PubMed Health⌕ Search

Biomedical subjects

M J Arthur

Publications and source records attributed to M J Arthur.

At least 19 recordsLinked to original sources
PubMed1997

Pathogenesis of liver fibrosis.

1. Liver fibrosis is a common sequel to diverse liver injuries. It is characterized by an accumulation of interstitial collagens and other matrix components. The hepatic stellate cell is pivotal to the pathogenic process. Fibrotic liver injury results in activation of the hepatic stellate cell which undergoes a phenotypic change to a proliferative myofibroblast-like cell which synthesizes excess interstitial collagens and other matrix components. 2. The process of initiation of activation and its perpetuation result from complex, often interrelated series of signalling mechanisms which converge on this effector cell. Such mechanisms include alterations in matrix resulting in changed cell-matrix interactions and stimulation by cytokines released from damaged hepatocytes, infiltrating inflammatory cells, Kupffer cells and matrix. Foremost among the profibrotic cytokines is transforming growth factor beta 1. 3. Once the hepatic stellate cell is activated the preceding matrix changes and recurrent injurious stimuli will perpetuate the activated state. 4. Despite the accumulation of excess collagens, the liver retains a capacity for matrix degradation. This capacity may be overwhelmed and any secreted matrix remodelling enzymes may be inhibited by the concurrently secreted tissue inhibitors of metallo-proteinase-1 and alpha 2-macroglobulin. 5. Our understanding of the molecular pathogenesis of liver fibrosis is increasing. It is anticipated that this knowledge will provide novel therapeutic avenues to treat this disease process.

Cell Differentiation↗
PubMed1996

Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver injury and fibrosis.

Liver fibrosis results from a relative imbalance between synthesis and degradation of matrix proteins. We have previously described release of the protein collagenase inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), by culture-activated human hepatic stellate cells (HSCs). In this study, we have investigated the relative expression of TIMP-1 and interstitial collagenase in culture-activated rat HSCs and rat models of liver injury and fibrosis. The complementary DNA (cDNA) for rat TIMP-1 was obtained by homology polymerase chain reaction (PCR) and sequenced. By Northern analysis using this probe, TIMP-1 messenger RNA (mRNA) expression was up-regulated with HSC activation by culture on plastic as defined by cellular expression of procollagen-1. Interstitial collagenase mRNA was expressed in early 1. Interstitial collagenase mRNA was expressed in early culture (<4 days) but became undetectable in more activated cells (7-21 days). By activity assay of serum-free cell-conditioned media, TIMP-1 was found to be released in increasingly concentrations with duration of culture on plastic. Expression of TIMP-1 interstitial collagenase, and procollagen-1 mRNAs were studied in rat models of biliary and parenchymal injury (bile duct ligation and CC14 administration) by ribonuclease protein assay. TIMP-1 mRNA expression was increased at 6, 24 hours, and 3 days after bile duct ligation and was also shown to rise in acute CC14 liver injury and remain elevated as the liver became fibrotic. TIMP-1 expression preceded procollagen-1 expression in both models. In contrasts, interstitial collagenase mRNA levels remained similar to control values throughout both models of liver injury. Total cellular RNA from hepatocytes, HSCs, and kupffer cells freshly isolated from livers after acute CC14 injury was subjected to Northern analysis. TIMP-1 transcripts were observed in nonparenchymal cells only. We suggest that increased expression of TIMP-1 relative to interstitial collagenase by HSCs may promote progression of liver fibrosis in these rat models by preventing degradation of secreted collagens.

Amino Acid Sequence↗
PubMed1996

The plasminogen-activating system in hepatic stellate cells.

Urokinase plasminogen activator (uPA) generates plasmin, a process inhibited by plasminogen-activator inhibitor (PAI)-1 and localized to the cell surface by binding of uPA to a specific receptor. Plasmin degrades extracellular matrix (ECM) both directly and by activation of matrix metalloproteinases (MMPs). Because stellate cells play a central role in the pathogenesis of liver fibrosis both via production of ECM proteins and through secretion of MMPs, their contribution to plasmin generation was assessed. Stellate cells were prepared from rat liver and cultured on plastic. Northern analysis showed cellular expression of messenger RNA (mRNA) for PAI-1, uPA, and uPA receptor. Zymography/reverse zymography identified cell-surface-associated uPA activity and uPA and PAI-1 in culture media. Net uPA activity in culture media was maximal after 7 days in culture and then declined, whereas PAI-1 antigen levels remained consistently elevated between 7 and 21 days in culture. Stellate cell-mediated plasmin generation was also seen in in vitro cultures supplemented with plasminogen. Because hepatic stellate cells (HSCs) contain retinoids and release them on activation, the effect of retinoic acid on the plasminogen-activating system was also assessed. Treatment of cultured HSCs with retinoic acid (1 micromol/L) increased uPA secretion 2.6-fold but did not alter PAI-1. We conclude that stellate cells synthesize key components of the plasminogen-activating system and generate plasmin and therefore have the ability to regulate MMP activation. Upregulation of uPA synthesis by retinoic acid may have implications in matrix remodeling in sites of stellate cell activation in which high concentrations of retinoids may be achieved.

