Liver fibrosis: Insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors

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BASIC–LIVER, PANCREAS, AND BILIARY TRACT Liver Fibrosis: Insights Into Migration of Hepatic Stellate Cells in Response to Extracellular Matrix and Growth Factors CHANGQING YANG,* MICHAEL ZEISBERG,* BARBARA MOSTERMAN,* AKULAPALLI SUDHAKAR,* UDAYA YERRAMALLA,* KATHRYN HOLTHAUS,* LIEMING XU,‡ FRANCIS ENG,‡ NEZAM AFDHAL,* and RAGHU KALLURI* *Program in Matrix Biology, Gastroenterology and Renal Divisions, Department of Medicine and the Liver Center, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; and ‡Division of Liver Diseases, Mount Sinai School of Medicine, New York, New York

Background & Aims: In liver fibrosis, alterations within the space of Disse microenvironment occur and facilitate further progression of chronic liver disease. The normal basement membrane–like matrix present within the space of Disse converts to a matrix rich in fibrilforming collagens during fibrosis. Methods: To further understand the pathogenesis of liver fibrosis, we modified an in vitro Boyden chamber system to partially mimic in vivo conditions of hepatic stellate cells (HSCs) during health and disease. Results: Stimulation of HSCs with platelet-derived growth factor (PDGF)-BB, transforming growth factor (TGF)-␤1, and/or epithelial growth factor (EGF) resulted in an increase in their migratory capacity and up-regulated matrix metalloproteinase (MMP)-2 activity. Migration induced by PDGF-BB was associated with increased proliferation, whereas TGF␤1/EGF–induced migration was proliferation independent. COL-3, an inhibitor of MMP-2 and MMP-9, inhibited migration of HSCs induced by direct activation of PDGF-BB or TGF-␤1 but had no effect on migration induced by chemotactic stimuli without direct contact, suggesting 2 distinct MMP-dependent and MMP-independent mechanisms of PDGF-BB– or TGF-␤1–induced migration. Additionally, we show that type I collagen by itself induced migration of HSCs. Migration induced by PDGF-BB, TGF-␤1, and collagen I could be inhibited by ␣1- and/or ␣2-integrin blocking antibodies, collectively suggesting an integrin-dependent, MMP-2–mediated migration of HSCs. Conclusions: Basement membrane matrix integrity, composition, and cell-matrix interactions play an important role in anchoring HSCs and preventing them from spreading within the space of Disse and potentially elsewhere in the liver. Additionally, our data provide strong evidence for MMPs in regulation of HSCs migration.

iver cirrhosis is still the sixth most common cause of death among U.S. citizens during the most productive period of their lives (ages 25– 64 years).1 Hepatic

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failure due to cirrhosis is caused by a progressive fibrosis that ultimately results in nodular regeneration with loss of function.2– 4 Even though liver cirrhosis can be caused by metabolic, autoimmune, postnecrotic, and toxic injury, they all produce the same end effect and involve a similar final common pathogenic pathway.5,6 Hence, further understanding of liver fibrosis as the common pathway leading to liver cirrhosis might lead to new potential therapeutic targets. The cellular elements of the liver are organized within sinusoids, with the subendothelial space of Disse separating the hepatocytes from the sinusoidal endothelium.6,7 The space of Disse contains a basement membrane–like matrix consisting largely of type IV and type VI collagens, allowing maximized passage of molecules from the fenestrated sinusoidal endothelium to hepatocytes and providing structural integrity for the liver parenchyme.6,7 Early fibrotic events lead to conversion of the basement membrane–like matrix to one rich in fibrilforming matrix such as type I collagen, type III collagen, and fibronectin.2,8,9 This conversion results from both disruption of the normal matrix by enzymatic action and excess deposition of fibril-forming collagens.10,11 These changes create an environment that is not conducive to normal hepatocyte functioning, because the passage through the space of Disse is blocked by excess fibrillar collagens.12 Ultimately, liver failure caused by fibrosis Abbreviations used in this paper: bFGF, basic fibroblast growth factor; DMEM, Dulbecco’s modified Eagle medium; EGF, epidermal growth factor; FBS, fetal bovine serum; HSC, hepatic stellate cell; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor. © 2003 by the American Gastroenterological Association 0016-5085/03/$35.00 doi:10.1053/gast.2003.50012

