1. Volume 122, Issue 7, Pages 1924-1940 (June 2002)
Antidiabetic thiazolidinediones inhibit collagen synthesis and hepatic stellate cell activation in vivo and in vitro 
Andrea Galli, David W. Crabb, Elisabetta Ceni, Renata Salzano, Tommaso Mello, Gianluca Svegliati–Baroni, Francesco Ridolfi, Luciano Trozzi, Calogero Surrenti, Alessandro Casini 
Gastroenterology 
Volume 122, Issue 7, Pages (June 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Sirius red staining of rat liver at the end of treatment with (A) DMN alone, (B) DMN + rosiglitazone, (C) DMN + pioglitazone, (D) CCl4 alone, (E) CCl4 + rosiglitazone, (F) CCl4 + pioglitazone, (G) BDL, (H) BDL + rosiglitazone, and (I) BDL + pioglitazone. (A) A centrilobular collagen deposition with thick fibrotic septa bridging between terminal hepatic veins is evident after 3 weeks of DMN administration. Oral administration of both (B) rosiglitazone and (C) pioglitazone reduced collagen deposition with only incomplete fibrotic septa that form thin parenchymal extension without a nodular pattern. (D) Periportal and centrilobular deposition of collagen with thick fibrotic septa forming a complete cirrhosis was obtained 8 weeks after CCl4 administration. Only thin intralobular bands of collagen associated with mild portal fibrosis but without a significant alteration of the lobular architecture occurred when CCl4 was administered in combination with either (E) rosiglitazone or (F) pioglitazone. (G) Four weeks after BDL, the lobular architecture was completely modified by proliferating ductules accompanied by fibrotic tissue, whereas (H and I) BDL rats treated with TZD showed a limited accumulation of connective tissue limited to portal tract. Original magnification: A–F, 100×; G–I, 400×.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
(A) Northern blot analysis of total RNA (20 μg) extracted from HSC freshly isolated from rats treated with either DMN or CCl4 in the presence or absence of TZD (RGZ or PGZ). HSC were obtained from rats after 1 week of treatment with DMN and from animals after 4 weeks of treatment with CCl4 as described in Materials and Methods. Both DMN and CCl4 strongly increased the expression of procollagen type I (α1), FN, and TGF-β1 mRNA in HSC. These stimulatory effects were significantly reduced in HSC isolated from animals treated with either RGZ or PGZ. The hybridization bands were quantified by scanning laser densitometry, normalized to 36B4 bands (used as a housekeeping gene), and the ratios expressed as a percentage of the control (mean values ± SD; lower panel). Two transcripts were present for α1(I) procollagen (5.7 and 4.7 kb), whereas a single transcript of 9.1, 2.5, and 1.4 kb was present for FN, TGF-β1, and 36B4, respectively. These results are representative of 3 different experiments performed in triplicate in different cell preparations obtained from 3 different animals. Densitometric data, shown in B, report the average values (±SD) from all the experiments performed. *P < 0.01 or higher degree of significance vs. controls; °P < 0.02 or higher degree of significance vs. DMN; ♦P < or higher degree of significance vs. CCl4.
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
(A) Northern blot analysis of total RNA (20 μg) extracted from HSC freshly isolated from rats after BDL in the presence or absence of TZD (RGZ or PGZ). HSC were obtained from rats after 1 week of BDL as described in Materials and Methods. BDL strongly increased the expression of procollagen type I (α1), FN, and TGF-β1 mRNA in HSC. These stimulatory effects were significantly reduced in HSC isolated from animals treated with either RGZ or PGZ. The hybridization bands were quantified by scanning laser densitometry, normalized to 36B4 bands (used as a housekeeping gene), and the ratios expressed as a percentage of the control (mean values ± SD; lower panel). These results are representative of 3 different experiments performed in triplicate in different cell preparations obtained from 3 different animals. Densitometric data shown in B report the average values (±SD) from all the experiments performed. *P < 0.01 or higher degree of significance vs. controls; °P < 0.02 or higher degree of significance vs. BDL.
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
(A) PPRE binding activity of nuclear extract from HSC freshly isolated from rats treated with either DMN, CCl4, or BDL in the presence or absence of TZD treatment (RGZ or PGZ). HSC were obtained from rats after 1 week of treatment with DMN, 4 weeks of treatment with CCl4, and after 1 week of BDL surgery as described in Materials and Methods. EMSA were performed with a probe consisting of a double-strand oligonucleotide, which contains a copy of the PPARγ response element (ARE-7) from the adipocyte fatty acid binding protein (aP2) gene. Nuclear extract was incubated with 20,000 cpm of the probe and then analyzed by electrophoresis through a 4% polyacrylamide gel and autoradiography. Lanes 1 and 4 represent the binding complexes of nuclear extracts from HSC isolated from control rats. Lane 11 represents the binding complexes of nuclear extracts from HSC isolated from sham-operated rats. The cold competitor lane indicates the molar excess (200×) of unlabeled oligonucleotide added to the binding reaction. Lane 3 shows a diminution of the binding complex by incubation with anti-PPARγ antibodies. PPRE binding was appreciably reduced in HSC isolated from DMN-, CCl4-, and BDL-treated rats (lanes 5, 6, and 12). Both RGZ and PGZ administration prevented the reduction of PPRE binding activity in HSC isolated either from 4-week CCl4 (lanes 7 and 8) or 1-week BDL-treated rats (lanes 13 and 14). Restored binding activity by TZD was completely abolished by incubation with anti-PPARγ antibodies (lanes 9, 10, 15, and 16). (B) Expression of PPARγ gene in HSC freshly isolated from rats treated with either DMN, CCl4, and BDL in the presence or absence of TZD treatment (RGZ or PGZ). HSC were obtained from rats after 1 week of treatment with DMN, 4 weeks of treatment with CCl4, and after 1 week of BDL surgery as described in Materials and Methods. One microgram of total RNA was reverse transcribed using random hexamers and amplified by polymerase chain reaction using specific primers for PPARγ and β-actin. The reverse-transcription polymerase chain reaction products were electrophoresed on ethidium bromide–containing agarose gels. HSC from fibrotic rats have reduced PPARγ expression compared with controls (lanes 2, 3, and 7). Concomitant administration of TZD abrogated this reduction (lanes 4, 5, 8, and 9). These results are representative of 3 different experiments performed in triplicate in different cell preparations obtained from 3 different animals.
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Effect of 5 μmol/L of thiazolidinediones and 1 μmol/L of 15d-PGJ2 (PGJ2) on procollagen type I (pIp; A) and fibronectin (FN; B) production induced by TGF-β1 in human HSC. Cells were maintained in SFIF medium for 48 hours and then stimulated for an additional 24 hours with TGF-β1 (1 ng/mL) in the absence or in the presence of either TZD (RGZ or PGZ) or 15d-PGJ2. Procollagen type I and FN were measured in culture media by an enzyme-linked immunoassay method as described in Materials and Methods. TGF-β1 significantly stimulated pIp and FN production (3-fold and 2.5-fold, respectively) in cultured human HSC. Both RGZ and PGZ, as well as 15d-PGJ2, significantly reduced the TGF-β1–induced stimulation of procollagen I and FN. (A) These substances were also able to significantly reduce the basal production of pIp, whereas (B) the FN basal levels were not affected. Data, expressed as micrograms per microgram of cellular DNA, are mean values ± SD of 5 experiments performed in triplicate. (A) *P < 0.03 or higher degree of significance vs. baseline controls; °P < 0.01 or higher degree of significance vs. TGF-β1–treated controls. (B) *P < vs. baseline controls; °P h or higher degree of significance vs. TGF-β1–treated controls.
Gastroenterology  , DOI: ( /gast )
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7. Fig. 6
(A) Northern blot analysis of total RNA (10 μg) extracted from HSC isolated from wedge sections of normal human liver and cultured in vitro as described in Materials and Methods. Cells were maintained in SFIF medium for 48 hours and then stimulated for an additional 6 hours with TGF-β1 (1 ng/mL) in the absence or in the presence of either 5 μmol/L TZD (RGZ or PGZ) or 1 μmol/L 15d-PGJ2 (PGJ2). TGF-β1 significantly stimulated the mRNA expression of procollagen type I (α1), FN, and TGF-β1 (4-fold, 3.5-fold, and 5-fold, respectively) in cultured human HSC. Both RGZ and PGZ strongly reduced the TGF-β1 induction of procollagen I, FN, and TGF-β1. 15d-PGJ2 was also, although to a lesser extent, able to significantly reduce the TGF-β1 induction of collagen, FN, and TGF-β1 mRNA expression. (B) Laser densitometric analysis of the blots. The mRNA hybridization bands were quantified by scanning laser densitometry and normalized to the 36B4 bands, used as a housekeeping gene. Data (mean ± SD) are expressed as percentage of increase of controls and were obtained by scanning 9 different blots coming from 3 different experiments performed in triplicate in 3 different cell lines. The size of mRNA transcripts (expressed in kb) are indicated at right. *P < 0.05 or higher degree of significance vs. control; °P < 0.01 or higher degree of significance vs. TGF-β1 stimulation.
