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Collagen synthesis in explant cultures of normal and CCl4-treated mouse liver
TOXICOLOGY AND APPLIED PHARMACOLOGY
48, 131-137 (1979)
Collagen Synthesis in Explant Cultures of Normal and CCl4-Treated Mouse Liver 1 LEONARDO GALLIGANI, MILENA LONATI-GALEIGANI, AND GEORGE C. FULLER
Department of Pharmacology and Toxicology, University of Rhode Island, Kingston, Rhode Island 02881 Received July 12, 1978; accepted Augus t 23, 1978 Collagen Synthesis in Explant Cultures of Normal and CCl4-Treated Mouse Liver. GALLIGANI,L., LONATI-GALLIGANI,M., AND FULLER,G. C. (1979). Toxicol. Appl. Pharmacol. 48, 131-137. Acute liver damage was induced in mice by oral administration of a single dose of CC14. The activity of prolyl hydroxylase and the incorporation of [2,3-aH]proline into collagen and noncollagen proteins were significantly increased 3 days after CC14 administration when compared to controls. Collagen synthesis, measured as the percentage of total protein synthesis in primary organ cultures, was also significantly increased at this time. Explant cultures of damaged liver fragments were characterized by a rapid outgrowth of cells, while normal liver explants grew more slowly as indicated quantitatively by cell number present and radioactive thymidine incorporation into DNA. The rate of incorporation of radioactive proline into both collagen and noncollagen proteins measured at different intervals in the explant cultures increased in parallel with the rate of outgrowth of the cells from the tissue fragments. The amount of collagen produced per cell by cultures of injured liver was elevated at all time periods with a maximum 10-fold elevation observed after 11 days in culture. Maximum collagen production as a percentage of total protein synthesis was found to be associated with the early stages of culture growth where the proportion of collagen made increased in the initial phases of outgrowth and subsequently decreased despite the continuous proliferation of the cultures. This relative rate of collagen synthesis was significantly higher in the exptant cultures of damaged liver tissue compared to the controls for at least 11 days in culture. This study indicates that the growth in culture of cells committed to collagen production is stimulated by acute liver injury. This response may be an early marker of the mesenchymal cell reaction to liver injury which eventually leads to liver fibrosis.
Hepatic fibrosis is characterized by excessive deposition of fibrous connective tissue which causes a distortion of the liver architecture a n d plays a crucial role in the progression of the liver disease to cirrhosis. According to P o p p e r (1977), this a c c u m u l a t i o n of collagenous fibers in the hepatic p a r e n c h y m a characteristic of cirrhosis m a y be conseq u e n t to the collapse of pre-existing supportive connective tissue or to invasive for-
m a t i o n of collagen a n d other matrix comp o n e n t s such as proteoglycans. A l t h o u g h the events preceding cirrhosis have been identified to include hepatocellular necrosis, inflammation, regeneration, a n d fibrosis, the pathogenesis of this disease remains u n k n o w n (Popper, 1977). Further, little is k n o w n a b o u t the cellular mechanisms responsible for collagen deposition in the liver. I n attempts to study this p r o b l e m m a n y morphological IA portion of this work was presented at the a n d biochemical studies on liver fibrosis have annual FASEB meeting in Atlantic City, April 1978. been carried out on model systems of acute 004 I~)08X/79/040131-07 $02.00/0 131 Copyright © 1979 by Academic Press, Inc. All rights o f reproduction in any form reserved. Printed in Great Britain
132
GALLIGANI~ LONATI-GALLIGANI, AND FULLER
a n d chronic CCI 4 intoxication in rats or mice ( S h a b a et al., 1973; R o j k i n d and K e r s h e n o bich, 1975 ; K e r w a r et al., 1977; M c G e e et al., 1972; K e n t et al., 1976; Suzuki et al., 1976). These studies have indicated t h a t the increased collagen a n d p r o t e o g l y c a n deposition is due to the increased synthesis o f these m a t r i x c o m p o n e n t s ( R o j k i n d and K e r s h e n o bich, 1975; K e r w a r et al., 1977; Suzuki et al., 1976) a n d that the resting precursors of fibroblasts located in the p a r e n c h y m a might be responsible for this a c c u m u l a t i o n ( M c G e e et al., 1972; K e n t et al., 1976). However, the presence o f prolyl h y d r o x y l a s e a n d the f o r m a t i o n o f peptidyl b o u n d h y d r o x y p r o l i n e by a d u l t rat p r i m a r y hepatocytes in m o n o layer culture have been recently r e p o r t e d in a p r e l i m i n a r y c o m m u n i c a t i o n (Guzelian et al., 1978). The present investigation was c o n d u c t e d to study the d y n a m i c s o f cell g r o w t h a n d o f collagen synthesis in explant cultures o f n o r m a l a n d CCl4-treated m o u s e liver.
ascorbic acid, 100/tg/ml fl-aminopropionitrile fumarate (BAPN), and 12.5/iCi/ml of [2,3-3H]proline for 24hr. After incubation, the liver fragments were homogenized in their own medium, an aliquot of the homogenate (0.2 ml) was saved for DNA determination, and the remainder was exhaustively dialyzed against 0.01 M Tris-HCl buffer, pH 7.4. Quantitation of collagen was carried out by the collagenase digestion method of Peterkofski and Diegelmann (1971). Using this procedure the rate of collagen synthesis was expressed as cpm in collagenasedigestible protein per /tg of DNA. The bacterial collagenase used for this assay was chromatographically pure and confirmed to be free of nonspecific protease by its failure to degrade [14C]trytophan-labeled E. coli protein. The rate of DNA synthesis was measured by adding 2.5 #Ci of [methyl-3H]thymidine per ml of medium and determining the incorporation into TCA (57o)-insoluble material after 24 hr incubation (Rubin, 1971). Colorimetric DNA determinations were carried out by the diphenylamine reaction method of Burton (1956) and protein content was determined by the method of Lowry et al. (1951), using bovine serum albumin as standard.