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can be attributed to initial and subsequent alteration of the architecture of the hepatic space of Disse.7 Hepatic stellate cells (HSCs) are increasingly being recognized as the key mediators of progressive liver disease.5,13 HSCs account for about 15% of the total number of liver cells.5 In normal liver, quiescent HSCs within the space of Disse are the principal storage site for retinoids.14,15 In chronic liver disease, HSCs acquire an “activated” phenotype, which includes increased proliferation, contractility, fibrogenesis, matrix degradation, chemotaxis, and cytokine release.5,6,13,16 –18 The current paradigm for activation of HSCs during liver fibrosis postulates that activation of HSCs is initiated by stimuli such as platelet-derived growth factor (PDGF) and reactive oxygen intermediates, which are released by hepatocytes and HSCs.13,18 The activated state of HSCs is achieved through the transformed microenvironment, which is contributed to by growth factors and the fibrillar matrix.2,5,13,17–19 A major focus of this study was to investigate the migratory capacity of activated HSCs in relation to the different microenvironment within the space of Disse in normal and fibrotic liver conditions. Transforming growth factor (TGF)-␤1 is a multifunctional cytokine that plays many roles, including tissue response to injury in parenchymal organs such as the kidney and liver.20 –24 The role of TGF-␤1 in the liver is of particular interest, because this cytokine is believed to be the principal contributor to fibrosis.22,25 Nearly all cells of the liver have been shown to produce TGF-␤1, but most of the work on hepatic fibrosis has focused on the role of HSCs.20 –22,25,26 Here we show 2 distinct matrix metalloproteinase (MMP)-dependent and MMP-independent mechanisms for migration of activated HSCs. MMP-2– dependent migration by TGF-␤1 and epidermal growth factor (EGF) leads to a spread of HSCs within the space of Disse, perpetuating fibrotic disease. MMP-2–independent migration of HSCs into injured areas by chemoattractive stimulation from TGF-␤1 and type I collagen facilitates further migration of HSCs into diseased areas, facilitating progression of disease. We also show that basement membrane integrity, composition, and cellmatrix interactions play an important role in potentially anchoring HSCs and preventing them from spreading within the space of Disse and possibly elsewhere in the liver.

Materials and Methods Materials Recombinant human TGF-␤1, human EGF, human vascular endothelial growth factor (VEGF), and human basic

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fibroblast growth factor (bFGF) were purchased from R & D Systems (Minneapolis, MN). PDGF-BB was a gift from Dr. Andrius Kazlauskas (Schepens Eye Research Institute of Harvard Medical School, Boston, MA). Blocking ␣1-integrin antibody and ␣2-integrin antibody with known reactivity to human and rat were purchased from Chemicon Inc. (Temecula, CA). Mouse type I and type IV collagens were obtained from Becton Dickinson (Franklin Lakes, NJ). Soluble TGF-␤ type II receptor was a generous gift from Biogen, Inc. (Cambridge, MA) [3HThymidine] was the product of NEN Inc. (Boston, MA). Active MMP-2, active MMP-9, and MMP-2 and MMP-9 monoclonal antibodies were purchased from Oncogene Inc. (Cambridge, MA). Goat anti-mouse immunoglobulins/horseradish peroxidase was purchased from DAKO A/S (Glostrup, Denmark).

Cell Culture HSC-T6, a rat HSCs line, was established and cultured according to the recommended conditions as previously described14,27,28 and provided by Dr. Scott L. Friedman (Mount Sinai School of Medicine, New York, NY). Briefly, stellate cells were isolated from male retired Sprague–Dawley breeder rats and maintained in primary culture on plastic dishes for 15 days in the presence of 10% fetal bovine serum (FBS). On day 15 of culture, approximately 2 ⫻ 106 rat primary HSCs were transiently transfected for 24 hours with lipofectamine, containing a complementary DNA in which the expression of the large T antigen of SV40 is driven by the Rous sarcoma virus promoter. After 5–7 days, emerging clones were harvested and plated to limiting dilution, and single-cell clones were isolated and amplified in the absence of any antibiotic selection. One of more than 20 clones, designated HSC-T6, was expanded for further characterization based on preliminary cytoskeletal analysis showing a phenotype most closely resembling primary rat stellate cells. HSC-T6 retains most of the features of activated stellate cells, including expression of desmin, a smooth muscle actin, and glial acidic fibrillary protein, and it can esterify retinol into retinyl esters. The cells maintain a stable phenotype for at least 40 passages and can be cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS as previously described.14,27,28 In our experiments, rat HSCs were used from passages 6 to 16.

LX-2 Human Stellate Cell Isolation and Culture Human primary HSCs were isolated as previously described with some modifications.29,30 Briefly, liver tissue of about 15 g was harvested and immediately put into cold phosphate-buffered saline (PBS) at 4°C. For isolation of HSCs, the human liver tissue was placed in a sterile tray and warmed to 37°C by incubating for 15 minutes in warm L-15 salt solution in a 37°C water bath. The tissue was then perfused with heparin (2 IU/mL in warm L-15 salts) by repeated injections into all apparent vessel openings on the tissue surface using a 16-gauge intravenous catheter attached to a sterile 60-mL syringe. Once the tissue was cleared of residual blood and adequately warmed, the L-15 solution in the tray

