scholarly journals Molecular assembly, secretion, and matrix deposition of type VI collagen.

1986 ◽  
Vol 102 (3) ◽  
pp. 703-710 ◽  
Author(s):  
E Engvall ◽  
H Hessle ◽  
G Klier
Keyword(s):  
Extracellular Matrix ◽  
Culture Media ◽  
Molecular Assembly ◽  
Collagen Molecule ◽  
Type Vi Collagen ◽  
Cultured Fibroblasts ◽  
Intracellular Pool ◽  
Matrix Deposition ◽  
The Matrix ◽  
Tissue Form

Monoclonal antibodies reactive with the tissue form of type VI collagen were used to isolate the type VI collagen polypeptides from cultured fibroblasts and muscle cells. Two [35S]methionine-labeled polypeptides of 260 and 140 kD were found intracellularly, in the medium, and in the extracellular matrix of metabolically labeled cells. These polypeptides were disulfide cross-linked into very large complexes. The 260- and 140-kD polypeptides were intimately associated and could not be separated from each other by reduction without denaturation. In the absence of ascorbic acid, both polypeptides accumulated inside the cell, and their amounts in the medium and in the matrix were decreased. These results suggest that both the 260- and the 140-kD polypeptides are integral parts of the type VI collagen molecule. Examination of type VI collagen isolated from the intracellular pool by electron microscopy after rotary shadowing revealed structures corresponding to different stages of assembly of type VI collagen. Based on these images, a sequence for the intracellular assembly of type VI collagen could be discerned. Type VI collagen monomers are approximately 125 nm long and are composed of two globules separated by a thin strand. The monomers assemble into dimers and tetramers by lateral association. Only tetramers were present in culture media, whereas both tetramers and multimers were found in extracellular matrix extracts. The multimers appeared to have assembled from tetramers by end-to-end association into filaments that had prominent knobs and a periodicity of approximately 110 nm. These results show that, unlike other collagens, type VI collagen is assembled into tetramers before it is secreted from the cells, and they also suggest an extracellular aggregation mechanism that appears to be unique to this collagen.

scholarly journals Induction of fibronectin matrix assembly in human fibrosarcoma cells by dexamethasone.

1987 ◽  
Vol 104 (3) ◽  
pp. 601-610 ◽  
Author(s):  
P J McKeown-Longo ◽  
C A Etzler
Keyword(s):  
Extracellular Matrix ◽  
Surface Protein ◽  
Order Rate Constant ◽  
Binding Activity ◽  
Matrix Assembly ◽  
Cultured Fibroblasts ◽  
Fibrosarcoma Cells ◽  
The Matrix ◽  
Fibronectin Binding ◽  
Human Fibrosarcoma Cells

Previous studies have suggested that the assembly of fibronectin into the extracellular matrix of cultured fibroblasts is mediated by specific matrix assembly receptors that recognize a binding site in the amino terminus of the fibronectin molecule (McKeown-Longo, P.J., and D.F. Mosher, 1985, J. Cell Biol., 100:364-374). In the presence of dexamethasone, human fibrosarcoma cells (HT-1080) acquired the ability to specifically bind exogenous plasma fibronectin and incorporate it into a detergent-insoluble extracellular matrix. Dexamethasone-induced fibronectin binding to HT-1080 cells was time dependent, dose dependent, and inhibited by cycloheximide. Saturation binding curves indicated that dexamethasone induced the appearance of 7.7 X 10(4) matrix assembly receptors per cell. The induced receptors exhibited a dissociation constant (KD) for soluble fibronectin of 5.0 X 10(-8) M. In parallel experiments, normal fibroblasts exhibited 4.1 X 10(5) receptors (KD = 5.3 X 10(-8) M) per cell. In the presence of cycloheximide, the induced fibronectin-binding activity on HT-1080 cells returned to uninduced levels within 12 h. In contrast, fibronectin-binding activity on normal fibroblasts was stable in the presence of cycloheximide for up to 54 h. The first-order rate constant (Kt = 2.07 X 10(-4) min-1) for the transfer of receptor-bound fibronectin to extracellular matrix was four- to fivefold less than that for normal fibroblasts (Kt = 1.32 X 10(-3) min-1). Lactoperoxidase-catalyzed iodination of HT-1080 monolayers indicated that a 48,000-mol-wt cell surface protein was enhanced with dexamethasone. The results from these experiments suggest that dexamethasone induces functional matrix assembly receptors on the surface of HT-1080 cells; however, the rate of incorporation of fibronectin into the matrix is much slower than that of normal fibroblasts.