RESULTS AND DISCUSSION METHODS Acute liver damage was induced in albino COBS CD-1 male mice (30 40 g) by oral administration of a single dose of 0.1 ml of 40 ~ C C I 4 in mineral oil. The same amount of mineral oil was administered to control animals. Three days after CC14 administration the animals were sacrificed, and their livers were quickly removed under sterile conditions and washed in ice2cold phosphate-buffered saline. Small fragments (1-mm cubes) were cut from each liver using a scalpel and were allowed to adhere to a tissue culture growth surface as explant cultures in 60-mm tissue culture dishes with 2 ml of Dulbecco's modified Eagle's medium (DME) containing 107oo fetal calf serum, 100 units/ml of penicillin, and 100/lg/ml of streptomycin. Cultures were maintained at 37°C in a humidified atmosphere of 5 ~ CO2-95 ~ air. A fragment of about 50-60 mg of each liver was homogenized in 10 volumes of 0.01 M Tris-HC1 buffer, pH 7.4, 10-5M EDTA, 10-4M DTT, and 0.1 ~ Triton X-100, and an aliquot of the whole homogenate (100~d) was assayed for the prolyl hydroxylase activity by the tritium release method of Hutton et al. (1966). For determination of collagen and noncollagen protein synthesis the explant cultures were incubated at 37°C in serum-free DME containing 100/~g/ml of
As previously r e p o r t e d by several l a b o r a tories, the activity o f liver prolyl h y d r o x y l a s e increases following CC14 p o i s o n i n g (Takeuchi et al., 1967; K e r w a r et al., 1977) or other types o f liver injury (Risteli a n d K i v i r i k k o , 1974; Risteli et al., 1976) a n d precedes the increase in collagen content. Since elevated prolyl hydroxylase activity occurs early in this m o d e l o f hepatic injury (Takeuchi et al., 1967), we have used this assay as an i n d i c a t o r of the efficacy of our CC14 t r e a t m e n t o f the animals. In a typical experiment (Table 1) prolyl hydroxylase activity was elevated 2.5-fold (p < 0.02) 3 days after CC14 treatment. W h e n the rates of i n c o r p o r a t i o n o f r a d i o active proline into collagen a n d noncollagen protein were c o m p a r e d for the first 24 hr after the establishment o f the exptant cultures (Table 2), significantly greater i n c o r p o r a t i o n o f proline into b o t h p r o t e i n pools was f o u n d in the CC14-treated liver ( p < 0 . 0 5 ) . In separate experiments it was f o u n d that the i n c o r p o r a t i o n o f labeled proline into rat liver
133
COLLAGEN SYNTHESIS IN CCI4-LIVER EXPLANTS
(needle biopsy tissue) collagen and noncollagen protein is linear over a 24-hr labeling period under the conditions used in this experiment where the tissue/media ratio does not exceed 35 mg/2 ml (Mann et al., 1978). The proportion of collagen made as a percentage of total protein synthesis, calculated after correction for the enrichment of collagen in proline residues according to the formula used by Peterkofsky and Diegelmann (1971), also increased significantly (p < 0.05). This increase in collagen synthesis is consistent with the higher values of prolyl hydroxylase activity found in the damaged liver and with previous reports by other authors (Shaba et al., 1973; Rojkind and Kershenobich, 1975; Kerwar et al., 1977). These data therefore confirm that, following
TABLE 1 PROLYL HYDROXYLASEACTIVITY IN NORMAL MOUSE LIVER AND 3 DAYS FOLLOWING CCI 4 TREATMENT
Treatment a
N
cpm aH20 released per mg of protein b
4 4
1228 + 159 3126+_ 513¢
Control CC14
a CC14 (0.1 ml of 407oo CC14 in mineral oil) given orally. Controls received 0.1 ml of mineral oil. b Each value represents the mean + SE. c Statistically different from the control at p < 0.02, Student's t test.
