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The cellular origin of extracellular matrix constituents
Journal of Hepatology, 1993; 19:1-3
1
© 1993 ElsevierScientificPublishers Ireland Ltd. All rights reserved. 0168-8278/93/$06.00 HEPAT 01490
Leader
The cellular origin of extracellular matrix constituents
P. B e d o s s a CNRS URA 1484 and Laboratoire d'Anatomie Pathologique. Hopital de Bicdtre. 94275-Le Kremlin-Bic~tre. France
Fibrogenesis and fibrosis are ubiquitous biological events that occur during wound healing and belong to the scarring process. Since hepatic fibrogenesis and fibrosis occur in the evolution "of most chronic liver diseases as a precursor of cirrhosis, this subject has been a major source of interest in liver research during the past 15 years. The characterisation of liver cell types involved in the production of the various extracellular matrix components has provoked many comprehensive studies. Although the clarification of this issue has taken advantage of the development of in situ techniques, liver cell purification and culture and the analysis of gene expression, investigations have led to conflicting results which have been the subject of recent debate. In this issue of the journal, Geerts et al. (1) show that in addition to perisinusoidal cells (Ito cells, fat storing cells), parenchymal cells might contribute significantly to the production of collagen. The results provided by Geerts and coworkers fall in between studies that found that parenchymal cells are the major source of hepatic collagen in the normal rat (2) and those which found that sinusoidal cells are the exclusive contributors of extracellular matrix protein production in hepatic fibrogenesis (3). The possibility of comparing studies in this area needs to be discussed considering the different procedures and models used in the published works. Also as close as possible to the situation of a living liver, all experimental procedures, even the more sophisticated, have limits.
The characterisation of the cellular origin of extracellular matrix components has been attempted through in situ or in vitro methods. In situ techniques are performed on a section of a liver sample which is quickly removed and frozen. The main advantage of these techniques is that they explore the cells within their ecosystem. As long as the removal and fixation procedures do not change the functional state of a cell, in situ techniques provide a valuable insight into the cellular source of a constituent. They can localise, either the protein (immuno-histochemistry or electron immunohistochemistry) or the mRNA (in situ hybridisation) in a cell and the origin can be characterised on a morphological basis. Unfortunately, results from these studies are dependent on technical conditions that explain, in some circumstances, the discrepancies. The specificity of immunohistochemical staining (optical or ultrastructural) is clearly related to the quality of the antibody. The sensitivity of immunodetection of an intracellular component is also dependent on fixation and membrane permeabilisation, a step which is necessary to detect an intracellular protein precursor (4). In addition, the immunodetection of an intracellular protein does not necessarily mean that it is produced locally, since intracellular labelling could be due to passive immunoadsorption or active reinternalisation of an extracellular component. Whatever the limits and potential pitfalls, these studies have offered initial insights into the cell types involved in extracellular matrix pro-
Correspondence to." P. Bedossa, Laboratoire d'Anatomie Pathologique. Hopital de Bic&re,94275-LeKremlin-Bictre,France.
2
tein production and provided evidence that virtually all cell types are associated with synthesis of some matrix constituents (4-7). These studies have shown that, the hepatocyte is the main source of fibronectin in the normal adult liver, that perisinusoidal cells and endothelial cells are highly involved in interstitial collagen production (Type I and III) and basement membrane components (Type IV collagen, laminine) and that Type I collagen is also detectable, at a lower level, in the cytoplasm of hepatocytes. Changes in immunostaining intensity were also detected and no major differences were noted from one species to another. The aim of in situ hybridisation is to localise intracellular mRNA. This technique has been recently developed to detect extracellular matrix component mRNAs and Milani and colleagues have localised collagens and laminin mRNA exclusively in non-parenchymal cells without any participation of hepatocytes either in normal or fibrotic liver (8,9). Although a valuable procedure, in situ hybridisation has its limits. Sensitivity to low level mRNAs has not been evaluated. The weak discrimination power of optical microscopy, the diffusion of the staining signal all over the cells (and their neighbours), and the drastic conditions needed to expose cellular mRNAs give only a rough approximation of the cell type of origin. With this technique, the different sinusoidal cell types can not be distinguished from one another. In the future, advances will occur in this technique with an increase in the detection threshold using a reverse transcription PCR step before hybridisation and better discrimination with the development of ultrastructural in situ hybridization (conversely with the development of cold probes). This might provide further information on the cellular origin of the extracellular matrix. However, at the same time the presence of mRNA in a wellcharacterized cell type does not imply that the protein will be produced. Finally, one major drawback of these in situ techniques is that they are not quantitative. The other approach used to resolve the key question of the cellular origin of extracellular matrix components is to isolate cells from the liver and to study their mRNA or protein production (1,3,10). A major technical advance has been the improvement of isolation and purification techniques which allow the isolation of a nearly pure cell type of both normal and fibrotic livers in animals and humans. In this case mRNA extraction and analysis provide a valuable and easy way to assess which extracellular matrix gene is expressed. A quantitative estimate can also be provided. These studies have provided evidence of the role of lipocytes as the primary source of the extracellular matrix in liver fibrosis but a signal
P. BEDOSSA
for mRNA collagen in hepatocytes has also been noted in certain studies (1,3,10). Once again, the discrepancies between studies might be due to technical factors such as the purity of cell population, the specificity of the cDNA probe or the sensitivity of the detection procedure. Assuming that mRNA collagen in purified parenchymal cells is not related to contamination by mesenchymal cells, as shown in the study by Geerts et al., (1) then the detection of mRNA of Type I and III collagen in the parenchymal cells of a normal liver, even at low levels, suggests that the participation of hepatocytes in collagen production is significant. In addition and considering that hepatocytes produced 15 times more RNA per cell than fat storing cells (1) and that parenchymal cells are 7 times more numerous than fat storing cells in a normal rat liver (15), then, and if a linear extrapolation is correct, hepatocytes become the major collagen-producing cell. One of the key questions of studies performed on isolated cells is whether cells retain their physiological functions after dissociation, isolation and culture. The answer is surely not. In culture, fat storing cells change their morphological and phenotypical characteristics and become transitional cells with the features of both fat storing cells and fibroblasts. Conversely, they change the rate and profile of the extracellular matrix components which are produced (i). Whether this process is similar to what happens in vivo during liver fibrosis needs to be proven. It is also clear that extracellular matrix gene expression is regulated by different complex mechanisms. Liver gene expression is clearly influenced by the extracellular matrix on which cells are cultivated (I 1). Cytokines and cell to cell interactions also modulate collagen gene expression (12). In vitro study cannot take into account all these complex mechanisms and, therefore, results only show the potential capacity of one cell type to produce extracellular matrix constituents in the experimental condition, which can then change according to the situation. Even by studying gene expression as soon as possible after cell isolation, the dissociation and purification procedure most probably impairs liver gene expression. Thus the ideal investigation procedure needs to be found. This procedure should take into account the liver ecosystem and specifically identify the cell type involved in extracellular matrix production. This has been done with an in vivo dual labelling method which measures hepatocyte specific collagen synthesis in normal and fibrotic rat liver in vivo (2). These studies have provided strong evidence for the role of parenchymal cells in col-
CELLULAR ORIGIN OF EXTRACELLULAR MATRIX lagen p r o d u c t i o n in b o t h n o r m a l (2) a n d fibrotic livers (13). H o w e v e r a n o t h e r s t u d y using a similar a p p r o a c h
3 3
o n l y detected a low p r o d u c t i o n o f collagen by rat hepatocytes (14). Clearly other studies are necessary before a n y c o n c l u s i o n c a n be reached. N o n e o f these studies c a n s i m u l t a n e o u s l y c o n s i d e r at the same time i n t r a c e l l u l a r m R N A
transcription and
4
5
m R N A stability, i n t r a c e l l u l a r a n d e x t r a c e l l u l a r p r o t e i n processing, the e x t r a c e l l u l a r d e g r a d a t i o n o f the protein, a n d finally the fibril f o r m a t i o n which is the o n l y hallm a r k o f i m b a l a n c e d synthesis a n d d e g r a d a t i o n mecha n i s m , with, as a result, liver fibrosis. A critical local threshold o f collagen p r o t e i n c o n c e n t r a t i o n m a y be necessary for collagen fibril f o r m a t i o n . In this case, even if hepatocytes are the m a j o r source o f collagen w h e n the liver is studied as a whole, the c o n c e n t r a t i o n o f hepatocytic collagen o n a per cell basis ( c o m p a r e d to p e r i s i n u s o i d a l cells) m i g h t be to low to i n d u c e fibre form a t i o n . A g a i n o t h e r studies are required to clarify this specific point. A l t h o u g h the cellular origin o f m a t r i x p r o t e i n s has o n l y b e g a n to be elucidated, recent studies have a l r e a d y characterised the cellular origin o f m a t r i x d e g r a d a t i n g enzymes. U s i n g the s a m e m e t h o d s described a b o v e , it has been s h o w n that the same cell (fat s t o r i n g cell) m a y p r o d u c e e x t r a c e l l u l a r m a t r i x c o n s t i t u e n t s a n d release m a t r i x p r o t e i n a s e (which selectively degrades extracellular m a t r i x c o n s t i t u e n t s ) (16) a n d i n h i b i t o r y molecules o f m a t r i x p r o t i n a s e (e.g. T I M P - - tissue i n h i b i t o r o f metalloproteinase) (17). T h u s lipocytes a p p e a r to regulate m a t r i x p r o d u c t i o n a n d t u r n o v e r at m a n y levels a n d provide a d d i t i o n a l s u p p o r t for these cells in liver i n j u r y a n d repair.
