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Thrombin stimulates proliferation of liver fat-storing cells and expression of monocyte chemotactic protein-1: Potential role in liver injury
Thrombin Stimulates Proliferation of Liver Fat-Storing Cells and Expression of Monocyte Chemotactic Protein-l: Potential Role in Liver Injury FABIO MARRA,1 GIUSEPPE GRANDALIANO,~ ANTHONY J. VALENTE,2 AND HANNA E. ABBOUD1
Liver fat-storing cells (FSC) proliferate and secrete extracellular matrix in experimental models of liver injury. In this study, w e d e t e r m i n e d if thrombin, a serine protease p r o d u c e d during acute and chronic tissue injury, modulates the functions of FSC. Thrombin stimulated DNA synthesis and proliferation of FSC, as assessed b y [3H]-thymidine incorporation assay and m e a s u r e m e n t of cell number, respectively. Thrombin also increased the secretion of m o n o c y t e chemotactic protein-1 (MCP-1) in a time- and d o s e - d e p e n d e n t fashion. The effect of t h r o m b i n o n both DNA synthesis and MCP-1 secretion was neutralized b y pretreatment of thrombin w i t h hirudin. The increased MCP-1 secretion was associated with increased steady-state levels of MCP-1 m e s s e n g e r RNA. Pretreatment of FSC w i t h 5 pmol/L retinol for 48 hours inhibited the mitogenic effects of t h r o m b i n but not the induction of MCP-1 secretion. FSC express specific transcripts e n c o d i n g for the h u m a n t h r o m b i n receptor, as s h o w n by N o r t h e r n blot analysis of poly(A) + RNA. Proteolytic activation of the thrombin receptor results in the formation of a n e w Nterminus that functions as a tethered ligand. We studied the effects of a t h r o m b i n receptor activating peptide (TRAP) c o r r e s p o n d i n g to the n e w l y formed N-terminus, on FSC. TRAP m i m i c k e d the effects of thrombin on [~H]t h y m i d i n e incorporation, MCP-1 secretion, and MCP-1 gene expression. This study suggests that t h r o m b i n m a y be i n v o l v e d in m o d u l a t i n g FSC proliferation and monocyte c h e m o t a x i s during h u m a n liver disease, t h r o u g h proteolytic activation of its receptor. (HEPATOLOGY 1995;22:780-787.)
Abbreviations: FSC, fat-storing cells; MCP-1, monocyte chemotactic protein-1; PDGF, platelet-derived growth factor; TRAP, thrombin receptor-activating peptide; SFIF, serum-free insulin-free. From the Departments of ~Medicine and 2Pathology, University of Texas Health Science Center at San Antonio, and Audie L. Murphy Memorial Veterans Administration Hospital, San Antonio, TX. Received August 18, 1994; accepted April 24, 1995. Supported by NIH grants DK-33665, DK-43988, and HL-26890, and by the VA Medical Research Service. G. Grandaliano is a recipient of a National Kidney Foundation Fellowship grant. Dr Marra is now at Istituto di Medicina Interna, Viale Morgagni, 85, 1-50134 Florence, Italy. Address reprint requests to: Hanna E. Abbeud, MD, Department of Medicine, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr, San Antonio, TX 78284. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2203-001353.00/0
Studies conducted over the past few years have identified fat-storing cells of the liver (FSC) as a key cellular element involved in liver tissue injury and repair processes. After acute or chronic liver injury, FSC undergo phenotypic transformation toward a matrix-producing or proliferative myofibroblastlike cell. 15 Studies in experimental models of liver injury have indicated that local cell proliferation m a y be an important mechanism responsible for the expansion of the FSC population, s'6 When the injury is acute, the activation of FSC is selflimited, whereas in chronic liver injury, persistent FSC activation results in liver fibrosis and cirrhosis. Cultured FSC have also been shown to produce macrophage-colony stimulating factor, which is involved in the proliferation and differentiation of mononuclear phagocytes, 7 and platelet-activating factor, a chemoattractant and activator of polymorphonuclear leukocytes, s We have recently reported 9 that h u m a n FSC secrete monocyte chemotactic protein-1 (MCP-1), a chemokine that specifically recruits monocytes from the bloodstream to the sites of tissue injury. 1°'~1 MCP1 gene expression is also upregulated in chronic liver disease. 9 The response of liver tissue to injury involves the activation of several soluble mediators, such as cytokines, that regulate cell proliferation, matrix production, and secretion of other mediators. ~ Thrombin is a serine protease that is activated by the proteolytic cleavage of prothrombin. 12 Attention has been focused recently on the role of thrombin as a mediator of the complex series of events that comprise tissue injury and repair processes. Thrombin is a potent mitogen for mesenchymal cells and stimulates secretion of platelet-derived growth factor (PDGF) by mesenchymal cells. 18-15 Treatment of cutaneous wounds with thrombin accelerates the healing process. TMLocal generation of thrombin in response to injury therefore represents not only a phlogistic mediator, b u t also a potential mechanism contributing to the process of tissue repair. 17 In the current study we report that thrombin stimulates FSC proliferation and secretion of MCP-1 through proteolytic activation of a receptor expressed in activated FSC. METHODS Reagents. Human thrombin was purchased from Ameri-
can Diagnostic (Greenwich, CT). Retinol (all trans) was from
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HEPATOLOGYVol. 22, No. 3, 1995 Fluka, Ronkonkoma, NY. Recombinant human interferonwas purchased from Collaborative Biomedical Products, Bedford, MA. Hirudin and amastatin were purchased from Sigma Chemical Co., St. Louis, MO. Neutralizing PDGF antibodies that recognize PDGF A and B chains were purchased from Collaborative Biomedical products (Bedford, MA). The thrombin receptor activating peptide (TRAP, sequence SFLLRNP) was synthesized by the Institutional Protein Core Facility, University of-Texas Health Science Center at San Antonio. The full-length complementary DNA encoding for the human thrombin receptor is was kindly provided by Dr S. R. Coughlin (University of California at San Francisco). Isolation a n d CultUre ofFSC. F S C w e r e i s o l a t e d f r o m sect i o n s of normal livers-unsuitable for transplantation. The procedures for isolation, culture, and characterization of the FSC h a v e b e e n :described in detail elsewhere. 9'19 FSC were cultured in Waymouth's medium supplemented with 17% fetal calf serum. Cells were cultured on plastic and used between passages 3 and 10, when transition toward a myofibroblastlike phenotype was evident. The cells were subcultured at a 1:3 split ratio. Measurement of DNA Synthesis. Confluent FSC were washed once in serum-free, insulin-free (SFIF) Waymouth's medium for 48 hours and incubated in the same medium before the addition of agonists. After 24 hours' incubation, the cells were pulsed with 1 #C~/wel] of [SH]thymidine for 4 hours, and trichloroacetic acid-precipitable radioactivity was determined as described elsewhere. 2° For PDGF neutralization experiments, cells were incubated with thrombin (3 U/ mL) in the presence or absence of neutralizing anti-PDGF antibodies (20 #g/mL). MCP-1 Radioimmunoassay. Near confluent FSC were preincubated in SFIF medium overnight before addition of agonists. After incubation for the appropriate length of time, the conditioned medium was removed and stored at -80°C until assayed. Remaining cells were trypsinized and counted. Measurement of MCP-1 was performed by radioimmunoassay as we described previously. 9 Briefly, 2 to 5 #g of the purified MCP-1 protein were labeled with 125I using the BoltonHunter reaction as described previously. 21 To 150 #L volume of the test solution were added 50 #L of 125I-MCP-1 (3 × 105 cpm/mL) and 100 #L of the rabbit antibody to MCP-1 (diluted 1:500). The tubes were incubated at 37°C for 2 hours, then 100 #L of calf serum (carrier) and 1 mL of 19.6% sodium sulfate were added and incubation continued for 30 minutes at room temperature. The tubes were centrifuged at 1,500g for 15 minutes, the supernatant was discarded, and the radioactivity in the precipitate was counted in a gamma counter. The limit of detection of this assay is approximately 3 to 5 ng/mL MCP-1. The range of the standard curve was 5 to 70 ng/mL. Northern Blot Analysis. RNA was isolated by one-step guanidinium thiocyanate-phenol chloroform extraction as described by Chomczynski and Sacchi. 22 The amount of RNA was determined by measuring 260 nm absorbance. Total RNA was fractionated by electrophoresis on 1% agaroseformaldehyde gel, and blotted on a nylon membrane (GeneScreen, New England Nuclear, Boston, MA). The filters were cross-linked using a UV-cross]inker (Stratagene, La Jolla, CA) according to the manufacturer's protocol, and prehybridized for 1 hour before hybridization overnight with 1 to 2 × 10~ cpm/mL of 32P-labeled probe at 42°C. Blots were then washed and autoradiographed using Kodak X-OMAT AR film (Rochester, NY). In some experiments, after removal of the MCP-1 probe, the same filters were hybridized with a a2p_
MARRA ET AL 781 1250
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FIG. 1. Thrombin stimulates DNA synthesis in human FSC. FSC were serum-starved for 48 hours before the addition of increasing doses of thrombin. After 24 hours' incubation, the cells were pulsed with 1 #Ci/wel] of [aH]thymidine and incubated for an additional 4 hours. Data are mean _+ SE (n = 11). *P < .05 or less vs. control unstimulated cells.
