [16].
displayed abundant microfilaments, dense bodies and were surrounded by a basal lamina-like structure (Fig. 1). Yield of Mfs was 1.1-2.2-106 per liver and viability by the trypan blue exclusion test was more than 84%. The yield of isolated FSCs from vitamin A-treated rat was 1.4-3.2.106 cells per liver and the viability was more than 85%. Most of the isolated FSCs contained multiple and large lipid droplets by oil-red-O staining and electron microscopic examination (Fig. 2). The growth of cultured FSCs was very slow and confluency was reached in 6 weeks in primary cultures and in 2-3 weeks in subcultures. The lipid droplets in the cytoplasm decreased gradually and could no longer be recognized by phase contrast microscopy 7-10 days after the initiation of the culture. However, by electron microscopy, the cells retained small
Fig. 2. Electron micrograph of isolated FSCs from vitamin Atreated rats. These cells contain multiple and large lipid droplets. (Uranyl acetate and lead citrate, x4300.)
DESMIN IN LIPOCYTES AND MYOFIBROBLASTS
271
Fig. 3. Electron micrograph of cultured FSCs (second passage). These cells have small lipid droplets (F), well-developed rough endoplasmic reticulum (rer) and smooth endoplasmic reticulum (ser). (Uranyl acetate and lead citrate, x 14 850.)
Fbs, as expected, were negative for desmin (Fig. 7a) and positive for vimentin (Fig. 7b). These fibroblasts maintained characteristic elongated shapes with strand cytoplasm. Table 1 summarizes the results of desmin and vimentin staining in cultured cells. In FSCs, the intensity of fluorescence in both reactions did not change after multiple passages. By contrast,
in SMCs, the reaction to desmin antibody was lost entirely in the first passage. The Fbs displayed reduced staining of vimentin antibody in secondary cultures. To examine the contamination of desmin-positive cells in cultured Mfs and the influence of enzyme digestion on desmin of SMCs, desmin staining in primary culture of Mfs and SMCs was carried out at the
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s. T A K A S E et al.
J
Fig. 4. Desmin and vimentin staining in the first passage of Mfs. The reaction to desmin antibody is virtually negative (a) but that to vimentin antibody is positive (b) (x 300).
O
Fig. 5. Desmin and vimentin staining in the 5th passage of FSCs. Reactions to both desmin (a) and vimentin (b) antibodies are positive ( x 300). Many fibrils of desmin (c) and vimentin (d) are clearly recognized in the cytoplasm at high magnification ( × 1070).
DESMIN IN LIPOCYTES AND MYOFIBROBLASTS
273
Fig. 6. Desmin and vimentin staining in primary culture and the first passage of SMCs. Reactions to both desmin (a) and vimentin (b) antibodies arc positive in primary culture. However, in the first passage, the reaction to desmin antibody is lost (c) whereas that to vimentin antibody is maintained (d) (×300).
Fig. 7. Desmin and vimentin staining in Fbs. Thc reaction to desmin antibody is negative (a) whereas that to vimentin antibody is positive (b) (x300).
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S. TAKASE ct al.
TABLE 1 INTENSITY OF FLUORESCENCE IN DESMIN AND VIMENTIN STAINING OF Mfs, FSCs, SMCs AND Fbs Cultured cells
Desmin
Vimentin
Mrs primary culturc 1st passage 5th passage
-
+ ++ ++
FSCs primary culture 1st passage 5th passage
++ ++ ++
++ ++ ++
SMCs primary culture 1st passage 5th passage
++ -
++ ++ +
Fbs primary culture 1st passage 5th passage
-
++ + +
Several types of cell have been considered to be involved in hepatic fibrogenesis. Mfs were described in granulation tissue by Gabbiani et al. [17,18], and were recognized in cirrhosis in man [19,20] and mice [21,22] and in noncirrhotic liver in alcoholics [1]. They were found to be desmin-negative in situ [13]. Although these ceils showed some similarities with smooth muscle cells, they were clearly differentiated from the latter by their morphologic features [23,24]. Some have postulated that myofibroblasts represent a reversible phenotype modulation of fibroblast-type cells to a contractile state [25], whereas others have
same time. Desmin-positive cells were recognized in 16.0 _+ 2.4% of cultured Mfs. In SMCs, 81.8 + 5.4% of cells showed a positive reaction to the desmin antibody (Table 2).
Discussion The present study revealed that in cultured rat
TABLE 2 PERCENT OF CELLS STAINING FOR DESMIN IN PRIMARY CULTURES OF Mfs AND SMCs OBTAINED BY THE ENZYME DIGESTION METHOD Values are means _+S.E. Cultured cells
Intensity of fluorescence -
+
cells, immunohistochemistry of intermediate filaments allows for the differentiation between FSCs, which are vimentin- and desmin-positive, and Mfs or Fbs, which are vimentin-positive but desmin-negative. SMCs are vimentin- and desmin-positive in primary cultures and become desmin-negative after the first passage.