6 7
8
9 10 11
12
13
14 15
16
References 1 Geerts A, Greenwel P, Cunningham M, et al. Identification of connective tissue gene transcripts in freshly isolated parenchymal, endothelial, Kupffer and fat-storing cells by Northern hybridization analysis. J Hepatol 1993: 19: . 2 Chojkier M. Hepatocyte collagen production in vivo in normal rats. J Clin Invest 1986; 78: 333-9.
17
Maher J J, McGuire R. Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibi'osis in vivo. J Clin In~,est 1990; 86: 1641-8. Clement B, Rissel M, Peyrol S, Mazurier Y. Grimaud JA, Guillouzo A. A procedure for light and electron microscopic intracellular immunolocalization of collagen and fibronectin in rat liver. J Histochem Cytochem 1985; 33: 407-14. Martinez-Hernandez A. The hepatic extracellular matrix. II Electron immunohistochemical studies in rats with CCI4-induced cirrhosis. Lab Invest 1985; 53: 166-86. Cl6ment B, Grimaud JA, Campion JP, Deugnier Y, Guillouzo A. Cell types involved in collagen and fibronectin production in normal and fibrotic human liver. Hepatology 1986; 6: 225-34. Grimaud JA, Druguet M, Peyrol S, Chevalier O, Herbage D, El Badrawy N. Collagen immunotyping in human liver: Light and electron microscope study. J Histochem Cytochem 1980; 28: 1145-56. Milani S, Herbst H, Schuppan D, Surrenti C, Riecken EO, Stein H. Cellular localization of Type 1. ii1 and IV procollagen gene transcripts in normal and fibrotic human liver. Am J Pathol 1990; 137: 59-70. Milani S, Herbst H, Schuppan D, Riecken EO, Stein H. Cellular localization of laminin gene transcripts in normal and fibrotic human liver. Am J Pathol 1989; 134:1175-82. Brenner DA. Alcorn JM, Feitelberg SP, Leffert HL, Chojkier M. Expression of collagen genes in the liver. Mol Biol Med 1990: 7: 105-15. Bucher NL, Robinson GS, Farmer SR. Effects of extracellular matrix on hepatocyte growth and gene expression: implications for hepatic regeneration and the repair of liver injury. Semin Liver Dis 1990; 10: 11-9. Gressner AM, Lotfi S, Gressner G, Lahme B. Identification and partial characterization of a hepatocyte-derived factor promoting proliferation of culturated fat-storing cells (parasinusoidal lipocytes). 1992: 16: 1250-66. Chojkier M, Lyche KD, Filip M. Increased production of collagen in vivo by hepatocytes and non parenchymal cells in rats with carbon tetrachloride-induced hepatic fibrosis. Hepatology 1988; 8: 808-14. Ogata 1, Mochida S, Tomiya T, Fujiwara K. Minor contribution of hepatocytes to collagen production in normal and early fibrotic rat liver. Hepatology 1991; 14: 361-7. Blaner W, Hendriks H. Brouwer A, De Leeuw A, Knook D, Goodman D. Retinoids, retinoid-binding proteins and retinyl palmitate hydrolase distributions in different types of rat liver cells. J Lipid Res 1985; 26: 1241-51. Herbst H, Heinrichs O, Schuppan D, Milani S, Stein H. Temporal and spatial patterns of Transin/Stromelysin RNA expression following toxic injury in rat liver. Virchows Arch B Cell Pathol 1991; 60: 295-300. Iredale JP, Murphy G, Hembry RM, Friedmann SL, Arthur MJP. Human hepatic lipocytes synthesize tissue inhibitor of metalloproteinase-l. Implication for regulation of matrix degradation in liver. J Clin Invest 1992; 90: 282-7.
1
© 1993 ElsevierScientificPublishers Ireland Ltd. All rights reserved. 0168-8278/93/$06.00 HEPAT 01490
Leader
The cellular origin of extracellular matrix constituents
P. B e d o s s a CNRS URA 1484 and Laboratoire d'Anatomie Pathologique. Hopital de Bicdtre. 94275-Le Kremlin-Bic~tre. France
Fibrogenesis and fibrosis are ubiquitous biological events that occur during wound healing and belong to the scarring process. Since hepatic fibrogenesis and fibrosis occur in the evolution "of most chronic liver diseases as a precursor of cirrhosis, this subject has been a major source of interest in liver research during the past 15 years. The characterisation of liver cell types involved in the production of the various extracellular matrix components has provoked many comprehensive studies. Although the clarification of this issue has taken advantage of the development of in situ techniques, liver cell purification and culture and the analysis of gene expression, investigations have led to conflicting results which have been the subject of recent debate. In this issue of the journal, Geerts et al. (1) show that in addition to perisinusoidal cells (Ito cells, fat storing cells), parenchymal cells might contribute significantly to the production of collagen. The results provided by Geerts and coworkers fall in between studies that found that parenchymal cells are the major source of hepatic collagen in the normal rat (2) and those which found that sinusoidal cells are the exclusive contributors of extracellular matrix protein production in hepatic fibrogenesis (3). The possibility of comparing studies in this area needs to be discussed considering the different procedures and models used in the published works. Also as close as possible to the situation of a living liver, all experimental procedures, even the more sophisticated, have limits.
The characterisation of the cellular origin of extracellular matrix components has been attempted through in situ or in vitro methods. In situ techniques are performed on a section of a liver sample which is quickly removed and frozen. The main advantage of these techniques is that they explore the cells within their ecosystem. As long as the removal and fixation procedures do not change the functional state of a cell, in situ techniques provide a valuable insight into the cellular source of a constituent. They can localise, either the protein (immuno-histochemistry or electron immunohistochemistry) or the mRNA (in situ hybridisation) in a cell and the origin can be characterised on a morphological basis. Unfortunately, results from these studies are dependent on technical conditions that explain, in some circumstances, the discrepancies. The specificity of immunohistochemical staining (optical or ultrastructural) is clearly related to the quality of the antibody. The sensitivity of immunodetection of an intracellular component is also dependent on fixation and membrane permeabilisation, a step which is necessary to detect an intracellular protein precursor (4). In addition, the immunodetection of an intracellular protein does not necessarily mean that it is produced locally, since intracellular labelling could be due to passive immunoadsorption or active reinternalisation of an extracellular component. Whatever the limits and potential pitfalls, these studies have offered initial insights into the cell types involved in extracellular matrix pro-
Correspondence to." P. Bedossa, Laboratoire d'Anatomie Pathologique. Hopital de Bic&re,94275-LeKremlin-Bictre,France.
2
tein production and provided evidence that virtually all cell types are associated with synthesis of some matrix constituents (4-7). These studies have shown that, the hepatocyte is the main source of fibronectin in the normal adult liver, that perisinusoidal cells and endothelial cells are highly involved in interstitial collagen production (Type I and III) and basement membrane components (Type IV collagen, laminine) and that Type I collagen is also detectable, at a lower level, in the cytoplasm of hepatocytes. Changes in immunostaining intensity were also detected and no major differences were noted from one species to another. The aim of in situ hybridisation is to localise intracellular mRNA. This technique has been recently developed to detect extracellular matrix component mRNAs and Milani and colleagues have localised collagens and laminin mRNA exclusively in non-parenchymal cells without any participation of hepatocytes either in normal or fibrotic liver (8,9). Although a valuable procedure, in situ hybridisation has its limits. Sensitivity to low level mRNAs has not been evaluated. The weak discrimination power of optical microscopy, the diffusion of the staining signal all over the cells (and their neighbours), and the drastic conditions needed to expose cellular mRNAs give only a rough approximation of the cell type of origin. With this technique, the different sinusoidal cell types can not be distinguished from one another. In the future, advances will occur in this technique with an increase in the detection threshold using a reverse transcription PCR step before hybridisation and better discrimination with the development of ultrastructural in situ hybridization (conversely with the development of cold probes). This might provide further information on the cellular origin of the extracellular matrix. However, at the same time the presence of mRNA in a wellcharacterized cell type does not imply that the protein will be produced. Finally, one major drawback of these in situ techniques is that they are not quantitative. The other approach used to resolve the key question of the cellular origin of extracellular matrix components is to isolate cells from the liver and to study their mRNA or protein production (1,3,10). A major technical advance has been the improvement of isolation and purification techniques which allow the isolation of a nearly pure cell type of both normal and fibrotic livers in animals and humans. In this case mRNA extraction and analysis provide a valuable and easy way to assess which extracellular matrix gene is expressed. A quantitative estimate can also be provided. These studies have provided evidence of the role of lipocytes as the primary source of the extracellular matrix in liver fibrosis but a signal
