Chorioallantoic placenta formation in the rat: II. Angiogenesis and maternal blood circulation in the mesometrial region of the implantation chamber prior to placenta formation.

Rat gestation sites were examined on days 7 through 9 of pregnancy by light microscopy and transmission and scanning electron microscopy to determine the extent of vascular modifications in the vicinity of the mesometrial part of the implantation chamber (mesometrial chamber). At a later time, the mesometrial chamber is, in conjunction with the uterine lumen, the site of chorioallantoic placenta formation. On day 7, in the vicinity of the mesometrial chamber, vessels derived from a subepithelial capillary plexus and venules draining the plexus were dilating. By early day 8, this network of thin-walled dilated vessels (sinusoids) was further enlarged and consisted primarily of hypertrophied endothelial cells with indistinct basal laminas. Sinusoids were frequently close to the mesometrial chamber's luminal surface which was devoid of epithelial cells but was lined by decidual cell processes and extracellular matrix. By late day 8, cytoplasmic projections of endothelial cells extended between healthy-appearing decidual cells and out onto the mesometrial chamber's luminal surface, and endothelial cells were sometimes found on the luminal surface indicating that endothelial cells were migrating. The presence of maternal blood cells in the mesometrial chamber lumen suggested that there was continuity between the chamber and blood-vessel lumens. On day 9, the mesometrial chamber was completely lined with hypertrophied endothelial cells, and sinusoid lumens were clearly continuous with the lumen of the mesometrial chamber. Mesometrial sinusoids and possibly the mesometrial chamber lumen were continuous with vessels in vicinity of the uterine lumen that were fed by mesometrial arterial vessels. Clearing of the mesometrial chamber lumen during perfusion fixation via the maternal vasculature indicated the patency of this luminal space and its confluence with mesometrial arterial vessels and sinusoids. The conceptus occupied an antimesometrial position in the implantation chamber on days 7 through 9, and it was not in direct contact with uterine tissues in the vicinity of the mesometrial chamber. These observations suggest that angiogenesis, not trophoblast invasion or decidual cell death, plays a major role in the opening of maternal vessels into the mesometrial chamber lumen before the formation of the chorioallantoic placenta.     

Immunomorphological research on the formation of the extracellular matrix in epithelial cell cultures

Formation of extracellular matrix structures in cultures of rat liver epithelial nontransformed cell line IAR2 was studied with antisera to fibronectin, laminin and type IV collagen by immunofluorescence and immunoelectron microscopy of platinum replicas. Fibronectin formed peripheral spots of variable size some of which outlined free cell edges, as well as fibrils located towards the center of single cells or of cellular islands. Similarly distributed structures were seen in isolated matrices. Codistribution of fibronectin and actin was observed only for the peripheral line of fibronectin spots and marginal circular actin bundle. Basement membrane components. laminin and type IV collagen, formed mainly spots of variable size predominantly beneath the cell or each cell in an island. Occasional fibrils were seen also. Essentially the same results were obtained by immunofluorescence and immunogold electron microscopy. Cytochalasin D treated cells displayed spots of both fibronectin and laminin. The relevance of previously postulated receptor-mediated assembly of extracellular matrix structures to the epithelial cells is discussed.     

Transforming growth factor-beta induces cytoskeleton and extracellular matrix modifications in FRTL-5 thyroid epithelial cells

The action of transforming growth factor-beta (TGF-beta) on the morphology, cytoskeleton and extracellular matrix was investigated in FRTL-5 thyroid epithelial cells. After treatment with TGF-beta, FRTL-5 cells became flat and developed straight and thick bundles of actin microfilaments. This effect of TGF-beta was observed even in the presence of thyrotropin, which has a strong microfilament disrupting action. TGF-beta also influenced some aspects of the extracellular matrix organization. Immunofluorescence staining of FRTL-5 cells revealed both the appearance of a fibrillar array of fibronectin in association with the basal plasma membrane and a change in the morphology of basally located laminin patches. TGF-beta induced the formation of adhesion structures at the ventral portion of the cell membrane. Vinculin was focally concentrated at the end of stress fibers in areas corresponding to focal adhesions as revealed by interference reflection microscopy (IRM). The ability to modulate cytoskeleton organization and extracellular matrix protein distribution might mediate some of the reported TGF-beta effects on the expression of specific functional properties in thyroid cells.     

