Drosophila PS integrins recognize vertebrate vitronectin and function as cell-substratum adhesion receptors in vitro

Development ◽  
1991 ◽  
Vol 113 (3) ◽  
pp. 1007-1016 ◽  
Author(s):  
S. Hirano ◽  
K. Ui ◽  
T. Miyake ◽  
T. Uemura ◽  
M. Takeichi
Keyword(s):  
Cell Attachment ◽  
Collagen Type I ◽  
Matrix Proteins ◽  
Type I ◽  
Rgd Peptides ◽  
Drosophila Cell ◽  
Type Iv ◽  
And Function

Using the Drosophila cell line MLDmBG-1, a monoclonal antibody aBG-1 that can inhibit not only cell clumping but also cell spreading was generated. This antibody immunoprecipitates a complex of molecules consisting of a major 120 × 10(3) Mr and other components. To characterize the 120 × 10(3) Mr component, we purified it, generated antibodies to it, and cloned its cDNA. Sequencing of this cDNA suggests that the 120 × 10(3) Mr molecule is identical to PS beta, a beta chain of Drosophila integrins. The other components immunoprecipitated included two alpha chains of Drosophila integrins, PS1 alpha and PS2 alpha, as revealed using specific antibodies to these molecules. These suggest that aBG-1 recognizes the PS beta associated with PS1 alpha or PS2 alpha. However, immunostaining of embryos and larvae with aBG-1 showed that the staining pattern is similar to that for PS2 alpha but not for PS beta, suggesting that the antibody preferentially recognizes the PS beta associated with particular alpha chains in situ. We then attempted to characterize the ligands for these integrin complexes, using culture dishes coated with various vertebrate matrix proteins. These cells spread very well on dishes coated with vitronectin and, to a lesser extent, on those with fibronectin. This spreading was partially inhibited by aBG-1, but not by other control antibodies or RGD peptides. The cell attachment to these substrata was not affected by the antibody. The cells also can attach to dishes coated with laminin but without spreading, and this attachment was not inhibited by aBG-1. Furthermore, they do not attach to dishes coated with collagen type I, type IV, and fibrinogen. These results indicate that Drosophila PS integrins can recognize vertebrate vitronectin, and also fibronectin with a weaker affinity, at sites other than RGD sequences, and thus can function in cell-substratum adhesion.

scholarly journals WNT-5A regulates TGF-β-related activities in liver fibrosis

2017 ◽  
Vol 312 (3) ◽  
pp. G219-G227 ◽  
Author(s):  
Leonie Beljaars ◽  
Sara Daliri ◽  
Christa Dijkhuizen ◽  
Klaas Poelstra ◽  
Reinoud Gosens
Keyword(s):  
Liver Fibrosis ◽  
Functional Role ◽  
Collagen Type ◽  
Collagen Type I ◽  
Matrix Proteins ◽  
Type I ◽  
Human Livers

WNT-5A is a secreted growth factor that belongs to the noncanonical members of the Wingless-related MMTV-integration family. Previous studies pointed to a connection between WNT-5A and the fibrogenic factor TGF-β warranting further studies into the functional role of WNT-5A in liver fibrosis. Therefore, we studied WNT-5A expressions in mouse and human fibrotic livers and examined the relation between WNT-5A and various fibrosis-associated growth factors, cytokines, and extracellular matrix proteins. WNT-5A gene and protein expressions were significantly increased in fibrotic mouse and human livers compared with healthy livers. Regression or therapeutic intervention in mice resulted in decreased hepatic WNT-5A levels paralleled by lower collagen levels. Immunohistochemical analysis showed WNT-5A staining in fibrotic septa colocalizing with desmin staining indicating WNT-5A expression in myofibroblasts. In vitro studies confirmed WNT-5A expression in this cell type and showed that TGF-β significantly enhanced WNT-5A expression in contrast to PDGF-BB and proinflammatory cytokines IL-1β and TNF-α. Additionally, TGF-β induces the expression of the WNT receptors FZD2 and FZD8. After silencing of WNT-5A, reduced levels of collagen type I, vimentin, and fibronectin in TGF-β-stimulated myofibroblasts were measured compared with nonsilencing siRNA-treated controls. Interestingly, the antifibrotic cytokine IFNγ suppressed WNT-5A in vitro and in vivo. IFNγ-treated fibrotic mice showed significantly less WNT-5A expression compared with untreated fibrotic mice. In conclusion, WNT-5A paralleled collagen I levels in fibrotic mouse and human livers. WNT-5A expression in myofibroblasts is induced by the profibrotic factor TGF-β and plays an important role in TGF-β-induced regulation of fibrotic matrix proteins, whereas its expression can be reversed upon treatment, both in vitro and in vivo. NEW & NOTEWORTHY This study describes the localization and functional role of WNT-5A in human and mouse fibrotic livers. Hepatic WNT-5A expression parallels collagen type I expression. In vivo and in vitro, the myofibroblasts were identified as the key hepatic cells producing WNT-5A. WNT-5A is under control of TGF-β and its activities are primarily profibrotic.

