scholarly journals Glycosaminoglycans facilitate the movement of fibroblasts through three-dimensional collagen matrices

1989 ◽  
Vol 92 (2) ◽  
pp. 263-270 ◽  
Author(s):  
R. Docherty ◽  
J.V. Forrester ◽  
J.M. Lackie ◽  
D.W. Gregory
Keyword(s):  
Cell Invasion ◽  
Type I Collagen ◽  
Chondroitin Sulphate ◽  
Three Dimensional ◽  
Collagen Matrix ◽  
Type I ◽  
Collagen Gels ◽  
Collagen Concentration ◽  
Collagen Matrices ◽  
Low Concentrations

The effect of glycosaminoglycans on the invasion of choroid fibroblasts into type I collagen gels was studied. Both hyaluronate and chondroitin sulphate, when incorporated into the gel, facilitated invasion of the collagen matrix, although hyaluronate was considerably more effective. Hyaluronate-induced fibroblast invasion was markedly concentration-dependent, being reduced at both high and low concentrations. Increased cell invasion appeared to correlate with denser packing of collagen fibrils within the gel, since the same effect could be achieved by increasing the collagen concentration of native, i.e. glycosaminoglycan-free gels. Scanning electron microscopy of the interior of the collagen gels suggested that changes in packing arrangement of fibrils in gels that had polymerized in the presence of glycosaminoglycans might account in part for different rates of cell invasion.

scholarly journals Inhibition of hydroxyapatite formation in collagen gels by chondroitin sulphate

1985 ◽  
Vol 228 (2) ◽  
pp. 463-469 ◽  
Author(s):  
G K Hunter ◽  
B L Allen ◽  
M D Grynpas ◽  
P T Cheng
Keyword(s):  
Type I Collagen ◽  
Chondroitin Sulphate ◽  
Collagen Gel ◽  
Calcium Pyrophosphate ◽  
Molar Ratio ◽  
Crystal Formation ◽  
Type I ◽  
Collagen Gels ◽  
High Concentrations ◽  
Hydroxyapatite Formation

Crystal growth in native collagen gels has been used to determine the role of extracellular matrix macromolecules in biological calcification phenomena. In this system, type I collagen gels containing sodium phosphate and buffered at pH 7.4 are overlayed with a solution containing CaCl2. Crystals form in the collagen gel adjacent to the gel-solution interface. Conditions were determined which permit the growth of crystals of hydroxyapatite [Ca10(PO4)6(OH)2]. At a Ca/P molar ratio of 2:1, the minimum concentrations of calcium and phosphate necessary for precipitation of hydroxyapatite are 10 mM and 5 mM, respectively. Under these conditions, precipitation is initiated at 18-24h, and is maximal between 24h and 6 days. Addition of high concentrations of chondroitin 4-sulphate inhibits the formation of hydroxyapatite in collagen gels; initiation of precipitation is delayed, and the final (equilibrium) amount of precipitation is decreased. Inhibition of hydroxyapatite formation requires concentrations of chondroitin sulphate higher than those required to inhibit calcium pyrophosphate crystal formation.

scholarly journals Thrombospondin gene expression by endothelial cells in culture is modulated by cell proliferation, cell shape and the substratum

1990 ◽  
Vol 268 (1) ◽  
pp. 225-230 ◽  
Author(s):  
A E Canfield ◽  
R P Boot-Handford ◽  
A M Schor
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Endothelial Cells ◽  
Cell Shape ◽  
Type I Collagen ◽  
Three Dimensional ◽  
Type I ◽  
Mrna Levels ◽  
Collagen Gels ◽  
Cells Cultured

Endothelial cells plated on the surface of a two-dimensional substratum (gelatin-coated dishes, dishes coated with native type I collagen or collagen gels) form a cobblestone monolayer at confluence, whereas cells plated within a three-dimensional gel matrix elongate into a sprouting morphology and self-associate into tube-like structures. In this study, we have compared the synthesis of thrombospondin by quiescent endothelial cells displaying (a) the same morphological phenotype (cobblestone) on different substrata (gelatin and collagen) and (b) different morphological phenotypes (cobblestone and sprouting) on the same substratum (collagen). We demonstrate that thrombospondin is a major biosynthetic product of confluent, quiescent cells cultured on dishes coated with either gelatin or collagen, and that the synthesis of this protein is markedly decreased when cells are plated on or in three-dimensional collagen gels. Moreover, we demonstrate that cells plated in gel (sprouting) secrete less thrombospondin than do cells plated on the gel surface (cobblestone). The regulation of thrombospondin synthesis is reversible and occurs at the level of transcription, as steady-state mRNA levels for thrombospondin decrease in a manner comparable with the levels of protein secreted by these cells. We also show that mRNA levels for laminin B2 chains are increased when cells are cultured on and in collagen gels compared with on gelatin-coated dishes, suggesting that the syntheses of thrombospondin and laminin are regulated by different mechanisms. When cells are cultured on gelatin- or collagen-coated dishes, thrombospondin gene expression is directly proportional to the proliferative state of the cultures. By contrast, the synthesis of thrombospondin by cells cultured on collagen gels remains at equally low levels whether they are labelled when they are sparse and rapidly proliferating or when they are confluent and quiescent. Fibronectin synthesis was found to increase with increasing confluency of the cells plated on all three substrata. These results demonstrate that thrombospondin gene expression is modulated by cell shape, cell proliferation and the nature of the substratum used for cell culture.

scholarly journals Arg-Gly-Asp-containing peptides expose novel collagen receptors on fibroblasts: implications for wound healing.

1991 ◽  
Vol 2 (12) ◽  
pp. 1035-1044 ◽  
Author(s):  
M V Agrez ◽  
R C Bates ◽  
A W Boyd ◽  
G F Burns
Keyword(s):  
Extracellular Matrix ◽  
Wound Healing ◽  
Type I Collagen ◽  
Regulatory Control ◽  
Type I ◽  
Collagen Gels ◽  
Collagen Matrices ◽  
Surface Receptors ◽  
Rgd Sequence ◽  
Collagen Receptors

Integrins are a family of cell-surface receptors intimately involved in the interactions of cells with their extracellular matrix. These receptors comprise an alpha and beta subunit in noncovalent association and many have been shown to recognize and bind an arginine-glycine-aspartate (RGD) sequence contained within their specific extracellular matrix ligand. Fibroblasts express integrin receptors belonging to two major subfamilies. Some of the members within the subfamily defined by beta 1 (VLA) are receptors for collagen but, perhaps surprisingly, the other major subfamily of integrins on fibroblasts--that defined by the alpha chain of the vitronectin receptor, alpha v--all appear to bind primarily vitronectin and/or fibronectin. In the present study we show that RGD-containing peptides expose cryptic binding sites on the alpha v-associated integrins enabling them to function as collagen receptors. The addition of RGD-containing peptides to fibroblasts cultured on type I collagen induced dramatic cell elongation and, when the cells were contained within collagen matrices, the peptides induced marked contraction of the gels. These processes were inhibited by Fab fragments of a monoclonal antibody against an alpha v integrin. Also, alpha v-associated integrins from cell lysates bound to collagen I affinity columns in the presence, but not in the absence, of RGD-containing peptides. These data suggest a novel regulatory control for integrin function. In addition, because the cryptic collagen receptors were shown to be implicated in the contraction of collagen gels, the generation of such binding forces suggests that this may be the major biological role for these integrins in processes such as wound healing.

scholarly journals Pancreatic Cancer Cells Respond to Type I Collagen by Inducing Snail Expression to Promote Membrane Type 1 Matrix Metalloproteinase-dependent Collagen Invasion