acute liver injury, the elevation in prolyl hydroxylase activity correlates with an increased activity of the cells committed to the production of collagen. It has been reported by other workers (Bakirov et al., 1969; Hays et al., 1975) that cells from CC14-damaged liver tissue grow rapidly in explant cultures compared to cells from normal liver tissue which grow very slowly. Bakirov et al. (1969) reported that connective tissue cells migrate out from the CC14-treated liver fragments during the first 4 days of cultivation and that, during the following days, proliferation of hepatic cells also occurs which form epithelial membranes. When explant cultures from normal and CC14-treated liver tissue were established, we observed the same phenomenon. A rapid outgrowth (3-4 days) of cells was observed from the damaged liver fragments, while a slow rate of outgrowth (10-15 days) was characteristic of the control cultures. The number of different cell types involved in this rapid proliferative response has not been completely identified. Electron microscopy studies in progress, however, indicate that nonparenchymal cells are predominant, at least in the early phases of this proliferation. These cells are characterized by prominent surface microvilli and sparse cytoplasmic organelles (unpublished results). In order to chemically define and quantitate the greater tendency of the cultures of CC14-
TABLE 2 COLLAGEN AND NONCOLLAGEN PROTEIN SYNTHESIS BY NORMAL AND CC14-TREATED MOUSE LIVER WITHIN 24 hr FROM THE ESTABLISHMENT OF EXPLANT CULTURES
[3H]Proline incorporation (cpm//lg DNA)" Treatment Control CC14-treated
N
Noncollagen
Collagen
Percentage collagen synthesisb
7 7
1462 -4-_317 2871 + 493c
7.8 + 2.3 30.0 + 5.8c
0.091 + 0.002 0.208 + 0.044c
" Each value represents the mean +_ME. b The percentage collagen synthesis was calculated according to the following formula (Peterkofski and Diegelmann, 1971) : percentage collagen = cpm [3H]proline in collagen x 100/(cpm pH]proline in noncollagen)x 5.4+cpm [3H]proline in collagen. c Statistically different from the control at p < 0.05, Student's t test.
134
GALLIGANI~ LONATI-GALLIGANI, AND FULLER
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FIG. 1. Time course of DNA synthesis in explant cultures of normal and CC]4-treated mouse liver. This figure illustrates a typical experiment where thymidine incorporation per /zg DNA is compared in control liver and liver obtained from a CCl4-treated mouse. The mean slope for three experiments was 50 + 5.3 for control compared to 192 + 44.2 for CCI4 treated (p < 0.05). damaged liver to proliferate compared to control cultures, thymidine incorporation into DNA was determined at several time periods after the establishment of explant cultures. The slopes of the two regression lines representing the rates of proliferation of the cells in explant cultures from a typical experiment are significantly different (p < 0.05) (Fig. 1). In this experimental design the rate of growth and rate of thymidine incorporation were determined for individual liver preparations and compared to control liver preparations. In each of several experiments, the difference in the slope of the curves indicated that the growth of cells from CC14-injured liver was three to four times more rapid than the growth of cells from normal liver explants. In order to test for the presence of specific growth-stimulating factors in the CC14-treated liver, normal liver explant cultures were grown in medium conditioned by CC14-damaged liver explants. There was no apparent difference in the growth of
normal liver cultures when exposed to conditioned medium (data not shown). The rate of incorporation of radioactive proline into collagen and noncollagen proteins was measured in control and CCI 4treated liver cultures on different days over a period of 3 weeks. In a typical experiment, the rate of incorporation of radioactive proline into noncollagen proteins (Fig. 2A) and collagen (Fig. 2B) increased in parallel with the rate of outgrowth of the cells from the tissue fragments of both normal and injured liver in culture. At all time periods the values obtained for the CC14-treated liver were higher than for the respective control liver with the increase in the rate of collagen synthesis progressing from a 4-fold difference on Day 1 to a maximum 10-fold difference on Day 11. The proportion of collagen to total protein synthesized (Fig. 2C) progressively increased, both in the cells outgrowing from damaged liver as well as in those outgrowing from the normal liver, from initial values of
135
COLLAGEN SYNTHESIS IN CC14-LIVER EXPLANTS
0.33 and 0.14~, respectively, to a maximum of 2.55 and 1.65~, respectively, after 7 days in culture. These values decreased slightly thereafter despite the continuous proliferation of the cultures. In order to permit statistical evaluation of these changes, data were pooled from separate experiments where cultures were labeled on Day 1, at approximately 1 week (6-11 days) and at 16-22 days (Table 3).
Collagen and noncollagen protein synthesis are significantly elevated at all time periods in the cultures of injured liver tissue compared to the controls. Collagen synthesis, measured as a percentage of total protein synthesis, increases during the early stage of culture growth of the damaged liver fragments from an initial value of 0.247 ~ to a maximum of 2.3 70 after 1 ~veek and is significantly higher than in the control cultures on
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FIG. 2. Comparison of the rates of incorporation of [2,3-3Hlproline into noncoUagen proteins (A) and collagen (B) and of the rates of relative collagen synthesis (C) between control (dotted lines-open circles) and CC14-treated (solid lines--closed circles) liver in explant cultures. The values are the mean of two nonreplicate determinations, each performed in triplicate.
