Maurizio Parola

Oxidative damage and fibrogenesis.

Various chronic disease processes are characterized by progressive accumulation of connective tissue under-going fibrotic degeneration. Evidence of oxidative reactions is often associated with fibrogenesis occurring in liver, lung, arteries, and nervous system. Moreover, an increasing bulk of experimental and clinical data supports a contributory role of oxidative stress in the pathogenesis of this kind of disease. Indeed, many etiological agents of fibrogenesis stimulate free radical reactions either directly or through inflammatory stimuli. Free radicals, as well as products of their reaction with biomolecules, appear to modulate the activity of the two cellular types mainly involved in the process, namely phagocytes and extracellular matrix-producing cells. Lipid peroxidation and certain lipid peroxidation products induce genetic overexpression of fibrogenic cytokines, the key molecules in the pathomechanisms of fibrosis, as well as increased transcription and synthesis of collagen. Both these events can be downregulated, at least in experimental models, by the use of antioxidants. The effect of oxidative stress on cytokine gene expression appears to be an important mechanism by which it promotes connective tissue deposition.

HNE interacts directly with JNK isoforms in human hepatic stellate cells.

4-Hydroxy-2,3-nonenal (HNE) is an aldehydic end product of lipid peroxidation which has been detected in vivo in clinical and experimental conditions of chronic liver damage. HNE has been shown to stimulate procollagen type I gene expression and synthesis in human hepatic stellate cells (hHSC) which are known to play a key role in liver fibrosis. In this study we investigated the molecular mechanisms underlying HNE actions in cultured hHSC. HNE, at doses compatible with those detected in vivo, lead to an early generation of nuclear HNE-protein adducts of 46, 54, and 66 kD, respectively, as revealed by using a monoclonal antibody specific for HNE-histidine adducts. This observation is related to the lack of crucial HNE-metabolizing enzymatic activities in hHSC. Kinetics of appearance of these nuclear adducts suggested translocation of cytosolic proteins. The p46 and p54 isoforms of c-Jun amino-terminal kinase (JNKs) were identified as HNE targets and were activated by this aldehyde. A biphasic increase in AP-1 DNA binding activity, associated with increased mRNA levels of c-jun, was also observed in response to HNE. HNE did not affect the Ras/ERK pathway, c-fos expression, DNA synthesis, or NF-kappaB binding. This study identifies a novel mechanism linking oxidative stress to nuclear signaling in hHSC. This mechanism is not based on redox sensors and is stimulated by concentrations of HNE compatible with those detected in vivo, and thus may be relevant during chronic liver diseases.

Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells

Leptin upregulates collagen expression in hepatic stellate cells (HSCs), but the possible modulation of other actions has not been elucidated. The aim of this study was to investigate the expression and function of leptin receptors (ObR) in human HSCs and the biological actions regulated by leptin. Exposure of HSCs to leptin resulted in upregulation of monocyte chemoattractant protein 1 (MCP‐1) expression. Leptin also increased gene expression of the proangiogenic cytokines vascular endothelial growth factor (VEGF) and angiopoietin‐1, and VEGF was also upregulated at the protein level. Activated HSCs express ObRb and possibly other ObR isoforms. Exposure to leptin increased the tyrosine kinase activity of ObR immunoprecipitates and resulted in activation of signal transducer and activator of transcription 3. Several signaling pathways were activated by leptin in HSCs, including extracellular‐signal–regulated kinase, Akt, and nuclear factor κB, the latter being relevant for chemokine expression. Leptin also increased the abundance of hypoxia‐inducible factor 1α, which regulates angiogenic gene expression, in an extracellular‐signal–regulated kinase– and phoshatidylinositol 3‐kinase–dependent fashion. In vivo, leptin administration induced higher MCP‐1 expression and more severe inflammation in mice after acute liver injury. Conversely, in leptin‐deficient mice, the increase in MCP‐1 messenger RNA and mononuclear infiltration was less marked than in wild‐type littermates. Finally, ObR expression colocalized with VEGF and α‐smooth muscle actin after induction of fibrosis in rats. In conclusion, ObR activation in HSCs leads to increased expression of proinflammatory and proangiogenic cytokines, indicating a complex role for leptin in the regulation of the liver wound‐healing response.(HEPATOLOGY 2005;42:1339–1348.)

β‐Site APP cleaving enzyme up‐regulation induced by 4‐hydroxynonenal is mediated by stress‐activated protein kinases pathways

4‐Hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, up‐regulates expression of the β‐site APP cleaving enzyme (BACE‐1), an aspartyl protease responsible for the β‐secretase cleavage of amyloid precursor protein (AβPP), and results in increased levels of amyloid β (Aβ) peptide. The mechanisms underlying this remain unclear but are of fundamental importance because prevention of BACE‐1 up‐regulation is viewed as an important therapeutic strategy. In this study, we exposed NT2 neurons to a range of HNE concentrations (0.5–5 µm) that elicited an up‐regulation of BACE‐1 expression, a significant increase in intracellular and secreted levels of Aβ peptides as well as apoptosis involving poly‐ADP ribose polymerase cleavage and activation of caspase 3. To delineate the molecular events involved in HNE‐mediated BACE‐1 activation, we investigated the involvement of stress‐activated protein kinases (SAPK), signal transducers and activators of transcription (STAT) and serine–threonine kinase B/phosphatidylinositol phosphate 3 kinase (Akt/PtdIns3K). Using specific pharmacological inhibitors, our results show that activation of c‐Jun N‐terminal kinases and p38MAPK., but not STAT or Akt/PtdIns3K, pathways mediate the HNE‐dependent up‐regulation of BACE‐1 expression. Therefore, HNE, an oxidative stress mediator detected in vivo in the brains of Alzheimers disease patients, may play a pathogenetic role in Alzheimers disease by selectively activating SAPK pathways and BACE‐1 that regulate the proteolytic processing of AβPP.

Redox mechanisms switch on hypoxia-dependent epithelial–mesenchymal transition in cancer cells

Epithelial-mesenchymal transition (EMT) and hypoxia are considered as crucial events favouring invasion and metastasis of many cancer cells. In this study, different human neoplastic cell lines of epithelial origin were exposed to hypoxic conditions in order to investigate whether hypoxia per se may trigger EMT programme as well as to mechanistically elucidate signal transduction mechanisms involved. The following human cancer cell lines were used: HepG2 (from human hepatoblastoma), PANC-1 (from pancreatic carcinoma), HT-29 (from colon carcinoma) and MCF-7 (from breast carcinoma). Cancer cells were exposed to carefully controlled hypoxic conditions and investigated for EMT changes and signal transduction by using morphological, cell and molecular biology techniques. All cancer cells responded to hypoxia within 72 h by classic EMT changes (fibroblastoid phenotype, SNAIL and beta-catenin nuclear translocation and changes in E-cadherin) and by increased migration and invasiveness. This was involving very early inhibition of glycogen synthase kinase-3beta (GSK-3beta), early SNAIL translocation as well as later and long-lasting activation of Wnt/beta-catenin-signalling machinery. Experimental manipulation, including silencing of hypoxia-inducible factor (HIF)-1alpha and the specific inhibition of mitochondrial generation of reactive oxygen species (ROS), revealed that early EMT-related events induced by hypoxia (GSK-3beta inhibition and SNAIL translocation) were dependent on transient intracellular increased generation of ROS whereas late migration and invasiveness were sustained by HIF-1alpha- and vascular endothelial growth factor (VEGF)-dependent mechanisms. These findings indicate that in cancer cells, early redox mechanisms can switch on hypoxia-dependent EMT programme whereas increased invasiveness is sustained by late and HIF-1alpha-dependent release of VEGF.

