1. Volume 125, Issue 1, Pages 178-191 (July 2003)
Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats 
Steven Dooley, Jafar Hamzavi, Katja Breitkopf, Eliza Wiercinska, Harun M Said, Johann Lorenzen, Peter Ten Dijke, Axel M Gressner 
Gastroenterology 
Volume 125, Issue 1, Pages (July 2003)
DOI: /S (03)

2. Figure 1
(A ) Time schedule of BDL, virus administration, and sample withdrawal. Virus was given immediately before BDL and then again after 1 and 2 weeks to examine the effect of Smad7 on fibrogenesis. Alternatively, AdSmad7 was delivered after 1 week for the first time to investigate its impact on established fibrosis (B, C, ‡). Blood and liver samples were taken at the indicated time points. (B) Smad7-dependent inhibition of ColIA2 expression in BDL-treated rat liver. Animals were treated with BDL or not and infected with virus as indicated. RT-PCR using the Light Cycler (Roche) as described in Materials and Methods was performed. As standard reaction, cDNA corresponding to 100 ng, 50 ng, or 25 ng total RNA of 1 sample was examined. At least 8 different rats were examined for each treatment, and 2 independent RT-PCR experiments were included. ∗Indicates P < 0.01 versus BDL control or Mock-infected animals. (C ) Smad7 reduces hydroxyproline content in experimental fibrosis. Rats were treated with BDL for 1 to 3 weeks and infected as indicated. Hydroxyproline content was measured spectrophotometrically as described31,32 and is shown as mean ± SD (n = 8). Three samples were analyzed from each rat liver. ∗Indicates P < 0.01 versus BDL control or Mock-infected animals.
Gastroenterology  , DOI: ( /S (03) )

3. Figure 2
Expression of Smad7 transgene in rat. (A ) Smad7 mRNA and protein expression was investigated in different rat tissues 7 and 14 days subsequent to infection with 1 × 109 pfu adenoviral Smad7 expression construct (injection via the tail vein) or in tissue from untreated controls (C, C1, C2). Ribosomal RNA signals were used as a loading reference. Smad7 protein expression was analyzed by anti-Flag antibody. HSCs, which were transfected with a Smad7 expression construct, were used as a positive control in both experiments. (B) Immunohistochemical detection of transgene expression in liver sections from AdSmad7- and AdLacZ-infected rats. Livers were prepared 7 days after infection, and antibodies detecting Smad7 and β-gal expression were used. Controls show sections that were treated with secondary antibody only. (C ) Lysates of purified HSC from 4 different AdSmad7 infected rats were investigated for Smad7 expression in Western blots (HSC∗) with anti-Flag antibody. HSC, isolated from 2 untreated rats (HSC) and those infected with AdSmad7 in culture (HSC∗∗), were used as controls.
Gastroenterology  , DOI: ( /S (03) )

4. Figure 2
Expression of Smad7 transgene in rat. (A ) Smad7 mRNA and protein expression was investigated in different rat tissues 7 and 14 days subsequent to infection with 1 × 109 pfu adenoviral Smad7 expression construct (injection via the tail vein) or in tissue from untreated controls (C, C1, C2). Ribosomal RNA signals were used as a loading reference. Smad7 protein expression was analyzed by anti-Flag antibody. HSCs, which were transfected with a Smad7 expression construct, were used as a positive control in both experiments. (B) Immunohistochemical detection of transgene expression in liver sections from AdSmad7- and AdLacZ-infected rats. Livers were prepared 7 days after infection, and antibodies detecting Smad7 and β-gal expression were used. Controls show sections that were treated with secondary antibody only. (C ) Lysates of purified HSC from 4 different AdSmad7 infected rats were investigated for Smad7 expression in Western blots (HSC∗) with anti-Flag antibody. HSC, isolated from 2 untreated rats (HSC) and those infected with AdSmad7 in culture (HSC∗∗), were used as controls.
Gastroenterology  , DOI: ( /S (03) )