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was discarded and the liver was sequentially perfused with pronase (600 mg in 200 mL Ham’s F12/DMEM) for 15 minutes and then with collagenase (55 mg in 225 mL Ham’s F12/DMEM) until the tissue appeared nearly liquefied. The softened liver section was transferred to a 10-cm culture dish containing Ham’s F12/DMEM, and the capsule was disrupted using 2 forceps. The cell suspension obtained was transferred to a 500-mL Erlenmeyer flask containing pronase (25 mg in 50 mL Ham’s F12/DMEM) and 5 mL of a deoxyribonuclease solution (2 mg in 25 mL Ham’s F12/DMEM), and the total volume was adjusted to 100 mL with Ham’s F12/DMEM and then agitated in a 37°C water bath for 30 minutes at 250 rpm. Afterward, the suspension was funnel filtered through sterile cotton gauze into two 50-mL conical polypropylene centrifuge tubes. The cells were pelleted (500g for 7 minutes) and washed 3 times with Ham’s F12/DMEM containing 1.6 ␮g/mL deoxyribonuclease at 4°C. A total of 43 mL of suspension was mixed with 17.4 mL of a 28.7% Nycodenz solution (in Hank’s balanced salt solution without sodium) and then divided (20 mL) into three 50-mL conical polypropylene centrifuge tubes. Hank’s balanced salt solution (10 mL) was gently layered on top of the Nycodenz mixed suspensions, and the cells were separated by centrifugation at 3200 rpm for 15 minutes at 4°C with the brake off. HSCs enriched at the Hank’s balanced salt solution/Nycodenz interface due to their lipid-rich buoyant density were recovered, washed once in Ham’s F12/DMEM, and plated in M199 media containing 20% FBS on uncoated plastic 60-mm dishes. HSCs were incubated in 5% CO2 at 37°C. The media was changed to M199 containing 10% FBS 24 hours later and every 3– 4 days thereafter. When the cultures reached confluence, they were trypsinized (0.05% trypsin/0.53 mmol/L ethylenediaminetetraacetic acid) and passaged at a ratio of 1:3. Subsequent passages were performed every 7–10 days. HSCs subcultured to passage 3 were trypsinized and plated at a density of approximately 40% confluence in a 10-cm dish. A single clone from the outgrowth was expanded and designated LX-2. The LX-2 cell line does not express the T antigen. In our experiments, human LX-2 HSCs were used from passages 5 to 9.

Migration Assay To mimic in vivo the microenvironment of the space of Disse, polyvinyl/pyrrolidone-free polycarbonate membranes with 8-␮m pores (Neuro Probes, Inc., Gaithersburg, MD) were coated with type IV collagen on the upper side (50 ␮g/mL) and with type I collagen on the lower side (50 ␮g/mL). The bottom wells of the chamber were filled with DMEM containing supplements according to the specific experimental protocol. Wells were covered with the coated membrane sheet, and 20,000 cells/well, which had been serum starved for 24 hours, were added into the upper chamber. The Boyden chamber was incubated for 4 hours at 37°C to allow possible migration of cells through the membrane into the lower chamber. Membranes were stained with Hema3 stain according to the manufacturer’s recommendations (Biochemical Sciences, Inc., Swedesboro, NJ). Cells that migrated

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through the membrane were counted using a counting grid, which was fitted into an eyepiece of a phase-contrast microscope. All experiments were repeated at least 3 times.

Proliferation Assay Proliferation assay was performed using [3H]-labeled thymidine as described previously.31 Subconfluent HSCs were serum starved for 24 hours, trypsinized, and resuspended with DMEM containing 0.1% FBS. A total of 3.5 ⫻ 105 cells per well were used in 24-well plates that were precoated with type IV collagen (50 ␮g/mL). Plates were incubated for 24 hours in a cell incubator. The following day, the medium was replaced with 1 mL DMEM/0.1% FBS containing PDGF-BB, TGF-␤1, EGF, VEGF, bFGF, or type I collagen according to the experimental protocol and the cells were incubated for an additional 12 hours. For these experiments, 0.1 ␮Ci of [3H]-thymidine was added into each well and the cells were incubated for an additional 24 hours. The next day, cells were lysed by incubation for 30 minutes at 60°C in 0.3 mol/L NaOH. The resulting solution was analyzed using a scintillation counter.

Zymography Cells (2 ⫻ 105 per well) were plated in 6-well plates and grown for 6 hours in 2 mL DMEM containing 10% FCS. Media were replaced with serum-free DMEM, supplemented with 3 ng/mL TGF-␤1 and/or 10 ng/mL EGF, 10 ng/mL VEGF, 5 ng/mL bFGF, 100 ␮g/mL type I collagen, and 100 ␮g/mL type IV collagen. After incubation for 48 hours, media were collected and precipitated with ammonium sulfate.32 The pellet was diluted with 0.5 mL PBS, and the solution was dialyzed overnight with PBS. Electrophoresis was performed using 15 ␮g of protein solution per lane in 10% gelatin zymogram ready-cast gels (BioRad, Hercules, CA). Active MMP-2 and MMP-9 enzymes were added respectively with 10 ng/lane. Gels were washed twice for 20 minutes at room temperature in renaturing buffer (2.5% vol/vol Triton X-100) and then incubated for 18 hours at 37°C in development buffer (40 mmol/L Tris, pH 7.5, 200 mmol/L NaCl, 5 mmol/L CaCl2 , 1 ␮mol/L ZnCl2 , 0.02% vol/vol Brij-35). Bands were visualized by staining the gel with 0.25% Coomassie blue in 30% methanol/10% glacial acetic acid for 20 minutes. Quantification of zymography gels and Western blots were performed using the NIH Image 1.62 software program.

