Expression of the novel extracellular matrix component tenascin in normal and diseased human liver. An immunohistochemical study.

The novel extracellular matrix glycoprotein tenascin was studied immunohistochemically in normal and fibrotic human liver. Its localization was compared to that of laminin, fibronectin and collagen type IV. In the normal liver, a weak staining for tenascin was detected along sinusoids, while portal tracts were negative. In both alcoholic and cholestatic liver disease and acute and chronic hepatitis, sinusoidal immunoreactivity for tenascin was variably increased as compared to the normal liver. Most striking, however, was the preferential accumulation of tenascin at connective tissue-parenchymal interfaces between proliferating ductules and in areas of piecemeal necrosis. As compared to laminin, fibronectin and collagen type IV, tenascin has the most restricted distribution. Our findings indicate that tenascin is a component of the extracellular matrix of the human liver. Its preferential expression at connective tissue-parenchymal interfaces in fibrosing areas in contrast to its absence from mature fibrous septa suggest a transient role in early matrix organization.     

Collagen isotypes, laminin, and fibronectin in granulomas of the liver and intestines of Schistosoma mansoni-infected mice

Patterns of fibrosis within hepatic and intestinal granulomas of Schistosoma mansoni-infected mice were analyzed by indirect immunofluorescence. Deposition of collagen isotypes, laminin, and fibronectin was evaluated semiquantitatively between 8 and 20 weeks of the infection. Liver granulomas were the largest at 8 weeks and contained low amounts of type I and higher amounts of type III collagen and fibronectin. Collagen deposition became pronounced as infection progressed. The relative amounts of type I collagen deposits rose and equalled that of type III. In the smaller immunomodulated granulomas at 20 weeks both types I and III were high, and type IV collagen deposition was observed. Fibronectin and laminin deposits were also detected. The small ileal granulomas did not change their size during the course of the infection. At 8 weeks, connective tissue matrix deposition was barely detectable within these lesions. Gradually, small deposits of types I and III appeared in equal amounts and attained highest levels by 20 weeks of the infection. Fibronectin deposits at that time were very prominent but laminin and type IV collagen were absent. Colon granulomas at 8 weeks of the infection were only somewhat smaller than those of the liver, yet contained very sparse deposits of types I and III collagen. During the ensuing weeks collagen deposits rose only slightly. By 20 weeks the granulomas diminished in size and within those lesions type III collagen was predominant. Whereas the presence of fibronectin was pronounced, type IV collagen and laminin were detectable only in trace amounts. These observations indicate the existence of important organ-related differences in the intragranulomatous deposition of connective tissue matrix.     

Isolation, characterization, and localization of cardiac collagen type VI. Associations with other extracellular matrix components.

We have isolated and characterized collagen type VI from murine, canine, and nonhuman primate hearts. In the three species studied, collagen type I was the major collagenous component of the cardiac interstitium (80% of total collagen), whereas collagen type VI represented approximately 5% of total collagen. To define the exact distribution of collagen type VI and its possible interactions with other components of the cardiac extracellular matrix, collagen types I, III, IV, and VI, laminin, and fibronectin were localized in the rat myocardium by immunohistochemistry, using monospecific antibodies. In the rat myocardium, collagen type VI was prevalent in the media and adventitia of muscular arteries, in fine connective tissue septa, in the area surrounding capillaries, and in the delicate endomysium in proximity to myocardial cells. When compared with the immunohistochemical localization of collagen types I, III, and IV, laminin, and fibronectin, the continuity and hierarchical organization of the cardiac extracellular matrix became apparent. The matrix forms a continuous network extending from the pericardium to the endocardium. Furthermore, there is an arborescent hierarchy in the system such that collagen type I is more prevalent in the wider septa, collagen type III being more obvious in medium-sized branches, and fibronectin and collagen type VI prevailing in the terminal (pericellular) aspects of the network. In this pericellular location, fibronectin and collagen type VI, by means of specific interactions, may act as anchor components linking the myocardial cell basement membranes not only to the extracellular matrix but also to the cardiac interstitial cells. This continuity, organization, and coupling of the cardiac extracellular matrix appears well suited to integrate and distribute the physical stress generated by the continuous contraction and relaxation of the myocardium.     

