Extracellular matrix components and somite chondrogenesis: A microscopic analysis

The stimulation of somite chondrogenesis by extracellular materials was studied using scanning and transmission electron microscopy and light microscopy. Analysis of control somite explants (no additives to the medium) cultured on Nuclepore filters for 24 h demonstrates cell processes extending to the undersurface of the filter. The cell processes secrete a matrix of fibers sparsely coated with granules which form amorphous sheets after 3 days in culture. Somite explants treated with proteoglycan complex, extracted from 13-day chick sterna, produce a dense matrix of fibers heavily coated with granules. Selective enzymatic digestions with chondroitinase ABC and purified collagenase demonstrate that the fibers are collagen and the granules are proteoglycans. Proteoglycan complex was separated into its components using cesium chloride density centrifugation. Each of these fractions was tested for its stimulating capacity in somite explants as analyzed using scanning electron microscopy. The importance of these components in relationship to the perinotochordal materials is discussed. When somite explants are cultured with the notochord, the matrix produced by somitic cells in the region of the notochord is similar to that of explants treated with proteoglycan complex. Away from the region of the notochord, the somitic cells produce a matrix similar to that of control explants. The evidence presented in this report suggests that it is the presence of the perinotochordal materials which creates the proper environment in vivo for the precise timing and phenotypic expression of somite chondrogenesis.     

Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts.

Transforming growth factor beta (TGF-beta) enhances the cell surface binding of 125I-fibronectin by cultured human fibroblasts. The effect of TGF-beta on cell surface binding was maximal after 2 h of exposure to TFG-beta and did not require epidermal growth factor or protein synthesis. The enhancement was dose dependent and was found with the 125I-labeled 70-kilodalton amino-terminal fragment of fibronectin as well as with 125I-fibronectin. Treatment of cultures with TGF-beta for 6 h resulted in a threefold increase in the estimated number of fibronectin binding sites. The increase in number of binding sites was accompanied by an increased accumulation of labeled fibronectin in detergent-insoluble extracellular matrix. The effect of TGF-beta was biphasic; after 6 h of exposure, less labeled fibronectin bound to treated cultures than to control cultures. Exposure of cells to TGF-beta for greater than 6 h caused a two- to threefold increase in the accumulation of cellular fibronectin in culture medium as detected by a quantitative enzyme-linked immunosorbent assay. The second phase of the biphasic effect and the increase in soluble cellular fibronectin were blocked by cycloheximide. Immunofluorescence staining of fibroblast cultures with antifibronectin revealed that TGF-beta caused a striking increase in fibronectin fibrils. The 70-kilodalton amino-terminal fragment of fibronectin, which blocks incorporation of fibronectin into extracellular matrix, blocked anchorage-independent growth of NRK-49F cells in the presence of epidermal growth factor. Our results show that an increase in the binding and rate of assembly of exogenous fibronectin is an early event preceding the increase in expression of extracellular matrix proteins. Such an early increase in cell surface binding of exogenous fibronectin may be a mechanism whereby TGF-beta can modify extracellular matrix characteristics rapidly after tissue injury or during embryonic morphogenesis.     

Long-term culture of renin containing tissue

Thin cortical tissue explants from kidneys of hydronephrotic mice were excised and incubated in different culture media containing growth and proliferation factors. Over a period of several months the content of renin in the explants and in the culture medium was repeatedly measured, to define the conditions necessary for the maintenance of renin production in a long-term culture. The best results were obtained when culturing the renal tissue in Dulbecco's medium (DMEM) with 10% fetal calf serum, 6 units/100 ml platelet-derived growth factor and 200 ng/ml glycylhistidyllysine. Renin was still present within the cells and in the culture medium after more than six months. Prevention of dedifferentiation, as evidenced in this case by the maintenance of renin production, seemed to be dependent on specific extracellular matrix proteins of renal origin. If the explants were dissociated from their matrix components by collagenase, a gradual loss of renin production was observed within 5 days. Complementation of the collagenase-digested cell suspension with different nonrenal extracellular matrix materials did not afford the stabilizing effect of the original pericellular matrix.     

Role of calcium in the insulin-dependent stimulation of DNA synthesis in mouse mammary gland in vitro

The role of extracellular Ca2+ in the control of DNA synthesis in mouse mammary tissue was studied using mammary gland explants maintained under chemically defined conditions in vitro. Chelation of calcium with ethyleneglycol-bis-(beta-aminoethyl ether) or omission of Ca2+ from the incubation media substantially reduced both basal and insulin-stimulated incorporation of [3H]thymidine into DNA. Addition of calcium to the Ca2+-deficient media restored DNA synthesis; other divalent cations could not be substituted for calcium. Insulin reduced by 5-fold the calcium concentration required to achieve half-maximal stimulation of DNA synthesis in explants, thus indicating that the Ca2+-related process may be involved in the mechanism by which insulin exerts its effect on cell multiplication. Evidence is presented that in mammary gland explants, calcium does not stimulate DNA synthesis by action on the thymidine pool size. Neither calcium nor insulin showed any effect on the activity of thymidine kinase in the mammary gland explants. On the other hand, calcium ions were shown to be necessary to maintain the activity of DNA polymerase-alpha, the enzyme involved in nuclear DNA replication.     

Long-term culture of renin containing tissue

Summary Thin cortical tissue explants from kidneys of hydronephrotic mice were excised and incubated in different culture media containing growth and proliferation factors. Over a period of several months the content of renin in the explants and in the culture medium was repeatedly measured, to define the conditions necessary for the maintenance of renin production in a long-term culture.The best results were obtained when culturing the renal tissue in Dulbecco's medium (DMEM) with 10% fetal calf serum, 6 units/100 ml platelet-derived growth factor and 200 ng/ml glycylhistidyllysine. Renin was still present within the cells and in the culture medium after more than six months. Prevention of dedifferentiation, as evidenced in this case by the maintenance of renin production, seemed to be dependent on specific extracellular matrix proteins of renal origin. If the explants were dissociated from their matrix components by collagenase, a gradual loss of renin production was observed within 5 days. Complementation of the collagenase-digested cell suspension with different nonrenal extracellular matrix materials did not afford the stabilizing effect of the original pericellular matrix.These studies were supported by the Deutsche Forschungsgemeinschaft-SFB 90-Cardiovasculäres System     

Protective effects of polydeoxyribonucleotides on cartilage degradation in experimental cultures

The capacity of cartilage self‐regeneration is considered to be limited. Joint injuries often evolve in the development of chronic wounds on the cartilage surface. Such lesions are associated with articular cartilage degeneration and osteoarthritis. Re‐establishing a correct micro/macro‐environment into damaged joints could stop or prevent the degenerative processes. This study investigated the effect of polydeoxyribonucleotides (PDRNs) on cartilage degradation in vitro and on cartilage extracted cells. The activities of matrix metalloproteinases 2 and 9 were measured in PDRN‐treated cells and in controls at days 0 and 30 of culture. Human nasal cartilage explants were cultured, and the degree of proteoglycan degradation was assessed by measuring the amount of glycosaminoglycans released into the culture medium. The PDRN properties compared with controls were tested on cartilage tissues to evaluate deposition of extracellular matrix. Chondrocytes treated with PDRNs showed a physiological deposition of extracellular matrix (aggrecan and type II collagen: Western blot, IFA, fluorescence activated cell sorting, Alcian blue and safranin O staining). PDRNs were able to inhibit proteoglycan degradation in cartilage explants. The activities of matrix metalloproteinases 2 and 9 were reduced in all PDRN‐treated samples. Our results indicate that PDRNs are suitable for a long‐term cultivation of in vitro cartilage and have therapeutic effects on chondrocytes by protecting cartilage. Copyright © 2012 John Wiley & Sons, Ltd.     

Thalidomide may inhibit proliferation of mesenchyme in human limb buds

Limb buds from 4- and 4.5-week-old human embryos were cultured on agar medium consisting of Medium 199, chick embryo extract and horse serum for 4 days with or without thalidomide (1-1.5 microgram/ml), and the direct effect of thalidomide was examined morphologically in histological preparations. In the explants treated with thalidomide, mitotic figures of mesenchymal cells were significantly decreased both in overall explant and in mesenchymal cell aggregates, but the extracellular matrix in the mesenchymal cell aggregates was seen in the experimental and control explants. These findings suggest that thalidomide affects undifferentiated and differentiated mesenchymal cell proliferation but not the chondrogenic capacity of the mesenchyme.     

Directional mesoderm cell migration in the Xenopus gastrula.

The movement of the dorsal mesoderm across the blastocoel roof of the Xenopus gastrula is examined. We show that different parts of the mesoderm which can be distinguished by their morphogenetic behavior in the embryo are all able to migrate independently on the inner surface of the blastocoel roof. The direction of mesoderm cell migration is determined by guidance cues in the extracellular matrix of the blastocoel roof and by an intrinsic tissue polarity of the mesoderm. The mesodermal polarity shows the same orientation as the external guidance cues and is strongly expressed in the more posterior mesoderm. The guidance cues of the extracellular matrix are recognized by all parts of the dorsal mesoderm and even by nonmesodermal cells from other regions of the embryo. The extracellular matrix consists of a network of fibronectin-containing fibrils. The adhesiveness of this matrix does not vary along the axis of mesoderm movement, excluding haptotaxis as a guidance mechanism in this system. However, an intact fibronectin fibril structure is necessary for directional mesoderm cell migration. When the assembly of fibronectin into fibrils is inhibited, mesoderm explants still migrate on the amorphous extracellular matrix, but no longer directionally. It is proposed that polarized extracellular matrix fibrils may normally guide the migrating mesoderm to its target region.     

Stimulation of collagen production in vitro by ascorbic acid released from explants of migrating avian neural crest

Embryonic neuronal tissues contain a collagen-stimulating factor, shown to enhance the hydroxylation and secretion of proline-containing macromolecules by cultured muscle cells. Here we report on a similar activity found during avian embryonic development in explants of migrating mesencephalic neural crest. The degree of proline hydroxylation of proteins secreted into the medium was stimulated 2.5-6-fold in neural crest-muscle and neural crest-somite cocultures, as compared with control cultures devoid of crest explants. No such stimulation occurred when cocultures were treated with the enzyme ascorbate oxidase (EC 1.10.3.3), suggesting that the active factor in neural crest explants was ascorbic acid or an ascorbate-like molecule. Further characterization of this molecule was performed in crest explants and other embryonic tissues by using HPLC with amperometric detection: this study revealed that migrating cephalic neural crest contains 1.5 micrograms ascorbic acid per mg protein. Our results suggest that ascorbic acid and/or related molecule(s) could act during development of the nervous system as a trigger for collagen production and subsequent assembly of an extracellular matrix.     

Heparin Prevents Intracellular Hyaluronan Synthesis and Autophagy Responses in Hyperglycemic Dividing Mesangial Cells and Activates Synthesis of an Extensive Extracellular Monocyte-adhesive Hyaluronan Matrix after Completing Cell Division

Growth-arrested rat mesangial cells (RMCs) at a G₀/G₁ interphase stimulated to divide in hyperglycemic medium initiate intracellular hyaluronan synthesis that induces autophagy/cyclin D3-induced formation of a monocyte-adhesive extracellular hyaluronan matrix after completing cell division. This study shows that heparin inhibits the intracellular hyaluronan synthesis and autophagy responses, but at the end of cell division it induces synthesis of a much larger extracellular monocyte-adhesive hyaluronan matrix. Heparin bound to RMC surfaces by 1 h, internalizes into the Golgi/endoplasmic reticulum region by 2 h, and was nearly gone by 4 h. Treatment by heparin for only the first 4 h was sufficient for its function. Streptozotocin diabetic rats treated daily with heparin showed similar results. Glomeruli in sections of diabetic kidneys showed extensive accumulation of autophagic RMCs, increased hyaluronan matrix, and influx of macrophages over 6 weeks. Hyaluronan staining in the glomeruli of heparin-treated diabetic rats was very high at week 1 and decreased to near control level by 6 weeks without any RMC autophagy. However, the influx of macrophages by 6 weeks was as pronounced as in diabetic glomeruli. The results are as follows: 1) heparin blocks synthesis of hyaluronan in intracellular compartments, which prevents the autophagy and cyclin D3 responses thereby allowing RMCs to complete cell division and sustain function; 2) interaction of heparin with RMCs in early G₁ phase is sufficient to induce signaling pathway(s) for its functions; and 3) influxed macrophages effectively remove the hyaluronan matrix without inducing pro-fibrotic responses that lead to nephropathy and proteinurea in diabetic kidneys.     

Activity-dependent formation and functions of chondroitin sulfate-rich extracellular matrix of perineuronal nets

Extracellular matrix molecules--including chondroitin sulfate proteoglycans, hyaluronan, and tenascin-R--are enriched in perineuronal nets (PNs) associated with subsets of neurons in the brain and spinal cord. In the present study, we show that similar cell type-dependent extracellular matrix aggregates are formed in dissociated cell cultures prepared from early postnatal mouse hippocampus. Starting from the 5th day in culture, accumulations of lattice-like extracellular structures labeled with Wisteria floribunda agglutinin were detected at the cell surface of parvalbumin-expressing interneurons, which developed after 2-3 weeks into conspicuous PNs localized around synaptic contacts at somata and proximal dendrites, as well as around axon initial segments. Physiological recording and intracellular labeling of PN-expressing neurons revealed that these are large fast-spiking interneurons with morphological characteristics of basket cells. To study mechanisms of activity-dependent formation of PNs, we performed pharmacological analysis and found that blockade of action potentials, transmitter release, Ca2+ permeable AMPA subtype of glutamate receptors or L-type Ca2+ voltage-gated channels strongly decreased the extracellular accumulation of PN components in cultured neurons. Thus, we suggest that Ca2+ influx via AMPA receptors and L-type channels is necessary for activity-dependent formation of PNs. To study functions of chondroitin sulfate-rich PNs, we treated cultures with chondroitinase ABC that resulted in a prominent reduction of several major PN components. Removal of PNs did not affect the number and distribution of perisomatic GABAergic contacts but increased the excitability of interneurons in cultures, implicating the extracellular matrix of PNs in regulation of interneuronal activity.     

Inhibition of "spontaneous," notochord-induced, and collagen-induced in vitro somite chondrogenesis by the calcium lonophore, A23187.

The present study represents a first step in investigating the possible involvement of calcium (Ca2+) in the stimulation of somite chondrogenesis elicited by extracellular matrix components produced by the embryonic notochord. The ionophore, A23187, a drug that facilitates Ca2+ uptake leading to elevation of cytoplasmic Ca2+ levels, at concentrations of 0.25-1.0 microgram/ml severely impairs "spontaneous" somite chondrogenesis, i.e., inhibits the formation of the small amount of cartilaginous matrix normally formed by embryonic somites in vitro in the absence of inducing tissues. This inhibition is reflected in a considerable reduction in sulfated glycosaminoglycan (GAG) accumulation by A23187-treated somite explants. Furthermore, A23187 inhibits the striking stimulation of cartilaginous matrix formation and sulfated GAG accumulation normally elicited by the embryonic notochord and collagen substrates. In fact, 1.0 microgram/ml of A23187 reduces sulfated GAG accumulation by somites cultured in association with notochord or on collagen to a level even below that accumulated by somites cultured in the absence of these inductive agents. Although these results must be interpreted with caution, they provide incentive for considering a possible regulatory role for Ca2+ in the chondrogenic response of somites to extracellular matrix components produced by the embryonic notochord.     

Embryonic induction and cation concentrations in amphibian embryos

Explanted ectoderm from early gastrulae of Triturus alpestris was treated with the Na-K ionophore gramicidin (10(-9) to 10(-5) M) and the Ca-ionophore A 23187 (10(-7) to 10(-5) M). The ectoderm developed almost exclusively to atypical epidermis as in the control explants. When the ectoderm was treated with ouabain (10(-4) M), intracellular Na+ increased about 4.4-fold and K+ was reduced by half. Mesenchyme cells in small number differentiated in about 40% of the ouabain-treated explants. The time course of total Na+ and K+ ion concentrations was measured over a period of 72 h in ectoderm of T. alpestris after induction with vegetalizing factor and in control explants. In the first 15 h after explantation, no significant differences between control and induced explants were found. Thereafter, the steady state concentration of K+ decreased in the induced explants, whereas the steady-state concentration of Na+ slightly increased. The membrane resting potential recorded intracellularly of ectoderm sandwiches from early gastrula stages was found to be -41.3 mV in control and -59.3 mV in induced explants. From the specific conductances and permeabilities of non-induced and induced cells it is concluded that the induction process leads to a differentiation of the cell membrane, which acquires the characteristics of ionic selectivity. Ectoderm from Ambystoma mexicanum forms neural or neuroid tissue, mesenchyme and melanophores after explantation in salt solution in up to 50% of the explants without any additions. Isolated Ambystoma ectoderm is therefore not suitable for test experiments.     

Extracellular ATP induces cell death in human intestinal epithelial cells

Background

Extracellular ATP is an endogenous signaling molecule released by various cell types and under different stimuli. High concentrations of ATP released into the extracellular medium activate the P2X7 receptor in most inflammatory conditions. Here, we seek to characterize the effects of ATP in human intestinal epithelial cells and to evaluate morphological changes in these cells in the presence of ATP.

Methods

We treated human intestinal epithelial cells with ATP and evaluated the effects of this nucleotide by scanning and transmission electron microscopy analysis and calcium measurements. We used flow cytometry to evaluate apoptosis. We collected human intestinal explants for immunohistochemistry, apoptosis by the TUNEL approach and caspase-3 activity using flow cytometry analyses. We also evaluated the ROS production by flow cytometry and NO secretion by the Griess technique.

Results

ATP treatment induced changes characteristic of cell death by apoptosis and autophagy but not necrosis in the HCT8 cell line. ATP induced apoptosis in human intestinal explants that showed TUNEL-positive cells in the epithelium and in the lamina propria. The explants exhibited a significant increase of caspase-3 activity when the colonic epithelial cells were incubated with IFN-gamma followed by ATP as compared to control cells. In addition, it was found that antioxidants were able to inhibit both the ROS production and the apoptosis induced by ATP in epithelial cells.

General significance

The activation of P2X7 receptors by ATP induces apoptosis and autophagy in human epithelial cells, possibly via ROS production, and this effect might have implications for gut inflammatory conditions.     

重组PAI－2对肿瘤细胞降解细胞外基质的影响

重组ＰＡＩ－２对肿瘤细胞降解细胞外基质的影响曹祥荣（南京师范大学生物系,２１００２４）关键词纤溶酶原激活剂抑制剂－２,基因重组,细胞外基质降解肿瘤细胞转移过程中最重要的因素是肿瘤细胞侵袭能力,其生化过程即为细胞外基质（ＥＯＭ）降解作用。纤溶酶系统（纤...     

Effect of a short-term disturbance of the cell contacts on the mesodermal differentiation of clawed toad embryos

The differentiation of the presumptive mesoderm explants from different sectors of the early gastrula marginal zone was compared with that of the identical explants placed just after explantation into a medium free of divalent cations for 30 s. The development of the treated dorsal explants differed from that of control explants by the presence of well differentiated forebrain structures and the development of the explants from more ventral zones by a decrease in the occurrence of blood cells and nondifferentiated endoderm and an increase in occurrence of epithelioid structures, which form frequently deep invaginations. The shape of the treated explants was more organized and differentiated than that of the control explants. A conclusion has been reached that the development of epithelioid and forebrain structures in the explants is stimulated after a short-term disturbance of cell contacts.     

Cell surface changes of the presumptive ectoderm following neural-inducing treatment by concanavalin A

Summary Scanning electron microscopic studies revealed that Concanavalin A (ConA) induces characteristic changes of the cell surface and the cell architecture of the presumptive ectoderm associated with differentiation into neural tissues. In Con A-treated cells, the filopodia with which cells were connected to each other disappeared from the interior (blastocoelic) surface and the cellular adhesivity decreased significantly. Thereafter, the cells underwent from those of the control explants. After cultivation for 60 h, a certain pattern of cell arrangement, which resembled the architecture of neural tissues, was observed among randomly arranged cells in the explants treated with Con A. The morphological changes specifically observed in Con A-treated explants were different from those found in explants treated with succinyl Con A (S-Con A) orDolichos biflorus agglutinin (DBA), which is unable to induce formation of the neural tissues. The molecular organization of the plasma membrane appears to be important in the mechanism of neural induction.