5. Figure 3
(A ) Immunohistological analysis of liver from rats treated with BDL and adenoviruses as indicated. Liver sections were immunostained with an antibody against α-SMA. Representative microphotographs are shown. AdLacZ-infected BDL rats displayed a similar intense staining as BDL controls, whereas AdSmad7-infected animals exhibited a significantly reduced α-SMA staining. (B) Equal amounts of protein lysate were examined for α-SMA expression in Western blots. Data from immunohistochemistry were confirmed, showing Smad7-dependent abrogation of α-SMA expression. A representative blot containing different samples as indicated is presented. Lysate from transdifferentiated HSCs (myofibroblasts, MFB) was used as a positive control. (C ) Smad7-expressing HSCs isolated from AdSmad7-infected rat remain in a quiescent stage and are α-SMA negative. Seven days after seeding, HSCs were stained for Smad7 (green) and α-SMA (red). Nuclei were stained with 4,6,-diamidino-2-phenylindole (blue). Ectopically expressed Smad7 is mainly located within the nuclei.
Gastroenterology  , DOI: ( /S (03) )

6. Figure 3
(A ) Immunohistological analysis of liver from rats treated with BDL and adenoviruses as indicated. Liver sections were immunostained with an antibody against α-SMA. Representative microphotographs are shown. AdLacZ-infected BDL rats displayed a similar intense staining as BDL controls, whereas AdSmad7-infected animals exhibited a significantly reduced α-SMA staining. (B) Equal amounts of protein lysate were examined for α-SMA expression in Western blots. Data from immunohistochemistry were confirmed, showing Smad7-dependent abrogation of α-SMA expression. A representative blot containing different samples as indicated is presented. Lysate from transdifferentiated HSCs (myofibroblasts, MFB) was used as a positive control. (C ) Smad7-expressing HSCs isolated from AdSmad7-infected rat remain in a quiescent stage and are α-SMA negative. Seven days after seeding, HSCs were stained for Smad7 (green) and α-SMA (red). Nuclei were stained with 4,6,-diamidino-2-phenylindole (blue). Ectopically expressed Smad7 is mainly located within the nuclei.
Gastroenterology  , DOI: ( /S (03) )

7. Figure 3
(A ) Immunohistological analysis of liver from rats treated with BDL and adenoviruses as indicated. Liver sections were immunostained with an antibody against α-SMA. Representative microphotographs are shown. AdLacZ-infected BDL rats displayed a similar intense staining as BDL controls, whereas AdSmad7-infected animals exhibited a significantly reduced α-SMA staining. (B) Equal amounts of protein lysate were examined for α-SMA expression in Western blots. Data from immunohistochemistry were confirmed, showing Smad7-dependent abrogation of α-SMA expression. A representative blot containing different samples as indicated is presented. Lysate from transdifferentiated HSCs (myofibroblasts, MFB) was used as a positive control. (C ) Smad7-expressing HSCs isolated from AdSmad7-infected rat remain in a quiescent stage and are α-SMA negative. Seven days after seeding, HSCs were stained for Smad7 (green) and α-SMA (red). Nuclei were stained with 4,6,-diamidino-2-phenylindole (blue). Ectopically expressed Smad7 is mainly located within the nuclei.
Gastroenterology  , DOI: ( /S (03) )

8. Figure 4
Smad7 abrogates fibrogenesis-dependent collagen expression. One or 3 week(s) after BDL and gene transfer, livers were resected and examined with sirius red staining. A representative histology of the different treatments is shown in part (A ) of the figure (100× magnification). The histology of untreated BDL rats was virtually the same as that of AdLacZ infected BDL rats, whereas AdSmad7-infected animals exhibited a significantly reduced staining. ‡Animals that received adenovirus 1 week after BDL. (B) Morphometric quantitation of sirius red staining as a measure of collagen deposition and fibrosis. Ten fields were selected randomly from every section of at least 8 rats from each group; OPENLAB improvision software (U.K.) was used. The graph shows the remaining percentage of collagen staining in AdSmad7-treated animals related to the corresponding AdLacZ-infected rats.
Gastroenterology  , DOI: ( /S (03) )

9. Figure 4
Smad7 abrogates fibrogenesis-dependent collagen expression. One or 3 week(s) after BDL and gene transfer, livers were resected and examined with sirius red staining. A representative histology of the different treatments is shown in part (A ) of the figure (100× magnification). The histology of untreated BDL rats was virtually the same as that of AdLacZ infected BDL rats, whereas AdSmad7-infected animals exhibited a significantly reduced staining. ‡Animals that received adenovirus 1 week after BDL. (B) Morphometric quantitation of sirius red staining as a measure of collagen deposition and fibrosis. Ten fields were selected randomly from every section of at least 8 rats from each group; OPENLAB improvision software (U.K.) was used. The graph shows the remaining percentage of collagen staining in AdSmad7-treated animals related to the corresponding AdLacZ-infected rats.
Gastroenterology  , DOI: ( /S (03) )

10. Figure 5
Smad7-dependent regulation of collagen I expression in primary-cultured HSCs. (A ) Representative Northern blot of ColIA1 mRNA expression in HSC isolated from normal rats. Cells were infected with 50 MOI adenovirus at day 3 of culture and harvested at day 6/7, as indicated. The expected 2 ColIA1 specific transcripts of 5.8 and 4.8 kb were detected. Northern blots were performed with 5 μg total RNA. Ethidiumbromide stained 28S and 18S band intensities were used as a loading reference. (B) Light Cycler RT-PCR was performed as described in Materials and Methods. HSCs were infected or not at day 2 with 50 MOI AdSmad7, AdCA-TβRI, or AdLacZ, and total RNA was purified after 5, 7, and 10 days. As standard reaction, cDNA corresponding to 100 ng, 50 ng, or 25 ng total RNA of 1 HSC sample was examined. One representative of 3 independent experiments (mean values ± SD of n = 4) is shown.
Gastroenterology  , DOI: ( /S (03) )

11. Figure 6
Smad7 inhibits transdifferentiation of primary-cultured HSCs. Nomarski differential interference contrast micrograph of rat liver HSC and myofibroblast-like cells during culture on plastic dishes. Two days after seeding, cells were infected with adenoviruses as indicated (infection rate >90 %). Transdifferentiation was monitored by storage of lipid droplets and the shape of the cells.
Gastroenterology  , DOI: ( /S (03) )

12. Figure 7
Smad7 prevents TGF-β signaling in HSCs. (A ) HSCs were infected with AdLacz, AdCA-TβRI, and AdSmad7 as indicated (+), and protein lysates were used for Western blot analysis. Ectopic protein expression was detected with an anti-Flag antibody (Smad7) or with an antibody against TβRI (V22, Santa Cruz). To prove the biological effects of ectopically expressed proteins, specific antisera against phosphorylated Smads 2 and 3 (PS2, PS124) were used. Parallel blots were probed with a polyclonal Smad2/3 specific antibody to confirm equal protein expression. (B and C ) Smad7 blocks nuclear translocation of Smads. (B) HSCs were subjected to adenoviral infections as indicated. Ectopically expressed Smad3/4 and phosphorylated endogenous Smad2 were detected with specific antisera as indicated. TGF-β1 induced nuclear translocation of activated Smad complexes. Nuclear staining was abolished by overexpression of Smad7. (C ) Western blot analysis, confirming the findings of part B. HSCs were infected with adenoviruses encoding Smads 2, 3, and 4. Where indicated, Smad7 was coexpressed. Cells were treated or not with TGF-β1 as described in Materials and Methods; 25 μg nuclear extracts were analyzed in Western blots using specific antisera as indicated. As a control, total lysate from AdSmad2/3/4-infected HSCs was used. The same blots were probed with anti- proliferating cell nuclear antigen to confirm equal loading. (D) Western blot analysis, showing that Smad7 has no impact on Smad2/3/4 expression; 20 μg (Smad2, 4) and 30 μg (Smad3) total lysates were used for PAGE, immunoblotted, and probed with Smad2,3,4-specific antisera as indicated in Materials and Methods. Hsp70 expression was analyzed as a loading control and lysates from Smad2,3,4 and 7 overexpressing cells were used as position control (C+). (E ) Analysis of the Smad7 effect on autocrine TGF-β signal transduction in HSCs. Primary-cultured HSCs were infected with 100 MOI of Ad(CAGA)9-MLP-Luc and various adenoviral expression constructs as indicated at day 2 after seeding (infection rate ≥90%). Lysates were prepared 24 hours later. Luciferase light counts per second (LCPS) are shown. Similar luciferase activity was obtained, when the cells were treated with 5 ng/mL TGF-β1 instead of AdCA-T βRI. The presented data are representative for at least three independent experiments (n = 4, ± SD).
Gastroenterology  , DOI: ( /S (03) )