Sodium Dodecyl Sulfate/Polyacrylamide Gel Electrophoresis and Western Blotting Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblotting were performed as described previously.31,33 Briefly, tissue culture supernatants were collected as described for zymography and 15 ␮g of protein solution per lane was used for sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The separated proteins were transferred to a nitrocellulose membrane and blocked with 5% milk solution for 30 minutes on a shaker at room temperature. After blocking, the blot paper was incubated with MMP-2 and MMP-9 antibodies in PBS containing 1% BSA. Subsequently, the blot was washed thor-

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Figure 1. Two-compartment Boyden chamber system. This in vitro system is an approximation and only partially represents in vivo conditions. The upper chamber represents the normal space of Disse that contains a basement membrane–like matrix (Matrigel), which is rich in type IV collagen and anchors resident HSCs (blue). (A and B) The lower chamber represents the diseased space of Disse, containing growth factors and fibrillar matrix, with type I collagen and fibronectin the most abundant components. (C) Without chemotactic stimuli from the lower chamber, only a few activated HSCs migrate through pores of the membrane (arrow). (D) When the medium in the lower compartment was supplemented with TGF-␤1 and EGF, activated HSCs showed an increased migratory response (arrows).

oughly with washing buffer and incubated with a secondary antibody conjugated to horseradish peroxidase for 1 hour at room temperature on a shaker. Positive reaction products were identified by enhanced chemiluminescence (Amersham, Piscataway, NJ) according to the manufacturer’s protocol.

Statistical Analysis Results are expressed as means ⫾ SD. Multiple comparisons were performed by one-way analysis of variance in SPSS 10.0 for Windows (Chicago, IL). P values ⬍0.05 were considered significant.

Results Schematic Illustration of the Experimental System To study the migratory properties of activated HSCs, we developed a novel 2-compartment Boyden chamber system that partially mimics the in vivo space of Disse in health (Figure 1A and C ) and disease (Figure 1B and D). The upper compartment mimics

the normal space of Disse microenvironment, which is mainly comprised of a basement membrane–like matrix (represented by type IV collagen or Matrigel coating of the upper side of the polycarbonate membrane). The lower compartment mimics inflamed areas of liver microenvironment, which is characterized by fibrillar matrix (represented by type I collagen or fibronectin coating of the lower side of the polycarbonate membrane). Hence, this experimental system allows the study of activated HSCs that are driven by a direct autocrine stimulation to invade adjacent areas of the space of Disse, which can potentially lead to spreading of fibrotic disease.5 The model also allows the study of chemotactic stimuli in the lower chamber, which can cause resident HSCs to migrate toward injured areas of the space of Disse, further contributing to progression of disease. Thus, in this model, the target area for migrating HSCs is the fibrillar extracellular matrix– containing mi-

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croenvironment, whereas the residential microenvironment is a basement membrane–like matrix. In this study, we only focused on 4 components in the subendothelial space of liver: the activated HSCs, basement membrane– like matrix, growth factors, and fibrillar matrix. Migratory Behavior of HSCs to Chemotactic Stimuli During liver fibrosis, changes in the space of Disse microenvironment occur.6,12 The basement membrane– like matrix is progressively replaced by fibrillar matrix and profibrogenic growth factors, which are released by hepatocytes, inflammatory cells, and activated HSCs. These cytokines/growth factors are PDGF-BB, TGF-␤1, EGF, bFGF, and VEGF.2,8,9,19 To delineate different properties of growth factors in facilitating migration of activated HSCs, we tested the migratory behavior of cells after direct stimulation in the upper chamber (mimicking HSCs direct stimulation) or in the lower chamber (mimicking chemotactic stimuli from the injured lower compartment). Chemotactic stimulation by PDGF-BB, TGF-␤1, or EGF induced HSCs to migrate into the lower chamber across a polycarbonate membrane that was precoated with type IV collagen on the upper side and type I collagen on the lower side (Figures 2A and 3D). Soluble TGF receptor and anti–TGF-␤1 neutralizing antibodies inhibited migration of rat HSCs that was induced by chemotactic stimulation with TGF-␤1 (Figure 2A). Chemotactic costimulation with a combination of TGF-␤1 and EGF had an additive effect and further enhanced migration of rat HSCs into the lower chamber (Figure 2A). Interestingly, soluble type I collagen, which is excessively deposited in the space of Disse during liver fibrosis and hence represents areas of tissue injury, was chemotactic for activated HSCs in a dose-dependent manner when added to the lower chamber (Figure 2B, black columns). Type IV collagen, the main component of the basement membrane–like matrix within the space of Disse during normal settings, was not chemotactic for activated HSCs (Figure 2B, gray columns). Similar experiments with human HSCs yielded identical results (data not shown). Migratory Behavior of Activated HSCs to Direct Stimulation With Growth Factors During liver fibrosis, activation of HSCs occurs, which is considered a major factor in the progression of chronic liver disease.5,6 Activated HSCs account for increased deposition of fibrillar extracellular matrix and enhanced turnover of basement membrane–like extracellular matrix and show increased proliferative activity.5,34,35 Cultured HSCs in vitro are generally regarded