136
GALLIGANI, LONATI-GALLIGANI,AND FULLER TABLE 3 COLLAGEN AND NONCOLLAGEN PROTEIN SYNTHESISBY NORMAL AND CCI4-TREATED MOUSE LIVER AT DIFFERENT TIME PERIODS IN EXPLANT CULTURE
[3H]Proline incorporation (cpm/pg DNA)" Days in culture 1 6-11 16-22
Treatment
Noncollagen
Control CC14 Control CC14 Control CC14
1123 _+315 (4) 2832+590 (4)b 1388_+509 (4) 8362_+ 1095 (3)c 1612_+470 (3) 24,088+3856(3) c
Collagen 5.4 + 1.8 (4) 40.7+7.3 (4)C 63.5+33.5 (4) 1505_+440(4)e 222+ 125.6 (4) 1620_+141(3)c
Percentage collagen synthesis 0.089 + 0.020 0.247_+0.031c 0.880_+0.400 2.300_+0.140b 1.080_+0.470 1.300_+0.270
a Each value represents the mean + SE with the number of values reported in parentheses. b Statistically different from the control at p < 0.05, Student's t test. c Statistically different from the control at p < 0.01, Student's t test. e Statistically different from the control at p < 0.02, Student's t test.
Day 1 and at 1 week. This value decreases despite the continuing outgrowth of the cells with no difference observed at 16-22 days. This decrease in the cellular commitment of injured liver cultures to collagen synthesis at 1622 days reflects the increased rate of collagen synthesis in control cultures with a concomitant increase in noncollagen protein synthesis occurring in the CC14-treated liver cultures. These data indicate that cells committed to collagen production are stimulated to grow and to produce collagen more rapidly in the cultures of injured liver tissue compared to control liver tissue. In both cases, maxim u m collagen production is associated w i t h the early stages of outgrowth of the cells from the tissue fragments. Watanabe (1966) observed in light and electron microscopy studies on primary explants of rat liver that proliferation of liver parenchymal cells in culture is preceded by the outgrowth of cells of mesenchymal origin. These cells may, in fact, be responsible in liver for the production of collagen (Kent, et al., 1976; McGee and Patrick, 1972; Shaba et al., 1973) and their proliferation may be an important component in the mesenchymal reaction to liver injury (Feher et al., 1975).
Our results indicate that during the early phase of this proliferation of cells, collagen is produced at its maximum rate. These studies, however, describe the response obtained with acute liver injury, not with long-term fibrosis. Fibrosis is characterized by connective tissue accumulation occurring in response not just to a single injury, but rather to a series of repeated injuries. Our results suggest that each time the liver is injured, mesenchymal cells are stimulated to proliferate and collagen is synthesized. Upon continuous stimuli, i.e., repeated injuries, collagen and other matrix components may accumulate progressively within the liver parenchyma at a rate which exceeds the ability of liver to degrade collagen thus resulting in septum formation in the precirrhotic and cirrhotic stages (Hirayama et al., 1969). In vivo and in vitro studies on the kinetics of collagen production in long-term fibrosis are currently in progress in our laboratory.
ACKNOWLEDGMENTS This project was supported in part by NIH grant AA01422. M. L. G. was recipient of a research fellowship of the University of Pavia (Italy).
COLLAGEN SYNTHESIS IN CCI4-LIVER EXPLANTS
137
MANN, S. W., VIDINS, E. I., RODIL, J. V., AND FULLER, G. C. (1978). Prolyl hydroxylase and collagen BAKIROV, R. D., ELISEEVA, T. A., FRIDENSHTEIN, synthesis in patients with alcoholic liver disease. A. YA., AND ABELEV,G. I. (1969). Organ cultures of Gastroenterology (in press). the liver of adult mice poisoned with CC14. Bull. MCGEE, J. O'D., AND PATRICK, R. S. (1972). The Exp. Biol. Med. 67, 531-533. roIe of perisinusoidai cells in hepatic fibrogenesis. BURTON, K. (1956). A study of the conditions and Lab. bwest. 26, 429-440. mechanisms of the diphenylamine reaction for the PETERKOFSKI, ]]., AND D1EGELMANN, R. (1971). Use colorimetric estimation of deoxyribonucleic acid. of a mixture of proteinase-free collagenases for the Biochem. J. 62, 315-323. specific assay of radioactive collagen in the presence FEHER, J., JAKAB, U, JOZSA, L., SZlLVAS1, I., AND of other proteins. Biochemistry I0, 988-994. PAPP, G. (1975). Mesenchymal reaction and serum POPPER, H. (1977). Pathologic aspects of cirrhosis. A glycoprotein concentration in chronic hepatitis and review. Amer. J. PathoL 87, 227-264. liver cirrhosis. Acta Morphol. Aead. Sci. Hung. 23, RISTELI, J., AND KIVIRIKKO, K. I. (1974). Activities of 319-326. prolyl hydroxylase, lysyl hydroxylase, collagen GUZELIAN, P. S., NIEHAUS, N. J., AND DIEGELMANN, galactosyltransferase and collagen glucosyltransR. F. (1978). Demonstration of collagen peptidyl ferase in the liver of rats with hepatic injury. proline hydroxylase activity (PPH) in primary