The Biofilm Matrix

The extracellular matrix is a complex and extremely important component of all biofilms, providing architectural structure and mechanical stability to the attached population. The matrix is composed of cells, water and secreted/released extracellular macromolecules. In addition, a range of enzymic and regulatory activities can be found within the matrix. Together, these different components and activities are likely to interact and in so doing create a series of local environments within the matrix which co-exist as a functional consortium. The matrix architecture is also subject to a number of extrinsic factors, including fluctuations in nutrient and gaseous levels and fluid shear. Together, these intrinsic and extrinsic factors combine to produce a dynamic, heterogeneous microenvironment for the attached and enveloped cells.     

Dual effect of nerve growth factor on cell death of PC12 cells Induced by serum deprivation

The effect of nerve growth factor (NGF) on the cell death of PC12 cells that is induced by serum deprivation was examined in the floating and attached cells to the extracellular matrix. NGF suppressed cell death occurred in the floating cells. The onset of cell death in the attached cells was much slower than in the floating cells. Moreover, the cell death in the attached cells was either accelerated in a high-density culture (over approximately 50% confluent), or inhibited in a low-density culture by NGF. While nucleosomal DNA fragmentation and poly (ADP-ribose) polymerase degradation was observed in both the floating and attached cells, the incidence of nuclear fragmentation and chromatin condensation was much lower in the attached cells than in the floating cells. The delayed onset of cell death in the attached cells was due to the signals that are generated from the extracellular matrix that is formed by PC12 cells, together with cell-to-cell interaction. The acceleration of cell death in the NGF-treated cells was anoikis, caused by the loss of the anchorage of the cell via the action of increased activities of matrix metalloproteinases (MMP2, MMP9). These results suggest that NGF has a different role in the cell death of PC12 cells that is induced by serum deprivation, depending on the cell-matrix, as well as the cell-cell interaction.     

Age-related changes in rat myocardium involve altered capacities of glycosaminoglycans to potentiate growth factor functions and heparan sulfate-altered sulfation

Glycosaminoglycans (GAGs) are essential components of the extracellular matrix, the natural environment from which cell behavior is regulated by a number or tissue homeostasis guarantors including growth factors. Because most heparin-binding growth factor activities are regulated by GAGs, structural and functional alterations of these polysaccharides may consequently affect the integrity of tissues during critical physiological and pathological processes. Here, we investigated whether the aging process can induce changes in the myocardial GAG composition in rats and whether these changes can affect the activities of particular heparin-binding growth factors known to sustain cardiac tissue integrity. Our results showed an age-dependent increase of GAG levels in the left ventricle. Biochemical and immunohistological studies pointed out heparan sulfates (HS) as the GAG species that increased with age. ELISA-based competition assays showed altered capacities of the aged myocardial GAGs to bind FGF-1, FGF-2, and VEGF but not HB EGF. Mitogenic assays in cultured cells showed an age-dependent decrease of the elderly GAG capacities to potentiate FGF-2 whereas the potentiating effect on VEGF(165) was increased, as confirmed by augmented angiogenic cell proliferation in Matrigel plugs. Moreover, HS disaccharide analysis showed considerably altered 6-O-sulfation with modest changes in N- and 2-O-sulfations. Together, these findings suggest a physiological significance of HS structural and functional alterations during aging. This can be associated with an age-dependent decline of the extracellular matrix capacity to efficiently modulate not only the activity of resident or therapeutic growth factors but also the homing of resident or therapeutic cells.     

Extracellular matrix components affect the pattern of protein synthesis of endothelial cells responding to hyperthermia

Summary The biosynthetic profile of endothelial cells responding to hyperthermia is altered by extracellular matrix components. The extracellular matrix components influence the quantitative expression of members of the HSP70 family and HSP90. The expression of several HSP70 mRNA species, which are strictly stress inducible, are modulated by extracellular matrix components. Both laminin and collagen type IV decrease the amount of HSP70 protein and mRNA expressed by endothelial cells exposed to hyperthermia relative to control cultures attached to virgin plastic. In contrast, both laminin and collagen type IV increased the amount of HSP90 mRNA constitutively expressed by endothelial cells at 37° C. When endothelial cells were exposed to elevated temperatures, these two extracellular matrix proteins decrease the amount of HSP90 mRNA relative to control cultures attached to virgin plastic. Our observations are consistent with the proposal that the extracellular matrix components regulate gene expression and cell behavior in regard to thermotolerance.     

Glia unglued: how signals from the extracellular matrix regulate the development of myelinating glia

The health and function of the nervous system relies on glial cells that ensheath neuronal axons with a specialized plasma membrane termed myelin. The molecular mechanisms by which glial cells target and enwrap axons with myelin are only beginning to be elucidated, yet several studies have implicated extracellular matrix proteins and their receptors as being important extrinsic regulators. This review provides an overview of the extracellular matrix proteins and their receptors that regulate multiple steps in the cellular development of Schwann cells and oligodendrocytes, the myelinating glia of the PNS and CNS, respectively, as well as in the construction and maintenance of the myelin sheath itself. The first part describes the relevant cellular events that are influenced by particular extracellular matrix proteins and receptors, including laminins, collagens, integrins, and dystroglycan. The second part describes the signaling pathways and effector molecules that have been demonstrated to be downstream of Schwann cell and oligodendroglial extracellular matrix receptors, including FAK, small Rho GTPases, ILK, and the PI3K/Akt pathway, and the roles that have been ascribed to these signaling mediators. Throughout, we emphasize the concept of extracellular matrix proteins as environmental sensors that act to integrate, or match, cellular responses, in particular to those downstream of growth factors, to appropriate matrix attachment.     

Mast cell fibroblastoid differentiation mediated by airway smooth muscle in asthma

Mast cell microlocalization to the airway smooth muscle (ASM) bundle is a key feature of asthma, but whether these mast cells have an altered phenotype is uncertain. In this paper, we report that in vivo, mast cells within the ASM bundle, in contrast to mast cells in the bronchial submucosa, commonly expressed fibroblast markers and the number of these cells was closely related to the degree of airway hyperresponsiveness. In vitro human lung mast cells and mast cell lines cultured with fibronectin or with primary human ASM cells acquired typical fibroblastic markers and morphology. This differentiation toward a fibroblastoid phenotype was mediated by ASM-derived extracellular matrix proteins, independent of cell adhesion molecule-1, and was attenuated by α5β1 blockade. Fibroblastoid mast cells demonstrated increased chymase expression and activation with exaggerated spontaneous histamine release. Together these data indicate that in asthma, ASM-derived extracellular matrix proteins mediate human mast cell transition to a fibroblastoid phenotype, suggesting that this may be pivotal in the development of airway dysfunction in asthma.     

The neuronal growth cone as a specialized transduction system.

Neuronal growth and remodelling are guided by both intracellular gene programs and extracellular stimuli. The growth cone is one site where the effects of these extrinsic and intrinsic factors converge upon the mechanical determinants of cell shape. We review the growth cone as a transduction device, converting extracellular signals into mechanical forces. A variety of soluble, extracellular matrix and membrane bound molecules control growth cone behavior. In addition, GAP-43 is discussed as a possible component of the intraneuronal gene program which modulates growth cone activity. The GTP-binding protein, Go, is a major growth cone membrane protein that may transduce signals not only from outside the cell, but from within as well. This may provide a molecular site in the growth cone for the coordination of a genetic growth program with environmental signals.     

Collagenous substrata regulate the nature and distribution of glycosaminoglycans produced by differentiated cultures of mouse mammary epithelial cells

We have investigated the influence of culture substrata upon glycosaminoglycans produced in primary cultures of mouse mammary epithelial cells isolated from the glands of late pregnant mice. Three substrata have been used for experiments: tissue culture plastic, collagen (type I) gels attached to culture dishes, and collagen (type I) gels that have been floated in the culture medium after cell attachment. These latter gels contract significantly. Cells cultured on all three substrata produce hyaluronic acid, heparan sulfate, chondroitin sulfates and dermatan sulfate but the relative quantities accumulated and their distribution among cellular and extracellular compartments differ according to the nature of the culture substratum. Notably most of the glycosaminoglycans accumulated by cells on plastic are secreted into the culture medium, while cells on floating gels incorporate almost all their glycosaminoglycans into an extracellular matrix fraction. Cells on attached collagen gels secrete approx. 30% of their glycosaminoglycans and assemble most of the remainder into an extracellular matrix. Hyaluronic acid is produced in significant quantities by cells on plastic and attached gels but in relatively reduced quantity by cells on floating gels. In contrast, iduronyl-rich dermatan sulfate is accumulated by cells on floating gels, where it is primarily associated with the extracellular matrix fraction, but is proportionally reduced in cells on plastic and attached gels. The results are discussed in terms of polarized assembly of a morphologically distinct basal lamina, a process that occurs primarily when cells are on floating gels. In addition, as these cultures secrete certain milk proteins only when cultured on floating gels, we discuss the possibility that cell synthesized glycosaminoglycans and proteoglycans may play a role in the maintenance of a differentiated phenotype.     

The extracellular matrix is instructive

The extracellular matrix does more than just blanket cells; it also provides informational cues which affect a variety of developmental and cellular maintenance activities. The constituents of the matrix provide the fabric for cell motility and cell shape as well as anchorage sites for bioactive factors which directly affect the cell's developmental pattern or mitotic activity. The influence of the extracellular matrix is controlled by the cell's responsiveness to these complex signals. The same matrix component, for example hyaluronic acid, can have completely different effects depending on the cell's lineage and developmental history. The functional interaction between the extracellular matrix and specific cell surface receptors provides cues which affect the control of development and the maintenance and aging events that affect specific cells and tissues.     

Extra-cellular matrix in vascular networks

The vascular network is a series of linked conduits of blood vessels composed of the endothelium, a monolayer of cells that adorn the vessel lumen and surrounding layer(s) of mesenchymal cells (vascular smooth muscle, pericytes and fibroblasts). In addition to providing structural support, the mesenchymal cells are essential for vessel contractility. The extracellular matrix is a major constituent of blood vessels and provides a framework in which these various cell types are attached and embedded. The composition and organization of vascular extracellular matrix is primarily controlled by the mesenchymal cells, and is also responsible for the mechanical properties of the vessel wall, forming complex networks of structural proteins which are highly regulated. The extracellular matrix also plays a central role in cellular adhesion, differentiation and proliferation. This review examines the cellular and extracellular matrix components of vessels, with specific emphasis on the regulation of collagen type I and implications in vascular disease.     

Blastocysts don't go it alone. Extrinsic signals fine-tune the intrinsic developmental program of trophoblast cells

The preimplantation embryo floats freely within the oviduct and is capable of developing into a blastocyst independently of the maternal reproductive tract. While establishment of the trophoblast lineage is dependent on expression of developmental regulatory genes, further differentiation leading to blastocyst implantation in the uterus requires external cues emanating from the microenvironment. Recent studies suggest that trophoblast differentiation requires intracellular signaling initiated by uterine-derived growth factors and integrin-binding components of the extracellular matrix. The progression of trophoblast development from the early blastocyst stage through the onset of implantation appears to be largely independent of new gene expression. Instead, extrinsic signals direct the sequential trafficking of cell surface receptors to orchestrate the developmental program that initiates blastocyst implantation. The dependence on external cues could coordinate embryonic activities with the developing uterine endometrium. Biochemical events that regulate trophoblast adhesion to fibronectin are presented to illustrate a developmental strategy employed by the peri-implantation blastocyst.     

Dystroglycan inside and out.

Dystroglycan connects the extracellular matrix and cytoskeleton. Key findings in the past year indicate that dystroglycan interacts with a wider repertoire of extracellular ligands than originally appreciated, that dystroglycan plays a critical role in organizing extracellular matrix molecules on the cell surface and in basement membranes, and that at least two human pathogens utilize dystroglycan to gain access to host cells. Together, these advances begin to help elucidate important biological roles for dystroglycan in development and disease.     

Esophageal muscle physiology and morphogenesis require assembly of a collagen XIX-rich basement membrane zone

Collagen XIX is an extremely rare extracellular matrix component that localizes to basement membrane zones and is transiently expressed by differentiating muscle cells. Characterization of mice harboring null and structural mutations of the collagen XIX (Col19a1) gene has revealed the critical contribution of this matrix protein to muscle physiology and differentiation. The phenotype includes smooth muscle motor dysfunction and hypertensive sphincter resulting from impaired swallowing-induced, nitric oxide-dependent relaxation of the sphincteric muscle. Muscle dysfunction was correlated with a disorganized matrix and a normal complement of enteric neurons and interstitial cells of Cajal. Mice without collagen XIX exhibit an additional defect, namely impaired smooth-to-skeletal muscle cell conversion in the abdominal segment of the esophagus. This developmental abnormality was accounted for by failed activation of myogenic regulatory factors that normally drive esophageal muscle transdifferentiation. Therefore, these findings identify collagen XIX as the first structural determinant of sphincteric muscle function, and as the first extrinsic factor of skeletal myogenesis in the murine esophagus.     

Extracellular Matrix Stiffness and Architecture Govern Intracellular Rheology in Cancer

Little is known about the complex interplay between the extracellular mechanical environment and the mechanical properties that characterize the dynamic intracellular environment. To elucidate this relationship in cancer, we probe the intracellular environment using particle-tracking microrheology. In three-dimensional (3D) matrices, intracellular effective creep compliance of prostate cancer cells is shown to increase with increasing extracellular matrix (ECM) stiffness, whereas modulating ECM stiffness does not significantly affect the intracellular mechanical state when cells are attached to two-dimensional (2D) matrices. Switching from 2D to 3D matrices induces an order-of-magnitude shift in intracellular effective creep compliance and apparent elastic modulus. However, for a given matrix stiffness, partial blocking of β1 integrins mitigates the shift in intracellular mechanical state that is invoked by switching from a 2D to 3D matrix architecture. This finding suggests that the increased cell-matrix engagement inherent to a 3D matrix architecture may contribute to differences observed in viscoelastic properties between cells attached to 2D matrices and cells embedded within 3D matrices. In total, our observations show that ECM stiffness and architecture can strongly influence the intracellular mechanical state of cancer cells.     

Extracellular matrix interactions 2: Extracellular matrix structure is important for growth factor localization and function

Summary The influence of extracellular matrix components and of extracellular matrix structure on in vitro cell growth was investigated in the UWOV2 (Pf), protein-free cell culture model. This cell line constitutively produces an ordered extracellular matrix in the absence of any exogenous protein or growth factor. Extracellular matrix from UWOV2 (Pf) cells was found to contain both transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF), which were shown to have an autostimulatory role for UWOV2 (Pf) cell growth. Matrix structure was shown to be important for allowing expression of the functional activity of these two growth factors. In addition, a nonuniform distribution of PDGF, embedded within the matrix structure, was demonstrated by immunoelectronmicroscopy. Apart from these two well-defined growth factors, additional but as yet unidentified growth stimulatory factor(s) were extractable from UWOV2 (Pf) extracellular matrix. These investigations indicate the potential role of extracellular matrix both as a mechanism for concentrating as well as modulating the function of cellular growth factors.     

A Novel Role for Lh3 Dependent ECM Modifications during Neural Crest Cell Migration in Zebrafish

During vertebrate development, trunk neural crest cells delaminate along the entire length of the dorsal neural tube and initially migrate as a non-segmented sheet. As they enter the somites, neural crest cells rearrange into spatially restricted segmental streams. Extracellular matrix components are likely to play critical roles in this transition from a sheet-like to a stream-like mode of migration, yet the extracellular matrix components and their modifying enzymes critical for this transition are largely unknown. Here, we identified the glycosyltransferase Lh3, known to modify extracellular matrix components, and its presumptive substrate Collagen18A1, to provide extrinsic signals critical for neural crest cells to transition from a sheet-like migration behavior to migrating as a segmental stream. Using live cell imaging we show that in lh3 null mutants, neural crest cells fail to transition from a sheet to a stream, and that they consequently enter the somites as multiple streams, or stall shortly after entering the somites. Moreover, we demonstrate that transgenic expression of lh3 in a small subset of somitic cells adjacent to where neural crest cells switch from sheet to stream migration restores segmental neural crest cell migration. Finally, we show that knockdown of the presumptive Lh3 substrate Collagen18A1 recapitulates the neural crest cell migration defects observed in lh3 mutants, consistent with the notion that Lh3 exerts its effect on neural crest cell migration by regulating post-translational modifications of Collagen18A1. Together these data suggest that Lh3–Collagen18A1 dependent ECM modifications regulate the transition of trunk neural crest cells from a non-segmental sheet like migration mode to a segmental stream migration mode.     

Bottom-up approaches in synthetic biology and biomaterials for tissue engineering applications

Synthetic biologists use engineering principles to design and construct genetic circuits for programming cells with novel functions. A bottom-up approach is commonly used to design and construct genetic circuits by piecing together functional modules that are capable of reprogramming cells with novel behavior. While genetic circuits control cell operations through the tight regulation of gene expression, a diverse array of environmental factors within the extracellular space also has a significant impact on cell behavior. This extracellular space offers an addition route for synthetic biologists to apply their engineering principles to program cell-responsive modules within the extracellular space using biomaterials. In this review, we discuss how taking a bottom-up approach to build genetic circuits using DNA modules can be applied to biomaterials for controlling cell behavior from the extracellular milieu. We suggest that, by collectively controlling intrinsic and extrinsic signals in synthetic biology and biomaterials, tissue engineering outcomes can be improved.     

delta 9-Tetrahydrocannabinol suppresses macrophage extrinsic antiherpesvirus activity.

The effect of delta 9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, on macrophage intrinsic and extrinsic antiherpesvirus activities was examined. THC had no effect on the capacity of the macrophage-like cells RAW264.7, J774A.1, and P388D1 to take up virus. In addition, replication of virus within macrophages did not occur regardless of drug treatment, indicating that THC had no effect on macrophage intrinsic antiviral activity. In contrast, the cannabinoid exerted a dose-dependent inhibition of macrophage extrinsic antiviral activity. This activity describes that macrophage function by which these cells suppress virus replication within xenogeneic cells in an interferon-independent manner. The inhibitory effect of THC on extrinsic antiviral activity was greatest on RAW264.7 and J774A.1 cells, followed by P388D1 cells. These macrophage-like cells regained their extrinsic antiviral activity in a time-related fashion following removal of the drug. These results indicate that THC inhibits macrophage extrinsic antiherpesvirus activity, but has no effect on intrinsic antiviral activity. However, the suppressive effect of THC on extrinsic antiviral activity is reversible upon removal of the drug.     

Fibronectin and laminin in the extracellular matrix and basement membrane of sea urchin embryos

Fibronectin and laminin have been found in the extracellular matrix and in the basement membrane of sea urchin embryos during early development. These glycoproteins are also found on the cell surfaces of the outer epithelial layer and on the secondary mesenchyme cells within the blastocoel. The similarity of functions of the extracellular matrix and basement membrane is discussed, as is the similarity of their molecular components. These observations suggest the possibility that fibronectin and laminin form a continuous matrix surrounding the cells which links the outer ECM (hyaline layer) to the inner ECM (basement membrane). Such a network could coordinate the various activities of the embryo during early morphogenesis.