Distribution of extracellular matrix proteins in pancreatic ductal adenocarcinoma and its influence on tumor cell proliferation in vitro

Affinity-purified antibodies to major components of the extracellular matrix (fibronectin and collagen type I) and basal lamina (laminin) were used in indirect immunofluorescence studies on frozen sections of 12 pancreatic ductal adenocarcinoma of the human and on sections of normal and inflamed pancreatic tissue of the same patients. Laminin-specific immunoreactivity was distributed in close correlation to the grade of differentiation of the tumor tissue. Intact basement membranes, also with some structural irregularities were found only in the highest grade of differentiation where tumor cells grew as tubular structures. With progressive dedifferentiation basal laminae were either absent or the laminin-positive material was focally distributed without spatial association with tumor cells. In all cases of pancreatic tumors a remarkable increase in interstitial connective tissue was observed, as demonstrated by antibodies specific for human collagen type I and for human serum fibronectin. Tumor extracts contained high amounts of collagen type I and V but no significant amount of collagen type III as visualized by analytical SDS gel electrophoresis. A similar distribution of collagen types was observed in lymph node and liver metastases, and in tumors xenografted into nude mice. Since previously a close correlation between grading and growth kinetics of primary tumors had been observed, in vitro experiments were performed analyzing the effect of purified extracellular matrix proteins on tumor cell proliferation. In vitro cultivation of two established cell lines of pancreatic carcinoma on collagen type I or on laminin resulted in an arrest of proliferation on laminin substrates, while normal proliferation comparable to growth on regular culture dishes was found using collagen type I and fibronectin as substrates. Fine structural studies demonstrated a higher degree of cell differentiation in the presence of laminin, as compared to collagen type I or fibronectin.     

Liver fibrosis and extracellular matrix

Liver fibrosis and extracellular matrix play a central role in liver function impairment. Little information is available on the dynamic aspects and the natural history of fibroplasia, even if there is growing evidence that extracellular matrix accumulation (collagen I, III, IV, fibronectin, laminin, proteoglycans, etc.) is not to be considered only a passive structural support for damaged hepatic tissue, but may actively modulate liver cell behaviour. Clinicians need to date liver fibrosis and to monitor connective tissue synthesis and degradation, but attempts to develop reliable serological markers for collagen metabolism are hampered by the absence of a well defined golden standard to validate them. Nevertheless, serum type III aminoterminal procollagen peptide, at the moment, seems to be the most acceptable parameter of fibrogenesis. The data concerning the mechanisms of collagen production-degradation are becoming so precise and numerous that even if they have not, to date, led to 'routine' advantages for patients, they will end up becoming important tools in the clinical practice and management of liver fibrosis.     

Modulation of extracellular matrix components during dimethylnitrosamine-induced cirrhosis

Liver fibrosis was induced in rats after administration of dimethylnitrosamine (DMN) intraperitoneally three times a week for 3 weeks. Incomplete septa appeared after 7 days and evidence of nodulation of the parenchyma was observed after 21 days. Both distribution of extracellular matrix components (collagen type I, type III and type IV, laminin, fibronectin, heparan sulphate proteoglycan) and the distribution of desmin as a marker of lipocytes (Ito cells) and of iso-alpha-smooth muscle actin were studied with immunoperoxidase. Changes in the distribution of extracellular matrix components outlined both the formation of septa and the development of nodules with changes in the sinusoidal pattern evoking aspects of capillarization. The number of desmin-positive cells increased in DMN-treated animals, showing a prominent reaction in the fibrous septa. In the normal liver, lipocytes were positive for laminin and negative for actin, but septal and juxta-septal lipocytes were positive for both antigens, suggesting the presence of transitional cells with mixed immunoreactivity. This was confirmed by ultrastructural studies which showed typical intraseptal myofibroblasts and other elements exhibiting the structural features of both myofibroblasts and lipocytes.     

Cell types involved in collagen and fibronectin production in normal and fibrotic human liver

Three collagen types (I, III and IV) and fibronectin were localized in normal and alcoholic human liver by light and electron microscopy using the indirect immunoperoxidase technique. In normal liver, most of the bundles of collagen fibers stained for type pro-III collagen while only a few reacted for type I. Basement membranes stained for type IV collagen which formed discontinuous discrete deposits in sinusoids. Only fibronectin appeared as an almost continuous layer in the space of Disse. At the intracellular level, hepatocytes were found to contain little type I collagen and large amounts of fibronectin. Fat-storing cells strongly stained for type IV collagen and expressed low amounts of types I and III collagen and fibronectin. Endothelial cells contained low amounts of all the components. Alcoholic livers were studied at three stages: steatosis, fibrosis and cirrhosis. Qualitative and quantitative differences were observed in extracellular and intracellular distributions of matrix proteins. Increased amounts of all components were usually found in fibrotic and cirrhotic livers compared to normal liver. In two fibrotic livers which contained numerous bundles of collagen in the sinusoids, fat-storing cells stained more intensely for type III collagen. In a cryptogenic fibrotic liver, abundant type IV collagen was observed in hepatocytes. These results suggest that hepatocytes, fat-storing cells and endothelial cells are engaged in production of extracellular matrix components in normal human liver. In fibrosis, hepatocytes which normally did not synthesize types III and IV collagen may produce these collagens.     

