Raffaella DeFranco

Expression of platelet-derived growth factor in newly formed cholangiocytes during experimental biliary fibrosis in rats.

BACKGROUND/AIMS Chronic cholestasis stimulates a fibroductular reaction which may progress to secondary biliary fibrosis and cirrhosis. Since platelet-derived growth factor has been indicated as a major fibrogenic factor in chronic liver disease, we analyzed its expression and that of its receptor beta subunit in a rat model of chronic cholestasis. METHODS Liver tissue samples collected at 7, 10, 21, and 28 days after induction of cholestasis obtained by bile duct ligation, were analyzed by immunohistochemistry, in situ hybridization and RNase protection assay for the expression of platelet-derived growth factor (PDGF)-B chain and receptor beta subunit. Furthermore, the expression of PDGF-B chain mRNA was analyzed in highly purified cholangiocytes from normal and cholestatic rat liver. RESULTS In cholestatic liver, platelet-derived growth factor-BB and B chain mRNA expression increased up to 4 weeks in epithelial cells of proliferating bile ducts, and periductular mesenchymal cells. The increased expression of PDGF-B chain mRNA was confirmed in highly purified cholangiocytes obtained from normal and cholestatic rat liver. The expression of the receptor beta subunit progressively increased after induction of cholestasis and was mainly localized to desmin-positive periductular hepatic stellate cells. CONCLUSIONS These data suggest that platelet-derived growth factor-B chain can be synthesized by cholangiocytes during chronic cholestasis. The presence of its receptor on periductular hepatic stellate cells raises the possibility that, in this experimental setting, this cytokine might contribute to fibrogenesis in vivo.

gp120 modulates the biology of human hepatic stellate cells: a link between HIV infection and liver fibrogenesis

Objective In patients with hepatitis C virus (HCV)/HIV co-infection, a faster progression of liver fibrosis to cirrhosis has been reported. In this study, an investigation was carried out to determine whether gp120, an HIV envelope protein, modulates the biology of human hepatic stellate cells (HSCs), key cell types in the pathogenesis of fibrosis. Methods Myofibroblastic HSCs were isolated from normal human liver tissue. Gene expression was measured by real-time PCR. Cell migration was assessed in Boyden chambers. Intracellular signalling pathways were evaluated using phosphorylation-specific antibodies or by transfection of a reporter plasmid. Results Transcripts for the chemokine receptors CCR5 and CXCR4, which bind gp120, were detectable in human HSCs. Upon exposure to M-tropic recombinant gp120, which binds CCR5, a significant increase in HSC chemotaxis was observed (1.6±0.3-fold, p=0.03). The effects of gp120 were prevented by protein inactivation. gp120 also resulted in a significant increase in secretion (1.5±0.3-fold, p=0.03) and gene expression (1.47±0.13-fold, p=0.02) of the proinflammatory chemokine monocyte chemoattractant protein-1, and in increased gene expression of tissue inhibitor of metalloprotease-1 and interleukin-6 (2.03±0.57-fold, p=0.02). gp120-induced migration required Akt activation. gp120 also induced activation of nuclear factor-κB (NF-κB) and p38MAPK. Preincubation of HSCs with TAK779, a CCR5 receptor antagonist, prevented gp120-mediated chemotaxis and monocyte chemoattractant protein-1 secretion. Expression of CCR5 was detectable in areas of inflammation and fibrogenesis in liver biopsies of patients with HCV/HIV co-infection. Conclusions This study shows that HIV gp120 modulates different aspects of HSC biology, including directional cell movement and expression of proinflammatory cytokines. These results identify a direct pathway possibly linking HIV infection with liver fibrogenesis via envelope proteins.

Up-regulated expression of fractalkine and its receptor CX3CR1 during liver injury in humans

BACKGROUND/AIMS Little is known about the role of fractalkine (CX3CL1) in the liver. The aim of this study was to investigate the expression patterns of fractalkine and its receptor CX3CR1 in normal human liver and in conditions of injury. METHODS Distribution and expression of fractalkine and its receptor were investigated using immunohistochemistry, in situ hybridization, flow cytometry and reverse transcriptase-polymerase chain reaction. In vitro experiments were conducted in HepG2 cells. RESULTS Both fractalkine and CX3CR1 were up-regulated during chronic injury, in areas of portal and lobular inflammation. In severe acute hepatitis, fractalkine and CX3CR1 were expressed at high levels not only in areas of inflammation but also in regenerating epithelial cells within bile duct-like structures, which showed co-expression of fractalkine and cytokeratin-7 or CX3CR1. The human hepatocarcinoma cell line HepG2 expressed fractalkine at the gene and protein level, and HepG2-conditioned medium was chemotactic for cells overexpressing CX3CR1. Transcripts for CX3CR1 were detected in HepG2, and exposure of these cells to recombinant fractalkine induced cell migration. CONCLUSIONS This study shows that the fractalkine system is up-regulated during liver damage, and suggests that fractalkine may play a role in the recruitment and adhesion of inflammatory cells and in the biology of liver epithelial cells.

Involvement of phosphatidylinositol 3‐kinase in the activation of extracellular signal‐regulated kinase by PDGF in hepatic stellate cells

Phosphatidylinositol 3‐kinase (PI 3‐K) is a lipid and protein kinase which associates with the activated platelet‐derived growth factor (PDGF) receptor and other tyrosine kinases. We studied the effects of wortmannin, a selective inhibitor of PI 3‐K, on the activation of extracellular‐signal regulated kinase (ERK) by PDGF in cultured hepatic stellate cells, mesenchymal cells responsible for extracellular matrix synthesis within the liver. Incubation with 100 nM wortmannin, a dose which almost completely blocks PI 3‐K, resulted in 50% reduction of ERK activity. Direct inhibition of ERK by wortmannin could not be considered responsible for this effect, since wortmannin did not inhibit ERK activity in vitro. Rather, inhibition of PI 3‐K acts on the kinase cascade that leads to ERK activation, since PDGF‐dependent phosphorylation of ERK was found to be reduced after incubation with wortmannin. Wortmannin also inhibited the increase in c‐fos mRNA induced by PDGF, which is dependent on ERK activation. The results of this study show that in hepatic stellate cells PI 3‐K is involved in ERK activation, although it is not necessary. These data indicate cross‐talk between PI 3‐K and the Ras/ERK pathway in PDGF‐stimulated cells.

Inhibition by pentoxifylline of extracellular signal‐regulated kinase activation by platelet‐derived growth factor in hepatic stellate cells

1 It has been proposed that pentoxifylline (PTF) acts an antifibrogenic agent by reducing the synthesis of extracellular matrix components, and this possibility has been confirmed in animal models of hepatic fibrosis. In this study the effects of PTF on the proliferation of extracellular matrix producing cells induced by platelet‐derived growth factor (PDGF) were evaluated. The study was performed on hepatic stellate cells, currently indicated as the major source of extracellular matrix in fibrotic liver. 2 PTF caused a dose‐dependent reduction of PDGF‐induced mitogenesis with an IC50 of 170 μm, identical to the EC50 for the increase in intracellular cyclic AMP levels. Preincubation with PTF did not affect either PDGF‐receptor autophosphorylation or phosphotidylinositol 3‐kinase activity, whereas it markedly reduced PDGF‐stimulated extracellular signal‐regulated kinase (ERK) activity and ERK isoform phosphorylation. PTF also reduced PDGF‐induced c‐fos mRNA expression, which is dependent on activation of the RAS/ERK pathway. In addition, the PDGF‐induced increase in cytsolic‐free calcium was almost completely prevented by pretreating the cells with PTF. 3 The results of the present study indicate that PTF, in addition to its effect on collagen deposition and degradation, may exert an antifibrogenic effect by reducing the PDGF‐induced proliferation of extracellular matrix producing cells. This effect appears to be mediated by a reduction of PDGF‐stimulated ERK activity as well as of other intracellular signalling pathways such as the PDGF‐induced elevation of cytosolic‐free calcium.

Effect of pentoxifylline on the degradation of procollagen type I produced by human hepatic stellate cells in response to transforming growth factor‐β1

1 Pentoxifylline (PTF) may act as a potential antifibrogenic agent by inhibiting cell proliferation and/or collagen deposition in cell type(s) responsible for the accumulation of extracellular matrix. The aim of the present study was to investigate at which level PTF may affect synthesis and degradation of type I collagen in human hepatic stellate cells (HSCs), a key source of connective tissue in fibrotic liver. 2 Procollagen type I synthesis and release were evaluated in cells maintained in serum free/insulin free medium for 48 h and then stimulated with transforming growth factor‐β1 (TGF‐β1) for different time periods in the presence or absence of PTF. TGF‐β1 caused an upregulation of procollagen I mRNA levels with a peak increase after 3–6 h of stimulation. This effect was followed by an increase in both the cell associated and the extracellular levels of the corresponding protein, with a peak effect at 9–12 h after the addition of TGF‐β1. Co‐incubation with PTF slightly but consistently reduced basal as well as stimulated procollagen I mRNA levels, with negligible effects on the cell‐associated expression of the corresponding protein. Conversely, PTF dose‐dependently reduced procollagen type I levels detected in supernatants from unstimulated and stimulated cells. 3 Pulse‐chase experiments employing L‐[3H]‐proline revealed that PTF was able to induce significantly the degradation of procollagen, mainly in the extracellular compartment. We next analysed the effect of PTF on the major pathway involved in type I collagen degradation. PTF did not affect the expression of metalloproteinase 1 (MMP‐1) mRNA both in basal and stimulated conditions, whereas it markedly reduced the expression of tissue inhibitor of metalloproteinase 1 (TIMP‐1) mRNA. Accordingly incubation with PTF increased the levels of ‘activated MMP‐1’ in cell supernatants in both basal and stimulated conditions. 4 These results suggest that the antifibrogenic action of PTF on human HSCs is mainly mediated by extracellular collagen degradation rather than by a reduction of collagen synthesis.

Ordered array of dendritic cells and CD8+ lymphocytes in portal infiltrates in chronic hepatitis C.

Ordered array of dendritic cells and CD8+ lymphocytes in portal infiltrates in chronic hepatitis C

Nuclear localization of the nerve growth factor (NGF) receptor, TRK-A in liver cells

the expression of MAT2A (p<O.OOl), a gene whose induction is associated with dedifferentiation of liver cells. In vitro studies showed that primary rat hepatocytes express WTl when stimulated with TGFbeta or when the cells undergo dedifferentiation in culture. Moreover, WTl down-regulates HNF-4 and up-regulates MAT2A, thus promoting reversion of hepatocytes to a more immature phenotype. Conclusions: we show that WTl, a developmental-restricted factor, is induced by TGFbeta and is reexpressed in the cirrhotic liver. WTl appears to have a negative effect on hepatocellular differentiation. facilitating the progression of liver cirrhosis to hepatic insufficiency.

The chemokine CCL21 modulates lymphocyte recruitment and fibrosis in chronic hepatitis C1 1The authors thank Wanda Delogu and Nadia Navari for skillful technical help, Dr. Roberto G. Romanelli for help in collecting liver biopsy specimens, and Dr. Mario Strazzabosco (Ospedali Riuniti di Bergamo, Italy) for providing part of the tissue samples with primary biliary cirrhosis.

BACKGROUND AND AIMS The chemokines CCL19 and CCL21 bind CCR7, which is involved in the organization of secondary lymphoid tissue and is expressed during chronic tissue inflammation. We investigated the expression of CCL21 and CCR7 in chronic hepatitis C. The effects of CCL21 on hepatic stellate cells (HSCs) were also studied. METHODS Expression of CCL21 was assessed by in situ hybridization and immunohistochemistry. CCR7 on T cells was analyzed by flow cytometry. Cultured human HSCs were studied in their activated phenotype. RESULTS In patients with chronic hepatitis C, expression of CCL21 and CCR7 was up-regulated. CCL21 was detected in the portal tracts and around inflammatory lymphoid follicles, in proximity to T lymphocytes and dendritic cells, which contributed to expression of this chemokine. Expression of CCR7 was also increased in patients with primary biliary cirrhosis. Intrahepatic CD8(+) T lymphocytes isolated from patients with chronic hepatitis C had a significantly higher percentage of positivity for CCR7 than those from healthy controls, and the expression of CCR7 was associated with that of CXCR3. Cultured HSCs expressed functional CCR7, the activation of which stimulated cell migration and accelerated wound healing in an in vitro model. Exposure of HSCs to CCL21 triggered several signaling pathways, including extracellular signal-regulated kinase, Akt, and nuclear factor kappaB, resulting in induction of proinflammatory genes. CONCLUSIONS Expression of CCL21 during chronic hepatitis C is implicated in the recruitment of T lymphocytes and the organization of inflammatory lymphoid tissue and may promote fibrogenesis in the inflamed areas via activation of CCR7 on HSCs.

Effects of nitric oxide (NO)-donors on activated human hepatic stellate cells (HSC): cellular basis for the treatment of portal hypertension

EFFECTS OF NITRIC OXIDE (NO)-DONORS ON ACTIVATED HUMAN HEPATIC STELLATE CELLS (HSC): CELLULAR BASIS FOR THE TREATMENT OF PORTAL HYPERTENSION. R.M.S. DeFranco, A. Calipiuri, *P. Failli, A. Gentilini, R. Romanelli, “A. Czsini, C. Tosti-Guerra, P. Gentilini, G. Laffi, and M. Pinzani. Istituto di. Medicina Interna, *Dipartimento di Farmacologia, and “Centro di Alcologia, Universita di Firenze, Firenze, Italy.