Effect of pentoxifylline on early proliferation and phenotypic modulation of fibrogenic cells in two rat models of liver fibrosis and on cultured hepatic stellate cells

doi:10.1016/S0168-8278(99)80192-5

Journal of Hepatology

Volume 30, Issue 4, April 1999, Pages 621-631

https://doi.org/10.1016/S0168-8278(99)80192-5 Get rights and content

Abstract

Background/Aims: During liver fibrosis, different fibroblastic cells, i.e. hepatic stellate cells (HSCs) or portal fibroblasts, are involved in the development of lesions, and acquire myofibroblastic differentiation. We investigated, in the rat, whether pentoxifylline can influence the early phase of fibrogenesis in two animal models of fibrosis induced by either carbon tetrachloride (CCl₄) plus acetone (given twice) or bile duct ligation.

Methods: The fibroproliferative response and myofibroblastic phenotypic modulation were evaluated by PCNA and alpha-smooth muscle (α-SM) actin immunohistochemistry, respectively, in livers taken 24 h after the last CCl₄ treatment or 72 h after bile duct ligation. Desmin expression was also measured, and inflammation was evaluated by ED-1 staining. Furthermore, proliferation and α-SM actin expression were studied in cultured HSCs after pentoxifylline treatment.

Results: In the CCl₄-acetone groups, pretreatment with pentoxifylline decreased the proliferative response and expression of α-SM actin in the HSCs. Similarly, pentoxifylline reduced the proliferation and myofibroblastic differentiation of portal fibroblasts after bile duct ligation. Pentoxifylline reduced ED-1 expression, particularly in the CCl₄ model, where there was significant inflammation. In cultured pentoxifylline-treated HSCs, both proliferation and α-SM actin expression were decreased.

Conclusions: In both animal models of fibrosis, during the early stages of tissue injury, pentoxifylline was able to reduce fibroproliferation and myofibroblastic differentiation and to reduce hepatocellular damage and the inflammatory response, particularly in the toxin-induced model. In culture, α-SM actin expression decreased in both growing and quiescent HSCs treated with pentoxifylline, indicating that the drug may also exert a direct effect on hepatic fibrogenic cells.

Section snippets

Animals

Male Sprague-Dawley rats (200–250 g body weight) were used, and divided into 7 experimental groups. In the first group (n=7), animals were given CCl₄ (5 mmol/kg per os) plus acetone (25 mmol/kg per os) on the 1st and 6th days. The acetone treatment protocol was used, since it has been shown to potentiate CCl₄-induced fibrosis (17). Acetone was administered 18 h before the toxin. In the second group (n=7), animals were subjected to common bile duct ligation as described before (3).

Biochemical analysis

As shown in Fig. 1, pentoxifylline induced a slight, but not significant, increase of serum SGPT compared with controls. In both models of fibrosis, liver cell damage was indicated by significant elevations in serum SGPT compared with controls (pFig. 1a, and decrease of 40% in the bile duct ligation model, Fig. 1b).

Hematoxylin-eosin examination

Twenty-four hours after the last CCl₄ dose, a marked inflammatory reaction and hepatocytic necrosis was observed around centrolobular veins (Fig. 2a). Pentoxifylline treatment

Discussion

During the development of fibrosis, fibroblastic cells proliferate and acquire myofibroblastic features, including the expression of α-SM actin (29). After liver injury, it is generally accepted that the HSC plays a major role in the progression of the lesions, and secretes extracellular matrix components, leading to the development of fibrosis 1., 2.. HSCs are mainly involved in repair, which develops around centrolobular veins after toxic injury (e.g. alcoholic fibrosis). Recently, it has

Acknowledgements

This work was supported by the Centre National de la Recherche Scientifique (France), the Association pour la Recherche sur le Cancer (France), the Swiss National Science Foundation no. 31–50568.97, the Medical Research Council of Canada, and the Centre Jacques Cartier, Lyon (France).

We thank Mr. M. Audet, Mrs M. Redard and A. Perea, for their excellent technical help.

References (44)

M Pinzani
Novel insight into the biology and physiology of the Ito cell
Pharmacol Ther
(1995)
TC Peterson et al.
Effect of pentoxifylline in rat and swine models of hepatic fibrosis: role of fibroproliferation in its mechanism
Immunopharmacology
(1996)
F Oberti et al.
Effects of simvastatin, pentoxifylline and spironolactone on hepatic fibrosis and portal hypertension in rats with bile duct ligation
J Hepatol
(1997)
B Berman et al.
Pentoxifylline inhibits normal human dermal fibroblasts in vitro proliferation, collagen, glycosaminoglycan, and fibronectin production, and increases collagenase activity
J Invest Dermatol
(1989)
MR Duncan et al.
Pentoxifylline, pentifylline, and interferons decrease type I and III procollagen mRNA levels in dermal fibroblasts: evidence for mediation by nuclear factor 1 down-regulation
J Invest Dermatol
(1995)
O Kocher et al.
Analysis of α-smooth-muscle actin mRNA expression in rat aortic smooth-muscle cells using a specific cDNA probe
Differentiation
(1987)
ML Bochaton-Piallat et al.
Cultured aortic smooth muscle cells from newborn and adult rats show distinct cytoskeletal features
Differentiation
(1992)
A Desmoulière et al.
α-Smooth muscle actin is expressed in a sub-population of cultured and cloned fibroblasts and is modulated by γ-interferon
Exp Cell Res
(1992)
MJ Dans et al.
Inhibition of collagen lattice contraction by pentoxifylline and interferon-alpha, -beta, and -gamma
J Invest Dermatol
(1994)
TC Peterson et al.
In vitro effect of platelet-derived growth factor on fibroproliferation and effect of cytokine antagonists
Immunopharmacology
(1994)

