Human tracheobronchial epithelial cells direct migration of lung fibroblasts in three-dimensional collagen gels

1992 ◽  
Vol 262 (5) ◽  
pp. L535-L541 ◽  
Author(s):  
M. D. Infeld ◽  
J. A. Brennan ◽  
P. B. Davis
Keyword(s):  
Epithelial Cells ◽  
Type I Collagen ◽  
Three Dimensional ◽  
Cell Number ◽  
Tracheobronchial Tree ◽  
Lung Fibroblasts ◽  
Type I ◽  
Collagen Gels ◽  
Directed Migration ◽  
Fibroblast Migration

Normal airway morphogenesis and repair after injury depend in part on the interaction between the mesenchymal and epithelial cells in the tracheobronchial tree. We cultured human lung fibroblasts between layers of type I collagen gel and examined sections through these three-dimensional matrices to assess fibroblast migration. The migration assay used in these experiments allowed simultaneous assessment of directed and random fibroblast migration as well as cell number. We tested the hypothesis that human tracheobronchial epithelial (HTBE) cells direct the migration of fibroblasts. When fibroblasts were cultured alone, migration was nearly equivalent in the upper and lower collagen layers. When HTBE cells were plated on the upper collagen lattice, there was a net migration of fibroblasts toward the HTBE cells. The differential migration was evident early in culture but became maximal after 1 wk. Differences increased at higher HTBE cell inoculation densities. No epithelial chemokinetic or mitogenic influence was evident: total cell migration and total fibroblast number were not significantly different between the control and coculture sections. HTBE fibronectin production may contribute to directed migration because fibronectin, added to the upper lattice, reproduced a portion of the directed migration seen in coculture. Our data support the hypothesis that epithelial cells direct fibroblast migration.

Stress-Dependent Effects of Platelet-Derived Growth Factor-BB on Fibroblast Migration and Traction Differ in Collagen and Fibrin

2000 ◽  
Author(s):  
David I. Shreiber ◽  
Paul A. J. Enever ◽  
Robert T. Tranquillo
Keyword(s):  
Type I Collagen ◽  
Cell Behavior ◽  
Environmental Cues ◽  
Type I ◽  
Collagen Gels ◽  
Fibrin Gels ◽  
Fibroblast Migration ◽  
Tissue Equivalents ◽  
Stress Dependent ◽  
Statistical Conclusion

Abstract We used our novel assays of cell behavior in tissue equivalents to study the dose-response effects of PDGF-BB on RDF migration and traction in mechanically stressed and stress-free type I collagen and fibrin gels. PDGF-BB increased fibroblast migration significantly in all assays, but the effects on traction depended on the presence of stress and the nature of the ECM. PDGF-BB decreased fibroblast traction in stressed collagen gels, but increased traction in stress-free gels. No statistical conclusion could be inferred for stressed fibrin gels, and increasing PDGF-BB decreased traction in stress-free fibrin gels. These results demonstrate the complex response of fibroblasts to environmental cues, and point to opportunities to orchestrate cell behavior to affect the outcome of wound healing.

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 Expression of extracellular matrix components is regulated by substratum.

1990 ◽  
Vol 110 (4) ◽  
pp. 1405-1415 ◽  
Author(s):  
C H Streuli ◽  
M J Bissell
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Type I Collagen ◽  
Basement Membranes ◽  
Type I ◽  
Collagen Gels ◽  
Extracellular Matrix Components ◽  
Matrix Components ◽  
Cells Cultured

Reconstituted basement membranes and extracellular matrices have been demonstrated to affect, positively and dramatically, the production of milk proteins in cultured mammary epithelial cells. Here we show that both the expression and the deposition of extracellular matrix components themselves are regulated by substratum. The steady-state levels of the laminin, type IV collagen, and fibronectin mRNAs in mammary epithelial cells cultured on plastic dishes and on type I collagen gels have been examined, as has the ability of these cells to synthesize, secrete, and deposit laminin and other, extracellular matrix proteins. We demonstrate de novo synthesis of a basement membrane by cells cultured on type I collagen gels which have been floated into the medium. Expression of the mRNA and proteins of basement membranes, however, are quite low in these cultures. In contrast, the levels of laminin, type IV collagen, and fibronectin mRNAs are highest in cells cultured on plastic surfaces, where no basement membrane is deposited. It is suggested that the interaction between epithelial cells and both basement membrane and stromally derived matrices exerts a negative influence on the expression of mRNA for extracellular matrix components. In addition, we show that the capacity for lactational differentiation correlates with conditions that favor the deposition of a continuous basement membrane, and argue that the interaction between specialized epithelial cells and stroma enables them to create their own microenvironment for accurate signal transduction and phenotypic function.