Immunohistochemistry of the hepatic extracellular matrix in acute viral hepatitis

The distribution of several extracellular matrix components in the liver of patients with acute viral hepatitis was studied by light and electron microscopy using indirect immunoperoxidase methods. Light microscopy revealed type III and type V collagen and fibronectin in the portal tracts and the area of focal necrosis, showing cell infiltration. Type III and type V collagen were more strongly stained in the periphery of focal necrosis. Type IV collagen was seen around the vessels and hepatocytes near the focal necrosis. Electron microscopy showed many transitional Ito cells in the area of focal necrosis and fibroblasts were observed in the portal tracts, showing collagen fiber deposition. Numerous collagen fibrils were observed around fibroblasts, Ito cells and hepatocytes. Using immunoelectron microscopy, type III and type IV collagen and fibronectin were observed in the rough endoplasmic reticulum of Ito cells and hepatocytes localized near the area of focal necrosis or fiber deposition. In addition, type IV collagen was seen in the rough endoplasmic reticulum of endothelial cells forming capillary vessels. These results suggest that several extracellular matrix components such as types III, IV and V collagen and fibronectin, produced by Ito cells, hepatocytes or endothelial cells, play important roles in the healing of liver damage in acute viral hepatitis.     

Extracellular matrix constituents affect superficial gastric epithelial cell adhesion

Abstract The interfoveolar and upper gastric pit cells become necrotic and slough off after superficial luminal injury to the gastric mucosa. The subsequent rapid epithelial restitution of the wound is dependent on an intact basal lamina upon which viable mucous cells migrate. Several lines of evidence suggest that migrating mucous cells recognize specific moieties in the basal lamina which would then affect restitution and the ability of the gastric mucosa to be repaired. Therefore, this study examined the effect of three individual protein constituents of the extracellular matrix, laminin, fibronectin and type IV collagen as well as a synthetic basal lamina, Matrigel, on adherence of mucous cells isolated from guinea-pig stomach to these substrates in culture. After 3 h, approximately 40% of the cells adhered to Matrigel, 25% to both collagen IV and fibronectin, but only about 10% to laminin and 3% to uncoated plastic substrates. Disruption of protein synthesis by pre-incubation with cyclohexamide significantly reduced adherence to Matrigel and collagen IV but not laminin, fibronectin or plastic substrates. These results suggest that gastric mucous cells have multiple receptors for extracellular matrix proteins (ligands) which influence the adherence and probably the migration of these cells. Furthermore, some of these receptors are synthesized in response to moieties in the substrate itself.     

Hepatocyte survival depends on beta1-integrin-mediated attachment of hepatocytes to hepatic extracellular matrix.

BACKGROUND: A major drawback of allogeneic hepatocyte transplantation is the lack of sustained survival of the transplanted cells in the recipient liver parenchyma. The purpose of this study was to determine the effect of the presence or absence of hepatic extracellular matrix (ECM) molecules on hepatocyte survival and function following hepatocyte isolation for transplantation purposes, and the role of beta1-integrin molecules therein. METHODS: Hepatocytes, either untreated or treated with anti-beta1 integrin antibodies or RGD peptides, were seeded on wells precoated with collagen type I, type IV, laminin, fibronectin or polyhydroxyethylmehacrylate. The extent of attachment and apoptosis was evaluated. RESULTS: When hepatocytes were added into wells precoated with either fibronectin, or collagen type IV, rapid spreading and prolonged survival occurred, in contrast to hepatocytes that were seeded in wells precoated with collagen type I or polyhydroxyethylmehacrylate. Pretreatment of the cells with anti-beta1-integrin antibodies resulted in reduction of cell attachment to laminin, fibronectin, collagen I, and collagen IV. Synthetic RGD (arginine-glycine-aspartate)-peptides and anti-beta1 antibodies inhibited apoptosis of cultured hepatocytes. CONCLUSIONS: Our findings indicate that embedding of hepatocytes within their normal liver ECM surroundings maintains their survival. When detached from their natural surrounding hepatocytes enter into apoptosis, unless treated with anti-beta1-integrin antibodies or RGD peptides. This knowledge will allow improvement of hepatocyte transplantation efficiency.     

Relative distribution of fibronectin and type I, III, IV, V collagens in normal and atherosclerotic intima of human arteries

Spatial distribution of fibronectin and type I, III, IV and V collagen has been investigated in normal arterial intima, fatty streaks, and atherosclerotic plaques by indirect immunofluorescence on transverse sections. Two distinct types of extracellular matrix were revealed in atherosclerotic lesions. The fibrous plaques consisted mostly of interstitial collagen types I and III, contained moderate amounts of type V and none of type IV collagen or fibronectin. In the extracellular matrix of the fatty streaks and in some areas of the fibrous plaques containing large amounts of subendothelial cells, some interstitial collagen was revealed, an increased amount of type IV, some type V collagen and a lot of fibronectin. Similarities of the extracellular matrix in atherosclerotic lesions and granulation tissues are discussed.     

Integrin receptors on aortic smooth muscle cells mediate adhesion to fibronectin, laminin, and collagen.

Extracellular matrix receptors on vascular smooth muscle cells help in anchoring the cells during contraction and in promoting cellular migration after vessel injury. We found that rat aortic smooth muscle cells attach to surfaces coated with fibronectin, laminin, and collagen types I and IV. Cell attachment to these substrates appears to be mediated by members of the beta 1 integrin family of extracellular matrix receptors. Antibodies to the beta 1 subunit not only demonstrated the presence of integrin complexes in focal adhesion plaques but also blocked cell adhesion to the different substrates. Ligand-affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis isolated a series of receptor complexes that were recognized by antisera to beta 1 integrin receptors. Each of the receptors appeared to be a heterodimer in which one of several alpha subunits shared a common 120-kDa (nonreduced) beta 1 subunit protein. The rat aortic smooth muscle cells had one alpha subunit (150 kDa nonreduced, 140 kDa reduced) that bound exclusively to fibronectin. There was a second alpha subunit (150 kDa nonreduced, 160 kDa reduced) that bound exclusively to collagen type I. In addition, there was a third alpha subunit (185 kDa nonreduced, 200 kDa reduced) that was promiscuous and bound to collagen types I and IV as well as to laminin; the 185-kDa alpha subunit appeared to bind to collagen more efficiently than it did to laminin. Thus, smooth muscle cells express multiple integrin receptors with different ligand specificities that appear to mediate cell interactions with the extracellular matrix.     

Serum type IV collagen in various liver diseases in comparison with serum 7S collagen,laminin, and type III procollagen peptide

The clinical significance of the immunoreactive triple helical domain of type IV collagen in serum was evaluated in 73 healthy controls and 161 patients with various biopsy-proven liver diseases. Although serum levels of type III procollagen peptide were increased in all liver diseases, those of type IV collagen, 7S collagen, and laminin were principally increased in chronic liver diseases associated with hepatic fibrogenesis/fibrosis. In both non-alcoholic and alcoholic liver diseases, 7S collagen was increased in serum, while type IV collagen and laminin in serum were particularly increased in alcoholic liver diseases and in hepatocellular carcinoma, in which latter the sensitivity was greater for type IV collagen than for laminin. Gel filtration analysis in Sephacryl S-400 revealed type IV collagen in serum to be a single molecular form with a molecular weight that correspond to type IV collagen, whereas 7S collagen was recognized as several heterogeneous macromolecules. These findings indicate that serum type IV collagen is derived from the type IV protocollagen pool, and is a sensitive marker for the fibrogenetic process in hepatic basement membranes.     

Immunolocalization of collagen types, laminin and fibronectin in the normal human pancreas

In order to define the connective matrix organization of the normal human pancreas collagen types I, III, pro-III and IV, laminin and fibronectin were labeled using specific, antihuman antibodies. Visualization was by indirect immunofluorescence. Collagen types I, III and pro-III were present within lobules: around acini, ducts and small vessels. Their immunofluorescence reaction was particularly obvious in septa and it also outlined interlobular vessels and ducts. The type III and pro-III fractions possessed a characteristic, branched appearance in many situations, when compared to the more linear type I reaction. Collagen type IV, laminin and fibronectin were closely applied to acini, ducts and vessels, but in contrast to the other collagen types were absent from septa.     

