The extracellular matrix of hydra is a porous sheet and contains type IV collagen

Hydra, as an early diploblastic metazoan, has a well-defined extracellular matrix (ECM) called mesoglea. It is organized in a tri-laminar pattern with one centrally located interstitial matrix that contains type I collagen and two sub-epithelial zones that resemble a basal lamina containing laminin and possibly type IV collagen. This study used monoclonal antibodies to the three hydra mesoglea components (type I, type IV collagens and laminin) and immunofluorescent staining to visualize hydra mesoglea structure and the relationship between these mesoglea components. In addition, hydra mesoglea was isolated free of cells and studied with immunofluorescence and scanning electron microscopy (SEM). Our results show that type IV collagen co-localizes with laminin in the basal lamina whereas type I collagen forms a grid pattern of fibers in the interstitial matrix. The isolated mesoglea can maintain its structural stability without epithelial cell attachment. Hydra mesoglea is porous with multiple trans-mesoglea pores ranging from 0.5 to 1 μm in diameter and about six pores per 100 μm² in density. We think these trans-mesoglea pores provide a structural base for epithelial cells on both sides to form multiple trans-mesoglea cell–cell contacts. Based on these findings, we propose a new model of hydra mesoglea structure.     

Lack of effect of thrombin on fibrin(ogen)-endothelial cell interaction

In the present study we investigate the fibrin(ogen)-endothelial cell binding and the effect of thrombin on the endothelial cells in relation to fibrin(ogen) binding capacity. Endothelial cell fibrinogen binding was concentration and time-dependent, reaching saturation at 1.4 M of added ligand. At equilibrium, the number of fibrinogen molecules bound per endothelial cell in the monolayer was 5.8±0.7×10⁶. When endothelial cells were activated by different concentrations of thrombin (0–0.1 NIH units ml^–1), no increase in fibrinogen binding capacity was observed at all the thrombin concentration tested. Whereas disruption of endothelial cell monolayers was observed at thrombin concentrations higher than 0.05 NIH units ml^–1, no increase in the amount of fibrinogen bound was observed. Therefore, resting and thrombin-activated endothelial cells show the same fibrinogen binding capacity.The adhesion of endothelial cells in suspension on immobilized fibrinogen or fibrin was studied to ascertain whether the behavior of fibrin is similar to that of fibrinogen. The extent of endothelial cell attachment to immobilized fibrinogen and fibrin was similar (4275±130 cells cm^–2 for fibrinogen and 4350±235 cells cm^–2 for fibrin) and represent approximately 40% of the added endothelial cells. However, endothelial cell adhesion to immobilized fibrin was significantly faster than endothelial cell adhesion to immobilized fibrinogen. The maximum binding rate was 66±9 and 46±8 cells cm^–2 min^–1 for fibrin and fibrinogen, respectively. Therefore, the fibrinopeptides released by thrombin from fibrinogen induce qualitative changes which enhance the fibrin interaction with the endothelial cells.     

Effects of Mutations in the Cytoplasmic Domain of Integrin β1 to Talin Binding and Cell Spreading

Integrins are transmembrane proteins linking the extracellular matrix or certain cell–cell contacts to the cytoskeleton. To study integrin–cytoskeleton interactions we wanted to relate talin–integrin interaction to integrin function in cell spreading and formation of focal adhesions. For talin-binding studies we used fusion proteins of glutathione S-transferase and the cytoplasmic domain of integrin β₁ (GST-cytoβ₁) expressed in bacteria. For functional studies chimeric integrins containing the extracellular and transmembrane parts of β₃ linked to the cytoplasmic domain of β₁ were expressed in CHO cells as a dimer with the α_IIb subunit. Point mutations in the amino acid sequence N⁷⁸⁵PIY⁷⁸⁸ of β₁ disrupted both the integrin–talin interaction and the ability of the integrin to mediate cell spreading. COOH-terminal truncation of β₁ at the amino acid position 797 disrupted its ability to mediate cell spreading, whereas the disruption of talin binding required deletion of five more amino acids (truncation at position 792). A synthetic peptide from this region of β₁ (W⁷⁸⁰DTGENPIYKSAV⁷⁹²) bound to purified talin and inhibited talin binding to GST-cytoβ₁. The ability of the mutants to mediate focal adhesion formation or to codistribute to focal adhesions formed by other integrins correlated with their ability to mediate cell spreading. These results confirm the previous finding that a talin-binding site in the integrin β₁ tail resides at or close to the central NPXY motif and suggest that the integrin–talin interaction is necessary but not sufficient for integrin-mediated cell spreading.     

Reestablishment of the heterogeneous distribution of hepatic glutamine synthetase during regeneration after CCl4-intoxication

Summary Intoxication of rats with CCl₄ (1 ml/kg) resulted in the almost complete loss of glutamine synthetase (GS) specific activity and immunologically detectable enzyme protein known to be expressed exclusively in some hepatocytes of the perivenous zone of the liver acinus. During regeneration the specific activity as well as the original number of GS-positive (GS⁺) hepatocytes were reestablished. However, while the GS⁺ hepatocytes in control livers were arranged in up to 3 cell layers surrounding the central veins the same number of GS⁺ hepatocytes in regenerated livers formed a single cell layer only, most likely because the central veins were enlarged in diameter. Investigation of the nuclear pattern of GS⁺ and GS^– hepatocytes of control animals in primary cultures revealed striking differences characterized by significantly more mononuclear diploid, binuclear diploid, and binuclear tetraploid cells among the GS⁺ hepatocytes and predominantly mononuclear tetraploid cells (70%) among the GS^– hepatocytes. Immediately after liver damage by CCl₄ and during regeneration small but significant changes in the nuclear pattern were noted for GS^– hepatocytes. However, the first GS⁺ cells appearing during early regeneration showed a pattern of ploidy classes close to the original one found for GS^– hepatocytes. These results indicate that new GS⁺ hepatocytes may be derived from formerly GS^– cells which are induced to express GS if they have reached the border of the central veins.     

Integrin α3β1 Engagement Disrupts Intercellular Adhesion

During tissue morphogenesis and tumor invasion, epithelial cells must undergo intercellular rearrangement in which cells are repositioned with respect to one another and the surrounding mesenchymal extracellular matrix. Using three-dimensional aggregates of squamous epithelial cells, we show that such intercellular rearrangements can be triggered by activation of β1 integrins after their ligation with extracellular matrices. On nonadherent substrates, multicellular aggregates (MCAs) formed rapidly via E-cadherin junctional complexes and over time became compacted spheroids exhibiting a more epithelial phenotype. After MCAs were replated on culture substrates, the spheroids collapsed to yield tightly arranged cell monolayers. Cell–cell contact induced rapid elevation in E-cadherin levels, which was due to an increase in the metabolic stability of junctional receptors. During MCA remodeling of cell–cell adhesions, and monolayer formation, their E-cadherin levels fell rapidly. Similar behavior was obtained regardless of which ECM ligand—collagen type I, fibronectin, or laminin 1—MCAs were seeded on. In contrast, when seeded onto a matrix elaborated by squamous epithelial cells, cells in the MCA attached, spread, lost cell–cell junctions, and dispersed. Analysis identified laminin 5 as the active ECM ligand in this matrix, and MCA dispersion required functional β1 integrin and specifically α3β1. Furthermore, substrate-immobilized anti-integrin antibody effectively reproduced the epithelial–mesenchymal-like transition induced by the laminin 5 matrix. During the early stages of aggregate rearrangement and collapse, cells on laminin 5 substrates, but not those on collagen I substrates, exhibited intense cortical arrays of F-actin, microspikes, and fascin accumulation at their peripheral surfaces. These results suggest that engagement of specific integrin–ligand pairs regulates cadherin junctional adhesions during events common to epithelial morphogenesis and tumor invasion.     

Fibroblast α11β1 Integrin Regulates Tensional Homeostasis in Fibroblast/A549 Carcinoma Heterospheroids

We have previously shown that fibroblast expression of α11β1 integrin stimulates A549 carcinoma cell growth in a xenograft tumor model. To understand the molecular mechanisms whereby a collagen receptor on fibroblast can regulate tumor growth we have used a 3D heterospheroid system composed of A549 tumor cells and fibroblasts without (α11^+/+) or with a deletion (α11^-/-) in integrin α11 gene. Our data show that α11^-/-/A549 spheroids are larger than α11^+/+/A549 spheroids, and that A549 cell number, cell migration and cell invasion in a collagen I gel are decreased in α11^-/-/A549 spheroids. Gene expression profiling of differentially expressed genes in fibroblast/A549 spheroids identified CXCL5 as one molecule down-regulated in A549 cells in the absence of α11 on the fibroblasts. Blocking CXCL5 function with the CXCR2 inhibitor SB225002 reduced cell proliferation and cell migration of A549 cells within spheroids, demonstrating that the fibroblast integrin α11β1 in a 3D heterospheroid context affects carcinoma cell growth and invasion by stimulating autocrine secretion of CXCL5. We furthermore suggest that fibroblast α11β1 in fibroblast/A549 spheroids regulates interstitial fluid pressure by compacting the collagen matrix, in turn implying a role for stromal collagen receptors in regulating tensional hemostasis in tumors. In summary, blocking stromal α11β1 integrin function might thus be a stroma-targeted therapeutic strategy to increase the efficacy of chemotherapy.     

Functions of lumican and fibromodulin: lessons from knockout mice

Lumican and fibromodulin are collagen-binding leucine-rich proteoglycans widely distributed in interstitial connective tissues. The phenotypes of lumican-null (Lum ^–/–), Fibromodulin-null (Fmod ^–/–) and compound double-null (Lum ^–/– Fmod ^–/–) mice identify a broad range of tissues where these two proteoglycans have overlapping and unique roles in modulating the extracellular matrix and cellular behavior. The lumican-deficient mice have reduced corneal transparency and skin fragility. The Lum ^–/– Fmod ^–/– mice are smaller than their wildtype littermates, display gait abnormality, joint laxity and age-dependent osteoarthritis. Misaligned knee patella, severe knee dysmorphogenesis and extreme tendon weakness are the likely cause for joint-laxity. Fibromodulin deficiency alone leads to significant reduction in tendon stiffness in the Lum ^+/+ Fmod ^–/– mice, with further loss in stiffness in a lumican gene dose-dependent way. At the level of ultrastructure, the Lum ^–/– cornea, skin and tendon show irregular collagen fibril contours and increased fibril diameter. The Fmod ^–/– tendon contains irregular contoured collagen fibrils, with increased frequency of small diameter fibrils. The tendons of Lum ^–/– Fmod ^–/– have an abnormally high frequency of small and large diameter fibrils indicating a de-regulation of collagen fibril formation and maturation. In tissues like the tendon, where both proteoglycans are present, fibromodulin may be required early in collagen fibrillogenesis to stabilize small-diameter fibril-intermediates and lumican may be needed at a later stage, primarily to limit lateral growth of fibrils Published in 2003.     

Cell attachment properties of collagen type VI and arg-gly-asp dependent binding to its α2(VI) and α3(VI) chains

Twelve of sixteen different cell types including fibroblasts and tumor cells were able to attach and spread on substrates of pepsin-solubilized or intact collagen VI, and on its triple helical domain. Attachment and spreading were independent of soluble mediator proteins (fibronectin, laminin) and collagen VI was distinct from collagens I, IV and V in the cells with which it interacted. Many of the same cells bound and spread on substrates prepared from unfolded α2(VI) and α3(VI) chains but not on the α1(VI) chain. The interactions with the chains were inhibited by low concentrations (10–100 μM) of synthetic RGDS and RGDT but not RGES peptides while the binding of cells to pepsin-solubilized collagen VI was more than 20-fold less sensitive to these peptides. The data incidate that cells have the ability to bind to collagen VI in a specific manner suggesting a similar function for collagen VI in situ.     

The role of let-7b in the inhibition of hepatic stellate cell activation by rSjP40

BackgroundHepatic stellate cells (HSCs) are one of the main cell types involved in liver fibrosis induced by many factors, including schistosomes. Previous studies in our lab have shown that recombinant P40 protein from Schistosoma japonicum (rSjP40) can inhibit HSC activation in vitro. Let-7b is a member of the let-7 microRNA family and plays an inhibitory role in a variety of diseases and inflammatory conditions. In this study, we investigated the role of let-7b in the inhibition of HSC activation by rSjP40.MethodsExpression of let-7b was detected by quantitative real-time PCR. A dual luciferase assay was used to confirm direct interaction between let-7b and collagen I. We also used western blot to assess protein levels of TGFβRI and collagen type I α1 (COL1A1).ResultsWe found that rSjP40 up-regulates expression of let-7b in HSCs. Let-7b inhibits collagen I expression by directly targeting the 3’UTR region of the collagen I gene. Furthermore, we discovered that let-7b inhibitor partially restores the loss of collagen I expression caused by rSjP40.ConclusionOur research clarifies the role of let-7b in the inhibition of HSC activation by rSjP40 and will provide new insights and ideas for the inhibition of HSC activation and treatment of liver fibrosis.     

Specific Insulin-Like Growth Factor-I Receptors on Circulating Bovine Mononuclear Cells

Abstract

We have examined the presence and properties of specific receptors for IGF-I on bovine mononuclear cells. Competitive binding studies showed that binding of [¹²⁵I]IGF-I to mononuclear cells was inhibited by unlabelled peptides with the rank of IGF-I > IGF-II > insulin. The binding of [¹²⁵I]IGF-I was a function of the cell concentration. Equilibrium dissociation constant and receptor concentration values for the average of 9 adult cows were 1.13 ± 0.11 nM and 108.9 ± 24.1 fMol/10⁷ cells, respectively. Moreover, IGF-I stimulated thymidine incorporation into bovine mononuclear cells in the absence of serum and phytohemagglutinin (PHA). The existence of specific and functional IGF-I receptors on circulating bovine mononuclear cells would provide an easily accessible source for studying IGF-I receptor changes in the bovine, both in physiologic and pathologic states.     

Nicotinamide adenine dinucleotides permeate through mitochondrial membranes in human Epstein-Barr virus-transformed lymphocytes

Human cultured cells are widely used for the investigation of respiratory chain disorders. Oxidative properties are generally investigated by means of polarographic studies carried out on detergent-permeabilized cells. By studying the oxidative properties of Epstein-Barr virus-transformed B lymphocytes, we found that the respiration was significantly decreased after 3–4 days of cell culture. Simultaneously, we observed that NAD⁺-dependent oxidations (malate, glutamate, pyruvate) became dependent upon the addition of exogenous NAD⁺. The effect of NAD⁺ was shown to be related to an influx of catalytic amount of NAD⁺ into the mitochondrial matrix. A full ability to oxidize NAD⁺-dependent substrates was restored less than 2 h after a change of the culture medium.These observations suggested: (a) the occurrence of fluxes of catalytic amounts of NAD⁺ through the mitochondrial inner membrane in human cells; (b) an early control of mitochondrial metabolism by matrix NAD⁺ content in cells grown under limiting growth conditions; (c) the possible confusion between complex I deficiency and a decrease content of matrix NAD⁺ when using human cultured cells. (Mol Cell Biochem 115–119, 1997)     

A novel method for quantifying cell migration through tissue engineering scaffolds: evaluation of modified collagen matrices

A novel method to quantify cell migration through potential tissue engineering 3-d scaffolds is described. The migration assay uses a dot-blotting apparatus into which the tissue engineering matrix is placed on top of a nitrocellulose membrane. This assay was used to evaluate human dermal fibroblast migration through four porcine collagen matrices with varying pore diameters and pitch lengths. Fibroblasts were placed on the matrix surface, at between 1 ×10³–3 × 10³ cells mm^–2, and left for 18 h to allow migration. The nitrocellulose membrane was stained with haematoxylin, the membrane digitised and the pixel intensity of the stained cells quantified. We showed that for all matrix variants, migration was more effective with a higher initial seeding density. The application of varying initial cell densities resulted in the greatest extent of cell migration through the matrix variant with pores of 30 m diameter and 400 m pitch length (i.e. 10.3% migration at 1 ×10³ cells mm^–2). This method was coupled with confocal microscopy to evaluate the depth of cell migration within the matrix. At a depth of 20 m cell numbers were similar to those on the matrix surface: at a depth of 100 m only a few cells were observed.     

Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

Chondroadherin (the 36-kD protein) is a leucine-rich, cartilage matrix protein known to mediate adhesion of isolated chondrocytes. In the present study we investigated cell surface proteins involved in the interaction of cells with chondroadherin in cell adhesion and by affinity purification. Adhesion of bovine articular chondrocytes to chondroadherin-coated dishes was dependent on Mg²⁺ or Mn²⁺ but not Ca²⁺. Adhesion was partially inhibited by an antibody recognizing β1 integrin subunit. Chondroadherin-binding proteins from chondrocyte lysates were affinity purified on chondroadherin-Sepharose. The β1 integrin antibody immunoprecipitated two proteins with molecular mass ~110 and 140 kD (nonreduced) from the EDTA-eluted material. These results indicate that a β1 integrin on chondrocytes interacts with chondroadherin. To identify the α integrin subunit(s) involved in interaction of cells with the protein, we affinity purified chondroadherin-binding membrane proteins from human fibroblasts. Immunoprecipitation of the EDTA-eluted material from the affinity column identified α2β1 as a chondroadherin-binding integrin. These results are in agreement with cell adhesion experiments where antibodies against the integrin subunit α2 partially inhibited adhesion of human fibroblast and human chondrocytes to chondroadherin. Since α2β1 also is a receptor for collagen type II, we tested the ability of different antibodies against the α2 subunit to inhibit adhesion of T47D cells to collagen type II and chondroadherin. The results suggested that adhesion to collagen type II and chondroadherin involves similar or nearby sites on the α2β1 integrin. Although α2β1 is a receptor for both collagen type II and chondroadherin, only adhesion of cells to collagen type II was found to mediate spreading.     

Soluble Integrin Ligands and Growth Factors Independently Rescue Neuroblastoma Cells from Apoptosis under Nonadherent Conditions

We have investigated the role of extracellular matrix (ECM) and growth factors in the survival of nonadherent human neuroblastoma cells (line SK-N-BE). Cells cultured in serum-free medium under nonadherent conditions died with apoptotic-like features (cromatin condensation and nuclear fragmentation). SK-N-BE cells underwent neuronal differentiation in response to retinoic acid (RA). While RA itself did not induce apoptosis, differentiation increased the susceptibility of SK-N-BE cells to detachment-induced apoptosis. The appearance of the apoptotic-like phenotype required the maintenance in suspension of SK-N-BE cells for at least 16 h (12.43 ± 1.40% of cells undergoing apoptosis) and the percentage increased up to 46.84 ± 3.15% after 24 h. Suspension-induced apoptosis did not depend on increased intracellular Ca²⁺levels nor onde novoprotein synthesis and was not associated with extensive DNA degradation. Stimulation by soluble collagen I rescued suspended cells from apoptosis, even in the absence of cell adhesion and spreading. The survival promoting effect of ECM was mediated by the integrin receptors, since ([1]) the protective effect of soluble collagen I was blocked by anti-integrin antibodies to β₁and α₁subunits and ([2]) the antibody-induced clustering of α₁, α₃, α_v, β₁, and β₃integrins rescued SK-N-BE cells cultured in suspension from apoptosis. As expected, adhesion on immobilized ECM proteins, collagen I, or laminin (0.1 to 10 μg/ml) also rescued SK-N-BE cells from apoptosis in a dose-dependent manner. Thede novoprotein synthesis was required to promote the survival effect of ECM, since cycloheximide completely abolished the protective effect of collagen I and protection from apoptosis by ECM or by anti-β₁antibody was associated with the increased expression ofbcl-2.In addition to integrin stimulation, serum, insulin, and nerve growth factor inhibited suspension-induced apoptosis of SK-N-BE cells. The survival effect of serum and growth factors did not require the synthesis of new proteins, unlike the ECM effect. These data show that matrix proteins can promote cell survival in neuronal cells via integrin receptors. This effect does not require cell adhesion and the subsequent changes in cell shape as it can be mediated by soluble integrin ligands in suspended cells and involves a signaling pathway different from that triggered by growth factors.     

Cell‐extracellular matrix interactions in the fluidic phase direct the topology and polarity of self‐organized epithelial structures

IntroductionIn vivo, cells are surrounded by extracellular matrix (ECM). To build organs from single cells, it is generally believed that ECM serves as scaffolds to coordinate cell positioning and differentiation. Nevertheless, how cells utilize cell‐ECM interactions for the spatiotemporal coordination to different ECM at the tissue scale is not fully understood.MethodsHere, using in vitro assay with engineered MDCK cells expressing H2B‐mCherry (nucleus) and gp135/Podocalyxin‐GFP (apical marker), we show in multi‐dimensions that such coordination for epithelial morphogenesis can be determined by cell‐soluble ECM interaction in the fluidic phase.ResultsThe coordination depends on the native topology of ECM components such as sheet‐like basement membrane (BM) and type I collagen (COL) fibres: scaffold formed by BM (COL) facilitates a close‐ended (open‐ended) coordination that leads to the formation of lobular (tubular) epithelium. Further, cells form apicobasal polarity throughout the entire lobule/tubule without a complete coverage of ECM at the basal side, and time‐lapse two‐photon scanning imaging reveals the polarization occurring early and maintained through the lobular expansion. During polarization, gp135‐GFP was converged to the apical surface collectively in the lobular/tubular structures, suggesting possible intercellular communications. Under suspension culture, the polarization was impaired with multi‐lumen formation in the tubules, implying the importance of ECM biomechanical microenvironment.ConclusionOur results suggest a biophysical mechanism for cells to form polarity and coordinate positioning at tissue scale, and in engineering epithelium through cell‐soluble ECM interaction and self‐assembly.     

Expansion and neural differentiation of embryonic stem cells in adherent and suspension cultures

The embryonic stem cell line, S25, is a genetically modified line that allows lineage selection of neural cells (M. Li, L. Lovell-Badge, A. Smith (1998) Current Biology 8: 971–974). Here, the growth parameters of this cell line were analysed. Serial passaging in adherent conditions enabled these cells to grow rapidly (average specific growth rates of 0.035 h^–1) and generate high viable cell densities (above 90%). The aggregation of the S25 cells into embryoid bodies (EBs) was also studied, indicating limited cell growth (maximum cell densities of 2.7×10⁵ cells ml^–1) and a high variability of aggregate size (70–400 m after 8 d). Enzymatic dissociation of EBs with 1% (v/v) trypsin gave highest cell viability (91%) and density (1.4×10⁴ cells ml^–1) and the cells thus obtained are able to differentiate into neurons.     

A Chondroitin/Dermatan Sulfate Form of CD44 Is a Receptor for Collagen XIV (Undulin)

Collagen XIV, a fibril-associated collagen with interrupted triple helices, is expressed in differentiated soft connective tissues and in cartilage. However, a cellular receptor for this protein has not been identified. Here we show that human placental collagen XIV, isolated by a mild and simple two-step method, serves as adhesive protein for a variety of mesenchymal and some epithelial cells. Cell adhesion could be inhibited by preincubation of the collagen XIV substrate with heparin or with the chondroitin/dermatan sulfate proteoglycan decorin and by pretreatment of cells with chondroitinase ABC or heparinase III, suggesting a cell membrane proteoglycan as receptor. Affinity chromatography of¹²⁵I-labeled fibroblast cell surface proteins on collagen XIV–Sepharose yielded a chondroitin/dermatan sulfate proteoglycan with a molecular mass of 97–105 kDa after chondroitinase ABC digestion and of 60–70 kDa after further treatment withN-glycosidase F. The eluates contained also some high-molecular-weight material that was susceptible to digestion with heparinase but no detectable integrins. Immunoprecipitation with a specific monoclonal antibody identified the prominent chondroitin/dermatan sulfate proteoglycan as a member of the CD44 family. The interaction between collagen XIV and cells appears to be finely tuned, since matrix-associated glycosaminoglycans, and particularly proteoglycans like decorin, could compete with cells for the binding site(s) on collagen XIV under physiological conditions.     

Effects of products from macrophages, blood mononuclear cells and of retinol on collagenase secretion and collagen synthesis in chondrocyte culture

Collagenase secretion was studied in cultures of rabbit articular chondrocytes. Differentiation of the cells was assessed by characterizing the type of ³H-labelled collagen produced during treatment with (1) conditioned media from rabbit peritoneal macrophages and human blood mononuclear cells, and (2) with retinol, a potent cartilage resorbing agent in tissue culture. Conditioned media stimulated collagenase secretion. Total collagen synthesis was reduced due to a decrease of synthesis of α₁ chains; the amount of α₂ chains synthesized was unchanged. This is thought to be due to a reduction in type II synthesis. Retinol did not stimulate collagenase secretion. Total collagen synthesis was reduced by retinol. α₂ chain synthesis, however, was significantly increased, suggesting a switch of collagen synthesis in favor of type I collagen and, therefore, dedifferentiation. These results demonstrate that dedifferentiation of chondrocytes with respect to collagen synthesis is not necessarily associated with a stimulation of collagenase secretion.     

Collagen synthesis by monkey arterial smooth muscle cells during proliferation and quiescence in culture

Monkey arterial smooth muscle cells (SMC) which are stimulated to proliferate in the presence of 5% monkey blood serum (MBS) and which remain quiescent in 5% monkey platelet-poor plasma serum (MPPPS) were examined for their ability to synthesize collagen in each of these conditions in culture. Collagen synthesis was measured by determining amounts of newly formed labeled hydroxyproline, following labelling in the presence of [³H]proline and ascorbic acid. Ascorbate requirements of SMC were examined to assure maximal hydroxylation. SMC synthesize the same amount of collagen/cell in 5% whole blood serum (MBS) during the early phase of rapid proliferation as during slow growth in later phases in culture. SMC grown in the presence of serum-lacking platelet factors synthesize 60–90% less collagen and 60–90% less non-collagen protein (per cell or per mg protein) than cells grown in MBS. Non-collagen protein synthesis was measured as incorporation of both [³H]proline and of [³H]leucine, determined as trichloroacetic acid (TCA)-precipitable material. Previous studies indicate that a factor derived from platelets is the principal mitogen present in whole blood serum for diploid cells such as SMC and fibroblasts in culture. Similarly derived factors are potent stimulators of both collagen and non-collagen protein synthesis by SMC. SMC, quiescent in medium lacking platelet derived material (MPPPS), is being used to investigate factors important in SMC proliferation since this is a significant event in atherogenesis in vivo. An increased deposition of collagen also occurs during atherogenesis. Consequently it will be useful to employ similar cultures of quiescent SMC to examine agents which affect production of this connective tissue matrix protein.     

Arg-Gly-Asp (RGD) sequence conjugated in a synthetic copolymer bearing a sugar moiety for improved culture of parenchymal cells (hepatocytes)

A copolymer, including a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence and sugar moieties, was synthesized for the culturing of parenchymal cells (hepatocytes). Hepatocyte cells attached to poly[N-p-vinylbenzyl-d-maltonamide-co-6-(p-vinylbenzamido)-hexanoic acid-GRGDS] [poly(VMA-co-VBRGD)]-coated dishes grew approximately 60% better than on other polymer-coated surface for 12 h. Also, about 80% greater albumin secretion (0.38 pg ml^–1) and about 70% greater urea synthesis (0.495 pg ml^–1) from hepatocytes were produced in this matrix as compared with unstimulated cells. The behaviour of hepatocytes on poly(VMA-co-VBGRGDS)-coated dishes was not distinct from those attached to a collagen. The conjugation of the adhesion molecules of the RGD peptide in the poly(VMA-co-VBGRGDS) copolymer therefore specifically interacts with integrin families on the hepatocyte cell membrane.