Giulia Pellegrini

Oxidative stress stimulates proliferation and invasiveness of hepatic stellate cells via a MMP2-mediated mechanism

Experimental evidence indicates that reactive oxygen species (ROS) are involved in the development of hepatic fibrosis; they induce hepatic stellate cells (HSC) proliferation and collagen synthesis. To address the role of matrix metalloproteinase (MMP)‐2 in promoting HSC proliferation during hepatic injury, we investigated whether oxidative stress modulates the growth and invasiveness of HSC by influencing MMP‐2 activation. Cell invasiveness and proliferation, which were studied using Boyden chambers and by counting cells under a microscope, were evaluated after treatment with a superoxide‐producing system, xanthine plus xanthine oxidase (X/XO), in the presence or absence of antioxidants and MMP inhibitors. Expression and activation of MMP‐2 were evaluated via gel zymography, immunoassay, and ribonuclease protection assay. The addition of X/XO induced proliferation and invasiveness of human HSC in a dose‐dependent manner. The addition of antioxidants as well as MMP‐2–specific inhibitors impaired these phenomena. X/XO treatment increased MMP‐2 expression and secretion appreciably and significantly induced members of its activation complex, specifically membrane‐type 1 MMP and tissue inhibitor metalloproteinase 2. To study the intracellular signaling pathways involved in X/XO‐induced MMP‐2 expression, we evaluated the effects of different kinase inhibitors. The inhibition of extracellular signal‐regulated kinase 1/2 (ERK1/2) and phosphatidyl inositol 3‐kinase (PI3K) abrogated X/XO‐elicited MMP‐2 upregulation and completely prevented X/XO‐induced growth and invasiveness of HSC. In conclusion, our findings suggest that MMP‐2 is required for the mitogenic and proinvasive effects of ROS on HSC and demonstrate that ERK1/2 and PI3K are the main signals involved in ROS‐mediated MMP‐2 expression. (HEPATOLOGY 2005;41:1074–1084.)

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.

Human hepatic stellate cells express class I alcohol dehydrogenase and aldehyde dehydrogenase but not cytochrome P4502E1

BACKGROUND/AIMS Alcohol dehydrogenase, cytochrome P4502E1 (CYP2E1), and aldehyde dehydrogenase are known to play an important role in alcohol metabolism in the liver. Although the ethanol oxidation pathways are mainly localized in hepatocytes, we examine whether human hepatic stellate cells might also metabolize ethanol and acetaldehyde. METHODS Hepatic stellate cells were isolated from normal human livers and exposed in vitro to 50 mmol/l ethanol or 85 micromol/l acetaldehyde for different periods of time. Alcohol dehydrogenase/aldehyde dehydrogenase activity and CYP2E1 protein expression were measured in hepatic stellate cells. Moreover, alcohol dehydrogenase and aldehyde dehydrogenase mRNA expression were evaluated in hepatic stellate cells. RESULTS Exposure of hepatic stellate cells to ethanol for 24 h resulted in a 5-fold increase in cell alcohol dehydrogenase activity. The effect of ethanol on alcohol dehydrogenase activity was paralleled by a significant increase in the alcohol dehydrogenase mRNA expression in hepatic stellate cells. Acetaldehyde significantly increased the activity of high affinity aldehyde dehydrogenase in hepatic stellate cells, whereas ethanol was devoid of any effect. Acetaldehyde also induced high affinity aldehyde dehydrogenase mRNA expression in hepatic stellate cells. CYP2E1 was not expressed in hepatic stellate cells either in basal condition or after ethanol/acetaldehyde exposure. CONCLUSIONS This study shows that human hepatic stellate cells have the capacity to metabolize both ethanol and acetaldehyde through a class I alcohol dehydrogenase- and an aldehyde dehydrogenase-oxidizing pathway. Conversely, no detectable levels of CYP2E1-associated proteins are expressed in these cells.

Transforming Growth Factor Beta-1 Stimulates Invasivity of Hepatic Stellate Cells by Engagement of the Cell-associated Fibrinolytic System

The activation of hepatic stellate cells (HSC) during liver fibrogenesis has been shown to be mediated by paracrine and autocrine loops involving transforming growth factor-β1 (TGF-β1) as the main fibrogenic mediator secreted by activated macrophages, endothelial cells and liberated by disintegrated platelets. The cell-associated plasminogen activation system regulates extracellular matrix (ECM) catabolism and cell movement. We have studied whether TGF-β1 could modulate the plasminogen activation system in human HSC and the role of such protease system in the activity of TGF-β1 on HSC. Urokinase plasminogen activator receptors (u-PAR), u-PA and plasminogen activator inhibitor type 1 (PAI-1) were determined by immunoassay and RNase protection assay. Cell migration, evaluated either as chemotaxis or as chemoinvasion, was studied in Boyden chambers after addition of TGF-β1, and inhibition with anti-u-PA and anti-u-PAR antagonists [antibodies against u-PA and u-PAR and antisense oligonucleotides (aODN) against u-PAR mRNA]. We have shown that TGF-β1 is not mitogenic for HSC, while it is a powerful motogen either in chemotaxis or chemoinvasion assays. TGF-β1 up-regulates the synthesis and expression of PAI-1, as well as u-PAR expression and exposure at the cell membrane, while it does not affect u-PA levels. TGF-β1-dependent chemoinvasion of reconstituted basement membrane exploits the cell-associated plasminogen activation system, since it is blocked by monoclonal antibodies against u-PA and against various u-PAR domains, as well as by anti-u-PAR aODN. We have also observed a cumulative effect of TGF-β1, b-FGF and PDGF in the invasion assay of HSC: in the presence of low amounts of TGF-β1 the chemoinvasive activity of PDGF and bFGF is dramatically increased. Also this cooperation requires u-PAR and is inhibited by monoclonal antibodies against u-PAR domains I, II and III.

Helicobacter pylori cag pathogenicity island is associated with enhanced interleukin-8 expression in human gastric mucosa.

BACKGROUND In vitro studies showed that Helicobacter pylori strains carrying the cag pathogenicity island are able to induce epithelial secretion of Interleukin-8. AIMS To evaluate the assessment of cag pathogenicity island and the expression of Interleukin-8 in the gastric mucosa of Helicobacter pylori-infected patients and correlate these data with the activity of gastritis and Helicobacter pylori density. METHODS cag status was determined by polymerase chain reaction directly on gastric biopsies from 13 Helicobacter pylori+ patients with non-ulcer dyspepsia and 13 Helicobacter pylori+ with duodenal ulcer. Interleukin-8 gene transcription and protein expression were analysed by in situ hybridization and immunofluorescence, respectively. Gastritis activity and Helicobacter pylori density were also investigated. RESULTS cag was present in 20/26 of Helicobacter pylori+ patients: in 7/13 non-ulcer dyspepsia (53.8%] and in 13/13 duodenal ulcer patients (100%), (p<0.05). Interleukin-8 mRNA and protein expression in epithelial and inflammatory cells was higher in cag+ than in cag- patients (p<0.005). Gastritis activity significantly correlated with cag (p<0.05) and Interleukin-8 expression (p<0.005]. Helicobacter pylori density was enhanced in cag+ [p<0.005] and correlated with Interleukin-8 expression (p<0.0051. CONCLUSIONS The present study demonstrates that in Helicobacter pylori-infected human gastric mucosa, cag+ infection is associated with enhanced Interleukin-8 expression, higher levels of active gastritis and bacterial density, and presence of duodenal ulcer.

Spatial and temporal pattern of expression of interstitial collagenase, stromelysin/transin, gelatinase A, and TIMP-1 during experimental gastric ulcer healing.

Background/Aim: Controlled proteolysis is a prerequisite for cell migration, angiogenesis, and matrix remodelling during gastric ulcer healing. We studied the temporal and spatial expression of three matrix metalloproteinases, gelatinase A (MMP-2), interstitial collagenase (MMP-13), stromelysin (MMP-3), and their major inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1) during experimental gastric ulcer healing induced in rats by acetic acid injection. Methods: Gastric tissue specimens were hybridized with antisense 35S-labelled RNA probes and the autoradiographic signal was analyzed by a computer aided image system. Gelatinase activity was analyzed by in situ and gel zymography. Results: During gastric ulcer healing, MMP-2, MMP-3, and MMP-13 RNA expression was increased in stromal cells of the gastric mucosa bordering the ulcer, suggesting a prevalent role of non-epithelial cells in pericellular proteolysis. Gelatinolytic activity was increased during ulcer healing and it was associated with extracellular matrix of the healing mucosa and newly formed vessels. In contrast to MMP-2 RNA, which was homogeneously distributed in all layers of the ulcer bed, MMP-3 and MMP-13 RNAs were confined to the upper layers of the granulation tissue. TIMP-1 RNA was detected in both epithelial and stromal cells of the gastric mucosa adjacent to the ulcer, as well as in the granulation tissue of the ulcer bed. Both MMP and TIMP-1 expression returned to basal levels during the late stages of tissue remodeling. Conclusion: Gastric ulcer repair is associated with a transient expression of specific metalloproteinases and their inhibitors in a distinct anatomical pattern pointing to complex cellular and cell/matrix interactions in the various layers of the healing mucosa.