Biochem. J. 144, 115-122. monolayer cultures of adult rat hepatocytes. RISTELI, J., TUDERMAN, L., AND KIVIRIKKO, K. I. Gastroenterology 74, 1165. (1976). Intracellular enzymes of collagen biosynHAYS, D. M., SERA, Y., KOGA, Y., NEUSTEIN,H. B., thesis in rat liver as a function of age and in HAYS, E. F., and NICHOLSON, M. O. (1975). hepatic injury induced by dimethylnitrosamine. Factors influencing growth of cells from regenerating Biochem. J. 158, 369-376. liver. In Gene Expression and Carcinogenesis in ROJKIND, M., AND KERSHENOBICH,D. (1975). Effect of Cultured Liver (L. E. Gerschenson and E. Brad colchicine on collagen, albumin and transferrin Thompson, eds.), pp. 282-285. Academic Press, synthesis by cirrhotic rat liver slices. Biochim. New York. Biophys. Acta 378, 415-423. HIRAYAMA, C., HIROSHIGE, K., AND MASUYA, T. (1969). Hepatic collagenolytic activity in rats after RUmN, H. (1971). pH and population density in the regulation of animal cell multiplication. J. Cell carbon tetrachloride poisoning. Biochem. J. 115, Biol. 51, 686-702. 843-847. HUTTON, J. J., JR., TAPPEL, A. L., AND UDENERIEND, SHABA, J. K., PATRICK, R. S., AND MCGEE, J. O'D. (1973). Collagen synthesis by mesenchymal cells S. (1966). A rapid assay for collagen proline isolated from normal and acutely damaged mouse hydroxylase. Anal. Biochem. 16, 384-394. liver. Brit. J. Exp. PathoL 54, 110-116. KENT, G., GAY, S., INOUYE, T., BAHU, R., M1NICK, O. T., AND POPPER, H. (1976). Vitamin A-con- SUZUKI, S., SUZUKI,S., NAKAMURA,N., AND KOIZUMI, T. (1976). The heterogeneity of dermatan sulfate taining lipocytes and formation of type III collagen and heparan sulfate in rat liver and a shift in the in liver injury. Proe. Natl. Acad. Sci. USA 73, glycosaminoglycan contents in carbon tetrachloride3719-3722. damaged liver. Biochim. Biophys. Acta 428, 166KERWAR, S. S., ORONSKY, A. L., CHOE, D., AND 181. ALVAREZ, B. (1977). Studies on the effect of 3,4-dehydroproline on collagen metabolism in carbon TAKEUCHI, T., KIVIRIKKO, K. I., AND PROCKOP, D. J. (1967). Increased protocollagen hydroxylase actitetrachloride-induced hepatic fibrosis. Arch. Biovity in the livers of rats with hepatic fibrosis. Biochem. Biophys. 182, 118-123. chem. Biophys. Res. Commun. 28, 940-944, LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL,R. J. (1951). Protein measurement with the WATANA~E, H. (1966). A fine structural study of liver Folin phenol reagent. J. Biol. Chem. 193, 265-275. culture. Exp. Cell. Res. 42, 685 699.
REFERENCES
48, 131-137 (1979)
Collagen Synthesis in Explant Cultures of Normal and CCl4-Treated Mouse Liver 1 LEONARDO GALLIGANI, MILENA LONATI-GALEIGANI, AND GEORGE C. FULLER
Department of Pharmacology and Toxicology, University of Rhode Island, Kingston, Rhode Island 02881 Received July 12, 1978; accepted Augus t 23, 1978 Collagen Synthesis in Explant Cultures of Normal and CCl4-Treated Mouse Liver. GALLIGANI,L., LONATI-GALLIGANI,M., AND FULLER,G. C. (1979). Toxicol. Appl. Pharmacol. 48, 131-137. Acute liver damage was induced in mice by oral administration of a single dose of CC14. The activity of prolyl hydroxylase and the incorporation of [2,3-aH]proline into collagen and noncollagen proteins were significantly increased 3 days after CC14 administration when compared to controls. Collagen synthesis, measured as the percentage of total protein synthesis in primary organ cultures, was also significantly increased at this time. Explant cultures of damaged liver fragments were characterized by a rapid outgrowth of cells, while normal liver explants grew more slowly as indicated quantitatively by cell number present and radioactive thymidine incorporation into DNA. The rate of incorporation of radioactive proline into both collagen and noncollagen proteins measured at different intervals in the explant cultures increased in parallel with the rate of outgrowth of the cells from the tissue fragments. The amount of collagen produced per cell by cultures of injured liver was elevated at all time periods with a maximum 10-fold elevation observed after 11 days in culture. Maximum collagen production as a percentage of total protein synthesis was found to be associated with the early stages of culture growth where the proportion of collagen made increased in the initial phases of outgrowth and subsequently decreased despite the continuous proliferation of the cultures. This relative rate of collagen synthesis was significantly higher in the exptant cultures of damaged liver tissue compared to the controls for at least 11 days in culture. This study indicates that the growth in culture of cells committed to collagen production is stimulated by acute liver injury. This response may be an early marker of the mesenchymal cell reaction to liver injury which eventually leads to liver fibrosis.
