1 Department of Chemical and Biological Engineering, Northwestern University, Evanston, 633 Clark Street, Chicago, IL 60208, USA
2 Laboratory of Cell Biophysics, École Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
3 Integrative Biosciences Institute, École Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
* Author for correspondence (e-mail: melody.swartz{at}epfl.ch)
Accepted 1 August 2005
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Summary |
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Key words: Fibrosis, -Smooth muscle actin, Transforming growth factor ß, ß1 Integrin, Shear stress
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Introduction |
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Myofibroblasts are generally characterized by expression of -smooth muscle actin (
-SMA) protein, the actin isoform typical of smooth muscle cells, conferring a high contractile activity to these cells (Hinz et al., 2001a
), although
-SMA is not required for collagen gel contraction in vitro (Grinnell, 1994
; Hinz and Gabbiani, 2003a
; Vanni et al., 2003
). The primary inducer of fibroblast-to-myofibroblast differentiation is transforming growth factor ß1 (TGF-ß1) (Desmouliere et al., 1993
; Ronnov-Jessen and Petersen, 1993
), acting either via paracrine release by inflammatory, epithelial or tumor cells (Werner and Grose, 2003
) or via autocrine regulation (Kim et al., 1990
). Mechanical factors that either provide resistance to matrix contraction or exert tensional forces on the fibroblast cytoskeleton can also modulate fibroblast differentiation. For example, when wound granulation tissue fibroblasts were subjected to mechanical tension in vivo by immobilizing the edges of full-thickness wounds,
-SMA expression was upregulated; tension release by frame removal led to stress fiber disassembly and downregulation of
-SMA expression (Hinz et al., 2001b
). In vitro, fibroblasts cultured in three-dimensional (3D) collagen gels exhibit increasing levels of
-SMA expression with increasing matrix stiffness and/or externally applied stretch (Grinnell et al., 2003
) but do not differentiate in free-floating gels (Arora et al., 1999
). Thus, mechanical forces are strongly implicated in myofibroblast differentiation.
Here, we demonstrate that low levels of interstitial flow (i.e. fluid flow through a 3D matrix) can itself induce collagen alignment and fibroblast-to-myofibroblast transition via autocrine upregulation of TGF-ß1. We previously reported that human dermal fibroblasts align under interstitial flow in 3D collagen gel cultures, perpendicular to the direction of flow (Ng and Swartz, 2003). As aligned fibroblasts and matrix fibers are often seen in wound and fibrotic tissues (Darby et al., 1990
; Hinz et al., 2001b
), we proposed that interstitial flow could itself contribute to fibrosis even in the absence of inflammatory cells as observed in idiopathic pulmonary fibrosis (Pardo and Selman, 2002
; Thannickal et al., 2004
). Interstitial flow is present in soft tissues as an important component of the microcirculation between blood and lymphatic vessels, and interstitial flow is increased during events such as inflammation and wound healing where an influx of inflammatory cells and active angiogenesis both contribute to increased fluid flux into the surrounding tissues. The levels of flow we imposed reflect probable pathological values, as they were three to ten times higher than those reported for normal tissue (Chary and Jain, 1989
). In the context of desmoplastic stroma, the high interstitial pressure of tumors may lead to an increased outflow of tumor interstitial fluid into the stromal tissues surrounding the tumors (Heldin et al., 2004
; Jain, 2001
; Swabb et al., 1974
). Thus, our findings suggest that the biomechanical environment associated with inflammation (which is accompanied by cytokines), vascularized tumors, remodeling blood vessels or increased lymphatic flow (which is not necessarily associated with cytokines), can itself stimulate myofibroblast differentiation.
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Materials and Methods |
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Preparation of fibroblast-populated matrices and application of interstitial flow
Collagen gels (2 mg/ml), seeded with 5x105 fibroblasts/ml, were cast in an interstitial flow chamber (Fig. 1A) as previously described (Ng and Swartz, 2003). The set-up was immersed in media overnight for cell attachment at 37°C, 5% CO2 in an incubator. For the induction of flow, the chamber was connected to a reservoir of growth medium via a peristaltic pump and a pressure manometer. The flow was delivered at 0.012 ml/minute, leading to a weighted average velocity of 6.3 µm/second. Medium surrounded the chamber and could diffuse through the outer and inner PE rings. Two static controls were used, one mechanically constrained and the other floating. In the first, the gel-filled chamber was set up as before but with no connection to the flow delivery apparatus. The mechanically relaxed static control consisted of the cell-populated gel seeded into an eight-well Lab-Tek coverslip chamber system (Nalge Nunc, Naperville, IL) and allowed to contract freely throughout the experiment. All cultures were maintained in a humidified 37°C, 5% CO2 incubator.
