Showing papers on "Extracellular matrix component published in 2005"

Tissue repair, contraction, and the myofibroblast.

Abstract: After the first description of the myofibroblast in granulation tissue of an open wound by means of electron microscopy, as an intermediate cell between the fibroblast and the smooth muscle cell, the myofibroblast has been identified both in normal tissues, particularly in locations where there is a necessity of mechanical force development, and in pathological tissues, in relation with hypertrophic scarring, fibromatoses and fibrocontractive diseases as well as in the stroma reaction to epithelial tumors It is now accepted that fibroblast/myofibroblast transition begins with the appearance of the protomyofibroblast, whose stress fibers contain only beta- and gamma-cytoplasmic actins and evolves, but not necessarily always, into the appearance of the differentiated myofibroblast, the most common variant of this cell, with stress fibers containing alpha-smooth muscle actin Myofibroblast differentiation is a complex process, regulated by at least a cytokine (the transforming growth factor-beta1), an extracellular matrix component (the ED-A splice variant of cellular fibronectin), as well as the presence of mechanical tension The myofibroblast is a key cell for the connective tissue remodeling that takes place during wound healing and fibrosis development On this basis, the myofibroblast may represent a new important target for improving the evolution of such diseases as hypertrophic scars, and liver, kidney or pulmonary fibrosis

An aspartic acid repeat polymorphism in asporin inhibits chondrogenesis and increases susceptibility to osteoarthritis.

Abstract: Osteoarthritis is the most common form of human arthritis. We investigated the potential role of asporin, an extracellular matrix component expressed abundantly in the articular cartilage of individuals with osteoarthritis, in the pathogenesis of osteoarthritis. Here we report a significant association between a polymorphism in the aspartic acid (D) repeat of the gene encoding asporin (ASPN) and osteoarthritis. In two independent populations of individuals with knee osteoarthritis, the D14 allele of ASPN is over-represented relative to the common D13 allele, and its frequency increases with disease severity. The D14 allele is also over-represented in individuals with hip osteoarthritis. Asporin suppresses TGF-β–mediated expression of the genes aggrecan (AGC1) and type II collagen (COL2A1) and reduced proteoglycan accumulation in an in vitro model of chondrogenesis. The effect on TGF-β activity is allele-specific, with the D14 allele resulting in greater inhibition than other alleles. In vitro binding assays showed a direct interaction between asporin and TGF-β. Taken together, these findings provide another functional link between extracellular matrix proteins, TGF-β activity and disease, suggesting new therapeutic strategies for osteoarthritis.

Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy.

Abstract: During vertebrate gastrulation, progenitor cells of different germ layers acquire specific adhesive properties that contribute to germ layer formation and separation. Wnt signals have been suggested to function in this process by modulating the different levels of adhesion between the germ layers, however, direct evidence for this is still lacking. Here we show that Wnt11, a key signal regulating gastrulation movements, is needed for the adhesion of zebrafish mesendodermal progenitor cells to fibronectin, an abundant extracellular matrix component during gastrulation. To measure this effect, we developed an assay to quantify the adhesion of single zebrafish primary mesendodermal progenitors using atomic-force microscopy (AFM). We observed significant differences in detachment force and work between cultured mesendodermal progenitors from wild-type embryos and from slb/wnt11 mutant embryos, which carry a loss-of-function mutation in the wnt11 gene, when tested on fibronectin-coated substrates. These differences were probably due to reduced adhesion to the fibronectin substrate as neither the overall cell morphology nor the cell elasticity grossly differed between wild-type and mutant cells. Furthermore, in the presence of inhibitors of fibronectin-integrin binding, such as RGD peptides, the adhesion force and work were strongly decreased, indicating that integrins are involved in the binding of mesendodermal progenitors in our assay. These findings demonstrate that AFM can be used to quantitatively determine the substrate-adhesion of cultured primary gastrulating cells and provide insight into the role of Wnt11 signalling in modulating cell adhesion at the single cell scale.

Transforming growth factor-beta 2 modulated extracellular matrix component expression in cultured human optic nerve head astrocytes.

Abstract: Purpose To study whether glaucomatous extracellular matrix (ECM) modifications in the lamina cribrosa might be induced by TGF-beta 2, the effect of TGF-beta 2 on the expression of collagen types I (Col1 alpha 1), III (Col3 alpha 1), and IV (Col4 alpha 2); fibronectin (FN); tissue transglutaminase (TGM2); connective tissue growth factor (CTGF); and thrombospondin (TSP-1) in cultured human optic nerve head (ONH) astrocytes was investigated. Methods Astrocytes were isolated from eyes of five human donors, and cultured monolayers were treated with 1.0 ng/mL TGF-beta 2 for 24 and 48 hours. Expression of Col1 alpha 1, Col3 alpha 1, Col4 alpha 2, FN, TGM2, CTGF, and TSP-1 was examined by semiquantitative RT-PCR and Northern and Western blot analyses. The effect of CTGF silencing on the TGF-beta 2-modulated expression of these genes was investigated by transfection of CTGF small interfering (si)RNA before TGF-beta 2 treatment. Results TGF-beta 2 treatment upregulated the expression of Col1 alpha 1, Col4 alpha 2, FN, CTGF, TGM2, and TSP-1 mRNA and protein in cultured astrocytes. Inductions ranged between 1.5- and 4-fold. Expression of Col3 alpha 1 remained unaffected. Transfection of 10 nM CTGF siRNA inhibited the TGF-beta 2-induced upregulation of CTGF, Col4 alpha 2, Col1 alpha 1, TGM2, and FN, whereas TSP-1 expression was not reduced. Conclusions TGF-beta 2 is capable of inducing the expression of ECM and basement membrane components in cultured ONH astrocytes via CTGF and upregulated TSP-1, a protein naturally involved in the activation of latent TGF-beta. Therefore, TGF-beta 2 could be a factor in the initiation of the modification of ECM in the glaucomatous ONH. In addition, TSP-1 induction may be a mechanism by which TGF-beta 2 amplifies its own activation.

The Two Isoforms of the Caenorhabditis elegans Leukocyte-Common Antigen Related Receptor Tyrosine Phosphatase PTP-3 Function Independently in Axon Guidance and Synapse Formation

Abstract: Leukocyte-common antigen related (LAR)-like phosphatase receptors are conserved cell adhesion molecules that function in multiple developmental processes. The Caenorhabditis elegans ptp-3 gene encodes two LAR family isoforms that differ in the extracellular domain. We show here that the long isoform, PTP-3A, localizes specifically at synapses and that the short isoform, PTP-3B, is extrasynaptic. Mutations in ptp-3 cause defects in axon guidance that can be rescued by PTP-3B but not by PTP-3A. Mutations that specifically affect ptp-3A do not affect axon guidance but instead cause alterations in synapse morphology. Genetic double-mutant analysis is consistent with ptp-3A acting with the extracellular matrix component nidogen, nid-1 , and the intracellular adaptor α-liprin, syd-2 . nid-1 and syd-2 are required for the recruitment and stability of PTP-3A at synapses, and mutations in ptp-3 or nid-1 result in aberrant localization of SYD-2. Overexpression of PTP-3A is able to bypass the requirement for nid-1 for the localization of SYD-2 and RIM. We propose that PTP-3A acts as a molecular link between the extracellular matrix and α-liprin during synaptogenesis.

Differentiation, maturation, and survival of dendritic cells by osteopontin regulation.

Abstract: Dendritic cells (DCs) are antigen-presenting cells with the ability to induce primary immune responses necessary in innate immunity and adaptive immunity. Osteopontin (OPN) is a secreted acidic phosphoprotein containing an arginine-glycine-aspartate sequence and has been suggested to play an important role in early cellular immune responses. The interaction between DCs and OPN has not been clarified. We hypothesized that there is an important interaction between DCs and OPN, which is an indispensable extracellular matrix component in early cellular immune responses. Human monocyte-derived DCs synthesized OPN especially during the differentiation from monocytes to immature DCs. By blocking of OPN with anti-OPN antibody, cultured DCs became smaller and expressed lower levels of costimulatory molecules and major histocompatibility complex class II antigens than untreated DCs. Furthermore, DCs treated with anti-OPN antibody easily underwent apoptosis. These results suggest that human DCs can produce OPN and that OPN may play a role in the differentiation, maturation, and survival of DCs by autocrine and/or paracrine pathways.

Epidermal growth factor receptor signaling is partially responsible for the increased matrix metalloproteinase-1 expression in ocular epithelial cells after UVB radiation.

Abstract: Pterygia are inflammatory, invasive, and proliferative lesions of the human ocular surface in which the matrix metalloproteinase (MMP) collagenase-1 (MMP-1) is highly expressed. Pterygia development may involve MMP-1 activity against interstitial fibrillar collagen, an abundant extracellular matrix component of the cornea, and its induction by ultraviolet light (UVB). We examined the pathways responsible for enhanced expression of MMP-1 in pterygium epithelial cells after UVB exposure and/or treatment with chemical inhibitors of mitogen-activated protein kinases or epidermal growth factor receptor. The induction of MMP-1 by UVB was comparable to that mediated by heparin-binding epidermal growth factor-like growth factor and epidermal growth factor. The epidermal growth factor receptor inhibitor PD153035 partially blocked the UVB-mediated induction of MMP-1 and totally abrogated its production after stimulation with either heparin-binding epidermal growth factor-like growth factor or epidermal growth factor. UVB exposure enhanced the phosphorylated form of ERK1/2 in a time-dependent manner whereas the ERK1/2 inhibitor PD98059 decreased this induction by at least fivefold. Transcripts for c-jun and c-fos were detected as early as 2 hours after UVB exposure and were suppressed by PD98059. The identification of a specific intracellular signaling pathway responsible for the enhanced production of a key enzyme that denatures intact fibrillar collagen has important implications for understanding the pathophysiology and future therapy for pterygia.

Defining the Interaction of the Treponema pallidum Adhesin Tp0751 with Laminin

Abstract: Various invasive pathogens attach to host tissues via the extracellular matrix component laminin, the major glycoprotein found within basement membranes. Previous investigations identified the laminin-binding adhesin Tp0751 within the spirochete bacterium Treponema pallidum. In the current study, Tp0751 was shown to attach to a variety of laminin isoforms that are widely distributed throughout the host, including laminins 1, 2, 4, 8, and 10. Such universal attachment is conducive for an adhesin present within a highly invasive pathogen that encounters a variety of tissue sites during the course of infection. Additional studies systematically identified the amino acid residues within Tp0751 that contribute to laminin binding using synthetic peptides designed from the mature protein sequence. The minimum laminin-binding region of the adhesin was localized to 10 amino acids; peptides containing these residues inhibited attachment of Tp0751 and T. pallidum to laminin. Further, Tp0751-specific antibodies inhibited attachment of T. pallidum to laminin. This study furthers our knowledge of the interaction of T. pallidum with laminin, an association that is proposed to facilitate bacterial traversal of basement membranes and subsequent entry into the circulation and tissue invasion. As such, these investigations will reveal new targets for possible prevention of bacterial dissemination and establishment of chronic infection.

Tissue morphogenesis and vascular stability require the Frem2 protein, product of the mouse myelencephalic blebs gene

Abstract: Adhesive properties of cells undergoing morphogenetic rearrangements can be regulated either at the cellular level or by altering the environment in which rearrangements occur. Here, we describe the identification of a mutation (myF11) in the mouse extracellular matrix component Frem2, and provide evidence that suggests Frem2 expression creates an environment conducive to morphogenetic events. Loss of Frem2 function results in defects in developmental events associated with morphogenetic rearrangements of the vasculature and of tissues arising from all germ layers. The Frem2 transcript is restricted both spatially and temporally and appears in advance of cell rearrangement events. Thus, expression of Frem2 may dynamically alter the extracellular matrix to provide a substrate for cell migration and rearrangements during embryogenesis.

The growth improvement of porcine esophageal smooth muscle cells on collagen-grafted poly(DL-lactide-co-glycolide) membrane.

Abstract: Synthetic polyester and the extracellular matrix component collagen are among the most widely used materials in tissue engineering. However, the integration of collagen into polyester scaffolds without loss of its biological function is a problem that has not been fully solved. This article investigates the covalent immobilization of collagen onto poly(DL-Lactide-co-Glycolide) (PLGA) membrane surfaces via a bridge of 1,8-diaminooctane and with glutaraldehyde as crosslinking agent. X-ray photoelectron spectroscopy (XPS) and fluorescence measurements confirmed the presence of bonded collagen. The effect of collagen grafting on cell behavior was investigated by comparing collagen-PLGA with unmodified PLGA sample and tissue culture polystyrene (TCPS) plates by using porcine esophageal smooth muscle cells (ESMC). DNA analysis showed that collagen-modified PLGA improved the overall proliferation of the ESMCs compared with unmodified PLGA and TCPS plates. Cells seeded on collagen-modified PLGA also showed a more extended morphology. Thus, we believe that collagen-modified PLGA shows good potential to be used as a scaffold material for tissue engineering of the esophagus.

Mutual paracrine effects of colorectal tumour cells and stromal cells: modulation of tumour and stromal cell differentiation and extracellular matrix component production in culture.

Abstract: Interactions of tumour and stromal cells influence tumour cell proliferation and differentiation, stromal cell phenotypic transdifferentiation and secretion of extracellular matrix (ECM) components. In this study, we established a monolayer and a three-dimensional cell-to-cell interaction model between canine mammary stromal cells and human colonic carcinoma cell lines (Caco-2 and HT-29) to investigate mutual paracrine effects of tumour cells and stromal cells on (i) tumour cell differentiation, (ii) production of ECM components and (iii) phenotypic transdifferentiation of stromal cells. We showed that when Caco-2 or HT-29 cells are cultured in collagen gels, they form a few small solid cell clusters with no lumina, but when cocultured with stromal cells, the tumour cells formed glandular structures with central lumina. This fibroblast-induced organization and differentiation of Caco-2 cells (not HT-29 cells) appeared to be mediated by transforming growth factor-beta (TGF-beta). Culturing of stromal cells, Caco-2 cells or HT-29 cells alone in both monolayers and gels resulted in weak tenascin-C expression in stromal cells and HT-29 cells and no expression in the Caco-2 cells. Coculturing of stromal cells with tumour cells resulted in increased tenascin-C expression in the stromal cells and HT-29 cells and induced expression of tenascin-C in the Caco-2 cells. This induction and increased expression of tenascin-C appeared to be mediated by TGF-beta. Culturing of stromal cells, Caco-2 cells or HT-29 cells alone on monolayers and in gels resulted in a weak expression of chondroitin sulfate (CS), chondroitin-6-sulfate (C-6-S) and versican in stromal cells and no expression in Caco-2 and HT-29 cells. Coculturing of stromal cells with tumour cells on monolayers and in gels resulted in increased CS, C-6-S and versican expression in stromal cells. This tumour cell-induced expression of CS, C-6-S and versican appeared to be mediated by TGF-beta and platelet-derived growth factor (PDGF). Coculturing of Caco-2 and HT-29 and stromal cells promoted the transdifferentiation of stromal cells into myofibroblasts, and this appeared to be mediated by TGF-beta. These results suggest that TGF-beta and PDGF are part of a paracrine system involved in stromal-epithelial cell interaction important in stromal cell differentiation and ECM component production.

Serum laminin is an independent prognostic factor in colorectal cancer

Abstract: We investigated laminin, an important extracellular matrix component, to elucidate mechanisms of invasion and metastasis in colorectal cancer, and whether preoperative serum laminin is a predictive marker of high-risk groups. We measured preoperative serum laminin levels using a two-step sandwich enzymeimmunassay (EIA) method in 205 patients with colorectal cancer, 109 with colon cancer and 96 with rectal cancer, 52 with hepatic metastases, and 153 with no hepatic metastases. Mean serum laminin in patients with colon cancer was 606.3±260.2 ng/ml, significantly higher than that of 258.0±92.0 ng/ml in normal controls (P<0.0001). The positive rate was higher at 89.3% for laminin vs. 38.0% for carcinoembryonic antigen (CEA) and 19.5% for CA19-9. Mean serum laminin in patients with hepatic metastases was 668.0±274.7 ng/ml, significantly higher than that of 585.2±252.5 ng/ml in patients without hepatic metastases (P=0.0472). Survival rates were significantly lower in the high (≥520 ng/ml) than in the low laminin group (<350 ng/ml; P=0.0451). Univariate and multivariate analysis, using Cox’s proportional hazard regression model, showed serum laminin is an independent prognostic factor in colorectal cancer, along with hepatic, pulmonary and peritoneal metastases. Preoperative serum laminin levels are a useful predictive marker of high-risk groups in colorectal cancer.

Cyclic RGD Peptides With High Affinity for α5β1 Integrin Protect Genetically Fat Zucker Rat Livers From Cold Ischemia/Reperfusion Injury

Abstract: We tested a hypothesis that interactions between fibronectin (FN), a key extracellular matrix component, and its integrin α5β1 receptor are important in the development of ischemia/reperfusion (I/R) injury of steatotic liver transplants. We examined the effect of a cyclic RGD peptide (cRGD), with high affinity for α5β1 integrin, in a well-established steatotic rat liver model of ex vivo cold ischemia followed by isotransplantation. In this model, cRGD peptides were administered through the portal vein of steatotic Zucker rat livers prior to and after cold ischemic storage. Lean Zucker recipients of fatty orthotopic liver transplants (OLTs) received an additional course of cRGD peptides 1 hour posttransplantation. cRGD peptide therapy significantly inhibited the recruitment of monocyte/macrophages, and repressed the expression of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ. Moreover, it resulted in selective inhibition of inducible nitric oxide synthase (iNOS), and MMP-9 expression. Importantly, cRGD peptide therapy improved the function and histologic preservation of steatotic liver grafts, extending their 14-day survival in lean recipients from 50% in untreated to 100% in cRGD-treated OLTs. Thus, cRGD peptide-mediated blockade of FN-α5β1 interaction protects against severe I/R injury otherwise experienced by steatotic OLTs.

Combined effects of estrogen and mechanical loading on anterior cruciate ligament fibroblast biosynthesis.

Abstract: For some time, estrogen has been suspected to play a negative role in anterior cruciate ligament (ACL) fibroblast biosynthesis; however, reports on this issue have been controversial. In a recent study, our group demonstrated a negative combined effect of estrogen and mechanical loading on the gene expression of major extracellular matrix component molecules in ACL fibroblasts.

v-Jun downregulates the alpha 2 (I) collagen target gene indirectly through Sp1/3.

Abstract: Transformation of chick embryo fibroblasts (CEFs) by the v-Jun oncoprotein correlates with a downregulation of the alpha 2 (I) collagen gene. To investigate whether this gene constitutes a direct target of v-Jun, an analysis of a large proximal fragment of the promoter, extending from position −1080 to +109, was performed. Transient transfections with −1080/+109 and deleted derivatives revealed that a short proximal fragment, −433/+11, is the target for repression by v-Jun. Extensive analysis, conducted in CEFs and in Sp1/3-deficient Drosophila SL2 cells, further showed that (i) high constitutive activity of −433/+11 requires a direct binding of the ubiquitous Sp1 and/or Sp3 transcription factors acting on two distinct motifs, that is, a proximal TCC-rich region and an upstream GC box, and that (ii) repression by v-Jun does not require any direct binding of the oncoprotein to the DNA, but an indirect binding within a v-Jun–Sp1/3–DNA chromatin-associated complex. This situation is reminiscent of a situation previously reported with the tata-less, SPARC (secreted protein, acidic, and rich in cysteine) target promoter that regulates the expression of another extracellular matrix component in the same model of cell transformation. Taken together, these data reinforce the view that, at least in CEFs, v-Jun downregulates a family of direct target genes by binding to the DNA indirectly through Sp1/3.

Anti-dermatopathy agent and skin care preparation for external use comprising the same

Abstract: PROBLEM TO BE SOLVED: To obtain an anti-dermatopathy agent reducing or ameliorating at least one or more dermatopathies of wrinkling caused by exposure to ultraviolet rays, pachymenia, sclerema, abnormal accumulation of an extracellular matrix component, formation of collagen crosslinking, and the like; and to provide a skin care preparation for external use, containing the agent as an active ingredient. SOLUTION: The anti-dermatopathy agent contains at least one or more kinds selected from extracts of plants selected from the group consisting of Chrysanthemum makinoi Matsum. et Nakai, Chrysanthemum boreale Makino, Chrysanthemum indicum L., Chrysanthemum pacificum, Chrysanthemum coronarium, Chrysanthemum leucanthemum L., and Chrysanthemum parthenium Peres. The skin care preparation for external use contains the agent as the active ingredient. COPYRIGHT: (C)2006,JPO&NCIPI

Method of treating cells for transplantation, cell suspension, prosthesis for transplantation and method of treating injured site

Abstract: It is intended to technically provide a prosthesis for transplantation aiming at quickly and favorably repairing a transplanted tissue or an injured biological tissue (in particular, tendon, ligament, skin, cartilage or bone) and a cell suspension. To achieve the above object, connective tissue cells such as fibroblasts are cultured in a medium containing at least one factor belonging to the TGF-β superfamily (in particular, TGF-β1) and thus the above-described prosthesis for transplantation and the cell suspension are obtained. In the case of the prosthesis for transplantation, in particular, the connective tissue cells are embedded in an extracellular matrix component such as collagen, fibrin or hyaluronic acid (in particular, fibrin) to give a prosthesis for transplantation in the form of a sheet. This prosthesis for transplantation is used in coating a transplanted tissue or an injured tissue site.

Heparin Modulates Human Osteosarcoma (MG-63) Activities on Collagen-Coated Substrata

Abstract: Collagen, the major extracellular matrix component, is a popular biomaterial due to its excellent biocompatibility. Collagen is widely used in sutures, corneal bandages, drug delivery gels, and as wound dressings, as well as in a newly emerging interdisciplinary biomedical field, tissue engineering. A better understanding of the interaction between cells and collagen substrata combining other ECM components should allow for a more rational scaffold design for tissue engineering. In this study, we explored the effects of heparin on the behavior of osteoblast-like cell lines (MG-63) on collagen-based substrates. Collagen mixture (0.1mg/mL) with heparin at different concentrations (0.02mg/mL, 0.1mg/mL, and 0.5mg/mL) was coated onto tissue culture polystyrene plates. We found that low concentration of heparin (0.02mg/mL) enhanced cell adhesion and spreading on collagen-coated plates, but did not influence cell growth and alkaline phosphatase activity. On the other hand, high doses of heparin (0.1 and 0.5mg/mL) decreased cell adhesion and spreading, as well as cell proliferation. Our results suggest that a small amount of heparin might support osteoblast attachment and spreading although high heparin concentration is not suitable for cell culture.

A Simple Apparatus and Process to Produce Novel Elastin Biomaterials

Abstract: Elastin is the extracellular matrix component responsible for elastic recoil of tissues such as blood vessels, lung, and skin. In our efforts to produce biomaterials for tissue repair, an apparatus and method for processing hot alkali derived porcine carotid elastin was developed. The apparatus and method was designed to remove water and condense the elastin fibrous network to improve mechanical strength and handling of NaOH extracted elastin. To achieve this, a sample holder and process was devised to cure elastin conduits in humidity, and temperature controlled environment in a nitrogen gas enriched atmosphere. Our efforts resulted in a novel biomaterial with significantly improved mechanical properties compared to non-cure processed, hot alkali isolated elastin.

Development and characterisation of 3D skeletal muscle constructs under defined mechanical regulation

Abstract: It has been shown that the IGF-I gene is spliced in response to mechanical signals producing forms of IGF-I which have different actions. To study the roles of environmental mechanical cues on gene programming and splicing in developing muscle, an in vitro 3D cell culture system was employed. C2C12 skeletal myoblasts were grown in 3D collagen cultures. The presence of this extracellular matrix component, the application of uniaxial strain (produced endogenously by the myoblasts) and a high concentration of cells was shown to enhance differentiation in these cultures. These differentiated myotube cultures were then subjected to different regimens of exogenous mechanical strain. IGF-IEa, which initiates the fusion of myoblasts to form myotubes, was found to be constitutively expressed in myoblasts and myotubes and its expression up-regulated by a single ramp stretch of one hour duration but reduced by repeated cyclical stretch. In contrast, MGF which is involved in the proliferation of mononucleated myoblasts, that are required for secondary myotube formation and to establish the muscle satellite (stem) cell pool, showed no significant constitutive expression in static cultures, but was up-regulated by a single ramp stretch and by cycling loading. The latter types of force simulate those generated in myoblasts by the first contractions of myotubes. These data indicate the importance of understanding the physiological signals that determine the ratios of splice variants of some growth factor/tissue factor genes in the early stages of development of skeletal muscle.