Role of carbohydrates in cell-substrate interactions during newt epidermal cell migration

The effect of several solubilized monosaccharides on epidermal cell migration from skin explants of the adult newt was examined. The ability of epidermal cells to migrate on substrates coated with these same sugars or with wheat germ agglutinin (WGA) was also determined. Adding 0.05 M N-acetyl-glucosamine (GlcNAc) to the medium inhibited epidermal cell migration in dishes coated with either type I collagen or fibrinogen. The same concentration of fucose, galactose, or mannose had no effect. In contrast to type I collagen, which supported considerable migration when dried onto the bottom of plastic dishes, epidermal cells were unable to migrate on dishes coated similarly with WGA, mucin (a protein high in sialic acid residues), or bovine serum albumin (BSA) conjugated to galactose, mannose, or GlcNAc. Red blood cell (RBC) binding assays showed that drying WGA onto plastic did not destroy its GlcNAc binding sites--nor did it damage the GlcNAc residues of BSA-GlcNAc. The RBC assay also verified that for both these proteins, substrates with distinctly different cell binding capacities had been tested in the migration experiments. In dishes coated with either WGA or BSA-GlcNAc, red cells bound to dish bottoms in a GlcNAc-specific manner right up to the margins of explants. Other control experiments showed that the failure of migration in WGA- and BSA-GlcNAc-coated dishes could not be explained by competition between adsorbed and desorbing protein for cell surface receptors. This work shows that adhesive bonds between epidermal cell surface GlcNAc and a GlcNAC-specific lectin on the substrate are not by themselves adequate to support cell migration. Nor is GlcNAc, sialic acid, galactose, or mannose alone on the substrate sufficient. In conjunction with our earlier work (Donaldson and Mahan: J. Exp. Zool., 231:211-219, '84; Donaldson, Mahan, Hasty, McCarthy, and Furcht: J. Cell. Biol., 101: 73-78, '85), these observations suggest that factors other than carbohydrate content or capacity to act as a lectin determine whether a given extracellular protein will support migration.     

Long-term culture of human esophageal explants and cells

Human esophageal epithelium obtained from intermediate autopsies (<12 h) was maintained as cell and explant cultures. In order to develop a serum-free, defined media culture model, several medias and additives were evaluated. The viability and differentiation of the epithelial cells cultured with serum-free, Keratinocyte Growth Media (KGM, Clonetics Co., San Diego, CA) was improved over that of esophageal cells and explants cultured in either serum-supplemented CMRL 1066 (OCM), serum-free additive-supplemented CMRL 1066, or cimetidine-supplemented CMRL 1066. The KGM component EGF was determined to be trophic for esophagus cells on the basis of findings of increased ³H-TdR tabelling in KGM cultures when compared to control cells grown in KGM without EGF (KBM). The morphologic pattern of the cytoskeletal proteins actin, keratin, and vimentin were characterized in isolated cell populations. The intermediate filaments, keratin, and vimentin were co-expressed in these epithelial cells. Esophageal explant viability, differentiation, and outgrowth from 15 cases were also evaluated in dishes coated with basement membrane associated proteins. Explants cultured in these dishes were equally well-preserved and differentiated. There were no significant differences in the explant histology when there was protein coating of the culture dishes, although one case showed improved outgrowth with laminin coating. A main advantage for using this culture system is that the same medium (KGM) can be used for both the culture of explants and isolated epithelial cells. Future applications of this model include determining: (1) the effect different concentrations of EGF and calcium in the media will have on esophageal proliferation and differentiation, and (2) the role of different basement membrane associated proteins on the plating efficiency of either isolated or outgrowth epithelial esophageal cells.This is publication #2544 from the Pathobiology Laboratory.     

Sucrose inhibits vascular smooth muscle cell proliferation/migration--protein synthesis is maintained

Abstract.  We have previously shown that the onset of smooth muscle cell proliferation in tissue cultures is triggered independently of serum. The aim of the present study was to investigate if this process was affected by osmotic stress. Vascular explants from 8-month-old male rats were cultured under serum-free conditions using collagen I as migration substrate. Sucrose was added to the culture medium in concentrations varying from 1 to 3% (30–90 mOsM). Cell migration from aortic explants onto the culture dishes was totally inhibited at a sucrose concentration of 90 mOsM. A significant dose-dependent decline in proliferation was shown for cells in explants pulse labelled with ³H-thymidine. In contrast, pulse labelling with ³⁵S-methionine revealed that protein synthesis was maintained in the presence of sucrose. The results indicate that osmotic pressure affects smooth muscle cell protein synthesis, proliferation and migration.     

Organotypic Culture of Human Skin to Study Melanocyte Migration

An ex vivo model system was developed to investigate melanocyte migration. Within this model system, melanocytes migrate among other epidermal cells in the epibolic outgrowth of skin explants. This process is initiated by loss of contact inhibition of epidermal cells at the rim of the explants and by locally produced chemotactic factors. Punch biopsies provided explants of reproducible diameter. Optimal culture conditions include medium consisting of Dulbecco's Minimal Essential Medium containing 10% inactivated normal human serum and placement of explants epidermal side up at the air-liquid interphase. Within 7 days, epidermal cells completely surround the explant. Approximately 3 days after the onset of keratinocyte migration, melanocytes distribute themselves within the newly formed epidermis. Throughout the 7-day culture period, melanocytes and keratinocytes show maintenance of subcellular morphology, and the dermo-epidermal junction remains intact. Melanocyte migration was quantified using immunoperoxidase staining in combination with light microscopy and computer-aided image analysis. Preliminary results using the model system to compare migration in control and nonlesional vitiligo skin indicate that no inherent migration defect is responsible for impaired repigmentation of vitiligo lesions. The organotypic culture model system allows for investigations on melanocytes within their environment of autologous epidermal and dermal components, closely resembling in vivo circumstances in human skin.     

The effect of cyclic AMP on neuritic outgrowth in explant cultures of developing chick olfactory epithelium

The effect of cyclic AMP (cAMP) analogs and phosphodiesterase (PDE) inhibitors on neurite outgrowth was studied in explant cultures of olfactory neurons. Nasal pits from 5- or 6-day-old chick embryos were minced, explanted into culture dishes, and grown in a serum-free medium. One of the cyclic AMP analogs, dibutyryl cyclic AMP (dbcAMP) or 8-bromo-cyclic AMP (8-Br-cAMP), or one of the PDE inhibitors, theophylline or isobutylmethylxanthine (IBMX), was added to the culture medium. The explants were examined for neurite outgrowth after 2 days in vitro. Db-cAMP increased the number of explants expressing neurites by 25-35% over control cultures, whereas 8-Br-cAMP had essentially no effect at the same concentrations. Addition of dibutyryl cyclic GMP (dbcGMP) gave no increase in neurite outgrowth, thus indicating that the effect of enhancing neuritic growth is specific to cAMP and not cyclic nucleotides in general. The resulting increase in neurite outgrowth is due to the cyclic nucleotide component of dbcAMP, since both IBMX and theophylline, which elevate intracellular cAMP, also increased neurite outgrowth significantly. When forskolin was added to the culture medium, there was a trend to increased neurite outgrowth; this was significantly enhanced when a subthreshold concentration of theophylline was added in addition to the forskolin.     

Neural crest migration in 3D extracellular matrix utilizes laminin, fibronectin, or collagen

The trunk neural crest originates by transformation of dorsal neuroepithelial cells into mesenchymal cells that migrate into embryonic interstices. Fibronectin (FN) is thought to be essential for the process, although other extracellular matrix (ECM) molecules are potentially important. We have examined the ability of three dimensional (3D) ECM to promote crest formation in vitro. Neural tubes from stage 12 chick embryos were suspended within gelling solutions of either basement membrane (BM) components or rat tail collagen, and the extent of crest outgrowth was measured after 22 hr. Fetal calf serum inhibits outgrowth in both gels and was not used unless specified. Neither BM gel nor collagen gel contains fibronectin. Extensive crest migration occurs into the BM gel, whereas outgrowth is less in rat tail collagen. Addition of fibronectin or embryo extract (EE), which is rich in fibronectin, does not increase the extent of neural crest outgrowth in BM, which is already maximal, but does stimulate migration into collagen gel. Removal of FN from EE with gelatin-Sepharose does not remove the ability of EE to stimulate migration. Endogenous FN is localized by immunofluorescence to the basal surface of cultured neural tubes, but is not seen in the proximity of migrating neural crest cells. Addition of the FN cell-binding hexapeptide GRGDSP does not affect migration into either the BM gel or the collagen gel with EE, although it does block spreading on FN-coated plastic. Thus, although crest cells appear to use exogenous fibronectin to migrate on planar substrata in vitro, they can interact with 3D collagenous matrices in the absence of exogenous or endogenous fibronectin. In BM gels, the laminin cell-binding peptide, YIGSR, completely inhibits migration of crest away from the neural tube, suggesting that laminin is the migratory substratum. Indeed, laminin as well as collagen and fibronectin is present in the embryonic ECM. Thus, it is possible that ECM molecules in addition to or instead of fibronectin may serve as migratory substrata for neural crest in vivo.     

Ouabain binding to preimplantation rabbit blastocysts

Ciliary ganglia (CG) from 8-day-old chick embryos were cultured as explants on a highly adhesive collagen substratum in the presence of the ciliary neuronotrophic factor (CNTF). A remarkable correlation was found between the formation of an outgrowth of ganglionic nonneuronal cells and the timing and extent of neuritic development outside the ganglion. Neurites were not seen to emerge from the ganglion before the onset (24 hr after explantation) of a nonneuronal cell outgrowth. After nonneurons began to migrate over the collagen substratum, neurites could be seen to extend up to, but not beyond the distal limit of the nonneuronal outgrowth. Time-lapse analysis showed that neuritic growth cones could move in synchrony with a nonneuron with which they were in contact as well as over the nonneuronal cell surface, but not on the collagen located distally to the external edge of the nonneuronal outgrowth.Freshly dissected CGs were also grown as secondary explants on preformed host monolayers of ganglionic nonneurons. These secondary explants showed considerable neuritic development within 24 hr, while control ganglia explanted on collagen had not produced neurites. Autoradiographic experiments indicated that this neuritic outgrowth occurred on nonneuronal cells emerging precociously from the secondary explant, rather than on the preexisting host nonneurons. Electron microscopy of 24-hr explants demonstrated that, inside the ganglion, neurites were also very closely associated with the surface of nonneuronal cells.Neuritic behavior in this nonneuron/collagen terrain is compared with previously described observations of CG explants on polyornithine (PORN) or dissociated CG neurons on PORN or collagen. These observations led to the identification of a PORN-bindable neurite promoting factor (PNPF) which does not bind to, and is not active on, collagen. The hypothesis is discussed that PNPF molecules are present on the surface of nonneuronal cells and that the cells owe to those molecules their competence as a suitable terrain for the elongation of neuritic processes.     

Intrinsic properties inhibit axonal outgrowth from neonatal rat spinal cord explant

Lumbar spinal cord explants, harvested from neonatal rat pups aged between postnatal day 0 (P0) and P6, were cultured for a period of 48 hrs in the chemically defined medium R(12) on a poly-ethylene-imine (PEI) and on poly-D-lysin (PDL) coated surface. The outgrowth outside the explant was quantified. Lumbar explants from the same rat and embedded in a collagen matrix, and cortical explants from a P0 rat were used as controls. Statistical analysis demonstrated a clear relation between age-at-explantation and the number of neurites in the corona surrounding the explant. The number of outgrowing neurites decreased sharply with age-at-explantation. The average number of neurites per explant obeyed to the expression log (n) = -0.736x + 3.294 on PEI, and log (n) = -0.721x + 2.295 on PDL; x epsilon in [P0 - P6] (n, the number of neurites per explant; x, the age-at-explantation expressed in postnatal days). A similar observed age-related decrease of outgrowth has been described when culturing the lumbar explant inside a collagen matrix. The phenomenon appears to be an intrinsic property of the explant. We review growth inhibitory properties in different models and propose that the phenomenon occurs here at the interface explant-world.     

Epidermal growth factor and transforming growth factor alpha regulate extracellular matrix production by embryonic mouse palatal mesenchymal cells cultured on a variety of substrata

Mouse embryonic palatal mesenchymal (MEPM) cells were cultured either on plastic tissue culture dishes or on the surface of three-dimensional collagen gels or within collagen gel matrices in DMEM/F12 medium containing 2.5% donor calf serum. MEPM cells proliferated exponentially when cultured on collagen or on plastic. Cells cultured within collagen gels did not proliferate but remained viable. Addition of 10 ng/ml epidermal growth factor (EGF) or transforming growth factor alpha (TGF) stimulated the proliferation of those cells cultured on plastic or on collagen but not those cultured within collagen gels. Immunocytochemical analysis revealed that MEPM cells synthesise collagen types I, III, IV, V, VI and IX; fibronectin, heparan sulphate proteoglycan, laminin and tenascin in vitro. These molecules are all present in the developing palate in vivo. EGF and TGF produced a generalised stimulation of extracellular matrix (ECM) synthesis by MEPM cells in vitro. Biochemical analysis indicated that cells cultured within collagen gels had the highest intrinsic rate of protein synthesis. On all substrata neither EGF nor TGF markedly altered the types of ECM molecules synthesised but rather caused a general increase in the total amount produced. This stimulation was most marked where the cells were cultured within collagen gels. The lack of stimulation of proliferation of MEPM cells cultured within collagen gels (i.e. in a physiologically-relevant environment) by EGF or TGF together with the marked stimulation of ECM synthesis suggests that these factors may act as differentiation signals via their effects on ECM production. Correspondence to: M.J. Dixon     

Extracellular matrix (ECM) modulates the EGF-induced migration of liver epithelial cells in serum-free,hormone-supplemented medium

Summary The influence of the extracellular matrix (ECM) glycoproteins collagen, IV laminin (LN), and fibronectin (FN) on the in vitro migration of epithelial cells was studied using the ECM migration track method (4) with preparations immunostained for LN and FN. The locomotion of rat liver epithelial cells stimulated to migrate in serum-free medium by epidermal growth factor (EGF) in the presence of the protein per cm². Neither LN nor collagen IV decreased the number of migrating cells, indicating that the inhibition is a specific effect of fibronectin. The data also indicate that the FN-mediated inhibition of migration is an additional and not alternative mechanism to the well-established contact inhibition of locomotion (1) which also occurs in liver epithelial cell cultures. The system is being used for a further analysis of the factors that influence migration of normal and neoplastic epithelial cells and the biochemical mechanisms underlying the migration reaction. Editor’s Statement This paper describes new and heretofore neglected aspects of EGF and fibronectin action on the migratory behavior of cultured cells. Gordon H. Sato     

Elimination of epiplakin by gene targeting results in acceleration of keratinocyte migration in mice

Epiplakin (EPPK) was originally identified as a human epidermal autoantigen. To identify the function of epiplakin, we generated epiplakin "knockout" mice. These mice developed normally, with apparently normal epidermis and hair. Electron microscopy after immunostaining revealed the presence of EPPK adjacent to keratin filaments in wild-type mice, suggesting that epiplakin might associate with keratin. The appearance and localization of keratin bundles in intact epidermal keratinocytes of EPPK-/- mice were similar to those in wild-type mice. Wounds on the backs of EPPK-/- mice closed more rapidly than those on the backs of wild-type and heterozygous mice. The outgrowth of keratinocytes from skin explants from knockout mice was enhanced compared to outgrowth from explants from wild-type mice, even in the presence of mitomycin C, suggesting that the difference in keratinocyte outgrowth might be due to a difference in the speed of migration of keratinocytes. At wound edges in wild-type mice, EPPK was expressed in proliferating keratinocytes in conjunction with keratin 6. In EPPK-/- mice, no similar proliferating keratinocytes were observed, but migrating keratinocytes weakly expressed keratin 6. EPPK was coexpressed with keratin 6 in some keratinocytes in explant cultures from wild mice. We propose that EPPK might be linked functionally with keratin 6.     

The growth of Vero cells in suspension as cell-aggregates in serum-free media

Vero cell lines, usually considered anchorage-dependent, could be grown as cell-aggregates in suspension culture with serum-free media. Several different combinations of base media gave growth results above 10⁶ cells/ml (NCTC 135:SFRE 199-1; NCTC 135:Waymouth MB 752/1; NCTC 135:RPMI 1640). Insulin was not essential for growth and Bovine Serum Albumin could be diluted out of the media if linoleic acid was present. The size and density of the aggregates formed varied depending on the media used.     

Serum-free organ culture of suckling rat jejunum: Effect of regulatory hormones

Summary To facilitate the study of regulators of differentiation and proliferation of small intestinal epithelium in the suckling rat we have developed a serum-free organ culture system and used it to examine epithelial responsiveness to various regulatory hormones. These hormones included the insulin-like growth factors (IGFs) whose action can be blocked by binding proteins in serum. Jejunal explants from 5-day-old suckling rats maintained better brush border enzyme activity and better histology when cultured under hyperbaric conditions for 24 h in serum-free Dulbecco’s modified Eagle’s medium/F12 medium than in RPMI 1640 plus 10% fetal bovine serum. Tissue responsiveness to various regulatory hormones was then tested in the serum-free medium. Insulin had no significant effect on morphology, proliferation rate, or enzyme activity in 5-day explants after 24 h in culture. However, insulin did increase lactase activity and induce the early appearance of sucrase in 10- and 12-day explants after 48 h culture. Dexamethasone increased specific activities of alkaline phosphatase (30%,P<0.001) and lactase (15%,P<0.001), and reduced shedding of alkaline phosphatase into the medium (P<0.001), in explants of 5-day-old rats cultured over 24 h. Dexamethasone combined with insulin had no obvious effect on the rate of protein or DNA synthesis but did increase villus height (P=0.04) and crypt depth (P=0.001) and acted synergistically to further increase lactase activity above levels obtained by either alone. IGF-I and IGF-II, des-(1–3)IGF-I, fibroblast growth factor (FGF), and growth hormone (GH) had no effect on morphology or biochemical activity of explants after 24 or 48 h culture. In conclusion, histology, enzyme activity, protein, and DNA synthesis of suckling rat jejunal explants were equivalent or better in serum-free than in serum-containing organ culture systems. Furthermore, biological responsiveness was demonstrated by dexamethasone and insulin altering the explants morphologically or biochemically. None of the IGFs or GH had any biological effects, raising doubts about their direct biological action on the developing intestinal epithelium.     

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues¹ and survival factors² located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons³. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons⁴. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro^5,6.Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol⁷) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival⁸.     

Down-regulation of cell adhesion molecules LFA-1 and ICAM-1 after in vitro treatment with the anti-TNF-alpha agent thalidomide.

The tumor necrosis factor-alpha (TNF-alpha) inhibitor thalidomide is known to be a potent modulator of host immunity, a potential treatment for autoimmune disorders such as rheumatoid arthritis (RA) and a treatment for complications of HIV-1 infection. RA is an autoimmune disease of the joints that has been associated with hyperactivity of lymphocytes and other leukocytes, over-expression of pro-inflammatory cytokines (TNF-alpha and IL-1) and chronic debilitating inflammation. Thalidomide may play a role in RA treatment by altering leukocyte function through down-modulation of cell adhesion molecules necessary for leukocyte migration to inflammatory sites. The present study investigates down-regulation of cell adhesion molecules (ICAM-1 and LFA-1) and decreases in cell-cell contacts between human T leukemic (CEM) cells and human umbilical vein endothelial cells (HUVEC) after thalidomide exposure. CEM cells were cultured in RPMI 1640 medium with 0, 10 or 50 microg/ml thalidomide, stained with fluorescent monoclonal antibodies specific to ICAM-1 and LFA-1 and expression was measured with flow cytometry. For cell-cell adhesion measurements, monolayers of HUVEC cultured in Kaign's F-12 medium were incubated with thalidomide treated CEM cells stained with calcein AM. Specific cell adhesion between the two cell types was visualized with fluorescence microscopy. Thalidomide treatment significantly reduced cell adhesion molecule expression in a dose-dependent fashion and inhibited HUVEC/CEM cell adhesion. These data support the hypothesis that thalidomide has modulatory actions on leukocyte functions through expression of cell adhesion molecules.     

Effects of neurotransmitters upon the discharge of secretory product from the cutaneous glands of the red-spotted newt.

The effects of sympathetic and parasympathetic agonists and antagonists on discharge of secretory product by the granular and mucous glands were examined in the red-spotted newt, Notopthalmus viridescens viridescens. Observations were made also on the South African clawed toad. Xenopus laevis, the grass frog, Rana pipiens, and the crested newt, Triturus cristatus. In contrast to the granular glands of the South African clawed toad and the grass frog, which were stimulated by alpha-adrenergic agents, those of the red-spotted newt discharge in response to acetylcholine, either in vitro when added to the Holtfreter's solution in which explants were incubated, or in vivo when injected subcutaneously. Granular glands of the crested newt were also dischared in response to subcutaneous injection of acetylcholine. Stimulation of the granular glands by acetylcholine was blocked by atopine but not by tubocurarie, indicating that the cholinergic receptors are muscarinic rather than nicotinic. The mucous glands of the red-spotted newt, on the other hand, did not discharge in response to either acetylcholine or to adrenergic agents.     

Optimisation of media for the culture of normal human epithelial cells from oesophageal mucosa

A technique has been developed which allows growth and histology/cytochemistry of primary oesophageal mucosal explant cultures to be monitored over four weeks. The paper describes experiments designed to optimise media and culture conditions. The results suggest that optimal growth can be obtained in media containing RPMI 1640, and 10% horse or newborn calf serum. McCoy's 5A medium or Dulbecco's modified MEM could be substituted for RPMI but Iscove's serum-free medium or Ham's nutrient mixture inhibited growth or promoted fibroblast contamination. The essential additive appeared to be insulin while selenium was highly toxic to the cells. Hydrocortisone or EGF improved growth slightly under some conditions. Neither transferrin nor cholera toxin had any beneficial effect. None of the cell culture flask coating agents improved attachment or growth.     

The promoting effect of pentadecapeptide BPC 157 on tendon healing involves tendon outgrowth, cell survival, and cell migration

Pentadecapeptide BPC 157, composed of 15 amino acids, is a partial sequence of body protection compound (BPC) that is discovered in and isolated from human gastric juice. Experimentally it has been demonstrated to accelerate the healing of many different wounds, including transected rat Achilles tendon. This study was designed to investigate the potential mechanism of BPC 157 to enhance healing of injured tendon. The outgrowth of tendon fibroblasts from tendon explants cultured with or without BPC 157 was examined. Results showed that BPC 157 significantly accelerated the outgrowth of tendon explants. Cell proliferation of cultured tendon fibroblasts derived from rat Achilles tendon was not directly affected by BPC 157 as evaluated by MTT assay. However, the survival of BPC 157-treated cells was significantly increased under the H(2)O(2) stress. BPC 157 markedly increased the in vitro migration of tendon fibroblasts in a dose-dependent manner as revealed by transwell filter migration assay. BPC 157 also dose dependently accelerated the spreading of tendon fibroblasts on culture dishes. The F-actin formation as detected by FITC-phalloidin staining was induced in BPC 157-treated fibroblasts. The protein expression and activation of FAK and paxillin were determined by Western blot analysis, and the phosphorylation levels of both FAK and paxillin were dose dependently increased by BPC 157 while the total amounts of protein was unaltered. In conclusion, BPC 157 promotes the ex vivo outgrowth of tendon fibroblasts from tendon explants, cell survival under stress, and the in vitro migration of tendon fibroblasts, which is likely mediated by the activation of the FAK-paxillin pathway.     

Collagen modulates gene activation of plasminogen activator system molecules

Skin extracellular matrix (ECM) molecules regulate a variety of cellular activities, including cell movement, which are central to wound healing and metastasis. Regulated cell movement is modulated by proteases and their associated molecules, including the serine proteases urinary-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) and their inhibitors (PAIs). As a result of wounding and loss of basement membrane structure, epidermal keratinocytes can become exposed to collagen. To test the hypothesis that during wounding, exposed collagen, the most abundant ECM molecule in the skin, regulates keratinocyte PA and PAI gene expression, we utilized an in vitro model in which activated keratinocytes were cultured in dishes coated with collagen or other ECM substrates. tPA, uPA, and PAI-1 mRNA and enzymatic activity were detected when activated keratinocytes attached to fibronectin, vitronectin, collagen IV, and RGD peptide. In contrast, adhesion to collagen I and collagen III completely suppressed expression of PAI-1 mRNA and protein and further increased tPA expression and activity. Similarly, keratinocyte adhesion to laminin-1 suppressed PAI-1 mRNA and protein expression and increased tPA activity. The suppressive effect of collagen I on PAI-1 gene induction was dependent on the maintenance of its native fibrillar structure. Thus, it would appear that collagen- and laminin-regulated gene expression of molecules associated with plasminogen activation provides an additional dimension in the regulation of cell movement and matrix remodeling in skin wound healing.     

Differentiation of pure chick embryo epidermis grown in primary serum-free culture

The differentiation of precocious embryonic epidermis in serum-free primary culture was analyzed by light and electron microscopic methods. Explants of 7-day chick embryo epidermis were grown on collagen or poly-L-lysine substrates in the absence of dermal mesenchyme. The serum substitute consisted of a mixture of insulin, transferrin, putrescine and seleneous acid together with (or without) Nerve Growth Factor. These culture conditions were shown to support proliferation, growth and development (evaluated using morphological criteria) of the epidermal explants up to 4-5 days; during this period, the epidermis underwent stratification; well-developed desmosomes as well as tonofilaments were formed and the epidermis achieved a morphology close to that of 10-11 day epidermis in ovo. However long-term survival of the explants was not obtained as cellular death, starting on day 5, progressively led to the necrosis of most parts of the explant. This morphological study demonstrates that the early phases of epidermal growth and maturation can occur to some extent in the virtual absence of dermal elements and serum factors. Chick embryo epidermal cells may thus possess the intrinsic ability to go through, at least for short periods in vitro, their differentiation programme. Then, at the onset of epidermal keratinization (12 days in ovo), they require specific exogenous factors to fully differentiate in vitro.