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The extracellular matrix (ECM) environment in connective tissues provides fibroblasts with a structural scaffold and modulates cell shape, but it also profoundly influences the fibroblast phenotype. Here we studied fibroblasts cultured in a three-dimensional network of native collagen, which was either mechanically stressed or relaxed. Mechanical load induces fibroblasts that synthesize abundant ECM and a characteristic array of cytokines/chemokines. This phenotype is reminiscent of late granulation tissue or scleroderma fibroblasts. By contrast, relaxed fibroblasts are characterized by induction of proteases and a subset of cytokines that does not overlap with that of mechanically stimulated cells. Thus, the biochemical composition and physical nature of the ECM exert powerful control over the phenotypes of fibroblasts, ranging from “synthetic” to “inflammatory” phenotypes. Interactions between fibroblasts and collagen fibrils are mostly mediated by a subset of β1 integrin receptors. Fibroblasts utilize α1β1, α2β1, and α11β1 integrins for establishing collagen contacts and transducing signals. In vitro assays and mouse genetics have demonstrated individual tasks served by each receptor, but also functional redundancy. Unraveling the integrated functions of fibroblasts, collagen integrin receptors, collagen fibrils, and mechanical tension will be important to understand the molecular mechanisms underlying tissue repair and fibrosis.
COX-2
cyclooxygenase-2
ECM
extracellular matrix
MCP-1/-3
monocyte chemoattractant protein-1/-3
MMP
matrix metalloproteinase
PAI
plasminogen activator inhibitor
Fibroblasts In Relaxed Versus Stressed Collagen Lattices
Tissue homeostasis is controlled by the interaction of resident cells with each other and with their extracellular environment. Cell–cell communication is achieved either by receptor–counter receptor binding or by the activity of diffusible mediators, for example growth factor, cytokines and chemokines, which act in paracrine or autocrine regulatory loops (Figure 1). These interactions are important and have been well characterized, but in this overview we will concentrate on the important regulatory functions of components of the extracellular matrix (ECM) and on the role of mechanical tension exerting stress on matrix structures and cells embedded therein. We will focus on dermal fibroblasts interacting with fibrillar type I collagen, which is the most abundant ECM component in connective tissues, and highlight how this interaction influences the genetic program of fibroblasts.
Figure 1Factors influencing tissue homeostasis. Cell phenotypes and biological responses are controlled by the interaction of one or several cell types via direct interaction or via soluble mediators and growth factors. Furthermore, some cell types, mainly mesenchymal in nature, interact with components of the ECM by way of specialized receptors, which are activated to elicit signal cascades that are particular to the individual ligand–receptor interaction; for example cell binding to collagen by integrin α1β1 elicits different responses than binding of collagen by α2β1 (see text). A further level of regulation is introduced by the physical nature of the ECM, for example by the mechanical load across the cell membrane.
To study fibroblast–collagen interactions in close to in vivo conditions, we as many other groups of researchers seed primary skin fibroblasts in three-dimensional collagen lattices. In this setting, the cells are surrounded on all surfaces by collagen fibrils, in contrast to monolayer cultures where one large part of the polarized cell is in contact with a rigid substrate of collagen monomers (no fibrils) and the other with culture medium. Casting the lattices in bacteriological dishes, which are not adhesive for either collagen fibrils nor cells, leads to relaxed lattices that float freely in the medium (Figure 2a) and shrink over time (
Integrin alpha 2 beta 1 is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils.
Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins.
). The result after 1 day is a compact disk-shaped tissue-like structure of about 1/10 of the original diameter with fibroblasts being densely packed in this reduced volume (Figure 2b).