Immunofluorescence staining
The entire gel was fixed by immersion in 2% paraformaldehyde in PBS for 30 minutes and permeabilized in 0.5% Triton X-100. To detect f-actin and -SMA, it was immersed overnight at 4°C in 150 nM Alexa 488-conjugated Phalloidin (Molecular Probes, Eugene, OR) and 5 µg/ml monoclonal Cy3-conjugated mouse anti-human
-SMA antibody (clone 1A4, Sigma). In some cases, the gels were also incubated in 500 nM TOTO-3 (Molecular Probes) for nuclear counterstaining. To detect proliferation, gels were incubated with 0.8 µg/ml monoclonal mouse anti-human Ki-67 (clone MIB-1, DakoCytomation, Carpinteria, CA) and then 10 µg/ml Alexa 546-conjugated rabbit anti-mouse IgG (Molecular Probes), followed by counterstaining with Phalloidin and TOTO-3. To visualize TGF-ß1 protein expression, gels were incubated with 20 µg/ml rabbit anti-human TGF-ß1 (Promega), followed by incubation with 2.5 µg/ml Alexa 647-conjugated goat anti-rabbit IgG (Molecular Probes).
Confocal fluorescence and reflectance microscopy
Images were taken using laser-scanning confocal microscopy (Leica LCS SP2 laser microscope system, Mannheim, Germany). Confocal reflectance contrast microscopy was performed to visualize collagen fibers using a 40x (1.25 NA) oil objective lens with modifications based on a previous protocol (Brightman et al., 2000; Friedl et al., 1997
). To detect Phalloidin and reflectance simultaneously, samples were excited with a 488 nm Ar laser and both the respective emission signal and reflected light passed through an RT 30/70 beam splitter and collected in two separate channels. Two other channels were used to detect emission signals from the
-SMA and TOTO-3 stains, which were excited by He-Ne lasers (543 nm and 633 nm respectively). Samples were vertically scanned from the bottom coverslip with a total depth of 20-100 µm and a pinhole diameter of 40-70 µm. The sequential images were collected at a step depth of 0.3-2.0 µm and reconstructed using Leica LCS (Leica) or Volocity (Improvision, Lexington, MA) software.
Bioneutralization studies
For bioneutralization studies, antibodies with known function-blocking activity were added to the cell suspension and incubated for 30 minutes at 37°C prior to seeding in the collagen matrices. Concentrations were chosen in accordance with previously demonstrated blocking concentrations. They were also maintained in the culture medium throughout the experiment at a lower concentration as indicated by pre-incubation and experiment: mouse anti-human 1ß1 integrin (clone SR84, BD Biosciences Pharmingen, 10 and 2 µg/ml) (Rettig et al., 1984
; Setty et al., 1998
), mouse anti-human
2ß1 (clone BHA2.1; Chemicon; 20 and 10 µg/ml) (Li et al., 2003
) and rabbit anti-human TGF-ß1 (Promega; 0.8 µg/ml) (Zatelli et al., 2000
).
Image analysis quantification
Fibroblast proliferation, density, spreading and expression of -SMA and TGF-ß1 were quantified using ImageJ (NIH, Bethesda, MD). All cells in each image (typically 50-200) were evaluated, using three images per experiment, with three to five experiments per condition. Particle counting was used to determine the number of proliferating (Ki67+) cells normalized to the total cell number (TOTO-3+).
-SMA and TGF-ß1 were quantified by calculating the projected areas of their signals and normalizing those to the f-actin signals. To quantify the projected areas, each image was first converted into a binary image using the threshold function with fixed limits determined from sample images; these were then despeckled, and the total area of signal (
-SMA or TGF-ß1) was divided by the total cell area (i.e. the total area of f-actin signal). Cell density (number of cells/mm3) was quantified directly from cell counts (TOTO-3+) whereas cell spreading was expressed as the fraction of projected cell area (f-actin signal) per total projected image area.