Adipocytes↗
PubMed1996

Expression of tissue inhibitor of metalloproteinases 1 and 2 is increased in fibrotic human liver.

BACKGROUND & AIMS: Tissue inhibitors of metalloproteinases may contribute to liver fibrosis by preventing remodeling of fibrillar collagens by interstitial collagenase. This hypothesis was investigated by comparing the relative expression of messenger RNA for interstitial collagenase, gelatinase A, and tissue inhibitor of metalloproteinases 1 and 2 in fibrotic and normal human liver. METHODS: Hepatic expression of metalloproteinases and their inhibitors was examined using ribonuclease protection assay, immunocytochemistry, and immunoassay. Lipocyte expression of tissue inhibitors of metalloproteinases was examined using Northern blotting and reverse zymography. RESULTS: Messenger RNA levels for tissue inhibitors of metalloproteinase 1 and 2 were elevated to 260%-526% of levels in normal liver in biliary atresia, primary biliary cirrhosis, and primary sclerosing cholangitis. In fibrotic livers, tissue inhibitor of metalloproteinase 1 protein was 367%-724% of normal. Gelatinase A messenger RNA level increased to 324%-430% of normal values in fibrotic liver, but interstitial collagenase messenger RNA level was not significantly altered. Normal human liver lipocytes activated by culture on plastic expressed messenger RNA and protein for tissue inhibitor of metalloproteinase 1 and 2. CONCLUSIONS: Increased expression of tissue inhibitors of metalloproteinases relative to interstitial collagenase may promote deposition of interstitial collagens in liver fibrosis. Our studies further suggest that lipocytes are an important source of tissue inhibitors of metalloproteinases in progressive liver fibrosis.

Adipocytes↗
PubMed1996

Iron overload and liver fibrosis.

The pathogenesis of liver fibrosis in genetic haemochromatosis and other iron overload states remains enigmatic. Recent advances in the cellular and molecular pathogenesis of liver fibrosis have determined a central role for hepatic stellate cells. These become activated to a myofibroblastic phenotype following most forms of liver injury and are the major cellular source of collagens and other matrix proteins laid down in fibrotic liver. Similar changes have now been reported in the liver in genetic haemochromatosis, with activation of stellate cells becoming more prominent with increasing hepatic iron concentration. In contrast to other liver diseases, this apparently occurs in the absence of significant necroinflammatory change. Unravelling the mechanism of liver fibrogenesis in iron overload states may, therefore, provide important general insights into the pathogenesis of liver fibrosis. The present article reviews current knowledge of this field with emphasis on the role of lipid peroxidation, sideronecrosis of hepatocytes and spillover of iron to Kupffer cells. An attempt is made to draw these observations together with previous studies of the mechanisms of stellate cell activation in other models and diseases. A unifying hypothesis emerges that helps to define some of the next research questions in the pathogenic mechanisms of liver fibrosis in iron overload.

Humans↗
PubMed1996

Augmentation by eosinophils of gelatinase activity in the airway mucosa: comparative effects as a putative mediator of epithelial injury.