Hepatic fibrosis is characterized by excessive deposition of fibrous connective tissue which causes a distortion of the liver architecture a n d plays a crucial role in the progression of the liver disease to cirrhosis. According to P o p p e r (1977), this a c c u m u l a t i o n of collagenous fibers in the hepatic p a r e n c h y m a characteristic of cirrhosis m a y be conseq u e n t to the collapse of pre-existing supportive connective tissue or to invasive for-
m a t i o n of collagen a n d other matrix comp o n e n t s such as proteoglycans. A l t h o u g h the events preceding cirrhosis have been identified to include hepatocellular necrosis, inflammation, regeneration, a n d fibrosis, the pathogenesis of this disease remains u n k n o w n (Popper, 1977). Further, little is k n o w n a b o u t the cellular mechanisms responsible for collagen deposition in the liver. I n attempts to study this p r o b l e m m a n y morphological IA portion of this work was presented at the a n d biochemical studies on liver fibrosis have annual FASEB meeting in Atlantic City, April 1978. been carried out on model systems of acute 004 I~)08X/79/040131-07 $02.00/0 131 Copyright © 1979 by Academic Press, Inc. All rights o f reproduction in any form reserved. Printed in Great Britain
132
GALLIGANI~ LONATI-GALLIGANI, AND FULLER
a n d chronic CCI 4 intoxication in rats or mice ( S h a b a et al., 1973; R o j k i n d and K e r s h e n o bich, 1975 ; K e r w a r et al., 1977; M c G e e et al., 1972; K e n t et al., 1976; Suzuki et al., 1976). These studies have indicated t h a t the increased collagen a n d p r o t e o g l y c a n deposition is due to the increased synthesis o f these m a t r i x c o m p o n e n t s ( R o j k i n d and K e r s h e n o bich, 1975; K e r w a r et al., 1977; Suzuki et al., 1976) a n d that the resting precursors of fibroblasts located in the p a r e n c h y m a might be responsible for this a c c u m u l a t i o n ( M c G e e et al., 1972; K e n t et al., 1976). However, the presence o f prolyl h y d r o x y l a s e a n d the f o r m a t i o n o f peptidyl b o u n d h y d r o x y p r o l i n e by a d u l t rat p r i m a r y hepatocytes in m o n o layer culture have been recently r e p o r t e d in a p r e l i m i n a r y c o m m u n i c a t i o n (Guzelian et al., 1978). The present investigation was c o n d u c t e d to study the d y n a m i c s o f cell g r o w t h a n d o f collagen synthesis in explant cultures o f n o r m a l a n d CCl4-treated m o u s e liver.
ascorbic acid, 100/tg/ml fl-aminopropionitrile fumarate (BAPN), and 12.5/iCi/ml of [2,3-3H]proline for 24hr. After incubation, the liver fragments were homogenized in their own medium, an aliquot of the homogenate (0.2 ml) was saved for DNA determination, and the remainder was exhaustively dialyzed against 0.01 M Tris-HCl buffer, pH 7.4. Quantitation of collagen was carried out by the collagenase digestion method of Peterkofski and Diegelmann (1971). Using this procedure the rate of collagen synthesis was expressed as cpm in collagenasedigestible protein per /tg of DNA. The bacterial collagenase used for this assay was chromatographically pure and confirmed to be free of nonspecific protease by its failure to degrade [14C]trytophan-labeled E. coli protein. The rate of DNA synthesis was measured by adding 2.5 #Ci of [methyl-3H]thymidine per ml of medium and determining the incorporation into TCA (57o)-insoluble material after 24 hr incubation (Rubin, 1971). Colorimetric DNA determinations were carried out by the diphenylamine reaction method of Burton (1956) and protein content was determined by the method of Lowry et al. (1951), using bovine serum albumin as standard.
RESULTS AND DISCUSSION METHODS Acute liver damage was induced in albino COBS CD-1 male mice (30 40 g) by oral administration of a single dose of 0.1 ml of 40 ~ C C I 4 in mineral oil. The same amount of mineral oil was administered to control animals. Three days after CC14 administration the animals were sacrificed, and their livers were quickly removed under sterile conditions and washed in ice2cold phosphate-buffered saline. Small fragments (1-mm cubes) were cut from each liver using a scalpel and were allowed to adhere to a tissue culture growth surface as explant cultures in 60-mm tissue culture dishes with 2 ml of Dulbecco's modified Eagle's medium (DME) containing 107oo fetal calf serum, 100 units/ml of penicillin, and 100/lg/ml of streptomycin. Cultures were maintained at 37°C in a humidified atmosphere of 5 ~ CO2-95 ~ air. A fragment of about 50-60 mg of each liver was homogenized in 10 volumes of 0.01 M Tris-HC1 buffer, pH 7.4, 10-5M EDTA, 10-4M DTT, and 0.1 ~ Triton X-100, and an aliquot of the whole homogenate (100~d) was assayed for the prolyl hydroxylase activity by the tritium release method of Hutton et al. (1966). For determination of collagen and noncollagen protein synthesis the explant cultures were incubated at 37°C in serum-free DME containing 100/~g/ml of
As previously r e p o r t e d by several l a b o r a tories, the activity o f liver prolyl h y d r o x y l a s e increases following CC14 p o i s o n i n g (Takeuchi et al., 1967; K e r w a r et al., 1977) or other types o f liver injury (Risteli a n d K i v i r i k k o , 1974; Risteli et al., 1976) a n d precedes the increase in collagen content. Since elevated prolyl hydroxylase activity occurs early in this m o d e l o f hepatic injury (Takeuchi et al., 1967), we have used this assay as an i n d i c a t o r of the efficacy of our CC14 t r e a t m e n t o f the animals. In a typical experiment (Table 1) prolyl hydroxylase activity was elevated 2.5-fold (p < 0.02) 3 days after CC14 treatment. W h e n the rates of i n c o r p o r a t i o n o f r a d i o active proline into collagen a n d noncollagen protein were c o m p a r e d for the first 24 hr after the establishment o f the exptant cultures (Table 2), significantly greater i n c o r p o r a t i o n o f proline into b o t h p r o t e i n pools was f o u n d in the CC14-treated liver ( p < 0 . 0 5 ) . In separate experiments it was f o u n d that the i n c o r p o r a t i o n o f labeled proline into rat liver
133
COLLAGEN SYNTHESIS IN CCI4-LIVER EXPLANTS
