1. Volume 117, Issue 5, Pages 1205-1221 (November 1999)
Rat liver myofibroblasts and hepatic stellate cells: Different cell populations of the fibroblast lineage with fibrogenic potential 
Thomas Knittel, Dominik Kobold, Bernhard Saile, Anka Grundmann, Katrin Neubauer, Fabio Piscaglia, Giuliano Ramadori 
Gastroenterology 
Volume 117, Issue 5, Pages (November 1999)
DOI: /S (99)
Copyright © 1999 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Morphology of HSCs and rat liver myofibroblasts. Phasecontrast microscopy of HSCs in primary culture 2 days (2d) and 7 days (7d) after plating, of HSCs at passage 1 (1.P) and 2 (2.P), and of rat liver myofibroblasts (rMF). In rat liver myofibroblast cultures, either a fibroblast-like (rMF: A and rMF: C) or a myofibroblast-like (rMF: B and rMF: D) growth pattern was detectable. (Bars = 66 μm [HSC 2d], 92 μm [HSC 7d, HSC 1.P, HSC 2.P, rMF: C, rMF: D], and 147 μm [rMF: A, rMF: B].)
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Expression of intermediate filaments and myofilaments in HSCs and rat liver myofibroblasts. (A) Detection of vimentin, SMA, and desmin by indirect immunofluorescence. HSCs 7 days after plating (HSC 7d) and rat liver myofibroblasts (rMF) were incubated with antibodies directed against vimentin, SMA, and desmin or with mouse Ig as a negative control (data not shown). Immunoreactive material was detected by using single, indirect immunofluorescence. Mouse Ig gave only faint background staining, which was even lower than the negative staining reaction shown for desmin of rMF-I. In the case of rat liver myofibroblasts, 3 different staining patterns were detected, which were classified as rMF-I (vimentin++, SMA−, desmin−), rMF-II (vimentin++, SMA+, desmin−), and rMF-II (vimentin++, SMA++, desmin+) (+, positive; ++, strongly positive; −, negative). To provide evidence that the negative staining patterns of desmin and SMA in rMF-I and rMF-II were not attributable to experimental problems, individual desmin (rMF-II)- or SMA (rMF-I)-positive cells within these populations are also shown (bar = 57 μm). (B) Detection of GFAP by the APAAP staining method. HSCs 7 days after plating (HSC 7d) and rat liver myofibroblasts were incubated with antibodies directed against GFAP. Immunoreactive material was detected by using the APAAP staining method. Mouse Ig as negative controls gave only a faint background staining (data not shown), which was comparable with the negative staining reaction shown for rat liver myofibroblasts (rMF) (bar = 43 μm). (C) Expression of vimentin, desmin, GFAP, and SMA as assessed by Western blot analysis. Freshly isolated HSCs (0d), HSCs at 2 days or 3 days (2/3) and 7 days after plating (7), HSCs at passage 1 (1.P), and rat liver myofibroblasts (rMF) from passages 1–5 (1.P–5.P) were solubilized as described in Materials and Methods. Twenty-five micrograms (desmin, GFAP) or 5 μg (vimentin, SM α-actin, β-actin) of cellular proteins was analyzed by 9% SDS-PAGE followed by Western blotting using the ECL detection system. To overcome differences within the individual cell preparations and cultures, cell lysates prepared from 3 different cell preparations were pooled for Western blot analysis. Molecular sizes are indicated at left. (D) Expression of desmin, GFAP, and SMA as assessed by Northern blot analysis. Total RNA was purified from freshly isolated HSCs (0d), from HSCs at 2 or 3 days (2/3), 4 or 5 days (4/5), 7 days (7), and 9 or 10 days (9/10) after plating, from HSCs from passage 1 (1.P), and from rat liver myofibroblasts (rMF) from passages 2–4 (2.P–4.P). Five micrograms of total RNA was size-selected by 1% agarose gel electrophoresis, and filters were hybridized by using specific cDNA probes.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