++
Mrs desmin staining control staining
84.3 + 1.7 100.0 + 0.0
13.5 + (I.8 0
2.5 _+0.6 0
SMCs desmin staining control staining
18.3 + 3.1 100.0 + (I.O
64.8 + 3.4 0
17.(I + 7.3 0
emphasized differences between myofibroblasts and fibroblasts [26]. After chronic alcohol consumption, the n u m b e r of Mfs and other mesenchymal cells increased [1,2]. FSCs, characterized by a b u n d a n t lipid droplets, have been considered to have a close correlation with fibrogenesis in the liver [3-6]. In baboons fed alcohol, the progression of hepatic fibrosis was a~sociated with the appearance of transitional cells characterized by a depletion of lipid droplets and a hypertrgphy of the rough endoplasmic reticulum [7]. Transitional cells have a b u n d a n t microfilaments, dense bodies and pinocytic vesicles and resemble the Mfs of the perivenular zone, but they can be distinguished by the lack of surrounding basal lamina [7]. De Leeuw et al. [10] described that in primary cultures and cell lines of FSCs from rat liver (established using metrizamide density centrifugation), collagen types l and III and laminin were present intracellularly in small granules. Friedman et al. [11] reported that cultured FSCs isolated from rat liver with Stractan density gradients secreted predominantly type I collagen, measured by E L I S A assay. Thus, both Mfs and FSCs have been incriminated in hepatic fibrogenests. However, the characteristic features or origin of these cells have not been fully elucidated. Our study demonstrates that in primary culture as well as
DESMIN IN LIPOCYTES AND MYOFIBROBLASTS subsequent passage of FSCs, reactions with antibodies to both desmin and vimentin were positive. Even after passage, the intermediate filaments in FSCs were maintained; because of their positivity to desrain and vimentin, FSCs could still be clearly distinguished from Mfs, SMCs and Fbs, which were desrain-negative. These findings suggest that although these cells have some similar morphological features, their cytoskeletal elements differ and that rat liver Mrs may be more closely related to Fbs than to SMCs. On the other hand, FSCs from rat liver are mesenchymal in origin and display some similarity with muscle cells. In studies of the intermediate filaments of vascular SMCs, conflicting data have been published: only vimentin and no desmin [27-29], or both vimentin and desmin [30], have been reported to be present. In addition, Travo et al. [31] described that in primary cultures of adult rat vascular SMCs derived from the thoracic aorta, there are three distinct cell types: cells rich in vimentin and lacking desmin, cells expressing both vimentin and desmin, and occasionally cells rich in desmin but lacking vimentin. In the present study, using monoclonal antibodies, coexistence of vimentin and desmin was demonstrated in primary cultures of SMCs derived from thoracic aorta. The disappearance of desmin in the first passage may sug-
References 1 Nakano M, Lieber CS. Ultrastructure of initial stages of perivenular fibrosis in alcohol-fed baboons. Am J Pathol 1982; 106: 145-155. 2 Nakano M, Worner TM, Lieber CS. Perivenular fibrosis in alcoholic liver injury: ultrastructure and histologic progression. Gastroenterology 1982; 83: 777-785. 3 Schnack H, Stockinger L, Wewalka F. Adventitious connective tissue cells in the space of Disse and their reaction to fibre formation. Rev Int Hepatol 1967; 17: 855-86//. 4 McGee JO'D, Patrick RS. The role of perisinusoidal cells in hepatic fibrogenesis. An electron microscopic study of acute carbon tetrachloride liver injury. Lab Invest 1972;26: 429-440. 5 Kent G, Gay S, lnouye T, Bahu R, Minick OT, Popper H. Vitamin A-containing lipocytes and formation of type III collagen in liver injury. Proc Natl Acad Sci USA 1976: 73: 3719-3722. 6 Minato Y, Hasumura Y, Takeuchi J. The role of fat-storing
275 gest that desmin is more unstable than vimentin in these cells. The reason for the selective loss of desmin is not known but it is not u n c o m m o n for cells to change some of their characteristic features upon passage from primary to secondary cultures. Voss et al. [32] studied SMCs in cultured tissue specimens from human fibrotic liver obtained by needle biopsy. Two cell types emerged from the tissue explants. From their morphology and biosynthetic products, they resembled SMCs and endothelial cells from blood vessel walls. Isolates of Mfs may be contaminated by SMCs and endothelial cells. However, in primary culture of the Mfs fraction, we found that only 16.0% of the cells were desmin positive. Also, SMCs obtained by the enzyme digestion method did not lose their reaction with antibody to desmin. Therefore, these findings show that the majority of cells isolated in the Mf fraction have characteristics which differ from SMCs of the thoracic aorta. SMCs were also shown to be desmin-positive in the wall of hepatic arteries [33,34] and, possibly, hepatic veins [35]. In summary, our results show that cultured FSCs maintain their desmin and vimentin, and that by desmin staining, FSCs can be differentiated from Mfs and Fbs, and from SMCs after the first passage in culture.