Fast Fourier image analysis and quantification of cell and collagen fiber orientation and alignment
Fast Fourier transform (FFT) analysis, an indirect method previously applied to quantify collagen fiber alignment in SEM and histological images of ligaments, sclerodermal lesions and scar tissues (Chaudhuri et al., 1987; Nishimura and Ansell, 2002
; Pourdeyhimi et al., 1997
; van Zuijlen et al., 2002
), was used here to evaluate the orientation distribution of structures in confocal images. We developed a MATLAB program to perform the analysis (Fig. 1B). First, an image was imported as a matrix array and Welch windowing was applied to reduce edge effects caused by discontinuities in the imperfect periodic images. The FFT algorithm then transformed the windowed image into a power spectrum, which was highly contrasted before the intensity frequencies were summed to determine the orientation intensity distribution histogram.
From each orientation histogram, the peak angle, or angle of highest frequency, was determined. However, although this indicates the angle at which the most cells or fibers are aligned, it does not reflect how many objects (cells or fibers) are aligned at this angle; if the objects were perfectly randomly oriented, the peak angle would be arbitrary and irrelevant. Thus, we also defined an alignment index to reveal the fraction of cells or fibers that were aligned within 20° of the peak angle and this was normalized to the fraction of randomly oriented fibers that would lie within this range (i.e. 40°/180°=0.22). A randomly aligned matrix would have an alignment index of 1; the higher the value, the higher the fraction of cells or fibers aligned near the peak angle.
Statistical analysis of parametric and non-parametric data
Normally distributed data were represented by bar graphs showing the mean and s.d., and unpaired Student's or Welch's t-tests were used to compare mean differences between data with unequal or equal variances, respectively (equality of the variances were assessed using an F-test). Comparisons of three groups or more were performed using ANOVA with Dunnett's post-test. For non-normally distributed data, which were represented by medians and 95% confidence intervals using box plots, the nonparametric Mann-Whitney test was used to compare median differences whereas the Krushal-Wallis test with Dunn's post test was used to compare three groups or more.
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Results |
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Furthermore, TGF-ß1 neutralization abolished flow-enhanced cell density and spreading without affecting cell alignment. Without the blocking antibody, cell density and spreading (i.e. projected cell area per cell) were both increased by interstitial flow after 2 days, but with TGF-ß1 neutralization, no increase in density or spreading was seen (Fig. 4D,E). Interestingly, blocking TGF-ß1 only slightly affected the flow-induced cell alignment: the alignment index was not changed (Fig. 4F) but there was a greater distribution in peak angle (Fig. 4G). In contrast, collagen fiber alignment was reduced (Fig. 4F), indicating that -SMA is required for the cells to align the matrix but not to align themselves. Taken together, these results indicate that interstitial flow causes an upregulation of TGF-ß1 expression, which induces
-SMA expression, which in turn causes matrix alignment.
Interstitial flow effects are mediated through 1ß1 integrin
Matrix remodeling depends on the transmission of intracellular contractile forces to the ECM at sites of integrin-type cell-matrix adhesions. Fibroblasts are known to mechanically interact with collagen fibers primarily through ß1 integrins, particularly 1ß1 and
2ß1 (Heino, 2000
). To investigate whether ligation and signaling through these ß1 integrins were important in mediating the fibroblast differentiation response to interstitial flow, blocking antibodies were used to specifically target
1ß1 and
2ß1 integrins. We found that although both reversed the effects of flow on
-SMA expression, cell density and cell spreading,
1ß1 integrin blocking completely neutralized
-SMA expression (Fig. 5A-D). The differences in
-SMA expression between blocking of
1ß1 and
2ß1 were significant (P=0.0002 using Mann-Whitney test), indicating that
1ß1 was a more potent regulator of
-SMA expression than
2ß1. Furthermore, blocking of
1ß1 integrin had a smaller effect than
2ß1 blocking on cell density and spreading (Fig. 5C,D). Importantly, specific blocking of
1ß1 had little effect on the ability of the fibroblasts to attach to the gel (Fig. 5A) and contract it (Fig. 5G), in contrast to blocking of
2ß1 integrins, which caused significant cell detachment and prevented matrix contraction (Fig. 5A,G). Finally, blocking
1ß1 prevented both cell and matrix alignment (Fig. 5E,F); the lack of change in cell alignment during
2ß1 blocking was probably due to the fact that very few cells remained attached to the matrix when
2ß1 is blocked. These results suggest that ligation of
1ß1 integrins is necessary for interstitial flow-induced myofibroblast differentiation and subsequent cell proliferation, cell alignment and matrix alignment.