13. Figure 7
Smad7 prevents TGF-β signaling in HSCs. (A ) HSCs were infected with AdLacz, AdCA-TβRI, and AdSmad7 as indicated (+), and protein lysates were used for Western blot analysis. Ectopic protein expression was detected with an anti-Flag antibody (Smad7) or with an antibody against TβRI (V22, Santa Cruz). To prove the biological effects of ectopically expressed proteins, specific antisera against phosphorylated Smads 2 and 3 (PS2, PS124) were used. Parallel blots were probed with a polyclonal Smad2/3 specific antibody to confirm equal protein expression. (B and C ) Smad7 blocks nuclear translocation of Smads. (B) HSCs were subjected to adenoviral infections as indicated. Ectopically expressed Smad3/4 and phosphorylated endogenous Smad2 were detected with specific antisera as indicated. TGF-β1 induced nuclear translocation of activated Smad complexes. Nuclear staining was abolished by overexpression of Smad7. (C ) Western blot analysis, confirming the findings of part B. HSCs were infected with adenoviruses encoding Smads 2, 3, and 4. Where indicated, Smad7 was coexpressed. Cells were treated or not with TGF-β1 as described in Materials and Methods; 25 μg nuclear extracts were analyzed in Western blots using specific antisera as indicated. As a control, total lysate from AdSmad2/3/4-infected HSCs was used. The same blots were probed with anti- proliferating cell nuclear antigen to confirm equal loading. (D) Western blot analysis, showing that Smad7 has no impact on Smad2/3/4 expression; 20 μg (Smad2, 4) and 30 μg (Smad3) total lysates were used for PAGE, immunoblotted, and probed with Smad2,3,4-specific antisera as indicated in Materials and Methods. Hsp70 expression was analyzed as a loading control and lysates from Smad2,3,4 and 7 overexpressing cells were used as position control (C+). (E ) Analysis of the Smad7 effect on autocrine TGF-β signal transduction in HSCs. Primary-cultured HSCs were infected with 100 MOI of Ad(CAGA)9-MLP-Luc and various adenoviral expression constructs as indicated at day 2 after seeding (infection rate ≥90%). Lysates were prepared 24 hours later. Luciferase light counts per second (LCPS) are shown. Similar luciferase activity was obtained, when the cells were treated with 5 ng/mL TGF-β1 instead of AdCA-T βRI. The presented data are representative for at least three independent experiments (n = 4, ± SD).
Gastroenterology  , DOI: ( /S (03) )

14. Figure 7
Smad7 prevents TGF-β signaling in HSCs. (A ) HSCs were infected with AdLacz, AdCA-TβRI, and AdSmad7 as indicated (+), and protein lysates were used for Western blot analysis. Ectopic protein expression was detected with an anti-Flag antibody (Smad7) or with an antibody against TβRI (V22, Santa Cruz). To prove the biological effects of ectopically expressed proteins, specific antisera against phosphorylated Smads 2 and 3 (PS2, PS124) were used. Parallel blots were probed with a polyclonal Smad2/3 specific antibody to confirm equal protein expression. (B and C ) Smad7 blocks nuclear translocation of Smads. (B) HSCs were subjected to adenoviral infections as indicated. Ectopically expressed Smad3/4 and phosphorylated endogenous Smad2 were detected with specific antisera as indicated. TGF-β1 induced nuclear translocation of activated Smad complexes. Nuclear staining was abolished by overexpression of Smad7. (C ) Western blot analysis, confirming the findings of part B. HSCs were infected with adenoviruses encoding Smads 2, 3, and 4. Where indicated, Smad7 was coexpressed. Cells were treated or not with TGF-β1 as described in Materials and Methods; 25 μg nuclear extracts were analyzed in Western blots using specific antisera as indicated. As a control, total lysate from AdSmad2/3/4-infected HSCs was used. The same blots were probed with anti- proliferating cell nuclear antigen to confirm equal loading. (D) Western blot analysis, showing that Smad7 has no impact on Smad2/3/4 expression; 20 μg (Smad2, 4) and 30 μg (Smad3) total lysates were used for PAGE, immunoblotted, and probed with Smad2,3,4-specific antisera as indicated in Materials and Methods. Hsp70 expression was analyzed as a loading control and lysates from Smad2,3,4 and 7 overexpressing cells were used as position control (C+). (E ) Analysis of the Smad7 effect on autocrine TGF-β signal transduction in HSCs. Primary-cultured HSCs were infected with 100 MOI of Ad(CAGA)9-MLP-Luc and various adenoviral expression constructs as indicated at day 2 after seeding (infection rate ≥90%). Lysates were prepared 24 hours later. Luciferase light counts per second (LCPS) are shown. Similar luciferase activity was obtained, when the cells were treated with 5 ng/mL TGF-β1 instead of AdCA-T βRI. The presented data are representative for at least three independent experiments (n = 4, ± SD).
Gastroenterology  , DOI: ( /S (03) )

15. Figure 7
Smad7 prevents TGF-β signaling in HSCs. (A ) HSCs were infected with AdLacz, AdCA-TβRI, and AdSmad7 as indicated (+), and protein lysates were used for Western blot analysis. Ectopic protein expression was detected with an anti-Flag antibody (Smad7) or with an antibody against TβRI (V22, Santa Cruz). To prove the biological effects of ectopically expressed proteins, specific antisera against phosphorylated Smads 2 and 3 (PS2, PS124) were used. Parallel blots were probed with a polyclonal Smad2/3 specific antibody to confirm equal protein expression. (B and C ) Smad7 blocks nuclear translocation of Smads. (B) HSCs were subjected to adenoviral infections as indicated. Ectopically expressed Smad3/4 and phosphorylated endogenous Smad2 were detected with specific antisera as indicated. TGF-β1 induced nuclear translocation of activated Smad complexes. Nuclear staining was abolished by overexpression of Smad7. (C ) Western blot analysis, confirming the findings of part B. HSCs were infected with adenoviruses encoding Smads 2, 3, and 4. Where indicated, Smad7 was coexpressed. Cells were treated or not with TGF-β1 as described in Materials and Methods; 25 μg nuclear extracts were analyzed in Western blots using specific antisera as indicated. As a control, total lysate from AdSmad2/3/4-infected HSCs was used. The same blots were probed with anti- proliferating cell nuclear antigen to confirm equal loading. (D) Western blot analysis, showing that Smad7 has no impact on Smad2/3/4 expression; 20 μg (Smad2, 4) and 30 μg (Smad3) total lysates were used for PAGE, immunoblotted, and probed with Smad2,3,4-specific antisera as indicated in Materials and Methods. Hsp70 expression was analyzed as a loading control and lysates from Smad2,3,4 and 7 overexpressing cells were used as position control (C+). (E ) Analysis of the Smad7 effect on autocrine TGF-β signal transduction in HSCs. Primary-cultured HSCs were infected with 100 MOI of Ad(CAGA)9-MLP-Luc and various adenoviral expression constructs as indicated at day 2 after seeding (infection rate ≥90%). Lysates were prepared 24 hours later. Luciferase light counts per second (LCPS) are shown. Similar luciferase activity was obtained, when the cells were treated with 5 ng/mL TGF-β1 instead of AdCA-T βRI. The presented data are representative for at least three independent experiments (n = 4, ± SD).
Gastroenterology  , DOI: ( /S (03) )

16. Figure 7
Smad7 prevents TGF-β signaling in HSCs. (A ) HSCs were infected with AdLacz, AdCA-TβRI, and AdSmad7 as indicated (+), and protein lysates were used for Western blot analysis. Ectopic protein expression was detected with an anti-Flag antibody (Smad7) or with an antibody against TβRI (V22, Santa Cruz). To prove the biological effects of ectopically expressed proteins, specific antisera against phosphorylated Smads 2 and 3 (PS2, PS124) were used. Parallel blots were probed with a polyclonal Smad2/3 specific antibody to confirm equal protein expression. (B and C ) Smad7 blocks nuclear translocation of Smads. (B) HSCs were subjected to adenoviral infections as indicated. Ectopically expressed Smad3/4 and phosphorylated endogenous Smad2 were detected with specific antisera as indicated. TGF-β1 induced nuclear translocation of activated Smad complexes. Nuclear staining was abolished by overexpression of Smad7. (C ) Western blot analysis, confirming the findings of part B. HSCs were infected with adenoviruses encoding Smads 2, 3, and 4. Where indicated, Smad7 was coexpressed. Cells were treated or not with TGF-β1 as described in Materials and Methods; 25 μg nuclear extracts were analyzed in Western blots using specific antisera as indicated. As a control, total lysate from AdSmad2/3/4-infected HSCs was used. The same blots were probed with anti- proliferating cell nuclear antigen to confirm equal loading. (D) Western blot analysis, showing that Smad7 has no impact on Smad2/3/4 expression; 20 μg (Smad2, 4) and 30 μg (Smad3) total lysates were used for PAGE, immunoblotted, and probed with Smad2,3,4-specific antisera as indicated in Materials and Methods. Hsp70 expression was analyzed as a loading control and lysates from Smad2,3,4 and 7 overexpressing cells were used as position control (C+). (E ) Analysis of the Smad7 effect on autocrine TGF-β signal transduction in HSCs. Primary-cultured HSCs were infected with 100 MOI of Ad(CAGA)9-MLP-Luc and various adenoviral expression constructs as indicated at day 2 after seeding (infection rate ≥90%). Lysates were prepared 24 hours later. Luciferase light counts per second (LCPS) are shown. Similar luciferase activity was obtained, when the cells were treated with 5 ng/mL TGF-β1 instead of AdCA-T βRI. The presented data are representative for at least three independent experiments (n = 4, ± SD).
Gastroenterology  , DOI: ( /S (03) )

17. Figure 8
Smad7 abrogates TGF-β-dependent proliferation inhibition in primary-cultured rat HSCs. Three-day-old HSCs were infected or not with AdLacZ and AdSmad7 and treated with different concentrations of TGF-β1 as indicated. 3H thymidine incorporation in newly synthesized DNA was measured during a labeling period of 24 hours as described in the Materials and Methods section.
Gastroenterology  , DOI: ( /S (03) )

18. Figure 9
(A ) α-SMA expression in activated HSC is not influenced by Smad7. α-SMA expression was analyzed in Western blots using 25 μg total lysate from primary-cultured HSCs, infected or not with AdLacZ or AdSmad7 at day 2 and cultured for different times as indicated. A representative experiment is shown, which was performed in triplicate. α-SMA signal intensity was evaluated densitometrically using Lumi Imager software (Roche). The data indicate that the expression is essentially unaltered. (B) Smad7 induces morphological changes of actin fiber organization in primary-cultured HSCs. Immunostaining was performed with a monoclonal anti α-SMA antibody. Representative light microscopical photomicrographs (LM) and confocal laser scanning microscopy images (LSM) of primary-cultured HSCs, infected or not with AdLacZ or AdSmad7, are shown. AdLacZ-infected cells display actin stress fibers; AdSmad7-infected cells, in contrast, have completely lost the ability to establish a fibrillar organization and display focal or diffuse staining within the cytoplasm (magnification 400×).
Gastroenterology  , DOI: ( /S (03) )

19. Figure 9
(A ) α-SMA expression in activated HSC is not influenced by Smad7. α-SMA expression was analyzed in Western blots using 25 μg total lysate from primary-cultured HSCs, infected or not with AdLacZ or AdSmad7 at day 2 and cultured for different times as indicated. A representative experiment is shown, which was performed in triplicate. α-SMA signal intensity was evaluated densitometrically using Lumi Imager software (Roche). The data indicate that the expression is essentially unaltered. (B) Smad7 induces morphological changes of actin fiber organization in primary-cultured HSCs. Immunostaining was performed with a monoclonal anti α-SMA antibody. Representative light microscopical photomicrographs (LM) and confocal laser scanning microscopy images (LSM) of primary-cultured HSCs, infected or not with AdLacZ or AdSmad7, are shown. AdLacZ-infected cells display actin stress fibers; AdSmad7-infected cells, in contrast, have completely lost the ability to establish a fibrillar organization and display focal or diffuse staining within the cytoplasm (magnification 400×).
Gastroenterology  , DOI: ( /S (03) )