P. BEDOSSA
for mRNA collagen in hepatocytes has also been noted in certain studies (1,3,10). Once again, the discrepancies between studies might be due to technical factors such as the purity of cell population, the specificity of the cDNA probe or the sensitivity of the detection procedure. Assuming that mRNA collagen in purified parenchymal cells is not related to contamination by mesenchymal cells, as shown in the study by Geerts et al., (1) then the detection of mRNA of Type I and III collagen in the parenchymal cells of a normal liver, even at low levels, suggests that the participation of hepatocytes in collagen production is significant. In addition and considering that hepatocytes produced 15 times more RNA per cell than fat storing cells (1) and that parenchymal cells are 7 times more numerous than fat storing cells in a normal rat liver (15), then, and if a linear extrapolation is correct, hepatocytes become the major collagen-producing cell. One of the key questions of studies performed on isolated cells is whether cells retain their physiological functions after dissociation, isolation and culture. The answer is surely not. In culture, fat storing cells change their morphological and phenotypical characteristics and become transitional cells with the features of both fat storing cells and fibroblasts. Conversely, they change the rate and profile of the extracellular matrix components which are produced (i). Whether this process is similar to what happens in vivo during liver fibrosis needs to be proven. It is also clear that extracellular matrix gene expression is regulated by different complex mechanisms. Liver gene expression is clearly influenced by the extracellular matrix on which cells are cultivated (I 1). Cytokines and cell to cell interactions also modulate collagen gene expression (12). In vitro study cannot take into account all these complex mechanisms and, therefore, results only show the potential capacity of one cell type to produce extracellular matrix constituents in the experimental condition, which can then change according to the situation. Even by studying gene expression as soon as possible after cell isolation, the dissociation and purification procedure most probably impairs liver gene expression. Thus the ideal investigation procedure needs to be found. This procedure should take into account the liver ecosystem and specifically identify the cell type involved in extracellular matrix production. This has been done with an in vivo dual labelling method which measures hepatocyte specific collagen synthesis in normal and fibrotic rat liver in vivo (2). These studies have provided strong evidence for the role of parenchymal cells in col-
CELLULAR ORIGIN OF EXTRACELLULAR MATRIX lagen p r o d u c t i o n in b o t h n o r m a l (2) a n d fibrotic livers (13). H o w e v e r a n o t h e r s t u d y using a similar a p p r o a c h
3 3
o n l y detected a low p r o d u c t i o n o f collagen by rat hepatocytes (14). Clearly other studies are necessary before a n y c o n c l u s i o n c a n be reached. N o n e o f these studies c a n s i m u l t a n e o u s l y c o n s i d e r at the same time i n t r a c e l l u l a r m R N A
transcription and
4
5
m R N A stability, i n t r a c e l l u l a r a n d e x t r a c e l l u l a r p r o t e i n processing, the e x t r a c e l l u l a r d e g r a d a t i o n o f the protein, a n d finally the fibril f o r m a t i o n which is the o n l y hallm a r k o f i m b a l a n c e d synthesis a n d d e g r a d a t i o n mecha n i s m , with, as a result, liver fibrosis. A critical local threshold o f collagen p r o t e i n c o n c e n t r a t i o n m a y be necessary for collagen fibril f o r m a t i o n . In this case, even if hepatocytes are the m a j o r source o f collagen w h e n the liver is studied as a whole, the c o n c e n t r a t i o n o f hepatocytic collagen o n a per cell basis ( c o m p a r e d to p e r i s i n u s o i d a l cells) m i g h t be to low to i n d u c e fibre form a t i o n . A g a i n o t h e r studies are required to clarify this specific point. A l t h o u g h the cellular origin o f m a t r i x p r o t e i n s has o n l y b e g a n to be elucidated, recent studies have a l r e a d y characterised the cellular origin o f m a t r i x d e g r a d a t i n g enzymes. U s i n g the s a m e m e t h o d s described a b o v e , it has been s h o w n that the same cell (fat s t o r i n g cell) m a y p r o d u c e e x t r a c e l l u l a r m a t r i x c o n s t i t u e n t s a n d release m a t r i x p r o t e i n a s e (which selectively degrades extracellular m a t r i x c o n s t i t u e n t s ) (16) a n d i n h i b i t o r y molecules o f m a t r i x p r o t i n a s e (e.g. T I M P - - tissue i n h i b i t o r o f metalloproteinase) (17). T h u s lipocytes a p p e a r to regulate m a t r i x p r o d u c t i o n a n d t u r n o v e r at m a n y levels a n d provide a d d i t i o n a l s u p p o r t for these cells in liver i n j u r y a n d repair.
6 7
8
9 10 11
12
13
14 15
16
References 1 Geerts A, Greenwel P, Cunningham M, et al. Identification of connective tissue gene transcripts in freshly isolated parenchymal, endothelial, Kupffer and fat-storing cells by Northern hybridization analysis. J Hepatol 1993: 19: . 2 Chojkier M. Hepatocyte collagen production in vivo in normal rats. J Clin Invest 1986; 78: 333-9.
17
Maher J J, McGuire R. Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibi'osis in vivo. J Clin In~,est 1990; 86: 1641-8. Clement B, Rissel M, Peyrol S, Mazurier Y. Grimaud JA, Guillouzo A. A procedure for light and electron microscopic intracellular immunolocalization of collagen and fibronectin in rat liver. J Histochem Cytochem 1985; 33: 407-14. Martinez-Hernandez A. The hepatic extracellular matrix. II Electron immunohistochemical studies in rats with CCI4-induced cirrhosis. Lab Invest 1985; 53: 166-86. Cl6ment B, Grimaud JA, Campion JP, Deugnier Y, Guillouzo A. Cell types involved in collagen and fibronectin production in normal and fibrotic human liver. Hepatology 1986; 6: 225-34. Grimaud JA, Druguet M, Peyrol S, Chevalier O, Herbage D, El Badrawy N. Collagen immunotyping in human liver: Light and electron microscope study. J Histochem Cytochem 1980; 28: 1145-56. Milani S, Herbst H, Schuppan D, Surrenti C, Riecken EO, Stein H. Cellular localization of Type 1. ii1 and IV procollagen gene transcripts in normal and fibrotic human liver. Am J Pathol 1990; 137: 59-70. Milani S, Herbst H, Schuppan D, Riecken EO, Stein H. Cellular localization of laminin gene transcripts in normal and fibrotic human liver. Am J Pathol 1989; 134:1175-82. Brenner DA. Alcorn JM, Feitelberg SP, Leffert HL, Chojkier M. Expression of collagen genes in the liver. Mol Biol Med 1990: 7: 105-15. Bucher NL, Robinson GS, Farmer SR. Effects of extracellular matrix on hepatocyte growth and gene expression: implications for hepatic regeneration and the repair of liver injury. Semin Liver Dis 1990; 10: 11-9. Gressner AM, Lotfi S, Gressner G, Lahme B. Identification and partial characterization of a hepatocyte-derived factor promoting proliferation of culturated fat-storing cells (parasinusoidal lipocytes). 1992: 16: 1250-66. Chojkier M, Lyche KD, Filip M. Increased production of collagen in vivo by hepatocytes and non parenchymal cells in rats with carbon tetrachloride-induced hepatic fibrosis. Hepatology 1988; 8: 808-14. Ogata 1, Mochida S, Tomiya T, Fujiwara K. Minor contribution of hepatocytes to collagen production in normal and early fibrotic rat liver. Hepatology 1991; 14: 361-7. Blaner W, Hendriks H. Brouwer A, De Leeuw A, Knook D, Goodman D. Retinoids, retinoid-binding proteins and retinyl palmitate hydrolase distributions in different types of rat liver cells. J Lipid Res 1985; 26: 1241-51. Herbst H, Heinrichs O, Schuppan D, Milani S, Stein H. Temporal and spatial patterns of Transin/Stromelysin RNA expression following toxic injury in rat liver. Virchows Arch B Cell Pathol 1991; 60: 295-300. Iredale JP, Murphy G, Hembry RM, Friedmann SL, Arthur MJP. Human hepatic lipocytes synthesize tissue inhibitor of metalloproteinase-l. Implication for regulation of matrix degradation in liver. J Clin Invest 1992; 90: 282-7.