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Discussion |
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Our results here suggest that interstitial flow alone may be sufficient to induce and sustain fibrosis, even in the absence of TGF-ß1 secretion by other cells such as inflammatory, epithelial or tumor cells, and correlates with key features of the progression of an inflammatory state to a fibrotic pathology. We show that fibroblasts in 3D collagen cultures undergoing somewhat superphysiological levels of interstitial flow (6 µm/second, or roughly 1-3 dyn/cm2 shear stress) exhibit features commonly observed in tissues of fibrotic phenotype such as scar tissue and the desmoplastic stroma around tumors: the cells proliferate, differentiate into myofibroblasts, and align parallel to each other. Our observation of cell and fiber alignment here is consistent with in vivo observations during wound healing where collagen fibers and fibroblasts become aligned within the wound bed (Ehrlich and Krummel, 1996; Tomasek et al., 2002
); furthermore, perpendicular cell alignment to fluid flow has previously been seen in 2D shear studies on smooth muscle cells (Lee et al., 2002
). The flow-induced increase in cell proliferation also mimics the pathological condition: whereas in normal wound repair, myofibroblasts eventually undergo apoptosis while the granulation tissue evolves into a scar with a sparse cell population (Tomasek et al., 2002
); in fibrotic conditions, they continue to proliferate and overproduce ECM, leading to elevated fibroblast density (Ehrlich and Krummel, 1996
).
To investigate the mechanism underlying this interstitial flow-induced myofibroblast differentiation, we examined the role of TGF-ß1, a potent and well-known inducer of -SMA expression (Arora et al., 1999
; Dugina et al., 2001
; Kunz-Schughart et al., 2003
; Vaughan et al., 2000
) as well as a stimulus of collagen production (Roberts et al., 1986
) and inhibitor of collagen proteolysis (Mutsaers et al., 1997
). First, we saw that interstitial flow triggered the autocrine production of TGF-ß1 in fibroblasts, correlating with the increased
-SMA expression, and that blocking TGF-ß1 with antibodies completely prevented the flow-induced
-SMA expression. Furthermore, this TGF-ß1 induction is probably responsible for the flow-enhanced cell proliferation, as TGF-ß1 has been shown to prevent apoptosis (Phan, 2002
; Zhang and Phan, 1999
) and induce cell proliferation, at least in vascular smooth muscle cells (although it also has been found to inhibit proliferation under certain conditions) (Gibbons, 1994).
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Thus, these studies suggest a mechanism of flow-induced myofibroblast differentiation and matrix remodeling as illustrated in Fig. 6. As for the fundamental mechanotransduction mechanism triggering these responses, it is still unclear exactly how the cell might sense such low levels of interstitial flow. It has been established that mechanical forces like stretch can drive fibroblast differentiation toward a myofibroblast phenotype (Arora and McCulloch, 1994; Hinz et al., 2001a
; Hinz and Gabbiani, 2003b
), although the mechanism remains unclear; furthermore, 2D shear stress can induce autocrine TGF-ß1 expression in vascular smooth muscle cells both in vitro on confluent cell monolayers (Ueba et al., 1997
) or in vivo after experimental artery injury (Song et al., 2000
). Here, the levels of interstitial flow imposed are extremely small: based on a measured average hydraulic conductivity of 1x109 cm2 at the beginning of the experiment that decreased to 2x1010 cm2 after 5 days of interstitial flow owing to matrix remodeling (Ng and Swartz, 2003
), we estimated the average fluid shear stress on the cells to vary between 0.15 and 0.33 dyn/cm2 (Wang and Tarbell, 1995
). It is not known whether such small shear stresses can be sensed by the cells, although 2D stresses as low as 0.1 dyn/cm2 imposed on endothelial cell monolayers can elicit gene upregulation (Barakat and Lieu, 2003
). Little to no stretch would be expected to be imposed on the cell as the ECM is anchored in all directions, although non-affine deformation behavior in a collagen matrix can lead to nonuniformly distributed strain; however, this would tend to decrease, rather than increase, imposed strain on the cell (Pedersen and Swartz, 2005
). Other possible mechanisms may include small changes in the local extracellular biochemical environments: for example, changes in extracellular distribution and transport of cell-secreted cytokines (Swartz, 2003
).
