PubMed Health⌕ Search

Biomedical subjects

S C Stamatoglou

Publications and source records attributed to S C Stamatoglou.

18 recordsLinked to original sources
PubMed1995

Differential expression of A and B laminin chains during rat liver regeneration.

We have studied the distribution and expression of laminin during rat liver regeneration by immunofluorescence and immunoblotting using affinity-purified laminin antibodies. Laminin was localized on sinusoidal surfaces in normal and regenerating hepatic parenchyma, but enhanced expression was detected during regeneration from 6 hours to 7 days after a partial hepatectomy. A and B laminin chains were specifically detected by Western blotting in highly purified plasma membrane fractions derived from the sinusoidal domain of hepatocytes. Analysis of laminin expression in whole homogenates from hepatectomized rats showed a differential expression of A and B laminin chains during regeneration: The A chain showed a progressive increase, whereas the B chain exhibited an early, prereplicative increase and then gradually declined to control levels at the end of restorative growth. The results suggest differential temporal requirements for A and B laminin chains during hepatic growth and tissue formation.

Animals↗
PubMed1994

Cell adhesion molecules in liver function and pattern formation.

In epithelia, adhesive interactions participate in specific histogenetic events and also instate a morphological polarization that is linked to the vectorial functioning of the epithelium. Cell polarity is a recurring event in differentiation, first observed in the preimplantation embryo and then during organogenesis, whereas in oncogenesis a breakdown of polarity may occur. We review here recent work on adhesive interactions in the liver parenchyma. We focus on the effect of cell-biomatrix interactions on hepatocyte function and polarization, on the integration of adhesive cell-cell and cell-matrix interactions, and on the correlation between structural differentiation and adhesion during hepatic development, regeneration, and oncogenesis. Although highlighting morphological differences between the liver and other epithelia, we stress that adhesion-mediated effects such as substratum regulation of differentiation, feedback control of matrix synthesis, and integration of intercellular and cell-matrix interactions, shown to transpire in the liver, most probably occur in other parenchymal tissues too.

Animals↗
PubMed1992

Dynamic interactions of hepatocytes with fibronectin substrata: temporal and spatial changes in the distribution of adhesive contacts fibronectin receptors, and actin filaments.

We have studied the interactions, over several hours, of adult rat hepatocytes with fibronectin substrata in vitro by interference reflection microscopy and by laser scanning confocal immunomicroscopy using antisera against the fibronectin receptors, integrin alpha 5 beta 1 and nonintegrin Agp110, together with phalloidin, a specific marker for filamentous actin. Distinct alterations in the pattern of cell-substratum adhesive contacts, in the distribution of receptors, and in actin organization were observed with time in culture. Cells examined 2 to 3 h after inoculation had formed focal contacts at symmetrically distributed microextensions of the basal cell periphery that contained integrin alpha 5 beta 1, AGp110, and termini of actin filaments. On more prolonged incubation, over 5 to 6 h, increasing numbers of cells displayed a lamellar structure in close juxtaposition to the substratum circumscribing more than half of the basal cell surface and long filopodia emanating from the remaining part of the cell periphery. Integrin alpha 5 beta 1 and AGp110 were mainly concentrated at the inner and, to a lesser extent, outer boundaries of the lamellae, and actin filaments close to the basal surface overlayed the inner boundary of the adhesive lamella colocalizing with the receptors. Filopodial extensions contained neither receptor.

Actins↗
PubMed1992

Temporal changes in the expression and distribution of adhesion molecules during liver development and regeneration.