Figure 2. Migratory response of activated rat HSCs to chemotactic stimuli. (A) Addition of TGF-␤1 (3 ng/mL) into the lower compartment of the Boyden chamber, mimicking injured areas of the space of Disse, induced migration of activated HSCs across the polycarbonate membrane, which was coated with type IV collagen on the upper side and with type I collagen on the lower side (6.4-fold increase compared with unstimulated control). Addition of TGF-␤1 neutralizing antibodies reversed the effect significantly (reduction by 64.3%). TGF-␤1–induced migration was also inhibited by the addition of soluble TGF-␤1 type II receptor. Similarly, addition of EGF (10 ng/mL) into the lower chamber increased migration of activated HSCs by 7.2-fold, whereas EGF neutralizing antibodies reversed the EGF-induced migratory response. Addition of TGF-␤1 and EGF into the lower chamber increased migration of activated HSCs by 12.7-fold. Addition of bFGF (5 ng/mL), VEGF (10 ng/mL), or bFGF/VEGF did not induce a significant migratory response of HSCs. (B) Addition of type I collagen into the lower chamber, which increases during fibrosis within the space of Disse, induced migration of activated HSCs in a dose-dependent manner (from 10 to 100 ␮g/mL) (■). Addition of type IV collagen into the lower chamber did not induce migration of activated HSCs ( ). *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001, #P ⬍ 0.05 compared with the control group.

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Figure 3. Regulation of migratory behavior of HSCs by MMP-2. (A) Addition of MMP-2, which degrades type IV collagen, into the upper chamber induced migration of HSCs. This effect was inhibited by COL-3. MMP-2 containing supernatant from HSCs that were stimulated with TGF-␤1 (3 ng/mL)/EGF (10 ng/mL) (TE-Sup) or PDGF-BB (10 ng/mL) (P-sup) were added into the upper compartment to HSCs in the Boyden chamber at various concentrations. It induced migration of HSCs across the type IV collagen/type I collagen– coated polycarbonate membrane in a dose-dependent manner. (B) Addition of 10 ␮g/mL TE-Sup led to a 2.8-fold increase in migration, and addition of 10 ␮g/mL P-sup led to a 2.3-fold increase in migration. Addition of COL-3 (10 ng/mL), an inhibitor of MMP-2 and MMP-9, significantly inhibited (51.1%) migration of HSCs that was induced by 10 ␮g/mL TE-Sup; Col-3 inhibited migration (34.7%) of HSCs that was induced by 10 ␮g/mL P-sup. *P ⬍ 0.05, **P ⬍ 0.01 compared with experiments without the Col-3 group. Col-3 has no inhibitory effect on chemotactic migration of HSCs induced by (C) TGF-␤1 (3 ng/mL) or (D) PDGF-BB (10 ng/mL). **P ⬍ 0.01, ***P ⬍ 0.001 compared with the control group.

to represent the activated phenotype due to their expression of ␣–smooth muscle actin.6,29,36 –38 Direct stimulation of cultured rat or human HSCs with PDGF-BB significantly enhanced their proliferation, whereas stimulation with TGF-␤1 (3 ng/mL), EGF (10 ng/mL), VEGF (10 ng/mL), bFGF (5 ng/mL), type I

collagen (100 ␮g/mL), and type IV collagen (100 ␮g/ mL) did not induce proliferation (Figure 4A and B, gray columns). Nonetheless, direct stimulation of rat or human HSCs with TGF-␤1, EGF, or type I collagen enhanced migration of HSCs across the polycarbonate membrane. VEGF, bFGF, and type IV collagen had an

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insignificant effect (Figure 4A and B, black columns). Together, these results suggest that an increased number of cells in the lower chamber is strictly due to migration of HSCs and not proliferation, contrary to migration induced by PDGF-BB, which depends partially on increased proliferation (Figure 4A and B). Also, these findings suggest that activated HSCs are nonresponsive to further mitotic stimulation with TGF-␤1 and/or EGF but can be stimulated to proliferate by PDGF-BB. Nevertheless, activated HSCs can be further activated by PDGF-BB, TGF-␤1, and/or EGF, resulting in an increased migratory capacity. Inhibition of HSCs Migration Through Blockade of ␣1 and ␣2 Integrins ␣1␤1 integrin is a receptor for type IV collagen, the major constituent of the subendothelial basement membrane–like extracellular matrix; however, it also binds to type I collagen,40 – 42 the major component of the fibrillar matrix.7 The ␣2␤1 integrin is predominantly a receptor for type I collagen with minor specificity to type IV collagen.42– 44 To investigate the importance of cell/extracellular matrix interaction for migration of HSCs, we incubated cells with blocking antibodies to integrins ␣1 and ␣2 in the upper chamber. Blocking of ␣1 integrin and ␣2 integrin inhibited migration of HSCs that was induced by chemotactic stimulation with type I collagen (Figure 5A). Interestingly, only ␣1-integrin blocking antibodies were sufficient to inhibit migration that was induced by TGF-␤1 or EGF, whereas blocking of ␣2 integrin had no significant effect (Figure 5A). In contrast, only ␣2-integrin blocking antibodies could inhibit migration that was induced by PDGF-BB (Figure 5A). These results collectively suggest that inhibition of aberrant interactions of HSCs with matrix via integrins can result in regulation of migration induced by collagen I, PDGF-BB, TGF-␤1, and EGF (Figure 5A). 39 – 41