labeled control probe encoding for the ribosomal protein 36B4. 23 Retinol Experiments. Retinol was dissolved in 100% ethanol to a final concentration of 5 mmol/L immediately before use. The cells were washed with phosphate-buffered saline, and SFIF medium containing retinol at a final concentration of 5/~mol/L was added. Ethanol was used as a control at the same final concentration (0.1%). The cells were incubated for 48 hours, and the agonists were then added. For MCP-1 experiments, after the 48-hour incubation the medium was replaced with fresh SFIF medium with or without retinol, containing the conditions to be tested. All the procedures involving retinol were performed under dim lighting. The retinol stock was stored in the dark under argon. RESULTS
T h r o m b i n S t i m u l a t e s F S C P r o l i f e r a t i o n . We first inv e s t i g a t e d the effects of t h r o m b i n on D N A synthesis. F S C were s e r u m s t a r v e d for 48 h o u r s a n d t h e n incub a t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s of h u m a n t h r o m bin for 24 h o u r s before p u l s i n g w i t h [3H]thymidine for a n a d d i t i o n a l 4 h o u r s (Fig. 1). S e r u m - s t a r v e d cells rem a i n a t t a c h e d w i t h m i n i m a l ( < 5 % ) cell a t t r i t i o n for periods as long as 72 hours. T h r o m b i n i n d u c e d a dosed e p e n d e n t increase in [aH]thymidine incorporation, w i t h a m a x i m a l fourfold increase in r e s p o n s e to 5 U/ mL. C o n c e n t r a t i o n s as low as 0.1 U / m L c o n s i s t e n t l y s t i m u l a t e d D N A synthesis. The specificity of t h r o m bin's effect w a s a s s e s s e d by u s i n g hirudin, a t h r o m b i n a n t a g o n i s t t h a t i n a c t i v a t e s t h r o m b i n by b i n d i n g the a n i o n exosite. 24 T h r o m b i n was p r e i n c u b a t e d at 37°C for 30 m i n u t e s w i t h or w i t h o u t h i r u d i n (3 h i r u d i n u n i t s / i t h r o m b i n unit) before addition to the cells. P r e i n c u b a tion w i t h h i r u d i n abolished t h e s t i m u l a t o r y effect of t h r o m b i n on D N A s y n t h e s i s ( d a t a not shown). To test if the effects of t h r o m b i n on t h y m i d i n e u p t a k e were p a r a l l e l e d by a n increase in cell n u m b e r , s u b c o n f l u e n t
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HEPATOLOGY S e p t e m b e r 1995
TABLE 1. E f f e c t s o f T h r o m b i n o n F S C P r o l i f e r a t i o n Cells ( x I O -3) T i m e (hrs)
Control
Thrombin
48 72
55.7 _+ 1.6 62.0 + 1.4
63.1 _+ 2.0* 72.4 + 1.49
N O T E . S u b c o n f l u e n t F S C w e r e i n c u b a t e d in S F I F m e d i u m in t h e p r e s e n c e or a b s e n c e of 2.5 U / m L t h r o m b i n for d i f f e r e n t periods. A t t h e e n d of t h e i n c u b a t i o n , cells w e r e t r y p s i n i z e d a n d counted. D a t a a r e m e a n _+ SE (n = 6). * P < .05 vs. control. P < .005 vs. control.
FSC were incubated in SFIF medium for 48 or 72 hours in the presence or absence of thrombin (2.5 U/mL). As shown in Table 1, incubation with thrombin resulted in a significant increase in the number of FSC, compared with control cells. These data demonstrate t h a t thrombin is a mitogen for cultured FSC, because the increased DNA synthesis is associated with an actual increase in cell number. Thrombin has been shown to induce PDGF secretion in mesenchymal cells. 14 To assess if the mitogenic effect of thrombin in FSC is media t e d at least in part by endogenous PDGF, we measured thrombin-induced DNA synthesis in the presence or absence of neutralizing PDGF antibodies. Coincubation with PDGF antibodies had no effect on DNA synthesis induced by thrombin (control 128 _+ 39; thrombin 437 _+ 137; thrombin + anti-PDGF antibodies 410 _+ 153 cpm/well; n = 8). Thrombin Upregulates MCP-1 Secretion and Gene Expression. Figure 2A shows a time course for the effect of thrombin (2.5 U/mL) on secretion of MCP-1 by FSC. Increased MCP-1 secretion was evident after 12 hours' incubation with thrombin. MCP-1 levels were more t h a n twofold higher t h a n control after a 24-hour incubation. The effect of thrombin was dose dependent. As depicted in Fig. 2B, 0.1 U/mL were sufficient to induce an increase in MCP-1 accumulation. Treatment of the cells with 5 U/mL thrombin resulted in twofold to threefold increase in MCP-1 levels. Preincubation with hirudin abolished thrombin-induced MCP-1 secretion (data not shown). The effect of thrombin on MCP1 gene expression is shown in Fig. 3. Incubation with thrombin increased the steady-state levels of MCP-1 messenger RNA. The MCP-1 messenger RNA levels were increased after 2 hours and peaked at 4 hours after incubation with thrombin. Differential Effects of Retinol on DNA Synthesis and MCP-1 Secretion. Retinol and retinoic acid inhibit the proliferation of FSC in culture, and it has been suggested t h a t the vitamin A content of quiescent FSC may contribute to their low tendency to proliferateY '26 We examined the effects ofretinol on thrombin-induced mitogenesis and MCP-1 secretion. Retinol was used at 5 mmol/L, a concentration t h a t has been shown to maximally inhibit growth-factor-induced mitogenesis in rat FSC. 26 Preliminary experiments showed t h a t this dose almost completely inhibits PDGF-induced DNA syn-
thesis also in h u m a n FSC (data not shown). FSC were preincubated in SFIF medium containing 5 #mol/L retinol for 48 hours. Thrombin was then added and cells incubated for 24 hours, then pulsed with [~H]-thymidine. Retinol pretreatment inhibited thrombin-induced DNA synthesis by 88% (Fig. 4A). To confirm the inhibitory effect of retinol on thrombin-induced FSC proliferation, FSC were treated with thrombin for 72 hours in the presence or absence of 5 mmol/L retinol, and the cells were counted at the end of the incubation. Pret r e a t m e n t with retinol completely inhibited the increase in cell number induced by 3 U/mL thrombin (control 39.0 _+ 13.1; thrombin 51.0 _+ 12.8; thrombin ÷ retino141.7 + 11.7 × 103 cells/well, n = 6). In contrast to its effects on cell proliferation, pretreatment with
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HEPATOLOGY Vol. 22, No. 3, 1995
MARRA ET AL
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(Fig. 6A), MCP-1 secretion (Fig. 6B), and MCP-1 gene expression (Fig. 7). For these experiments, cells were co-incubated with amastatin, an inhibitor of aminopeptidases, to prevent the hydrolysis of the oligopeptide. 2s Amastatin alone did not have any effect on FSC (Figs. 6 and 7). Of note is that the concentration of TRAP required to stimulate DNA synthesis or MCP-1 production is several orders of magnitude higher than the concentration of thrombin required to exert the same effect. This finding is probably related to the fact that activation of the TR by thrombin generates a ligand tethered to the receptor, thus requiring a lower number of molecules to exert a stimulatory effect. Moreover, TRAP does not possess the full anion-binding exosite, which is important for binding to the thrombin receptor. ~ 3500
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FIG. 3. Northern blot analysis of MCP-1 gene expression in response to thrombin in h u m a n FSC. Confluent h u m a n FSC were incubated in SFIF medium overnight followed by the addition of h u m a n thrombin for the indicated time periods. For Northern blot analysis, 15 ~g of total RNA were fractionated on a 1% agarose-formaldehyde gel, blotted on a nylon membrane, and hybridized with a baboon cDNA encoding for MCP-1. The same filters were hybridized with a probe encoding for the ribosomal protein 36B4 (control probe).
