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The selective cyclooxygenase-2 inhibitor SC-236 reduces liver fibrosis by mechanisms involving non-parenchymal cell apoptosis and PPAR activation

Anna Planagumà^*, Joan Clària^*,1, Rosa Miquel, Marta López-Parra^*, Esther Titos^*, Jaime L. Masferrer^*, Vicente Arroyo and Joan Rodés

^* DNA Unit,
Pathology Laboratory and
Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain

¹ Correspondence: DNA Unit, Hospital Clínic, Villarroel 170, Barcelona 08036, Spain. E-mail: jclaria{at}clinic.ub.es

SPECIFIC AIMS

There is growing recognition of the importance of inflammation in initiating the sequence of events leading to liver fibrosis. We have examined the effects of SC-236, a selective cyclooxygenase (COX)-2 inhibitor in experimental liver fibrosis. The investigation is comprised of in vivo studies of rats with carbon tetrachloride (CCl₄)-induced liver injury as well as in vitro studies of Kupffer cells and hepatic stellate cells (HSCs), the major players in liver inflammation and liver fibrogenesis, respectively.

PRINCIPAL FINDINGS

1. COX-2 protein expression is markedly increased in the liver of CCl₄-treated rats
Compared with controls, a marked induction of COX-2 protein expression was detected by immunohistochemistry in the liver of CCl₄-treated rats.

2. The selective COX-2 inhibitor SC-236 reduces liver fibrosis in CCl₄-treated rats
After CCl₄ administration, rats developed hepatic steatosis, inflammation, hepatocyte ballooning, and necrosis. Beyond the 6th wk of CCl₄ treatment, liver architecture was extensively disorganized as the sinusoids were no longer distinguishable; few areas of healthy hepatocytes were present and collagen deposition with septa bridging portal regions was detected. As shown in Fig. 1 A, administration of SC-236 to CCl₄-treated rats exerted a significant antifibrotic effect, as revealed by the histological analysis of liver sections and the presence of a lower hepatic hydroxyproline content (13.0±1.4 vs. 16.9±1.4 nmol/mg liver tissue, P<0.05), an established marker of collagen synthesis.

View larger version (77K): [in this window] [in a new window]   Figure 1. Antifibrotic effects of SC-236 in CCl4-treated rats. A) Histological assessment of liver fibrosis in CCl4-treated and control rats. The extent of matrix deposition was measured by Masson’s trichrome staining of liver tissue sections from control animals (left panels, magnification x40) and 10 wk CCl4-treated rats (right panels, x100) receiving either placebo (upper panels) or SC-236 (lower panels). Data are representative of results obtained from 10 control and 20 CCl4-treated rats. B) Detection of hepatic MMP-2 and MMP-9 activities by zymography. Gelatin polyacrylamide gel representative of changes in activity of MMP-2 (72 kDa) and MMP-9 (92 kDa) during the weeks of CCl4 administration (6, 8, and 10 wk) in CCl4-treated rats receiving placebo or SC-236. Lower panel: analysis by densitometry of MMP-2 activity in these samples. The results are the mean ± SE of 5 different experiments. *P < 0.05 vs. placebo. C) Effects of SC-236 on hepatic -SMA protein expression in CCl4-treated rats. Liver tissue samples from rats with CCl4-induced fibrosis treated with (+) or without (–) SC-236 were analyzed by Western blot using a specific antibody against -SMA. A representative blot is shown in the upper panel; the histogram in the lower panel shows the results obtained from the densitometric analysis of band intensities from 4 separate experiments (mean±SE). , placebo; •, SC-236; CT+, total protein from mesangial cells. **P< 0.01 vs. placebo.

3. SC-236 decreases matrix metalloproteinase (MMP)-2 and MMP-9 activities and -smooth muscle actin (-SMA) protein expression
Since MMPs are key enzymes in the regulation of extracellular matrix remodeling and their activity closely correlates with the severity of liver fibrosis, we determined hepatic MMP-2 and MMP-9 activities by zymography. SC-236 significantly reduced hepatic MMP-2 gelatinolytic activity and abolished MMP-9 activity in CCl₄-treated rats (Fig. 1B ). SC-236 dramatically suppressed -SMA protein expression, a well-established marker of HSC activation during liver fibrogenesis (Fig. 1C ).

4. SC-236 reduces hepatic levels of 15-deoxy-^12,14 prostaglandin (PG) J₂ (15d-PGJ₂) and restores peroxisome proliferator-activated receptor (PPAR) mRNA expression in the liver of CCl₄-treated rats
Levels of 15d-PGJ₂, the dehydration product of the major hepatic PG, PGD₂, were markedly increased whereas PPAR mRNA, as determined by reverse transcription real-time quantitative PCR, was significantly abrogated in the liver of CCl₄-treated rats. SC-236 normalized 15d-PGJ₂ levels and restored PPAR expression in the liver.

5. SC-236 reduces cell growth and induces apoptosis in HSCs and Kupffer cells
To characterize the cellular mechanisms underlying the hepatic antifibrotic effects of SC-236, we performed additional studies in HSCs and Kupffer cells. HSCs are the major cell type involved in liver fibrogenesis; Kupffer cells are primarily responsible for COX-2 induction and eicosanoid biosynthesis in the liver. As shown in Fig. 2 A, B, SC-236 reduced, in a concentration- and time-dependent manner, HSC proliferation, an effect associated with an increased number of nuclei with densely compacted chromatin characteristic of apoptotic cells (Fig. 2C ). Similar findings were observed in Kupffer cells. The effect of SC-236 on HSC proliferation was similar to that previously reported for PPAR ligands. In HSCs, SC-236 inhibited the proliferative response induced by PDGF to an extent similar to that of the PPAR ligand rosiglitazone.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effects of SC-236 on HSC growth and survival. Concentration and time responses to SC-236. HSCs were incubated with increasing concentrations of SC-236 (0, 15, and 25 µM) for 24 h (A) or grown for 0, 6, 12, and 24 h with 15 µM SC-236 (B); cell number was determined by the MTT assay. Results are mean ± SE of 3 different experiments with duplicate determinations. *P< 0.05, **P< 0.01 vs. untreated cells. C) Changes in nuclear morphology associated with programmed cell death were assessed by fluorescence microscopy in HSCs stained with propidium iodide after 6 h of incubation at 37°C with vehicle (0.1% ethanol), SC-236 (25 µM), and 15d-PGJ2 (10 µM) alone or in combination with SC-236 (25 µM).

6. SC-236 increases PPAR mRNA expression in HSCs and activates PPAR in a cell-based trans-activation assay
We assessed the effect of increasing concentrations of SC-236 on PPAR expression in HSCs by reverse transcription and real-time quantitative PCR, and found that SC-236 significantly increases PPAR mRNA levels in these cells. We explored whether SC-236 is able to bind and activate PPAR. To avoid interference from the endogenous receptor, we used a cell-based PPAR and luciferase cotransfection reporter assay. SC-236 acted in a concentration-dependent manner as a PPAR agonist. This effect was very potent and comparable to the induction exerted by 15d-PGJ₂, the natural ligand and activator of PPAR. These findings suggest that the antifibrotic effect exerted by SC-236 in CCl₄-treated rats is mediated at least in part by PPAR activation.

CONCLUSIONS AND SIGNIFICANCE

Inflammation plays a major role in the pathogenesis of liver fibrosis and precedes or coexists with the development of extracellular matrix alterations in this organ. Several anti-inflammatory strategies aimed to efficiently prevent liver fibrosis have been tested. Unfortunately, anti-inflammatory therapies such as glucocorticoids and nonsteroidal anti-inflammatory drugs (NSAIDs) have not proved consistently effective and are not devoid of side effects (i.e., NSAIDs are not recommended in patients with chronic liver disease because of renal side effects). Therefore, novel therapeutic approaches are needed.

A novel strategy to treat liver inflammation and related hepatic disorders may involve the use of selective COX-2 inhibitors. This new class of anti-inflammatory compounds has an efficacy similar to traditional NSAIDs but with a lower incidence of adverse gastrointestinal and renal effects. In the current study, we provide evidence that the selective COX-2 inhibitor, SC-236, exerts a significant antifibrotic effect in rats with CCl₄-induced liver fibrosis. Our results open new avenues for the applicability of these anti-inflammatory compounds as a novel therapy for liver inflammation and fibrosis. Such a strategy has been shown to be effective in preventing other fibrotic disorders, including renal interstitial fibrosis, fibrosis of the testes, and oral submucous fibrosis.

Our study also provides molecular mechanisms for the antifibrotic effects of SC-236 in the liver of CCl₄-treated animals. Our results support the concept that the antifibrogenic effects of SC-236 are mediated at least in part by PPAR (Fig. 3 ). In fact, SC-236 increased PPAR expression in HSCs and restored its expression to normal levels in the liver of CCl₄-treated rats. PPAR is a ligand-activated transcription factor with a DNA binding domain that recognizes response elements in the promoter region of specific target genes linked to inflammation, cell proliferation, apoptosis, and differentiation. PPAR plays a pivotal role in the progression of liver fibrosis since HSC activation is associated with a reduction in both expression and transcriptional activity of this nuclear receptor. In culture-activated HSCs, synthetic PPAR ligands restore PPAR and reverse the activated HSCs to the quiescent phenotype. In vivo, administration of synthetic PPAR ligands (i.e., antidiabetic thiazolidinediones) to animal models of liver fibrosis effectively reduces HSC trans-differentiation and collagen deposition. The results obtained in our trans-activation reporter assay are indicative that SC-236 not only restores PPAR expression, but also works as a PPAR ligand. This effect was very potent and comparable to the induction exerted by 15d-PGJ₂, the natural ligand and activator of PPAR. These findings together with the observation that the antiproliferative effects of SC-236 were similar to those exerted by the PPAR ligand rosiglitazone support the concept that the antifibrotic properties of SC-236 are related to PPAR.

View larger version (57K): [in this window] [in a new window]   Figure 3. Summary of the proposed mechanism by which SC-236 attenuates liver fibrosis. A) Liver injury induces COX-2 overexpression in Kupffer cells (KC) and initiates a cascade of events, including massive production of cytokines and growth factors, leading to hepatic stellate cell (HSC) activation and proliferation, and consequently to liver fibrosis. During fibrosis progression, KC proliferate, thereby amplifying the release of proinflammatory mediators. B) The selective COX-2 inhibitor dampens liver inflammation and fibrosis by mechanisms involving PPAR activation, down-regulation of -SMA expression and MMP-2 and -9 activities and induction of KC and HSC apoptosis.

The results of our study also indicate that SC-236 exhibits proapoptotic and growth-inhibitory properties in HSCs and Kupffer cells (Fig. 3) . Identification of SC-236 as an apoptotic stimulus for nonparenchymal liver cells may have pathophysiological implications. Activated HSCs are central to liver fibrosis as the major source of collagens I and III. During fibrotic injury, these retinoid-rich, nonparenchymal cells proliferate and undergo a phenotypic transformation to myofibroblast-like cells, a process termed activation. Therefore, the control of HSC proliferation and apoptosis is a key event in regulating the progression of liver fibrosis. Strategies based on inhibition of HSC proliferation and induction of HSC apoptosis have proved to be potential antifibrotic approaches. On the other hand, Kupffer cells, the resident macrophages of the liver, are well recognized for their activity in liver inflammation and to promote activation of HSCs through release of paracrine factors. The presence of an increased number of activated Kupffer cells is considered to be critical in the initiation of the inflammatory cascade leading to liver fibrosis. Consistent with their role in liver inflammation, selective depletion of macrophages during progression of injury or reduction of Kupffer cell survival by inhibition of the 5-lipoxygenase pathway are associated with a remarkable antifibrotic effect. Results of the current study suggest that inhibition of Kupffer cell survival by SC-236 contributes to the dampening of liver fibrogenesis. Although we did not establish the exact mechanisms underlying the proapoptotic proper-ties of SC-236, several molecular mechanisms, including COX-dependent and COX-independent pathways, are likely to be implicated.

The results of the current study bring to light the antifibrogenic potential of the selective COX-2 inhibitor SC-236 and open new avenues for the development of novel anti-inflammatory strategies leading to an effective therapy for liver fibrosis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2753fje; doi: 10.1096/fj.04-2753fje

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