Polyanionic carbosilane dendrimers prevent hepatitis C virus infection in cell culture.

Hepatitis C virus (HCV) infection is a major biomedical problem worldwide. Although new direct antiviral agents (DAAs) have been developed for the treatment of chronic HCV infection, the potential emergence of resistant virus variants and the difficulties to implement their administration worldwide make the development of novel antiviral agents an urgent need. Moreover, no effective vaccine is available against HCV and transmission of the virus still occurs particularly when prophylactic measures are not taken. We used a cell-based system to screen a battery of polyanionic carbosilane dendrimers (PCDs) to identify compounds with antiviral activity against HCV and show that they inhibit effective virus adsorption of major HCV genotypes. Interestingly, one of the PCDs irreversibly destabilized infectious virions. This compound displays additive effect in combination with a clinically relevant DAA, sofosbuvir. Our results support further characterization of these molecules as nanotools for the control of hepatitis C virus spread.

Apolipoprotein E, but Not Apolipoprotein B, Is Essential for Efficient Cell-to-Cell Transmission of Hepatitis C Virus.

UNLABELLED: Hepatitis C virus (HCV) infects hepatocytes through two different routes: (i) cell-free particle diffusion followed by engagement with specific cellular receptors and (ii) cell-to-cell direct transmission mediated by mechanisms not well defined yet. HCV exits host cells in association with very-low-density lipoprotein (VLDL) components. VLDL particles contain apolipoproteins B (ApoB) and E (ApoE), which are required for viral assembly and/or infectivity. Based on these precedents, we decided to study whether these VLDL components participate in HCV cell-to-cell transmission in vitro. We observed that cell-to-cell viral spread was compromised after ApoE interference in donor but not in acceptor cells. In contrast, ApoB knockdown in either donor or acceptor cells did not impair cell-to-cell viral transmission. Interestingly, ApoB participated in the assembly of cell-free infective virions, suggesting a differential regulation of cell-to-cell and cell-free HCV infection. This study identifies host-specific factors involved in these distinct routes of infection that may unveil new therapeutic targets and advance our understanding of HCV pathogenesis. IMPORTANCE: This work demonstrates that cell-to-cell transmission of HCV depends on ApoE but not ApoB. The data also indicate that ApoB is required for the assembly of cell-free infective particles, strongly suggesting the existence of mechanisms involving VLDL components that differentially regulate cell-free and cell-to-cell HCV transmission. These data clarify some of the questions regarding the role of VLDL in HCV pathogenesis and the transmission of the virus cell to cell as a possible mechanism of immune evasion and open the door to therapeutic intervention.

Clathrin mediates infectious hepatitis C virus particle egress.

UNLABELLED: Although it is well established that hepatitis C virus (HCV) entry into hepatocytes depends on clathrin-mediated endocytosis, the possible roles of clathrin in other steps of the viral cycle remain unexplored. Thus, we studied whether cell culture-derived HCV (HCVcc) exocytosis was altered after clathrin interference. Knockdown of clathrin or the clathrin adaptor AP-1 in HCVcc-infected human hepatoma cell cultures impaired viral secretion without altering intracellular HCVcc levels or apolipoprotein B (apoB) and apoE exocytosis. Similar reductions in HCVcc secretion were observed after treatment with specific clathrin and dynamin inhibitors. Furthermore, detergent-free immunoprecipitation assays, neutralization experiments, and immunofluorescence analyses suggested that whereas apoE associated with infectious intracellular HCV precursors in endoplasmic reticulum (ER)-related structures, AP-1 participated in HCVcc egress in a post-ER compartment. Finally, we observed that clathrin and AP-1 knockdown altered the endosomal distribution of HCV core, reducing and increasing its colocalization with early endosome and lysosome markers, respectively. Our data support a model in which nascent HCV particles associate with apoE in the ER and exit cells following a clathrin-dependent transendosomal secretory route. IMPORTANCE: HCV entry into hepatocytes depends on clathrin-mediated endocytosis. Here we demonstrate for the first time that clathrin also participates in HCV exit from infected cells. Our data uncover important features of HCV egress, which may lead to the development of new therapeutic interventions. Interestingly, we show that secretion of the very-low-density lipoprotein (VLDL) components apoB and apoE is not impaired after clathrin interference. This is a significant finding, since, to date, it has been proposed that HCV and VLDL follow similar exocytic routes. Given that lipid metabolism recently emerged as a potential target for therapies against HCV infection, our data may help in the design of new strategies to interfere specifically with HCV exocytosis without perturbing cellular lipid homeostasis, with the aim of achieving more efficient, selective, and safe antivirals.

Apicobasal polarity controls lymphocyte adhesion to hepatic epithelial cells.

Loss of apicobasal polarity is a hallmark of epithelial pathologies. Leukocyte infiltration and crosstalk with dysfunctional epithelial barriers are crucial for the inflammatory response. Here, we show that apicobasal architecture regulates the adhesion between hepatic epithelial cells and lymphocytes. Polarized hepatocytes and epithelium from bile ducts segregate the intercellular adhesion molecule 1 (ICAM-1) adhesion receptor onto their apical, microvilli-rich membranes, which are less accessible by circulating immune cells. Upon cell depolarization, hepatic ICAM-1 becomes exposed and increases lymphocyte binding. Polarized hepatic cells prevent ICAM-1 exposure to lymphocytes by redirecting basolateral ICAM-1 to apical domains. Loss of ICAM-1 polarity occurs in human inflammatory liver diseases and can be induced by the inflammatory cytokine tumor necrosis factor alpha (TNF-α). We propose that adhesion receptor polarization is a parenchymal immune checkpoint that allows functional epithelium to hamper leukocyte binding. This contributes to the haptotactic guidance of leukocytes toward neighboring damaged or chronically inflamed epithelial cells that expose their adhesion machinery.

Modulation of PI3K-LXRα-dependent lipogenesis mediated by oxidative/nitrosative stress contributes to inhibition of HCV replication by quercetin.

There is experimental evidence that some antioxidant flavonoids show therapeutic potential in the treatment of hepatitis C through inhibition of hepatitis C virus (HCV) replication. We examined the effect of treatment with the flavonols quercetin and kaempferol, the flavanone taxifolin and the flavone apigenin on HCV replication efficiency in an in vitro model. While all flavonoids studied were able to reduce viral replication at very low concentrations (ranging from 0.1 to 5 µM), quercetin appeared to be the most effective inhibitor of HCV replication, showing a marked anti-HCV activity in replicon-containing cells when combined with interferon (IFN)α. The contribution of oxidative/nitrosative stress and lipogenesis modulation to inhibition of HCV replication by quercetin was also examined. As expected, quercetin decreased HCV-induced reactive oxygen and nitrogen species (ROS/RNS) generation and lipoperoxidation in replicating cells. Quercetin also inhibited liver X receptor (LXR)α-induced lipid accumulation in LXRα-overexpressing and replicon-containing Huh7 cells. The mechanism underlying the LXRα-dependent lipogenesis modulatory effect of quercetin in HCV-replicating cells seems to involve phosphatidylinositol 3-kinase (PI3K)/AKT pathway inactivation. Thus, inhibition of the PI3K pathway by LY294002 attenuated LXRα upregulation and HCV replication mediated by lipid accumulation, showing an additive effect when combined with quercetin. Inactivation of the PI3K pathway by quercetin may contribute to the repression of LXRα-dependent lipogenesis and to the inhibition of viral replication induced by the flavonol. Combined, our data suggest that oxidative/nitrosative stress blockage and subsequent modulation of PI3K-LXRα-mediated lipogenesis might contribute to the inhibitory effect of quercetin on HCV replication.

Carcinoma-associated fibroblasts derive from mesothelial cells via mesothelial-to-mesenchymal transition in peritoneal metastasis.

Peritoneal dissemination is a frequent metastatic route for cancers of the ovary and gastrointestinal tract. Tumour cells metastasize by attaching to and invading through the mesothelial cell (MC) monolayer that lines the peritoneal cavity. Metastases are influenced by carcinoma-associated fibroblasts (CAFs), a cell population that derives from different sources. Hence, we investigated whether MCs, through mesothelial-mesenchymal transition (MMT), were a source of CAFs during peritoneal carcinomatosis and whether MMT affected the adhesion and invasion of tumour cells. Biopsies from patients with peritoneal dissemination revealed the presence of myofibroblasts expressing mesothelial markers in the proximity of carcinoma implants. Prominent new vessel formation was observed in the peritoneal areas harbouring tumour cells when compared with tumour-free regions. The use of a mouse model of peritoneal dissemination confirmed the myofibroblast conversion of MCs and the increase in angiogenesis at places of tumour implants. Treatment of omentum MCs with conditioned media from carcinoma cell cultures resulted in phenotype changes reminiscent of MMT. Adhesion experiments demonstrated that MMT enhanced the binding of cancer cells to MCs in a ß1-integrin-dependent manner. Scanning electron microscopy imaging showed that the enhanced adhesion was mostly due to increased cell-cell interaction and not to a mere matrix exposure. Invasion assays suggested a reciprocal stimulation of the invasive capacity of tumour cells and MCs. Our results demonstrate that CAFs can derive from mesothelial cells during peritoneal metastasis. We suggest that MMT renders the peritoneum more receptive for tumour cell attachment/invasion and contributes to secondary tumour growth by promoting its vascularization.

Functional relevance of the switch of VEGF receptors/co-receptors during peritoneal dialysis-induced mesothelial to mesenchymal transition.

Vascular endothelial growth factor (VEGF) is up-regulated during mesothelial to mesenchymal transition (MMT) and has been associated with peritoneal membrane dysfunction in peritoneal dialysis (PD) patients. It has been shown that normal and malignant mesothelial cells (MCs) express VEGF receptors (VEGFRs) and co-receptors and that VEGF is an autocrine growth factor for mesothelioma. Hence, we evaluated the expression patterns and the functional relevance of the VEGF/VEGFRs/co-receptors axis during the mesenchymal conversion of MCs induced by peritoneal dialysis. Omentum-derived MCs treated with TGF-ß1 plus IL-1ß (in vitro MMT) and PD effluent-derived MCs with non-epithelioid phenotype (ex vivo MMT) showed down-regulated expression of the two main receptors Flt-1/VEGFR-1 and KDR/VEGFR-2, whereas the co-receptor neuropilin-1 (Nrp-1) was up-regulated. The expression of the Nrp-1 ligand semaphorin-3A (Sema-3A), a functional VEGF competitor, was repressed throughout the MMT process. These expression pattern changes were accompanied by a reduction of the proliferation capacity and by a parallel induction of the invasive capacity of MCs that had undergone an in vitro or ex vivo MMT. Treatment with neutralizing anti-VEGF or anti-Nrp-1 antibodies showed that these molecules played a relevant role in cellular proliferation only in naïve omentum-derived MCs. Conversely, treatment with these blocking antibodies, as well as with recombinant Sema-3A, indicated that the switched VEGF/VEGFRs/co-receptors axis drove the enhanced invasion capacity of MCs undergoing MMT. In conclusion, the expression patterns of VEGFRs and co-receptors change in MCs during MMT, which in turn would determine their behaviour in terms of proliferation and invasion in response to VEGF.

Matrigel-embedded 3D culture of Huh-7 cells as a hepatocyte-like polarized system to study hepatitis C virus cycle.

Hepatocytes are highly polarized cells where intercellular junctions, including tight junctions (TJs), determine the polarity. Recently, the TJ-associated proteins claudin-1 and occludin have been implicated in hepatitis C virus (HCV) entry and spread. Nevertheless, cell line-based experimental systems that exhibit hepatocyte-like polarity and permit robust infection and virion production are not currently available. Thus, we sought to determine whether cell line-based, Matrigel-embedded cultures could be used to study hepatitis C virus (HCV) infection and virion production in a context of hepatocyte-like polarized cells. In contrast to standard bidimensional cultures, Matrigel-cultured Huh-7 cells adopted hepatocyte polarization features forming a continuous network of functional proto-bile canaliculi structures. These 3D cultures supported HCV infection by JFH-1 virus and produced infective viral particles which shifted towards lower densities with higher associated specific infectivity. In conclusion, our findings describe a novel use of Matrigel to study the entire HCV cycle in a more relevant context.

Interplay among cellular polarization, lipoprotein metabolism and hepatitis C virus entry.

Hepatitis C virus (HCV) infects more than three million new individuals worldwide each year. In a high percentage of patients, acute infections become chronic, eventually progressing to fibrosis, cirrhosis, and hepatocellular carcinoma. Given the lack of effective prophylactic or therapeutic vaccines, and the limited sustained virological response rates to current therapies, new approaches are needed to prevent, control, and clear HCV infection. Entry into the host cell, being the first step of the viral cycle, is a potential target for the design of new antiviral compounds. Despite the recent discovery of the tight junction-associated proteins claudin-1 and occludin as HCV co-receptors, which is an important step towards the understanding of HCV entry, the precise mechanisms are still largely unknown. In addition, increasing evidence indicates that tools that are broadly employed to study HCV infection do not accurately reflect the real process in terms of viral particle composition and host cell phenotype. Thus, systems that more closely mimic natural infection are urgently required to elucidate the mechanisms of HCV entry, which will in turn help to design antiviral strategies against this part of the infection process.

Blocking TGF-ß1 protects the peritoneal membrane from dialysate-induced damage.

During peritoneal dialysis (PD), mesothelial cells undergo mesothelial-to-mesenchymal transition (MMT), a process associated with peritoneal-membrane dysfunction. Because TGF-ß1 can induce MMT, we evaluated the efficacy of TGF-ß1-blocking peptides in modulating MMT and ameliorating peritoneal damage in a mouse model of PD. Exposure of the peritoneum to PD fluid induced fibrosis, angiogenesis, functional impairment, and the accumulation of fibroblasts. In addition to expressing fibroblast-specific protein-1 (FSP-1), some fibroblasts co-expressed cytokeratin, indicating their mesothelial origin. These intermediate-phenotype (Cyto(+)/FSP-1(+)) fibroblasts had features of myofibroblasts with fibrogenic capacity. PD fluid treatment triggered the appearance of CD31(+)/FSP-1(+) and CD45(+)/FSP-1(+) cells, suggesting that fibroblasts also originate from endothelial cells and from cells recruited from bone marrow. Administration of blocking peptides significantly ameliorated fibrosis and angiogenesis, improved peritoneal function, and reduced the number of FSP-1(+) cells, especially in the Cyto(+)/FSP-1(+) subpopulation. Conversely, overexpression of TGF-ß1 in the peritoneum by adenovirus-mediated gene transfer led to a marked accumulation of fibroblasts, most of which derived from the mesothelium. Taken together, these results demonstrate that TGF-ß1 drives the peritoneal deterioration induced by dialysis fluid and highlights a role of TGF-ß1-mediated MMT in the pathophysiology of peritoneal-membrane dysfunction.

Expression of pituitary tumor-transforming gene 1 (PTTG1)/securin in hepatitis B virus (HBV)-associated liver diseases: evidence for an HBV X protein-mediated inhibition of PTTG1 ubiquitination and degradation.

Chronic infection with hepatitis B virus (HBV) is strongly associated with hepatocellular carcinoma (HCC), and the viral HBx protein plays a crucial role in the pathogenesis of liver tumors. Because the protooncogene pituitary tumor-transforming gene 1 (PTTG1) is overexpressed in HCC, we investigated the regulation of this protein by HBx. We analyzed PTTG1 expression levels in liver biopsies from patients chronically infected with HBV, presenting different disease stages, and from HBx transgenic mice. PTTG1 was undetectable in biopsies from chronic hepatitis B patients or from normal mouse livers. In contrast, hyperplastic livers from transgenic mice and biopsies from patients with cirrhosis, presented PTTG1 expression which was found mainly in HBx-expressing hepatocytes. PTTG1 staining was further increased in HCC specimens. Experiments in vitro revealed that HBx induced a marked accumulation of PTTG1 protein without affecting its messenger RNA levels. HBx expression promoted the inhibition of PTTG1 ubiquitination, which in turn impaired its degradation by the proteasome. Glutathione S-transferase pull-down and co-immunoprecipitation experiments demonstrated that the interaction between PTTG1 and the Skp1-Cul1-F-box ubiquitin ligase complex (SCF) was partially disrupted, possibly through a mechanism involving protein-protein interactions of HBx with PTTG1 and/or SCF. Furthermore, confocal analysis revealed that HBx colocalized with PTTG1 and Cul1. We propose that HBx promotes an abnormal accumulation of PTTG1, which may provide new insights into the molecular mechanisms of HBV-related pathogenesis of progressive liver disease leading to HCC development.

The tight junction-associated protein occludin is required for a postbinding step in hepatitis C virus entry and infection.

The precise mechanisms regulating hepatitis C virus (HCV) entry into hepatic cells remain unknown. However, several cell surface proteins have been identified as entry factors for this virus. Of these molecules, claudin-1, a tight junction (TJ) component, is considered a coreceptor required for HCV entry. Recently, we have demonstrated that HCV envelope glycoproteins (HCVgp) promote structural and functional TJ alterations. Additionally, we have shown that the intracellular interaction between viral E2 glycoprotein and occludin, another TJ-associated protein, could be the cause of the mislocalization of TJ proteins. Herein we demonstrated, by using cell culture-derived HCV particles (HCVcc), that interference of occludin expression markedly reduced HCV infection. Furthermore, our results with HCV pseudotyped particles indicated that occludin, but not other TJ-associated proteins, such as junctional adhesion molecule A or zonula occludens protein 1, was required for HCV entry. Using HCVcc, we demonstrated that occludin did not play an essential role in the initial attachment of HCV to target cells. Surface protein labeling experiments showed that both expression levels and cell surface localization of HCV (co)receptors CD81, scavenger receptor class B type I, and claudin-1 were not affected upon occludin knockdown. In addition, immunofluorescence confocal analysis showed that occludin interference did not affect subcellular distribution of the HCV (co)receptors analyzed. However, HCVgp fusion-associated events were altered after occludin silencing. In summary, we propose that occludin plays an essential role in HCV infection and probably affects late entry events. This observation may provide new insights into HCV infection and related pathogenesis.

Hepatitis C virus NS5A and core proteins induce oxidative stress-mediated calcium signalling alterations in hepatocytes.

BACKGROUND/AIMS: The hepatitis C virus (HCV) structural core and non-structural NS5A proteins induce in liver cells a series of intracellular events, including elevation of reactive oxygen and nitrogen species (ROS/RNS). Since oxidative stress is associated to altered intracellular Ca(2+) homeostasis, we aimed to investigate the effect of these proteins on Ca(2+) mobilization in human hepatocyte-derived transfected cells, and the protective effect of quercetin treatment. METHODS: Ca(2+) mobilization and actin reorganization were determined by spectrofluorimetry. Production of ROS/RNS was determined by flow cytometry. RESULTS: Cells transfected with NS5A and core proteins showed enhanced ROS/RNS production and resting cytosolic Ca(2+) concentration, and reduced Ca(2+) concentration into the stores. Phenylephrine-evoked Ca(2+) release, Ca(2+) entry and extrusion by the plasma membrane Ca(2+)-ATPase were significantly reduced in transfected cells. Similar effects were observed in cytokine-activated cells. Phenylephrine-evoked actin reorganization was reduced in the presence of core and NS5A proteins. These effects were significantly prevented by quercetin. Altered Ca(2+) mobilization and increased calpain activation were observed in replicon-containing cells. CONCLUSIONS: NS5A and core proteins induce oxidative stress-mediated Ca(2+) homeostasis alterations in human hepatocyte-derived cells, which might underlie the effects of both proteins in the pathogenesis of liver disorders associated to HCV infection.

Hepatitis C virus envelope components alter localization of hepatocyte tight junction-associated proteins and promote occludin retention in the endoplasmic reticulum.

UNLABELLED: Hepatocyte tight junctions (TJ) play key roles in characteristic liver functions, including bile formation and secretion. Infection by hepatitis C virus (HCV) may cause alterations of the liver architecture and disruption of the bile duct, which ultimately can lead to cholestasis. Herein, we employed the HCV replicon system to analyze the effect of HCV on TJ organization. TJ-associated proteins occludin, claudin-1, and Zonula Occludens protein-1 (ZO-1) disappeared from their normal localization at the border of adjacent cells in Huh7 clones harboring genomic but not subgenomic replicons expressing only the nonstructural proteins. Furthermore, cells containing genomic replicons showed a cytoplasmic accumulation of occludin in the endoplasmic reticulum (ER). TJ-associated function, measured as FITC-dextran paracellular permeability, of genomic replicon-containing cells, was also altered. Interestingly, clearance of the HCV replicon by interferon-alpha (IFN-alpha) treatment and by short hairpin RNA (shRNA) significantly restored the localization of TJ-associated proteins. Transient expression of all HCV structural proteins, but not core protein alone, altered the localization of TJ-associated proteins in Huh7 cells and in clones with subgenomic replicons. Confocal analysis showed that accumulation of occludin in the ER partially co-localized with HCV envelope glycoprotein E2. E2/occludin association was further confirmed by co-immunoprecipitation and pull-down assays. Additionally, using a cell culture model of HCV infection, we observed the cytoplasmic dot-like accumulation of occludin in infected Huh7 cells. CONCLUSION: We propose that HCV structural proteins, most likely those of the viral envelope, promote alterations of TJ-associated proteins, which may provide new insights for HCV-related pathogenesis.

The hepatitis B virus X protein induces paracrine activation of human hepatic stellate cells.

UNLABELLED: Chronic hepatitis B virus (HBV) infection is a major cause of liver fibrosis, eventually leading to cirrhosis and hepatocellular carcinoma. Although the involvement of the X protein of HBV (HBx) in viral replication and tumor development has been extensively studied, little is known about its possible role in the development of fibrosis. In this work we show that expression of HBx in hepatocytes results in paracrine activation and proliferation of hepatic stellate cells (HSCs), the main producers of extracellular matrix proteins in the fibrotic liver. Both human primary HSCs and rat HSCs exposed to conditioned medium from HBx-expressing hepatocytes showed increased expression of collagen I, connective tissue growth factor, alpha smooth muscle actin, matrix metalloproteinase-2, and transforming growth factor-beta (TGF-beta), together with an enhanced proliferation rate. We found that HBx induced TGF-beta secretion in hepatocytes and that the activation of HSCs by conditioned medium from HBx-expressing hepatocytes was prevented by a neutralizing anti-TGF-beta antibody, indicating the involvement of this profibrotic factor in the process. CONCLUSION: Our results propose a direct role for HBx in the development of liver fibrosis by the paracrine activation of stellate cells and reinforce the indication of antiviral treatment in patients with advanced HBV-related chronic liver disease and persistent liver replication.

Thymidylate synthase expression pattern is a prognostic factor in patients of colorectal cancer treated with 5-fluorouracil.

High intratumoral expression of thymidylate synthase (TS) has been reported as a factor of poor prognosis in patients with advanced colorectal cancer (CRC), but such association is unclear in some studies. Also, TS has been stated as a typical cytosolic enzyme, but nuclear location has been occasionally reported, and data on the clinical meaning of TS intracellular location are scarce. A retrospective study was performed in paraffin-embedded sections of primary tumor from 77 CRC patients treated with surgical resection and adjuvant 5-FU-based chemotherapy. TS levels and expression patterns were determined by immunohistochemistry (IHQ) using TS-106 antibody. Qualitative and quantitative variables were compared respectively by chi2 and Kruskal-Wallis tests; overall survival (OS) and disease-free survival (DFS) were analyzed with the Kaplan-Meier method and compared using the log-rank and Wilcoxon tests. TS was cytoplasmic in 27.1% of positive tumors and both, nuclear and cytoplasmic in 72.9%; specimens from seven patients (9.1%) lacked TS expression. TS levels were high in 21.6% of tumors with nuclear expression and low in 5.6%, whereas 68.4% of cytoplasmic ones showed low immunostaining intensity (p=0.02); cytoplasmic pattern was also associated to longer OS (p<0.009) and DFS (p=0.003). In patients with nuclear expression, low TS expression was associated to shorter OS (p<0.003) and DFS (p<0.04). These results indicate that, in our study, TS immunostaining patterns were related with OS and DFS, the best prognostic corresponding to cytoplasmic one, and, within the subset of patients with nuclear expression, low TS levels were associated to worse clinical outcome.

The mitogenic activity of the liver growth factor is mediated by tumor necrosis factor alpha in rat liver.

BACKGROUND/AIMS: Liver growth factor (LGF) is a hepatic mitogen, however, the hepatic stimulation pathway remains to be characterized. The aim of this study was to determine whether tumor necrosis factor alpha (TNF-alpha) stimulation constitutes a step in the mitogenic pathway of LGF. METHODS: Rats were injected with 4.5 microg LGF/rat, and LGF activity was measured both by liver DNA synthesis stimulation and "proliferating cell nuclear antigen (PCNA)-positive" hepatocytes in rats injected with LGF or +anti-TNF-alpha. TNF-alpha expression was evaluated by reverse-transcription polymerase chain reaction. TNF-alpha-producing cells were immunodetected. Human endothelial cells (HUVEC) were stimulated by LGF. TNF-alpha was detected in the supernatant, and the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial adhesion molecule-1 (VCAM-1) by flow cytometry analysis. RESULTS: LGF-injected rats showed higher intrahepatic TNF-alpha expression. DNA synthesis and PCNA-positive hepatocytes induced by LGF were inhibited by anti-TNF-alpha, PCNA-positive hepatocytes being especially abundant around the central vein when LGF was injected alone, but TNF-alpha exhibited increased signal intensity in endothelial cells of the portal vein. LGF stimulated TNF-alpha secretion in HUVEC, but did not stimulate ICAM-1 or VCAM-1 up-regulation. CONCLUSIONS: The mitogenic cascade initiated by LGF in rat liver in vivo depends, at least in part, on TNF-alpha stimulation. Portal vein endothelial cells seem to be a source of TNF-alpha.

Hepatocyte growth factor activates endothelial proangiogenic mechanisms relevant in chronic hepatitis C-associated neoangiogenesis.

BACKGROUND: Angiogenesis occurs in inflamed portal tracts of chronic hepatitis C (CHC) patients. AIMS: To characterize this phenomenon, by investigating the molecular mechanisms involved in neovessel formation in the livers of CHC patients and the angiogenic effects of hepatocyte growth factor (HGF) on human endothelial cells. METHODS: Vascular endothelial growth factor (VEGF), VE-cadherin and alphavbeta3 integrin were determined in CHC biopsies by Western blot and immunohistochemistry. Effects of HGF on VEGF and cell adhesion molecules expression by cultured human microvascular endothelial cells were evaluated by Western blot, Northern blot or immunofluorescence. HGF effects on cell proliferation were assessed by [(3)H]thymidine incorporation. RESULTS: VEGF, VE-cadherin and alphavbeta3 integrin were increased in CHC liver samples. In cultured endothelial cells, HGF transcriptionally increased VEGF expression, an effect which was blocked by an anti-VEGF receptor antibody. HGF transiently decreased VE-cadherin expression and its associated cytoskeleton-linking molecule beta-catenin, thus weakening intercellular contacts. HGF increased alphavbeta3 integrin at focal contacts, and cell proliferation, an effect which was inhibited by an anti-VEGF receptor antibody. CONCLUSIONS: Our results show that HGF and VEGF modulate the expression of cell adhesion and migration molecules and induce proliferation in endothelial cells, mechanisms through which these factors may contribute to CHC-associated liver angiogenesis.