We have compared by immunocytochemistry and immunoblotting the expression and distribution of adhesion molecules participating in cell-matrix and cell-cell interactions during embryonic development and regeneration of rat liver. Fibronectin and the fibronectin receptor, integrin alpha 5 beta 1, were distributed pericellularly and expressed at a steady level during development from the 16th day of gestation and in neonate and adult liver. AGp110, a nonintegrin fibronectin receptor was first detected on the 17th day of gestation in a similar, nonpolarized distribution on parenchymal cell surfaces. At that stage of development haemopoiesis is at a peak in rat liver and fibronectin and receptors alpha 5 beta 1 and AGp110 were prominent on the surface of blood cell precursors. During the last 2 d of gestation (20th and 21st day) hepatocytes assembled around lumina. AGp110 was initially depolarized on the surface of these acinar cells but then confined to the lumen and to newly-formed bile canaliculi. At birth, a marked increase occurred in the canalicular expression of AGp110 and in the branching of the canalicular network. Simultaneously, there was enhanced expression of ZO-1, a protein component of tight junctions. On the second day postpartum, presence of AGp110 and of protein constituents of desmosomes and intermediate junctions, DGI and E-cadherin, respectively, was notably enhanced in cellular fractions insoluble in nonionic detergents, presumably signifying linkage of AGp110 with the cytoskeleton and assembly of desmosomal and intermediate junctions. During liver regeneration after partial hepatectomy, AGp110 remained confined to apical surfaces, indicating a preservation of basic polarity in parenchymal cells. A decrease in the extent and continuity of the canalicular network occurred in proliferating parenchyma, starting 24 h after resection in areas close to the terminal afferent blood supply of portal veins and spreading to the rest of the liver within the next 24 h. Distinct acinar structures, similar to the ones in prenatal liver, appeared at 72 h after hepatectomy. Restoration of the normal branching of the biliary tree commenced at 72 h. At 7 d postoperatively acinar formation declined and one-cell-thick hepatic plates, as in normal liver, were observed.

Animals↗
PubMed1991

Affinity of integrin alpha 1 beta 1 from liver sinusoidal membranes for type IV collagen.

Hepatic sinusoidal membranes isolated from adult rats were extracted with detergent and fractionated on a wheat germ agglutinin affinity column. Bound glycoproteins were eluted with N-acetyl glucosamine and chromatographed on a type IV collagen affinity column. Recovery of the bound fraction by EDTA and analysis by SDS-PAGE revealed two glycoproteins with apparent molecular weights of 180,000 and 117,000. These were identified immunologically by Western blotting as the alpha and beta subunits of integrin alpha 1 beta 1.

Animals↗
PubMed1991

Distribution of fibronectin and fibronectin-binding proteins, AGp110 and integrin alpha 5 beta 1, during chemically induced hepatocarcinogenesis in adult rats.

We have used single- and double-label immunocytochemistry to examine the distribution of AGp110, integrin alpha 5 beta 1 and fibronectin in adult rat liver during carcinogenesis induced by aflatoxin B1 or diethylnitrosamine. In normal liver fibronectin and the fibronectin integrin receptor alpha 5 beta 1 are localized on all three domains of the parenchymal cell surface: sinusoidal, lateral and canalicular. In contrast, AGp110, a non-integrin monomeric glycoprotein with fibronectin receptor properties, is confined to the bile canalicular (apical) plasma membrane of hepatocytes. Hepatocarcinogenesis induced by aflatoxin B1 causes altered cell foci to form in the parenchyma, followed by enlargement of these foci to form pre-neoplastic nodules and finally hepatocellular carcinomas of either poorly differentiated, trabecular or adenocarcinoma morphology. Expression of AGp110 decreased to a minimal level, at first selectively in altered cell foci, from the 9th week of treatment, and then indiscriminately in poorly differentiated carcinomas. The same lesions that were deficient in AGp110 also displayed a reduced level of fibronectin and alpha 5 beta 1, although the observed change in AGp110 demarcated altered foci and poorly differentiated tumour lesions more sharply, since expression of alpha 5 beta 1 and fibronectin, though substantially reduced, was still faintly apparent on the cell surface. Small acinar structures, observed in late hyperplastic nodules and in trabecular carcinomas, exhibited even, pericellular staining of fibronectin and alpha 5 beta 1, including prominent staining of the lumen area, whereas staining of AGp110 appeared to be confined to the lumen. In larger ducts of overt adenocarcinomas, fibronectin and alpha 5 beta 1 were distributed along the basal surface of the epithelium and AGp110 on the apical domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Identification of a novel glycoprotein (AGp110) involved in interactions of rat liver parenchymal cells with fibronectin.

We have identified an integral membrane glycoprotein in rat liver that mediates adhesion of cultured hepatocytes on fibronectin substrata. The protein was isolated by affinity chromatography of detergent extracts on wheat germ lectin-Agarose followed by chromatography of the WGA binding fraction on fibronectin-Sepharose. The glycoprotein (AGp110), eluted at high salt concentrations from the fibronectin column, has a molecular mass of 110 kD and a pI of 4.2. Binding of immobilized AGp110 to soluble rat plasma fibronectin required Ca2+ ions but was not inhibited by RGD peptides. Fab' fragments of immunoglobulins raised in rabbits against AGp110 reversed the spreading of primary hepatocytes attached onto fibronectin-coated substrata, but had no effect on cells spread on type IV collagen or laminin substrata. The effect of the antiserum on cell spreading was reversible. AGp110 was detected by immunofluorescence around the periphery of the ventral surface of substratum attached hepatocytes, and scattered on the dorsal surface. Immunohistochemical evidence and Western blotting of fractionated liver plasma membranes indicated a bile canalicular (apical) localization of AGp110 in the liver parenchyma. Expression of AGp110 is tissue specific: it was found mainly in liver, kidney, pancreas, and small intestine but was not detected in stomach, skeletal muscle, heart, and large intestine. AGp110 could be labeled by lactoperoxidase-catalyzed surface iodination of intact liver cells and, after phase partitioning of liver plasma membranes with the detergent Triton X-114, it was preferentially distributed in the hydrophobic phase. Treatment with glycosidases indicated extensive sialic acid substitution in at least 10 O-linked carbohydrate chains and 1-2 N-linked glycans. Immunological comparisons suggest that AGp110, the integrin fibronectin receptor and dipeptidyl peptidase IV, an enzyme involved in fibronectin-mediated adhesion of hepatocytes on collagen, are distinct proteins.

Amino Acid Sequence↗
PubMed1990

An endogenous carbohydrate-binding protein of baby hamster kidney (BHK21 C13) cells. Temporal changes in cellular expression in the developing kidney.

Asialofetuin (ASF) coupled to Sepharose has been used to isolate a Mr 30,000 protein from Triton X-100 extracts of the baby hamster kidney cell line BHK21 C13. Binding to ASF-Sepharose was specific for terminal beta-galactosyl residues. The lectin requires detergent for optimal solubilization and binding is independent of Ca2+ or reducing reagents. The lectin was labelled in a lactoperoxidase-catalysed iodination of intact BHK21 C13 cells, suggesting that it is associated with the cell surface. Antibodies to the lectin identify in Western blotting cross-reactive components in established cell lines of kidney (MDCK, NRK) and non-kidney (L, CHO, 3T3) origin. In young adult hamsters, the lectin is expressed in colon and duodenum and in lesser amounts in ileum, stomach, lung, liver and testes but is absent in kidney. The lectin is expressed in late embryonic and newborn hamster kidney but expression declines during 14 days after birth. Immunofluorescent staining of cryostat sections of newborn hamster kidney and intestine show that the lectin is expressed at apical epithelial surfaces. The presence of the lectin at the luminal surface of kidney tubules suggests a tubular origin for the BHK21 C13 cell line. Possible functions of the Mr 30,000 lectin in kidney development are discussed.

Animals↗
PubMed1990

Localization of two fibronectin-binding glycoproteins in rat liver and primary hepatocytes. Co-distribution in vitro of integrin (alpha 5 beta 1) and non-integrin (AGp110) receptors in cell-substratum adhesion sites.

We have compared the localization of integrin alpha 5 beta 1 and AGp110 (apical glycoprotein of Mr 110 x 10(3]] in rat liver parenchyma and in primary hepatocyte cultures. Integrin alpha 5 beta 1 is a heterodimeric fibronectin receptor. AGp110 is a newly described monomeric glycoprotein of the apical (bile canalicular) membrane domain of liver parenchyma that binds in an RGD-independent manner to fibronectin and mediates spreading of hepatocytes onto fibronectin-coated substrata. Using Western blotting of fractionated liver membranes and immunocytochemistry of liver sections at light- and electron-microscope levels, we have confirmed that AGp110 is a canalicular glycoprotein and have established that integrin is located in approximately equal proportions in the sinusoidal, lateral and canalicular membrane domains. In the canalicular surface domain both glycoproteins are associated with microvilli. Examination of immunolabelled primary hepatocytes spread on fibronectin-coated substrata by light and laser scanning confocal microscopy revealed colocalization of AGp110, integrin, actin and vinculin in substratum-attached microextensions at the periphery of the basal cell surface. Actin filaments that terminated at these cell processes originated from circular sub-cortical actin fibres. Interference reflection microscopy revealed focal adhesive contacts at the edge of the basal cell periphery at the same location where AGp110 and integrin were observed by immunofluorescence. In vitro, a proportion of the primary hepatocytes seeded onto fibronectin-coated substrata aggregated into colonies of several cells with intercellular contacts between neighbouring cells. Cell-substratum contacts containing integrin, AGp110, actin and vinculin followed the contours of these colonies in the same manner as they delineated the basal periphery of single, substratum-attached cells. We conclude that both integrin and AGp110 contribute to hepatocyte-fibronectin adhesive interactions and that intercellular adhesion and cooperation among hepatocytes in their response to fibronectin matrices leads to colony formation and morphological differentiation of parenchymal cell monolayers in vitro.

Actins↗
PubMed1987

Rat hepatocytes in serum-free primary culture elaborate an extensive extracellular matrix containing fibrin and fibronectin.

Adult rat hepatocytes cultured on type IV collagen, fibronectin, or laminin and maintained in serum-free medium were examined by indirect immunofluorescence using polyclonal antibodies against extracellular matrix proteins. An extensive fibrillar matrix containing fibronectin and fibrin was detected in all hepatocyte cultures irrespective of the exogenous matrix substratum used to support cell adhesion. Fibrils radiated from the cell periphery and covered the entire culture substratum. In addition, thicker fibers or bundles of fibers were localized on top of hepatocytes. This matrix did not contain laminin or the major types of collagen found in the liver biomatrix (types I, III, and IV). Isolation of the fibrillar matrix and analysis on polyacrylamide gels under reducing conditions demonstrated a major 58-kD polypeptide, derived from beta-fibrinogen as indicated by immunoblotting and two-dimensional peptide mapping. Plasmin rapidly dissolved the matrix. Deposition of the fibrin matrix in hepatocyte cultures was arrested by hirudin, by specific heparin oligosaccharides that potentiate thrombin inhibition by antithrombin III, and by dermatan sulfate, an activator of heparin cofactor II-mediated inhibition of thrombin. The results indicate that hepatocytes in culture synthesize and activate coagulation zymogens. In the absence of inhibitory and fibrinolytic mechanisms, a fibrin clot is formed by the action of thrombin on fibrinogen. Fibronectin attaches to this fibrin clot but fails to elaborate a fibrillar matrix on its own in the presence of coagulation inhibitors.

Animals↗
PubMed1987

Adhesive interactions and the metabolic activity of hepatocytes.

The nature and influence of adhesive interactions of rat hepatocytes with components of the extracellular matrix has been studied in culture. Hepatocytes interact with different kinetics to substrata composed of collagen type IV, laminin or fibronectin and adopt significantly different morphologies. The receptors mediating these various responses appear to be specific, according to the matrix, and in the case of fibronectin are complex, implicating several components of the hepatocyte surface. Collagen type IV maintains a differentiated phenotype more efficiently than fibronectin or laminin as measured by the production of adult hepatocyte markers such as albumin and repression of alpha-foetoprotein synthesis. Formation of matrix components is also influenced by the substratum: synthesis and secretion of fibronectin or collagen type IV is down-regulated when cells are cultured on the homologous substratum. Hepatocytes cultured in vitro secrete components of the coagulation cascade and also mediate fibrinolysis on addition of exogenous plasmin. The results are discussed in relation to the normal phenotype of the mature hepatocyte in vivo.

Animals↗
PubMed1986

Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes.

Hepatocytes isolated by perfusion of adult rat liver and cultured on substrata consisting of one or more of the major components of the liver biomatrix (fibronectin, laminin, type IV collagen) have been examined for the synthesis of defined proteins. Under these conditions, tyrosine amino transferase, a marker of hepatocyte function, is maintained at similar levels in response to dexamethasone over 5 days in culture on each substratum, and total cellular protein synthesis remains constant. By contrast, there is a rapid decrease in synthesis and secretion of albumin and a 3-7-fold increase in synthesis and secretion of alpha-fetoprotein which are most marked on a laminin substratum, but least evident on type IV collagen, and an increased synthesis of fibronectin and type IV collagen. The newly synthesized matrix proteins are present in the cell layer as well as in cell secretions. The enhanced synthesis of fibronectin is less in cells seeded onto a fibronectin substratum than on laminin or type IV collagen substrata, and its synthesis by hepatocytes seeded onto a mixed substratum of laminin and fibronectin is down-regulated by fibronectin in a dose-related manner. Similarly, type IV collagen synthesis is less when the cells are seeded on the homologous matrix protein substratum than on heterologous substrata. These results indicate that hepatocytes cultured in serum-free medium on substrata composed of components of the liver biomatrix maintain certain functions of the differentiated state (tyrosine amino transferase), lose others (albumin secretion) and switch to increased synthesis of matrix components as well as fetal markers such as alpha-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured.

Animals↗
PubMed1986

Interactions of rat hepatocytes with type IV collagen, fibronectin and laminin matrices. Distinct matrix-controlled modes of attachment and spreading.

We have examined the interaction of adult rat hepatocytes in primary culture, to type IV collagen, fibronectin, and laminin, the major basement membrane proteins of normal rat liver. Culture substrata consisted of glass coverslips, which were covalently derivatized with individual purified basement membrane constituents at varying densities of protein. The attachment of freshly prepared hepatocytes was examined after incubation at 37 degrees C for 30 min as a function of the amount of protein on the coverslips. For each of the three types of substratum under study, distinct modes of cell attachment were observed, with the apparent affinity of hepatocytes for type IV collagen being three-fold greater than for fibronectin and ten-fold greater than for laminin. Cell attachment exhibited saturation on all substrata. Hepatocyte spreading was measured by scanning electron microscopy of cells incubated at 37 degrees for 2 h on similarly prepared coverslips. A five-fold greater surface density of type IV collagen was required for maximal spreading compared with attachment. For cells on fibronectin or laminin the maximal cell spreading reached on type IV collagen did not occur even at coverslip protein densities 10 to 20 times those providing for maximal cell attachment. A very similar qualitative pattern of cell proteins was secreted within a few hours of plating on the various substrata and further studies failed to reveal any evidence that attachment and spreading was mediated by endogenously produced matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Ultrastructure of ventral membranes of rat hepatocytes spread on type IV collagen.

Rat hepatocytes obtained by means of liver perfusion with collagenase were allowed to spread on type IV collagen coated coverslips for 20 h. Interference reflection microscopy revealed a peripheral ring of dark spots. Carbon replicas of ventral membranes left attached to coverslips after lysis and squirting provided high resolution information on the ultrastructure of the protoplasmic surface. Correlative light and electron microscopy of the same ventral membrane showed that the area of the peripheral 'adhesion annulus' was rich in clathrin-coated structures (sheets, pits and vesicles). In vertical thin sections of hepatocyte monolayers numerous small smooth vesicles were observed piled up below the peripheral portion of the cell. These findings suggest high cytotic activity at the cell periphery during spreading. No bundles of microfilaments were observed in cells after squirting or in sections, but a ring of filaments at the cell periphery could be seen in many cells in whole mount preparations after treatment with Triton X-100. The absence of microfilaments associated with the points of adhesion indicates a cytoskeleton independent adhesion mechanism in hepatocytes during the first 20 h of spreading.

Animals↗
PubMed1983

Correlation between cell substrate attachment in vitro and cell surface heparan sulfate affinity for fibronectin and collagen.