2011 ◽  
Vol 286 (12) ◽  
pp. 10495-10504 ◽  
Author(s):  
Mario A. Shields ◽  
Surabhi Dangi-Garimella ◽  
Seth B. Krantz ◽  
David J. Bentrem ◽  
Hidayatullah G. Munshi
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Type I Collagen ◽  
Epithelial Mesenchymal Transition ◽  
Three Dimensional ◽  
Type I ◽  
Collagen Gels ◽  
Mesenchymal Transition ◽  
Pancreatic Cancer Cells ◽  
Membrane Type

Pancreatic ductal adenocarcinoma (PDAC) is characterized by pronounced fibrotic reaction composed primarily of type I collagen. Although type I collagen functions as a barrier to invasion, pancreatic cancer cells have been shown to respond to type I collagen by becoming more motile and invasive. Because epithelial-mesenchymal transition is also associated with cancer invasion, we examined the extent to which collagen modulated the expression of Snail, a well known regulator of epithelial-mesenchymal transition. Relative to cells grown on tissue culture plastic, PDAC cells grown in three-dimensional collagen gels induced Snail. Inhibiting the activity or expression of the TGF-β type I receptor abrogated collagen-induced Snail. Downstream of the receptor, we showed that Smad3 and Smad4 were critical for the induction of Snail by collagen. In contrast, Smad2 or ERK1/2 was not involved in collagen-mediated Snail expression. Overexpression of Snail in PDAC cells resulted in a robust membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14)-dependent invasion through collagen-coated transwell chambers. Snail-expressing PDAC cells also demonstrated MT1-MMP-dependent scattering in three-dimensional collagen gels. Mechanistically, Snail increased the expression of MT1-MMP through activation of ERK-MAPK signaling, and inhibiting ERK signaling in Snail-expressing cells blocked two-dimensional collagen invasion and attenuated scattering in three-dimensional collagen. To provide in vivo support for our findings that Snail can regulate MT1-MMP, we examined the expression of Snail and MT1-MMP in human PDAC tumors and found a statistically significant positive correlation between MT1-MMP and Snail in these tumors. Overall, our data demonstrate that pancreatic cancer cells increase Snail on encountering collagen-rich milieu and suggest that the desmoplastic reaction actively contributes to PDAC progression.

Sodium nitroprusside augments human lung fibroblast collagen gel contraction independently of NO-cGMP pathway

2000 ◽  
Vol 278 (5) ◽  
pp. L1032-L1038 ◽  
Author(s):  
X. D. Liu ◽  
C. M. Skold ◽  
T. Umino ◽  
J. R. Spurzem ◽  
D. J. Romberger ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Human Lung ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Lung Fibroblast ◽  
Type I ◽  
Collagen Gels ◽  
Human Lung Fibroblast ◽  
Collagen Gel Contraction ◽  
Cgmp Pathway

Nitric oxide (NO) relaxes vascular smooth muscle in part through an accumulation of cGMP in the target cells. We hypothesized that a similar effect may also exist on collagen gel contraction mediated by human fetal lung (HFL1) fibroblasts, a model of wound contraction. To evaluate this, HFL1 cells were cultured in three-dimensional type I collagen gels and floated in serum-free DMEM with and without various NO donors. Gel size was measured with an image analyzer. Sodium nitroprusside (SNP, 100 μM) significantly augmented collagen gel contraction by HFL1 cells (78.5 ± 0.8 vs. 58.3 ± 2.1, P < 0.01), whereas S-nitroso- N-acetylpenicillamine, 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride, NONOate, and N G-monomethyl-l-arginine did not affect the contraction. Sodium ferricyanide, sodium nitrate, or sodium nitrite was not active. The augmentory effect of SNP could not be blocked by 1 H-[1,2,4]-oxadiazolo-[4,3- a]-quinoxalin-1-one, whereas it was partially reversed by 8-(4-chlorophenylthio) (CPT)-cGMP. To further explore the mechanisms by which SNP acted, fibronectin and PGE2 production were measured by immunoassay after 2 days of gel contraction. SNP inhibited PGE2 production and increased fibronectin production by HFL1 cells in a concentration-dependent manner. CPT-cGMP had opposite effects on fibronectin and PGE2 production. Addition of exogenous PGE2 blocked SNP-augmented contraction and fibronectin production by HFL1 cells. Therefore, SNP was able to augment human lung fibroblast-mediated collagen gel contraction, an effect that appears to be independent of NO production and not mediated through cGMP. Decreased PGE2 production and augmented fibronectin production may have a role in this effect. These data suggest that human lung fibroblasts in three-dimensional type I collagen gels respond distinctly to SNP by mechanisms unrelated to the NO-cGMP pathway.

scholarly journals In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices.

1983 ◽  
Vol 97 (5) ◽  
pp. 1648-1652 ◽  
Author(s):  
R Montesano ◽  
L Orci ◽  
P Vassalli
Keyword(s):  
Endothelial Cells ◽  
Three Dimensional ◽  
Collagen Matrix ◽  
Collagen Gels ◽  
Collagen Matrices ◽  
Narrow Lumen ◽  
Capillary Endothelial Cells ◽  
Vitro Model

We have studied the behavior of cloned capillary endothelial cells grown inside a three dimensional collagen matrix. Cell monolayers established on the surface of collagen gels were covered with a second layer of collagen. This induced the monolayers of endothelial cells to reorganize into a network of branching and anastomosing capillary-like tubes. As seen by electron microscopy, the tubes were formed by at least two cells (in transverse sections) delimiting a narrow lumen. In addition, distinct basal lamina material was present between the abluminal face of the endothelial cells and the collagen matrix. These results showed that capillary endothelial cells have the capacity to form vessel-like structures with well-oriented cell polarity in vitro. They also suggest that an appropriate topological relationship of endothelial cells with collagen matrices, similar to that occurring in vivo, has an inducive role on the expression of this potential. This culture system provides a simple in vitro model for studying the factors involved in the formation of new blood vessels (angiogenesis).

scholarly journals Influence of collagen lattice on the metabolism of small proteoglycan II by cultured fibroblasts

1990 ◽  
Vol 269 (1) ◽  
pp. 149-155 ◽  
Author(s):  
H Greve ◽  
P Blumberg ◽  
G Schmidt ◽  
W Schlumberger ◽  
J Rauterberg ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Type I ◽  
Collagen Gels ◽  
Dermatan Sulphate ◽  
Cultured Fibroblasts ◽  
Monolayer Cultures ◽  
Collagen Lattice ◽  
Small Proteoglycan

Small dermatan sulphate proteoglycan II from cultured human skin fibroblasts interacts with type I collagen in vitro and in vivo. When fibroblasts are maintained in a type I collagen lattice the proteoglycan remains exclusively within the lattice, and its association with fibrils can be demonstrated immunocytochemically. On the basis of [35S]sulphate incorporation, small proteoglycan II comprises about 80% of total proteoglycans secreted by cells in monolayer culture. In a collagen lattice, fibroblasts down-regulate its synthesis to the level of large chondroitin sulphate/dermatan sulphate and of heparan sulphate proteoglycans, the synthesis of which remains unaffected. Compared with the product from monolayer cultures, small proteoglycan II from collagen gels contained a longer polysaccharide chain which is characterized by a larger proportion of disulphated and a smaller proportion of monosulphated glucuronic acid-containing disaccharides. The half-life varied between 60 and 110 h. It is suggested that the compositional differences between the proteoglycan from monolayer cultures and from cells in a collagen lattice are related to the slower intracellular trafficking of the proteoglycan under the latter culture conditions.

scholarly journals Physiological type I collagen organization induces the formation of a novel class of linear invadosomes

2012 ◽  
Vol 23 (2) ◽  
pp. 297-309 ◽  
Author(s):  
Amélie Juin ◽  
Clotilde Billottet ◽  
Violaine Moreau ◽  
Olivier Destaing ◽  
Corinne Albiges-Rizo ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Type I Collagen ◽  
Three Dimensional ◽  
Collagen Matrix ◽  
Matrix Metalloproteases ◽  
Collagen I ◽  
Matrix Metalloproteinase 2 ◽  
Type I ◽  
Fibrillar Collagen ◽  
Functional Relationships

Invadosomes are F-actin structures capable of degrading the matrix through the activation of matrix metalloproteases. As fibrillar type I collagen promotes pro-matrix metalloproteinase 2 activation by membrane type 1 matrix metalloproteinase, we aimed at investigating the functional relationships between collagen I organization and invadosome induction. We found that fibrillar collagen I induced linear F-actin structures, distributed along the fibrils, on endothelial cells, macrophages, fibroblasts, and tumor cells. These structures share features with conventional invadosomes, as they express cortactin and N-WASP and accumulate the scaffold protein Tks5, which proved essential for their formation. On the basis of their ability to degrade extracellular matrix elements and their original architecture, we named these structures “linear invadosomes.” Interestingly, podosomes or invadopodia were replaced by linear invadosomes upon contact of the cells with fibrillar collagen I. However, linear invadosomes clearly differ from classical invadosomes, as they do not contain paxillin, vinculin, and β1/β3 integrins. Using knockout mouse embryonic fibroblasts and RGD peptide, we demonstrate that linear invadosome formation and activity are independent of β1 and β3 integrins. Finally, linear invadosomes also formed in a three-dimensional collagen matrix. This study demonstrates that fibrillar collagen I is the physiological inducer of a novel class of invadosomes.

Stress-relaxation and contraction of a collagen matrix induces expression of TGF-β and triggers apoptosis in dermal fibroblasts

2000 ◽  
Vol 78 (4) ◽  
pp. 427-436 ◽  
Author(s):  
M Varedi ◽  
E E Tredget ◽  
A Ghahary ◽  
P G Scott
Keyword(s):  
Mechanical Properties ◽  
Growth Factor ◽  
Type I Collagen ◽  
Transforming Growth Factor ◽  
Collagen Matrix ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Collagen Matrices ◽  
Matrix Metalloproteinase 1 ◽  
Tgf Β1

Extracellular matrix serves as a scaffold for cells and can also regulate gene expression and ultimately cell behaviour. In this study, we compared the effects of three forms of type I collagen matrix, which differed only in their mechanical properties, and plastic on the expression of transforming growth factor-β1 (TGF-β1), matrix metalloproteinase-1 (collagenase), and type I collagen and on the growth and survival of human dermal fibroblasts. These effects were correlated with alterations in cell morphology and organization of intracellular actin. Cells in detached or stress-relaxed matrices were spherical, lacked stress fibres, and showed increased TGF-β1 mRNA compared to the cells in anchored collagen matrices or on plastic, which were polygonal or bipolar and formed stress fibres. The levels of TGF-β measured by bioassay were higher in detached and stress-relaxed collagen matrices, than in anchored collagen matrices. Cells on plastic contained little or no immunoreactive TGF-β, while most cells in collagen matrices were stained. The levels of collagenase mRNA were significantly higher in all the collagen matrix cultures compared to those on plastic, but there were no statistically significant differences between them. Levels of mRNA for procollagen type I were not significantly affected by culture in the collagen matrices. Apoptotic fibroblasts were detected by the TUNEL assay in detached (5.7%) and to a lesser extent in stress-relaxed (2.2%) matrices, but none were observed in anchored collagen matrices or on plastic. These results show that alterations in the mechanical properties of matrix can induce the expression of TGF-β and trigger apoptosis in dermal fibroblasts. They further suggest that inability to reorganize this matrix could be responsible for the maintenance of the fibroproliferative phenotype associated with fibroblasts in hypertrophic scarring. Key words: transforming growth factor-β, apoptosis, fibroblasts.

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