Volume 124, Issue 1, Pages 147-159 (January 2003) 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, Raghu Kalluri  Gastroenterology  Volume 124, Issue 1, Pages 147-159 (January 2003) DOI: 10.1053/gast.2003.50012 Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 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-β1 and EGF, activated HSCs showed an increased migratory response (arrows). Gastroenterology 2003 124, 147-159DOI: (10.1053/gast.2003.50012) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 2 Migratory response of activated rat HSCs to chemotactic stimuli. (A) Addition of TGF-β1 (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-β1 neutralizing antibodies reversed the effect significantly (reduction by 64.3%). TGF-β1–induced migration was also inhibited by the addition of soluble TGF-β1 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-β1 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 μg/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. Gastroenterology 2003 124, 147-159DOI: (10.1053/gast.2003.50012) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 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-β1 (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 μg/mL TE-Sup led to a 2.8-fold increase in migration, and addition of 10 μg/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 μg/mL TE-Sup; Col-3 inhibited migration (34.7%) of HSCs that was induced by 10 μg/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-β1 (3 ng/mL) or (D) PDGF-BB (10 ng/mL). **P < 0.01, ***P < 0.001 compared with the control group. Gastroenterology 2003 124, 147-159DOI: (10.1053/gast.2003.50012) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 4 Proliferation and migration of activated rat and human HSCs to direct stimulation with growth factors. Stimulation of rat HSCs with TGF-β1 (3 ng/mL), EGF (10 ng/mL), TGF-β1/EGF, VEGF (10 ng/mL), bFGF (5 ng/mL), type I collagen (100 μg/mL), or type IV collagen (100 μg/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-β1 (4.9-fold increase compared with unstimulated control), EGF (4.5-fold increase), TGF-β1, 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-β1 (3 ng/mL), EGF (10 ng/mL), TGF-β1/EGF, VEGF (10 ng/mL), bFGF (5 ng/mL), type I collagen (100 μg/mL), type IV collagen (100 μg/mL), fibronectin (100 μg/mL), or Matrigel (100 μg/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-β1 (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 (■). Gastroenterology 2003 124, 147-159DOI: (10.1053/gast.2003.50012) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 5 Regulation of activated human LX-2 HSCs migration by integrins and extracellular matrix. (A) α1-Integrin blocking antibodies (5 μg/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-β1 (48.2%), 10 ng/mL EGF (44.7%), TGF-β1 + EGF (65.4%), or 100 μg/mL type I collagen (40.1%). Blocking of α2 integrin inhibited migration that was induced by 10 ng/mL PDGF-BB (52.1%) or type I collagen (46.2%). □, Control; ●, anti-α1; ▨, anti-α2. (B) The effect of different matrix on the migration of activated HSCs was estimated by their migratory response to chemotactic stimulation to TGF-β1 (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 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. Gastroenterology 2003 124, 147-159DOI: (10.1053/gast.2003.50012) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 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-β1 (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-β1/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 μg/mL) or fibronectin (100 μg/mL) resulted in an increase in MMP-2 activity (lanes 4 and 6) compared with control (lane 7). Collagen IV (100 μg/mL) or Matrigel (100 μg/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-β1 (lane 2), and TGF-β1/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). Gastroenterology 2003 124, 147-159DOI: (10.1053/gast.2003.50012) Copyright © 2003 American Gastroenterological Association Terms and Conditions