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Collagen type V modulates fibroblast behavior dependent on substrate stiffness
Biochemical and Biophysical Research Communications 380 (2009) 425–429
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Collagen type V modulates fibroblast behavior dependent on substrate stiffness Roel G.M. Breuls a, Darinka D. Klumpers a, Vincent Everts b, Theo H. Smit a,* a
Department of Physics and Medical Technology, VU University Medical Center, Research Institute MOVE, Van der Boechorststraat 7, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands b
a r t i c l e
i n f o
Article history: Received 5 January 2009 Available online 23 January 2009
Keywords: Collagen V Collagen I Cell spreading Cell shape Fibroblast Actin Focal adhesions Tissue development Wound healing ECM
a b s t r a c t Collagen type V is highly expressed during tissue development and wound repair, but its exact function remains unclear. Cell binding to collagen V affects various basic cell functions and increased collagen V levels alter the structural organization and the stiffness of the ECM. We studied the combined effects of collagen V and substrate stiffness on the morphology, focal adhesion formation, and actin organization of fibroblasts. We found that a hybrid collagen I/V coating impairs fibroblast spreading on soft substrates (<10 kPa), but not on stiffer substrates (68 kPa or glass). In sharp contrast, a pure collagen I coating does not impair cell spreading on soft substrates. The impairment of cell spreading by collagen V is accompanied by diffuse actin staining patterns and small focal adhesions. These observations suggest that collagen V plays an essential role in modifying cell behavior during development and remodeling, when very soft tissues are present. Ó 2009 Elsevier Inc. All rights reserved.
Collagen V is a member of the fibrillar subfamily of collagens and is a minor component of the extracellular matrix (ECM) in connective tissue. In healthy tissue, collagen V constitutes about 1–5% of the total collagen content [1]. Although collagen V is usually buried within collagen I fibers, cells bind to collagen V when it becomes transiently available during extracellular remodeling [2,3]. In vitro studies indicate that collagen V may affect cell morphology, growth kinetics, protein synthesis and migration [1,4–8]. It was recently reported that collagen V is highly expressed in a broad variety of developing tissues [9], during inflammation and in wound healing [1]. Some diseased tissues also show increased collagen V levels compared to their healthy counterparts, both in terms of total amount of collagen V expressed and as a percentage of total collagen content [7]. For example, increased collagen V levels are found in skin and colon tumors [10–12], atherosclerotic plaques [13–15], and scar tissue [16]. These findings suggest that collagen V plays an important role in the modeling and remodeling of the ECM; however, its exact function in these processes remains unknown. Although it has long been appreciated that cells respond to different constituents of the ECM, more recently it has become clear
* Corresponding author. E-mail address: [email protected] (T.H. Smit). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.01.110
that cells are also surprisingly sensitive to small changes in stiffness of the surrounding matrix [17–21]. This is very relevant to collagen V, since increased collagen V levels appear to modify the stiffness of the ECM by changing ECM fibril organization [7,22– 25]. This is related to the role of collagen V in regulating the diameter of collagen I fibers during fibrillogenesis [23–25]. Given the biological effect of collagen V on basic cell function and its effect on the stiffness of the ECM we investigated the independent and combined effects of collagen V and substrate stiffness on the morphology, focal adhesion formation and actin organization of fibroblasts.