4. Fig. 2
Expression of intermediate filaments and myofilaments in HSCs and rat liver myofibroblasts. (A) Detection of vimentin, SMA, and desmin by indirect immunofluorescence. HSCs 7 days after plating (HSC 7d) and rat liver myofibroblasts (rMF) were incubated with antibodies directed against vimentin, SMA, and desmin or with mouse Ig as a negative control (data not shown). Immunoreactive material was detected by using single, indirect immunofluorescence. Mouse Ig gave only faint background staining, which was even lower than the negative staining reaction shown for desmin of rMF-I. In the case of rat liver myofibroblasts, 3 different staining patterns were detected, which were classified as rMF-I (vimentin++, SMA−, desmin−), rMF-II (vimentin++, SMA+, desmin−), and rMF-II (vimentin++, SMA++, desmin+) (+, positive; ++, strongly positive; −, negative). To provide evidence that the negative staining patterns of desmin and SMA in rMF-I and rMF-II were not attributable to experimental problems, individual desmin (rMF-II)- or SMA (rMF-I)-positive cells within these populations are also shown (bar = 57 μm). (B) Detection of GFAP by the APAAP staining method. HSCs 7 days after plating (HSC 7d) and rat liver myofibroblasts were incubated with antibodies directed against GFAP. Immunoreactive material was detected by using the APAAP staining method. Mouse Ig as negative controls gave only a faint background staining (data not shown), which was comparable with the negative staining reaction shown for rat liver myofibroblasts (rMF) (bar = 43 μm). (C) Expression of vimentin, desmin, GFAP, and SMA as assessed by Western blot analysis. Freshly isolated HSCs (0d), HSCs at 2 days or 3 days (2/3) and 7 days after plating (7), HSCs at passage 1 (1.P), and rat liver myofibroblasts (rMF) from passages 1–5 (1.P–5.P) were solubilized as described in Materials and Methods. Twenty-five micrograms (desmin, GFAP) or 5 μg (vimentin, SM α-actin, β-actin) of cellular proteins was analyzed by 9% SDS-PAGE followed by Western blotting using the ECL detection system. To overcome differences within the individual cell preparations and cultures, cell lysates prepared from 3 different cell preparations were pooled for Western blot analysis. Molecular sizes are indicated at left. (D) Expression of desmin, GFAP, and SMA as assessed by Northern blot analysis. Total RNA was purified from freshly isolated HSCs (0d), from HSCs at 2 or 3 days (2/3), 4 or 5 days (4/5), 7 days (7), and 9 or 10 days (9/10) after plating, from HSCs from passage 1 (1.P), and from rat liver myofibroblasts (rMF) from passages 2–4 (2.P–4.P). Five micrograms of total RNA was size-selected by 1% agarose gel electrophoresis, and filters were hybridized by using specific cDNA probes.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

5. Fig. 2
Expression of intermediate filaments and myofilaments in HSCs and rat liver myofibroblasts. (A) Detection of vimentin, SMA, and desmin by indirect immunofluorescence. HSCs 7 days after plating (HSC 7d) and rat liver myofibroblasts (rMF) were incubated with antibodies directed against vimentin, SMA, and desmin or with mouse Ig as a negative control (data not shown). Immunoreactive material was detected by using single, indirect immunofluorescence. Mouse Ig gave only faint background staining, which was even lower than the negative staining reaction shown for desmin of rMF-I. In the case of rat liver myofibroblasts, 3 different staining patterns were detected, which were classified as rMF-I (vimentin++, SMA−, desmin−), rMF-II (vimentin++, SMA+, desmin−), and rMF-II (vimentin++, SMA++, desmin+) (+, positive; ++, strongly positive; −, negative). To provide evidence that the negative staining patterns of desmin and SMA in rMF-I and rMF-II were not attributable to experimental problems, individual desmin (rMF-II)- or SMA (rMF-I)-positive cells within these populations are also shown (bar = 57 μm). (B) Detection of GFAP by the APAAP staining method. HSCs 7 days after plating (HSC 7d) and rat liver myofibroblasts were incubated with antibodies directed against GFAP. Immunoreactive material was detected by using the APAAP staining method. Mouse Ig as negative controls gave only a faint background staining (data not shown), which was comparable with the negative staining reaction shown for rat liver myofibroblasts (rMF) (bar = 43 μm). (C) Expression of vimentin, desmin, GFAP, and SMA as assessed by Western blot analysis. Freshly isolated HSCs (0d), HSCs at 2 days or 3 days (2/3) and 7 days after plating (7), HSCs at passage 1 (1.P), and rat liver myofibroblasts (rMF) from passages 1–5 (1.P–5.P) were solubilized as described in Materials and Methods. Twenty-five micrograms (desmin, GFAP) or 5 μg (vimentin, SM α-actin, β-actin) of cellular proteins was analyzed by 9% SDS-PAGE followed by Western blotting using the ECL detection system. To overcome differences within the individual cell preparations and cultures, cell lysates prepared from 3 different cell preparations were pooled for Western blot analysis. Molecular sizes are indicated at left. (D) Expression of desmin, GFAP, and SMA as assessed by Northern blot analysis. Total RNA was purified from freshly isolated HSCs (0d), from HSCs at 2 or 3 days (2/3), 4 or 5 days (4/5), 7 days (7), and 9 or 10 days (9/10) after plating, from HSCs from passage 1 (1.P), and from rat liver myofibroblasts (rMF) from passages 2–4 (2.P–4.P). Five micrograms of total RNA was size-selected by 1% agarose gel electrophoresis, and filters were hybridized by using specific cDNA probes.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