RA Isbrucker et al.

Platelet-derived growth factor and pentoxifylline modulation of collagen synthesis in myofibroblasts

Toxicol Appl Pharmacol

(1998)

B Berman et al.

Pentoxifylline inhibits certain constitutive and tumor necrosis factor induced activities of human normal dermal fibroblasts

J Invest Dermatol

(1992)

K Kozaki et al.

Effects of pentoxifylline pretreatment on Kupffer cells in rat liver transplantation

Hepatology

(1995)

G Ramadori

The stellate cell (Ito-cell, fat-storing cell, lipocyte, perisinusoidal cell) of the liver. New insights into pathophysiology of an intriguing cell

Virchows Arch B Cell Pathol

(1991)

B Tuchweber et al.

Proliferation and phenotypic modulation of portal fibroblasts in the early stages of cholestatic fibrosis in the rat

Lab Invest

(1996)

TC Peterson

Pentoxifylline prevents fibrosis in an animal model and inhibits platelet-derived growth factor-driven proliferation of fibroblasts

Hepatology

(1993)

PD Hodgson et al.

Effect of pentoxifylline in a heterologous serum model of fibrosis in the rat

Hepatology

(1995)

M Rubio et al.

Pentoxifylline prevents liver fibrosis but induces cellular damage in carbon tetrachloride injured rats

J Hepatol

(1994)

G Boigk et al.

Antifibrotic effect of pentoxifylline in rat biliary fibrosis due to bile duct obstruction

J Hepatol

(1995)

EJ Park et al.

Antifibrotic effects of a polysaccharide extracted from Ganoderma lucidum, glycyrrhizin, and pentoxifylline in rats with cirrhosis induced by biliary obstruction

Biol Pharm Bull

(1997)

C Raetsch et al.

Pentoxifyllin inhibits hepatic collagen deposition in early but not advanced rat biliary fibrosis

Gastroenterology

(1996)

B Berman et al.

Pentoxifylline inhibits the proliferation of human fibroblasts derived from keloid, scleroderma and morphoea skin and their production of collagen, glycosaminoglycans and fibronectin

Br J Dermatol

(1990)

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Effect of pentoxifylline on early proliferation and phenotypic modulation of fibrogenic cells in two rat models of liver fibrosis and on cultured hepatic stellate cells

Abstract

Section snippets

Animals

Biochemical analysis

Hematoxylin-eosin examination

Discussion

Acknowledgements

Pharmacol Ther

Immunopharmacology

J Hepatol

J Invest Dermatol

J Invest Dermatol

Differentiation

Differentiation

Exp Cell Res

J Invest Dermatol

Immunopharmacology

Toxicol Appl Pharmacol

J Invest Dermatol

Hepatology

The stellate cell (Ito-cell, fat-storing cell, lipocyte, perisinusoidal cell) of the liver. New insights into pathophysiology of an intriguing cell

Virchows Arch B Cell Pathol

Proliferation and phenotypic modulation of portal fibroblasts in the early stages of cholestatic fibrosis in the rat

Lab Invest

Pentoxifylline prevents fibrosis in an animal model and inhibits platelet-derived growth factor-driven proliferation of fibroblasts

Hepatology

Effect of pentoxifylline in a heterologous serum model of fibrosis in the rat

Hepatology

Pentoxifylline prevents liver fibrosis but induces cellular damage in carbon tetrachloride injured rats

J Hepatol

Antifibrotic effect of pentoxifylline in rat biliary fibrosis due to bile duct obstruction

J Hepatol

Antifibrotic effects of a polysaccharide extracted from Ganoderma lucidum, glycyrrhizin, and pentoxifylline in rats with cirrhosis induced by biliary obstruction

Biol Pharm Bull

Pentoxifyllin inhibits hepatic collagen deposition in early but not advanced rat biliary fibrosis

Gastroenterology

Pentoxifylline inhibits the proliferation of human fibroblasts derived from keloid, scleroderma and morphoea skin and their production of collagen, glycosaminoglycans and fibronectin

Br J Dermatol