(needle biopsy tissue) collagen and noncollagen protein is linear over a 24-hr labeling period under the conditions used in this experiment where the tissue/media ratio does not exceed 35 mg/2 ml (Mann et al., 1978). The proportion of collagen made as a percentage of total protein synthesis, calculated after correction for the enrichment of collagen in proline residues according to the formula used by Peterkofsky and Diegelmann (1971), also increased significantly (p < 0.05). This increase in collagen synthesis is consistent with the higher values of prolyl hydroxylase activity found in the damaged liver and with previous reports by other authors (Shaba et al., 1973; Rojkind and Kershenobich, 1975; Kerwar et al., 1977). These data therefore confirm that, following
TABLE 1 PROLYL HYDROXYLASEACTIVITY IN NORMAL MOUSE LIVER AND 3 DAYS FOLLOWING CCI 4 TREATMENT
Treatment a
N
cpm aH20 released per mg of protein b
4 4
1228 + 159 3126+_ 513¢
Control CC14
a CC14 (0.1 ml of 407oo CC14 in mineral oil) given orally. Controls received 0.1 ml of mineral oil. b Each value represents the mean + SE. c Statistically different from the control at p < 0.02, Student's t test.
acute liver injury, the elevation in prolyl hydroxylase activity correlates with an increased activity of the cells committed to the production of collagen. It has been reported by other workers (Bakirov et al., 1969; Hays et al., 1975) that cells from CC14-damaged liver tissue grow rapidly in explant cultures compared to cells from normal liver tissue which grow very slowly. Bakirov et al. (1969) reported that connective tissue cells migrate out from the CC14-treated liver fragments during the first 4 days of cultivation and that, during the following days, proliferation of hepatic cells also occurs which form epithelial membranes. When explant cultures from normal and CC14-treated liver tissue were established, we observed the same phenomenon. A rapid outgrowth (3-4 days) of cells was observed from the damaged liver fragments, while a slow rate of outgrowth (10-15 days) was characteristic of the control cultures. The number of different cell types involved in this rapid proliferative response has not been completely identified. Electron microscopy studies in progress, however, indicate that nonparenchymal cells are predominant, at least in the early phases of this proliferation. These cells are characterized by prominent surface microvilli and sparse cytoplasmic organelles (unpublished results). In order to chemically define and quantitate the greater tendency of the cultures of CC14-
TABLE 2 COLLAGEN AND NONCOLLAGEN PROTEIN SYNTHESIS BY NORMAL AND CC14-TREATED MOUSE LIVER WITHIN 24 hr FROM THE ESTABLISHMENT OF EXPLANT CULTURES
[3H]Proline incorporation (cpm//lg DNA)" Treatment Control CC14-treated
N
Noncollagen
Collagen
Percentage collagen synthesisb
7 7
1462 -4-_317 2871 + 493c
7.8 + 2.3 30.0 + 5.8c
0.091 + 0.002 0.208 + 0.044c
" Each value represents the mean +_ME. b The percentage collagen synthesis was calculated according to the following formula (Peterkofski and Diegelmann, 1971) : percentage collagen = cpm [3H]proline in collagen x 100/(cpm pH]proline in noncollagen)x 5.4+cpm [3H]proline in collagen. c Statistically different from the control at p < 0.05, Student's t test.
134
GALLIGANI~ LONATI-GALLIGANI, AND FULLER
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FIG. 1. Time course of DNA synthesis in explant cultures of normal and CC]4-treated mouse liver. This figure illustrates a typical experiment where thymidine incorporation per /zg DNA is compared in control liver and liver obtained from a CCl4-treated mouse. The mean slope for three experiments was 50 + 5.3 for control compared to 192 + 44.2 for CCI4 treated (p < 0.05). damaged liver to proliferate compared to control cultures, thymidine incorporation into DNA was determined at several time periods after the establishment of explant cultures. The slopes of the two regression lines representing the rates of proliferation of the cells in explant cultures from a typical experiment are significantly different (p < 0.05) (Fig. 1). In this experimental design the rate of growth and rate of thymidine incorporation were determined for individual liver preparations and compared to control liver preparations. In each of several experiments, the difference in the slope of the curves indicated that the growth of cells from CC14-injured liver was three to four times more rapid than the growth of cells from normal liver explants. In order to test for the presence of specific growth-stimulating factors in the CC14-treated liver, normal liver explant cultures were grown in medium conditioned by CC14-damaged liver explants. There was no apparent difference in the growth of
normal liver cultures when exposed to conditioned medium (data not shown). The rate of incorporation of radioactive proline into collagen and noncollagen proteins was measured in control and CCI 4treated liver cultures on different days over a period of 3 weeks. In a typical experiment, the rate of incorporation of radioactive proline into noncollagen proteins (Fig. 2A) and collagen (Fig. 2B) increased in parallel with the rate of outgrowth of the cells from the tissue fragments of both normal and injured liver in culture. At all time periods the values obtained for the CC14-treated liver were higher than for the respective control liver with the increase in the rate of collagen synthesis progressing from a 4-fold difference on Day 1 to a maximum 10-fold difference on Day 11. The proportion of collagen to total protein synthesized (Fig. 2C) progressively increased, both in the cells outgrowing from damaged liver as well as in those outgrowing from the normal liver, from initial values of
135
COLLAGEN SYNTHESIS IN CC14-LIVER EXPLANTS
0.33 and 0.14~, respectively, to a maximum of 2.55 and 1.65~, respectively, after 7 days in culture. These values decreased slightly thereafter despite the continuous proliferation of the cultures. In order to permit statistical evaluation of these changes, data were pooled from separate experiments where cultures were labeled on Day 1, at approximately 1 week (6-11 days) and at 16-22 days (Table 3).