scholarly journals Binding and degradation of platelet thrombospondin by cultured fibroblasts.

1984 ◽  
Vol 98 (1) ◽  
pp. 22-28 ◽  
Author(s):  
P J McKeown-Longo ◽  
R Hanning ◽  
D F Mosher
Keyword(s):  
Extracellular Matrix ◽  
Cell Layer ◽  
Extracellular Fluid ◽  
Human Platelets ◽  
Lung Fibroblasts ◽  
Human Embryonic Lung ◽  
Cultured Fibroblasts ◽  
The Matrix ◽  
Specific Receptors ◽  
Or Organization

Thrombospondin was purified from human platelets and labeled with 125I, and its metabolism was quantified in cell cultures of human embryonic lung fibroblasts. 125I-Thrombospondin bound to the cell layer. The binding reached an apparent steady state within 45 min. Trichloroacetic acid-soluble radioactivity was detected in the medium after 30 min of incubation; the rate of degradation of 125I-thrombospondin was linear for several hours thereafter. Degradation of 125I-thrombospondin was saturable. The apparent Km and Vmax for degradation at 37 degrees C were 6 X 10(-8) M and 1.4 X 10(5) molecules per cell per minute, respectively. Degradation was inhibited by chloroquine or by lowering the temperature to 4 degrees C. Experiments in which cultures were incubated with thrombospondin for 45 min and then incubated in medium containing no thrombospondin revealed two fractions of bound thrombospondin. One fraction was localized by indirect immunofluorescence to punctate structures; these structures were lost coincident with the rapid degradation of 50-80% of bound 125I-thrombospondin. The second fraction was localized to a trypsin-sensitive, fibrillar, extracellular matrix. 125I-Thrombospondin in the matrix was slowly degraded over a period of hours. Binding of 125I-thrombospondin to the extracellular matrix was not saturable and indeed was enhanced at thrombospondin concentrations greater than 3 X 10(-8) M. The ability of 125I-thrombospondin to bind to extracellular matrix was diminished tenfold by limited proteolytic cleavage with trypsin. Degradation of trypsinized 125I-thrombospondin was also diminished, although to a lesser extent than matrix binding. Heparin inhibited both degradation and matrix binding. These results suggest that thrombospondin may play a transitory role in matrix formation and/or organization and that specific receptors on the cell surface are responsible for the selective removal of thrombospondin from the extracellular fluid and matrix.

scholarly journals Modulation of collagen synthesis by a growth factor and by the extracellular matrix: comparison of cellular response to two different stimuli.

1983 ◽  
Vol 97 (3) ◽  
pp. 803-809 ◽  
Author(s):  
S C Tseng ◽  
N Savion ◽  
D Gospodarowicz ◽  
R Stern
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Cellular Response ◽  
Collagen Synthesis ◽  
Basal Surface ◽  
Matrix Deposition ◽  
The Matrix ◽  
The Media ◽  
In Cells