Figure 2Fibroblasts in freely contracting and tethered collagen lattices. Shown are collagen lattice systems that either lack (b) or generate mechanical tension (c and d). Fibroblasts are lifted off the culture dish and mixed with neutralized collagen solution and fetal calf serum. (a) This mixture is cast in bacteriological (non-adhesive) dishes, resulting in a gel with collagen fibrils after about 30 minutes at 37°C. (b) Within 1 day, the cells attach to the fibrils and remodel the lattice to form a dense, tissue-like structure. This is a model system largely devoid of mechanical tension acting on the cells. (c) Shows a lattice that is identical in composition to (b), however, in (c) tension is generated by cells in the lattice being held attached to the nylon thread, which is placed at the inner perimeter of the dish. (d) The culture force monitor system is depicted. The rectangular lattice is seen between two bars on either long side of the gel. One of these bars is fixed, while the other one is connected to a force transducer, which records the forces over time.
In contrast, fibroblasts are subjected to mechanical tension when the matrix to which they adhere and on which they exert tractional forces, cannot be contracted, because it is tethered to a nylon ring placed inside the dish and lining the periphery (Figure 2c) (
). In another model, the lattice is cast between two rectangular bars to which it adheres. One bar is fixed, while the other one is connected to a force transducer capable of picking up the force generated by fibroblasts within the collagen matrix (
). We used the circular system (Figure 2c) to monitor changes in the gene expression profile and fibroblast morphology in comparison to the relaxed system (Figure 2b), and the culture force monitor (Figure 2d) to assess magnitude of forces generated (
To obtain a comprehensive list of genes activated by mechanical stress, we performed a cDNA microarray screen of fibroblasts cultured in stressed collagen lattices or in relaxed collagen lattices at 20 hours, when force generation in stressed fibroblasts had reached plateau levels and contraction in the relaxed system was maximal (
Induction of an “inflammatory” Fibroblast Phenotype in a Mechanically Relaxed Ecm Environment
Since long we know that taking fibroblasts from monolayer cultures to freely contracting, relaxed collagen lattices induces a pronounced change in the expression of selected genes. Cells undergo apoptosis by about the time when contraction is macroscopically terminated (
Downregulation of collagen synthesis in fibroblasts within three- dimensional collagen lattices involves transcriptional and posttranscriptional mechanisms.
Differential regulation of transcription and transcript stability of pro-alpha 1(I) collagen and fibronectin in activated fibroblasts derived from patients with systemic scleroderma.
). However, cells do not go into general quiescence, as seen by more than 20-fold induced synthesis and acitvation of matrix metalloproteinase (MMP)-1 (
The microarray analysis confirmed downregulation of collagen, induction of MMPs as well as of genes related to apoptosis, and furthermore indicated induction of transcripts associated with inhibition of proliferation and of a distinct subset of growth factors and stress response genes (
Expression of pro-inflammatory markers by human dermal fibroblasts in a three-dimensional culture model is mediated by an autocrine interleukin-1 loop.
). Among the latter were cyclooxygenase-2 (COX-2), IL-1 and IL-6, ICAM-1, keratinocyte growth factor and the mitochondrial superoxide dismutase. COX-2 was further studied because its relative induction was the highest in the entire screen, suggesting high prostaglandin synthesis in response to inflammatory mediators in this system. In a time course study, interestingly, COX-2 expression was similar to that of IL-1, and blocking IL-1 expression by IL-1 receptor antagonist abrogated COX-2 induction. This result suggested an autocrine IL-1 loop in fibroblasts in the relaxed system which is causally involved in COX-2 induction. From these and other data, we concluded that a mechanically relaxed collagen environment induces a phenotype in fibroblasts that is characterized by production of high IL-1 levels, which stimulate synthesis of IL-6 and COX-2 (Figure 3). These cells are clearly not the quiescent cell type often pictured, but by releasing immunomodulatory substances, they are active players in an inflammatory setting. Probably this phenotype is required during early phases of tissue repair.
Figure 3Fibroblasts are key modulators of inflammatory responses. Fibroblasts in contact with a relaxed three-dimensional collagen network produce abundant IL-1, which upregulates the production of further inflammatory mediators, for example IL-6 and COX-2, key mediators of inflammatory reactions. COX-2 induction is abrogated by inhibition of IL-1, indicating a hierarchy of cytokine action.
Induction of a “synthetic” Fibroblast Phenotype in an Ecm Environment Under Mechanical Tension
Fibroblasts cultured in a collagen lattice that is identical in composition but differs in that cells cannot contract the matrix because it is tethered to a ring placed at the periphery of the dish (Figure 2c) assume a different phenotype from those cultured in contracting lattices (
). Phase contrast inspection shows bipolar, elongated cells of almost 100 μm in length in contrast to the stellate appearance of fibroblasts in relaxed lattices. Most of these cells express α-smooth muscle actin-positive stress fibers that terminate in well-defined focal adhesions (
). This differentiated, specialized fibroblast cell type is not observed in contracting lattices, but is a result of forces acting across the cell membrane via transmembrane receptors, for example integrins.
In accordance with the morphology, the comparative microarray analysis revealed upregulation of α-smooth muscle actin and vinculin and further cytoskeletal and focal adhesion components. In agreement with previous findings (
), fibroblasts in stressed conditions proliferate and express proliferation-associated genes, and they synthesize elevated levels of numerous ECM components, including collagens, tenascin, and hyaluronan among others. In addition, protease inhibitors, such as plasminogen activator inhibitor (PAI)-1 and -2, some of the tissue inhibitors of matrix metalloproteinases and others were distinctly upregulated, creating an environment of ECM accumulation. In line with this observation, we detected upregulation of mediators that are known to stimulate ECM production, for example transforming growth factor-β1 and -β3, connective tissue growth factor and the closely related Cyr61, and monocyte chemoattractant protein-1 (MCP-1) (Figure 4).
Figure 4Induction of different fibroblast phenotypes by mechanical tension. Fibroblasts interact with surrounding collagen fibrils using mainly receptors of the β1 family of integrins. Lack of mechanical load in this system produces an “inflammatory” phenotype that is characterized by high expression of inflammatory mediators and proteases along with low proliferation. In contrast, mechanical load induces an “activated” proliferating fibroblast phenotype that synthesizes abundant ECM and protease inhibitors as well as fibrogenic mediators. Presumably, the relaxed fibroblast is encountered in early wound in loose forming granulation tissue. The latter, activated type is thought to reside in later stage wounds and in fibrotic skin.
Since the circular lattice system is not suited to measure the magnitude of forces that the cells generate in this system, we used a culture force monitor with a force transducer connected to one of the long sides of the rectangular lattice (Figure 2d). This detects reduction in gel width due to fibroblast-mediated lattice shrinkage, while the length remains constant (
). The forces produced by primary human skin fibroblasts were on the order of 1 mN (using 2.5 × 105 cells per ml and 1.75 mg collagen per ml), which is less than the forces developed by smooth muscle cells but more than mouse skin fibroblasts can mount.
In summary, fibroblasts in a three-dimensional collagen network under mechanical tension turn into an activated, matrix producing cell, a phenotype, which is required at later stages of wound repair. This phenotype could also be responsible for excessive matrix deposition in fibrotic diseases. It is further characterized by α-smooth muscle actin-positive stress fibers, reminiscent of the specialized myofibroblast, which is also seen in mid to late phases of healing wounds and in some fibrotic tissues (
Similar Characteristic Properties in Mechanically Stimulated Fibroblasts and Scleroderma Fibroblasts
Scleroderma is a chronic fibrosing disease that involves the skin but also many internal organs. It is characterized by excessive deposition of ECM, mainly collagen, throughout the entire dermis including the subcutaneous tissue (
), presumably by a combination of cell–cell contact with endothelial and immune cells and fibroproliferative, and fibrogenic mediators released by the environment. Our question was whether these activated scleroderma fibroblasts share phenotypic similarities with the mechanically stimulated fibroblasts seen in tethered collagen lattices. In the following, three examples will be outlined that confirm such similarities.
First, we demonstrated that connective tissue growth factor is abundantly expressed by fibroblasts in lesional skin, in particular by fibroblasts which also overexpress collagen type I (
). Connective tissue growth factor is a chemotactic, fibroproliferative and fibrogenic mediator, that strongly upregulates collagen production in fibrotic skin either following induction by transforming growth factor-β or on its own. Similar findings were reported by others (
). MCP-1 belongs to the family of C–C chemokines and is produced by a wide variety of cell types. It is a potent chemoattractant for monocytes and macrophages, which are recruited in vivo to sites of inflammation in many pathological conditions. We found abundant MCP-1 signals in scleroderma, but not in normal skin and moreover demonstrated that cultured scleroderma fibroblasts are more sensitive to added MCP-1 than control fibroblasts. Thus it is conceivable that in scleroderma, activated fibroblasts release high levels of MCP-1, to which they react in an autocrine fashion. In addition, MCP-1 recruits monocytes/macrophages, which further activate fibroblasts, for example by transforming growth factor-β, to produce more ECM. In this way, fibroblasts are thought to play an important role in modulating the inflammatory response in early phases of the disease, and that they contribute significantly to the sustained fibrotic tissue response. MCP-1 was also detected at elevated levels in the serum of scleroderma patients (
Augmented production of chemokines (monocyte chemotactic protein-1 (MCP- 1), macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta) in patients with systemic sclerosis: MCP-1 and MIP-1alpha may be involved in the development of pulmonary fibrosis.
The third example is PAI-2, which was among the transcripts most strongly induced by mechanical forces. It blocks the conversion of plasminogen to plasmin and thereby indirectly inhibits the activity of plasmin-activated MMPs in the extracellular space, resulting in decreased matrix degradation. Probably it is also involved in regulating the activity of matrix-bound cytokines. By regulating protease activity, PAI-2 is thought to modulate ECM metabolism and in particular to favor ECM accumulation. Interestingly, a similar plasmin inhibitor was detected at elevated levels in scleroderma fibroblasts, protease nexin-1, which, due to binding plasmin, may also inhibit MMP activation (
). Our findings demonstrated high PAI-2 RNA and protein levels in scleroderma fibroblasts, but immunoblotting and staining clearly showed that PAI-2 was confined to the cell cytoplasm (
). This result speaks against an extracellular MMP inhibitory activity, and at present it is unclear which function could be fulfilled by cytoplasmic PAI-2. Possibly it might be involved in the regulation of mRNA stability.
Role of Collagen-binding Integrin Receptors in Fibroblast–collagen Interactions
We think that the contact of cells with surrounding collagen is critical for the generation of the different phenotypes discussed above, and that this interaction is mediated by receptors of the β1 integrin family. Native collagens are recognized by integrins α1β1, α2β1, α10β1, and α11β1 (
). Of these four, fibroblasts express α2β1 and α11β1, which bind with relatively high affinity to collagen type I, and α1β1, which has a preference for basement membrane collagen IV (Table 1), while α10β1 is expressed in almost undetectable amounts. A combination of experimental approaches involving antibody perturbation (
Integrin alpha 2 beta 1 is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils.
Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins.
) showed that these two receptors are involved in different processes, sometimes acting antagonistically (Table 1). Integrin α1β1 is required for optimal fibroblast proliferation in culture (
Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins.
Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins.
Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins.
). The phenotype of both knockout mouse models is rather mild, indicating a backup system of functional compensation by other collagen-binding integrins. Functions of α11β1 are only recently emerging with roles in cell migration (
Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins.
Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins.
Integrin alpha 2 beta 1 is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils.
Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins.
Mice completely deficient in α2β1 live and reproduce, which came as a surprise in view of the manifold in vitro functions ascribed to this receptor. We therefore analyzed three different cell types in functional in vitro assays and in vivo. These are platelets, keratinocytes, and fibroblasts, which express abundant α2β1 and the function of which was thought to rely on α2β1.
Platelet function appeared normal, as no prolonged bleeding was observed in α2-deficient mice. Platelet counts were normal, but in vitro aggregation of platelets in response to collagen was delayed (
). This observation could be of clinical importance, if GPVI is targeted by antithrombotic therapy. Given that α2β1 levels vary considerably in healthy humans (
), such anti-GPVI treatment could have serious side effects in patients with low α2β1 expression.
In contrast to platelets and fibroblasts, epidermal keratinocytes express α2β1 as sole collagen receptor. Accordingly, primary keratinocytes isolated from α2β1 knockout mice clearly failed to adhere to either collagen I or IV in vitro (
). Surprisingly, closure of skin wounds in these mice proceeded at rates comparable to wild-type wounds. In addition, rates of wound re-epithelialization were not significantly altered, indicating that wound keratinocytes either up-regulate a collagen receptor that is normally not expressed, or that they do not migrate on collagens in the wound bed (M. Zweers, T. Krieg and B. Eckes, manuscript in preparation).
In contrast to keratinocytes, adhesion to collagen I by fibroblasts lacking α2β1 was slightly reduced in long-term assays, but short-term adhesion clearly required α2β1 (
) (Table 2). Blocking β1 subunit function in α2-deficient fibroblasts abrogated adhesion, while blocking α1 in these cells had no major effect. These results point to α11β1 as a compensatory receptor for collagen I in these assays. Interestingly, adhesion to collagen IV was found to require not only α1β1, but also α2β1. Thus, binding of fibroblasts to collagen I can proceed to some extent in the absence of the α2β1 that was so far considered the major receptor mediating this interaction. Can other fibroblast functions that were thought to require α2β1 be equally fulfilled without this receptor? We found that collagen lattice contraction by α2 null fibroblasts was delayed and impaired (Table 2), similar to contraction by fibroblasts treated with antibodies blocking α2 and β1 (
Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins.
). We think that these dynamic functions are impaired in fibroblasts lacking α2β1, because these cells display higher levels of active RhoA than wild type. A similar correlation was previously observed in fibroblasts expressing constitutively active RhoA, which were equally defective in collagen lattice contraction (
). We hypothesize that high levels of active RhoA inhibit regular focal adhesion turnover, resulting in impaired dynamic fibroblast functions which rely on fast formation and resolution of matrix attachment points.
Table 2Phenotype of primary fibroblasts derived from integrin α2β1-deficient mice
Comparing mice lacking integrin α2β1 or α1β1 clearly demonstrates that both receptors serve different functions, some of which can be compensated by each other or by α11β1. Analysis of mutants deficient in two α-subunits or even all three will enable to delineate the precise contribution of the individual receptors of the collagen-binding β1 integrins.
Conflict of Interest
The authors state no conflict of interest.
ACKNOWLEDGMENTS
We thank Daniela Kessler-Becker, Olaf Holtkötter, Frank Hirche, Toshiyuki Yamamoto, and Bernhard Nieswandt for fruitful discussion, Gabriele Scherr, Nicole Brüggenolte, and Monika Pesch for excellent technical assistance. These studies were supported by the Deutsche Forschungsgemeinschaft (SFB 589 and KR558/13), the Deutsche Zentrum für Luft- und Raumfahrt (50WB0321), the Center for Molecular Medicine in Cologne (B1, B6), and the Köln Fortune Program/Medical Faculty of the University of Cologne.
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Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro.
Downregulation of collagen synthesis in fibroblasts within three- dimensional collagen lattices involves transcriptional and posttranscriptional mechanisms.
Differential regulation of transcription and transcript stability of pro-alpha 1(I) collagen and fibronectin in activated fibroblasts derived from patients with systemic scleroderma.
Augmented production of chemokines (monocyte chemotactic protein-1 (MCP- 1), macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta) in patients with systemic sclerosis: MCP-1 and MIP-1alpha may be involved in the development of pulmonary fibrosis.
Expression of pro-inflammatory markers by human dermal fibroblasts in a three-dimensional culture model is mediated by an autocrine interleukin-1 loop.
Integrin alpha 2 beta 1 is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils.
Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins.
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