Rat Hepatic Stellate Cell Line Research Articles

Pentoxifylline downregulates α (I) collagen expression by the inhibition of Iκbα degradation in liver stellate cells

Overproduction of collagen (I) by activated hepatic stellate cells is a critical step in the development of liver fibrosis. It has been established that these cells express interleukin (IL)-6 and respond to this cytokine with an increase in alpha(I) collagen. Pentoxifylline, a methylxanthine derivate, has been reported to have antifibrotic properties, but the mechanism responsible for this effect is unknown. The aim of this study was to determine the effect of pentoxifylline on acetaldehyde-induced collagen production in a rat hepatic stellate cell line (CFSC-2G cells). Cells were treated with 100 microM acetaldehyde and 200 microM pentoxifyline for 3 h. IL-6 and alpha(I) collagen messenger RNA (mRNA) were determined by reverse transcriptase polymerase chain reaction (RT-PCR) assay. NFkappaB activation was determined by electrophoretic mobility shift assay. To corroborate NFkappaB participation in pentoxifylline effect, cells were pretreated with 10 microM TPCK, a NFkappaB inhibitor. IkappaBalpha was determined by Western blot. IL-6 expression decreased significantly in acetaldehyde-pentoxifylline-treated cells. Acetaldehyde-treated cells pretreated with an anti-IL-6 monoclonal antibody did not show any increase in alpha (I) collagen expression. Acetaldehyde-treated cells increased 1.48 times NFkappaB activation, whereas acetaldehyde-pentoxifylline-treated cells decreased NFkappaB activation to control values. TPCK pretreated acetaldehyde cells did not present NFkappaB activation. To corroborate NFkappaB participation in pentoxifylline effect, IkappaBalpha was determined. IkappaBalpha protein level decreased 50% in acetaldehyde-treated cells, while acetaldehyde-pentoxifylline-treated cells showed IkappaBalpha control cells value. The data suggest that acetaldehyde induced alpha(I) collagen and IL-6 expression via NFkappaB activation. Pentoxifylline prevents acetaldehyde-induced alpha(I) collagen and IL-6 expression by a mechanism dependent on IkappaBalpha degradation, which in turn blocks NFkappaB activation.

The Effect of Ras Inhibition on the Proliferation, Apoptosis and Matrix Metalloproteases Activity in Rat Hepatic Stellate Cells

In vivo inhibition of Ras by its antagonist farnesylthiosalicylic acid (FTS) prevents and reverses liver fibrosis in a rat model. In this study we showed the in vitro effects of Ras inhibition in a rat hepatic stellate cell line, HSC-T6. The IC(50) of FTS that inhibited PDGF-induced proliferation was 15 microM. FTS, by itself or in combination with PDGF, induced a three- to fivefold increase in the number of apoptotic stellate cells but did not induce apoptosis in cells cultured with TGFbeta1. We observed increased activity of MMP-9 and MMP-2 induced by FTS in combination with PDGF or TGFbeta. FTS, alone or in the presence of PDGF and TGFbeta, reduced collagen I mRNA expression. In conclusion, the in vivo amelioration of liver fibrosis by FTS may be explained by its ability to inhibit hepatic stellate cell proliferation, induce apoptosis and MMP-2 and MMP-9 activity, and decrease collagen I expression.

Studies on Antiproliferative Effects of Phthalides from Ligusticum chuanxiong in Hepatic Stellate Cells

Suppression of hepatic stellate cell (HSC) growth and activation, and induction of apoptosis, have been proposed as therapeutic strategies for the treatment and prevention of liver fibrosis. Our previous study showed that the Chinese herb Ligusticum chuanxiong (LC) inhibits platelet-derived growth factor (PDGF-BB)-induced HSC proliferation. The present study was designed to investigate the active principles and their action mechanisms. With a bioactivity-directed fractionation approach, DNA synthesis (bromodeoxyuridine (BrdU) incorporation), cell cycle related proteins and apoptosis markers were determined to evaluate the inhibitory effects of active principles of LC. Two phthalides, Z,Z'-6,8',7,3'-diligustilide (1) and levistolide A (2), from LC significantly abrogated PDGF-BB-induced proliferation in both rat and human HSC lines. These inhibitory effects of compounds 1 and 2 were associated with reduction of alpha-smooth muscle actin and collagen expressions. The cell cycle promoting proteins, cyclins D1, D2, E, A and B1, were downregulated while the inhibitory proteins p21 and 27 were up-regulated. JNK phosphorylation was up-regulated by compounds 1 and 2. In HSC-T6, the two compounds induced apoptosis through the activation of caspases 9 and 3, increase in cytosolic cytochrome c release, and downregulation of Bcl-2 and Akt phosphorylation. Moreover, neither phthalides caused direct cytotoxicity to either HSCs or rat primary hepatocytes under experimental concentrations. These results indicate that two phthalides from LC inhibited PDGF-BB-activated HSC proliferation possibly through cell cycle inhibition and apoptosis mechanisms. They might be potential anti-fibrotic drugs for the treatment and prevention of hepatic fibrosis.

Studies on anti-proliferative effects of phthalides from Ligusticum chuanxiong in hepatic stellate cells

Aims: Suppression of hepatic stellate cell (HSC) growth and activation and induction of apoptosis have been proposed as therapeutic strategies for the treatment and prevention of liver fibrosis. Our previous study showed that a Chinese herb Ligusticum chuanxiong (LC) inhibits platelet-derived growth factor (PDGF-BB)-induced HSC proliferation. The present study was designed to investigate the active principles and their action mechanisms. With a bioactivity-directed fractionation approach, DNA synthesis (bromodeoxyuridine (BrdU) incorporation), cell cycle related proteins and apoptosis markers were determined to evaluate the inhibitory effects of active principles of LC. Two phthalides, Z,Z'-6,8',7,3'-diligustilide (1) and levistolide A (2), from LC significantly abrogated PDGF-BB-induced proliferation in both rat and human HSC lines. These inhibitory effects of compounds 1 and 2 were associated with reduction of α-smooth muscle actin and collagen expressions. The cell cycle promoting proteins, cyclins D1, D2, E, A and B1, were downregulated while the inhibitory proteins p21 and 27 up-regulated. JNK phosphorylation was up-regulated by compounds 1 and 2. In HSC-T6, the two compounds induced apoptosis through the activation of caspases 9 and 3 with cytochrome c release, and downregulation of Bcl-2 and Akt phosphorylation. Moreover, both phthalides did not cause direct cytotoxicity to either HSCs or rat primary hepatocytes under experimental concentrations. Conclusion: These results indicate that two phthalides from LC inhibited PDGF-BB-activated HSC proliferation possibly through cell cycle inhibition and apoptosis mechanisms. They might be potential anti-fibrotic drugs for the treatment and prevention of hepatic fibrosis.

Inhibitory Effect of Emodin on Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) Expression in Rat Hepatic Stellate Cells

Emodin inhibited expression of both transforming growth factor beta1 (TGFbeta1)- and phorbol ester (PMA)-induced tissue inhibitors of metalloproteinase-1 (TIMP-1) in an immortalized rat hepatic stellate cell line, HSC-T6, by Western blot and reverse transcription polymerase chain reaction. Reporter gene assays showed that emodin reduced both basal and PMA-induced activated protein-1 (AP-1) promoter activities. Electrophoretic mobility shift assay revealed that emodin reduced AP-1 DNA binding activities in HSC-T6 cells. AP-1 components analysis showed that emodin also attenuated JunD mRNA expression. Furthermore, emodin markedly inhibited TGFbeta1-induced p42/p44 mitogen-activated protein kinase phosphorylation but did not alter PMA induction. We conclude that emodin effectively inhibits PMA- and TGFbeta1-stimulated TIMP-1 expression in hepatic stellate cells by suppressing the AP-1 signaling pathway and extracellular signal-regulated kinase activation, respectively. These data provide new insight into the cellular and molecular mechanisms of emodin against liver fibrosis.

Adenosine A2A receptors play a role in the pathogenesis of hepatic cirrhosis

1. Adenosine is a potent endogenous regulator of inflammation and tissue repair. Adenosine, which is released from injured and hypoxic tissue or in response to toxins and medications, may induce pulmonary fibrosis in mice, presumably via interaction with a specific adenosine receptor. We therefore determined whether adenosine and its receptors contribute to the pathogenesis of hepatic fibrosis. 2. As in other tissues and cell types, adenosine is released in vitro in response to the fibrogenic stimuli ethanol (40 mg dl(-1)) and methotrexate (100 nM). 3. Adenosine A(2A) receptors are expressed on rat and human hepatic stellate cell lines and adenosine A(2A) receptor occupancy promotes collagen production by these cells. Liver sections from mice treated with the hepatotoxins carbon tetrachloride (CCl(4)) (0.05 ml in oil, 50 : 50 v : v, subcutaneously) and thioacetamide (100 mg kg(-1) in PBS, intraperitoneally) released more adenosine than those from untreated mice when cultured ex vivo. 4. Adenosine A(2A) receptor-deficient, but not wild-type or A(3) receptor-deficient, mice are protected from development of hepatic fibrosis following CCl(4) or thioacetamide exposure. 5. Similarly, caffeine (50 mg kg(-1) day(-1), po), a nonselective adenosine receptor antagonist, and ZM241385 (25 mg kg(-1) bid), a more selective antagonist of the adenosine A(2A) receptor, diminished hepatic fibrosis in wild-type mice exposed to either CCl(4) or thioacetamide. 6. These results demonstrate that hepatic adenosine A(2A) receptors play an active role in the pathogenesis of hepatic fibrosis, and suggest a novel therapeutic target in the treatment and prevention of hepatic cirrhosis.

Novel Inhibitors of Prolyl 4-Hydroxylase; Solid-phase Synthesis of 2,2-Dimethyl-3,4-Dialkoxy-Substituted 6-Aminobenzopyran Derivatives

2,2-Dimethyl-3,4-dialkoxy-substituted 6-aminobenzopyran analogues (eg., 7 and 8) were identified as prolyl 4-hydroxylase inhibitors via a screening process using HSC-T6 and LI 90 cells that express an immortalized rat hepatic stellate cell line and as part of a test of the type I collagen contents employing the ELISA method. A subsequent lead optimization effort based on solid-phase parallel synthesis led to the identification of 2,2-dimethyl-3,4-dialkoxy-substituted 6-aminobenzopyrans as potent inhibitors of prolyl 4-hydroxylase.

Halofuginone Induces Matrix Metalloproteinases in Rat Hepatic Stellate Cells via Activation of p38 and NFκB

The semisynthetic plant alkaloid halofuginone (HAL) was reported to prevent and partly reverse experimental liver fibrosis. However, its mechanisms of action are poorly understood. We therefore aimed to determine the antifibrotic potential of HAL and to characterize involved signal transduction pathways in hepatic stellate cells (HSCs). Results were compared with its in vivo effects in a rat model of reversal of established liver fibrosis induced by thioacetamide. In vitro HAL inhibited HSC proliferation and migration dose dependently at submicromolar concentrations. HAL (200 nm) up-regulated matrix metalloproteinase (MMP)-3 and MMP-13 expression between 10- and 50-fold, resulting in a 2- to 3-fold increase of interstitial collagenase activity. Procollagen alpha1(I) and MMP-2 transcript levels were suppressed 2- to 3-fold, whereas expression of other profibrogenic mRNAs remained unaffected. p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappaB(NFkappaB) pathways were activated by HAL, and specific inhibitors of p38 MAPK and NFkappaB dose dependently inhibited MMP-13 induction. Treatment with HAL did not affect HSC viability, and observed effects were reversible after its removal. In vivo HAL up-regulated MMP-3 and -13 mRNA expression 1.5- and 2-fold, respectively, in cirrhotic rats, whereas tissue inhibitor of metalloproteinase-1 was suppressed by 50%. In conclusion, submicromolar concentrations of HAL inhibit HSC proliferation and migration and up-regulate their expression of fibrolytic MMP-3 and -13 via activation of p38 MAPK and NFkappaB. The remarkable induction of MMP-3 and -13 makes HAL a promising agent for antifibrotic combination therapies.

Growth Arrest and Decrease of α-SMA and Type I Collagen Expression by Palmitic Acid in the Rat Hepatic Stellate Cell Line PAV-1

Liver fibrosis is characterized by an activation of hepatic stellate cells (HSC). During primary culture HSC evolve from a quiescent into an activated phenotype which is characterized by alpha-smooth muscle actin (alpha-SMA) up-regulation, increase in cell growth, and extracellular matrix secretion. HSC culture with trans-resveratrol can lead to deactivation of myofibroblast-like HSC. We used an HSC line, PAV-1, to check the role of retinol and palmitic acid in the deactivation process of HSC. Using mass and metabolic-based methods, Western blot and immunocytochemistry assays, we demonstrated that treatment with palmitic acid (75 muM) alone or in combination with retinol (2 muM) significantly decreased cell proliferation and alpha-SMA expression. We also established that the association of both compounds strongly decreased collagen type I expression. Our results suggest the potential use of palmitic acid alone or in combination with retinol to induce HSC deactivation.

722. Silencing of Transforming Growth Factor-β Receptor II by RNA Interference Suppresses Extracellular Matrix Production in Rat Hepatic Stellate Cells

BACKGROUND: Transforming growth factor-β (TGF-β) is a major pro-fibrogenic cytokine that mediates the activation of hepatic stellate cells (HSC) and their over-production of extracellular matrix (ECM) components, the two key events during hepatic fibrogenesis. Signal transduction of TGF-β is initiated by binding of the ligand to TGF-β type II receptor (TGFβRII). The aim of this study is to reduce ECM production in rat HSC by knocking-down TGFβRII with RNA interference (RNAi) strategies. METHODS: Selected siRNA were transfected in a rat HSC line, SBC10, using TransIT TKO transfecting reagent.

GFAP promoter directs lacZ expression specifically in a rat hepatic stellate cell line

The GFAP was traditionally considered to be a biomarker for neural glia (mainly astrocytes and non-myelinating Schwann cells). Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells (HSC). In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. The GFAP-lacZ transgene was transfected into various cell lines (HSC, hepatocyte, and other non-HSC cell types). The transgene expression specificity was determined by X-gal staining of the beta-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-beta1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-beta1 by upregulation in a dose- and time-dependent manner, similar to the endogenous GFAP. In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.

Anti-proliferative and pro-apoptotic effects of herbal medicine on hepatic stellate cell

Hepatic stellate cells (HSC) play a central role in hepatic fibrosis and compounds that promote apoptosis in HSC may have anti-fibrotic potentials. Herbal medicine has long been used in chronic liver disease but there is little scientific evidence for their actions. The present study investigated the effects of 14 commonly used herbs on cellular proliferation and apoptosis of a rat hepatic stellate cell line, HSC-T6 and the underlying mechanism of herb-induced apoptosis. HSC-T6 cell were incubated with herbal extracts and their proliferation was assessed by colorimetric assay. Apoptosis was measured and confirmed by flow cytometry, terminal transferase uridyl nick end labeling (TUNEL) assay and morphological features in hematoxylin and eosin staining. Apoptotic pathways involving Fas receptor and Bcl-2 family were investigated by Western blot. Five herbs, namely Angelica sinensis (AS), Carthamus tinctorius (CT), Ligusticum chuanxiong (LC), Salvia miltiorrhiza (SM) and Stephania tetrandra (ST) demonstrated both anti-proliferative and pro-apoptotic activities in HSC-T6. The highest potency was detected in SM and ST with 51.63 and 44.52% of HSC-T6 showing apoptotic changes, respectively. This was associated with upregulation of Fas and Bax and down-regulation of Bcl-xL in HSC. Fas ligand and Bcl2 expressions remained unchanged. The potential anti-fibrotic effect of herbal medicine warrants further evaluation.

Three-dimensional co-culture of hepatocytes and stellate cells

Hepatocytes self-assemble in culture to form compacted spherical aggregates, or spheroids, that mimic the structure of the liver by forming tight junctions and bile canalicular channels. Hepatocyte spheroids thus resemble the liver to a great extent. However, liver tissue contains other cell types and has bile ducts and sinusoids formed by endothelial cells. Reproducing 3-D co-culture in vitro could provide a means to develop a more complex tissue-like structure. Stellate cells participate in revascularization after liver injury by excreting between hepatocytes a laminin trail that endothelial cells follow to form sinusoids. In this study we investigated co-culture of rat hepatocytes and a rat hepatic stellate cell line, HSC-T6. HSC-T6, which does not grow in serum-free spheroid medium, was able to grow under co-culture conditions. Using a three-dimensional cell tracking technique, the interactions of HSC-T6 and hepatocyte spheroids were visualized. The two cell types formed heterospheroids in culture, and HSC-T6 cell invasion into hepatocyte spheroids and subsequent retraction was observed. RT-PCR revealed that albumin and cytochrome P450 2B1/2 expression were better maintained in co-culture conditions. These three-dimensional heterospheroids provide an attractive system for in vitro studies of hepatocyte-stellate cell interactions.

Zinc pretreatment prevents hepatic stellate cells from cadmium-produced oxidative damage

Pretreatment with zinc produces tolerance to several cadmium toxic effects. This study was performed to further elucidate the mechanism of zinc-induced tolerance to cadmium cytotoxicity in a rat hepatic stellate cell line (CFSC-2G). Twenty four hours after seeding, cells were treated with 60 micromol/L ZnCl2 for 24 h. Following zinc pretreatment, cells were exposed to 3 micromol/L and 5 micromol/L CdCl2 for an additional 24 h. The toxicity of cadmium was significantly reduced in the zinc-pretreated cells. Zinc pretreatment produced a decrease in lipid peroxidation damage of cadmium-treated cells. Glutathione cell content diminished 33% and 43% as a result of 3 micromol/L and 5 micromol/ L CdCl2 treatment, respectively. Cell pretreatment with zinc recovered glutathione content at control cells level. Catalase and glutathione peroxidase activities were also recovered to control values with zinc pretreatment. Cadmium (5 micromol/L) was able to induce 39% the expression of alpha1 collagen (I) gene after 1 h treatment, while zinc pretreatment prevented this cadmium profibrogenic effect. We also examined the production of heat shock protein 70 (Hsp70) as a cellular response to oxidative stress produced by cadmium. By Western blot analysis, a 1.3 and 3 times increment in Hsp70, with 3 micromol/L and 5 micromol/L CdCl2 treatment, respectively, was observed. Zinc pretreatment prevented the production of Hsp70. Metallothionein-II (MT-II) gene expression was induced by cadmium, but the induction was unaffected with zinc pretreatment. These data suggest that zinc-induced protection against the cytotoxicity of cadmium in stellate cells may be related to the maintenance of normal redox balance inside the cell.

Regulatory role of vHL/HIF-1α in hypoxia-induced VEGF production in hepatic stellate cells

Activated hepatic stellate cells (HSCs) produce cyclooxygenase-2 (COX-2) protein to induce vascular endothelial growth factor (VEGF) production that participates in angiogenesis in injured liver. To reveal the unknown regulatory mechanism, we used hypoxic atmosphere mimicking injured-tissue microenvironment to induce VEGF expression in a rat hepatic stellate cell line (T6-HSCs). The present study showed that hypoxia up-regulated the protein levels of COX-2 and hypoxia-inducible factor-1-α (HIF-1α), but rapidly effected degradation of von Hippel–Lindau (vHL) protein. As a result, expression of VEGF in HSCs was markedly elevated; and pretreatment with COX-2 inhibitors (nimesulide or indomethacin) could significantly ameliorate the angiogenic event. Collectively, hypoxic HSCs increased accumulation of HIF-1α protein and induced VEGF expression in a time-dependent manner. Inhibition of COX-2 activities would prevent vHL protein from degradation and suppress HIF-1α up-regulation. Thus, vHL/HIF-1α has a regulatory role in COX-2-mediated VEGF production in hypoxic stellate cells in injured liver.

PAR1 antagonism protects against experimental liver fibrosis. Role of proteinase receptors in stellate cell activation.

In fibroblasts, thrombin induces collagen deposition through activation of a G-protein-coupled receptor, proteinase-activated receptor 1 (PAR(1)). In the current study, we examined whether PAR(1) antagonism inhibits hepatic stellate cell (HSC) activation in vitro and whether it protects against fibrosis development in a rodent model of cirrhosis. A rat HSC line was used for in vitro studies whereas cirrhosis was induced by bile duct ligation (BDL). The current results demonstrated that HSCs express PAR(1), as well as proteinase-activated receptors 2 (PAR(2)) and 4 (PAR(4)), and that all three PARs were up-regulated in response to exposure to growth factor in vitro. Exposure to thrombin and to SFLLRN-(SF)-NH(2), a PAR(1) agonist, and GYPGKF (GY)-NH(2), a PAR(4) agonist, triggered HSC proliferation and contraction, as well as monocyte chemotactic protein-1 (MCP-1) production and collagen I synthesis and release. These effects were inhibited by the PAR(1) antagonist. Administration of this antagonist, 1.5 mg/kg/d, to BDL rats reduced liver type I collagen messenger RNA (mRNA) expression and surface collagen by 63%, as measured by quantitative morphometric analysis. Similarly, hepatic and urinary excretion of hydroxyproline was reduced significantly by the PAR(1) antagonist. In conclusion, PAR(s) regulates HSC activity; development of PAR antagonists might be a feasible therapeutic strategy for protecting against fibrosis in patients with chronic liver diseases.

Establishment and identification of a novel immortalized rat hepatic stellate cell line HSC-PQ

Establishment and identification of a novel immortalized rat hepatic stellate cell line HSC-PQ

Triiodothyronine and interleukin-6 (IL-6) induce expression of HGF in an immortalized rat hepatic stellate cell line.

Despite its being considered a primary mitogen for hepatocytes, triiodothyronine (T3) has no effect on the proliferation of hepatocytes in vitro, and in our studies, induces significant in vivo hepatocyte proliferation only during liver injury. We hypothesized that T3 may affect hepatocytes proliferation indirectly, by inducing other cells in the liver to secrete hepatic mitogens. In vivo studies: Lipopolysaccharide, T3 and a combination of the two were injected into rats, and hepatocyte proliferation was determined by PCNA staining and mitotic index. a rat hepatic stellate cell line (HSC-6T) was cultured with T3, IL-6 and a combination of the two, and we assessed the effect of these cytokine/hormone combinations on the cell proliferation and on secretion of IL-6 and HGF, measured by ELISA. Expression of thyroid hormone receptors was assessed by RT-PCR. In vivo: T3, together with lipopolysaccharide, enhances PCNA staining and the mitotic index of hepatocytes in the treated rats. In vitro: the hepatic stellate cell line expresses thyroid hormone receptor alpha 1, but not beta 1. Proliferation of stellate cells is not affected by T3, with or without IL-6. T3 has no effect on secreted levels of IL-6 in the stellate cell line. Hepatic stellate cells cultured with T3 and IL-6 show significantly increased amounts of secreted HGF after 48 h in culture. T3 may induce hepatocyte proliferation in vivo during injury by turning on expression of HGF in stellate cells and acting together with IL-6.

Proangiogenic role of tumor-activated hepatic stellate cells in experimental melanoma metastasis

Myofibroblasts infiltrate malignant liver tumors, although their pathogenic implications are unclear. Immunohistochemical detection of α-smooth muscle actin, glial fibrillary acidic protein (GFAP), and CD31 and CD34 expression was used to analyze the contribution of myofibroblasts to angiogenesis in hepatic metastasis produced by intrasplenically-injected B16 melanoma (B16M). Because activated hepatic stellate cells (HSCs) are oxygen-sensing myofibroblasts producing vascular endothelial growth factor (VEGF), the effect of B16M and human A375 melanoma supernatants on VEGF production by immortalized rat HSC line T6 and primary cultured human HSCs also was studied under an hypoxic atmosphere mimicking a tumor microenvironment. Myofibroblast infiltration preceded endothelium recruitment in avascular micrometastasis and generated specific stroma for sinusoidal-type and portal-type angiogeneses. Thereafter, myofibroblasts and endothelial cells colocalized within both angiogenic patterns and their numerical densities correlated with metastasis development. Myofibroblasts often were GFAP-positive, suggesting an HSC origin. Melanoma supernatants stimulated VEGF messenger RNA and protein synthesis by HSCs. These effects were potentiated by hypoxia. VEGF up-regulation was accompanied by increased expression of cyclooxygenase type 2 (COX-2) and PGE2 synthesis. HSC production of VEGF decreased under COX-2 inhibition, whereas it was increased by exogenous PGE2. The high VEGF expression in HSCs induced by melanoma factors and hypoxia resulted in mitogenic, antiapoptotic, and motogenic stimulation of both murine hepatic sinusoidal endothelium and human umbilical vein endothelium. In conclusion, temporal and positional relationships evolve between myofibroblast and endothelium recruitment during metastasis development. Mechanistically, hypoxic induction of VEGF in tumor-activated HSCs may create a proangiogenic microenvironment, facilitating endothelial cell recruitment and survival during hepatic metastasis transition from an avascular to a vascular stage. (H EPATOLOGY 2003;37:674-685.)

PENTOXIFYLLINE DIMINISHED ACETALDEHYDE-INDUCED COLLAGEN PRODUCTION IN HEPATIC STELLATE CELLS BY DECREASING INTERLEUKIN-6 EXPRESSION

The effect of pentoxifylline (PTX), a methylxanthine derivative, on collagen induction and secretion and on the production of mRNA of two fibrogenic cytokines: interleukin-6 and transforming growth factor- β 1 (IL-6 and TGF- β 1) in a rat hepatic stellate cell line (CFSC-2G) exposed to acetaldehyde was studied. CFSC-2G cells were treated with 175 μM acetaldehyde for 24 h. The cells were then exposed to a medium containing 200 μM PTX. Collagen secretion, increased 2.6 times in acetaldehyde treated cells. Cells exposed to acetaldehyde and treated with PTX diminished collagen secretion to control values and decreased α 1(I) collagen mRNA by 15%. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays of TGF- β 1 mRNA showed no variation in different experimental conditions. However, PTX induced a decrease of 32% in IL-6 mRNA in acetaldehyde-treated cells. CFSC-2G cells treated with anti-IL-6 monoclonal antibody, 15 min before acetaldehyde was added, did not present an increase in α 1(I) collagen mRNA. These results show that PTX inhibits the expression of α 1(I) collagen via the inhibition of IL-6 in acetaldehyde treated cells. The effect herein reported on IL-6 and α 1(I) collagen mRNA adds to the previously described effect of PTX, which could be useful in the fibrogenic process induced by acetaldehyde.