Bacterial Endotoxin Inhibits Migration, Attachment, and Orientation of Human Gingival Fibroblasts in vitro and Delays Collagen Gel Contraction

1987 ◽  
Vol 66 (9) ◽  
pp. 1449-1455 ◽  
Author(s):  
S. Pitaru ◽  
M. Soldinger ◽  
D. Madgar ◽  
Z. Metzger
Keyword(s):  
Collagen Type ◽  
Cell Attachment ◽  
Collagen Gel ◽  
Collagen Type I ◽  
Human Gingival Fibroblasts ◽  
Bacterial Endotoxin ◽  
Type I ◽  
Gingival Fibroblasts ◽  
Collagen Gels

The purpose of this study was to assess the effect of endotoxin adsorbed to dental surfaces and to collagen type I on the migration, attachment, and orientation of human gingival fibroblasts (HGF). Transversely cut porcine tooth root slices (RS), 200 μm thick, were prepared. Half of the RS obtained were partially demineralized in EDTA. Half of the demineralized and non-demineralized RS were incubated with 400 μg/mL of endotoxin for 24 hr, whereas the other half were maintained in PBS and served as controls. Experimental and control RS were placed on confluent layers of HFG and cultured for six days. Cell migration toward and cell attachment to the periphery of the RS and the formation of oriented cell sheets were assessed by means of photographic techniques. Additionally, six-day-old cultures were fixed and processed for SEM observation. In separate experiments, the effect of endotoxin on cell attachment to collagen type I and on contraction of three-dimensional collagen gels was assessed. It was found that: (i) bacterial endotoxin inhibited migration and attachment of HGF to both demineralized and non-demineralized cementum and interfered with the development of oriented cellular structure ; (ii) the inhibitory effect was significantly more pronounced for non-demineralized than for demineralized cementum; (iii) the morphology of HGF attached to endotoxin-treated dental surfaces was altered compared with that of their controls; and (iv) bacterial endotoxin inhibited cell attachment to collagen type I and delayed the contraction of collagen gel.

Type I collagen gene regulation and the molecular pathogenesis of cirrhosis

1993 ◽  
Vol 264 (4) ◽  
pp. G589-G595 ◽  
Author(s):  
D. A. Brenner ◽  
J. Westwick ◽  
M. Breindl
Keyword(s):  
Protein Interactions ◽  
Type I Collagen ◽  
Collagen Type ◽  
Collagen Type I ◽  
Molecular Pathogenesis ◽  
Matrix Proteins ◽  
Type I ◽  
And Function ◽  
Collagen Genes ◽  
Insight Into

Cirrhosis is characterized by an increased deposition of extracellular matrix proteins, including type I collagen. Type I collagen is a product of two genes, alpha 1(I) and alpha 2(I), which are generally coordinately regulated. Since expression of type I collagen genes is increased during cirrhosis, understanding the structure and function of the regulatory components of the type I collagen genes should provide insight into the molecular pathogenesis of cirrhosis. This review will analyze the collagen alpha 1(I) gene with respect to chromatin structure, DNA methylation, regulation by agonists, and DNA-protein interactions.

scholarly journals Correlation between cell substrate attachment in vitro and cell surface heparan sulfate affinity for fibronectin and collagen.

1983 ◽  
Vol 96 (6) ◽  
pp. 1820-1823 ◽  
Author(s):  
S C Stamatoglou ◽  
J M Keller
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Cell Attachment ◽  
Myeloma Cell ◽  
Collagen Type I ◽  
Type I ◽  
Heparan Sulfates ◽  
Sepharose 4B

Heparan sulfate glycosaminoglycan, isolated from the cell surface of nonadhering murine myeloma cells (P3X63-Ag8653), does not bind to plasma fibronectin, but binds partially to collagen type I, as assayed by affinity chromatography with proteins immobilized on cyanogen bromide-activated Sepharose 4B. Identical results were obtained when myeloma heparan sulfate was cochromatographed, on the same fibronectin and collagen columns, with cell surface heparan sulfates collagen columns, with cell surface heparan sulfates from adhering Swiss mouse 3T3 and SV3T3 cells. These latter heparan sulfates do, however, bind to both fibronectin and collagen, as reported earlier (Stamatoglou, S.C., and J.M. Keller, 1981, Biochim. Biophys. Acta., 719:90-97). Cell adhesion assays established that hydrated collagen substrata can support myeloma cell attachment, but fibronectin cannot. Saturation of the heparan sulfate binding sites on the collagen substrata with heparan sulfate or heparin, prior to cell inoculation, abolished the ability to support cell adhesion, whereas chondroitin 4 sulfate, chondroitin 6 sulfate, and hyaluronic acid had no effect.

scholarly journals Collagen metabolism in cultured osteoblasts from osteogenesis imperfecta patients

1992 ◽  
Vol 286 (1) ◽  
pp. 73-77 ◽  
Author(s):  
M Mörike ◽  
R E Brenner ◽  
G B Bushart ◽  
W M Teller ◽  
U Vetter
Keyword(s):  
Osteogenesis Imperfecta ◽  
Type I Collagen ◽  
Collagen Type ◽  
Bone Cells ◽  
Collagen Type I ◽  
Collagen Metabolism ◽  
Type I ◽  
Type Iii ◽  
Type Iv

Collagen produced in vitro by bone cells isolated from 19 patients with different forms of osteogenesis imperfecta (OI) was analysed. Clinically, four patients were classified as OI type I, 10 patients as OI type III and five patients as OI type IV. Bone cells of 12 of the 19 OI patients produced structurally abnormal type I collagen. Electrophoretically uniformly slower migrating collagen type I alpha-chains were found in one case of OI type I, in seven cases of OI type III and in one case of OI type IV; two cultures of OI type III produced two different populations of collagen type I alpha-chains, and one culture of OI type IV showed reduction-sensitive dimer formation of alpha 1(I) chains, resulting from the inadequate incorporation of a cysteine residue into the triple helical domain of alpha 1(I). Quantitative analysis of collagen metabolism led to the distinction of two groups of cultured OI osteoblasts. In osteoblasts of OI type I, mainly production of collagen was decreased, whereas secretion, processing and pericellular accumulation of (pro)collagen type I was similar to that in control osteoblasts. In contrast, in osteoblasts of OI types III and IV, production as well as secretion, processing and pericellular accumulation of (pro)collagen type I were significantly decreased. Low levels of type I collagen were found irrespective of the presence or absence of structural abnormalities of collagen type I in all OI types.

scholarly journals Evaluation of candidate markers for the peritubular myoid cell lineage in the developing mouse testis

Reproduction ◽  
2005 ◽  
Vol 130 (4) ◽  
pp. 509-516 ◽  
Author(s):  
Angela Jeanes ◽  
Dagmar Wilhelm ◽  
Megan J Wilson ◽  
Josephine Bowles ◽  
Peter J McClive ◽  
...  
Keyword(s):  
Early Stage ◽  
Cell Lineage ◽  
Collagen Type I ◽  
Marker Genes ◽  
Type I ◽  
Myoid Cell ◽  
Fetal Testis ◽  
And Function ◽  
Peritubular Myoid Cell

Despite the importance of peritubular myoid (PM) cells in the histogenesis of the fetal testis, understanding the origin and function of these cells has been hampered by the lack of suitable markers. The current study was aimed at identifying molecular markers for PM cells during the early stages of testis development in the mouse embryo. Expression of candidate marker genes was tested by section in situ hybridisation, in some instances followed by immunofluorescent detection of protein products. Collagen type-I, inhibinβA, caldesmon 1 and tropomyosin 1 were found to be expressed by early-stage PM cells. These markers were also expressed in subsets of interstitial cells, most likely reflecting their common embryological provenance from migrating mesonephric cells. Although not strictly specific for PM cells, these markers are likely to be useful in studying the biology of early PM cells in the fetal testis.

3D Model of Thrombopoiesis.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1609-1609 ◽  
Author(s):  
Isabella Pallotta ◽  
Michael L. Lovett ◽  
David L. Kaplan ◽  
Alessandra Balduini
Keyword(s):  
Bone Marrow ◽  
Type I Collagen ◽  
Blood Platelets ◽  
Type I ◽  
Type Iv ◽  
Vascular Niche ◽  
Factor Type ◽  
And Function ◽  
Von Willebrand

Abstract Abstract 1609 Background. The mechanisms that regulate megakaryocytic (Mk) development within the bone marrow environment remain poorly understood. The underlying relationships between Mk maturation and bone marrow components are key factors in this process. Mk development occurs in a complex microenvironment where extracellular matrices are fundamental regulatory components. The first events occur in the osteoblastic niche and include commitment of the hemopoietic progenitor cell to Mk, arrest of proliferation and initiation of endomitosis. The second step is Mk maturation and is associated with rapid cytoplasm expansion and intense synthesis of proteins. Finally Mks, which migrate to the vascular niche, convert the bulk of their cytoplasm into multiple long processes called proplatelets that protrude through the vascular endothelium into the sinusoid lumen, where the platelets are released. Hypothesis. The hypothesis for the present work is that a complex in vitro 3D bone marrow-like environment can be used to gain fundamental mechanistic insight into cell signalling and matrix-cell interactions in the bone marrow niche related to Mk development. Methods. We propose the first 3D model for Mk function in the bone marrow environment, by refining a recently proposed bioreactor platform (Lovett et al., 2007). These bioreactors consist of 3 wells (10 mm × 15 mm × 5 mm) within a PDMS block (25 mm × 60 mm × 5 mm) which is plasma bonded to cover glass for imaging. Each bioreactor well was perfused by 23 G stainless steel needles, spanned by porous silk microtubes as blood vessel scaffolds (640 μm inner diameter), positioned approximately 500–750 μm from the bottom of the bioreactor and connected to tubing for media perfusion using a programmable syringe pump. These microtubes were prepared by dipping several times straight lengths of stainless steel wire into 10–14% (w/v) aqueous silk fibroin to obtain blood vessel scaffolds with a wall thickness of around 50 mm. Defined pore sizes of 6–8 μm were obtained by adding 6 w/t % poly(ethylene oxide) (PEO) to the silk fibroin. The perfused silk tubes comprised the vascular niche and were embedded within a cell-seeded hydrogel which comprises the osteoblastic niche. The silk microtubes were coated with a combination of fibrinogen, von Willebrand Factor, type IV collagen and SDF-1 alpha, to better establish the composition of the vascular niche. Control experiments were performed by coating silk microtubes with type I collagen. After staining human umbilical cord blood derived Mks, the cell suspension was added to the hydrogel and Mk migration was analyzed in a time-dependent manner using confocal microscopy analysis. Further, flow effluent through the vascular tubes in the bioreactor was collected at regular time intervals and platelet numbers and function were analyzed by flow cytometry and microscopy. Culture released platelets were counted as CD61+ events with the same scatter properties of human blood platelets. Results. Our results showed that Mks migrated towards the vascular microtube coated with Fibrinogen, von Willebrand Factor, type IV collagen and SDF-1. Mks were also able to complete their maturation in the proximity of the microtube by extending proplatelets. Interestingly, confocal microscopy analysis revealed that Mks were able to extend proplatelets through the vascular microtube wall and release CD61+ platelet-like particles inside the vascular microtube. Cytofluorimentric analysis demonstrated that the particles collected in the flow effluent of the vascular microtube were CD61+ cells with the same scatter properties of human peripheral blood platelets. Finally, upon coating with only type I collagen Mks did not migrate towards the vascular microtube or extend proplatelets to release platelets. Thus, by mimicking the relationship between Mks and the bone marrow environment, a model to reproduce the different steps of Mk development, such as Mk migration, proplatelet formation and platelet release, is established. This is a first significant step towards relevant systems for the study of these cellular processes in detail as well as toward potentially useful in vitro platelet production systems. Conclusions. In this work we developed a new 3D bone marrow system in vitro that could represent a new tool to understand the mechanistic basis for Mk development and function, and the diseases related to these cells. Disclosures: No relevant conflicts of interest to declare.

The attachment of intrinsic and extrinsic, mobilized and immobilized adhesion cells to collagen and fibronectin

2011 ◽  
Vol 37 (6) ◽  
pp. 564-572 ◽  
Author(s):  
O. A. Branford ◽  
D. A. Lee ◽  
K. J. Rolfe ◽  
A. O. Grobbelaar
Keyword(s):  
Cell Attachment ◽  
Flexor Tendon ◽  
Zealand White Rabbit ◽  
Tendon Healing ◽  
Collagen Type I ◽  
Matrix Proteins ◽  
Type I ◽  
Tendon Cells ◽  
Flexor Tendon Injuries ◽  
Cell Groups

This study investigated the attachment of intrinsic and extrinsic, mobilized and immobilized adhesion cells to the extracellular matrix. Five New Zealand White rabbit forepaws were dissected to isolate the flexor tendon core, tendon surface and synovial sheath, which were explanted separately. A further 10 animals were subjected to flexor tendon injuries, randomized to either mobilization or immobilization, and adhesions were explanted at 2 weeks. Cell groups were tested for attachment to collagen type-I or fibronectin and morphometric analysis was made. The attachment of intrinsic tendon cells and adhesion cells from mobilized tendons to both matrix proteins was statistically significantly greater than that of extrinsic tendon cells and adhesion cells from immobilized tendons. Adhesion cells from mobilized tendons were statistically significantly more elongated, which may correlate with the deposition of a more organized matrix. Because the synovial sheath cells were least attached to matrix proteins, selective treatments that reduce cell attachment may be used to exclude them, without inhibiting intrinsic tendon healing.

Collagen Matrix Alignment Using Inkjet Printer Technology

2008 ◽  
Vol 1094 ◽  
Author(s):  
Sandra Deitch ◽  
Catherine Kunkle ◽  
Xiaofeng Cui ◽  
Thomas Boland ◽  
Delphine Dean
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Extra Cellular Matrix ◽  
Matrix Proteins ◽  
Type I ◽  
Alignment Technique ◽  
Inkjet Printer ◽  
Collagen Alignment

AbstractCollagen fiber orientation plays an important role in many cell properties and actions in vivo. Collagen and other matrix proteins are aligned in many tissues during normal functioning. For example, cardiomyocytes align in the heart to produce a synchronously beating tissue. The extra-cellular matrix environment, including collagen, is aligned along the cells. This matrix helps with cell adhesion and the alignment of the fibers also contributes to the anisotropic mechanical property of the tissue. While it is easy to replicate randomly oriented collagen in vitro, it is much more difficult to create aligned collagen matrices for cell culture. In this work, a novel inkjet printer-based collagen alignment technique was established. A 1 mg/ml rat tail collagen type I solution was printed, using a modified HP DeskJet 500 printer, onto plasma cleaned and UV sterilized glass slides. The collagen was printed in an eight line pattern, designed in Microsoft Word with 87.5 μm by 23.1 mm lines. The pattern was printed three successive times on each slide to complete the alignment. Immunofluorescence imaging of primary antibodies specific to collagen type I indicated that the heat involved in the printing process was not great enough to denature the collagen. The extent of collagen alignment was quantified using atomic force microscopy and compared to random collagen films and collagen films aligned using a mechanical scraping method. Additionally, neonatal rat cardiomyocytes were cultured on the aligned matrices. These cells require extracellular matrix alignment to maintain their in vivo-like phenotype during in vitro culture. The cells grew along the lines of collagen and coordinated beating, indicating the success of the aligned matrix. This collagen alignment technique is cheap, fast, precise, and easy to use in comparison to other current techniques. It can be used to align collagen on any type of substrate, such as a gel, which makes it a useful tool in many applications. This technique may also be used to align other extra-cellular matrix proteins and could even be used to create a three dimensional construct with varying fiber orientations.

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