Three-dimensional modeling of mechanical forces in the extracellular matrix during epithelial lumen formation

Mechanical interactions between cells and extracellular matrix (ECM) mediate epithelial cyst formation. This work relies on the combination of numerical modeling with live cell imaging, to piece together a novel nonintrusive method for determining three-dimensional (3D) mechanical forces caused by shape changes of a multicellular aggregate at the early stages of epithelial cyst formation. We analyzed the evolution of Madin-Darby canine kidney cells in 3D cultures using time-lapse microscopy, with type I collagen gel forming the ECM. The evolving 3D interface between the ECM and the cell aggregate was obtained from microscopy images, and the stress on the surface of a proliferating aggregate and in the surrounding ECM was calculated using the finite element method. The viscoelastic properties of the ECM (a needed input for the finite element method solver) were obtained through oscillatory shear flow experiments on a rheometer. For validation purpose, the forces exerted by an aggregate on a force-sensor array were measured and compared against the computational results.     

Identification of components of the extracellular matrix in trophoblastic cell columns of human placenta]

The distribution of five components of the extracellular matrix was studied in human placenta (9-12 and 39-40 weeks of gestation) by an indirect immunofluorescence method with polyclonal monospecific antibodies. In trophoblastic cell columns fibronectin, collagen types IV and V formed homogeneous deposits, whereas collagen types I and II comprised small conglomerates and scanty, discrete granules. The origin of these macromolecules was discussed.     

Role of the extracellular matrix in epithelial morphogenesis

The extracellular matrix (ECM) plays an essential role in organizing tissues, defining their shapes or in presenting growth factors. Their components have been well described in most species, but our understanding of the mechanisms that control ECM remodeling remains limited. Likewise, how the ECM contributes to cellular mechanical responses has been examined in few cases. Here, I review how studies performed in C. elegans have brought several significant advances on those topics. Focusing only on epithelial cells, I discuss basement membrane invasion by the anchor cell during vulva morphogenesis, a process that has greatly expanded our knowledge of ECM remodeling in vivo. I then discuss the ECM role in a novel mechanotransduction process, whereby muscle contractions stimulate the remodeling of hemidesmosome-like junctions in the epidermis, which highlights that these junctions are mechanosensitive. Finally, I discuss progress in defining the composition and potential roles of the apical ECM covering epidermal cells in embryos.     

Biphasic effect of the mitotoxin bFGF-saporin on bovine lens epithelial cell growth: effect of cell density and extracellular matrix.

We have studied the effect of a specific FGF receptor suicide antagonist on the growth of bovine epithelial cells (BEL cells) in culture. This basic fibroblast growth factor-saporin conjugate (bFGF-SAP) has a biphasic effect on bovine lens epithelial cells (BEL cells). Whereas 0.01 nM and 0.1 nM bFGF-SAP stimulate BEL cells proliferation, 1 nM and 10 nM bFGF-SAP have the predicted toxic effects on BEL cell growth. The toxicity of bFGF-SAP is observed 2 to 3 days after the initial treatment and depends on cell density. Accordingly, the sensitivity of confluent cells to bFGF-SAP is reduced compared to sparse cells. A time course analysis reveals that bFGF-SAP is effective after a short exposure to cells and that its effects are not increased with longer treatments. Cell growth on bFGF-SAP pretreated extracellular matrix (ECM) or posterior lens capsule (PLC) is also affected. Basic FGF-SAP has been shown to bind to the extracellular material, allowing a modulation of lens cells migration and survival by a single treatment in vitro. This finding raises the possibility of its use in vivo to prevent capsules invasion by lens cells after cataract surgery.     

Casein gene expression in mouse mammary epithelial cell lines: dependence upon extracellular matrix and cell type

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D line. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of beta-casein mRNA in the presence or absence of prolactin. The heterogeneous COMMA-D line, but none of the clonal lines, was induced by the presence of prolactin to produce significantly increased levels of beta-casein MRNA. The inducibility of beta-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. Individual matrix components of laminin, fibronectin, heparan sulfate, heparan, or hyaluronic acid were not effective as substrata for the induction of beta-casein mRNA. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.     