Figure 4. Proliferation and migration of activated rat and human HSCs to direct stimulation with growth factors. Stimulation of rat HSCs with TGF-␤1 (3 ng/mL), EGF (10 ng/mL), TGF-␤1/EGF, VEGF (10 ng/mL), bFGF (5 ng/mL), type I collagen (100 ␮g/mL), or type IV collagen (100 ␮g/mL) did not induce proliferative activity compared with unstimulated control HSCs as determined by thymidine incorporation assay. (A) All proliferation assays were performed with cells grown in 24-well plates coated with type IV collagen ( ). In migration assay, direct stimulation of cells in the upper chamber with TGF-␤1 (4.9-fold increase compared with unstimulated control), EGF (4.5-fold increase), TGF-␤1, and EGF (7.1-fold increase) or type I collagen (2.6-fold increase) induced a migratory response. Direct stimulation with bFGF (5 ng/mL), VEGF (10 ng/mL), or type IV collagen did not induce migration. *P ⬍ 0.05, ***P ⬍ 0.001 vs. unstimulated control (■). Human LX-2 HSCs in vitro are considered to represent an activated phenotype when cultured for more than 7 days. (B) Stimulation of HSCs with TGF-␤1 (3 ng/mL), EGF (10 ng/mL), TGF-␤1/EGF, VEGF (10 ng/mL), bFGF (5 ng/mL), type I collagen (100 ␮g/mL), type IV

Š collagen (100 ␮g/mL), fibronectin (100 ␮g/mL), or Matrigel (100 ␮g/mL) did not induce proliferative activity compared with unstimulated control HSCs as determined by thymidine incorporation assay. All proliferation assays were performed with cells grown in 24-well plates coated with type IV collagen ( ). However, PDGF-BB (10 ng/ mL) induced proliferation (2.3-fold). In migration assays, direct stimulation of cells in the upper chamber with PDGF-BB (6.4-fold increase compared with unstimulated control), TGF-␤1 (4.1-fold increase), EGF (3.1-fold increase), type I collagen (2.5-fold increase), or fibronectin (2.3-fold increase) induced a migratory response. Direct stimulation with bFGF (5 ng/mL), VEGF (10 ng/mL), type IV collagen, or Matrigel did not induce migration. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 vs. unstimulated control (■).

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Regulation of HSCs Migration Is Influenced by Extracellular Matrix Integrity

Figure 5. Regulation of activated human LX-2 HSCs migration by integrins and extracellular matrix. (A) ␣1-Integrin blocking antibodies (5 ␮g/mL) inhibited chemotactic migration of activated HSCs across a polycarbonate membrane coated with type IV collagen on the upper side and type I collagen on the lower side. Migration was induced by chemotactic stimulation from the lower chamber with 3 ng/mL TGF-␤1 (48.2%), 10 ng/mL EGF (44.7%), TGF-␤1 ⫹ EGF (65.4%), or 100 ␮g/mL type I collagen (40.1%). Blocking of ␣2 integrin inhibited migration that was induced by 10 ng/mL PDGF-BB (52.1%) or type I collagen (46.2%). 䊐, Control; , anti-␣1; , anti-␣2. (B) The effect of different matrix on the migration of activated HSCs was estimated by their migratory response to chemotactic stimulation to TGF-␤1 (3 ng/mL)/EGF (10 ng/mL) in the lower chamber. Activated HSCs had the strongest migratory response across a noncoated polycarbonate membrane. Coating with type IV collagen on both sides or Matrigel on

During hepatic fibrosis, the normal subendothelial basement membrane–like matrix is replaced by fibril-forming collagens.2,8,9 We speculated that the normal space of Disse microenvironment, which consists largely of type IV collagen,6,45 might provide stability for HSCs and also “activated” HSCs. Additionally, we considered whether migration of activated HSCs is directly associated with degradation of basement membrane–like matrix, enabling the cells to spread within the space of Disse and potentially elsewhere in the liver. Coating of the polycarbonate membrane with type IV collagen on both sides or Matrigel on the upper side significantly inhibited migration of activated HSCs when a combination of TGF-␤1 and EGF were used as chemoattractant stimuli in the lower chamber (Figure 5B). Migration of activated HSCs was significantly increased when the membrane was coated with type I collagen or fibronectin compared with type IV collagen (Figure 5B). These findings suggest that type IV collagen provides a protective environment for activated HSCs, whereas type I collagen, which is up-regulated in liver fibrosis, is more permissive for migration of activated cells. Activated HSCs are known to be major sources of MMPs in vivo; therefore, we examined MMP production after direct stimulation of HSCs with PDGF-BB, TGF␤1, EGF, VEGF, and bFGF. Stimulation of activated HSCs with PDGF-BB and TGF-␤1 resulted in a robust up-regulation of MMP-2 with an insignificant increase of MMP-9. EGF alone induced an increase in MMP-9 levels (Figure 6A and C), whereas bFGF and VEGF, which do not induce rat or human HSCs migration, had no effect on MMP-2 and MMP-9 levels in these cells (Figure 6A and C). Costimulation with TGF-␤1 and EGF, which induced the strongest migratory response, further enhanced MMP-2 levels compared with stimulation with TGF-␤1 or EGF alone (Figure 6A and C). Interestingly, type I collagen and fibronectin (both fibrotic matrix molecules) also induced MMP-2 in activated HSCs (Figure 6B and D). Next, we attempted to further delineate the role of MMPs and growth factors in the migration of HSCs. Addition of active MMP-2 enzyme in the upper chamber directly enhanced rat HSCs migration across the collagenŠ the upper side significantly inhibited migration. The inhibitory effect when type I collagen or fibronectin was coated on both sides was significantly less. Coating with type IV collagen on the upper side and with type I collagen on the lower side had an intermediate inhibitory effect. *P ⬍ 0.05, **P ⬍ 0.01, #P ⬍ 0.05.