cells in Dissc space fibrogenesis in alcoholic liver disease. Hepatology 1983; 3: 559-566. 7 Mak KM, Leo MA, Lieber CS. Alcoholic liver injury in baboons: transformation of lipocytes to transitional cells. Gastroenterology 1984: 87: 188-21111. 8 Savolainen E-R, Leo MA, Timpl R. Lieber CS. Acetaldehyde and lactate stimulate collagen synthesis of cultured baboon liver myofibroblasts. Gastroenterology 1984: 87: 777-787. 9 Leo MA, Mak KM, Savolainen E-R, Licber CS. Isolation and culture of myofibroblasts from rat liver. Proc Soc Exp Biol Med 1985: 180:382-391. 10 De Leeuw AM. McCarthy SP, Geerts A, Knook DL. Purified rat liver fat-storing cells in culture dividc and contain collagen. Hepatology 1984; 4: 392-41/3. 11 Friedman SL, Roll FJ, Boyles J, Bissell DM. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci USA 1985: 82:8681-8685. 12 Yokoi Y, Namihisa T, Kuroda H, ct al. Immunocytochemical detection of desmin in fat-storing cells (Ito cells). Hep-
276 atology 1984: 4: 7119-714. 13 Schi,irch W, Seemayer TA, Lagac6 R, Gabbiani G. The intermediate filament cytoskeleton of myofibroblasts: an immunofluorescence and ultrastructural study. Virchows Arch 1984; 41)3: 323-336. 14 Ross R. The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol 1971; 50: 172-186. 15 Barone LM. Faris B, Chipman SD. Toselli P. Oakes BW, Franzblau C. Alteration of the extracellular matrix of smooth muscle cells by ascorbate treatment. Biochim Biophys Acta 1985: 84(1: 245-254. 16 James-Kracke MR, Sloane BF, Shuman H, Somlyo AP. kysosomal composition in cultured vascular smooth muscle cells: electron probe analysis. Proc Natl Acad Sci USA 1979; 76: 6461-6465. 17 Gabbiani G. Ryan GB, Majno G. Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction. Experientia 1971; 27: 549-550. 18 Gabbiani G, Hirschel BJ, Ryan GB. Granulation tissue as a contractile organ: a study of structure and function. J Exp Med 1972; 135: 719-734. 19 Bhathal PS. Presence of modified fibroblasts in cirrhotic livers in man. Pathology 1972; 4: 139-144. 20 Rudolph R, McClure WJ, Woodward M. Contractile fibroblasts in chronic alcoholic cirrhosis. Gastroenterology 1979; 76: 7114-7(19. 21 Irle C, Kocher O. Gabbiani G. Contractility of myofibroblasts during experimental liver cirrhosis. J Submicrosc Cytol 1980; 12: 209-217. 22 Hay ED, Hasty DE, Kiehnau KL. Morphological investigation of fibers derived from various types: fine structure of collagens and their relation to glucosaminoglycans (GAG). In: Gastpar H, K/.ihn K, Marx R, eds. Collagen-Platelet Interaction. New York: Stuttgart-Schattauer Verlag, 1978: 129-162. 23 Lipper S, Kai LB, Reddick RL. The myofibroblast. Pathol Annu 1980; 443-470 (year book). 24 Barsky SH, Green WR, Grotendorst GR, Liotta LA. Desmoplastic breast carcinoma as a source of human myofibroblasts. Am J Pathol 1984; 115: 329-333.
S. TAKASE et al. 25 Bellows CG, Melcher AH, Bhargava U, Aubin JE. Fibroblasts contrasting three-dimensional collagen gels exhibit ultrastructure consistent with either contraction or protein secretion. J Ultrastruct Res 1982: 78: 178-192. 26 Van de Berg JS, Rudolph R., Woodward M. Comparative growth dynamics and morphology between cultured myofibroblasts from granulating wounds and dermal fibroblasts. Am J Pathol 1984; 114: 187-200. 27 Gabbiani G, Schmid E, Winter S, et al. Vascular smooth muscle cells differ from other smooth muscle cells: predominance of vimentin filaments and a specific a-type actin. Proc Natl Acad Sci USA 1981 ; 78: 298-3(12. 28 Frank ED, Warren L. Aortic smooth muscle cells contain vimentin instead of desmin. Cell Biol 1981 ; 78: 3020-3024. 29 Yaoita E, Kazama T. Kawasaki K, Miyazaki S, Yamamoto T, Kihara I. In vitro characteristics of rat mesangial cells in comparison with aortic smooth muscle cells and derman fibroblasts. Virchows Arch 1985; 49: 285-294. 30 Schmid E, Osborn M, Rungger-Brfindle E, Gabbiani G, Weber K, Franke WW. Distribution of vimentin and desrain filaments in smooth muscle tissue of mammalian and avian aorta. Exp Cell Res 1982; 137: 329-340. 31 Travo P, Weber K, Osborn M. Co-existence of vimentin and desmin type intermediate filaments in a subpopulation of adult rat vascular smooth muscle cells growing in primary culture. Exp Cell R.es 1982; 139: 87-94. 32 Voss B. Rauterberg J, Pott G, et al. Nonparenchymal cells cultivated from explants of fibrotic liver resemble endothelial and smooth muscle cells from blood vessel walls. Hepatology 1982; 2: 19-28. 33 Yokoi Y, Namihisa T, Kuroda H, et al. lmmunocytochemical detection of desmin in fat-storing cells (lto cells). Hepatology 1984; 4: 709-714. 34 Burt AD, MacSween RNM. Immunolocalization of desmin in fixed rat and human liver. In: Kim A, Knook DL, Wisse E, eds. Cells of the Hepatic Sinusoid. Rijswijk, The Netherlands: the Kupffer Cell Foundation, 1986; 253-254. 35 Burt AD, R.obertson JL, Heir J, MacSween RNM. Desmin-/containing stellate cells in rat liver; distribution in normal animals and response to experimental acute liver injury. J Pathol 1986; 150: 29-35.
267
Elsevier HEP 00406
Desmin distinguishes cultured fat-storing cells from myofibroblasts, smooth muscle cells and fibroblasts in the rat
Shujiro Takase, Maria A. Leo, Toshihiko Nouchi and Charles S. Lieber Section of Liver Disease and Nutrition, Mount Sinai School of Medicine (CUNY), and Alcohol Research and Treatment Center, Bronx Veterans Administration Medical Center, New York, N Y (U.S.A.)
(Received 12 November 1987) (Accepted 19 January 1988)
Summary To differentiate cultured rat liver myofibroblasts, fat-storing cells, aortic smooth muscle cells and skin fibroblasts from each other, desmin and vimentin stainings were undertaken by indirect immunofluorescence using monoclonal antibodies. In myofibroblasts, the reaction with antibodies to vimentin was positive but that with antibodies to desmin was virtually negative. In primary cultures as well as subsequent passage of fat-storing cells, reactions with antibodies to both desmin and vimentin were positive. In primary culture of smooth muscle cells, both reactions were positive, but in the first passage, smooth muscle cells lost the reactivity with antibodies to desmin. Fibroblasts showed a positive reaction with antibodies to vimentin and a negative one with antibodies to desmin. Thus, immunohistochemistry of intermediate filaments allows for the differentiation between fat-storing cells, which are desmin- and vimentin-positive, and myofibroblasts or fibroblasts, which are desmin-negative but vimentin-positive. Smooth muscle cells are also vimentin-positive and become desmin-negative after the first passage.
Introduction Deposition of collagen in the Disse space and in the pericellular interstitium of the liver is one of the characteristic features of alcoholic liver injury. Myo-
fibroblasts (Mfs) are associated with the deposition of collagen in the perivenular area of the liver in alcohol-fed baboons [1] and in alcoholic patients [2]. Fatstoring cells (FSCs), also called lipocytes or Ito cells and located in the space of Disse, have been con-
Supported, in part, by the Veterans Administration and DHHS grants AA 03508 and AM 32810. Correspondence: Charles S. Lieber, M.D., Alcohol Research and Treatment Center, Veterans Administration Medical Center, 130 West Kingsbridge Road, Bronx, New York 10468. U.S.A. Tel: (212) 933 5666. 0168-8278/88/$03.50 (~) 1988 Elsevier Science Publishers B.V. (Biomedical Division)
268 sidered to have a close correlation with fibrogenesis in the liver [3-6]. It also has been suggested that the progression of hepatic fibrosis is associated with transformation of FSCs to transitional cells characterized by depletion of lipid droplets and a hypertrophy of the rough endoplasmic reticulum [7]. Recently, isolation and culture of Mfs from baboon [8] and rat [9] livers were achieved and the capacity of these cells to synthesize types I, llI and IV collagens and laminin was demonstrated. FSCs also were isolated and cultured from rat liver, and their fibrogenic role was determined [10,11]. However, the character or origin of these cells has not been fully elucidated. In studies of the cytoskeleton, Yokoi et al. [12] reported that FSCs of rat liver contained desmin, and De Leeuw et al. [10] observed actin, tubulin and vimentin in the first passage of cultured FSCs. Also, Schtirch et al. [13] demonstrated that human Mfs of infiltrating ductal mammary carcinomas lack desmin and prekeratin, and contain only vimentin. The present study was undertaken to establish differential features of cultured liver Mrs and FSCs, aortic smooth muscle cells (SMCs) and skin fibroblasts (Fbs) in the rat by using antibodies against the intermediate filaments desmin and vimentin. This differentiation may help to establish the respective roles of these cells in the process of fibrogenesis and in defining the purity of cell culture systems. Such studies have been hampered thus far by the lack of relatively convenient methods of identification in vitro.
Methods
Arabinogalactan (Stractan), retinol acetate, collagenase type I, protease type XIV, elastase type lII and DNAase were purchased from Sigma Chemical Company, St. Louis, MO, and Percoll density gradient material from Pharmacia Fine Chemicals AB, Uppsala, Sweden. Tissue culture media and fetal bovine serum were obtained from Grand Island Biological Company (Grand Island, NY). We purchased fluorescein isothiocyanate (FITC)-conjugated antibodies from Cappel Laboratories (Westchester, PA), and mouse monoclonal antibodies (IgG class) to des-
s. TAKASE ct al. min and vimentin from Labsystems (Helsinki, Finland). The latter had been lyophilized in diluted ascites fluid containing phosphate-buffered saline (PBS), and 0.1% thiomersal as a preservative. The lyophilized material was reconstituted by adding 0.25 m[ of distilled water.
Animal procedures Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) weighing about 250-300 g were fed Purina Chow diet. For isolation of FSCs, the rats were injected subcutaneously with 60 000 IU of retinol acetate three times a week for 2 weeks. Retinol acetate was dissolved in corn oil and sonicated.