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retinol did not inhibit MCP-1 secretion in response to thrombin (Fig. 4B). Retinol did not reduce the levels of MCP-1 also when FSC were incubated with interferon~, a more potent agonist (data not shown). Expression of the Human Thrombin Receptor in FSC. The recently cloned thrombin receptor belongs to the "seven t r a n s m e m b r a n e domain" family of G-protein-linked receptors, is We used a full-length complem e n t a r y DNA clone encoding for the h u m a n thrombin receptor to test its expression in FSC. Hybridization of poly(A)+ RNA isolated from h u m a n FSC (Fig. 5) showed the presence of two specific transcripts of approximately 3.5 and 4.0 kb. Interestingly, the 4.0 kb transcript has not been reported in other cell lines, and its significance remains to be determined. The size of the two bands is very similar, and it is possible that under different conditions the two transcripts appear as a single band. T R A P Mimics the Actions of Thrombin in FSC. The thrombin receptor is characterized by a unique mechanism of activation. Thrombin proteolytically cleaves the receptor, exposing a new amino terminus that functions as a tethered ligand that activates the receptor, is We used a thrombin receptor activating peptide (TRAP, sequence SFLLRNP) corresponding to the aminoacid sequence of the cleaved h u m a n TR, ls'27 to investigate if the effects of thrombin on FSC are mediated by a specific receptor. One hundred micromolar TRAP mimicked the thrombin-induced increase in DNA synthesis
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FIG. 4. Differential effects of retinol on DNA synthesis and MCPi secretion in response to thrombin. (A) FSC in 24-well plates were incubated in the presence or absence of 5 #mol/L retinol for 48 hours in SFIF medium, t h e n 2.5 U/mL of thrombin were added, and DNA synthesis was measured as described in legend to Fig. 1. Data are mean _+ SE (n = 5). *P < .05 vs. thrombin alone. (B) FSC were incubated in the presence or absence of 5 #mol/L retinol for 48 hours in SFIF medium. Medium was then replaced with fresh SFIF medium containing thrombin or retinel and cells were incubated for additional 24 hours. MCP-1 levels were measured in the conditioned medium. Data are mean ± SE (n = 6). No statistically significant differences were observed comparing thrombin and thrombin plus retinol.
784
MARRA ET AL
HEPATOLOGY September 1995
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likely to occur in human liver d i s e a s e Y Expansion of FSC population may ultimately lead to increased deposition ofextracellular matrix, and therefore factors that stimulate FSC proliferation may contribute to liver fibrosis. The mechanism by which thrombin stimulates DNA synthesis is not precisely identified. Although thrombin has been shown to induce secretion of PDGF in other mesenchymal cells, ~4 the increase in DNA synthesis in FSC was not inhibited by neutralizing PDGF antibodies. This observation indicates that thrombin stimulates FSC mitogenesis independent of endogenous PDGF secretion. Among several factors, only PDGF is more potent than thrombin in stimulating DNA synthesis in human FSC. It is likely that thrombin in concert with other cytokines secreted during liver injury contributes to the increase in FSC number in vivo. 700
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The pivotal role of thrombin in coagulation has been known for several decades. In recent years, several studies have emphasized the role of thrombin as a mediator of injury and repair in different biological systerns. 1~'~9~ However, there is very little information pertinent to the potential role of thrombin in liver injury. Tissue injury results in the formation of the prothrombin activating complex through the extrinsic pathway, most likely through damaged cell membranes. ~e This mechanism is likely to be operating also in the injured liver, because fibrin deposition has been demonstrated in areas of hepatocellular necrosis during acute hepatitis and in several models of liver injury. ~e-~ This study demonstrates that thrombin stimulates FSC proliferation and secretion of MCP-1, a potent monocyte chemoattractant. These properties of thrombin are somehow unique among agents that stimulate FSC, because PDGF, a very strong mitogen for FSC, ~° weakly stimulates MCP-1 secretion, whereas interleukin-la strongly induces MCP-19 without affecting FSC mitogenesis. Proliferation of FSC is a wellrecognized event after experimental liver injury and is
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Amastatin FIG. 6. A thrombin receptor activating peptide (TRAP) mimics thrombin effects on DNA synthesis and MCP-1 secretion. (A) FSC were serum-starved for 48 hours before the addition of 10 /~mol/L amastatin with or without 100 #mol/L TRAP. DNA synthesis was measured as described in legend to Fig. 1. Data are mean _+ SE (n = 9). *P < .05 vs amastatin alone. (B) FSC were incubated for 24 hours in SFIF medium in the presence of 2.5 U/mL thrombin, or 10 #mol/L amastatin with or without 100 #mol/L TRAP. MCP-1 secre~ tion was measured in the conditioned medium. Data are mean _+ SE (n = 5). *P < .05 vs. control; **P < .05 vs. amastatin alone.
HEPATOLOGY Vol. 22, No. 3, 1995
MARRA ET AL
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FIG. 7. TRAP stimulates MCP-1 gene expression in h u m a n FSC. Confluent h u m a n FSC were incubated in SFIF medium overnight before the addition of 10 pmol/L amastatin with or without 100 #mol/ L TRAP for 4 hours. Northern blot analysis ofMCP-1 gene expression was carried out as described in legend to Fig. 3.
Thrombin is chemotactic for monocytes, 37 and stimulates calcium transients and secretion of platelet activating factor by h u m a n FSC. s'3s In this study we show that thrombin is potentially able to induce monocyte recruitment and activation by stimulating MCP-1 secretion and gene expression. MCP-1 is a major monocyte chemoattractant secreted by FSC, because more than 80% of the monocyte chemotactic activity secreted is neutralized by MCP-1 antibodies. 9 Additionally, MCP-1 gene is upregulated during liver disease. 9 Activated monocytes highly express tissue factor, 39 which participates in the formation of the prothrombinase complex, thus resulting in further generation of thrombin, and therefore amplifying the inflammatory response. Acquisition of an "activated" phenotype by FSC is associated with upregulation and or de novo expression of receptors for cytokines, such as PDGF or transforming growth factor /~.4o.41 In this study, we show that the TR is expressed in cultured FSC. FSC were studied in culture after several passages, a condition that results in acquisition of an activated phenotype. It remains to be determined if this receptor is also expressed in "quiescent" FSC present in the normal liver or if it is upregulated during liver injury. Activation of the TR is mediated by the proteolytic activity of thrombin, which cleaves the receptor,
785
exposing a new amino terminus that functions as a tethered ligand that activates the receptor. Is As a consequence, oligopeptides with sequences identical to the new amino terminus of the TR mimic the actions of thrombin in the absence of proteolytic cleavage of the receptor. Is'27 However, it has been reported that some but not all effects of thrombin can be reproduced by these peptides, suggesting that alternative mechanisms or additional receptors mediate the effects of t h r o m b i n Y Thrombin and TRAP may elicit different effects on intracellular calcium concentration in osteoblastlike cells. 43 In this study, all the effects of thrombin on FSC could be reproduced by TRAP in the presence of an aminopeptidase inhibitor, which prevents the peptide from degradationY The elucidation of the mechanism(s) by which thrombin modulates the biological functions of FSC m a y have therapeutic implications. Synthetic "mutant" peptides that resist cleavage by thrombin inhibit the activation of the receptor by thrombin. 44 Such peptides m a y have a role in blocking the effects of thrombin. In the normal liver, "quiescent" FSC contain vitamin A droplets, and the phenotypic transformation toward myofibroblastlike cells that occurs during liver injury is associated with decreased retinoid content. 45'4~It has been reported that vitamin A inhibits FSC proliferation stimulated by serum, PDGF, or epidermal growth factor. 25'26 In this article we show that retinol inhibits thrombin-induced DNA synthesis but not MCP-1 secretion. These data indicate that retinol does not exert a nonspecific inhibitory effect on FSC function. Rather, retinol selectively inhibits certain FSC functions such as mitogenesis or secretion of collagen and transforming growth factor-~. 2~ The molecular mechanisms by which retinoids exert their inhibitory effects are only partially known. Retinoic acid does not seem to influence the early signaling pathways that follow PDGF receptor activation in F S C . 47 The involvement of downstream pathways is also suggested by our studies, where the effects of an agonist such as thrombin, which does not activate a receptor tyrosine kinase, can also be inhibited by retinol. Because quiescent FSC have a high retinoid content, one m a y speculate that thrombin produced at the site of liver injury is able to induce secretion of MCP-1 by quiescent FSC and initiate an inflammatory response to injury through monocyte chemotaxis. In summary, this study demonstrates that thrombin stimulates mitogenesis of FSC and secretion of MCPI through a proteolitically activated receptor. These data suggest involvement of thrombin in modulating pathologic events that follow liver injury. Thrombin may be involved in monocyte recruitment and FSC proliferation, thus contributing to tissue remodeling. However, in the context of a chronic tissue injury, thrombin, together with other cytokines, may establish a vicious circle leading to functional and structural damage. Acknowledgment: We thank Dr M. Pinzani for generously providing the fat-storing cell lines used for
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HEPATOLOGYSeptember 1995
these studies, and Dr S. R. Coughlin for the kind gift of the human thrombin receptor cDNA. The expert technical help of K. A. Woodruff and S. Garcia is also acknowledged. REFERENCES
1. Friedman SL, Roll FJ, Boyles J, Bissel DM. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci USA 1985;82:8681-8685. 2. Maher JJ, McGuire RF. Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo. J Clin Invest 1990;86:1641-1648. 3. Geerts A, Lazou JM, De Bleser P, Wisse E. Tissue distribution, quantitation and proliferation kinetics of fat-storing cells in carbon tetrachloride-injured rat liver. HEPATOLOGY1991;13:11931202. 4. Bissel DM. Lipocyte activation and hepatic fibrosis. Gastroenterology 1992; 102:1803-1805. 5. Friedman SL. The cellular basis of hepatic fibrosis: mechanisms and treatment strategies. N Engl J Med 1993;328:1828-1835. 6. Mancini R, Jezequel AM, Benedetti A, Paolucci F, Trozzi L, Orlandi F. Quantitative analysis of proliferating sinusoidal cells in dimethylnitrosamine-inducedcirrhosis. J Hepatol 1992; 15:361366. 7. Pinzani M, Abboud HE, Gesua]do L, Abboud SL. Regulation of macrophage colony-stimulating factor in liver fat storing cells by peptide growth factors. Am J Physiol 1992;262:C876-C881. 8. Pinzani M, Carloni V, Marra F, Riccardi D, Laffi G, Gentilini P. Biosynthesis of platelet-activating factor and its 10-acyl analogue by liver fat-storing cells. Gastroenterology 1994; 106:13011311. 9. Marra F, Valente AJ, Pinzani M, Abboud HE. Cultured human liver fat-storing cells produce monocyte chemotactic protein. 1. Regulation by proinfiammatory cytokines. J Clin Invest 1993;92:1674-1680. 10. Valente AJ, Graves DT, Vialle-Valentin CE, Delgado R, Schwartz CJ. Purification of a monocyte chemotactic factor secreted by nonhuman primate vascular cells in culture. Biochemistry 1988;27:4162-4168. 11. Graves DT, Jiang YL, Williamson MJ, Valente AJ. Identification of monocyte chemotactic activity produced by malignant cells. Science 1989;245:1490-1493. 12. Esmon CT. Cell mediated events that control blood coagulation and vascular injury. Annu Rev Cell Biol 1993;9:1-26. 13. Glenn KC, Carney DH, Fenton JW II, Cunningham DD. Thrombin active site regions required for fibroblast receptor binding and initiation of cell division. J Biol Chem 1980;255:6609-6616. 14. Shultz PJ, Knauss TC, Mene P, Abboud HE. Mitogenic signals for thrombin in mesangial cells: regulation of phospholipase C and PDGF genes. Am J Physiol 1989;257:F366-F374. 15. McNamara CA, Sarembock IJ, Gimple LW, Fenton JW II, Coughlin SR, Owens GK. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest 1993;91:94-98. 16. Carney DH, Mann R, Redin WR, Pernia SD, Berry D, Heggers JP, Hayward PG, et al. Enhancement of incisional wound healing and neovascularization in rats by thrombin and synthetic thrombin receptor activating peptides. J Clin Invest 1992;89:1469-1477. 17. Stiernberg J, Redin WR, Warner WS, Carney DH. The role of thrombin and thrombin receptor activating peptide (TRAP-508) in initiation of tissue repair. Thromb Haemost 1993;70:158-162. 18. Vu T-KH, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991;64:1057-1068. 19. Casini A, Pinzani M, Milani S, Grappone C, Galli G, Jezequel AM, Sehuppan D, et al. Regulation of extracellular matrix synthesis by transforming growth factor fl~ in human fat-storing cells. Gastroenterology 1993; 105:245-253. 20. Pinzani M, Gesnaldo L, Sabbah GM, Abboud HE. Effects of platelet-derived growth factor and other polypeptide mitogens on
21. 22. 23.
24. 25. 26.
27.
28. 29. 30. 31. 32. 33. 34.
35. 36. 37. 38.
39.
40.
41.
DNA synthesis and growth of cultured rat liver fat-storing cells. J Clin Invest 1989;84:1786-1793. Valente AJ, Rozek MM, Schwartz CJ, Graves DT. Characterization of monocyte chemotactic protein-1 binding to human monocytes. Biochem Biophys Res Commun 1991;176:309-314. Chomczynski P, Sacehi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156-159. Basset P, Bellocq JP, Wolf C, Stoll I, Hutin P, Limacher JM, Podhajcer OL, et al. A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas. Nature 1990; 348:699-704. Rydel TJ, Rabichandran KG, Tulinski A, Bode W, Huber R, Roitsc C, Fenton JW II. The structure of a complex of recombinant hirudin and human a-thrombin. Science 1990; 249:277-280. Davis BH, Kramer RT, Davidson NO. Retinoid acid modulates rat Ito cell proliferation, collagen, and transforming growth factor fl production. J Clin Invest 1990;86:2026-2070. Pinzani M, Gentilini P, Abboud HE. Phenotypical modulation of liver fat-storing cells by retinoids. Influence on unstimulated and growth factor-induced cell proliferation. J Hepatol 1992; 14:211220. Gerstzen RE, Chen J, Ishil M, Ishil K, Wang L, Nanevicz T, Turck CW, et al. Specificity of the thrombin receptor for agonist peptide is defined by its extracellular surface. Nature 1994;368:648-651. Zacharias U, He C-J, Nguyen G, Sraer JD, Rondeau E. Longterm effects ofthrombin require sustained activation of the functional thrombin receptor. FEBS Lett 1993;334:225-228. Esmon CT, Taylor FB, Snow TR. Inflammation and coagulation: linked processes potentially regulated through a common pathway mediated by protein C. Thromb Haemost 1991;66:160-165. Cunningham DD, Pulliam L, Vaughan PJ. Protease nexin-1 and thrombin: injury-related processes in the brain. Thromb Haemost 1993;70:168-171. Morris R, Winyard PG, Blake DR, Morris CJ. Thrombin in inflammation and healing: relevance to rheumatoid arthritis. Ann Rheum Dis 1994;53:72-79. Popper H, Franklin M. Viral versus toxic hepatic necrosis. Arch Pathol 1948;46:338-376. Rake MO, Flute PT, Shilkin KB, Lewis ML, Winch J, Williams R. Early and intensive therapy of intravascular coagulation in acute liver failure. Lancet 1971;ii:1215-1218. Rake MO, Flute PT, Pannell G, Shilkin KB, Williams R. Experimental hepatic necrosis: studies on coagulation abnormalities, plasma clearance, and organ distribution of 125I-labeledfibrinogen. Gut 1973;14:574-580. Hillenbrand P, Parbhoo SP, Jedrichowski A, Sherlock S. Significance of intravascular coagxtlation and fibrinolysis in acute hepatic failure. Gut 1974;15:83-88. Hirata K, Ogata I, Ohta Y, Fujiwara K. Hepatic sinusoidal cell destruction in the development of intravascular coagulation in acute liver failure of rats. J Pathol 1989; 158:157-165. Bar-Shavit R, Kahn A, Wilner GD, Fenton JW II. Monocyte chemotaxis: stimulation by specific exosite region in thrombin. Science 1983;220:728~731. Pinzani M, Failli P, Ruocco C, Casini A, Milani S, Baldi E, Giotti A, et al. Fat-storing cells as liver specific pericytes: spatial dynamics of agonist-stimulated intracellular calcium transients. J Clin Invest 1992;90:642-646. Edwards RL, Levine JB, Green R, Duffy M, Mathews E, Brande W, Rickles FR. Activation of blood coagulation in Crohn's disease: increased plasma fibrinopeptide A and enhanced generation of monocyte tissue factor activity. Gastroenterology 1987; 92:329-337. Friedman SL, Arthur MJP. Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium: direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors. J Clin Invest 1989;84:1780-1785. Friedman SL, Yamasaki G, Wong L. Modulation of transforming growth factor fl receptors of rat lipocytes during the hepatic
HEPATOLOGYVol. 22, No. 3, 1995 wound healing response: enhanced binding and reduced gene expression accompany cellular activation in culture and in vivo. J Biol Chem 1994;269:10551-10588. 42. Vouret-Craviari V, Van Obberghen-SchillingE, Rasmussen UB, Pavirani A, Lecocq J-P, Poyssegur J. Synthetic a-thrombin receptor peptides activate G protein coupled signaling pathways but are unable to induce mitogenesis. Mol Biol Cell 1992;3: 95-102. 43. Jenkins AL, Bootman MD, Berridge MJ, Stone SR. Differences in intracellular calcium signaling after activation of the thrombin receptor by thrombin and agonist peptide in osteoblast-like cells. J Biol Chem 1994;269:17104-17110. 44. Hung D, Vu T-KH, Wheaton VI, Charo IF, Nelken NA, Esmon N, Esmon CT, et al. "Mirror image" antagonists of thrombin-
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induced platelet activation based on thrombin receptor structure. J Clin Invest 1992; 89:444-450. 45. Takahara T, Kojima T, Miyabayashi C, Inoue K, Sasaki H, Muragaki Y, Ooshima A. Collagen production in fat-storing cells after carbon tetrachloride intoxication in the rat: immunoelectron microscopic observation of type I, type III collagens and prolyl hydrolase. Lab Invest 1988;59:509-521. 46. Friedman SL, Wei S, Blaner WS. Retinol release by activated rat lipocytes: regulation by Kupffer cell-conditionedmedium and PDGF. Am J Physiol 1993;264:G947-G952. 47. Davis BH, Coll D, Beno DWA. Retinoic acid suppresses the response to platelet-derived growth factor in human hepatic Itocell-like myofibroblasts: a post-receptor mechanism independent of raf/fos/jun/egr activation. Biochem J 1993;294:785-791.
Liver fat-storing cells (FSC) proliferate and secrete extracellular matrix in experimental models of liver injury. In this study, w e d e t e r m i n e d if thrombin, a serine protease p r o d u c e d during acute and chronic tissue injury, modulates the functions of FSC. Thrombin stimulated DNA synthesis and proliferation of FSC, as assessed b y [3H]-thymidine incorporation assay and m e a s u r e m e n t of cell number, respectively. Thrombin also increased the secretion of m o n o c y t e chemotactic protein-1 (MCP-1) in a time- and d o s e - d e p e n d e n t fashion. The effect of t h r o m b i n o n both DNA synthesis and MCP-1 secretion was neutralized b y pretreatment of thrombin w i t h hirudin. The increased MCP-1 secretion was associated with increased steady-state levels of MCP-1 m e s s e n g e r RNA. Pretreatment of FSC w i t h 5 pmol/L retinol for 48 hours inhibited the mitogenic effects of t h r o m b i n but not the induction of MCP-1 secretion. FSC express specific transcripts e n c o d i n g for the h u m a n t h r o m b i n receptor, as s h o w n by N o r t h e r n blot analysis of poly(A) + RNA. Proteolytic activation of the thrombin receptor results in the formation of a n e w Nterminus that functions as a tethered ligand. We studied the effects of a t h r o m b i n receptor activating peptide (TRAP) c o r r e s p o n d i n g to the n e w l y formed N-terminus, on FSC. TRAP m i m i c k e d the effects of thrombin on [~H]t h y m i d i n e incorporation, MCP-1 secretion, and MCP-1 gene expression. This study suggests that t h r o m b i n m a y be i n v o l v e d in m o d u l a t i n g FSC proliferation and monocyte c h e m o t a x i s during h u m a n liver disease, t h r o u g h proteolytic activation of its receptor. (HEPATOLOGY 1995;22:780-787.)
Abbreviations: FSC, fat-storing cells; MCP-1, monocyte chemotactic protein-1; PDGF, platelet-derived growth factor; TRAP, thrombin receptor-activating peptide; SFIF, serum-free insulin-free. From the Departments of ~Medicine and 2Pathology, University of Texas Health Science Center at San Antonio, and Audie L. Murphy Memorial Veterans Administration Hospital, San Antonio, TX. Received August 18, 1994; accepted April 24, 1995. Supported by NIH grants DK-33665, DK-43988, and HL-26890, and by the VA Medical Research Service. G. Grandaliano is a recipient of a National Kidney Foundation Fellowship grant. Dr Marra is now at Istituto di Medicina Interna, Viale Morgagni, 85, 1-50134 Florence, Italy. Address reprint requests to: Hanna E. Abbeud, MD, Department of Medicine, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr, San Antonio, TX 78284. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2203-001353.00/0
Studies conducted over the past few years have identified fat-storing cells of the liver (FSC) as a key cellular element involved in liver tissue injury and repair processes. After acute or chronic liver injury, FSC undergo phenotypic transformation toward a matrix-producing or proliferative myofibroblastlike cell. 15 Studies in experimental models of liver injury have indicated that local cell proliferation m a y be an important mechanism responsible for the expansion of the FSC population, s'6 When the injury is acute, the activation of FSC is selflimited, whereas in chronic liver injury, persistent FSC activation results in liver fibrosis and cirrhosis. Cultured FSC have also been shown to produce macrophage-colony stimulating factor, which is involved in the proliferation and differentiation of mononuclear phagocytes, 7 and platelet-activating factor, a chemoattractant and activator of polymorphonuclear leukocytes, s We have recently reported 9 that h u m a n FSC secrete monocyte chemotactic protein-1 (MCP-1), a chemokine that specifically recruits monocytes from the bloodstream to the sites of tissue injury. 1°'~1 MCP1 gene expression is also upregulated in chronic liver disease. 9 The response of liver tissue to injury involves the activation of several soluble mediators, such as cytokines, that regulate cell proliferation, matrix production, and secretion of other mediators. ~ Thrombin is a serine protease that is activated by the proteolytic cleavage of prothrombin. 12 Attention has been focused recently on the role of thrombin as a mediator of the complex series of events that comprise tissue injury and repair processes. Thrombin is a potent mitogen for mesenchymal cells and stimulates secretion of platelet-derived growth factor (PDGF) by mesenchymal cells. 18-15 Treatment of cutaneous wounds with thrombin accelerates the healing process. TMLocal generation of thrombin in response to injury therefore represents not only a phlogistic mediator, b u t also a potential mechanism contributing to the process of tissue repair. 17 In the current study we report that thrombin stimulates FSC proliferation and secretion of MCP-1 through proteolytic activation of a receptor expressed in activated FSC. METHODS Reagents. Human thrombin was purchased from Ameri-
can Diagnostic (Greenwich, CT). Retinol (all trans) was from
780
HEPATOLOGYVol. 22, No. 3, 1995 Fluka, Ronkonkoma, NY. Recombinant human interferonwas purchased from Collaborative Biomedical Products, Bedford, MA. Hirudin and amastatin were purchased from Sigma Chemical Co., St. Louis, MO. Neutralizing PDGF antibodies that recognize PDGF A and B chains were purchased from Collaborative Biomedical products (Bedford, MA). The thrombin receptor activating peptide (TRAP, sequence SFLLRNP) was synthesized by the Institutional Protein Core Facility, University of-Texas Health Science Center at San Antonio. The full-length complementary DNA encoding for the human thrombin receptor is was kindly provided by Dr S. R. Coughlin (University of California at San Francisco). Isolation a n d CultUre ofFSC. F S C w e r e i s o l a t e d f r o m sect i o n s of normal livers-unsuitable for transplantation. The procedures for isolation, culture, and characterization of the FSC h a v e b e e n :described in detail elsewhere. 9'19 FSC were cultured in Waymouth's medium supplemented with 17% fetal calf serum. Cells were cultured on plastic and used between passages 3 and 10, when transition toward a myofibroblastlike phenotype was evident. The cells were subcultured at a 1:3 split ratio. Measurement of DNA Synthesis. Confluent FSC were washed once in serum-free, insulin-free (SFIF) Waymouth's medium for 48 hours and incubated in the same medium before the addition of agonists. After 24 hours' incubation, the cells were pulsed with 1 #C~/wel] of [SH]thymidine for 4 hours, and trichloroacetic acid-precipitable radioactivity was determined as described elsewhere. 2° For PDGF neutralization experiments, cells were incubated with thrombin (3 U/ mL) in the presence or absence of neutralizing anti-PDGF antibodies (20 #g/mL). MCP-1 Radioimmunoassay. Near confluent FSC were preincubated in SFIF medium overnight before addition of agonists. After incubation for the appropriate length of time, the conditioned medium was removed and stored at -80°C until assayed. Remaining cells were trypsinized and counted. Measurement of MCP-1 was performed by radioimmunoassay as we described previously. 9 Briefly, 2 to 5 #g of the purified MCP-1 protein were labeled with 125I using the BoltonHunter reaction as described previously. 21 To 150 #L volume of the test solution were added 50 #L of 125I-MCP-1 (3 × 105 cpm/mL) and 100 #L of the rabbit antibody to MCP-1 (diluted 1:500). The tubes were incubated at 37°C for 2 hours, then 100 #L of calf serum (carrier) and 1 mL of 19.6% sodium sulfate were added and incubation continued for 30 minutes at room temperature. The tubes were centrifuged at 1,500g for 15 minutes, the supernatant was discarded, and the radioactivity in the precipitate was counted in a gamma counter. The limit of detection of this assay is approximately 3 to 5 ng/mL MCP-1. The range of the standard curve was 5 to 70 ng/mL. Northern Blot Analysis. RNA was isolated by one-step guanidinium thiocyanate-phenol chloroform extraction as described by Chomczynski and Sacchi. 22 The amount of RNA was determined by measuring 260 nm absorbance. Total RNA was fractionated by electrophoresis on 1% agaroseformaldehyde gel, and blotted on a nylon membrane (GeneScreen, New England Nuclear, Boston, MA). The filters were cross-linked using a UV-cross]inker (Stratagene, La Jolla, CA) according to the manufacturer's protocol, and prehybridized for 1 hour before hybridization overnight with 1 to 2 × 10~ cpm/mL of 32P-labeled probe at 42°C. Blots were then washed and autoradiographed using Kodak X-OMAT AR film (Rochester, NY). In some experiments, after removal of the MCP-1 probe, the same filters were hybridized with a a2p_
MARRA ET AL 781 1250
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FIG. 1. Thrombin stimulates DNA synthesis in human FSC. FSC were serum-starved for 48 hours before the addition of increasing doses of thrombin. After 24 hours' incubation, the cells were pulsed with 1 #Ci/wel] of [aH]thymidine and incubated for an additional 4 hours. Data are mean _+ SE (n = 11). *P < .05 or less vs. control unstimulated cells.
labeled control probe encoding for the ribosomal protein 36B4. 23 Retinol Experiments. Retinol was dissolved in 100% ethanol to a final concentration of 5 mmol/L immediately before use. The cells were washed with phosphate-buffered saline, and SFIF medium containing retinol at a final concentration of 5/~mol/L was added. Ethanol was used as a control at the same final concentration (0.1%). The cells were incubated for 48 hours, and the agonists were then added. For MCP-1 experiments, after the 48-hour incubation the medium was replaced with fresh SFIF medium with or without retinol, containing the conditions to be tested. All the procedures involving retinol were performed under dim lighting. The retinol stock was stored in the dark under argon. RESULTS
T h r o m b i n S t i m u l a t e s F S C P r o l i f e r a t i o n . We first inv e s t i g a t e d the effects of t h r o m b i n on D N A synthesis. F S C were s e r u m s t a r v e d for 48 h o u r s a n d t h e n incub a t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s of h u m a n t h r o m bin for 24 h o u r s before p u l s i n g w i t h [3H]thymidine for a n a d d i t i o n a l 4 h o u r s (Fig. 1). S e r u m - s t a r v e d cells rem a i n a t t a c h e d w i t h m i n i m a l ( < 5 % ) cell a t t r i t i o n for periods as long as 72 hours. T h r o m b i n i n d u c e d a dosed e p e n d e n t increase in [aH]thymidine incorporation, w i t h a m a x i m a l fourfold increase in r e s p o n s e to 5 U/ mL. C o n c e n t r a t i o n s as low as 0.1 U / m L c o n s i s t e n t l y s t i m u l a t e d D N A synthesis. The specificity of t h r o m bin's effect w a s a s s e s s e d by u s i n g hirudin, a t h r o m b i n a n t a g o n i s t t h a t i n a c t i v a t e s t h r o m b i n by b i n d i n g the a n i o n exosite. 24 T h r o m b i n was p r e i n c u b a t e d at 37°C for 30 m i n u t e s w i t h or w i t h o u t h i r u d i n (3 h i r u d i n u n i t s / i t h r o m b i n unit) before addition to the cells. P r e i n c u b a tion w i t h h i r u d i n abolished t h e s t i m u l a t o r y effect of t h r o m b i n on D N A s y n t h e s i s ( d a t a not shown). To test if the effects of t h r o m b i n on t h y m i d i n e u p t a k e were p a r a l l e l e d by a n increase in cell n u m b e r , s u b c o n f l u e n t
782
MARRA ET AL
HEPATOLOGY S e p t e m b e r 1995
TABLE 1. E f f e c t s o f T h r o m b i n o n F S C P r o l i f e r a t i o n Cells ( x I O -3) T i m e (hrs)
Control
Thrombin
48 72
55.7 _+ 1.6 62.0 + 1.4
63.1 _+ 2.0* 72.4 + 1.49
N O T E . S u b c o n f l u e n t F S C w e r e i n c u b a t e d in S F I F m e d i u m in t h e p r e s e n c e or a b s e n c e of 2.5 U / m L t h r o m b i n for d i f f e r e n t periods. A t t h e e n d of t h e i n c u b a t i o n , cells w e r e t r y p s i n i z e d a n d counted. D a t a a r e m e a n _+ SE (n = 6). * P < .05 vs. control. P < .005 vs. control.
FSC were incubated in SFIF medium for 48 or 72 hours in the presence or absence of thrombin (2.5 U/mL). As shown in Table 1, incubation with thrombin resulted in a significant increase in the number of FSC, compared with control cells. These data demonstrate t h a t thrombin is a mitogen for cultured FSC, because the increased DNA synthesis is associated with an actual increase in cell number. Thrombin has been shown to induce PDGF secretion in mesenchymal cells. 14 To assess if the mitogenic effect of thrombin in FSC is media t e d at least in part by endogenous PDGF, we measured thrombin-induced DNA synthesis in the presence or absence of neutralizing PDGF antibodies. Coincubation with PDGF antibodies had no effect on DNA synthesis induced by thrombin (control 128 _+ 39; thrombin 437 _+ 137; thrombin + anti-PDGF antibodies 410 _+ 153 cpm/well; n = 8). Thrombin Upregulates MCP-1 Secretion and Gene Expression. Figure 2A shows a time course for the effect of thrombin (2.5 U/mL) on secretion of MCP-1 by FSC. Increased MCP-1 secretion was evident after 12 hours' incubation with thrombin. MCP-1 levels were more t h a n twofold higher t h a n control after a 24-hour incubation. The effect of thrombin was dose dependent. As depicted in Fig. 2B, 0.1 U/mL were sufficient to induce an increase in MCP-1 accumulation. Treatment of the cells with 5 U/mL thrombin resulted in twofold to threefold increase in MCP-1 levels. Preincubation with hirudin abolished thrombin-induced MCP-1 secretion (data not shown). The effect of thrombin on MCP1 gene expression is shown in Fig. 3. Incubation with thrombin increased the steady-state levels of MCP-1 messenger RNA. The MCP-1 messenger RNA levels were increased after 2 hours and peaked at 4 hours after incubation with thrombin. Differential Effects of Retinol on DNA Synthesis and MCP-1 Secretion. Retinol and retinoic acid inhibit the proliferation of FSC in culture, and it has been suggested t h a t the vitamin A content of quiescent FSC may contribute to their low tendency to proliferateY '26 We examined the effects ofretinol on thrombin-induced mitogenesis and MCP-1 secretion. Retinol was used at 5 mmol/L, a concentration t h a t has been shown to maximally inhibit growth-factor-induced mitogenesis in rat FSC. 26 Preliminary experiments showed t h a t this dose almost completely inhibits PDGF-induced DNA syn-
thesis also in h u m a n FSC (data not shown). FSC were preincubated in SFIF medium containing 5 #mol/L retinol for 48 hours. Thrombin was then added and cells incubated for 24 hours, then pulsed with [~H]-thymidine. Retinol pretreatment inhibited thrombin-induced DNA synthesis by 88% (Fig. 4A). To confirm the inhibitory effect of retinol on thrombin-induced FSC proliferation, FSC were treated with thrombin for 72 hours in the presence or absence of 5 mmol/L retinol, and the cells were counted at the end of the incubation. Pret r e a t m e n t with retinol completely inhibited the increase in cell number induced by 3 U/mL thrombin (control 39.0 _+ 13.1; thrombin 51.0 _+ 12.8; thrombin ÷ retino141.7 + 11.7 × 103 cells/well, n = 6). In contrast to its effects on cell proliferation, pretreatment with
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HEPATOLOGY Vol. 22, No. 3, 1995
MARRA ET AL
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retinol did not inhibit MCP-1 secretion in response to thrombin (Fig. 4B). Retinol did not reduce the levels of MCP-1 also when FSC were incubated with interferon~, a more potent agonist (data not shown). Expression of the Human Thrombin Receptor in FSC. The recently cloned thrombin receptor belongs to the "seven t r a n s m e m b r a n e domain" family of G-protein-linked receptors, is We used a full-length complem e n t a r y DNA clone encoding for the h u m a n thrombin receptor to test its expression in FSC. Hybridization of poly(A)+ RNA isolated from h u m a n FSC (Fig. 5) showed the presence of two specific transcripts of approximately 3.5 and 4.0 kb. Interestingly, the 4.0 kb transcript has not been reported in other cell lines, and its significance remains to be determined. The size of the two bands is very similar, and it is possible that under different conditions the two transcripts appear as a single band. T R A P Mimics the Actions of Thrombin in FSC. The thrombin receptor is characterized by a unique mechanism of activation. Thrombin proteolytically cleaves the receptor, exposing a new amino terminus that functions as a tethered ligand that activates the receptor, is We used a thrombin receptor activating peptide (TRAP, sequence SFLLRNP) corresponding to the aminoacid sequence of the cleaved h u m a n TR, ls'27 to investigate if the effects of thrombin on FSC are mediated by a specific receptor. One hundred micromolar TRAP mimicked the thrombin-induced increase in DNA synthesis
-
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FIG. 4. Differential effects of retinol on DNA synthesis and MCPi secretion in response to thrombin. (A) FSC in 24-well plates were incubated in the presence or absence of 5 #mol/L retinol for 48 hours in SFIF medium, t h e n 2.5 U/mL of thrombin were added, and DNA synthesis was measured as described in legend to Fig. 1. Data are mean _+ SE (n = 5). *P < .05 vs. thrombin alone. (B) FSC were incubated in the presence or absence of 5 #mol/L retinol for 48 hours in SFIF medium. Medium was then replaced with fresh SFIF medium containing thrombin or retinel and cells were incubated for additional 24 hours. MCP-1 levels were measured in the conditioned medium. Data are mean ± SE (n = 6). No statistically significant differences were observed comparing thrombin and thrombin plus retinol.