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Figure 6. Zymography and immunoblot of supernatants treated from activated human LX-2 HSCs. MMP-2 and MMP-9 in tissue culture supernatants of human LX-2 HSCs were analyzed by zymography and immunoblot. (A) Stimulation of human HSCs with PDGF-BB (10 ng/mL) and TGF-␤1 (3 ng/mL) resulted in a significant increase in MMP-2 activity (lanes 3 and 4) compared with control (lane 9). Stimulation with EGF (10 ng/mL) resulted in a moderate increase in MMP-9 (lane 5). Costimulation with TGF-␤1/EGF had an additive effect on both MMP-2 and MMP-9 up-regulation (lane 6). Stimulation with VEGF or bFGF had no significant effect on MMP-2 activity in supernatants (lanes 7 and 8). (B) Stimulation of HSCs with type I collagen (100 ␮g/mL) or fibronectin (100 ␮g/mL) resulted in an increase in MMP-2 activity (lanes 4 and 6) compared with control (lane 7). Collagen IV (100 ␮g/mL) or Matrigel (100 ␮g/mL) had no significant effect on MMP-2 activity in supernatants (lanes 3 and 5). (C) Immunoblot analysis for MMP-2 and MMP-9 confirmed an increase in MMP-2 after direct stimulation with PDGF-BB (lane 1), TGF-␤1 (lane 2), and TGF-␤1/EGF (lane 4). (D) Immunoblot analysis for MMP-2 and MMP-9 confirmed an increase in MMP-2 after direct stimulation with type I collagen (lane 2) and fibronectin (lane 4) compared with control (lane 5), whereas collagen IV or Matrigel had no significant effect on expression of MMP-2 and MMP-9 (lanes 1 and 3).

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coated membrane, whereas COL-3, an inhibitor of MMP-2 and MMP-9,46 – 48 completely reversed this effect (Figure 3A). Human HSCs also exhibited similar properties (data not shown). To test the hypothesis that TGF-␤1 and EGF stimulated HSCs migration, we collected supernatant from PDGF-BB– or TGF-␤1/EGF–treated HSCs, respectively. Supernatant from PDGF-BB– and supernatant from TGF␤1/EGF–treated HSCs were precipitated using ammonium sulfate and added in various concentrations to HSCs in the upper chamber after resolubilization in PBS at known concentrations. These MMP-containing supernatants induced migration of human HSCs into the lower chamber across the type IV collagen/type I collagen– coated polycarbonate membrane in a dose-dependent manner (Figure 3B). Addition of COL-3, at an optimized concentration of 10 ng/mL, to the supernatant in the upper chamber significantly inhibited migration of human HSCs, suggesting a direct role of MMP-2 in facilitating migration of human HSCs (Figure 3B). Experiments with rat HSCs also showed similar properties (data not shown). Addition of supernatant from PDGF-BB– or supernatant from TGF-␤1/EGF–treated HSCs to the upper chamber enhanced chemotactic migration of activated HSCs. COL-3 reversed this effect of MMPcontaining supernatant but had no effect on chemotactic stimulation (Figure 3B–D). Collectively, these results suggest 2 distinct MMP-independent and MMP-dependent mechanisms of migratory behavior of activated HSCs. These findings further suggest that integrity of the basement membrane–like microenvironment in the space of Disse is important for HSCs and could potentially contribute to preventing them from spreading within the space of Disse and possibly other hepatic compartments.

Discussion Basement membrane–like matrix in the space of Disse provides a tightly regulated microenvironment that allows optimized passage of macromolecules from the sinusoids to the hepatocytes and provides functional and structural integrity for hepatocytes.6,49 Changes in the space of Disse microenvironment, which are accounted mainly by activated HSCs, play an important role in facilitating progression of liver fibrosis.13,16 –18 Migration of resident HSCs within the space of Disse and potentially in other compartments is considered important for progression of liver fibrosis because it accounts for increased numbers of activated HSCs in areas of inflammation.18,50 In the present study, we established a 2-chamber system partially mimicking the normal space of Disse microenvironment, which consists largely of type IV collagen, and the chronically injured space of Disse,

