A three-dimensional collagen-fiber network model of the extracellular matrix for the simulation of the mechanical behaviors and micro structures

The extracellular matrix (ECM) provides structural and biochemical support to cells and tissues, which is a critical factor for modulating cell dynamic behavior and intercellular communication. In order to further understand the mechanisms of the interactive relationship between cell and the ECM, we developed a three-dimensional (3D) collagen-fiber network model to simulate the micro structure and mechanical behaviors of the ECM and studied the stress–strain relationship as well as the deformation of the ECM under tension. In the model, the collagen-fiber network consists of abundant random distributed collagen fibers and some crosslinks, in which each fiber is modeled as an elastic beam and a crosslink is modeled as a linear spring with tensile limit, it means crosslinks will fail while the tensile forces exceed the limit of spring. With the given parameters of the beam and the spring, the simulated tensile stress–strain relation of the ECM highly matches the experimental results including damaged and failed behaviors. Moreover, by applying the maximal inscribed sphere method, we measured the size distribution of pores in the fiber network and learned the variation of the distribution with deformation. We also defined the alignment of the collagen-fibers to depict the orientation of fibers in the ECM quantitatively. By the study of changes of the alignment and the damaged crosslinks against the tensile strain, this paper reveals the comprehensive mechanisms of four stages of ‘toe’, ‘linear’, ‘damage’ and ‘failure’ in the tensile stress–strain relation of the ECM which can provide further insight in the study of cell-ECM interaction.     

Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells

Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function.     

Dynamic expression of miR-126 and its effects on proliferation and contraction of hepatic stellate cells

In our previous study, miR-126 was identified as one of the leading miRNAs that is downregulated during activation of hepatic stellate cells (HSCs). However, the roles and related mechanisms of miR-126 in HSCs are not understood. In this study, we compared expression of miR-126 during HSC activation both in vitro and in vivo. We also applied RNA interference to analyze the role and mechanism of miR-126^∗ in the activation of HSCs. Restoring HSCs with Lv-miR-126^∗ resulted in decreased proliferation, accumulation of extracellular matrix components, and cell contraction, while also negatively regulating the vascular endothelial growth factor (VEGF) signal transduction pathways by partially targeted VEGF-A. Thus, we postulate that miR-126 may be a biological marker for the activation of HSCs, and useful for reducing intrahepatic vascular resistance and improving the sinusoidal microcirculation in chronic liver diseases.     

Measurement of free and bound fractions of extracellular ATP in biological solutions using bioluminescence.

Measurement of extracellular ATP in biological solutions is complicated by protein-binding and rapid enzymatic degradation. We hypothesized that the concentration of extracellular ATP could be determined luminometrically by limiting degradation and measuring the free and protein-bound fractions. ATP was added (a) at constant concentration to solutions containing varying albumin concentrations; (b) at varying concentrations to a physiological albumin solution (4 gm/dL); (c) at varying concentrations to plasma. After centrifugation, a fraction of each supernatant was heated. ATP in heated and unheated samples was measured luminometrically. Blood was drawn into saline or an ATP-stabilizing solution and endogenous plasma ATP measured. ATP-albumin binding was a linear function of albumin concentration (3.5% ATP bound at 100 micromol/L to 33.2% ATP bound at 1000 micromol/L) but independent of ATP concentration (29.3%, 10-1000 nmol/L ATP in 602 micromol/L albumin). Heating released the majority of bound ATP from albumin-containing solutions (94.8 +/- 1.7%) and plasma (97.6 +/- 5.1%). Total endogenous plasma ATP comprised 93 +/- 27 nmol/L (free) and 150 +/- 40 nmol/L (total fraction). Without stabilizing solution, degradation of free endogenous plasma ATP occurred. Within a physiological range (10-1000 nmol/L), ATP binds albumin independently of ATP concentration. Heating releases bound ATP, enabling accurate luminometric measurement of total extracellular ATP (free and bound) in biological samples.     

The Fibronectin Domain ED-A Is Crucial for Myofibroblastic Phenotype Induction by Transforming Growth Factor-β1

Transforming growth factor-β1 (TGFβ1), a major promoter of myofibroblast differentiation, induces α-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes α-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFβ1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of α-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se α-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFβ1-triggered enhancement of α-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A–containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFβ1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.     

cDNA cloning of an extracellular dermal glycoprotein of carrot and its expression in response to wounding

Suspension-cultured cells of carrot (Daucus carota L.) synthesize and secrete a glycoprotein that is normally found only in dermal tissues (epidermis, endodermis and periderm). This protein, previously called GP57, is now referred to as EDGP (E xtracellular D ermal G lyco P rotein). We purified sufficient quantities of EDGP to obtain amino-acid sequences on two internal tryptic peptides and screened a cDNA library of young carrot roots with antiserum to EDGP and with oligonucleotides corresponding to the peptides. Here we report the derived amino-acid sequence of EDGP. Sequence comparisons show that it has 40% amino-acid sequence identity with 7S basic globulin, a protein that is released when soybean seeds are soaked in hot water for a few hours. We suggest that these two proteins belong to a new family of dermal proteins. As far as we know, this is the first reported derived amino-acid sequence for protein that is specific to the epidermis and other dermal tissues. The level of EDGP mRNA is low in dry seeds, but increases rapidly in growing seedlings as they develop dermal tissues. The level of mRNA is low in storage roots, but increases rapidly in response to wounding. The presence of EDGP in dermal tissues and its up-regulation in response to wounding indicate a role in the response of plants to biotic and-or abiotic stresses. An unusual feature of the amino-acid sequence of EDGP is that it contains a short motif, which is present at the active site of aspartyl proteases such as pepsin and chymosin.Abbreviations cDNA copy DNA - 2,4-D 2,4-dichlorophen-oxyacetic acid - EDGP extracellular dermal glycoprotein - 7SBG 7S basic globulin Supported by a contract from the United States Department of Energy (Energy Biosciences) (to M.J.C.) and a Grant-in-Aid for Special Research on Priority Areas (01660002, Cellular and Molecular Basis for Reproductive Processes in Plants) from the Ministry of Education, Science and Culture, and by the Fund from Basic Research Core System of Science and Technology Agency, Japan (to S.S.).     

Matrix loading: assembly of extracellular matrix collagen fibrils during embryogenesis

Nothing in biology stimulates the imagination like the development of a single fertilized egg into a newborn child. Consequently, a major focus of biomedical research is aimed at understanding cell differentiation, proliferation, and specialization during child health and human development. However, the fact that the increase in size and shape of the growing embryo has as much to do with the extracellular matrix (ECM) as with the cells themselves, is largely overlooked. Cells in developing tissues are surrounded by a fiber-composite ECM that transmits mechanical stimuli, maintains the shape of developing tissues, and functions as a scaffold for cell migration and attachment. The major structural element of the ECM is the collagen fibril. The fibrils, which are indeterminate in length, are arranged in different tissues in exquisite supramolecular architectures, including parallel bundles, orthogonal lamellae, and concentric weaves. This article reviews our current understanding of the synthesis and assembly of collagen fibrils, and discusses challenging questions about how cells assemble an organized ECM during embryogenesis.     

Slr4, a newly identified S-layer protein from marine Gammaproteobacteria,is a major biofilm matrix component

S-layers are paracrystalline proteinaceous lattices that surround prokaryotic cells, forming a critical interface between the cells and their extracellular environment. Here, we report the discovery of a novel S-layer protein present in the Gram-negative marine organism, Pseudoalteromonas tunicata D2. An uncharacterized protein (EAR28894) was identified as the most abundant protein in planktonic cultures and biofilms. Bioinformatic methods predicted a beta-helical structure for EAR28894 similar to the Caulobacter S-layer protein, RsaA, despite sharing less than 20% sequence identity. Transmission electron microscopy revealed that purified EAR28894 protein assembled into paracrystalline sheets with a unique square lattice symmetry and a unit cell spacing of ~9.1 nm. An S-layer was found surrounding the outer membrane in wild-type cells and completely removed from cells in an EAR28894 deletion mutant. S-layer material also appeared to be “shed” from wild-type cells and was highly abundant in the extracellular matrix where it is associated with outer membrane vesicles and other matrix components. EAR28894 and its homologs form a new family of S-layer proteins that are widely distributed in Gammaproteobacteria including species of Pseudoalteromonas and Vibrio, and found exclusively in marine metagenomes. We propose the name Slr4 for this novel protein family.     

A citrus polymethoxyflavonoid, nobiletin, is a novel MEK inhibitor that exhibits antitumor metastasis in human fibrosarcoma HT-1080 cells

The activation of mitogen-activated protein/extracellular signal-regulated kinase (MEK) is well known to be associated with tumor invasion and metastasis. We previously reported that a polymethoxyflavonoid, nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone), derived from Citrus depressa (Hayata), inhibits the phosphorylation of MEK and thereby suppresses matrix metalloproteinase (MMP) expression in a tumor-metastasis stimulator, 12-O-tetradecanoyl phorbol 13-acetate (TPA)-stimulated human fibrosarcoma HT-1080 cells [Mol. Cancer Ther. 3 (2004) 839-847]. In the present study, we investigated whether or not nobiletin might directly influence MEK activity to exhibit the antitumor metastatic activity in vitro. MEK kinase assay using myelin basic protein (MBP) revealed that TPA-augmented MEK activity in HT-1080 cells and that the augmented MEK activity was diminished by nobiletin treatment. In addition, the decrease in MEK activity caused by nobiletin was found to inhibit the phosphorylation of extracellular regulated kinases (ERK), a downstream signaling factor for MEK. Furthermore, when an immunoprecipitated active MEK was incubated with nobiletin under cell-free conditions, nobiletin was found to inhibit the MEK-mediated MBP phosphorylation. In contrast, other citrus polymethoxyflavonoids such as 3-hydroxy-5,6,7,8,3′,4′-hexamethoxyflavone (natsudaidain) and 3,5,6,7,8,3′,4′-heptamethoxyflavone, did not directly inhibit MEK activity. Moreover, natsudaidain and 3,5,6,7,8,3′,4′-heptamethoxyflavone exhibited no or less inhibitory effect than nobiletin on the proMMP-9/progelatinase B production in HT-1080 cells. Therefore, these results provide novel evidence that nobiletin directly inhibits MEK activity and decreases the sequential phosphorylation of ERK, exhibiting the antitumor metastatic activity by suppressing MMP expression in HT-1080 cells.     

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.