Cell isolation and culture Isolation and culture of Mrs from rat liver was carried out by the method of Leo et al. [9]. Primary cultures were established in Falcon 50-ml plastic flasks (Falcon Labware, Oxnard, CA) with Ham's medium containing 10% fetal bovine serum, 100 units of penicillin, and 100 pg of streptomycin per ml of medium and the subcultures were grown in Dulbecco's modified Eagle's (DME) medium supplemented as noted above. FSCs were isolated and cultured by the method of Friedman et al. [ll]. The liver was first perfused through the portal vein in situ with Ca -'+- and Mg 2+free Hanks" balanced salt solution (HBSS) at 37 °C for 10 rain at a flow rate of 15 ml/min. Then, 0.03% collagenase type I in Leibovitz L-15 medium (200 ml) and 0.1% protease type XIV in the same medium (100 ml) at 37 °C was used. After the liver capsule and big vessels were removed, the liver was cut into small pieces and incubated in 100 ml of medium containing 0.04% protease type XIV and 1 mg of DNAase at 37 °C for 30 min. The pH was kept at about 7.4 with 1 N NaOH. At the end of the incubation period, the cell suspension was filtered through nylon gauze and the filtrate centrifuged at 450 x g for 7 min. The cell pellet was washed twice with medium, resuspended and dispersed through a 20 gauge needle to further dissociate the cells. The non-parenchymal cell suspension was centrifuged on a discontinuous
DESMIN IN LIPOCYTES AND MYOFIBROBLASTS gradient of 6, 8, 12 and 20% Stractan at 20 000 rpm for 30 rain using a swinging bucket rotor (Beckman, SW 41Ti). The FSC fraction was collected from the top interface of the gradient and washed twice with medium. Primary and subcultures were carried out in DME medium containing 20% fetal bovine serum, 100 units of penicillin and 100 pg of streptomycin per ml of medium. Cultures of aortic SMCs and skin Fbs from rats were carried out as follows [14]: The thoracic aorta was dissected and rinsed in HBSS. The adventitia and intima were scraped off with a scalpel and the inner layer of the media was cut into 1 mm 3 pieces. Skin including epidermis and dermis was taken out from the back of the rat under sterile conditions. Biopsied skin was minced into 3 - 4 mm 3 pieces. These tissue pieces were placed in culture chambers with sufficient D M E medium with 10% fetal bovine serum to prevent drying. Explants attached on to the coverslips or the culture flasks within a few days, at which time additional medium was added. After cell migration around the explants was recognized, tissue pieces were removed from culture chambers and cell cultures were maintained in D M E medium with 10% fetal bovine serum. In addition, isolation and culture of SMCs were carried out by the method of collagenase-elastase digestion [15]. A collagenase (type I, 0.4%)-elastase (type Ill, 0.05%) mixture in D M E medium without fetal bovine serum was added to small pieces of aorta. The enzyme digestion was carried out with gentle stirring at 37 °C for 60 rain. The resulting cell suspension was centrifuged at 400 x g for 5 min, and the cell pellet obtained was washed twice with D M E medium containing 10% fetal bovine serum. For subculture, the cultures were washed at confluency in Ca 2+- arid M f + - f r e e HBSS followed by trypsinization for 10 min in Ca 2+- and Mg2+-free HBSS containing trypsin (0.5%) and E D T A - 4 N a (0.2%). Trypsinization was stopped by adding fetal bovine serum and the cell suspension was centrifuged at 450 x g for 4 rain. The supernatant was removed and the cell pellet was resuspended in D M E medium. The culture was carried out at 37 °C in a humidified atmosphere containing 5% CO 2 in air.
269
Morphology For electron microscopy, the cell monolayers were scraped off, fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and post-fixed in 1% OsO4 in 0.1 M cacodylate buffer (4.5% sucrose was added to both fixatives). The cells were dehydrated in graded ethanol and embedded in Epon 812. Thin sections were cut and examined in a Zeiss EM 10-C electron microscope (Carl Zeiss, Inc., Thornwood, NY).
Indirect immunofluorescence For indirect immunofluorescence staining of desmin and vimentin, the cells were grown on coverslips. The cells were washed with HBSS (to remove the culture medium) and with PBS three times. Fixation of cultured cells was carried out with acetone at - 2 0 °C for 5 min. After three washes with PBS the cells were covered with normal rabbit serum for 20 rain in order to reduce non-specific background fluorescence. Then, the cells were incubated with mouse monoclohal antibodies to desmin and vimentin (diluted 1:10 with PBS), at room temperature for 1 h in a humidified chamber, and washed carefully with PBS. The cells were then incubated with FITC-conjugated anti-mouse IgG at room temperature for 1 h, and finally washed with PBS. After mounting with 90% glycerol in PBS (pH 8-9) containing 1% o-phenylenediamine [10], the slides were examined and photographed using a Zeiss fluorescence microscope equipped with epiillumination. Contro.s were carried out by omitting the first antibody and including instead either PBS or a preimmune mouse serum or mouse ascites fluid at the same dilution as used for the monoclonal antibody. Negative results were obtained with these controls.
Results
Cell growth of Mfs was observed within 4 - 5 days after the initiation of the culture, and confluency was reached within 1 month. The Mfs in secondary cultures reached confluency in about 2 weeks. By transmission electron microscopy, Mfs in primary culture
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Fig. 1. Electron micrograph of cultured Mfs in primary culture. These cells display abundant microfilaments (mf), dense bodies (db). ~,nd basal lamina-like structures (bl). (Uranyl acetate and lead citrate. ×4160.)
lipid droplets in the second passage and also a welldeveloped rough endoplasmic reticulum (Fig. 3). In cultures of SMCs and Fbs, cell migration around the tissue explants was recognized within 4-5 days; confluency was reached in 10-14 days, and subcultures were carried out each week. Cultures of SMCs obtained by the enzyme digestion method displayed the same growth as noted above. In primary cultures and 1st and 5th passage of Mrs, the reaction with the antibody to desmin was negative or virtually negative (Fig. 4a), but that with antibody to vimentin was positive (Fig. 4b). Passaged myofibroblasts maintained these characteristic features, as described before [8,9]. By contrast, in FSCs, reactions with antibodies to both desmin and vimentin were positive in primary cultures as well as subsequent passages (Fig. 5a and b). Numerous fibrils of desmin or vimentin were clearly recognized in the cytoplasm, and the same reactive pattern with each antibody was observed (Fig. 5c and d). In primary cultures of SMCs, both reactions were positive (Fig. 6a and b), but in the first passage, SMCs lost reactivity to desmin antibody (Fig. 6c) and maintained that to vimentin antibody (Fig. 6d). One morphologic feature of these SMCs was the abundance of microfilaments and lysosomes, as previously reported [16].