Mouse submandibular salivary epithelial cell growth and differentiation in long-term culture: Influence of the extracellular matrix

Summary The adult mouse submandibular salivary gland provides a good model system to study gene regulation during normal and abnormal cell behavior because it synthesizes functionally distinct products ranging from growth factors and digestive enzymes to factors of relevance to homeostatic mechanisms. The present study describes the long-term growth and differentiation of submandibular salivary epithelial cells from adult male mice as a function of the culture substratum. Using a two-step partial dissociation procedure, it was possible to enrich for ductal cells of the granular convoluted tubules, the site of epidermal growth factor synthesis. Long-term cell growth over a period of 2 to 3 mo. with at least 3 serial passages was obtained only within three-dimensional collagen gels. Cells grew as ductal-type structures, many of which generated lumens with time in culture. Electron microscopic analysis in reference to the submandibular gland in vivo revealed enrichment for and maintenance of morphologic features of granular convoluted tubule cells. Reactivity with a keratin-specific monoclonal antibody established the epithelial nature of the cells that grew within collagen. Maintenance of cell differentiation, using immunoreactivity for epidermal growth factor as criterion, was determined by both cytochemical and biochemical approaches and was found to be dependent on the collagen matrix and hormones. Greater than 50% of the cells in primary collagen cultures contained epidermal growth factor only in the presence of testosterone and triiodothyronine. In contrast, cells initially seeded on plastic or cycled to plastic from collagen gels were virtually negative for epidermal growth factor. Biochemical analysis confirmed the presence of a protein with an apparent molecular weight of 6000 which comigrated with purified mouse epidermal growth factor. Epidermal growth factor was also present in detectable levels in Passage 1 cells. This culture system should permit assessment of whether modulation of submandibular gland ductal cell growth can be exerted via a mechanism that in itself includes epidermal growth factor and its receptor and signal transduction pathway. This work was supported by Public Health Service grant DE07766 from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD.     

The regulation of cancer cell death and metabolism by extracellular matrix attachment

The metastasis of cancer cells to distant sites is responsible for the vast majority of cancer mortalities yet the molecular mechanisms underlying this extraordinarily complicated process have yet to be sufficiently elucidated. Recently, it has become clear that cancer cells need to inhibit anoikis, a cell death program induced by loss of attachment to the extracellular matrix (ECM), in order to successfully metastasize. These studies have motivated additional research into the relationship between ECM-detachment and cell viability, much of which reveals integral connections between ECM-detachment and cell metabolism. This review serves to thoroughly discuss the signaling pathways and metabolic changes that are induced by ECM-detachment. In addition, the molecular mechanisms by which cancer cells can alter signaling and metabolism to survive in the absence of ECM-attachment will be highlighted. Furthermore, cell death mechanisms that have been observed or implicated in cells detached from the ECM will also be examined. In aggregate, the studies discussed in this review reveal that ECM-detachment can regulate cancer cell metabolism in a variety of distinct cell types and suggest that interfering with metabolism in ECM-detached cells may be a novel and effective chemotherapeutic approach to selectively inhibit tumor progression.     

Mammary epithelial cell: influence of extracellular matrix composition and organization during development and tumorigenesis

Stromal-epithelial interactions regulate mammary gland development and are critical for the maintenance of tissue homeostasis. The extracellular matrix, which is a proteinaceous component of the stroma, regulates mammary epithelial growth, survival, migration and differentiation through a repertoire of transmembrane receptors, of which integrins are the best characterized. Integrins modulate cell fate by reciprocally transducing biochemical and biophysical cues between the cell and the extracellular matrix, facilitating processes such as embryonic branching morphogenesis and lactation in the mammary gland. During breast development and cancer progression, the extracellular matrix is dynamically altered such that its composition, turnover, processing and orientation change dramatically. These modifications influence mammary epithelial cell shape, and modulate growth factor and hormonal responses to regulate processes including branching morphogenesis and alveolar differentiation. Malignant transformation of the breast is also associated with significant matrix remodeling and a progressive stiffening of the stroma that can enhance mammary epithelial cell growth, perturb breast tissue organization, and promote cell invasion and survival. In this review, we discuss the role of stromal-epithelial interactions in normal and malignant mammary epithelial cell behavior. We specifically focus on how dynamic modulation of the biochemical and biophysical properties of the extracellular matrix elicit a dialogue with the mammary epithelium through transmembrane integrin receptors to influence tissue morphogenesis, homeostasis and malignant transformation.     

Cellular growth modulation. I. Effects of extracellular matrix and tumor cell conditioned medium

Earlier studies reported the enzymatic modulation of the cell surface in malignant transformation of human normal mammary epithelial cells and in conversion of mammary carcinoma. Carcinoembryonic antigen (CEA) is a neoplasm-associated antigen, its production and release is used to monitor changes in cell phenotype. The present study shows that CEA production and release by human colon carcinoma (CCC), and by colon cells from patients with familial polyposia coli (FPC) and ulcerative colitis (UCC) is inhibited when the cells are cultured in contact with confluent normal colon epithelial (HNCEC) cell monolayer. Footprints left behind and/or conditioned media from HNCEC cells inhibited, whereas footprints left behind and/or conditioned media from CCC, FPC or Ucc enhanced CEA release. During sequential passages of HNCEC cells grown on footprints and/or in spent media from CCC cultures, HNCEC cells acquire the ability to produce and release CEA, and to develop tumors in athymic Nu/Nu mice. On the other hand, during sequential passages, CCC, FPC or UCC grown in spent media, or on footprints left behind HNCEC cells, showed significant decrease in CEA production and release, and in oncologic ability in athymic mice. It is concluded that both the extracellular matrix, and a growth-regulating factor(s) in the spent medium modulate cellular transformation. Quantitative data on CEA-release indicate that FPC and UCC represent an intermediary stage between normal colon epithelial cells and colon carcinoma cells, i.e. a preneoplastic stage.     

Response of basal epithelial cell surface and Cytoskeleton to solubilized extracellular matrix molecules

Corneal epithelium removed from underlying extracellular matrix (ECM) extends numerous cytoplasmic processes (blebs) from the formerly smooth basal surface. If blebbing epithelia are grown on collagen gels or lens capsules in vitro, the basal surface flattens and takes on the smooth contour typical of epithelium in contact with basal lamina in situ. This study examines the effect of soluble extracellular matrix components on the basal surface. Corneal epithelia from 9- to 11-d-old chick embryos were isolated with trypsin-collagenase or ethylenediamine tetraacetic acid, then placed on Millipore filters (Millipore Corp., Bedford, Mass.), and cultured at the medium-air interface. Media were prepared with no serum, with 10% of calf serum, or with serum from which plasma fibronectin was removed. Epithelia grown on filters in this medium continue to bleb for the duration of the experiments (12-14 h). If soluble collagen, laminin, or fibronectin is added to the medium, however, blebs are withdrawn and by 2-6 h the basal surface is flat. Epithelia grown on filters in the presence of albumin, IgG, or glycosaminoglycans continue to bleb. Epithelia cultured on solid substrata, such as glass, also continue to bleb if ECM is absent from the medium. The basal cell cortex in situ contains a compact cortical mat of filaments that decorate with S-1 myosin subfragments; some, if not all, of these filaments point away from the plasmalemma. The actin filaments disperse into the cytoplasmic processes during blebbing and now many appear to point toward the plasmalemma. In isolated epithelia that flatten in response to soluble collagens, laminin, and fibronectin, the actin filaments reform the basal cortical mat typical or epithelial in situ. Thus, extracellular macromolecules influence and organize not only the basal cell surface but also the actin-rich basal cell cortex of epithelial cells.     

Role of the extracellular matrix in epithelial morphogenesis: A view from C. elegans

The extracellular matrix (ECM) plays an essential role in organizing tissues, defining their shapes or in presenting growth factors. Their components have been well described in most species, but our understanding of the mechanisms that control ECM remodeling remains limited. Likewise, how the ECM contributes to cellular mechanical responses has been examined in few cases. Here, I review how studies performed in C. elegans have brought several significant advances on those topics. Focusing only on epithelial cells, I discuss basement membrane invasion by the anchor cell during vulva morphogenesis, a process that has greatly expanded our knowledge of ECM remodeling in vivo. I then discuss the ECM role in a novel mechanotransduction process, whereby muscle contractions stimulate the remodeling of hemidesmosome-like junctions in the epidermis, which highlights that these junctions are mechanosensitive. Finally, I discuss progress in defining the composition and potential roles of the apical ECM covering epidermal cells in embryos.     

Apoptosis as the mode of uterine epithelial cell death during embryo implantation in mice and rats

An ultrastructural study of mouse and rat embryo implantation sites was undertaken to determine whether the uterine luminal epithelial cells surrounding the blastocyst exhibited the morphologic characteristics of apoptotic or necrotic cell death. In both species the epithelial cells exhibited all of the characteristics of apoptosis, including surface blebbing, shrinkage and fragmentation of the cells, condensation of chromatin, and indentation and fragmentation of nuclei. Cytoplasmic organelles remained morphologically intact, and the cytoplasm maintained normal or increased staining density. Also, the epithelial cells and cell fragments were phagocytosed by the adjacent trophoblast cells. The epithelial cells did not exhibit the characteristics of necrotic cell death, such as swollen cells and mitochondria, damaged surface membranes, and disintegrated cytoplasmic organelles. We conclude that uterine epithelial cells surrounding mouse and rat embryos during implantation undergo apoptotic cell death leading to their phagocytosis by trophoblast cells.     

Cyclic AMP stimulation of transferrin secretion by breast cancer cell grown on extracellular matrix or in two-compartment culture chambers.

Extrahepatic synthesis and secretion of transferrin (Tf), the major iron-carrying protein, have been described in normal and tumoral tissues suggesting a potential role for paracrine or autocrine function. In breast tumor cell MCF-7, we have previously shown a Tf secretion stimulated by estradiol which might confer selective growth advantages of these rapidly proliferating cells. The present work refers to possible additional Tf functions related to differentiation of breast tumor cells. We induced MCF-7 cell differentiation by the cyclic AMP derivative, dibutyryl cAMP (dB cAMP) and studied Tf secretion in different culture conditions after labeling with [35S] methionine. Our results demonstrate that dB cAMP stimulates Tf secretion only in culture environment that permits access to the basolateral surface and caters to the polarity requirements of the cell. These results suggest that Tf may also act as a modulator of cellular differentiation in breast cancer cells.     

Hormonal regulation of mummy is needed for apical extracellular matrix formation and epithelial morphogenesis in Drosophila

Many epithelia produce apical extracellular matrices (aECM) that are crucial for organ morphogenesis or physiology. Apical ECM formation relies on coordinated synthesis and modification of constituting components, to enable their subcellular targeting and extracellular assembly into functional matrices. The exoskeleton of Drosophila, the cuticle, is a stratified aECM containing ordered chitin polysaccharide lamellae and proteinaceous layers, and is suited for studies of molecular functions needed for aECM assembly. Here, we show that Drosophila mummy (mmy) mutants display defects in epithelial organisation in conjunction with aberrant deposition of the cuticle and an apical matrix needed for tracheal tubulogenesis. We find that mmy encodes the UDP-N-acetylglucosamine pyrophosphorylase, which catalyses the production of UDP-N-acetylglucosamine, an obligate substrate for chitin synthases as well as for protein glycosylation and GPI-anchor formation. Consequently, in mmy mutants GlcNAc-groups including chitin are severely reduced and modification and subcellular localisation of proteins designated for extracellular space is defective. Moreover, mmy expression is selectively upregulated in epithelia at the time they actively deposit aECM, and is altered by the moulting hormone 20-Hydroxyecdysone, suggesting that mmy is part of a developmental genetic programme to promote aECM formation.     

Dissecting the influence of cellular senescence on cell mechanics and extracellular matrix formation in vitro

Tissue formation and healing both require cell proliferation and migration, but also extracellular matrix production and tensioning. In addition to restricting proliferation of damaged cells, increasing evidence suggests that cellular senescence also has distinct modulatory effects during wound healing and fibrosis. Yet, a direct role of senescent cells during tissue formation beyond paracrine signaling remains unknown. We here report how individual modules of the senescence program differentially influence cell mechanics and ECM expression with relevance for tissue formation. We compared DNA damage-mediated and DNA damage-independent senescence which was achieved through over-expression of either p16^Ink4a or p21^Cip1 cyclin-dependent kinase inhibitors in primary human skin fibroblasts. Cellular senescence modulated focal adhesion size and composition. All senescent cells exhibited increased single cell forces which led to an increase in tissue stiffness and contraction in an in vitro 3D tissue formation model selectively for p16 and p21-overexpressing cells. The mechanical component was complemented by an altered expression profile of ECM-related genes including collagens, lysyl oxidases, and MMPs. We found that particularly the lack of collagen and lysyl oxidase expression in the case of DNA damage-mediated senescence foiled their intrinsic mechanical potential. These observations highlight the active mechanical role of cellular senescence during tissue formation as well as the need to synthesize a functional ECM network capable of transferring and storing cellular forces.     

Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones

Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET). These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC) did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction.