Synergistic neutrophil elastase-cytokine interaction degrades collagen in three-dimensional culture

2001 ◽  
Vol 281 (4) ◽  
pp. L868-L878 ◽  
Author(s):  
Y. K. Zhu ◽  
X. D. Liu ◽  
C. M. Sköld ◽  
T. Umino ◽  
H. J. Wang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Type I Collagen ◽  
Mononuclear Cells ◽  
Gelatin Zymography ◽  
Collagen Degradation ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Lung Fibroblasts ◽  
Type I ◽  
Collagen Gels ◽  
Hydroxyproline Content

Proteolytic degradation of extracellular matrix is thought to play an important role in many lung disorders. In the current study, human lung fibroblasts were cast into type I collagen gels and floated in medium containing elastase, cytomix (combination of tumor necrosis factor-α, interleukin-1β, and interferon-γ), or both. After 5 days, gel collagen content was determined by measuring hydroxyproline. Elastase alone did not result in collagen degradation, but in the presence of fibroblasts, elastase reduced hydroxyproline content to 75.2% ( P < 0.01), whereas cytomix alone resulted in reduction of hydroxyproline content to 93% ( P < 0.05). The combination of elastase and cytomix reduced hydroxyproline content to 5.2% ( P < 0.01). α1-Proteinase inhibitor blocked this synergy. Gelatin zymography and Western blot revealed that matrix metalloproteinase (MMP)-1, -3, and -9 were induced by cytomix and activated in the presence of elastase. Tissue inhibitor of metalloproteinase (TIMP)-1 and -2 were also induced by cytomix but were cleaved by elastase. We conclude that a synergistic interaction between cytomix and elastase, mediated through cytokine induction of MMP production and elastase-induced activation of latent MMPs and degradation of TIMPs, can result in a dramatic augmentation of collagen degradation. These findings support the notion that interaction among inflammatory mediators secreted by mononuclear cells and neutrophils can induce tissue cells to degrade extracellular matrix. Such a mechanism may contribute to the protease-anti-protease imbalance in emphysema.

Surfactant components modulate fibroblast apoptosis and type I collagen and collagenase-1 expression

2000 ◽  
Vol 279 (5) ◽  
pp. L950-L957 ◽  
Author(s):  
Luis Vázquez De Lara ◽  
Carina Becerril ◽  
Martha Montaño ◽  
Carlos Ramos ◽  
Vilma Maldonado ◽  
...  
Keyword(s):  
Growth Rate ◽  
Type I Collagen ◽  
Protein A ◽  
Surfactant Protein ◽  
Northern Blotting ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Lung Fibroblasts ◽  
Type I ◽  
Chromogenic Assay ◽  
Collagen Accumulation

During lung injury, fibroblasts migrate into the alveolar spaces where they can be exposed to pulmonary surfactant. We examined the effects of Survanta and surfactant protein A (SP-A) on fibroblast growth and apoptosis and on type I collagen, collagenase-1, and tissue inhibitor of metalloproteinase (TIMP)-1 expression. Lung fibroblasts were treated with 100, 500, and 1,000 μg/ml of Survanta; 10, 50, and 100 μg/ml of SP-A; and 500 μg/ml of Survanta plus 50 μg/ml of SP-A. Growth rate was evaluated by a formazan-based chromogenic assay, apoptosis was evaluated by DNA end labeling and ELISA, and collagen, collagenase-1, and TIMP-1 were evaluated by Northern blotting. Survanta provoked fibroblast apoptosis, induced collagenase-1 expression, and decreased type I collagen affecting mRNA stability ∼10-fold as assessed with the use of actinomycin D. Collagen synthesis and collagenase activity paralleled the gene expression results. SP-A increased collagen expression ∼2-fold and had no effect on collagenase-1, TIMP-1, or growth rate. When fibroblasts were exposed to a combination of Survanta plus SP-A, the effects of Survanta were partially reversed. These findings suggest that surfactant lipids may protect against intraluminal fibrogenesis by inducing fibroblast apoptosis and decreasing collagen accumulation.

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 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 Micromechanics of cellularized biopolymer networks

2015 ◽  
Vol 112 (37) ◽  
pp. E5117-E5122 ◽  
Author(s):  
Christopher A. R. Jones ◽  
Matthew Cibula ◽  
Jingchen Feng ◽  
Emma A. Krnacik ◽  
David H. McIntyre ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Optical Tweezers ◽  
Collagen Fiber ◽  
Type I Collagen ◽  
Qualitative Difference ◽  
Type I ◽  
Dependent Manner ◽  
Adhesion Forces ◽  
Collagen Gels ◽  
Fiber Network

Collagen gels are widely used in experiments on cell mechanics because they mimic the extracellular matrix in physiological conditions. Collagen gels are often characterized by their bulk rheology; however, variations in the collagen fiber microstructure and cell adhesion forces cause the mechanical properties to be inhomogeneous at the cellular scale. We study the mechanics of type I collagen on the scale of tens to hundreds of microns by using holographic optical tweezers to apply pN forces to microparticles embedded in the collagen fiber network. We find that in response to optical forces, particle displacements are inhomogeneous, anisotropic, and asymmetric. Gels prepared at 21 °C and 37 °C show qualitative difference in their micromechanical characteristics. We also demonstrate that contracting cells remodel the micromechanics of their surrounding extracellular matrix in a strain- and distance-dependent manner. To further understand the micromechanics of cellularized extracellular matrix, we have constructed a computational model which reproduces the main experiment findings.

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.

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.

scholarly journals Evaluation of Dental Pulp Stem Cell Heterogeneity and Behaviour in 3D Type I Collagen Gels

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Amr Alraies ◽  
Rachel J. Waddington ◽  
Alastair J. Sloan ◽  
Ryan Moseley
Keyword(s):  
Dental Pulp ◽  
Type I Collagen ◽  
Gelatin Zymography ◽  
Population Doubling ◽  
Significant Heterogeneity ◽  
Type I ◽  
Collagen Gels ◽  
Phenotypic Properties ◽  
Cell Counts

Dental pulp stem cells (DPSCs) are increasingly being advocated for regenerative medicine-based therapies. However, significant heterogeneity in the genotypic/phenotypic properties of DPSC subpopulations exist, influencing their therapeutic potentials. As most studies have established DPSC heterogeneity using 2D culture approaches, we investigated whether heterogeneous DPSC proliferative and contraction/remodelling capabilities were further evident within 3D type I collagen gels in vitro. DPSC subpopulations were isolated from human third molars and identified as high/low proliferative and multipotent/unipotent, following in vitro culture expansion and population doubling (PD) analysis. High proliferative/multipotent DPSCs, such as A3 (30 PDs and 80 PDs), and low proliferative/unipotent DPSCs, such as A1 (17 PDs), were cultured in collagen gels for 12 days, either attached or detached from the surrounding culture plastic. Collagen architecture and high proliferative/multipotent DPSC morphologies were visualised by Scanning Electron Microscopy and FITC-phalloidin/Fluorescence Microscopy. DPSC proliferation (cell counts), contraction (% diameter reductions), and remodelling (MMP-2/MMP-9 gelatin zymography) of collagen gels were also evaluated. Unexpectedly, no proliferation differences existed between DPSCs, A3 (30 PDs) and A1 (17 PDs), although A3 (80 PDs) responses were significantly reduced. Despite rapid detached collagen gel contraction with A3 (30 PDs), similar contraction rates were determined with A1 (17 PDs), although A3 (80 PDs) contraction was significantly impaired. Gel contraction correlated to distinct gelatinase profiles. A3 (30 PDs) possessed superior MMP-9 and comparable MMP-2 activities to A1 (17 PDs), whereas A3 (80 PDs) had significantly reduced MMP-2/MMP-9. High proliferative/multipotent DPSCs, A3 (30 PDs), further exhibited fibroblast-like morphologies becoming polygonal within attached gels, whilst losing cytoskeletal organization and fibroblastic morphologies in detached gels. This study demonstrates that heterogeneity exists in the gel contraction and MMP expression/activity capabilities of DPSCs, potentially reflecting differences in their abilities to degrade biomaterial scaffolds and regulate cellular functions in 3D environments and their regenerative properties overall. Thus, such findings enhance our understanding of the molecular and phenotypic characteristics associated with high proliferative/multipotent DPSCs.

scholarly journals Calreticulin Regulates Transforming Growth Factor-β-stimulated Extracellular Matrix Production

2013 ◽  
Vol 288 (20) ◽  
pp. 14584-14598 ◽  
Author(s):  
Kurt A. Zimmerman ◽  
Lauren V. Graham ◽  
Manuel A. Pallero ◽  
Joanne E. Murphy-Ullrich
Keyword(s):  
Extracellular Matrix ◽  
Er Stress ◽  
Type I Collagen ◽  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Transforming Growth Factor Β ◽  
Lung Fibroblasts ◽  
Type I ◽  
Reporter Activity ◽  
Stimulation Of

Endoplasmic reticulum (ER) stress is an emerging factor in fibrotic disease, although precise mechanisms are not clear. Calreticulin (CRT) is an ER chaperone and regulator of Ca2+ signaling up-regulated by ER stress and in fibrotic tissues. Previously, we showed that ER CRT regulates type I collagen transcript, trafficking, secretion, and processing into the extracellular matrix (ECM). To determine the role of CRT in ECM regulation under fibrotic conditions, we asked whether CRT modified cellular responses to the pro-fibrotic cytokine, TGF-β. These studies show that CRT−/− mouse embryonic fibroblasts (MEFs) and rat and human idiopathic pulmonary fibrosis lung fibroblasts with siRNA CRT knockdown had impaired TGF-β stimulation of type I collagen and fibronectin. In contrast, fibroblasts with increased CRT expression had enhanced responses to TGF-β. The lack of CRT does not impact canonical TGF-β signaling as TGF-β was able to stimulate Smad reporter activity in CRT−/− MEFs. CRT regulation of TGF-β-stimulated Ca2+ signaling is important for induction of ECM. CRT−/− MEFs failed to increase intracellular Ca2+ levels in response to TGF-β. NFAT activity is required for ECM stimulation by TGF-β. In CRT−/− MEFs, TGF-β stimulation of NFAT nuclear translocation and reporter activity is impaired. Importantly, CRT is required for TGF-β stimulation of ECM under conditions of ER stress, as tunicamycin-induced ER stress was insufficient to induce ECM production in TGF-β stimulated CRT−/− MEFs. Together, these data identify CRT-regulated Ca2+-dependent pathways as a critical molecular link between ER stress and TGF-β fibrotic signaling.

Repair of critical size rat calvarial defects using extracellular matrix protein gels

1995 ◽  
Vol 83 (4) ◽  
pp. 710-715 ◽  
Author(s):  
Thomas M. Sweeney ◽  
Lynne A. Opperman ◽  
John A. Persing ◽  
Roy C. Ogle
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Bone Repair ◽  
Type I Collagen ◽  
Critical Size ◽  
Type I ◽  
Collagen Gels ◽  
Content Type ◽  
Calvarial Defects ◽  
Fine Print

✓ In this study the authors examined the capacity of gels of reconstituted basement membrane, laminin, and type I collagen to mediate repair of critical size defects in rat calvaria. Although autografts are widely used to repair bone defects caused by trauma or surgical treatment of congenital malformations, neoplasms, and infections, an adequate quantity of graft is not always available. Allogenic bone is readily available, but its use is associated with an increased incidence of nonunion, fatigue fracture, and rejection. Biologically active, purified components of basement membranes, which have been shown to promote osteogenic differentiation and angiogenesis in vitro and type I collagen (the major constituent of bone extracellular matrix) can be formed into native isotonic space-filling gels. In this study critical size calvarial defects were created in retired male Sprague-Dawley rats. Thirty-six animals were divided into seven groups. Group 1 (control) received no treatment for the defects. Group 2 animals were implanted with methylcellulose. Groups 3, 4, 5, and 6 were implanted with gels of type I collagen, reconstituted basement membrane, or laminin, respectively. The last group of three animals (Group 7) was implanted with 100 µg of type I collagen gels (identical to Group 3) and sacrificed at 20 weeks following a single CT scan to determine if complete healing could be obtained with this method given sufficient time. Except for rats in the type I collagen group that was evaluated by multiple computerized tomography (CT) scans biweekly from 2 to 12 weeks, bone repair was evaluated using CT at 12 weeks. Healing was quantified using three-dimensional reconstruction of CT. Following the final CT scan in each experimental group, animals were sacrificed, and a sample of tissues was evaluated by conventional histology. Animals treated with type I collagen gels showed 87.5% repair of the area of the defects at 12 weeks and 92.5% repair by 20 weeks. Increasing the gel volume 1.5 × accelerated complete repair to 3 months. Murine-reconstituted basement membrane and laminin gels induced 55.5% and 46.3% repair, respectively, at 3 months. In untreated control animals 7% repair of the area of the defects showed at 3 months. Histological analysis confirmed new bone formation in partial and completely healed defects. Bioengineered native collagen gels may have wide applicability for bone repair as an alternative bone graft material alone, in combination with autograft or marrow aspirate, or as a delivery system for osteogenic growth factors.

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