6. Fig. 3
Expression of cell adhesion molecules in HSCs and rat liver myofibroblasts. (A) Detection of ICAM-1, VCAM-1, and NCAM by the APAAP staining method. HSCs at day 7 after plating (HSC 7d) and rat liver myofibroblasts (rMF) were incubated with antibodies directed against cell adhesion molecules or mouse Igs as negative controls (data not shown). Immunoreactive material was detected by using the APAAP staining method. Mouse Igs gave only faint background staining, which was comparable with the negative staining reaction shown for rMF-II (VCAM-1, NCAM). For rat liver myofibroblasts, 2 different staining patterns were detected, classified as rMF-I (ICAM-1+, VCAM-1+, NCAM+) and rMF-II (ICAM-1+, VCAM-1−, NCAM−). Arrow indicates a single VCAM-positive rat liver myofibroblast (bar = 43 μm). (B) Expression of ICAM-1, VCAM-1, and NCAM as assessed by Northern blot analysis. Total RNA was purified from freshly isolated HSCs (0d), from HSCs at days 2 or 3 (2/3), 4 or 5 (4/5), 7 (7), and 9 or 10 (9/10) after plating, from HSCs from passage 1 (1.P), and from rat liver myofibroblasts (rMF) from passages 2–4 (2.P–4.P). Five micrograms of total RNA was size-selected by 1% agarose gel electrophoresis, and filters were hybridized by using specific cDNA probes.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

7. Fig. 3
Expression of cell adhesion molecules in HSCs and rat liver myofibroblasts. (A) Detection of ICAM-1, VCAM-1, and NCAM by the APAAP staining method. HSCs at day 7 after plating (HSC 7d) and rat liver myofibroblasts (rMF) were incubated with antibodies directed against cell adhesion molecules or mouse Igs as negative controls (data not shown). Immunoreactive material was detected by using the APAAP staining method. Mouse Igs gave only faint background staining, which was comparable with the negative staining reaction shown for rMF-II (VCAM-1, NCAM). For rat liver myofibroblasts, 2 different staining patterns were detected, classified as rMF-I (ICAM-1+, VCAM-1+, NCAM+) and rMF-II (ICAM-1+, VCAM-1−, NCAM−). Arrow indicates a single VCAM-positive rat liver myofibroblast (bar = 43 μm). (B) Expression of ICAM-1, VCAM-1, and NCAM as assessed by Northern blot analysis. Total RNA was purified from freshly isolated HSCs (0d), from HSCs at days 2 or 3 (2/3), 4 or 5 (4/5), 7 (7), and 9 or 10 (9/10) after plating, from HSCs from passage 1 (1.P), and from rat liver myofibroblasts (rMF) from passages 2–4 (2.P–4.P). Five micrograms of total RNA was size-selected by 1% agarose gel electrophoresis, and filters were hybridized by using specific cDNA probes.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

8. Fig. 4
Expression of ECM proteins by HSCs and rat liver myofibroblasts. Total RNA was purified from freshly isolated HSCs (0d), from HSCs 2 or 3 days (2/3), 4 or 5 days (4/5), 7 days (7), and 9 or 10 days (9/10) after plating, from HSCs from passage 1 (1.P), and from rat liver myofibroblasts (rMF) from passages 2–4 (2.P–4.P). Five micrograms of total RNA was size-selected by 1% agarose gel electrophoresis, and filters were hybridized by using specific cDNA probes. A normalization of the samples using β-actin and GAPDH are provided in Figures 2D and 5, respectively.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

9. Fig. 5
Expression of protease and protease inhibitors by HSCs and rat liver myofibroblasts. Total RNA was purified from freshly isolated HSCs (0d), from HSCs 2 or 3 days (2/3), 4 or 5 days (4/5), 7 days (7), and 9 or 10 days (9/10) after plating, from HSCs from passage 1 (1.P), and from rat liver myofibroblasts (rMF) from passages 2–4 (2.P–4.P). Five micrograms of total RNA was size-selected by 1% agarose gel electrophoresis, and filters were hybridized by using specific cDNA probes.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