784
MARRA ET AL
HEPATOLOGY September 1995
kb
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likely to occur in human liver d i s e a s e Y Expansion of FSC population may ultimately lead to increased deposition ofextracellular matrix, and therefore factors that stimulate FSC proliferation may contribute to liver fibrosis. The mechanism by which thrombin stimulates DNA synthesis is not precisely identified. Although thrombin has been shown to induce secretion of PDGF in other mesenchymal cells, ~4 the increase in DNA synthesis in FSC was not inhibited by neutralizing PDGF antibodies. This observation indicates that thrombin stimulates FSC mitogenesis independent of endogenous PDGF secretion. Among several factors, only PDGF is more potent than thrombin in stimulating DNA synthesis in human FSC. It is likely that thrombin in concert with other cytokines secreted during liver injury contributes to the increase in FSC number in vivo. 700
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FIG. 5. Expression of thrombin receptor mRNA by human FSC. Poly (A) ÷ RNA was isolated from FSC grown in complete medium. Fifteen micrograms ofpoly(A) ÷ RNA were separated on a 1% agarose/ formaldehyde gel, blotted, and hybridized with a ~P-labeled fulllength cDNA probe for the human thrombin receptor.
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B DISCUSSION
The pivotal role of thrombin in coagulation has been known for several decades. In recent years, several studies have emphasized the role of thrombin as a mediator of injury and repair in different biological systerns. 1~'~9~ However, there is very little information pertinent to the potential role of thrombin in liver injury. Tissue injury results in the formation of the prothrombin activating complex through the extrinsic pathway, most likely through damaged cell membranes. ~e This mechanism is likely to be operating also in the injured liver, because fibrin deposition has been demonstrated in areas of hepatocellular necrosis during acute hepatitis and in several models of liver injury. ~e-~ This study demonstrates that thrombin stimulates FSC proliferation and secretion of MCP-1, a potent monocyte chemoattractant. These properties of thrombin are somehow unique among agents that stimulate FSC, because PDGF, a very strong mitogen for FSC, ~° weakly stimulates MCP-1 secretion, whereas interleukin-la strongly induces MCP-19 without affecting FSC mitogenesis. Proliferation of FSC is a wellrecognized event after experimental liver injury and is
-~
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I %% % %%
% %
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% % %%
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x % ~ x , s ] 2 x % % % % , / t x / % u x x ,
% x x % % ,
o Control
Thrombin
Amastatin
TRAP
+
Amastatin FIG. 6. A thrombin receptor activating peptide (TRAP) mimics thrombin effects on DNA synthesis and MCP-1 secretion. (A) FSC were serum-starved for 48 hours before the addition of 10 /~mol/L amastatin with or without 100 #mol/L TRAP. DNA synthesis was measured as described in legend to Fig. 1. Data are mean _+ SE (n = 9). *P < .05 vs amastatin alone. (B) FSC were incubated for 24 hours in SFIF medium in the presence of 2.5 U/mL thrombin, or 10 #mol/L amastatin with or without 100 #mol/L TRAP. MCP-1 secre~ tion was measured in the conditioned medium. Data are mean _+ SE (n = 5). *P < .05 vs. control; **P < .05 vs. amastatin alone.
HEPATOLOGY Vol. 22, No. 3, 1995
MARRA ET AL
p: z
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0
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+
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-
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FIG. 7. TRAP stimulates MCP-1 gene expression in h u m a n FSC. Confluent h u m a n FSC were incubated in SFIF medium overnight before the addition of 10 pmol/L amastatin with or without 100 #mol/ L TRAP for 4 hours. Northern blot analysis ofMCP-1 gene expression was carried out as described in legend to Fig. 3.
Thrombin is chemotactic for monocytes, 37 and stimulates calcium transients and secretion of platelet activating factor by h u m a n FSC. s'3s In this study we show that thrombin is potentially able to induce monocyte recruitment and activation by stimulating MCP-1 secretion and gene expression. MCP-1 is a major monocyte chemoattractant secreted by FSC, because more than 80% of the monocyte chemotactic activity secreted is neutralized by MCP-1 antibodies. 9 Additionally, MCP-1 gene is upregulated during liver disease. 9 Activated monocytes highly express tissue factor, 39 which participates in the formation of the prothrombinase complex, thus resulting in further generation of thrombin, and therefore amplifying the inflammatory response. Acquisition of an "activated" phenotype by FSC is associated with upregulation and or de novo expression of receptors for cytokines, such as PDGF or transforming growth factor /~.4o.41 In this study, we show that the TR is expressed in cultured FSC. FSC were studied in culture after several passages, a condition that results in acquisition of an activated phenotype. It remains to be determined if this receptor is also expressed in "quiescent" FSC present in the normal liver or if it is upregulated during liver injury. Activation of the TR is mediated by the proteolytic activity of thrombin, which cleaves the receptor,
785
exposing a new amino terminus that functions as a tethered ligand that activates the receptor. Is As a consequence, oligopeptides with sequences identical to the new amino terminus of the TR mimic the actions of thrombin in the absence of proteolytic cleavage of the receptor. Is'27 However, it has been reported that some but not all effects of thrombin can be reproduced by these peptides, suggesting that alternative mechanisms or additional receptors mediate the effects of t h r o m b i n Y Thrombin and TRAP may elicit different effects on intracellular calcium concentration in osteoblastlike cells. 43 In this study, all the effects of thrombin on FSC could be reproduced by TRAP in the presence of an aminopeptidase inhibitor, which prevents the peptide from degradationY The elucidation of the mechanism(s) by which thrombin modulates the biological functions of FSC m a y have therapeutic implications. Synthetic "mutant" peptides that resist cleavage by thrombin inhibit the activation of the receptor by thrombin. 44 Such peptides m a y have a role in blocking the effects of thrombin. In the normal liver, "quiescent" FSC contain vitamin A droplets, and the phenotypic transformation toward myofibroblastlike cells that occurs during liver injury is associated with decreased retinoid content. 45'4~It has been reported that vitamin A inhibits FSC proliferation stimulated by serum, PDGF, or epidermal growth factor. 25'26 In this article we show that retinol inhibits thrombin-induced DNA synthesis but not MCP-1 secretion. These data indicate that retinol does not exert a nonspecific inhibitory effect on FSC function. Rather, retinol selectively inhibits certain FSC functions such as mitogenesis or secretion of collagen and transforming growth factor-~. 2~ The molecular mechanisms by which retinoids exert their inhibitory effects are only partially known. Retinoic acid does not seem to influence the early signaling pathways that follow PDGF receptor activation in F S C . 47 The involvement of downstream pathways is also suggested by our studies, where the effects of an agonist such as thrombin, which does not activate a receptor tyrosine kinase, can also be inhibited by retinol. Because quiescent FSC have a high retinoid content, one m a y speculate that thrombin produced at the site of liver injury is able to induce secretion of MCP-1 by quiescent FSC and initiate an inflammatory response to injury through monocyte chemotaxis. In summary, this study demonstrates that thrombin stimulates mitogenesis of FSC and secretion of MCPI through a proteolitically activated receptor. These data suggest involvement of thrombin in modulating pathologic events that follow liver injury. Thrombin may be involved in monocyte recruitment and FSC proliferation, thus contributing to tissue remodeling. However, in the context of a chronic tissue injury, thrombin, together with other cytokines, may establish a vicious circle leading to functional and structural damage. Acknowledgment: We thank Dr M. Pinzani for generously providing the fat-storing cell lines used for
786
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HEPATOLOGYSeptember 1995
these studies, and Dr S. R. Coughlin for the kind gift of the human thrombin receptor cDNA. The expert technical help of K. A. Woodruff and S. Garcia is also acknowledged. REFERENCES
1. Friedman SL, Roll FJ, Boyles J, Bissel DM. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci USA 1985;82:8681-8685. 2. Maher JJ, McGuire RF. Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo. J Clin Invest 1990;86:1641-1648. 3. Geerts A, Lazou JM, De Bleser P, Wisse E. Tissue distribution, quantitation and proliferation kinetics of fat-storing cells in carbon tetrachloride-injured rat liver. HEPATOLOGY1991;13:11931202. 4. Bissel DM. Lipocyte activation and hepatic fibrosis. Gastroenterology 1992; 102:1803-1805. 5. Friedman SL. The cellular basis of hepatic fibrosis: mechanisms and treatment strategies. N Engl J Med 1993;328:1828-1835. 6. Mancini R, Jezequel AM, Benedetti A, Paolucci F, Trozzi L, Orlandi F. Quantitative analysis of proliferating sinusoidal cells in dimethylnitrosamine-inducedcirrhosis. J Hepatol 1992; 15:361366. 7. Pinzani M, Abboud HE, Gesua]do L, Abboud SL. Regulation of macrophage colony-stimulating factor in liver fat storing cells by peptide growth factors. Am J Physiol 1992;262:C876-C881. 8. Pinzani M, Carloni V, Marra F, Riccardi D, Laffi G, Gentilini P. Biosynthesis of platelet-activating factor and its 10-acyl analogue by liver fat-storing cells. Gastroenterology 1994; 106:13011311. 9. Marra F, Valente AJ, Pinzani M, Abboud HE. Cultured human liver fat-storing cells produce monocyte chemotactic protein. 1. Regulation by proinfiammatory cytokines. J Clin Invest 1993;92:1674-1680. 10. Valente AJ, Graves DT, Vialle-Valentin CE, Delgado R, Schwartz CJ. Purification of a monocyte chemotactic factor secreted by nonhuman primate vascular cells in culture. Biochemistry 1988;27:4162-4168. 11. Graves DT, Jiang YL, Williamson MJ, Valente AJ. Identification of monocyte chemotactic activity produced by malignant cells. Science 1989;245:1490-1493. 12. Esmon CT. Cell mediated events that control blood coagulation and vascular injury. Annu Rev Cell Biol 1993;9:1-26. 13. Glenn KC, Carney DH, Fenton JW II, Cunningham DD. Thrombin active site regions required for fibroblast receptor binding and initiation of cell division. J Biol Chem 1980;255:6609-6616. 14. Shultz PJ, Knauss TC, Mene P, Abboud HE. Mitogenic signals for thrombin in mesangial cells: regulation of phospholipase C and PDGF genes. Am J Physiol 1989;257:F366-F374. 15. McNamara CA, Sarembock IJ, Gimple LW, Fenton JW II, Coughlin SR, Owens GK. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest 1993;91:94-98. 16. Carney DH, Mann R, Redin WR, Pernia SD, Berry D, Heggers JP, Hayward PG, et al. Enhancement of incisional wound healing and neovascularization in rats by thrombin and synthetic thrombin receptor activating peptides. J Clin Invest 1992;89:1469-1477. 17. Stiernberg J, Redin WR, Warner WS, Carney DH. The role of thrombin and thrombin receptor activating peptide (TRAP-508) in initiation of tissue repair. Thromb Haemost 1993;70:158-162. 18. Vu T-KH, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991;64:1057-1068. 19. Casini A, Pinzani M, Milani S, Grappone C, Galli G, Jezequel AM, Sehuppan D, et al. Regulation of extracellular matrix synthesis by transforming growth factor fl~ in human fat-storing cells. Gastroenterology 1993; 105:245-253. 20. Pinzani M, Gesnaldo L, Sabbah GM, Abboud HE. Effects of platelet-derived growth factor and other polypeptide mitogens on
21. 22. 23.
24. 25. 26.
27.
28. 29. 30. 31. 32. 33. 34.
35. 36. 37. 38.
39.
40.
41.
DNA synthesis and growth of cultured rat liver fat-storing cells. J Clin Invest 1989;84:1786-1793. Valente AJ, Rozek MM, Schwartz CJ, Graves DT. Characterization of monocyte chemotactic protein-1 binding to human monocytes. Biochem Biophys Res Commun 1991;176:309-314. Chomczynski P, Sacehi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156-159. Basset P, Bellocq JP, Wolf C, Stoll I, Hutin P, Limacher JM, Podhajcer OL, et al. A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas. Nature 1990; 348:699-704. Rydel TJ, Rabichandran KG, Tulinski A, Bode W, Huber R, Roitsc C, Fenton JW II. The structure of a complex of recombinant hirudin and human a-thrombin. Science 1990; 249:277-280. Davis BH, Kramer RT, Davidson NO. Retinoid acid modulates rat Ito cell proliferation, collagen, and transforming growth factor fl production. J Clin Invest 1990;86:2026-2070. Pinzani M, Gentilini P, Abboud HE. Phenotypical modulation of liver fat-storing cells by retinoids. Influence on unstimulated and growth factor-induced cell proliferation. J Hepatol 1992; 14:211220. Gerstzen RE, Chen J, Ishil M, Ishil K, Wang L, Nanevicz T, Turck CW, et al. Specificity of the thrombin receptor for agonist peptide is defined by its extracellular surface. Nature 1994;368:648-651. Zacharias U, He C-J, Nguyen G, Sraer JD, Rondeau E. Longterm effects ofthrombin require sustained activation of the functional thrombin receptor. FEBS Lett 1993;334:225-228. Esmon CT, Taylor FB, Snow TR. Inflammation and coagulation: linked processes potentially regulated through a common pathway mediated by protein C. Thromb Haemost 1991;66:160-165. Cunningham DD, Pulliam L, Vaughan PJ. Protease nexin-1 and thrombin: injury-related processes in the brain. Thromb Haemost 1993;70:168-171. Morris R, Winyard PG, Blake DR, Morris CJ. Thrombin in inflammation and healing: relevance to rheumatoid arthritis. Ann Rheum Dis 1994;53:72-79. Popper H, Franklin M. Viral versus toxic hepatic necrosis. Arch Pathol 1948;46:338-376. Rake MO, Flute PT, Shilkin KB, Lewis ML, Winch J, Williams R. Early and intensive therapy of intravascular coagulation in acute liver failure. Lancet 1971;ii:1215-1218. Rake MO, Flute PT, Pannell G, Shilkin KB, Williams R. Experimental hepatic necrosis: studies on coagulation abnormalities, plasma clearance, and organ distribution of 125I-labeledfibrinogen. Gut 1973;14:574-580. Hillenbrand P, Parbhoo SP, Jedrichowski A, Sherlock S. Significance of intravascular coagxtlation and fibrinolysis in acute hepatic failure. Gut 1974;15:83-88. Hirata K, Ogata I, Ohta Y, Fujiwara K. Hepatic sinusoidal cell destruction in the development of intravascular coagulation in acute liver failure of rats. J Pathol 1989; 158:157-165. Bar-Shavit R, Kahn A, Wilner GD, Fenton JW II. Monocyte chemotaxis: stimulation by specific exosite region in thrombin. Science 1983;220:728~731. Pinzani M, Failli P, Ruocco C, Casini A, Milani S, Baldi E, Giotti A, et al. Fat-storing cells as liver specific pericytes: spatial dynamics of agonist-stimulated intracellular calcium transients. J Clin Invest 1992;90:642-646. Edwards RL, Levine JB, Green R, Duffy M, Mathews E, Brande W, Rickles FR. Activation of blood coagulation in Crohn's disease: increased plasma fibrinopeptide A and enhanced generation of monocyte tissue factor activity. Gastroenterology 1987; 92:329-337. Friedman SL, Arthur MJP. Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium: direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors. J Clin Invest 1989;84:1780-1785. Friedman SL, Yamasaki G, Wong L. Modulation of transforming growth factor fl receptors of rat lipocytes during the hepatic
HEPATOLOGYVol. 22, No. 3, 1995 wound healing response: enhanced binding and reduced gene expression accompany cellular activation in culture and in vivo. J Biol Chem 1994;269:10551-10588. 42. Vouret-Craviari V, Van Obberghen-SchillingE, Rasmussen UB, Pavirani A, Lecocq J-P, Poyssegur J. Synthetic a-thrombin receptor peptides activate G protein coupled signaling pathways but are unable to induce mitogenesis. Mol Biol Cell 1992;3: 95-102. 43. Jenkins AL, Bootman MD, Berridge MJ, Stone SR. Differences in intracellular calcium signaling after activation of the thrombin receptor by thrombin and agonist peptide in osteoblast-like cells. J Biol Chem 1994;269:17104-17110. 44. Hung D, Vu T-KH, Wheaton VI, Charo IF, Nelken NA, Esmon N, Esmon CT, et al. "Mirror image" antagonists of thrombin-
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induced platelet activation based on thrombin receptor structure. J Clin Invest 1992; 89:444-450. 45. Takahara T, Kojima T, Miyabayashi C, Inoue K, Sasaki H, Muragaki Y, Ooshima A. Collagen production in fat-storing cells after carbon tetrachloride intoxication in the rat: immunoelectron microscopic observation of type I, type III collagens and prolyl hydrolase. Lab Invest 1988;59:509-521. 46. Friedman SL, Wei S, Blaner WS. Retinol release by activated rat lipocytes: regulation by Kupffer cell-conditionedmedium and PDGF. Am J Physiol 1993;264:G947-G952. 47. Davis BH, Coll D, Beno DWA. Retinoic acid suppresses the response to platelet-derived growth factor in human hepatic Itocell-like myofibroblasts: a post-receptor mechanism independent of raf/fos/jun/egr activation. Biochem J 1993;294:785-791.