Cultured bovine corneal endothelial cells can be grown in three ways: on plastic, on plastic with fibroblast growth factor present in the media, and on their own preformed extracellular matrix. On plastic alone, cells grow in a disorderly fashion and secrete matrix on all cell surfaces. Cells grown on plastic with growth factor or on a matrix, at confluence, have matrix deposition only on the basal surface of the cells and an orderly contact-inhibited pattern of growth. This correlates with the polarity they demonstrate histologically. This cell-matrix pattern resembles the pattern observed in vivo. Both the soluble growth factor and the extracellular matrix are able to modulate the pattern of collagen synthesis and deposition by cells, but they do so in two entirely different ways. In cells grown on the extracellular matrix, total collagen synthesis is lower but more efficient. Collagen is deposited primarily into the cell layer even at the early sparse stage of culture. In cells grown on plastic with growth factor in the media, collagen is initially secreted into the media and does not become incorporated into the matrix. The deposition of collagen on the basal surface of cell occurs only late in the culture, and is achieved by increments in a stepwise manner. The in vivo-like pattern is not manifest until confluence has been reached. Thus, the extracellular matrix functions not only as a structural support, but is also instructional to the cells plated on it. In this case, the matrix regulates the level of collagen synthesis in the cells and modulates the pattern of collagen deposition. Soluble growth factors may act in part by enhancing a cell's ability to elaborate an appropriate matrix pattern necessary for the cell's own growth and accurate function.

scholarly journals Type VI collagen and glycoprotein MFPI are distinct components of the extracellular matrix

1986 ◽  
Vol 236 (1) ◽  
pp. 299-302 ◽  
Author(s):  
S Ayad ◽  
C A Chambers ◽  
L Berry ◽  
C A Shuttleworth ◽  
M E Grant
Keyword(s):  
Extracellular Matrix ◽  
Physicochemical Characteristics ◽  
Type Vi Collagen ◽  
Cultured Fibroblasts

Two collagenous glycoproteins, Mr 140,000 and Mr 150,000, are synthesized and secreted into the medium of cultured fibroblasts. The glycoprotein of Mr 140,000 is identical with the 140K(VI) component of type VI collagen by both immunological and physicochemical criteria. The glycoprotein of Mr 150,000 is immunologically distinct and exhibits the physicochemical characteristics of the putative elastic microfibrillar glycoprotein MFPI.

scholarly journals The role of intermolecular disulfide bonding in deposition of GP140 in the extracellular matrix.

1984 ◽  
Vol 99 (1) ◽  
pp. 105-114 ◽  
Author(s):  
W G Carter
Keyword(s):  
Extracellular Matrix ◽  
Culture Media ◽  
Molecular Forms ◽  
Cell Protein ◽  
Cell Contact ◽  
Relative Molecular Mass ◽  
Disulfide Bonding ◽  
Sequence Of Events ◽  
The Matrix ◽  
Matrix Precipitation

Human WI-38 fibroblasts in cultures synthesized at least three molecular forms of the major, extracellular matrix glycoprotein (GP), GP140: (a) cytoplasmic GP140 (1.2 ng of GP140/micrograms of cell protein) was detergent-soluble, underglycosylated, and possessed detectable levels of intermolecular disulfide bonding; (b) matrix GP140 (3.6 ng of GP140/micrograms of cell protein) was detergent-insoluble, more highly glycosylated and polymerized by intermolecular disulfide bonding, and co-distributed in the extracellular matrix with fibronectin; and (c) released GP140 (2 ng of GP140/micrograms of cell protein per 24 h) was recovered in the conditioned culture media and lacked intermolecular disulfide bonding. Cytoplasmic GP140 was the immediate biosynthetic precursor of the matrix form of GP140. In addition, various human adult and fetal tissues contained a form of GP140 that resembled the fibroblast matrix GP140 in the degree of intermolecular disulfide bonding, relative molecular mass, and immunological reactivity. Analysis of the sequence of events in assembly of GP140 and fibronectin in the extracellular matrix detected the following: (a) fibronectin was first to appear in the extracellular matrix; (b) GP140 accumulated in the cytoplasm, then deposited in the extracellular matrix and co-aligned with the established fibronectin; and (c) maturation of the extracellular matrix proceeded by continued intermolecular disulfide bonding. To evaluate possible roles for intermolecular disulfide bonding in cell interactions, a unique assay system was utilized based on the ability of labeled cells to incorporate radioactive matrix components into a biotinylated exogenous matrix. Precipitation of the biotinylated matrix from extracts of the cultures using avidin indicated: (a) disulfide bonding of radioactive GP140 and fibronectin into the exogenous biotinylated matrix required cell contact with the matrix. The newly deposited GP140 and fibronectin derived from the cells and not from GP140 and fibronectin present in the conditioned culture media. (b) Pro-alpha 1 and Pro-alpha 2 procollagens, present in the culture media, bound to the exogenous matrix in a noncovalent manner and were independent of cell contact. (c) SV40 transformed cells (WI-38 VA13) synthesized released form GP140 but did not deposit GP140 into the biotinylated matrix.

Abstract 359: The Production of Acellular Cardiac Extracellular Matrix from P3 Murine Heart

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Stefan M Kren ◽  
Daniel J Garry ◽  
Mary G Garry
Keyword(s):  
Extracellular Matrix ◽  
Cardiac Tissue ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Culture Media ◽  
Heart Tissue ◽  
Cell Nuclei ◽  
Dapi Staining ◽  
Neonatal Heart ◽  
The Matrix

Introduction: Understanding the role of extracellular matrix (ECM) in the creation of the cellular microenvironment during tissue formation and regeneration could be vital in extending this capability to injured adult tissue. To date the only confirmed mammalian heart tissue regeneration/regrowth has been in neonatal murine heart, with the regenerative capacity ceasing after day P3. By focusing on ECM from P3 heart tissue we hope to elucidate its contribution to regenerative plasticity and transfer this capacity to injured adult myocardium. Hypothesis: Detergent decellularization protocols used in adult tissues can be modified to function on a neonatal scale, serving to remove the cellular components of the neonatal heart, leaving structurally intact ECM to serve as a scaffold for the generation of cardiac tissue equivalents. Materials and Methods: Murine P3 hearts were perfused with 1% SDS in water at 20mm Hg for 12 hrs. Following detergent decellularization, perfusion with water, 1% Triton X-100, PBS and culture media restored biocompatibility to the isolated ECM. P1 and P7 primary cardiomyocytes expressing the mCherry red fluorescent protein reporter under control of the alpha myosin promoter were isolated by enzymatic disassociation and cultured in the heart matrix in a perfusion based bioreactor. Results: The decellularized ECM demonstrated removal of 97% of native DNA when compared to control by pico-green dsDNA binding assay. Histologic analysis demonstrated an absence of cell nuclei by H & E and DAPI staining. The preservation of the matrix structure and the maintenance of matrix immunoreactivity (collagen IV) were also demonstrated histologically. Following infusion of P1 or P7 mCherry positive cells, contractile behavior of the recellularized heart constructs was observed, and markers of cardiac linage (alpha-actinin in mCherry positive cells) were present. Conclusions: Neonatal heart matrix can be effectively decellularized. With appropriate modification of perfusion parameters, pediatric ECM structure can be preserved. This isolated matrix can serve as a scaffold for growth and maintenance of immature and mature cardiomyocytes, supporting continued contractility of cultured cells.

Synthesis of fibronectin and laminin by type II pulmonary epithelial cells

1996 ◽  
Vol 270 (2) ◽  
pp. L215-L223 ◽  
Author(s):  
S. E. Dunsmore ◽  
Y. C. Lee ◽  
C. Martinez-Williams ◽  
D. E. Rannels
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Primary Culture ◽  
Messenger Rna ◽  
Type Ii ◽  
Matrix Deposition ◽  
Pulmonary Epithelial Cells ◽  
The Matrix ◽  
Average Proportion ◽  
Type Ii Cell

Previous investigations demonstrated that type II pulmonary epithelial cells regulate extracellular matrix deposition as a function of time in primary culture. In those studies, the matrix fraction was analyzed as a whole. The present work focused on two components of the type II cell matrix, fibronectin and laminin. These glycoproteins have differing effects on differentiation of type II cells in primary culture. Fibronectin synthesis was quantitated between day 1 and day 6 in the cells, matrix, and medium; laminin synthesis was quantitated only in the cells. Although total fibronectin synthesis was regulated as a function of time in culture, reaching its greatest value on day 2, the average proportion of newly synthesized fibronectin in the cells (35%), medium (50%), and matrix (15%) remained constant over a 6-day interval. Between day 2 and day 6, the relative abundance of fibronectin messenger RNA increased 6.5-fold. Rates of cellular laminin synthesis did not vary with time in culture. These results support a role for differential regulation of fibronectin and laminin synthesis to determine the composition of the type II cell extracellular matrix.

Enhanced assembly of basement membrane matrix by endodermal cells in response to fibronectin substrata

1991 ◽  
Vol 99 (2) ◽  
pp. 443-451
Author(s):  
M.R. Austria ◽  
J.R. Couchman
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Basement Membranes ◽  
Binding Domain ◽  
Type I ◽  
Matrix Assembly ◽  
Matrix Deposition ◽  
Membrane Matrix ◽  
The Matrix

Basement membranes are complex extracellular matrices contributing to the regulation of growth, migration and differentiation of many cell types. However, little is known about the mechanisms regulating the deposition and assembly of basement membrane from its constituents. We have investigated the role of extracellular matrix molecules in the control of basement membrane matrix assembly by cultured endodermal (PFHR-9) cells. In the presence of fibronectin-depleted serum, substrata of fibronectin or laminin induced an increase in deposition of laminin, type IV collagen and proteoglycans by PFHR-9 cells, in comparison to cells adherent to type I collagen-coated, vitronectin-coated or uncoated substrata. Direct effects of fibronectin or laminin on the degree of cell spreading or rate of proliferation were not responsible for enhanced matrix deposition. The effect did not result from a redirection of basement membrane components to the matrix, since there was no decrease in matrix constituents released to the culture supernatants. Furthermore, the synthesis and release of other molecules that are not basement membrane constituents was unaltered in response to different extracellular matrix substrata. Experiments with fibronectin fragments showed that a 105 × 10(3) Mr ‘cell’-binding domain (containing the cell attachment sequence Arg-Gly-Asp-Ser) was an important contributor to enhanced matrix deposition, while the N-terminal 29 × 10(3) Mr heparin-binding domain also contributed to the effect, particularly with respect to heparan sulfate proteoglycan deposition. It seems that fibronectin has a dual role of action in promoting basement membrane matrix assembly, through direct cell surface interactions, and through the binding of fibronectin to other matrix components that may nucleate or stabilize the matrix assembly.

Extracellular matrix: the gatekeeper of tumor angiogenesis

2019 ◽  
Vol 47 (5) ◽  
pp. 1543-1555 ◽  
Author(s):  
Maurizio Mongiat ◽  
Simone Buraschi ◽  
Eva Andreuzzi ◽  
Thomas Neill ◽  
Renato V. Iozzo
Keyword(s):  
Extracellular Matrix ◽  
Tumor Angiogenesis ◽  
Signaling Cascades ◽  
Cancer Growth ◽  
Cell Behavior ◽  
Metastatic Progression ◽  
Surface Receptors ◽  
The Matrix ◽  
Multiple Signaling

Abstract The extracellular matrix is a network of secreted macromolecules that provides a harmonious meshwork for the growth and homeostatic development of organisms. It conveys multiple signaling cascades affecting specific surface receptors that impact cell behavior. During cancer growth, this bioactive meshwork is remodeled and enriched in newly formed blood vessels, which provide nutrients and oxygen to the growing tumor cells. Remodeling of the tumor microenvironment leads to the formation of bioactive fragments that may have a distinct function from their parent molecules, and the balance among these factors directly influence cell viability and metastatic progression. Indeed, the matrix acts as a gatekeeper by regulating the access of cancer cells to nutrients. Here, we will critically evaluate the role of selected matrix constituents in regulating tumor angiogenesis and provide up-to-date information concerning their primary mechanisms of action.

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