Materials and methods Polyacrylamide gels and protein attachment. Polyacrylamide (PA) gels with tunable stiffness were prepared according to published protocols [17] with some small modifications regarding the pH of the solutions. Briefly, stock solutions of 40% acrylamide (Acryl) and 2% N,N0 -methylene-bis-acrylamide (Bis), both from Bio-Rad Laboratories (Hercules, CA), were mixed in 10 mM Hepes pH 7.4 to obtain final concentrations of 5%/0.03%, 5%/0.3%, and 10%/0.5% Acryl/Bis (v/v%). PA gel cross-linking was induced by adding 10% ammonium persulfate and TEMED (both from Bio-Rad). For each gel, 25 ll of the acryl–bisacryl solution was pipetted into a Lab-
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TekTM Chamber SlideTM (Nalge Nunc, Rochester, NY) and covered with a 18-mm glass coverslip. After removing the coverslips, the PA gels were functionalized by covalently attaching either collagen I, collagen V, or a mixture of collagen I/V using the cross-linker sulfo-SANPAH (Pierce Biotechnology, Rockford, IL). Sulfo-SANPAH (0.5 M) was dissolved in Hepes 50 mM, added to the gels (100 ll) and treated with UV light to couple the cross-linker to the gel. Subsequently, rat tail collagen I (BD Biosciences, Bedford, MA) and/or collagen V from human placenta (Sigma, St. Louis, MO) were diluted in PBS to obtain a 0.05 mg/ml solution whereby 0.5 M NaOH was added to set the pH to 7.4. The protein solutions (100 ll) were added to the gels and incubated overnight. It was assessed that a pH of 7.4 did not adversely affect the coupling reaction but improved the homogeneity of the coatings. As a control, glass chamber slides were coated using the same coating solutions. The coatings are termed 100/0, 95/5, 80/20, and 0/100 to indicate the v/v percentages of collagen I and V in the coating solution, i.e. a 100/0-coating contains 100% collagen I and 0% collagen V. Characterization of the mechanical properties of PA gels and collagen coatings. The bulk macroscopic elastic properties (Young’s moduli) of the PA gels were quantified using a commercial rheometer (Paar Physica MCR501, Anton Paar GmbH, Graz, Austria). Gel solution (300 ll) was polymerized in the sample holder with a cone and plate geometry while monitoring storage and loss moduli at 0.5% strain and 0.5 Hz. Polymerization was induced at room temperature. When polymerization was finished, as indicated by steady state values of the storage and loss moduli, the temperature was raised to 37 °C and the material properties were measured. The attachment of collagen I, collagen V, and hybrid collagen I/V coatings to the PA gels was examined using fluorescently labeled antibodies against collagen I and collagen V. Briefly, samples were washed with PBS and incubated with 2% glycine/1%BSA in PBS for 30 min. To stain for collagen I, the samples were incubated with 2.5 lg/ml rabbit-anti-Rat/Hu type I collagen (Abcam, Cambridge, UK) for 2 h, washed with PBS, and stained with 10 lg/ml donkey anti-rabbit Alexa 488 (Invitrogen Ltd., Paisley, UK) for 1 h. Likewise, samples were incubated with 10 lg/ml goat-anti-human type V collagen, biotin conjugated (Southern Biotech) and 25 lg/ml Streptavidin-Alexa Fluor 635 (Invitrogen) using the same incubation times. Images of the coating were taken with a confocal microscope after which the mean fluorescence intensity per image was calculated using the image processing toolbox of Matlab (The MathWorks, Natick, MA). For each gel stiffness, three gels were analyzed from which 5 images were taken at randomly chosen positions. The mean fluorescence intensity of the images from a medium gel was arbitrarily set to 1 after which other images were scaled accordingly. Cell culture. Human periodontal ligament fibroblasts were isolated as previously described [26]. Informed consent was obtained from each individual. Before use, cells were cultured in growth medium consisting of DMEM (Gibco BRL, Paisly, Schotland) + 10% FCS (HyClone, Logan, UT) + antibiotics (100 U/ml penicillin, 100 lg/ml streptomycin, and 250 ng/ml amphotericin B). Cells from passages 5–7 were trypsinized at subconfluency and plated on the PA gels at a density of 5000 cells/cm2. Confocal microscopy. Cells were visualized with a Bio-Rad MRC1000 UV Leica confocal system attached to a Leica inverted microscope (Leica Microsystems, Wetzlar, Germany). F-actin staining and paxillin staining. To stain for F-actin, cells were fixed with 4% formaldehyde and treated with 2% glycine/1% BSA in PBS for 30 min and subsequently stained with Alexa Fluor 488 phalloidin (Molecular Probes, Leiden, The Netherlands) dissolved in PBS-0.1% BSA (1:40) for 2 h at room temperature. To stain for paxillin, cells were fixed for 8 min with PHEM buffer (60 mM Pipes, 25 mM Hepes, 5 nM EGTA, 1 mM MgCl2, 3% sucrose, 0.1% Triton X-100) containing 4% formaldehyde. After washing with PBS,
