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Hepatic stellate cells (HSC) coordinate the liver wound-healing response through secretion of several cytokines and chemokines, including CCL2 (formerly known as monocyte chemoattractant protein-1). In this study, we evaluated the role of different proteins of the MAPK family (ERK, p38MAPK, and JNK) in the regulation of CCL2 expression by HSC, as an index of their proinflammatory activity. Several mediators activated all three MAPK, including TNF, IL-1, and PDGF. To assess the relative role of the different MAPKs, specific pharmacological inhibitors were used; namely, SB203580 (p38MAPK), SP600125 (JNK), and PD98059 (MEK/ERK). The efficacy and specificity of the different inhibitors in our cellular system were verified analyzing the enzymatic activity of the different MAPKs using in vitro kinase assays and/or testing the inhibition of phosphorylation of downstream substrates. SB203580 and SP600125 dose-dependently inhibited CCL2 secretion and gene expression induced by IL-1 or TNF. In contrast, inhibition of ERK did not affect the upregulation of CCL2 induced by the two cytokines. Finally, activin A was also found to stimulate CCL2 expression and to activate ERK, JNK, p38, and their downstream targets. Unlike in cells exposed to proinflammatory cytokines, all three MAPKs were required to induce CCL2 secretion in response to activin. We conclude that members of the MAPK family differentially regulate cytokine-induced chemokine expression in human HSC.

Background Several interrelated host and hepatitis C virus (HCV) associated factors have been proposed to explain the variable outcomes in HCV recurrence. Recent evidence suggests that cytomegalovirus (CMV) infection not only is co-factor in progression of HCV recurrence but may precipitate allograft rejection. We investigated whether short-term CMV viremia influences HCV recurrence, the number and grade of acute rejection episodes, and the histological course of HCV recurrence during the first year after orthotopic liver transplantation (OLT) for HCV-related cirrhosis. Methods A cohort of 39 patients transplanted for cirrhosis HCV-related was analyzed. Patients were evaluated twice weekly for CMV infection by a blood polymerase chain reaction (PCR) assay. Triple therapy with cyclosporine or tacrolimus, azathioprine and prednisolone was the initial immunosuppressive regimen. Preemptive treatment with ganciclovir was started when two consecutive PCRs for CMV were positive. Liver biopsies were performed on day 7 after OLT or when indicated. A 3-day IV 1 g methilprednisolone was given to patients with moderate or severe rejection. Ishak's score was used to grade inflammation and to stage fibrosis. Results Neither CMV viremia nor CMV disease after OLT for HCV-related cirrhosis adversely influenced the incidence and grade of acute rejection episodes nor the histological outcome of post transplant HCV recurrence, during the first year after liver transplantation. Conclusion CMV viremia as detected by PCR does not affect the progression of HCV recurrence in liver grafts.

Cirrhosis due to hepatitis C is now the commonest indication for liver transplantation in Western Europe and in the United States. Graft reinfection is almost universal. The natural history of recurrent hepatitis C ranges from minimal damage to cirrhosis in a few months or years. Different virus and host immune factors are involved in the pathogenesis of hepatitis and are determinants of the outcome. The association between immunosuppression and severity of HCV recurrence is conflicting and remains to be evaluated fully. The treatment of recurrent HCV disease with IFN or ribavirin, as monotherapy, is ineffective. Preliminary results from combination therapy, however, are encouraging. Currently, a reasonable approach would be to treat patients with histological and clinical disease progression. New approaches for the prophylaxis of recurrent hepatitis C are under evaluation but whether this treatment will influence the severity of liver disease or the outcome of recurrence is still unknown.

We investigated whether activation of integrin receptors could modulate the expression of monocyte chemotactic protein-1 (MCP-1) in human hepatic stellate cells (HSC), mesenchymal cells responsible for extracellular matrix synthesis within the liver. When compared to non-adherent cells, HSC plated on collagen types I or IV, or fibronectin, showed increased MCP-1 gene expression and protein secretion in the conditioned medium. Increased MCP-1 secretion was also observed when cells were plated on dishes coated with a monoclonal antibody directed against the β1-integrin subunit, demonstrating that ligation of β1-integrins is sufficient to stimulate MCP-1 expression. Conversely, integrin-independent cell adhesion on poly-l-lysine did not modify MCP-1 secretion. Disruption of the actin cytoskeleton by cytochalasin D blocked the collagen-dependent increase in MCP-1 secretion. Chemotactic assay of HSC-conditioned medium showed that HSC plated on collagen secrete higher amounts of chemotactic factors for lymphomonocytes, and that MCP-1 accounts for the great majority of this effect. These findings indicate a novel mechanism of MCP-1 regulation possibly relevant in those conditions where HSC interact with an altered extracellular matrix.

Bacterial infections have been postulated as a trigger for variceal bleeding in cirrhotic patients, and impair coagulation evaluated by thrombelastography (TEG). Endogenous heparinoids have been detected after variceal bleeding and during liver transplantation in some cirrhotics using heparinase-modified-TEG.To assess if bacterial infection is associated with endogenous heparinoids in cirrhotics, thus impairing coagulation.Native and heparinase-modified-TEG (cleavage of heparin and heparan-sulphate) was performed in 60 cirrhotics (Grade A, 2; B, 30; C, 28): 30 infected [septicaemia, 6 (culture positive); 6 (culture negative); spontaneous bacterial peritonitis, 10; chest infection, 4; others, 4], 30 not infected, and five infected patients without liver diseases, comparing TEG parameters r, alpha, and ma. Eight cirrhotics were studied before and after infection. The diagnosis of presence and type of infection was based on international standard criteria.A significant heparin effect was found only in infected cirrhotics (28 of 30) with significant changes in r (P=0.0003), alpha (P0.0001), and ma (P0.0001), but in none of those not infected. This effect completely reversed in the eight evaluated after resolution of infection. There was no heparin effect in infected non-cirrhotics.A heparin effect was only found in cirrhotic patients with infection, further confirming that infection significantly modifies coagulation in cirrhotic patients.

PPAR-gamma belongs to the nuclear hormone receptor superfamily and its ligands include antidiabetic drugs of the thiazolidindione class, and endogenous molecules, including eicosanoids and fatty acids. PPAR-gamma is involved in the pathophysiology of obesity and type II diabetes. More recently, accumulating evidence suggests its role in atherosclerosis, inflammation and cancer. Recent data obtained in cellular models of liver fibrosis indicate that PPAR-gamma activation results in the inhibition of the processes leading to the development of liver fibrosis. These studies identify potential novel therapeutic strategies for the treatment of liver fibrosis.

Activated hepatic stellate cells (HSC) regulate the liver "wound-healing" response through expression of chemokines, including monocyte chemoattractant protein-1 (MCP-1), which participate in the formation of the inflammatory infiltrate during liver injury. Cyclooxygenase (COX) catalyzes the conversion of arachidonic acid into prostaglandins, which may contribute to the inflammatory response. In this study, we investigated the effects of COX inhibitors on the expression of MCP-1 in cultured HSC. Pretreatment of HSC with nonspecific COX inhibitors such as indomethacin or ibuprofen markedly reduced the expression of MCP-1 caused by exposure to tumor necrosis factor alpha (TNF-alpha) or interleukin-1alpha (IL-1alpha). NS-398, a specific COX-2 inhibitor, also resulted in a dose-dependent inhibition of MCP-1 gene and protein expression. These effects were dependent on reduced MCP-1 transcription, as established using a reporter plasmid. In contrast, the up-regulation of MCP-1 expression caused by interferon gamma (IFN-gamma) was not sensitive to COX inhibitors. Quiescent HSC did not show detectable expression of COX-2, which became evident after activation in culture, and while TNF-alpha and IL-1alpha markedly increased the expression of COX-2, IFN-gamma did not have any effects. Pretreatment of HSC with the stable cyclic adenosine monophosphate (cAMP) analog, 8-bromo cAMP, reverted the effects of the COX-2 inhibitor, but not of a nuclear factor-kappaB (NF-kappaB) inhibitor, demonstrating that prostaglandins modulate MCP-1 expression via production of cAMP. On the other hand, the action of NF-kappaB inhibitors was negligible in IFN-gamma-stimulated cells. These findings indicate that cross-talk between cytokines and a prostaglandin-cAMP pathway differentially regulates the proinflammatory potential of HSC, contributing to the modulation of liver tissue inflammation.

Hepatic artery thrombosis (HAT) is one of the principal causes of morbidity and graft loss following liver transplantation. There are several risk factors for the development of HAT; technical aspects of the arterial anastomosis are important particularly for early thrombosis, but the improvement of surgical technique has lessened this problem. Apart from technical causes, other risk factors include a variety of conditions such as low donor/recipient age ratio, immunologic factors, clotting abnormalities, tobacco use, and infections. In particular, cytomegalovirus (CMV) infection of endothelial cells has been recently suggested as an infective cause of HAT, as it is known to be followed by a rapid procoagulant response. Thus, latent CMV in an allograft may become activated and promote or contribute to vascular thrombosis. This review evaluates these aspects, focusing on data relating CMV infection or viremia to HAT following liver transplantation.

Following liver injury, hepatic stellate cells (HSC) undergo proliferation and migrate into damaged areas in response to chemotactic factors. HSC have been shown to regulate leukocyte trafficking by secreting monocyte chemotactic protein-1 (MCP-1), a chemokine that recruits monocytes and lymphocytes. In this study, we explored whether MCP-1 exerts biological actions on HSC. HSC were isolated from normal human livers, cultured on plastic, and studied in their myofibroblast-like phenotype, and three different cells lines were used. Chemotaxis was measured in modified Boyden chambers. Phosphatidylinositol 3-kinase (PI 3-K) was assayed on phosphotyrosine immunoprecipitates. Exposure of HSC to MCP-1 stimulated migration of HSC in a dose-dependent fashion. Maximal stimulation was obtained with 250 ng/mL MCP-1, which resulted in a 3- to 4-fold stimulation of cell migration. Checkerboard analysis showed that the increase in cell migration was almost completely a result of chemotaxis rather than chemokinesis. In contrast, in quiescent HSC, MCP-1 did not exert any effect on cell migration. In leukocytes, MCP-1 activates the pertussis toxin-sensitive CCR2 receptor. However, transcripts for CCR2 could not be shown in HSC, and pertussis toxin only modestly inhibited MCP-1-induced migration. Exposure of HSC to MCP-1 was associated with an increase in cytosolic calcium concentration, PI 3-K activity, protein tyrosine phosphorylation. Blocking calcium influx or pretreatment of HSC with the PI 3-K inhibitor wortmannin markedly reduced cell migration. This study shows, for the first time, a potential direct profibrogenic action of MCP-1 via HSC chemotaxis. MCP-1-dependent signals in these cells are not transduced by CCR2 and may be mediated by alternative chemokine receptors. (HEPATOLOGY 1999;29:140-148.)

AIM: To investigate the effects of troglitazone (TGZ), an anti-diabetic drug which activates peroxisome proliferator-activated receptor-γ (PPAR-γ), for liver tissue repair, and the development of ductular reaction, following common bile duct ligation (BDL) in rats. METHODS: Rats were supplemented with TGZ (0.2% w/w in the pelleted food) for 1 wk before BDL or sham operation. Animals were killed at 1, 2, or 4 wk after surgery. RESULTS: The development of liver fibrosis was reduced in rats receiving TGZ, as indicated by significant decreases of procollagen type I gene expression and liver hydroxy-proline levels. Accumulation of α-smooth-muscle actin (SMA)-expressing cells surrounding newly formed bile ducts following BDL, as well as total hepatic levels of SMA were partially inhibited by TGZ treatment, indicating the presence of a reduced number and/or activation of hepatic stellate cells (HSC) and myofibroblasts. Development of the ductular reaction was inhibited by TGZ, as indicated by histochemical evaluation and hepatic activity of γ-glutamyl-transferase (GGT). CONCLUSION: Treatment with thiazolidinedione reduces ductular proliferation and fibrosis in a model of chronic cholestasis, and suggests that limiting cholangiocyte proliferation may contribute to the lower development of scarring in this system.