The control of DNA synthesis in primary cultures of hepatocytes from adult and young rats: interactions of extracellular matrix components, epidermal growth factor, and the cell cycle

Hepatocytes from adult and 4-week-old rats cultured on one of several extracellular matrix components were stimulated to replicate by epidermal growth factor (EGF). DNA synthesis was increased at 44-48 hr in adult hepatocytes and at 24, 48, and 72 hr in hepatocytes from young rats when EGF was added 2 hr after explantation. When EGF was added at 24 hr, maximal DNA synthesis of adult hepatocytes was observed at 48 hr, whereas that of 4-week-old hepatocytes was seen at 48 and 72 hr. Ten ng EGF per ml was the optimal concentration for maximal DNA synthesis in both adult and young cells. DNA synthesis decreased with increasing cell density, but this effect was less in hepatocytes from young than in those from adults. When hepatocytes were cultured on substrata consisting of individual extracellular matrix components, neither the time that adult cells needed to respond to EGF nor the time from stimulation by EGF to the peak of maximal DNA synthesis was altered in either adult or young cells. The optimal EGF concentration for maximal DNA synthesis and the cell density control of replication were also not altered by the substrata used. Substrata made from each of the extracellular matrix components studied enhanced DNA synthesis of adult and young hepatocytes stimulated by EGF in the following decreasing order: fibronectin, type IV collagen, type I collagen, and laminin. In both adult and young hepatocytes the enhancement of DNA synthesis was greatest when cultured on fibronectin. Thus the initiation and magnitude of DNA synthesis in primary cultures of rat hepatocytes were altered both by the age of the donor and the substratum on which the cells were explanted.     

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)     

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.     

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.     

Expression of extracellular matrix components is regulated by substratum

Reconstituted basement membranes and extracellular matrices have been demonstrated to affect, positively and dramatically, the production of milk proteins in cultured mammary epithelial cells. Here we show that both the expression and the deposition of extracellular matrix components themselves are regulated by substratum. The steady-state levels of the laminin, type IV collagen, and fibronectin mRNAs in mammary epithelial cells cultured on plastic dishes and on type I collagen gels have been examined, as has the ability of these cells to synthesize, secrete, and deposit laminin and other, extracellular matrix proteins. We demonstrate de novo synthesis of a basement membrane by cells cultured on type I collagen gels which have been floated into the medium. Expression of the mRNA and proteins of basement membranes, however, are quite low in these cultures. In contrast, the levels of laminin, type IV collagen, and fibronectin mRNAs are highest in cells cultured on plastic surfaces, where no basement membrane is deposited. It is suggested that the interaction between epithelial cells and both basement membrane and stromally derived matrices exerts a negative influence on the expression of mRNA for extracellular matrix components. In addition, we show that the capacity for lactational differentiation correlates with conditions that favor the deposition of a continuous basement membrane, and argue that the interaction between specialized epithelial cells and stroma enables them to create their own microenvironment for accurate signal transduction and phenotypic function.     

Ontogenesis of the murine hepatic extracellular matrix: an immunohistochemical study

To define the role of the extracellular matrix (ECM) in hepatogenesis, we examined the temporal and spatial deposition of fibronectin, laminin and collagen types I and IV in 12.5-21.5 day fetal and 1, 7 and 14 day postnatal rat livers. In early fetal liver, discontinuous deposits of the four ECM components studied were present in the perisinusoidal space, with laminin being the most prevalent. All basement membrane zones contained collagen type IV and laminin, including those of the capsule (mesothelial), portal vein radicles and bile ductules. Fibronectin had a distribution similar to that of collagen type IV early in gestation. However, at later gestational dates, fibronectin distribution in the portal triads approached that of collagen type I, being present in the interstitial connective tissues; whereas, collagen type IV and laminin were restricted to vascular and biliary basement membrane zones in those regions. The cytoplasm of some sinusoidal lining cells and hepatocytes reacted with antibodies to extracellular matrix components. By electron microscopy the immunoreactive material was localized in the endoplasmic reticulum, indicating the ability of these cells to synthesize these ECM proteins. Biliary ductular cells had prominent intracytoplasmic staining for laminin and collagen type IV from day 19.5 gestation until 7 days of postnatal life, but lacked demonstrable fibronectin or collagen type I. These results demonstrate that by 12.5 days of gestation the rat liver anlage has deposited a complex extracellular matrix in the perisinusoidal space. The prevalence of laminin in the developing hepatic lobules suggests a possible role for this glycoprotein in hepatic morphogenesis. In view of the intimate association of the hepatic lobular extracellular matrix with the developing vasculature, we hypothesize that laminin provides a scaffold of the developing liver, but once the ontogenesis is complete, intrahepatic perisinusoidal laminin expression is suppressed.     

Effects of laminin, fibronectin and type IV collagen on liver cell cultures

The effects on mouse liver cells of laminin, fibronectin and type IV collagen, all of which are the main matrix of the basement membrane, were studied. Laminin, a glycoprotein isolated from cultures of rat yolk sac carcinoma cells, promoted the attachment of mouse fetal liver cells to laminin-coated dishes, but did not have a strong influence upon the attachment of normal adult liver cells. On the other hand, fibronectin which was purified from mouse plasma promoted the attachment of adult liver cells but not that of fetal liver cells. The number of neonatal liver cells attached to the surfaces coated was intermediate between those of fetal and adult liver cells in each matrix. DNA synthesis and cell proliferation during the culture of full-term fetal liver cells in laminin-coated dishes were higher than those in fibronectin- or type IV collagen-coated dishes. The amount of alpha-fetoprotein secreted in the laminin-coated dishes was more than in other groups. No differences in secretion of albumin into media, however, were observed in either group. These results suggest that laminin may be necessary for cell growth, tissue organization and cell differentiation during the normal development of liver in vivo.     

Characterization of calcium accumulation in the brain of rats administered orally calcium: The significance of energy-dependent mechanism

Primary cultures of rat hepatocytes maintained on different matrix proteins such as collagen (Co IV) fibronectin (Fn), Laminin (Ln) or different tissue biomatrices were metabolically labelled with ³⁵[S]-SO₄ and the synthesis of sulphated proteoglycans was studied. The incorporation of the label into total glycosaminoglycan (GAG) was significantly higher in cells maintained on Co IV compared to those maintained on Fn or Ln. Similarly the incorporation of label was maximum in those cells maintained on the aortic biomatrix compared to liver or mammary gland biomatrix. About 80–95% of the GAG synthesised and secreted by cells maintained on individual matrix proteins and liver biomatrix was heparan sulphate (HS). But in the case of cells maintained on collagen IV aortic or mammary biomatrix in addition to HS, significant amount of chondroitin sulphate (CS) was also found. Nearly 50% of the total ³⁵[S]-GAG was associated with the cell layer after 24 h in culture in the case of cells maintained on individual matrix protein while those maintained on tissue biomatrix, retained about 70% of the ³⁵[S]-labelled proteoglycans (PG) with the cell layer. Analysis of the cell surface ³⁵[S]-labelled proteoglycans isolated from cells maintained on different biomatrix showed that it is a hybrid proteoglycan consisting of CS and HS. While the PG isolated from cells maintained on liver biomatrix consists of HS and CS in the ratio of 3:2 that from cells maintained on aorta or mammary gland matrix was about 2:3 indicating an alteration in the nature of the cell surface PGs produced by cells maintained on different tissue biomatrix. These results indicate that depending on the nature of the matrix substratum with which the cells are in contact, the nature and quantity of sulphated proteoglycans produced by hepatocytes vary.     

Vitamin A modulation of basement membrane production by purified testicular myoid cells.

Purified myoid cells, isolated from prepubertal rat testes, cultured in a chemically defined medium for up to 1 week do not change their metabolic activities, evaluated as protein synthesis and secretion, during the culture time. We report that fibronectin, collagen IV, and laminin are synthesized and secreted by myoid cells as demonstrated by immunocytochemical and biochemical methods. The deposition of all three proteins was spotty, with different regional localizations. The effect of vitamin A on the synthesis and the secretion of the basement membrane components was also evaluated. Retinol supplementation induces a higher synthesis of fibronectin and laminin, whereas it does not change collagen IV synthesis and secretion. The secretion of the other two molecules is differentially regulated by retinol; in fact fibronectin secretion is increased, whereas laminin secretion is reduced. Similar results were obtained utilizing retinoic acid. The data we report in this paper show, for the first time, that purified testicular myoid cells synthesize and secrete fibronectin, collagen IV, and laminin and that synthesis and secretion of these components of the basement membrane are regulated by retinol. These findings reveal a new effect of vitamin A in the regulation of mammalian spermatogenesis.     

Roles of extracellular matrix components in differentiating teratocarcinoma cells

F9 embryonal carcinoma cells treated with 5 X 10(-8) M retinoic acid and cultured in suspension for 8 days form aggregates consisting of an outer epithelial layer of alpha-fetoprotein-producing visceral endoderm cells. We have previously shown (Grover, A., Oshima, R. G., and Adamson, E. D. (1983) J. Cell Biol. 96, 1690-1696) that the differentiation of F9 cells to visceral endoderm is accompanied by the activation of several genes, and increased laminin synthesis is one of the earliest events. Here we analyze in detail the syntheses and secretion of fibronectin, type IV collagen, and laminin during the 8-day process. Employing immunoprecipitation and enzyme-linked immunosorbent assay, we show that the levels of all three components change with different patterns. Unstimulated F9 cells synthesize and secrete relatively high levels of fibronectin and low levels of type IV collagen. Fibronectin synthesis and secretion decreases to 10% of its original level whereas type IV collagen synthesis rises approximately 3-fold during the differentiation process. Laminin synthesis also rises at least 2-fold, and the proportions of its subunits change as the syntheses of B1 and A accelerate starting on day 2. However, unlike fibronectin and type IV collagen, laminin is largely accumulated in the aggregates. The data suggest that fibronectin has a role in aggregation whereas laminin is important in the differentiation process.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix

This study examined the role of extracellular matrix in regulating matrix phenotype of hepatic lipocytes, the major source of matrix in liver. Lipocytes (Ito, stellate, or fat-storing cells) were purified from normal rat liver and established in primary culture on either uncoated plastic, plastic coated with individual matrix proteins, or a "complete" gel matrix, a basement membrane-like matrix derived from the Engelbreth-Holm-Swarm (EHS) murine tumor. The ultrastructure of lipocytes cultured on the gel matrix resembled that of cells in normal liver, whereas lipocytes on plastic had dispersed nuclear chromatin and expanded rough endoplasmic reticulum, consistent with active proliferation and secretion. Lipocytes on the gel matrix exhibited no proliferative activity; cells maintained on plastic proliferated and produced type I collagen predominantly. Total collagen secretion by lipocytes on the gel matrix was 29% of that of cells on plastic, and consisted of type III collagen only. This difference extended to proteoglycan production, which was less than 5% of the amount produced by cells in conventional culture on plastic. The effects of the EHS gel were not reproduced by the individual components of the gel (laminin, type IV collagen, and heparan sulfate proteoglycan) or by a type I collagen gel. They were also reversible upon transfer of the cells to conventional culture. In contrast to lipocytes, collagen synthesis by hepatocytes was similar whether cultured on EHS gel or on plastic. These results show that the extracellular matrix can modulate matrix protein production by lipocytes and imply that, in early hepatic inflammation, changes in the hepatic subendothelial matrix may underlie stimulation of lipocyte matrix production and progression of the fibrotic process.     

Epithelial-mesenchymal interactions: effects of a dental biomatrix on odontoblasts

The functional differentiation of odontoblasts requires specific interactions between these cells and the extracellular matrix. To further analyze these phenomena we studied the effects of a "dental papillae biomatrix" on isolated dental papillae cultured in vitro. The dental papillae biomatrix was extracted from EDTA-dissociated day-18 mouse dental papillae by homogenization, NaCl and enzymatic treatments, and deposited on Millipore filters. This biomatrix was studied by means of transmission electron microscopy and indirect immunofluorescence: it contained collagen fibrils, type IV collagen, fibronectin and laminin; cellular residues were also observed. The dental papillae were isolated by trypsin treatment of homologous tooth germs and cultured on uncoated (control) and coated filters. As shown by histological and cytological data, odontoblast-like cells never differentiated in control cultures. In presence of biomatrix and serum, polarized functional cells were observed. The functional state of these cells was enhanced by the addition of ascorbic acid to the culture media. Study of the incorporation of 3H-proline in cultured dental papillae and in macromolecules secreted into the culture media corroborated the morphological findings.     

Differential expression of extracellular matrix components in rat Sertoli cells.

We studied expression of laminin, fibronectin, and Type IV collagen in the testis by means of immunofluorescence and immunoblot analysis and also examined gene expression of fibronectin using the ribonuclease protection assay. By immunofluorescence on sections from 20-day-old rats, laminin, fibronectin, and Type IV collagen were found in the basement membrane of the seminiferous tubules and in the interstitial regions of the testis. No localization of any extracellular matrix components was found inside the sectioned cells. However, when Sertoli cells were cultured on glass coverslips, laminin and Type IV collagen were both found inside the cells, suggesting new synthesis. In cultured peritubular cells, Type IV collagen, laminin, and fibronectin were found within the cells. When examined by immunoblot analysis, freshly isolated Sertoli and peritubular cells from 20-day-old rats did not demonstrate production of laminin or fibronectin. After 5 days in culture, peritubular cells produced both laminin and fibronectin, whereas cultured Sertoli cells produced only laminin. In contrast, freshly isolated and cultured Sertoli and peritubular cells all produced Type IV collagen. Moreover, the ribonuclease protection assay indicated that the bulk of fibronectin gene expression occurs within the first 10 days of postnatal development, with lower maintenance levels occurring thereafter. These results indicate that in the testis the highest levels of expression of laminin and fibronectin occur during development and in primary cell culture, whereas expression of Type IV collagen is higher at later stages.     

Extracellular matrix (ECM) synthesis in muscle cell cultures: quantitative and qualitative studies during myogenesis

When the synthesis of extracellular matrix components was examined in G8-1 murine skeletal muscle cells as a function of differentiation, non-collagen and to an even greater extent collagen synthesis was increased. Specifically, collagen types I, III, IV, laminin and fibronectin were identified by SDS-PAGE. Immunoprecipitation, with specific antibodies revealed that both the cell layer and medium of differentiated multinucleated myotubes contained increased levels of type IV collagen and laminin, decreased levels of type III collagen and fibronectin and equivalent levels of type I collagen compared to mononuclear myoblasts.     

Signal transduction for chemotaxis and haptotaxis by matrix molecules in tumor cells

Transduction of signals initiating motility by extracellular matrix (ECM) molecules differed depending on the type of matrix molecule and whether the ligand was in solution or bound to a substratum. Laminin, fibronectin, and type IV collagen stimulated both chemotaxis and haptotaxis of the A2058 human melanoma cell line. Peak chemotactic responses were reached at 50-200 nM for laminin, 50-100 nM for fibronectin, and 200-370 nM for type IV collagen. Checkerboard analysis of each attractant in solution demonstrated a predominantly directional (chemotactic) response, with a minor chemokinetic component. The cells also migrated in a concentration-dependent manner to insoluble step gradients of substratum-bound attractant (haptotaxis). The haptotactic responses reached maximal levels at coating concentrations of 20 nM for laminin and type IV collagen, and from 30 to 45 nM for fibronectin. Pretreatment of cells with the protein synthesis inhibitor, cycloheximide (5 micrograms/ml), resulted in a 5-30% inhibition of both chemotactic and haptotactic responses to each matrix protein, indicating that de novo protein synthesis was not required for a significant motility response. Pretreatment of cells with 50-500 micrograms/ml of synthetic peptides containing the fibronectin cell-recognition sequence GRGDS resulted in a concentration-dependent inhibition of fibronectin-mediated chemotaxis and haptotaxis (70-80% inhibition compared to control motility); negative control peptide GRGES had only a minimal effect. Neither GRGDS nor GRGES significantly inhibited motility to laminin or type IV collagen. Therefore, these results support a role for the RGD-directed integrin receptor in both types of motility response to fibronectin. After pretreatment with pertussis toxin (PT), chemotactic responses to laminin, fibronectin, and type IV collagen were distinctly different. Chemotaxis to laminin was intermediate in sensitivity; chemotaxis to fibronectin was completely insensitive; and chemotaxis to type IV collagen was profoundly inhibited by PT. In marked contrast to the inhibition of chemotaxis, the hepatotactic responses to all three ligands were unaffected by any of the tested concentrations of PT. High concentrations of cholera toxin (CT; 10 micrograms/ml) or the cAMP analogue, 8-Br-cAMP (0.5 mM), did not significantly affect chemotactic or haptotactic motility to any of the attractant proteins, ruling out the involvement of cAMP in the biochemical pathway initiating motility in these cells. The sensitivity of chemotaxis induced by laminin and type IV collagen, but not fibronectin, to PT indicates the involvement of a PT-sensitive G protein in transduction of the signals initiating motility to soluble laminin and type IV collagen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Basement Membrane Proteins Influence Brain Capillary Endothelial Barrier Function In Vitro

Abstract: The influence of basement membrane proteins on cellular barrier properties of primary cultures of porcine brain capillary endothelial cells grown on permeable filter inserts has been investigated. Measurements of transcellular electrical resistance (TER) by impedance spectroscopy were performed with cells cultured on type IV collagen, fibronectin, laminin, and one-to-one mixtures of these proteins. Moreover, a one-to-one combination of type IV collagen and SPARC (secreted protein acidic and rich in cysteine) has been studied. Rat tail collagen has been used as a reference substratum. If TERs of cells from a given preparation were low (∼350 Ω× cm²) on the reference substratum, type IV collagen, fibronectin, and laminin as well as one-to-one combinations of these proteins elevated transcellular resistances significantly (2.3- to 2.9-fold) compared with rat tail collagen. TER of cells exhibiting a high reference level (∼1,000 Ω× cm²) could, by contrast, be increased only 1.1- to 1.2-fold. The type IV collagen/SPARC mixture did not elevate TER. Our findings suggest that type IV collagen, fibronectin, and laminin are involved in tight junction formation between cerebral capillary endothelial cells. The differential effects observed for individual preparations probably reflect more or less dedifferentiated states of the endothelium, in which basement membrane proteins can influence cellular differentiation more or less strongly. However, our results indicate that type IV collagen, fibronectin, and laminin enhance the reliability and suitability of primary microvascular endothelial cell cultures as an in vitro model of the blood-brain barrier.     

Extracellular matrix synthesis is required for the movement of sclerotome and neural crest cells on collagen

During early embryogenesis cells of several different populations disperse by active cell movement from one location to another. Preexisting extracellular materials are major determinants of these dispersal patterns, but the cells are also able to modify their substrata by synthesizing and secreting extracellular matrix molecules as they move. In order to determine the contribution made by these deposited materials, several tissues from the early chick embryo have been cultured in the presence of inhibitors of extracellular matrix synthesis and secretion. The tissues examined were sclerotome cells from differentiated somites and neural crest cells. For comparison, undifferentiated somites were also cultured. The movement of these cells was compared in type I collagen gel culture and in conventional culture on artificial substrata. Inhibitors of collagen synthesis were used (cis-hydroxy proline and L-azetidine-2-carboxylic acid) in addition to a proteoglycan inhibitor (p-nitrophenyl-xylopyranoside) and a secretion inhibitor (monensin). Results indicate that sclerotome cells require collagen synthesis for movement in a collagen matrix. Reversal of the effects of collagen inhibitors, by proline and type II collagen, suggest that sclerotome cells normally condition the type I matrix in order to move in it. Inhibition of proteoglycan synthesis produced the greatest effect on the movement of neural crest cells regardless of the substratum, confirming an important role for these molecules in the crest migratory routes. The attachment of all cells to collagen was highly sensitive to the presence of monensin, which is known to reduce the deposition of glycosaminoglycans and fibronectin. These results suggest that conditioning of the extracellular matrix by newly synthesized material is required for cell attachment and movement during early development.     

Connective tissue biomatrix: its isolation and utilization for long- term cultures of normal rat hepatocytes

A new procedure is introduced for the isolation of connective tissue fibers, called biomatrix, containing a significant portion of the extracellular matrix (basement membrane components and components of the ground substance). Biomatrix isolated from normal rat liver contains >90% of the tissue's collagens and all of the known collagen types, including types I and III and basement membrane collagens. The purified collagenous fibers are associated with noncollagenous acidic proteins (including fibronectins and possibly small amounts of glycosaminoglycans). Procedures are also described for preparing tissue culture substrates with these fibers by either smearing tissue culture dishes with frozen sections or by shredding the biomatrix into small fibrils with a homogenizer. The biomatrix as a substrate has a remarkable ability to sustain normal rat hepatocytes long-term in culture. The hepatocytes, which on tissue culture plastic or on type I collagen gels do not survive more than a few weeks, have been maintained for more than 5 mo in vitro when cultured on biomatrix. These cells cultured on rat liver biomatrix show increased attachment and survival efficiencies, long-term survival (months) and retention of some hepatocyte-specific functions.     

The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells

Laminin and fibronectin are glycoproteins that influence cell behavior and mediate cell/substratum adhesion. We have examined the interaction of these macromolecules with the serine protease plasminogen activator (PA) in two types of extracellular matrices; one produced by the murine Engelbreth-Holm-Swarm (EHS) tumor (Matrigel), and another by normal kidney epithelial cells in culture. Matrigel was found to contain significant quantities of tissue-type PA (tPA). Two of the major components of Matrigel, laminin and type IV collagen, were also examined. Tissue-type PA was associated with purified preparations of laminin; however, it was not found associated with type IV collagen. Normal kidney epithelial cells in culture secrete large amounts of urokinase (UK) and deposit a subepithelial matrix containing both laminin and fibronectin. These matrix macromolecules were isolated from the deposited matrix by immunoprecipitation, examined by zymography, and found to contain UK. The potential role of this interaction in the mechanisms of cell migration and matrix remodeling is discussed.