Human mesenchymal stem cells as a two-edged sword in hepatic regenerative medicine: engraftment and hepatocyte differentiation versus profibrogenic potential

Background and aim: Mesenchymal stem cells from bone marrow (MSCs) may have the potential to differentiate in vitro and in vivo into hepatocytes. We investigated whether transplanted human MSCs (hMSCs) may engraft the liver of non-obese diabetic severe combined immuno-deficient (NOD/SCID) mice and differentiate into cells of hepatic lineage. Methods: Ex vivo expanded, highly purified and functionally active hMSCs from bone marrow were transplanted (caudal vein) in sublethally irradiated NOD/SCID mice that were either exposed or not to acute liver injury or submitted to a protocol of chronic injury (single or chronic intraperitoneal injection of CCl4, respectively). Chimeric livers were analysed for expression of human transcripts and antigens. Results: Liver engraftment of cells of human origin was very low in normal and acutely injured NOD/SCID mice with significantly higher numbers found in chronically injured livers. However, hepatocellular differentiation was relatively rare, limited to a low number of cells (ranging from less than 0.1% to 0.23%) as confirmed by very low or not detectable levels of human transcripts for α-fetoprotein, CK18, CK19 and albumin in either normal or injured livers. Finally, a significant number of cells of human origin exhibited a myofibroblast-like morphology. Conclusions: Transplanted hMSCs have the potential to migrate into normal and injured liver parenchyma, particularly under conditions of chronic injury, but differentiation into hepatocyte-like cells is a rare event and pro-fibrogenic potential of hMSC transplant should be not under-evaluated.

Phosphatidylinositol 3-kinase is required for platelet-derived growth factor's actions on hepatic stellate cells.

BACKGROUND & AIMS Platelet-derived growth factor (PDGF) is the most potent mitogen for hepatic stellate cells (HSCs) in vitro. The aim of this study was to investigate the role of phosphatidylinositol 3-kinase (PI 3-K) activation in mediating the biological effects of PDGF on cultured HSCs and its involvement in vivo. METHODS HSCs were isolated from normal human livers. PI 3-K was assayed on phosphotyrosine or PDGF-receptor immunoprecipitates by in vitro kinase assay. RESULTS Incubation of HSCs with PDGF caused a time-dependent increase in PI 3-K activity. Immunoprecipitation of PDGF-alpha and -beta receptors showed that both subunits associate with active PI 3-K in PDGF-stimulated HSCs. Wortmannin, a specific PI 3-K inhibitor, dose-dependently blocked PI 3-K activity induced by PDGF and inhibited DNA synthesis. PDGF (homodimer)-BB also stimulated HSC chemotaxis, which was inhibited by pretreatment with wortmannin. To explore the potential role of PI 3-K in vivo, liver homogenates from rats treated with CCl4 and from control rats were immunoprecipitated with anti-PDGF-beta-receptor antibodies. Liver injury was associated with increased PDGF-beta-receptor autophosphorylation, and greater PI 3-K activity associated with the receptor itself. CONCLUSIONS This study shows that in cultured HSCs, PI 3-K activation is necessary for both mitogenesis and chemotaxis induced by PDGF and that this pathway is up-regulated during liver injury in vivo.

H2o2 and 4-hydroxynonenal mediate amyloid β-induced neuronal apoptosis by activating jnks and p38mapk

Amyloid beta peptides (Abeta) may be neurotoxic during the progression of Alzheimers disease by eliciting oxidative stress. Exposure of neuronally differentiated SK-N-BE cells to Abeta(25-35) fragment as well as to full-length Abeta(1-40) and Abeta(1-42) induces early and time-dependent generation of oxidative stress that has been evaluated by carefully monitoring generation of hydrogen peroxide (H(2)O(2)), 4-hydroxynonenal (HNE), thiobarbituric acid reactive substances (TBARS), and fluorescent chromolipids. Abeta treatment also results in the activation of c-Jun aminoterminal kinases (JNKs) and p38(MAPK) and is followed by characteristic nuclear changes of apoptosis as evaluated by DAPI staining and TUNEL technique. To reproduce the relationships between oxidative stress and Abeta apoptosis we found that only the simultaneous administration of HNE and H(2)O(2), at concentrations similar to those generated within the first 3 h of Abeta exposure, can fully mimic Abeta-dependent activation of JNKs and p38(MAPK) and occurrence of apoptosis. Antioxidants such as alpha-tocopherol and N-acetylcysteine prevent completely either neuronal apoptosis or activation of JNKs and p38(MAPK) elicited by Abeta or by simultaneous HNE and H(2)O(2) addition. Finally, direct evidence that activation of these kinases is required for cell death induced by Abeta has been obtained by pretreating cell with specific inhibitors of JNKs and p38(MAPK). These results suggest the existence of a sequence of events in Abeta-induced apoptosis involving simultaneous generation of HNE and H(2)O(2) and oxidative stress-dependent activation of JNKs and p38(MAPK).

The up-regulation of BACE1 mediated by hypoxia and ischemic injury: role of oxidative stress and HIF1α

While it is well established that stroke and cerebral hypoperfusion are both significant risk factors for Alzheimer’s disease, the molecular link between ischemia and amyloid precursor protein processing has only been recently established. Specifically, hypoxia significantly increases β‐site APP cleaving enzyme (BACE1) gene transcription through the over‐expression of hypoxia inducible factor 1α, resulting in increased BACE1 secretase activity and amyloid‐β production. In this study, we significantly extend these findings both in vitro, in differentiated SK‐N‐BE neuroblastoma cells, and in vivo, in rats subjected to cerebral ischemia, showing that hypoxia up‐regulates BACE1 expression through a biphasic mechanism. The early post‐hypoxic up‐regulation of BACE1 depends on the production of reactive oxygen species mediated by the sudden interruption of the mitochondrial electron transport chain, while the later expression of BACE1 is caused by hypoxia inducible factor 1α activation. The involvement of reactive oxygen species released by mitochondria in the BACE1 up‐regulation was confirmed by the complete protection exerted by complex I inhibitors such as rotenone and diphenyl‐phenylen iodonium. Moreover, the oxidative stress‐mediated up‐regulation of BACE1 is mediated by c‐jun N terminal kinase pathway as demonstrated by the protection exerted by the silencing of c‐jun N‐terminal kinase isoforms 1 and 2. Our study strengthens the hypothesis that oxidative stress is a basic common mechanism of amyloid‐β accumulation.

On the role of lipid peroxidation in the pathogenesis of liver damage induced by long-standing cholestasis

Previous studies have suggested a possible involvement of free radical reactions in the pathogenesis of cholestatic liver injury as well as in the modulation of hepatic fibrogenesis. In this study we investigated whether lipid peroxidation is involved in the development of chronic liver damage induced by long-standing cholestasis. For this purpose we have used the rat model of bile duct ligation (BDL), which leads to liver fibrosis and cirrhosis. Using this model we observed that the development of chronic liver damage was associated with the onset of lipid peroxidation, as pointed out by detection of carbonyl compounds, 4-hydroxynonenal (HNE) and malondialdehyde (MDA), in BDL livers and of fluorescent adducts between MDA and serum proteins. Lipid peroxidation was a relatively late event (starting after 1-2 weeks of BDL) and was unrelated to the early development of liver necrosis and cholestasis (already evident after 72 h after BDL). A positive significant linear correlation between the kinetic of infiltration of neutrophils and of a monocyte/macrophage population in BDL livers and MDA and HNE generation in the same organs is presented, indicating a close link between lipid peroxidation and the activation of inflammatory cells. We also observed that a positive linear correlation exists between collagen deposition in these livers and hepatic production of MDA and HNE. This event, which is accompanied by an increase in the number of fat storing cells (FSC, the cells that produce collagen in fibrotic liver), suggests that lipid peroxidation in this model may contribute to stimulate collagen synthesis by proliferating FSC.