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where type I collagen is the most abundant constituent of extracellular matrix.6,51 We show that chemotactic stimulation by PDGF-BB or a combination of TGF-␤1 and EGF induce HSCs to migrate. Also, type I collagen and fibronectin can serve as a chemoattractant stimulus for HSCs, whereas type IV collagen does not exhibit this property. These results suggest that growth factors that are potentially released in adjacent areas of injury by inflammatory cells, activated HSCs, and hepatocytes induce resident HSCs to migrate into wider areas, where they further enhance progression of disease. These results further suggest that the major structural constituent of fibrotic extracellular matrix, type I collagen, also induces HSCs to invade the fibrillar matrix, which is capable of further perpetuating HSC activation. Direct stimulation with TGF-␤1 and EGF results in an increased migratory response of “activated” HSCs, whereas no further stimulation of proliferation is observed. These results suggest that activated HSCs can be further activated to acquire an enhanced phenotype, which shows enhanced migratory capacity, even though they are not susceptible to further mitotic stimuli. These results also suggest that autocrine release of growth factors by activated HSCs can drive them into wider areas in the liver, further mediating the spread of disease. Whereas the combination of TGF-␤1 and EGF induced migration of rat and human stellate cells without any increase in proliferation, PDGF-BB induced migration associated with proliferation of rat and human HSCs. Therefore, it is very likely that the promigratory properties of TGF-␤1 and PDGF-BB are mediated by 2 distinct mechanisms, although both mechanisms involve the up-regulation of MMP-2. The diverse intracellular mechanism induced by TGF-␤1/EGF and PDGF-BB needs to be sorted out in the future. The present study provides valuable insights into the role of space of Disse microenvironment in regulating HSCs behavior. In our experiments, growth factors that induce MMP-2 and potentially facilitate degradation of the normal space of Disse microenvironment52,53 induce migration. Also, MMP-2 and MMP-9 containing supernatant from TGF-␤1– and EGF-stimulated rat or human HSCs induce migration, suggesting that migration of activated HSCs is clearly associated with degradation of the basement membrane–like matrix. Interestingly, COL-3 inhibits PDGF-BB– or TGF-␤1–induced MMP-2– and MMP-9 –mediated migration, but it has no effect on migration toward chemotactic stimulation by PDGF-BB or TGF-␤1. These findings suggest that MMP-dependent migration of HSCs, which is driven by direct autocrine stimulation, possibly plays a role in an early

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phase of fibrosis, when activated HSCs migrate into areas still containing intact basement membrane. MMP-independent migration of HSCs, which is induced by chemotactic stimulation by either type I collagen, fibronectin, PDGF-BB, or TGF-␤1, is possibly more important in later phases of fibrosis, when injured areas of disease expand and spread HSCs to further enhance injury. Our experiments strongly suggest that initial influences for migration of activated HSCs must include matrix/basement membrane degradation and/or manipulation of the space of Disse architecture. This could partially be achieved by up-regulated MMPs, as shown in the present study. Once this process is in operation, type I collagen and the potentially higher concentrations of TGF-␤1/ EGF outside the interstitium can further influence the migration of HSCs by chemotactic mechanisms depending ideally on favorable concentration gradients, which are established by type I collagen and TGF-␤1. Cell-surface integrins mediate cell behavior and signaling via interaction with basement membrane and extracellular matrix microenvironment.54 –56 ␣1␤1 and ␣2␤1 integrins have been reported to mediate adhesion, collagen-dependent proliferation, migration, and matrix remodeling in many organs.57– 60 In our experiments, we found that ␣1 integrin but not ␣2 integrin is necessary for TGF-␤1/EGF–mediated migration of HSCs and that ␣2 integrin but not ␣1 integrin is necessary for PDGFBB–mediated migration of HSCs, whereas both of these integrins mediate migration that is induced by type I collagen. These results suggest that PDGF-BB–mediated migration and proliferation is mediated by ␣2␤1 integrin and TGF-␤1/EGF–mediated migration without proliferation is mediated by ␣1␤1 integrin. These observations provide insights into the possible role of ␣2␤1 integrin in proliferation-dependent migration of HSCs and proliferation-independent migration mediated by ␣1␤1 integrin. It has been known for some time that primary HSCs can be activated in culture; just as in transformed HSCs,6,29,38 this “activation” leads to irreversible hyperproliferation of HSCs, enhances their capacity to produce an abundance of matrix molecules, and also leads to several phenotypic changes such as increased contractility, cytokine release, and expression of ␣–smooth muscle actin, as shown in other studies.16 –18,61– 63 In this study, we show that “activated” HSCs display irreversibly altered patterns of cellular migration and require persistent TGF-␤1/EGF stimulation. This newly acquired phenotype is reversible; when the stimuli are removed, they return back to their basal migratory state. We recognize that studies in this report were performed with immortalized cells, and thus this needs further validation with

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use of primary HSCs. Whereas the TGF-␤1/EGF combination results in proliferation-independent migration, PDGF-BB induces proliferation-dependent migration, both of which are reversible. Additionally, we show that this state can also be achieved by direct or indirect stimulation by collagen I. Migration of activated HSCs is mediated by MMPs and integrins ␣1␤1 and ␣2␤1 , further implicating a crucial role of matrix microenvironment in the regulation of migratory behavior HSCs.

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Received January 18, 2002. Accepted September 19, 2002. Address requests for reprints to: Raghu Kalluri, Ph.D., Harvard Medical School, Department of Medicine, Dana 514, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, Massachusetts 02215. e-mail: [email protected]; fax: (617) 975-5663. Supported by the Espinosa Fibrosis Fund and in part by research grants DK51711 and DK55001 from the National Institutes of Health. C.Y. was supported by a postdoctoral fellowship from the Espinosa Fibrosis Fund and Beth Israel Deaconess Medical Center Liver Center. The authors thank Biogen, Inc., for their generous research gift and Collagenex, Inc., for their gift of COL-3. Rat hepatic stellate cell line and human primary LX-2 stellate cells were gifts from Dr. Scott L. Friedman (Mount Sinai School of Medicine, New York, NY).