10. Fig. 6
Synthesis of α2-macroglobulin, fibronectin, and collagens by HSCs and rat liver myofibroblasts. (A) Immunoprecipitation: HSCs at 2 days (2d) and 7 days (7d) of primary culture, HSCs from passage 1 (1.P), and rat liver myofibroblasts (rMF) from passages 2 and 4 (2.P and 4.P) were pulse-labeled with [35S]methionine for 4 hours. Aliquots of cell layer lysates (int) and culture supernatants (ext) containing the same amount of protein-bound radioactivity (int, 2 × 106 cpm; ext, 2 × 105 cpm) were immunoprecipitated with antibodies directed against α2-macroglobulin (α2-M), fibronectin (Fn), and collagen type IV. Samples were subjected to 5% SDS-PAGE followed by autoradiography. Molecular size is indicated at left. (B) Western blot analysis: HSCs at 2 or 3 days (2/3) and 7 days (7) after plating and rat liver myofibroblasts (rMF) from passage 2 or 4 (2.P, 4.P) were solubilized as described in Materials and Methods. Twenty micrograms of cellular proteins was analyzed by 5% SDS-PAGE followed by Western blotting by using the ECL detection system. Samples used in this analysis were identical with those used in Figures 2C and 9B. Molecular sizes are indicated at left.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

11. Fig. 7
Expression of cytokines and cell membrane receptors by HSCs and rat liver myofibroblasts. Total RNA was purified from freshly isolated HSCs (0d), from HSCs at 2 or 3 days (2/3), 4 or 5 days (4/5), 7 days (7), and 9 or 10 days (9/10) after plating, from HSCs from passage 1 (1.P), and from rat liver myofibroblasts (rMF) from passages 2–4 (2.P–4.P). Five micrograms of total RNA was size-selected by 1% agarose gel electrophoresis, and filters were hybridized by using specific cDNA probes.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

12. Fig. 8
Detection of IL-6 mRNA–expressing cells in myofibroblast cultures. Myofibroblasts from passage 4 were hybridized with 35S-labeled IL-6–specific antisense RNA probes and, to detect unspecific signals, with sense RNA probes. Arrows indicate a few IL-6–negative rat liver myofibroblasts, whereas most of the rat liver myofibroblasts are IL-6 positive (bar = 43 μm).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

13. Fig. 8
Detection of IL-6 mRNA–expressing cells in myofibroblast cultures. Myofibroblasts from passage 4 were hybridized with 35S-labeled IL-6–specific antisense RNA probes and, to detect unspecific signals, with sense RNA probes. Arrows indicate a few IL-6–negative rat liver myofibroblasts, whereas most of the rat liver myofibroblasts are IL-6 positive (bar = 43 μm).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

14. Fig. 9
Expression of fibulin 2 by rat liver myofibroblasts and HSCs. (A) Expression of fibulin 2 as assessed by Northern blot analysis. Total RNA was purified from freshly isolated HSCs (0d), HSCs at 2 days or 3 days (2/3), 4 days or 5 days (4/5), or 7 days (7) after plating, and from HSCs from passage 1 (1.P) and rat liver myofibroblasts (rMF) from passages 1–6 (1.P–6.P). Five micrograms of total RNA was size-selected by 1% agarose gel electrophoresis, and filters were hybridized by using specific cDNA probes. (B) Expression of vimentin, desmin, GFAP, and SMA as assessed by Western blot analysis. HSCs at 2 or 3 days (2/3) and 7 days after plating (7) and rat liver myofibroblasts (rMF) from passages 2–4 (2.P–4.P) were solubilized as described in Materials and Methods. Twenty-five micrograms (fibulin 2) or 5 μg (β-actin) of cellular protein was analyzed by SDS-PAGE followed by Western blotting using the ECL detection system. To overcome differences within the individual cell preparations and cultures, cell lysates prepared from 3 different cell preparations were pooled for Western blot analysis. Molecular sizes are indicated at left. (C) Detection of fibulin 2, SMA, and desmin by indirect immunofluorescence. HSCs at 7 days after plating (HSC 7d) and rat liver myofibroblasts (rMF) from passage 2 were incubated with antibodies directed against fibulin 2, SMA, and desmin, with mouse or rabbit Ig as negative controls. Immunoreactive material was detected by using indirect immunofluorescence. Panels 3–8 show double immunofluorescence against fibulin 2 and SMA (panels 3, 4, and 7) or against fibulin 2 and desmin (panels 5, 6, and 8). Mouse or rabbit Ig gave only faint background staining (data not shown), which was even lower than the negative staining reaction shown for fibulin 2 of HSCs 7d (panel 1). White arrows indicate fibulin 2–positive cells in HSC cultures present as cell clusters (panel 3) or single positive cells (panels 5 and 6); open arrows indicate the corresponding desmin- or SMA-positive cells (bar = 114 μm [panels 1–4] and 57 μm [panels 5–8]).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions