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Biomedical subjects

M Pinzani

Publications and source records attributed to M Pinzani.

At least 19 recordsLinked to original sources
PubMed2006

Overexpression of Bcl-2 by activated human hepatic stellate cells: resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans.

BACKGROUND AND AIMS: Myofibroblast-like cells, originating from activation of hepatic stellate cells (HSC/MFs), play a key role in liver fibrosis, a potentially reversible process that may rely on induction of HSC/MFs apoptosis. While this possibility has been shown in cultured rat HSC, very limited data are currently available for human HSC/MFs. METHODS: Cultured human HSC/MFs were exposed to several proapoptotic stimuli, including those known to induce apoptosis in rat HSC/MFs, and induction of cell death and related mechanisms were investigated using morphology, molecular biology, and biochemical techniques. RESULTS: In this study we report that fully activated human HSC/MFs did not undergo spontaneous apoptosis and survived to prolonged serum deprivation, Fas activation, or exposure to nerve growth factor, tumour necrosis factor alpha (TNF-alpha), oxidative stress mediators, doxorubicin, and etoposide. Induction of caspase dependent, mitochondria driven apoptosis in HSC/MFs was observed only when protein synthesis or transcription were inhibited. Importantly, the process of HSC activation was accompanied by changes in expression of a set of genes involved in apoptosis control. In particular, activated human HSC/MFs in culture overexpressed Bcl-2. The role of Bcl-2 was crucial as Bcl-2 silenced cells became susceptible to TNF-alpha induced apoptosis. Finally, Bcl-2 was markedly expressed in HSC/MFs present in liver tissue obtained from patients with hepatitis C virus related cirrhosis. CONCLUSIONS: Human activated HSC/MFs are resistant to most proapoptotic stimuli due to Bcl-2 overexpression and this feature may play a key role in the progression of fibrosis in chronic liver diseases.

Apoptosis↗
PubMed2005

Dose dependent and divergent effects of superoxide anion on cell death, proliferation, and migration of activated human hepatic stellate cells.

BACKGROUND AND AIMS: Activated myofibroblast-like cells, originating from hepatic stellate cells (HSC/MFs) or other cellular sources, play a key profibrogenic role in chronic liver diseases (CLDs) that, as suggested by studies in animal models or rat HSC/MFs, may be modulated by reactive oxygen intermediates (ROI). In this study, human HSC/MFs, exposed to different levels of superoxide anion (O(2)(.-)) and, for comparison, hydrogen peroxide (H(2)O(2)), were analysed in terms of cytotoxicity, proliferative response, and migration. METHODS: Cultured human HSC/MFs were exposed to controlled O(2)(.-) generation by hypoxanthine/xanthine oxidase systems or to a range of H(2)O(2) concentrations. Induction of cell death, proliferation, and migration were investigated using morphology, molecular biology, and biochemical techniques. RESULTS: Human HSC/MFs were shown to be extremely resistant to induction of cell death by O(2)(.-) and only high rates of O(2)(.-) generation induced either necrotic or apoptotic cell death. Non-cytotoxic low levels of O(2)(.-), able to upregulate procollagen type I expression (but not tissue inhibitor of metalloproteinase 1 and 2), stimulated migration of human HSC/MFs in a Ras/extracellular regulated kinase (ERK) dependent, antioxidant sensitive way, without affecting basal or platelet derived growth factor (PDGF) stimulated cell proliferation. Non-cytotoxic levels of H(2)O(2) did not affect Ras/ERK or proliferative response. A high rate of O(2)(.-) generation or elevated levels of H(2)O(2 )induced cytoskeletal alterations, block in motility, and inhibition of PDGF dependent DNA synthesis. CONCLUSIONS: Low non-cytotoxic levels of extracellularly generated O(2)(.-) may stimulate selected profibrogenic responses in human HSC/MFs without affecting proliferation.

Apoptosis↗
PubMed2004

Liver fibrosis: from the bench to clinical targets.

Progressive liver fibrosis is the main cause of organ failure in chronic liver diseases of any aetiology. Fibrosis develops with different spatial patterns and is a consequence of different prevalent mechanisms according to the diverse causes of parenchymal damage. Indeed, fibrosis, observed as a consequence of chronic viral infection is initially concentrated within and around the portal tract, while fibrosis secondary to toxic/metabolic damage is located mainly in the centrolobular areas. In addition, it is increasingly evident that different cell types are involved in the deposition of fibrillar extracellular matrix during active hepatic fibrogenesis: hepatic stellate cells are mainly involved when hepatocellular damage is limited or concentrated within the liver lobule, whereas portal myofibroblasts and fibroblasts provide a predominant contribution when the damage is located in the proximity of the portal tracts. In the later stages of evolution (septal fibrosis) it is likely that all extracellular matrix-producing cells contribute to fibrogenesis. Recruitment and activation of extracellular matrix-producing cells to the site of tissue damage can be due to different major mechanisms: (1) Chronic activation of the tissue repair process. In this case, as a consequence of the reiterated damage, accumulation of fibrillar extracellular matrix reflects the impossibility of an effective remodelling and regeneration. (2) Effect of oxidative stress products, including reactive oxygen intermediates and reactive aldehydes. These products, whose concentration become critical in toxic/metabolic liver injury, are able to induce the synthesis of fibrillar extracellular matrix even in the absence of significant hepatocyte damage and inflammation. (3) Derangement of normal the epithelial/mesenchymal interaction. This typically occurs in all conditions characterised by cholangiocyte damage/proliferation, where a consensual proliferation of extracellular matrix-producing cells and progressive fibrogenesis is commonly observed. A major advancement towards the understanding of the molecular mechanisms of fibrogenesis is derived from a consistent number of in vitro studies investigating the biological role of growth factors/cytokines and other soluble factors and their intracellular signalling pathways. The relevance of these factors has been confirmed by studies performed on animal models and by studies performed on pathological human liver. Along these lines, the elucidation of a consistent number of cellular and molecular mechanisms responsible for the progression of liver fibrosis has provided sound basis for the development of pharmacological strategies able to modulate this important pathophysiological process. Finally, there are several clinically relevant issues that need re-evaluation and/or further investigation, and in particular: (1) the need of an accurate and effective monitoring of the fibrotic progression of chronic liver diseases and of the effectiveness of the currently proposed treatments; (2) the identification of general or individual factors potentially relevant for a faster progression of the disease.

Apoptosis↗
PubMed2004

Expression of somatostatin receptors in normal and cirrhotic human liver and in hepatocellular carcinoma.

BACKGROUND: Somatostatin analogues have been used with conflicting results to treat advanced hepatocellular carcinoma (HCC). The aim of this study was to investigate expression of somatostatin receptor (SSTR) subtypes in human liver, and to examine the effect of selective SSTR agonists on proliferation, apoptosis, and migration of hepatoma cells (HepG2, HuH7) and hepatic stellate cells (HSCs). METHODS: Expression of SSTRs in cell lines, normal and cirrhotic liver, and HCC was examined by immunohistochemistry and reverse transcription-polymerase chain reaction. Effects of SSTR agonists on proliferation and apoptosis of tumour cells and HSCs were assessed by the 5-bromo-2' deoxyuridine and TUNEL methods, respectively. The influence of SSTR agonists on migration was investigated using Boyden chambers. RESULTS: In normal liver, both hepatocytes and HSCs were negative for all five SSTRs. Cirrhotic liver and HCC as well as cultured hepatoma cells and HSCs expressed all five SSTRs, both at the protein and mRNA levels, except for HuH7 cells which did not immunoreact with SSTR3. None of the agonists influenced proliferation or apoptosis. However, compared with untreated cells, L-797,591, an SSTR1 agonist, reduced migration of HepG2, HuH7, and HSCs significantly to 88 (7)% (p<0.05), 83 (11)% (p<0.05), and 67 (13)% (p<0.01), respectively. CONCLUSIONS: Cirrhotic liver and HCC express SSTRs. Although the somatostatin analogues used in this study did not affect proliferation and apoptosis, stimulation of SSTR1 may decrease invasiveness of HCC by reducing migration of hepatoma cells and/or HSCs. Clinical trials evaluating somatostatin analogues for the treatment of HCC should take these findings into account.

Apoptosis↗
PubMed2001

Signal transduction by the chemokine receptor CXCR3: activation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/Akt controls cell migration and proliferation in human vascular pericytes.

Hepatic stellate cells (HSC) and glomerular mesangial cells (MC) are tissue-specific pericytes involved in tissue repair, a process that is regulated by members of the chemokine family. In this study, we explored the signal transduction pathways activated by the chemokine receptor CXCR3 in vascular pericytes. In HSC, interaction of CXCR3 with its ligands resulted in increased chemotaxis and activation of the Ras/ERK cascade. Activation of CXCR3 also stimulated Src phosphorylation and kinase activity and increased the activity of phosphatidylinositol 3-kinase and its downstream pathway, Akt. The increase in ERK activity was inhibited by genistein and PP1, but not by wortmannin, indicating that Src activation is necessary for the activation of the Ras/ERK pathway by CXCR3. Inhibition of ERK activation resulted in a decreased chemotactic and mitogenic effect of CXCR3 ligands. In MC, which respond to CXCR3 ligands with increased DNA synthesis, CXCR3 activation resulted in a biphasic stimulation of ERK activation, a pattern similar to the one observed in HSC exposed to platelet-derived growth factor, indicating that this type of response is related to the stimulation of cell proliferation. These data characterize CXCR3 signaling in pericytes and clarify the relevance of downstream pathways in the modulation of different biologic responses.

Androstadienes↗
PubMed2001

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

AIMS: Despite the importance of dendritic cells in stimulating primary and secondary immune responses by presenting antigens to T-lymphocytes in draining lymph nodes and peripheral tissues, respectively, very limited information is available on the presence and localization of these cells in hepatitis C virus (HCV)-related chronic active hepatitis. Therefore, we addressed the ultrastructure, immunophenotype, distribution and relationships to lymphatics of dendritic cells in portal infiltrates of this disease. METHODS AND RESULTS: Part of percutaneous diagnostic liver biopsies (Knodell's histological assessment index: 9-13) was processed for electron microscopy and for immunohistochemical detection of immune system cell membrane antigens and of the lymphatic endothelium marker podoplanin. In portal infiltrates, cells with electron microscopical and cell marker features of dendritic cells and expressing the activation markers CD54, CD80, CD83 and CD86 were organized in a discontinuous network, that embedded CD8+ lymphocytes in close contact with dendritic cells and came in contact with hepatocytes, sometimes infiltrating beyond the limiting plate. Also, dendritic cells were found within newly formed lymphatic capillaries in thin, infiltrated septa among hepatocytes. CONCLUSIONS: This evidence strongly suggests a critical role of dendritic cells and newly formed lymphatics in the pathogenesis and organization of the immune infiltrate that characterizes HCV-related chronic active hepatitis.

Adult↗
PubMed2001

Agonist-specific regulation of monocyte chemoattractant protein-1 expression by cyclooxygenase metabolites in hepatic stellate cells.

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.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2001

Cytokine receptors and signaling in hepatic stellate cells.

Following acute or chronic liver tissue damage, hepatic stellate cells (HSCs) undergo a process of activation toward a phenotype characterized by increased proliferation, motility, contractility, and synthesis of extracellular matrix components. Activation of HSCs is regulated by several soluble factors, including growth factors, cytokines, chemokines, and products of oxidative stress, as well as by extensive changes in the composition and organization of the ECM. Different groups of soluble factors may be classified according to their prevalent biological effect: (a) factors promoting HSC proliferation and/or migration (i.e., platelet-derived growth factor, basic fibroblast growth factor, insulin-like growth factor-1); (b) factors promoting fibrillar ECM accumulation, particularly transforming growth factor-beta1; (c) factors with a prevalent contractile effect on HSCs, such as endothelin-1, thrombin, angiotensin-II and vasopressin, although all these agents also may promote HSC proliferation; (d) proinflammatory cytokines and chemokines; and (e) cytokines with a prominent antiinflammatory/antifibrogenic activity, such as interleukin-10 and interferon-gamma. Additional important issues are represented by the relationship between cytokine and integrin signaling, and by the effects of oxidative stress-related molecules on cytokine signaling. In the past decade the major intracellular signaling pathways elicited by these factors in HSCs have been greatly elucidated.

Cytokines↗
PubMed2001

Hepatology 2001.

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Journal Article↗
PubMed2000

Interaction between 4-hydroxy-2,3-alkenals and the platelet-derived growth factor-beta receptor. Reduced tyrosine phosphorylation and downstream signaling in hepatic stellate cells.

Hepatic stellate cells (HSC) undergo activation toward myofibroblast-like cells during early stages of liver injury associated with fibrogenesis. Platelet-derived growth factor (PDGF), particularly its BB isoform, has been identified as the most potent mitogen for HSC. 4-Hydroxy-2,3-nonenal and related 4-hydroxy-2, 3-alkenals (HAKs) have been suggested to modulate the process of HSC activation. In this study we investigated the relationship between HAKs and PDGF receptor activation in human HSC. By employing noncytotoxic concentrations (10(-6) m) of HAKs, we observed a significant inhibition of PDGF-BB-dependent DNA synthesis. HAKs inhibited relevant pathways of PDGF-BB-dependent mitogenic signaling, including autophosphorylation of PDGF receptor (PDGF-R) beta subunits and activation of phosphatidylinositol 3-kinase and extracellular regulated kinases 1/2. Inhibition of DNA synthesis was reversible, and recovery of PDGF-mediated mitogenic signaling occurred within 24-48 h and was associated with HAKs-induced up-regulation of PDGF-R beta gene expression. 4-Hydroxy-2,3-nonenal, used as a model HAK, inhibited the intrinsic tyrosine kinase activity associated with the PDGF-R beta subunit, whereas binding of PDGF to its receptor was unaffected. This study identifies a novel regulatory mechanism of reactive aldehydes on PDGF receptor signaling and biologic actions, which may be relevant in several pathophysiological conditions, including liver fibrosis.

Aldehydes↗
PubMed2000

Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on ras in human hepatic stellate cells.

Focal adhesion kinase (FAK) is a widely expressed nonreceptor tyrosine kinase found in focal adhesions. FAK has been indicated as a point of convergence of other signaling pathways including platelet-derived growth factor (PDGF) receptors, and recently, FAK tyrosine phosphorylation has been shown to be stimulated by PDGF. In the present study we assessed the role of Ras as a possible intermediate protein regulating PDGF-induced FAK tyrosine phosphorylation in human hepatic stellate cells (HSCs), liver-specific pericytes primarily involved in the pathogenesis of liver fibrosis. For this purpose, cells were first subjected to retroviral-mediated gene transfer with a dominant-negative mutant of Ras (N17Ras). This resulted in a marked inhibition of PDGF-induced FAK tyrosine phosphorylation together with the expected reduction of PDGF-induced extracellular signal-regulated kinase activity (ERK). Afterward, the effects of pharmacological agents potentially affecting Ras isoprenylation were evaluated. PDGF-induced FAK tyrosine phosphorylation, ERK activity and intracellular calcium increase, as well as the biological effects of this growth factor, (i.e., mitogenesis and cell migration) were effectively blocked by GGTI-298, an inhibitor of geranylgeranyltransferase I. Inhibition of Ras processing obtained with FTI-277, an inhibitor of farnesyltransferase, resulted in detectable effects only at high doses. Taken together, these results establish that Ras operates as a protein-linking PDGF-beta receptor to FAK in human HSCs, and that signaling molecules requiring geranylgeranylation may also be involved in this process.

Alkyl and Aryl Transferases↗
PubMed2000

Phosphatidylinositol-3 kinase and extracellular signal-regulated kinase mediate the chemotactic and mitogenic effects of insulin-like growth factor-I in human hepatic stellate cells.

BACKGROUND/AIM: Several studies have shown that proliferation of hepatic stellate cells is stimulated by insulin-like growth factor-I. The aim of this study was to investigate the effect of insulin-like growth factor-I on human hepatic stellate cells chemotaxis and the intracellular pathways involved in both mitogenic and chemotactic effects. METHODS/RESULTS: Insulin-like growth factor-I, at the concentration of 100 ng/ml, was able to induce a 2- to 3-fold increase in human hepatic stellate cells migration in a modified Boyden chamber system. This effect was associated with a marked activation of phosphatidylinositol 3-kinase by insulin-like growth factor-I, as evaluated by measurement of phosphatidylinositol 3-kinase activity in phosphotyrosine immunoprecipitates In order to establish a functional link between these observations, we then performed experiments employing two selective phosphatidylinositol 3-kinase inhibitors, namely wortmannin and LY294002. These compounds blocked activation of phosphatidylinositol 3-kinase and inhibited insulin-like growth factor-I-induced hepatic stellate cells migration. Since phosphatidylinositol 3-kinase activation has been shown to be necessary for platelet-derived growth factor-induced mitogenesis in hepatic stellate cells, we verified the effects of phosphatidylinositol 3-kinase inhibition on insulin-like growth factor-I-induced DNA synthesis. Incubation with either wortmannin or LY294002, dose-dependently reduced the mitogenic potential of insulin-like growth factor-I. Since phosphatidylinositol 3-kinase is involved, at least in part, in the activation of the Ras/extracellular signal-regulated kinase pathway in hepatic stellate cells, the role of extracellular signal-regulated kinase activation in mediating the biological effects of insulin-like growth factor-I was explored. Insulin-like growth factor-I induced mitogenesis and chemotaxis were markedly reduced by pre-incubation of hepatic stellate cells with PD-98059, a selective inhibitor of MEK. CONCLUSIONS: Activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase is required for both insulin-like growth factor-I-dependent hepatic stellate cells proliferation and chemotaxis. Insulin-like growth factor-I, together with other soluble mediators, may contribute to the hepatic wound-healing response by modulating hepatic stellate cells migration and proliferation.

Androstadienes↗