1. We have studied the release of gelatin-degrading enzymes from isolated sheets of bronchial mucosa in the presence and absence of eosinophils. 2. Isolated sheets of bovine bronchial mucosa released gelatin-degrading activity in similar amounts from both the apical and basolateral aspects of the tissue. Gelatinolytic activity could not be increased by treatment of the mucosal sheets with calcium ionophore, A23187. 3. The activity of the released gelatinases could be inhibited by chelation of divalent cations or by the matrix metalloproteinase inhibitors, BB-94 and BB-250. However, inhibitors of serine proteinases, or of cysteine proteinases were without effect. In zymography, major bands of gelatin-degrading activity consistent with gelatinases A and B were identified. 4. Addition of guinea-pig eosinophils to the basolateral aspect of bronchial mucosa for 60 min resulted in an increase in the gelatinolytic activity of the conditioned medium, irrespective of whether the eosinophils were stimulated with ionophore A23187 or not. However, only ionophore-stimulated eosinophils reacted to produce sufficient tissue damage to increase the transepithelial flux of serum albumin. 5. Purified eosinophils were a poor source of gelatinolytic activity, indicating that when interacting with the bronchial mucosa their effect is to increase the apparent release and/or activation of gelatinases derived from the airway mucosa. 6. After organomercurial activation, recombinant human progelatinase A increased the permeability of the bronchial mucosa to mannitol. However, the activity of enzyme and duration of exposure required to do this were greater than the amounts of gelatinase activity detected during eosinophil-mediated injury. Sheets of airway mucosa were also resistant to injury evoked by high concentrations of hydrogen peroxide or plasmin. 7. Collectively, these results suggest that if gelatinases are involved in eosinophil-mediated injury and repair of the bronchial mucosa, they require other mediators to act in concert to bring about outright epithelial cell detachment. This does not preclude the possibility that gelatinases are crucial in rendering the airway mucosa hyperfragile.

Animals↗
PubMed1995

Rat hepatic lipocytes synthesize and secrete transin (stromelysin) in early primary culture.

BACKGROUND & AIMS: Hepatic lipocyte proliferation and activation are pivotal in liver fibrosis. Disruption of normal lipocyte-matrix interactions may contribute to this process. The synthesis of transin, which degrades normal liver matrix, by culture-activated hepatic lipocytes was investigated. METHODS: Lipocytes were isolated by pronase/collagenase perfusion, density gradient centrifugation, and centrifugal elutriation. Transin messenger RNA in lipocytes was analyzed by Northern blotting. Transin activity was analyzed by zymography, Western blotting, immunocytochemistry, and quantitative [14C]beta-casein degradation assay. RESULTS: Transin messenger RNA was detected in early primary culture (3-5 days) but not in freshly isolated lipocytes or late primary culture. Zymography of lipocyte medium showed caseinolytic activity (relative molecular weight, 57 kilodaltons and 60 kilodaltons) inhibited by ethyl-enediaminetetraacetic acid but not thiol or serine protease inhibitors. Immunoblotting and immunocytochemistry confirmed the presence of transin in media and cells. Quantitative transin activity decreased progressively with increasing duration of primary lipocyte culture and myofibroblastic transformation. CONCLUSION: Rat hepatic lipocytes express the transin gene and secrete its product during the early phase of lipocyte activation in primary culture. Because this enzyme degrades a wide spectrum of normal basement membrane proteins and activates progelatinase B and interstitial collagenase, it may have an important role in liver injury and fibrosis.

Adipocytes↗
PubMed1995

Tissue inhibitor of metalloproteinase-I and interstitial collagenase expression in autoimmune chronic active hepatitis and activated human hepatic lipocytes.

1. Activated hepatic lipocytes are central to the pathogenesis of liver fibrosis as the principal source of both interstitial collagens and matrix-degrading metalloproteinases. In progressive fibrosis there is a failure to degrade interstitial collagens with a reported decrease in collagenase activity. In these studies we investigate expression of the potent collagenase inhibitor, tissue inhibitor of metalloproteinase-1, and interstitial collagenase in end-stage autoimmune chronic active hepatitis and activated human hepatic lipocytes in culture. 2. Messenger RNA transcripts for interstitial collagenase and tissue inhibitor of metalloproteinase-1 in explanted human liver were quantified by ribonuclease protection assay and densitometric analysis. This indicated that tissue inhibitor of metalloproteinase-1 and interstitial collagenase expression in autoimmune chronic active hepatitis were also coordinately up-regulated. 3. Using Northern analysis of RNA from human lipocytes in primary culture on plastic, mRNA for interstitial collagenase could not be detected in unstimulated cells but was present after stimulation with tumour necrosis factor alpha. Tissue inhibitor of metalloproteinase-1 mRNA was present in unstimulated lipocytes and up-regulated fivefold in response to tumour necrosis factor alpha. Using activity assay of serum-free conditioned media, interstitial collagenase could not be detected in unstimulated primary cultures, primary cultures stimulated with tumour necrosis factor alpha or transforming growth factor beta-1 (n = 3 and n = 4 respectively) or in passaged lipocytes (n = 6). In contrast, free tissue inhibitor of metalloproteinase-1 activity was present in unstimulated and passaged cultures and this was increased in response to tumour necrosis factor alpha and transforming growth factor beta-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoimmune Diseases↗
PubMed1995

Role of Ito cells in the degradation of matrix in liver.