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.

Human bronchial epithelial cells can contract type I collagen gels

1998 ◽  
Vol 274 (1) ◽  
pp. L58-L65 ◽  
Author(s):  
Xiangde Liu ◽  
Takeshi Umino ◽  
Marty Cano ◽  
Ronald Ertl ◽  
Tom Veys ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Type I Collagen ◽  
Bronchial Epithelial Cells ◽  
Image Analyzer ◽  
Type I ◽  
Collagen Gels ◽  
Human Bronchial Epithelial Cells ◽  
Maximal Contraction ◽  
Bronchial Epithelial ◽  
Contract Type

Fibroblasts can contract collagen gels, a process thought to be related to tissue remodeling. Because epithelial cells are also involved in repair responses, we postulated that human bronchial epithelial cells (HBECs) could cause contraction of collagen gels. To evaluate this, HBECs were plated on the top of native type I collagen gels and were incubated for 48 h. After this, the gels were released and floated in LHC-9-RPMI 1640 for varying times, and gel size was measured with an image analyzer. HBECs caused a marked contraction of the gels within 24 h; the area was reduced by 88 ± 4% ( P < 0.01). The degree of gel contraction was dependent on cell density; 12,500 cells/cm2 resulted in maximal contraction, and half-maximal contraction occurred at 7,500 cells/cm2. Contraction varied inversely with the collagen concentration (91 ± 1% with 0.5 mg/ml collagen vs. 43 ± 5% with 1.5 mg/ml collagen). In contrast to fibroblasts that contract gels most efficiently when cast into the gel, HBEC-mediated contraction was significantly ( P < 0.01) more efficient when cells were on top of the gels rather than when cast into the gels. Anti-β1-integrin antibody blocked HBEC-mediated contraction by >50%, whereas anti-α2-, anti-α3-, anti-αv-, anti-αvβ5-, anti-β2-, or anti-β4-integrin antibody was without effect. The combination of anti-β1-integrin antibody and an anti-α-subfamily antibody completely blocked gel contraction induced by HBECs. In contrast, anti-cellular fibronectin antibody did not block HBEC-induced gel contraction, whereas it did block fibroblast-mediated gel contraction. In summary, human airway epithelial cells can contract type I collagen gels, a process that appears to require integrins but may not require fibronectin. This process may contribute to airway remodeling.

Bradykinin augments fibroblast-mediated contraction of released collagen gels

2001 ◽  
Vol 281 (1) ◽  
pp. L164-L171 ◽  
Author(s):  
Tadashi Mio ◽  
Xiangde Liu ◽  
Myron L. Toews ◽  
Yuichi Adachi ◽  
Debra J. Romberger ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Type I Collagen ◽  
Inflammatory Diseases ◽  
Fetal Lung ◽  
Lung Fibroblasts ◽  
Type I ◽  
Dependent Manner ◽  
Collagen Gels ◽  
Protein Kinase C Inhibitors

Bradykinin is a multifunctional mediator of inflammation believed to have a role in asthma, a disorder associated with remodeling of extracellular connective tissue. Using contraction of collagen gels as an in vitro model of wound contraction, we assessed the effects of bradykinin tissue on remodeling. Human fetal lung fibroblasts were embedded in type I collagen gels and cultured for 5 days. After release, the floating gels were cultured in the presence of bradykinin. Bradykinin significantly stimulated contraction in a concentration- and time-dependent manner. Coincubation with phosphoramidon augmented the effect of 10−9 and 10−8 M bradykinin. A B2 receptor antagonist attenuated the effect of bradykinin, whereas a B1 receptor antagonist had no effect, suggesting that the effect is mediated by the B2 receptor. An inhibitor of intracellular Ca2+mobilization abolished the response; addition of EGTA to the culture medium attenuated the contraction of control gels but did not modulate the response to bradykinin. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase C inhibitors staurosporine and GF-109203X attenuated the responses. These data suggest that by augmenting the contractility of fibroblasts, bradykinin may have an important role in remodeling of extracellular matrix that may result in tissue dysfunction in chronic inflammatory diseases, such as asthma.

Cell-matrix and cell-cell interactions modulate apoptosis of bronchial epithelial cells

1997 ◽  
Vol 272 (1) ◽  
pp. L28-L37 ◽  
Author(s):  
K. Aoshiba ◽  
S. I. Rennard ◽  
J. R. Spurzem
Keyword(s):  
Epithelial Cells ◽  
Type I Collagen ◽  
Bronchial Epithelium ◽  
Cell Aggregation ◽  
Cell Number ◽  
Bronchial Epithelial Cells ◽  
Cell Interactions ◽  
Type I ◽  
Bronchial Epithelial ◽  
Cell Cell

Apoptosis is an important process maintaining cell number and tissue structure. To determine whether cell-extracellular matrix (ECM) and cell-cell interactions modulate apoptosis in bronchial epithelium, we cultured human bronchial epithelial cells in different conditions and evaluated the cells for apoptosis. We found that plating cells in conditions that prevent cell-ECM adhesion induced apoptosis. Plating cells on type I collagen, fibronectin, and biosynthesized matrix prevented apoptosis, due at least in part to integrin-mediated adhesion. When cells were cultured at high density but under conditions preventing cell-substratum adhesion, aggregation occurred. Apoptosis was inversely correlated with aggregation. Cell-cell adhesion in these conditions was mediated at least partly by integrins containing alpha v. Cell aggregation was not associated with activation of a signaling pathway that is usually activated by cell-ECM adhesion, phosphorylation of focal adhesion kinase, but was associated with Bcl-2 protein expression, consistent with the concept that Bcl-2 protects against apoptosis. We conclude that both cell-ECM and cell-cell interactions, likely mediated in part by integrins, modulate apoptosis in bronchial epithelium.

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 Phenotypic modulation of rat glomerular visceral epithelial cells by culture substratum.

1995 ◽  
Vol 5 (8) ◽  
pp. 1591-1599
Author(s):  
C K Abrass ◽  
A K Berfield
Keyword(s):  
Electron Microscopy ◽  
Epithelial Cells ◽  
Cellular Proliferation ◽  
Type I Collagen ◽  
Gene Activation ◽  
Random Pattern ◽  
Specific Gene ◽  
Type I ◽  
Collagen Gels ◽  
The Matrix

The interaction of cells with their supporting extracellular matrix influences cellular phenotype, cellular proliferation, protein synthetic profile, and specific gene activation. To examine the ability of culture substratum to modulate the phenotype expressed by glomerular epithelial cells (GEC) in culture, GEC were grown on plastic culture plates coated with collagen gels (Type I collagen, Vitrogen) or a complex matrix from the Englebreth-Holm-Swarm tumor (Matrigel). Cultures were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). On untreated plastic, GEC grew in a random pattern. Cells were flat and thin with many filamentous processes. When grown on collagen I gels, GEC grew to confluence as a tight monolayer with typical cobblestone appearance. These cells demonstrated surface microvilli and a central cilium. TEM showed an epithelial appearance with tight junctions. When plated on the surface of Matrigel, GEC formed nests of cells that gradually burrowed into the gel. Proliferation on this matrix was extremely slow. TEM demonstrated that there are surface projections that abut the matrix and that the nests of cells are hollow with a central lumen. SEM demonstrated nests of cells that formed a sphere. Surface microvilli were not as abundant as cells grown on Vitrogen, and cilia were not seen. Cells could be removed from one surface, plated onto another, and would shift phenotype to that observed for subcultures primarily plated onto that surface. Cells on each complex substrate, as well as GEC plated on tissue culture plates coated with individual matrix proteins were biosynthetically labeled with (35S)methionine. The profile and rate of protein synthesis were modified by the plating substrate. These observations demonstrate that rat GEC can be induced to display variable phenotypes in culture that are determined by the plating substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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