Collagen and noncollagen protein synthesis are significantly elevated at all time periods in the cultures of injured liver tissue compared to the controls. Collagen synthesis, measured as a percentage of total protein synthesis, increases during the early stage of culture growth of the damaged liver fragments from an initial value of 0.247 ~ to a maximum of 2.3 70 after 1 ~veek and is significantly higher than in the control cultures on
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FIG. 2. Comparison of the rates of incorporation of [2,3-3Hlproline into noncoUagen proteins (A) and collagen (B) and of the rates of relative collagen synthesis (C) between control (dotted lines-open circles) and CC14-treated (solid lines--closed circles) liver in explant cultures. The values are the mean of two nonreplicate determinations, each performed in triplicate.
136
GALLIGANI, LONATI-GALLIGANI,AND FULLER TABLE 3 COLLAGEN AND NONCOLLAGEN PROTEIN SYNTHESISBY NORMAL AND CCI4-TREATED MOUSE LIVER AT DIFFERENT TIME PERIODS IN EXPLANT CULTURE
[3H]Proline incorporation (cpm/pg DNA)" Days in culture 1 6-11 16-22
Treatment
Noncollagen
Control CC14 Control CC14 Control CC14
1123 _+315 (4) 2832+590 (4)b 1388_+509 (4) 8362_+ 1095 (3)c 1612_+470 (3) 24,088+3856(3) c
Collagen 5.4 + 1.8 (4) 40.7+7.3 (4)C 63.5+33.5 (4) 1505_+440(4)e 222+ 125.6 (4) 1620_+141(3)c
Percentage collagen synthesis 0.089 + 0.020 0.247_+0.031c 0.880_+0.400 2.300_+0.140b 1.080_+0.470 1.300_+0.270
a Each value represents the mean + SE with the number of values reported in parentheses. b Statistically different from the control at p < 0.05, Student's t test. c Statistically different from the control at p < 0.01, Student's t test. e Statistically different from the control at p < 0.02, Student's t test.
Day 1 and at 1 week. This value decreases despite the continuing outgrowth of the cells with no difference observed at 16-22 days. This decrease in the cellular commitment of injured liver cultures to collagen synthesis at 1622 days reflects the increased rate of collagen synthesis in control cultures with a concomitant increase in noncollagen protein synthesis occurring in the CC14-treated liver cultures. These data indicate that cells committed to collagen production are stimulated to grow and to produce collagen more rapidly in the cultures of injured liver tissue compared to control liver tissue. In both cases, maxim u m collagen production is associated w i t h the early stages of outgrowth of the cells from the tissue fragments. Watanabe (1966) observed in light and electron microscopy studies on primary explants of rat liver that proliferation of liver parenchymal cells in culture is preceded by the outgrowth of cells of mesenchymal origin. These cells may, in fact, be responsible in liver for the production of collagen (Kent, et al., 1976; McGee and Patrick, 1972; Shaba et al., 1973) and their proliferation may be an important component in the mesenchymal reaction to liver injury (Feher et al., 1975).
Our results indicate that during the early phase of this proliferation of cells, collagen is produced at its maximum rate. These studies, however, describe the response obtained with acute liver injury, not with long-term fibrosis. Fibrosis is characterized by connective tissue accumulation occurring in response not just to a single injury, but rather to a series of repeated injuries. Our results suggest that each time the liver is injured, mesenchymal cells are stimulated to proliferate and collagen is synthesized. Upon continuous stimuli, i.e., repeated injuries, collagen and other matrix components may accumulate progressively within the liver parenchyma at a rate which exceeds the ability of liver to degrade collagen thus resulting in septum formation in the precirrhotic and cirrhotic stages (Hirayama et al., 1969). In vivo and in vitro studies on the kinetics of collagen production in long-term fibrosis are currently in progress in our laboratory.
ACKNOWLEDGMENTS This project was supported in part by NIH grant AA01422. M. L. G. was recipient of a research fellowship of the University of Pavia (Italy).
COLLAGEN SYNTHESIS IN CCI4-LIVER EXPLANTS
137
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REFERENCES