Liver fibrosis is a dynamic process caused by changes in not only the synthesis of matrix proteins but also their degradation. Current evidence indicates that Ito cells, when activated to a myofibroblastic phenotype, play a very active role in regulating matrix degradation in liver. This is mediated via their ability to synthesize and release several members of the matrix metalloproteinase family, a class of enzymes which are responsible for degradation of matrix proteins in the extracellular space. Activated Ito cells have been demonstrated to release prostromelysin, progelatinase A and the pro-enzyme form of interstitial collagenase. In addition, these cells can express appropriate systems for cleaving pro-metalloproteinases to active forms (e.g. the plasminogen activator system, urokinase) as well as specific tissue inhibitors of the activated metalloproteinases (TIMP). In the early phases of liver injury, enzymes with the ability to degrade components of normal liver matrix are expressed (stromelysin and gelatinase A). In contrast, in the fibrotic phase of liver injury, during which fibrillar collagens accumulate, there is little (if any) expression of interstitial collagenase but marked expression of TIMP. These findings suggest that metalloproteinase and their inhibitors play a significant role in liver injury and fibrosis.

Animals↗
PubMed1995

Kupffer cell-derived 95-kd type IV collagenase/gelatinase B: characterization and expression in cultured cells.

Release of 92-kd type IV collagenase/gelatinase, also known as gelatinase B, by inflammatory and tumor cells is increasingly recognized and is believed to facilitate cellular migration across basement membranes. It has been implicated in the pathogenesis of many diseases, but little is known of its cellular origin(s) and function in liver. In this study we have demonstrated synthesis and release of gelatinase B by human and rat Kupffer cells in primary culture. Northern analysis of RNA extracted from Kupffer cells stimulated with phorbol ester demonstrated a 2.8 kb transcript for gelatinase B. Immunoblotting and zymography of serum-free Kupffer cell-conditioned media demonstrated extracellular release of immunoreactive enzyme and gelatinase activity, Mr 92,000 (95,000 from rat cells). The organomercurial 4-aminophenyl mercuric acetate (APMA) activated the enzyme in vitro, indicating secretion primarily as a proenzyme. Stimulation of Kupffer cells by phorbol ester markedly induced gelatinase B release, which was inhibited by cycloheximide. In contrast, cycloheximide had no effect on constitutive secretion in culture, suggesting that there is some intracellular storage. Kupffer cell-derived gelatinase B was also partially purified and characterized. After separation by gelatin sepharose and gel filtration chromatogrpahy, gelatin-degrading activities of 95, 88, 75, and 65 kd were detected, the three lower-molecular-weight species probably representing activated forms. Enzyme activity was inhibited by ethyl-enediaminetetra-acetic acid (EDTA), but not by serine- and thiol-protease inhibitors, and was restored by zinc. Activity was also inhibited by tissue inhibitor of metalloproteinase-1 (TIMP-1) and alpha-2 macroglobulin. The partially purified enzyme rapidly degraded denatured collagens (gelatin) as well as native types III, IV, and V collagens, but had no activity against casein, types I and VI collagens.

Animals↗
PubMed1995

Collagenases and liver fibrosis.

Hepatic lipocytes which are activated to a myofibroblastic phenotype synthesize many of the metalloproteinases and their inhibitors, particularly TIMP-1. The available evidence suggests that this enzyme/inhibitor system for regulating matrix degradation is important in liver in two respects; (i) degradation of the normal liver matrix by the gelatinases (A and B) and stromelysin and the role this has in the pathogenesis of liver injury, and (ii) failure of matrix degradation consequent upon the relative expression of interstitial collagenase and TIMP-1 by hepatic lipocytes and the role this has in the progression of liver fibrosis. Recent progress in this field provides a clear indication that liver fibrosis is dynamic, involving a balance between matrix synthesis and regulated matrix degradation. These observations offer opportunities for the development of new therapeutic strategies in the management of liver fibrosis.

Animals↗
PubMed1994

Degradation of matrix proteins in liver fibrosis.