Heparan sulfate glycosaminoglycan, isolated from the cell surface of nonadhering murine myeloma cells (P3X63-Ag8653), does not bind to plasma fibronectin, but binds partially to collagen type I, as assayed by affinity chromatography with proteins immobilized on cyanogen bromide-activated Sepharose 4B. Identical results were obtained when myeloma heparan sulfate was cochromatographed, on the same fibronectin and collagen columns, with cell surface heparan sulfates collagen columns, with cell surface heparan sulfates from adhering Swiss mouse 3T3 and SV3T3 cells. These latter heparan sulfates do, however, bind to both fibronectin and collagen, as reported earlier (Stamatoglou, S.C., and J.M. Keller, 1981, Biochim. Biophys. Acta., 719:90-97). Cell adhesion assays established that hydrated collagen substrata can support myeloma cell attachment, but fibronectin cannot. Saturation of the heparan sulfate binding sites on the collagen substrata with heparan sulfate or heparin, prior to cell inoculation, abolished the ability to support cell adhesion, whereas chondroitin 4 sulfate, chondroitin 6 sulfate, and hyaluronic acid had no effect.

Animals↗
PubMed1982

Interactions of cellular glycosaminoglycans with plasma fibronectin and collagen.

The interactions of metabolically radiolabelled glycosaminoglycans, isolated from Swiss mouse 3T3 and SV3T3 cells, with plasma fibronectin and collagen were studied by affinity column chromatograph in Hepes-buffered saline (150 mM NaCl in 10 mM Hepes buffer). Cell surface heparan sulfate glycosaminoglycan bound to plasma fibronectin and to native calf skin collagen in the absence of Ca2+ and Mg2+ with maximal binding at slightly acidic pH. Elution should be effected with elevated ionic strength. 3T3 and SV3T3 heparan sulfate coeluted when exposed to a salt concentration gradient. The core protein of the heparan sulfate proteoglycan did not bind to either fibronectin or collagen. Reference standard chondroitin 4-sulfate and chondroitin 6-sulfate had no effect upon heparan sulfate binding, whereas 90% of the heparan sulfate bound to fibronectin or collagen was eluted with heparin and 60% removed from fibronectin with dermatan sulfate. Cell surface chondroitin sulfate proteoglycan bound only to collagen at acidic pH and no interaction was seen with fibronectin. Trypsin treatment of chondroitin sulfate proteoglycan reduced the binding to collagen. Cell surface hyaluronic acid did not display any affinity for either fibronectin or collagen.

Animals↗
PubMed1978

Ruthenium red-positive surface layer, extracellular filamentous material and intercellular junctions in hybrids between tumour and normal cells: abundant gap junctions correlate with density-dependent inhibition of growth.

The cell surface and intercellular junctions of transformed and non-transformed hybrid cells, obtained from fusion of murine mammary adenocarcinoma cells (TA3B) and normal rat embryofibroblasts (REF), were compared with those of the parental cells, using ruthenium red (RR) staining of cells fixed in situ. An RR-positive layer of variable thickness was found on the surface of all cell types. Measurements of the thickness of this layer on the free surface of the cell cultures deomonstrated a significant difference between transformed and non-transformed cells. The thickness distribution of the RR layer was similar on the surface of REF and non-transformed hybrid cells, but there was significant variation among all the transformed cell lines. Extracellular filamentous RR-positive material, usually in direct contact with the cell surface, was present in confluent cultures of REF and non-transformed hybrid cells, but was absent in the transformed cell lines. Gap junctions were few in both parental cell lines and rare or absent in transformed hybrids; a large increase in number and size of gap junctions was found in non-transformed hybrids. Abundance of long gap junctions was correlated with density-dependent inhibition of growth, since non-transformed hybrids grew in single layers whereas both normal and tumour parental cell types, and transformed hybrids grew in multilayers. Tight junctions were frequently encountered in TA3B and transformed hybrid cells but were not seen in REF cells and only occasionally seen in non-transformed hybrids. Intermediate-type junctions occurred in all cells, but desmosomes were found only rarely in TA3B cells and never in the other cell types.

Adenocarcinoma↗