Transformation of sinusoids into capillaries in a rat model of selenium-induced nodular regenerative hyperplasia: An immunolight and immunoelectron microscopic study

The oral administration of selenium (Se) to young rats induces, over a 2-month period, the formation of nodular regenerative hyperplasia with sinusoidal damage around nodules. Perinodular areas located in zone 1 comprise atrophic hepatocytes and capillarized sinusoids without fibrosis. We used this unique model of capillarization without fibrosis to investigate the temporal relationship between the process of capillarization and changes occurring in the deposition of components of the extracellular matrix. After 2 weeks of intoxication, type III collagen and fibronectin were stable, but laminin and type IV collagen had increased in zone 1, resulting in the formation of septae between portal tracts. Even at 8 weeks, these two components still formed the principal deposits in perinodular zones. Electron microscopy showed already at 1 week in zone 1 that part of the endothelial wall had detached from hepatocytes. Sinusoidal endothelial cells progressively acquired certain of the characteristics of a vascular endothelium, some proliferated, and perisinusoidal cells transformed into myofibroblasts, surrounded by deposits of laminin and type IV collagen. These results indicate that both laminin and type IV collagen are involved in capillarization without fibrosis and in angiogenesis; fibronectin would not seem to play a role.     

Sequential connective matrix changes in experimental acute pancreatitis. An immunohistochemical and biochemical assessment in the rat

The effects of acute pancreatitis on the rat pancreatic connective tissue matrix were studied following intraductal pancreatic injection of trypsin solution and serial killing of the animals. Pancreatic tissue was examined using light microscopy, hydroxyproline measurement and indirect immunofluorescence, using antibodies against collagen types I, III, IV, procollagen III, fibronectin and laminin. Light microscopy revealed that acute pancreatitis was present for up to four days after injection and that perilobular and intralobular fibrosis appeared at four days and subsequently regressed. Immunofluorescence studies demonstrated an abnormal fibronectin deposit at one day in acute pancreatitis. At four days this deposit was co-located with fibrosis which was composed of collagen and procollagen type III. By eight days the immunofluorescence and light microscopic changes were minimal. Biochemical analysis confirmed a significant rise in hydroxyproline concentration at four days, which was maximal at eight days, subsequently decreasing. This peak at eight days probably reflects collagen breakdown products.     

Extracellular matrix proteins involved in pseudoislets formation

Extracellular matrix proteins are known to mediate, through integrins, cell adhesion and are involved in a number of cellular processes, including insulin expression and secretion in pancreatic islets. We investigated whether expression of some extracellular matrix proteins were implied in islets-like structure formation, named pseudoislets. For this purpose, we cultured the β-cell line, RINm5F, during 1, 3, 5 and 7 days of culture on treated or untreated culture plate to form adherent cells or pseudoislets and analysed insulin, collagen IV, fibronectin, laminin 5 and β1-integrin expression. We observed that insulin expression and secretion were increased during pseudoislets formation. Moreover, we showed by immunohistochemistry an aggregation of insulin secreting cells in the centre of the pseudoislets. Peripheral β-cells of pseudoislets did not express insulin after 7 days of culture. RT-PCR and immunohistochemistry studies showed a transient expression of type IV collagen in pseudoislets for the first 3 days of culture. Study of fibronectin expression indicated that adherent cells expressed more fibronectin than pseudoislets. In contrast, laminin 5 was more expressed in pseudoislets than in adherent cells. Finally, expression of β1-integrin was increased in pseudoislets as compared to adherent cells. In conclusion, laminin 5 and collagen IV might be implicated in pseudoislets formation whereas fibronectin might be involved in cell adhesion. These data suggested that extracellular matrix proteins may enhance the function of pseudoislets.     

Extracellular matrix proteins involved in pseudoislets formation

Extracellular matrix proteins are known to mediate, through integrins, cell adhesion and are involved in a number of cellular processes, including insulin expression and secretion in pancreatic islets. We investigated whether expression of some extracellular matrix proteins were implied in islets-like structure formation, named pseudoislets. For this purpose, we cultured the β-cell line, RINm5F, during 1, 3, 5 and 7 days of culture on treated or untreated culture plate to form adherent cells or pseudoislets and analysed insulin, collagen IV, fibronectin, laminin 5 and β1-integrin expression. We observed that insulin expression and secretion were increased during pseudoislets formation. Moreover, we showed by immunohistochemistry an aggregation of insulin secreting cells in the centre of the pseudoislets. Peripheral β-cells of pseudoislets did not express insulin after 7 days of culture. RT-PCR and immunohistochemistry studies showed a transient expression of type IV collagen in pseudoislets for the first 3 days of culture. Study of fibronectin expression indicated that adherent cells expressed more fibronectin than pseudoislets. In contrast, laminin 5 was more expressed in pseudoislets than in adherent cells. Finally, expression of β1-integrin was increased in pseudoislets as compared to adherent cells. In conclusion, laminin 5 and collagen IV might be implicated in pseudoislets formation whereas fibronectin might be involved in cell adhesion. These data suggested that extracellular matrix proteins may enhance the function of pseudoislets.