Hepatic fibrosis occurs as a consequence of net accumulation of matrix proteins (particularly collagen types I and III) in liver. Current concepts of the pathogenesis of liver fibrosis place major emphasis on the activation of hepatic lipocytes (fat-storing or Ito cells) to a myofibroblast-like phenotype with a consequent increase in their synthesis of matrix proteins. While this is an important factor, there is increasing evidence to indicate that liver fibrosis is a dynamic pathologic process in which altered matrix degradation may also play a significant role. Extracellular degradation of matrix proteins is regulated by a family of enzymes called the matrix metalloproteinases, which is subdivided into three groups; collagenases which degrade interstitial collagens (types I, II and III), type IV collagenases/gelatinases which degrade basement membrane (type IV) collagen and gelatins and stromelysins which degrade a broad range of substrates including proteoglycans, laminin, gelatins and fibronectin. The extracellular activity of these enzymes is regulated by several mechanisms which include alterations in gene transcription and proenzyme synthesis, cleavage of secreted proenzymes to active forms, and specific inhibition of activated forms by tissue inhibitor(s) of metalloproteinases (TIMPs). In liver, current evidence indicates that activated hepatic lipocytes and Kupffer cells play a central role in synthesis of matrix metalloproteinases. Under defined conditions they synthesize interstitial collagenase, 72 kDa and 95 kDa type IV collagenase/gelatinase and possibly stromelysin. Moreover, lipocytes also contribute to regulation of the extracellular activity of these enzymes by secretion of TIMP-1 and alpha 2-macroglobulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Differential cellular synthesis of insulin-like growth factor binding protein-1 (IGFBP-1) and IGFBP-3 within human liver.

The two major insulin-like growth factor-binding protein (IGFBP) species in the human circulation, IGFBP-1 and IGFBP-3, are synthesized in large amounts by liver. To determine which hepatic cell populations in human liver were responsible for the synthesis and release of these IGFBPs, we 1) performed in situ hybridization with specific complementary RNA (RNA) probes for human IGFBP-1 or-3 or performed immunohistochemical analysis to reveal the sites of messenger RNA (mRNA) presence and peptide translation, respectively, in sections of normal liver derived from organ donors; and 2) examined the release of IGFBP species by Western ligand and immunoblots of medium conditioned by isolated cultures of hepatocytes, lipocytes, and Kupffer cells. In situ hybridization showed that IGFBP-1 mRNA was distributed widely among the parenchymal cell population, which also showed immunohistochemical staining for IGFBP-1 peptide. Conversely, IGFBP-3 mRNA and immunoreactive peptide were mainly localized to Kupffer cells, which were positively identified by immunoreactivity with antiserum against the glycoprotein marker, CD68. Isolated hepatocytes released two species of IGFBP of 28 and 30-32 kilodaltons, which were recognized immunologically as IGFBP-1. Isolated Kupffer cells released only a 43- to 46-kilodalton IGFBP immunologically recognized as IGFBP-3. Lipocyte cultures released no detectable IGFBP species. The results suggest that IGFBP-1 and IGFBP-3 are derived from separate cell populations in human liver.

Adolescent↗
PubMed1992

Secretion of 72 kDa type IV collagenase/gelatinase by cultured human lipocytes. Analysis of gene expression, protein synthesis and proteinase activity.

The matrix metalloproteinases play an important role in matrix degradation, but there is limited information about this family of enzymes in either normal or diseased human liver. In this study, we have examined the synthesis of a 72 kDa type IV collagenase/gelatinase by human hepatic lipocytes in primary culture. Hepatic lipocytes were isolated from wedges of normal human donor liver by Pronase/collagenase perfusion, purified by density-gradient centrifugation, and established in primary culture on uncoated plastic. By Northern-blot analysis, the total RNA extracted from cultured human lipocytes was found to contain 3.4 kb mRNA for the 72 kDa type IV collagenase/gelatinase. Low levels of expression of this mRNA were observed in freshly isolated lipocytes but expression increased with the duration of lipocyte culture. Using anti-human 72 kDa type IV collagenase/gelatinase IgG, synthesized enzyme was immunolocalized to monensin-treated human lipocyte cultures. De novo synthesis and secretion of 72 kDa type IV collagenase/gelatinase were confirmed by immunoprecipitation of radiolabelled enzyme from medium obtained from [35S]methionine-treated cells. Activity of the secreted enzyme was demonstrated by gelatin-zymography and by degradation of soluble, radiolabelled [14C]gelatin. The enzyme was released both in active and latent pro-enzyme forms and its inhibition profile was that of a metalloproteinase. These studies indicate that cultured human hepatic lipocytes express the gene for the 72 kDa type IV collagenase/gelatinase, and secrete this enzyme, particularly in prolonged primary culture. As this enzyme exhibits degradative activity against basement membrane collagen, its release by activated hepatic lipocytes in the space of Disse could lead to disruption of the normal subendothelial liver matrix. It is suggested that this enzyme may have an important role in human liver injury and fibrosis.

Adipose Tissue↗