displayed abundant microfilaments, dense bodies and were surrounded by a basal lamina-like structure (Fig. 1). Yield of Mfs was 1.1-2.2-106 per liver and viability by the trypan blue exclusion test was more than 84%. The yield of isolated FSCs from vitamin A-treated rat was 1.4-3.2.106 cells per liver and the viability was more than 85%. Most of the isolated FSCs contained multiple and large lipid droplets by oil-red-O staining and electron microscopic examination (Fig. 2). The growth of cultured FSCs was very slow and confluency was reached in 6 weeks in primary cultures and in 2-3 weeks in subcultures. The lipid droplets in the cytoplasm decreased gradually and could no longer be recognized by phase contrast microscopy 7-10 days after the initiation of the culture. However, by electron microscopy, the cells retained small
Fig. 2. Electron micrograph of isolated FSCs from vitamin Atreated rats. These cells contain multiple and large lipid droplets. (Uranyl acetate and lead citrate, x4300.)
DESMIN IN LIPOCYTES AND MYOFIBROBLASTS
271
Fig. 3. Electron micrograph of cultured FSCs (second passage). These cells have small lipid droplets (F), well-developed rough endoplasmic reticulum (rer) and smooth endoplasmic reticulum (ser). (Uranyl acetate and lead citrate, x 14 850.)
Fbs, as expected, were negative for desmin (Fig. 7a) and positive for vimentin (Fig. 7b). These fibroblasts maintained characteristic elongated shapes with strand cytoplasm. Table 1 summarizes the results of desmin and vimentin staining in cultured cells. In FSCs, the intensity of fluorescence in both reactions did not change after multiple passages. By contrast,
in SMCs, the reaction to desmin antibody was lost entirely in the first passage. The Fbs displayed reduced staining of vimentin antibody in secondary cultures. To examine the contamination of desmin-positive cells in cultured Mfs and the influence of enzyme digestion on desmin of SMCs, desmin staining in primary culture of Mfs and SMCs was carried out at the
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J
Fig. 4. Desmin and vimentin staining in the first passage of Mfs. The reaction to desmin antibody is virtually negative (a) but that to vimentin antibody is positive (b) (x 300).
O
Fig. 5. Desmin and vimentin staining in the 5th passage of FSCs. Reactions to both desmin (a) and vimentin (b) antibodies are positive ( x 300). Many fibrils of desmin (c) and vimentin (d) are clearly recognized in the cytoplasm at high magnification ( × 1070).
DESMIN IN LIPOCYTES AND MYOFIBROBLASTS
273
Fig. 6. Desmin and vimentin staining in primary culture and the first passage of SMCs. Reactions to both desmin (a) and vimentin (b) antibodies arc positive in primary culture. However, in the first passage, the reaction to desmin antibody is lost (c) whereas that to vimentin antibody is maintained (d) (×300).
Fig. 7. Desmin and vimentin staining in Fbs. Thc reaction to desmin antibody is negative (a) whereas that to vimentin antibody is positive (b) (x300).
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TABLE 1 INTENSITY OF FLUORESCENCE IN DESMIN AND VIMENTIN STAINING OF Mfs, FSCs, SMCs AND Fbs Cultured cells
Desmin
Vimentin
Mrs primary culturc 1st passage 5th passage
-
+ ++ ++
FSCs primary culture 1st passage 5th passage
++ ++ ++
++ ++ ++
SMCs primary culture 1st passage 5th passage
++ -
++ ++ +
Fbs primary culture 1st passage 5th passage
-
++ + +
Several types of cell have been considered to be involved in hepatic fibrogenesis. Mfs were described in granulation tissue by Gabbiani et al. [17,18], and were recognized in cirrhosis in man [19,20] and mice [21,22] and in noncirrhotic liver in alcoholics [1]. They were found to be desmin-negative in situ [13]. Although these ceils showed some similarities with smooth muscle cells, they were clearly differentiated from the latter by their morphologic features [23,24]. Some have postulated that myofibroblasts represent a reversible phenotype modulation of fibroblast-type cells to a contractile state [25], whereas others have
same time. Desmin-positive cells were recognized in 16.0 _+ 2.4% of cultured Mfs. In SMCs, 81.8 + 5.4% of cells showed a positive reaction to the desmin antibody (Table 2).
Discussion The present study revealed that in cultured rat
TABLE 2 PERCENT OF CELLS STAINING FOR DESMIN IN PRIMARY CULTURES OF Mfs AND SMCs OBTAINED BY THE ENZYME DIGESTION METHOD Values are means _+S.E. Cultured cells
Intensity of fluorescence -
+
cells, immunohistochemistry of intermediate filaments allows for the differentiation between FSCs, which are vimentin- and desmin-positive, and Mfs or Fbs, which are vimentin-positive but desmin-negative. SMCs are vimentin- and desmin-positive in primary cultures and become desmin-negative after the first passage.
++
Mrs desmin staining control staining
84.3 + 1.7 100.0 + 0.0
13.5 + (I.8 0
2.5 _+0.6 0
SMCs desmin staining control staining
18.3 + 3.1 100.0 + (I.O
64.8 + 3.4 0
17.(I + 7.3 0
emphasized differences between myofibroblasts and fibroblasts [26]. After chronic alcohol consumption, the n u m b e r of Mfs and other mesenchymal cells increased [1,2]. FSCs, characterized by a b u n d a n t lipid droplets, have been considered to have a close correlation with fibrogenesis in the liver [3-6]. In baboons fed alcohol, the progression of hepatic fibrosis was a~sociated with the appearance of transitional cells characterized by a depletion of lipid droplets and a hypertrgphy of the rough endoplasmic reticulum [7]. Transitional cells have a b u n d a n t microfilaments, dense bodies and pinocytic vesicles and resemble the Mfs of the perivenular zone, but they can be distinguished by the lack of surrounding basal lamina [7]. De Leeuw et al. [10] described that in primary cultures and cell lines of FSCs from rat liver (established using metrizamide density centrifugation), collagen types l and III and laminin were present intracellularly in small granules. Friedman et al. [11] reported that cultured FSCs isolated from rat liver with Stractan density gradients secreted predominantly type I collagen, measured by E L I S A assay. Thus, both Mfs and FSCs have been incriminated in hepatic fibrogenests. However, the characteristic features or origin of these cells have not been fully elucidated. Our study demonstrates that in primary culture as well as
DESMIN IN LIPOCYTES AND MYOFIBROBLASTS subsequent passage of FSCs, reactions with antibodies to both desmin and vimentin were positive. Even after passage, the intermediate filaments in FSCs were maintained; because of their positivity to desrain and vimentin, FSCs could still be clearly distinguished from Mfs, SMCs and Fbs, which were desrain-negative. These findings suggest that although these cells have some similar morphological features, their cytoskeletal elements differ and that rat liver Mrs may be more closely related to Fbs than to SMCs. On the other hand, FSCs from rat liver are mesenchymal in origin and display some similarity with muscle cells. In studies of the intermediate filaments of vascular SMCs, conflicting data have been published: only vimentin and no desmin [27-29], or both vimentin and desmin [30], have been reported to be present. In addition, Travo et al. [31] described that in primary cultures of adult rat vascular SMCs derived from the thoracic aorta, there are three distinct cell types: cells rich in vimentin and lacking desmin, cells expressing both vimentin and desmin, and occasionally cells rich in desmin but lacking vimentin. In the present study, using monoclonal antibodies, coexistence of vimentin and desmin was demonstrated in primary cultures of SMCs derived from thoracic aorta. The disappearance of desmin in the first passage may sug-
References 1 Nakano M, Lieber CS. Ultrastructure of initial stages of perivenular fibrosis in alcohol-fed baboons. Am J Pathol 1982; 106: 145-155. 2 Nakano M, Worner TM, Lieber CS. Perivenular fibrosis in alcoholic liver injury: ultrastructure and histologic progression. Gastroenterology 1982; 83: 777-785. 3 Schnack H, Stockinger L, Wewalka F. Adventitious connective tissue cells in the space of Disse and their reaction to fibre formation. Rev Int Hepatol 1967; 17: 855-86//. 4 McGee JO'D, Patrick RS. The role of perisinusoidal cells in hepatic fibrogenesis. An electron microscopic study of acute carbon tetrachloride liver injury. Lab Invest 1972;26: 429-440. 5 Kent G, Gay S, lnouye T, Bahu R, Minick OT, Popper H. Vitamin A-containing lipocytes and formation of type III collagen in liver injury. Proc Natl Acad Sci USA 1976: 73: 3719-3722. 6 Minato Y, Hasumura Y, Takeuchi J. The role of fat-storing
275 gest that desmin is more unstable than vimentin in these cells. The reason for the selective loss of desmin is not known but it is not u n c o m m o n for cells to change some of their characteristic features upon passage from primary to secondary cultures. Voss et al. [32] studied SMCs in cultured tissue specimens from human fibrotic liver obtained by needle biopsy. Two cell types emerged from the tissue explants. From their morphology and biosynthetic products, they resembled SMCs and endothelial cells from blood vessel walls. Isolates of Mfs may be contaminated by SMCs and endothelial cells. However, in primary culture of the Mfs fraction, we found that only 16.0% of the cells were desmin positive. Also, SMCs obtained by the enzyme digestion method did not lose their reaction with antibody to desmin. Therefore, these findings show that the majority of cells isolated in the Mf fraction have characteristics which differ from SMCs of the thoracic aorta. SMCs were also shown to be desmin-positive in the wall of hepatic arteries [33,34] and, possibly, hepatic veins [35]. In summary, our results show that cultured FSCs maintain their desmin and vimentin, and that by desmin staining, FSCs can be differentiated from Mfs and Fbs, and from SMCs after the first passage in culture.
cells in Dissc space fibrogenesis in alcoholic liver disease. Hepatology 1983; 3: 559-566. 7 Mak KM, Leo MA, Lieber CS. Alcoholic liver injury in baboons: transformation of lipocytes to transitional cells. Gastroenterology 1984: 87: 188-21111. 8 Savolainen E-R, Leo MA, Timpl R. Lieber CS. Acetaldehyde and lactate stimulate collagen synthesis of cultured baboon liver myofibroblasts. Gastroenterology 1984: 87: 777-787. 9 Leo MA, Mak KM, Savolainen E-R, Licber CS. Isolation and culture of myofibroblasts from rat liver. Proc Soc Exp Biol Med 1985: 180:382-391. 10 De Leeuw AM. McCarthy SP, Geerts A, Knook DL. Purified rat liver fat-storing cells in culture dividc and contain collagen. Hepatology 1984; 4: 392-41/3. 11 Friedman SL, Roll FJ, Boyles J, Bissell DM. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci USA 1985: 82:8681-8685. 12 Yokoi Y, Namihisa T, Kuroda H, ct al. Immunocytochemical detection of desmin in fat-storing cells (Ito cells). Hep-
276 atology 1984: 4: 7119-714. 13 Schi,irch W, Seemayer TA, Lagac6 R, Gabbiani G. The intermediate filament cytoskeleton of myofibroblasts: an immunofluorescence and ultrastructural study. Virchows Arch 1984; 41)3: 323-336. 14 Ross R. The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol 1971; 50: 172-186. 15 Barone LM. Faris B, Chipman SD. Toselli P. Oakes BW, Franzblau C. Alteration of the extracellular matrix of smooth muscle cells by ascorbate treatment. Biochim Biophys Acta 1985: 84(1: 245-254. 16 James-Kracke MR, Sloane BF, Shuman H, Somlyo AP. kysosomal composition in cultured vascular smooth muscle cells: electron probe analysis. Proc Natl Acad Sci USA 1979; 76: 6461-6465. 17 Gabbiani G. Ryan GB, Majno G. Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction. Experientia 1971; 27: 549-550. 18 Gabbiani G, Hirschel BJ, Ryan GB. Granulation tissue as a contractile organ: a study of structure and function. J Exp Med 1972; 135: 719-734. 19 Bhathal PS. Presence of modified fibroblasts in cirrhotic livers in man. Pathology 1972; 4: 139-144. 20 Rudolph R, McClure WJ, Woodward M. Contractile fibroblasts in chronic alcoholic cirrhosis. Gastroenterology 1979; 76: 7114-7(19. 21 Irle C, Kocher O. Gabbiani G. Contractility of myofibroblasts during experimental liver cirrhosis. J Submicrosc Cytol 1980; 12: 209-217. 22 Hay ED, Hasty DE, Kiehnau KL. Morphological investigation of fibers derived from various types: fine structure of collagens and their relation to glucosaminoglycans (GAG). In: Gastpar H, K/.ihn K, Marx R, eds. Collagen-Platelet Interaction. New York: Stuttgart-Schattauer Verlag, 1978: 129-162. 23 Lipper S, Kai LB, Reddick RL. The myofibroblast. Pathol Annu 1980; 443-470 (year book). 24 Barsky SH, Green WR, Grotendorst GR, Liotta LA. Desmoplastic breast carcinoma as a source of human myofibroblasts. Am J Pathol 1984; 115: 329-333.
S. TAKASE et al. 25 Bellows CG, Melcher AH, Bhargava U, Aubin JE. Fibroblasts contrasting three-dimensional collagen gels exhibit ultrastructure consistent with either contraction or protein secretion. J Ultrastruct Res 1982: 78: 178-192. 26 Van de Berg JS, Rudolph R., Woodward M. Comparative growth dynamics and morphology between cultured myofibroblasts from granulating wounds and dermal fibroblasts. Am J Pathol 1984; 114: 187-200. 27 Gabbiani G, Schmid E, Winter S, et al. Vascular smooth muscle cells differ from other smooth muscle cells: predominance of vimentin filaments and a specific a-type actin. Proc Natl Acad Sci USA 1981 ; 78: 298-3(12. 28 Frank ED, Warren L. Aortic smooth muscle cells contain vimentin instead of desmin. Cell Biol 1981 ; 78: 3020-3024. 29 Yaoita E, Kazama T. Kawasaki K, Miyazaki S, Yamamoto T, Kihara I. In vitro characteristics of rat mesangial cells in comparison with aortic smooth muscle cells and derman fibroblasts. Virchows Arch 1985; 49: 285-294. 30 Schmid E, Osborn M, Rungger-Brfindle E, Gabbiani G, Weber K, Franke WW. Distribution of vimentin and desrain filaments in smooth muscle tissue of mammalian and avian aorta. Exp Cell Res 1982; 137: 329-340. 31 Travo P, Weber K, Osborn M. Co-existence of vimentin and desmin type intermediate filaments in a subpopulation of adult rat vascular smooth muscle cells growing in primary culture. Exp Cell R.es 1982; 139: 87-94. 32 Voss B. Rauterberg J, Pott G, et al. Nonparenchymal cells cultivated from explants of fibrotic liver resemble endothelial and smooth muscle cells from blood vessel walls. Hepatology 1982; 2: 19-28. 33 Yokoi Y, Namihisa T, Kuroda H, et al. lmmunocytochemical detection of desmin in fat-storing cells (lto cells). Hepatology 1984; 4: 709-714. 34 Burt AD, MacSween RNM. Immunolocalization of desmin in fixed rat and human liver. In: Kim A, Knook DL, Wisse E, eds. Cells of the Hepatic Sinusoid. Rijswijk, The Netherlands: the Kupffer Cell Foundation, 1986; 253-254. 35 Burt AD, R.obertson JL, Heir J, MacSween RNM. Desmin-/containing stellate cells in rat liver; distribution in normal animals and response to experimental acute liver injury. J Pathol 1986; 150: 29-35.