Gastroenterology  , DOI: ( /gast )
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8. Fig. 7
Increased cellular levels of PPARγ protein inhibit TGF-β1–induced collagen synthesis. (A) Quiescent human HSC were transfected with increasing amounts of pCMX-hPPARγ expression plasmid or pCMX empty vector by the calcium phosphate procedure. The total amount of DNA transfected in each set of cells was maintained constant by using a carried DNA (pcDNA3.1). Twenty-four hours after transfection, cells were treated with TGF-β1 for an additional 24 hours. Procollagen type I was determined in culture media by an enzyme-linked immunoassay method as described in Materials and Methods. Data, expressed as micrograms per microgram of cellular DNA, are mean values ± SD of 5 experiments performed in triplicate. First bar on the left represents basal value. *P < 0.01 or higher degree of significance vs. control. (B) Levels of immunoreactive PPARγ in human HSC were detected to monitored transfection efficiency. Fifty micrograms of nuclear protein extract was fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Receptor proteins were detected by incubating the filter with specific anti-PPARγ antibodies.
Gastroenterology  , DOI: ( /gast )
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9. Fig. 8
Effect of BADGE on TZD-inhibited collagen synthesis. (A) Cells were maintained in SFIF medium for 48 hours and then stimulated for an additional 24 hours with TGF-β1 (1 ng/mL), in absence or presence of TZD (RGZ or PGZ) with or without varying concentrations of the PPARγ antagonist BADGE. Procollagen type I was measured in culture media by enzyme-linked immunosorbent assay as described in Materials and Methods. BADGE abrogated the inhibitory effect of RGZ and PGZ on the TGF-β1–induced collagen synthesis in a dose-dependent manner. (B) Effect of BADGE on TZD-induced PPARγ transactivation. Quiescent human HSC were cotransfected with PPRE3-tk-luc reporter plasmid and with β-galactosidase expression vector (pSV-β-Gal) as an internal control for transfection efficiency. Twelve hours after transfection, cells were treated with TGF-β1 (1 ng/mL), in absence or presence of TZD (RGZ or PGZ) with or without varying concentrations of BADGE. After 24 hours of incubation, cells were harvested for luciferase and β-galactosidase assay. BADGE abrogated the TZD induction of PPRE-tk-luc reporter activity. The data are mean ± SD for 4 experiments performed in triplicate. (A) *P < 0.05 or higher degree of significance vs. control. °P < 0.05 vs. TGF-β1. (B) *P < 0.05 or higher degree of significance vs. control. ♢P < 0.01 or higher degree of significance vs. TGF-β1 + TZD (+RGZ or +PGZ).
Gastroenterology  , DOI: ( /gast )
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10. Fig. 9
Effect of PPARγ transcriptional activation on the pro-α2(1) collagen promoter in cultured human HSC. Quiescent human HSC were cotransfected with pro-α2(1) collagen promoter–CAT reporter construct (pMS-3.5/CAT) and PPRE3-tk-LUC reporter. Cells were also cotransfected with either pCMX-hPPARγ expression plasmid or pCMX (empty vector), and with the β-galactosidase expression vector pSV-β-Gal as an internal control of transfection efficiency. The total amount of DNA transfected in each set of cells was maintained constant by using a carrier DNA (pcDNA3.1). Twelve hours after transfection, cells were treated with TGF-β1 (1 ng/mL) in the absence or in the presence of either 5 μmol/L of TZD (RGZ or PGZ) or 1 μmol/L of 15d-PGJ2 (PGJ2). After 24 hours of incubation, cells were harvested for CAT, luciferase, and β-galactosidase assay. (A) The expression of pro-α2(1) collagen promoter construct is reported as the ratio of CAT activity to β-galactosidase activity. (B) PPARγ-induced transcription of PPRE3-tk-LUC reporter was expressed as the ratio of luciferase activity to β-galactosidase activity. PPARγ transcriptional activation reduces pro-α2(1) collagen promoter expression in ligand-dependent and -independent fashion. The data are mean ± SD for 3 or 4 experiments performed in triplicate. (A) *P < or higher degree of significance vs. control; °P < 0.05 or higher degree of significance vs. TGF-β1 effect in human HSC transfected with pCMX empty vector; ♦P < 0.01 vs. TGF-β1 effect in human HSC transfected with pCMX-hPPARγ; ♢P < 0.03 vs. TGF-β1 effect in human HSC transfected with pCMX empty vector. (B) *P < vs. controls; °P < 0.01 or higher degree of significance vs. human HSC transfected with pCMX-hPPARγ without ligand exposure (black bar).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions