Successful targeting to rat hepatic stellate cells using albumin modified with cyclic peptides that recognize the collagen type VI receptor.

The key pathogenic event in liver fibrosis is the activation of hepatic stellate cells (HSC). Consequently, new antifibrotic therapies are directed toward an inhibition of HSC activities. The aim of the present study was to develop a drug carrier to HSC, which would allow cell-specific delivery of antifibrotic drugs thus enhancing their effectiveness in vivo. We modified human serum albumin (HSA) with 10 cyclic peptide moieties recognizing collagen type VI receptors (C*GRGDSPC*, in which C* denotes the cyclizing cysteine residues) yielding pCVI-HSA. In vivo experiments showed preferential distribution of pCVI-HSA to both fibrotic and normal rat livers (respectively, 62 +/- 6 and 75 +/- 16% of the dose at 10 min after intravenous injection). Immunohistochemical analysis demonstrated that pCVI-HSA predominantly bound to HSC in fibrotic livers (73 +/- 14%). In contrast, endothelial cells contributed mostly to the total liver accumulation in normal rats. In vitro studies showed that pCVI-HSA specifically bound to rat HSC, in particular to the activated cells, and showed internalization of pCVI-HSA by these cells. In conclusion, pCVI-HSA may be applied as a carrier to deliver antifibrotic agents to HSC, which may strongly enhance the effectiveness and tissue selectivity of these drugs. This approach has the additional benefit that such carriers may block receptors that play a putative role in the pathogenesis of liver fibrosis.

Glutathione levels discriminate between oxidative stress and transforming growth factor-beta signaling in activated rat hepatic stellate cells.

Reactive oxygen species are implicated in the pathogenesis of several diseases, including Alzheimer's disease, multiple sclerosis, human immunodeficiency virus, and liver fibrosis. With respect to liver fibrosis, we have investigated differences in antioxidant enzymes expression in stellate cells (SCs) and parenchymal cells from normal and CCl(4)-treated rat livers. We observed an increase in the expression of catalase in activated SCs. Treatment with transforming growth factor-beta (TGF-beta) increased the production of H(2)O(2). Treatment with catalase decreased TGF-beta expression. Addition of H(2)O(2) resulted in increased TGF-beta production. 3-Amino-1,2,4-triazole abolished the capacity of SCs to remove H(2)O(2). A paradoxical increase in capacity was observed when the cells were pretreated with diethyl maleate. Treatment with 3-amino-1, 2,4-triazole increased TGF-beta production. A paradoxical decrease of TGF-beta production was observed with diethyl maleate. Treatment of the cells with N-acetylcysteine resulted in increased TGF-beta production. TGF-beta decreased the capacity of the SCs to remove H(2)O(2.) An increase in the capacity to remove H(2)O(2) was observed when TGF-beta was removed by neutralizing antibodies. In conclusion, our results suggest: 1) a link between cellular GSH levels and TGF-beta production and 2) that cellular GSH levels discriminate whether H(2)O(2) is the result of oxidative stress or acts as second messenger in the TGF-beta signal transduction pathway.

Insufficient TGF-beta 1 production inactivates the autocrine growth suppressive circuit in human ovarian cancer cell lines.

TGF-beta 1 is a secreted polypeptide that elicits an antiproliferanve response in many cell types. However, many epithelial cancer cell lines are resistant to TGF-beta 1 growth inhibition. We investigated the in vitro growth suppressive effect of TGF-beta 1 on five ovarian cancer cell lines. Two of these (OVCAR-3 and AZ364) were growth inhibited by TGF-beta 1. The other three cell lines (SKOV-3, AZ224 and AZ547), were resistant to the antiproliferative action of the cytokine. All five cell lines produce TGF-beta 1 mRNA at very different levels and also secrete the TGF-beta 1 polypeptide, but mainly in a biologically latent form as tested by ELISA; this probably explains the fact that the TGF-beta 1 autocrine growth inhibition circuit is not active, even in sensitive cell lines. Even complete activation of the in vitro secreted latent form would be insufficient to induce growth arrest when compared to the levels of exogenous TGF-beta 1 needed to induce growth arrest in sensitive cell lines. The TGF-beta 1 receptor type I mRNA is expressed by all five ovarian cancer cell lines, but two of them (AZ224 and AZ547) lack detectable TGF-beta 1 receptor type II mRNA expression. Since TGF-beta 1 signaling requires both receptor types, the lack of receptor type II in two cell lines may explain their resistance to growth inhibition. Further experiments should be carried out on receptors and downstream components to pinpoint the cause of resistance in the SKOV cell line.

Localization and cellular sources of activins in normal and fibrotic rat liver.

UNLABELLED: Activins are dimeric proteins, members of the transforming growth factor beta (TGF-beta) gene superfamily, consisting of the beta-subunits of inhibin (betaA and betaB). Recently, it was shown that activin A (betaA:betaA) inhibits DNA replication and induces apoptosis in rat parenchymal cells in vitro and in vivo. Cryostat sections of normal livers and livers of rats treated with intraperitoneal injections of CCl4 were stained for the different inhibin subunits and desmin, a marker for stellate cells (SC). Staining for inhibin-alpha was invariably negative, both in normal and fibrotic rat liver. In normal liver, inhibin-betaA subunit immunoreactivity was localized in parenchymal cells (PC). Staining for inhibin-betaB was weaker but similarly distributed. In fibrotic livers, connective tissue septa were strongly immunoreactive for inhibin-betaA. Desmin-positive stellate cells (SC) accumulated in areas strongly immunoreactive for inhibin-betaA and several cells were positive for both desmin and inhibin-betaA. Staining for inhibin-betaB was weaker but similarly distributed. As above data pointed to a possible role for PC and SC, we examined by Northern blot analysis, the expression of inhibin-alpha, -betaA, and -betaB messenger RNA (mRNA) in total RNA extracted from freshly isolated SC and PC of normal and CCl4-treated liver and in cultured SC. Inhibin-betaA mRNA was predominantly expressed in PC of normal liver. Expression was lost in PC of CCl4-treated liver. Inhibin-betaB mRNA expression was induced in SC of CCl4-treated liver. Inhibin-betaA mRNA, and to a lesser extent, inhibin-betaB mRNA expression was rapidly induced in cultured SC. The presence of activin A in conditioned media of cultured SC was shown by Western blotting. Apoptotic cells, identified by terminal deoxy-transferase mediated X-dUTP nick end labeling (TUNEL)-staining, were found predominantly in and near the fibrotic septa. IN CONCLUSION: 1) while activin A was constitutively expressed in PC of normal liver, its expression was lost in PC of fibrotic liver; 2) expression of activins was induced in activated SC; and 3) apoptotic cells were found predominantly near the septa, in support of the hypothesis that activin A, derived from activated SC in the septa, contributes to the induction of cell death in neighboring PC.

Transforming growth factor-beta gene expression in normal and fibrotic rat liver.

BACKGROUND/AIMS: Transforming growth factor-beta (TGF-beta) is considered to be an important mediator in the development of fibrosis in several chronic liver diseases. To understand the mechanism(s) by which TGF-beta exerts its action(s), we investigated the cellular distribution of TGF-beta(1,2,3) transcripts in normal and carbon tetrachloride (CCl4)-induced fibrotic rat liver. METHODS: Parenchymal, sinusoidal endothelial, Kupffer and stellate cells were isolated and purified. The exact cellular composition of each isolate was determined by transmission electron microscopy. Expression of TGF-beta(1,2,3) transcripts was investigated using Northern hybridization analysis. Hybridization signals were quantified by scanning densitometry and corrected for: (i) differences in extractable RNA per cell type, (ii) signal contribution from contaminating cells, and (iii) differences in loading, capillary transfer and hybridization. RESULTS: In normal liver, TGF-beta1 mRNA was predominantly expressed in Kupffer cells, exhibiting values approximately 9-fold higher than those in stellate cells. No expression was found in endothelial and parenchymal cells. Signals for TGF-beta2 and TGF-beta3 were much weaker when compared to TGF-beta1. In Kupffer cells, the level of TGF-beta2 was approximately 4-fold higher than in stellate cells. Little expression was found in endothelial cells. TGF-beta3 expression could only be detected in stellate cells. TGF-beta2 and TGF-beta3 was not expressed in parenchymal cells. In fibrotic liver, TGF-beta1 mRNA was strongly expressed in all the sinusoidal cells. TGF-beta2 and TGF-beta3 could no longer be detected. When compared to the level of expression in normal stellate cells, the level of TGF-beta1 increased 12-fold in stellate cells from fibrotic livers, and 6-fold in endothelial cells. In Kupffer cells, the level of expression remained unchanged. CONCLUSIONS: (i) In both normal and fibrotic liver, TGF-beta1 is the most abundant isoform, (ii) in normal liver, TGF-beta1 is expressed strongly by Kupffer cells and moderately by stellate cells, TGF-beta2 expression is highest in Kupffer cells, followed by stellate cells and endothelial cells. TGF-beta3 is expressed by stellate cells, (iii) in fibrotic liver, the level of TGF-beta1 expression increases selectively in stellate cells and endothelial cells. This suggests an important role, not only for stellate, but also for endothelial cells in fibrogenesis.

Insulin-like growth factor II/mannose 6-phosphate-receptor expression in liver and serum during acute CCl4 intoxication in the rat.

The liver is reported to be the main source of soluble insulin-like growth factor-II/mannose 6-phosphate (IGF-II/M6P) receptor in adults. In view of the role of this receptor in the activation of transforming growth factor beta (TGF-beta) during hepatic fibrogenesis, we have investigated the correlation between serum levels and tissue expression of the receptor during acute CCl4 intoxication of the rat. Sixteen hours after CCl4, injection, the level of the soluble receptor in serum, as measured by radioimmunoassay (RIA), increased threefold. At 24 hours, values almost returned to normal, but increased again by twofold at 48 hours. By 96 hours, nearly normal values were obtained. Northern blot analysis showed peaks in tissue IGF-II/M6P receptor messenger RNA (mRNA) levels at 24 hours and at 48 hours. In normal liver, immunostaining for IGF-II/M6P receptor showed weak positivity in parenchymal cells. CCl4-induced hydropic changes appeared in centrilobular parenchymal cells (PCs) at 8 hours. These changes extended to the midzonal region at 16 hours. Hydropic cells were devoid of receptor staining. The hydropic wave became extinct at 32 hours. At 48 hours, we observed a collapse of PCs in the centrilobular zone, coinciding with strongly positive staining for IGF-II/M6P receptor in fat-storing cells (FSCs), identified by dual IGF-II/M6P receptor and desmin immunostaining. Between 48 and 72 hours, the liver gradually regained its normal appearance. As shown by Western blotting, in vitro differentiated FSCs released soluble receptor in the medium. Northern blot analysis showed this release to be preceded by an increased receptor-mRNA expression, whereas immunostaining showed an increase of intracellular receptor. In conclusion, we have shown that acute CCl4 intoxication induces two peaks in serum levels of soluble receptor. While the first peak at 16 hours coincides with the loss of receptor-staining in hydropically damaged PCs, the second peak at 48 hours is paralleled by an increase in positive staining in FSCs and tissue mRNA level. Differentiated FSCs shed soluble receptor in vitro. As a consequence, these cells might contribute to the serum levels of soluble receptor in vivo. These results indicate that measuring serum soluble IGF-II/M6P receptor might useful in the diagnosis of early acute liver damage.

Comparison of glial fibrillary acidic protein and desmin staining in normal and CCl4-induced fibrotic rat livers.

Fat-storing cells are the major producers of extracellular matrix in the liver. A good immunocytochemical marker is, however, still lacking for this cell type. Desmin, frequently used by most investigators, fails to stain many pericentral fat-storing cells in normal rat liver. The aim of the present study is to evaluate glial fibrillary acidic protein (GFAP) as an alternative marker of fat-storing cells. In normal rat liver, immunostaining of GFAP revealed numerous fat-storing cells with characteristic cytoplasmic extensions. Unlike desmin, which was preferentially expressed in periportal fat-storing cells, GFAP-positive fat-storing cells were distributed more evenly in the lobules. In a narrow periportal zone, however, GFAP-positive cells were occasionally absent. Dual GFAP/desmin staining revealed colocalization of these markers, but fat-storing cells positive only for GFAP or desmin were also present. Chronic carbon tetrachloride exposure induced a spatial change in the expression of GFAP and desmin. At 3 weeks, accumulation of GFAP/desmin double-positive cells was observed in developing fibrotic septa. At 8 weeks, the GFAP positivity in the septa persisted but became weak, while desmin expression became stronger. In contrast, the expression of GFAP within the lobule was gradually decreased as fibrosis progressed. We conclude that GFAP is expressed by a subpopulation of fat-storing cells, which differs partially from the population that expresses desmin. Because in normal rat liver desmin-negative fat-storing cells can be identified by GFAP staining and vice versa, dual GFAP/desmin staining allows more complete identification of fat-storing cells. In chronically injured liver, GFAP may not be as useful as in normal rat liver. The coexpression of GFAP/desmin in developing septa and the subsequent downregulation of GFAP in an advanced stage of fibrosis may reflect different stages of fat-storing cell activation. Further investigation is required to determine the functional significance of alteration of GFAP expression in fat-storing cells.

Dexamethasone alters messenger RNA levels but not synthesis of collagens, fibronectin, or laminin by cultured rat fat-storing cells.

Glucocorticoids have been shown to suppress collagen synthesis and gene expression by fibroblasts. However, little is known about their effects on fat-storing cells, the major matrix-producing cells in liver fibrosis. In this study we investigated the effect of dexamethasone on the extracellular matrix expression by cultured rat fat-storing cells. Fat-storing cells were isolated from male Wistar rats by collagenase/pronase digestion and purified by density gradient centrifugation. Fat-storing cells in early primary culture (3-day-old, representing a relatively quiescent phenotype) and in subculture (one passage, about 2-week-old, representing an activated phenotype) were treated with 10(-6) mol/L dexamethasone for messenger RNA (mRNA) study or with 10(-8) to 10(-6) mol/L dexamethasone for protein study. Expression of collagen type I, III, IV, fibronectin, and laminin was analyzed at the mRNA level by Northern hybridization, and at the protein level by metabolic labeling and immunoprecipitation. Dexamethasone had a variable effect on the expression of collagen alpha1(I) mRNA level. While a tendency for modest suppression was observed (5%-50%) in primary cells, the difference was not statistically significant. Variable response was observed in subcultured cells. Collagen alpha1(III) mRNA level showed a tendency for stimulation. Dexamethasone stimulated the expression of collagen alpha1 (IV), fibronectin, and laminin B1 mRNA levels by 1.4-, 2.4-, and 1.6-fold respectively, in primary fat-storing cells. Subcultured cells showed a similar response, but the magnitude of stimulation was more variable than that of primary cells. Unexpectedly, at the protein level dexamethasone had no effect on the expression of these proteins. Our results indicate that glucocorticoids do not possess a net suppressive effect on extracellular matrix synthesis by fat-storing cells. Beneficial effects of glucocorticoids may be attributable to other mechanisms of action, such as their anti-inflammatory effect.

Insulinlike growth factor-II/mannose 6-phosphate receptor is expressed on CCl4-exposed rat fat-storing cells and facilitates activation of latent transforming growth factor-beta in cocultures with sinusoidal endothelial cells.

Transforming growth factor beta (TGF-beta), a potent fibrogenic cytokine, is secreted in latent form. We examined which cell type in both normal and carbon tetrachloride (CCl4)-induced fibrotic rat liver bears surface type II IGF/mannose 6-phosphate (IGF-II/M6P) receptor, known to facilitate activation of TGF-beta. In addition, the role of the IGF-II/M6P receptor in activation of latent TGF-beta was investigated in a coculture system with sinusoidal endothelial cells. Northern hybridization analysis for IGF-II/M6P receptor messenger RNA (mRNA) was performed on total RNA of different isolated and purified liver cell types. In normal liver, cells expressed little IGF-II/M6P receptor mRNA. In fibrotic liver, we found significant expression only in fat-storing cells. The presence of IGF-II/M6P receptors was established by [125I]IGF-II binding assays on freshly isolated fat-storing cells from normal and CCl4-exposed rat livers. We found specific binding of [125I]IGF-II only on CCl4 exposed fat-storing cells. As determined by polyacrylamide gel electrophoresis after affinity labeling, the specific binding involved 220 kD type II IGF receptors. Scatchard analysis revealed the presence of 3,043 +/- 1,378 IGF-II/M6P high-affinity receptors/fat-storing cell, with a Kd of 387 = 165 pmol/L. With a mink lung epithelial cell (Mv1Lu) proliferation inhibition assay, inhibition of proliferation (a measure of active TGF-beta function) was determined using conditioned media of activated fat-storing cells, cocultures of fat-storing cells, and endothelial cells and pure endothelial cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)