In conclusion, our data demonstrate the influence of interstitial flow on myofibroblast differentiation, fibroblast proliferation and matrix alignment; all of which are distinct and important characteristics of fibroblasts in fibrotic tissues. Its strong ability to induce myofibroblast differentiation occurs without exogenous inflammatory mediators. This suggests that interstitial flow may help to modulate fibroblast phenotypes and drive the progression of fibrotic diseases, including organ fibrosis (lung, liver, renal or heart), defective wound healing like Duputryen's contacture (Tomasek et al., 2002) and connective tissue diseases like artherosclerosis, scleroderma and asthma. Interestingly, we have previously found that the same range of interstitial flow also enhances endothelial morphogenesis (Ng et al., 2004
), which is another essential component of the wound healing process (Ehrlich and Krummel, 1996
). Taken together, these results suggest that the biophysical environment of tissues undergoing chronic inflammation and/or swelling may significantly affect long-term tissue remodeling towards a fibrotic state. Our results also have a potential use in regenerative medicine and may be useful in the design of therapeutic approaches to prevent fibrotic disease or to promote wound healing.
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Acknowledgments |
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References |
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Arora, P. D. and McCulloch, C. A. G. (1994). Dependence of collagen remodeling on alpha-smooth muscle actin expression by fibroblasts. J. Cell. Physiol. 159, 161-175.[CrossRef][Medline]
Arora, P. D., Narani, N. and McCulloch, C. A. G. (1999). The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts. Am. J. Pathol. 154, 871-882.
Barakat, A. and Lieu, D. (2003). Differential responsiveness of vascular endothelial cells to different types of fluid mechanical shear stress. Cell Biochem. Biophys. 38, 323-343.[CrossRef][Medline]
Brightman, A. O., Rajwa, B. P., Sturgis, J. E., McCallister, M. E., Robinson, J. P. and Voytik-Harbin, S. L. (2000). Time-lapse confocal reflection microscopy of collagen fibrillogenesis and extracellular matrix assembly in vitro. Biopolymers 54, 222-234.[CrossRef][Medline]
Campisi, C. and Boccardo, F. (2002). Lymphedema and microsurgery. Microsurgery 22, 74-80.[CrossRef][Medline]
Chary, S. R. and Jain, R. K. (1989). Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching. Proc. Natl. Acad. Sci. USA 86, 5385-5389.
Chaudhuri, S., Nguyen, H., Rangayyan, R. M., Walsh, S. and Frank, C. B. (1987). A Fourier domain directional filtering method for analysis of collagen alignment in ligaments. IEEE Trans. Biomed. Eng. 34, 509-518.[Medline]
Chiquet, M., Reneda, A. S., Huber, F. and Fluck, M. (2003). How do fibroblasts translate mechanical signals into changes in extracellular matrix production? Matrix Biol. 22, 73-80.[CrossRef][Medline]
Danen, E. H. J. and Sonnenberg, A. (2003). Integrins in regulation of tissue development and function. J. Pathol. 201, 632-641.[CrossRef][Medline]
Darby, I., Skalli, O. and Gabbiani, G. (1990). Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound-healing. Lab. Invest. 63, 21-29.[Medline]
Desmouliere, A., Geinoz, A., Gabbiani, F. and Gabbiani, G. (1993). Transforming growth factor beta-1 induces alpha-smooth muscle actin expression in granulation-tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J. Cell Biol. 122, 103-111.[Abstract]
Desmouliere, A., Redard, M., Darby, I. and Gabbiani, G. (1995). Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar. Am. J. Pathol. 146, 56-66.[Abstract]
Desmouliere, A., Darby, I. A. and Gabbiani, G. (2003). Normal and pathologic soft tissue remodeling: Role of the myofibroblast, with special emphasis on liver and kidney fibrosis. Lab. Invest. 83, 1689-1707.[CrossRef][Medline]
Desmouliere, A., Guyot, C. and Gabbiani, C. (2004). The stroma reaction myofibroblast: a key player in the control of tumor cell behavior. Int. J. Dev. Biol. 48, 509-517.[CrossRef][Medline]
Dugina, V., Fontao, L., Chaponnier, C., Vasiliev, J. and Gabbiani, G. (2001). Focal adhesion features during myofibroblastic differentiation are controlled by intracellular and extracellular factors. J. Cell Sci. 114, 3285-3296.[Medline]
Eckes, B., Kessler, D., Aumailley, M. and Krieg, T. (1999). Interactions of fibroblasts with the extracellular matrix: implications for the understanding of fibrosis. Sem. Immunopathol. 21, 415-429.
Ehrlich, H. P. and Krummel, T. M. (1996). Regulation of wound healing from a connective tissue perspective. Wound Repair Regen. 4, 203-210.[CrossRef]
Friedl, P., Maaser, K., Klein, C. E., Niggemann, B., Krohne, G. and Zanker, K. S. (1997). Migration of highly aggressive MV3 melanoma cells in 3-dimensional collagen lattices results in local matrix reorganization and shedding of alpha2 and beta1 integrins and CD44. Cancer Res. 57, 2061-2070.[Abstract]
Gabbiani, G. (2003). The myofibroblast in wound healing and fibrocontractive diseases. J. Pathol. 200, 500-503.[CrossRef][Medline]
Gibbons, G. H. and Dzau, V. J. (1994). The emerging concept of vascular remodeling. New Engl. J. Med. 330, 1431-1438.
Grinnell, F. (1994). Fibroblasts, myofibroblasts, and wound contraction. J. Cell Biol. 124, 401-404.[CrossRef][Medline]
Grinnell, F., Ho, C. H., Tamariz, E., Lee, D. J. and Skuta, G. (2003). Dendritic fibroblasts in three-dimensional collagen matrices. Mol. Biol. Cell 14, 384-395.
Heino, J. (2000). The collagen receptor integrins have distinct ligand recognition and signaling functions. Matrix Biol. 19, 319-323.[CrossRef][Medline]
Heldin, C. H., Rubin, K., Pietras, K. and Ostman, A. (2004). High interstitial fluid pressure an obstacle in cancer therapy. Nat. Rev. Cancer 4, 806-813.[CrossRef][Medline]
Hinz, B., Celetta, G., Tomasek, J. J., Gabbiani, G. and Chaponnier, C. (2001a). Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Mol. Biol. Cell 12, 2730-2741.
Hinz, B., Mastrangelo, D., Iselin, C. E., Chaponnier, C. and Gabbiani, G. (2001b). Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation. Am. J. Pathol. 159, 1009-1020.
Hinz, B. and Gabbiani, C. (2003a). Mechanisms of force generation and transmission by myofibroblasts. Curr. Opin. Biotechnol. 14, 538-546.[CrossRef][Medline]
Hinz, B. and Gabbiani, G. (2003b). Cell-matrix and cell-cell contacts of myofibroblasts: role in connective tissue remodeling. Thromb. Haemost. 90, 993-1002.[Medline]
Jain, R. K. (2001). Delivery of molecular and cellular medicine to solid tumors. Adv. Drug Deliv. Rev. 46, 149-168.[CrossRef][Medline]
Jenkins, G., Redwood, K. L., Meadows, L. and Green, M. R. (1999). Effect of gel reorganization and tensional forces on alpha 2 beta 1 integrin levels in dermal fibroblasts. Eur. J. Biochem. 263, 93-103.
Kim, S. J., Angel, P., Lafyatis, R., Hattori, K., Kim, K. Y., Sporn, M. B., Karin, M. and Roberts, A. B. (1990). Autoinduction of transforming growth factor-beta-1 is mediated by the Ap-1 complex. Mol. Cell. Biol. 10, 1492-1497.[Medline]
Kunz-Schughart, L. A., Wenninger, S., Neumeier, T., Seidel, P. and Knuechel, R. (2003). Three-dimensional tissue structure affects sensitivity of fibroblasts to TGF-beta 1. Am. J. Physiol. 284, C209-C219.
Langholz, O., Rockel, D., Mauch, C., Kozlowska, E., Bank, I., Krieg, T. and Eckes, B. (1995). Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins. J. Cell Biol. 131, 1903-1915.[Abstract]
Lee, A. A., Graham, D. A., Dela Cruz, S., Ratcliffe, A. and Karlon, W. J. (2002). Fluid shear stress-induced alignment of cultured vascular smooth muscle cells. J. Biomech. Eng. 124, 37-43.[CrossRef][Medline]
Li, S. H., Van den Diepstraten, C., D'Souza, S. J., Chan, B. M. C. and Pickering, J. G. (2003). Vascular smooth muscle cells orchestrate the assembly of type I collagen via alpha 2 beta 1 integrin, RhoA, and fibronectin polymerization. Am. J. Pathol. 163, 1045-1056.
Martin, P. (1997). Wound healing aiming for perfect skin regeneration. Science 276, 75-81.
Mueller, M. M. and Fusenig, N. E. (2004). Friends or foes bipolar effects of the tumour stroma in cancer. Nat. Rev. Cancer 4, 839-849.[CrossRef][Medline]
Mutsaers, S. E., Bishop, J. E., McGrouther, G. and Laurent, G. J. (1997). Mechanisms of tissue repair: from wound healing to fibrosis. Int. J. Biochem. Cell Biol. 29, 5-17.[CrossRef][Medline]
Ng, C. P. and Swartz, M. A. (2003). Fibroblast alignment under interstitial fluid flow using a novel 3-D tissue culture model. Am. J. Physiol. 284, H1771-H1777.
Ng, C. P., Helm, C. E. and Swartz, M. A. (2004). Interstitial flow differentially stimulates blood and lymphatic endothelial cell morphogenesis in vitro. Microvasc. Res. 68, 258-264.[CrossRef][Medline]
Nishimura, T. and Ansell, M. P. (2002). Fast Fourier transform and filtered image analyses of fiber orientation in OSB. Wood Sci. Technol. 36, 287-307.[CrossRef]
Pardo, A. and Selman, M. (2002). Idiopathic pulmonary fibrosis: new insights in its pathogenesis. Int. J. Biochem. Cell Biol. 34, 1534-1538.[CrossRef][Medline]
Pedersen, J. A. and Swartz, M. A. (2005). Mechanobiology in the 3rd Dimension. Ann. Biomed. Eng. 33, 1-22..[CrossRef]
Phan, S. H. (2002). The myofibroblast in pulmonary fibrosis. Chest 122, 286S-289S.
Pourdeyhimi, B., Dent, R. and Davis, H. (1997). Measuring fiber orientation in nonwovens: 3. Fourier transform. Textile Res. J. 67, 143-151.
Racine-Samson, L., Rockey, D. C. and Bissell, D. M. (1997). The role of alpha 1 beta 1 integrin in wound contraction: a quantitative analysis of liver myofibroblasts in vivo and in primary culture. J. Biol. Chem. 272, 30911-30917.
Rettig, W. J., Dracopoli, N. C., Goetzger, T. A., Spengler, B. A., Biedler, J. L., Oettgen, H. F. and Old, L. J. (1984). Somatic cell genetic analysis of human cell surface antigens chromosomal assignments and regulation of expression in rodent human hybrid cells. Proc. Natl. Acad. Sci. USA 81, 6437-6441.
Roberts, A. B., Sporn, M. B., Assoian, R. K., Smith, J. M., Roche, N. S., Wakefield, L. M., Heine, U. I., Liotta, L. A., Falanga, V., Kehrl, J. H. et al. (1986). Transforming growth factor type beta rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc. Natl. Acad. Sci. USA 83, 4167-4171.
Ronnov-Jessen, L. and Petersen, O. W. (1993). Induction of alpha-smooth muscle actin by transforming growth factor beta-1 in quiescent human breast gland fibroblasts implications for myofibroblast generation in breast neoplasia. Lab. Invest. 68, 696-707.[Medline]
Schiro, J. A., Chan, B. M., Roswit, W. T., Kassner, P. D., Pentland, A. P., Hemler, M. E., Eisen, A. Z. and Kupper, T. S. (1991). Integrin alpha 2 beta 1 (VLA-2) mediates reorganization and contraction of collagen matrices by human cells. Cell 67, 403-410.[CrossRef][Medline]
Serini, G. and Gabbiani, G. (1999). Mechanisms of myofibroblast activity and phenotypic modulation. Exp. Cell Res. 250, 273-283.[CrossRef][Medline]
Setty, S., Kim, Y., Fields, G. B., Clegg, D. O., Wayner, E. A. and Tsilibary, E. C. (1998). Interactions of type IV collagen and its domains with human mesangial cells. J. Biol. Chem. 273, 12244-12249.
Song, R. H., Kocharyan, H. K., Fortunato, J. E., Glagov, S. and Bassiouny, H. S. (2000). Increased flow and shear stress enhance in vivo transforming growth factor-beta 1 after experimental arterial injury. Arterioscler. Thromb. Vasc. Biol. 20, 923-930.
Swabb, E. A., Wei, J. and Gullino, P. M. (1974). Diffusion and convection in normal and neoplastic tissues. Cancer Res. 34, 2814-2822.[Medline]
Swartz, M. A. (2003). Signaling in morphogenesis: transport cues in morphogenesis. Curr. Opin. Biotech. 14, 547-550.[CrossRef][Medline]
Tamariz, E. and Grinnell, F. (2002). Modulation of fibroblast morphology and adhesion during collagen matrix remodeling. Mol. Biol. Cell 13, 3915-3929
Thannickal, V. J., Toews, G. B., White, E. S., Lynch, J. P. and Martinez, F. J. (2004). Mechanisms of pulmonary fibrosis. Ann. Rev. Med. 55, 395-417.[CrossRef][Medline]
Tomasek, J. J., Gabbiani, G., Hinz, B., Chaponnier, C. and Brown, R. A. (2002). Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat. Rev. Mol. Cell. Biol. 3, 349-363.[CrossRef][Medline]
Ueba, H., Kawakami, M. and Yaginuma, T. (1997). Shear stress as an inhibitor of vascular smooth muscle cell proliferation Role of transforming growth factor-beta 1 and tissue-type plasminogen activator. Arterioscler. Thromb. Vasc. Biol. 17, 1512-1516.
van Zuijlen, P. P. M., de Vries, H. J. C., Lamme, E. N., Coppens, J. S. E., van Marle, J., Kreis, R. W. and Middelkoop, E. (2002). Morphometry of dermal collagen orientation by Fourier analysis is superior to multi-observer assessment. J. Pathol. 198, 284-291.[CrossRef][Medline]
Vanni, S., Lagerholm, B. C., Otey, C., Taylor, D. L. and Lanni, F. (2003). Internet-based image analysis quantifies contractile behavior of individual fibroblasts inside model tissue. Biophys. J. 84, 2715-2727.
Varga, J. and Jimenez, S. A. (1995). Modulation of collagen gene expression its relation to fibrosis in systemic sclerosis and other disorders. Ann. Int. Med. 122, 60-62.
Vaughan, M. B., Howard, E. W. and Tomasek, J. J. (2000). Transforming growth factor-beta1 promotes the morphological and functional differentiation of the myofibroblast. Exp. Cell Res. 257, 180-189.[CrossRef][Medline]
Walker, R. A. (2001). The complexities of breast cancer desmoplasia. Breast Cancer Res. 3, 143-145.[CrossRef][Medline]
Wang, D. M. and Tarbell, J. M. (1995). Modeling interstitial flow in an artery wall allows estimation of wall shear stress on smooth muscle cells. J. Biomech. Eng. 117, 358-363.[Medline]
Werner, S. and Grose, R. (2003). Regulation of wound healing by growth factors and cytokines. Physiol. Rev. 83, 835-870.
Zatelli, M. C., Rossi, R. and Degli Uberti, E. C. (2000). Androgen influences transforming growth factor-beta 1 gene expression in human adrenocortical cells. J. Clin. Endocrinol. Metabol. 85, 847-852.
Zhang, H. Y. and Phan, S. H. (1999). Inhibition of myofibroblast apoptosis by transforming growth factor beta(1). Am. J. Resp. Cell Mol. Biol. 21, 658-665.
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