samples were blocked in blocking buffer (PBS containing 5% fetal calf serum, 5% glycine, and 0.1% Triton X-100) and incubated overnight with 1:40 rabbit anti-human Phospho-Paxillin (Tyr118) antibody (Cell Signaling Technology, Inc., Danvars, MA). After 3 washing steps, cells were incubated with 1:250 dilution donkey anti-rabbit Alexa 488 (Invitrogen) in blocking buffer for 60 min. Cell shape quantification. Cell shape was quantified by determining a cell shape factor S, as defined by: S = 4pA/P2 in which A is the cell area and P the perimeter [27]. The shape factor is equal to 1 for a perfectly round cell and approaches 0 for an elongated cell. Shape factors were determined from 2D projections of single cells in a phase contrast image using custom-made software written in MatLab (The MathWorks, Natick, MA). Only solitary cells were evaluated. Statistics. Paired t-test were used to evaluate differences in fluorescence intensity of gels with different rigidities and to evaluate differences in cell shape factors for different coatings and gel rigidities. Mean values of the fluorescence intensities and cell shape factors were calculated in Matlab (The MathWorks, Natick, MA). Differences were considered significant if p < 0.05. Results Characterization of substrate stiffness and coatings Polyacrylamide (PA) hydrogels with different rigidities were coated with collagen I, collagen V, or mixtures of the two collagens. Rheometric measurements revealed that gels prepared with 5% acrylamide/0.03% bisacrylamide, 5% acrylamide/0.3% bisacrylamide and 10% acrylamide/0.5% bisacrylamide had elastic moduli of 2.1 ± 0.04, 9.7 ± 0.3 and 68 ± 0.6 kPa, respectively (mean ± SD) as shown in Fig. 1A. Each value is statistically significant from the others (p < 0.001). Immunofluorescent labeling of collagen I and V in 100/0- and 0/ 100-coatings (see methods for terminology) confirmed the attachment and uniformity of these coatings (Fig 1B and C). Fig. 1D shows a representative image of a hybrid 80/20-coating, indicating that collagen V is homogeneously distributed and mixed with the collagen I. The fluorescence intensity of the coatings on the 2.1, 9.7 and 68 kPa gels was quantified (Fig. 1E and F), whereby a normalized value of 1 represents a theoretical value of 2.5 lg/cm2. The fluorescence intensity of the 100/0- and 0/100-coatings is uniform, regardless of the gel stiffness (Fig. 1E). The fluorescence intensities of collagen I and V in a hybrid 80/20-coating (Fig. 1F) are approximately 80% and 20% of the pure collagen I and V coatings (Fig. 1E) and again similar for the different gels stiffnesses. Noteworthy, it was previously demonstrated that attached collagen coatings in the PA gel system do not affect the gel elasticity as sensed by the cells [28]. Taken together, these results indicate that the PA gel system can be used to independently study the effects of coating composition and gel stiffness. Effect of collagen V concentration on cell morphology and F-actin staining We investigated whether collagen V affected cell morphology by plating fibroblasts onto 9.7 kPa gels coated with collagen I, collagen V, or a mixture of collagen I and V. On the 100/0-coated gels, cells were elongated and showed abundant F-actin stress fibers (Fig. 2A). The 95/5-coated gels showed a mixed population of elongated and more rounded cells (Fig. 2B), whereas almost all cells were rounded on the 80/20-coated gels (Fig. 2C). The cells on the 80/20-coated gels showed diffuse actin staining (Fig. 2C, insert). On 0/100-coated gels hardly any cells attached, suggesting that a pure collagen V coating does not allow cell attachment (data not shown). Gels coated with the same amount of collagen I as used in the 80/20-coating (but which lacked collagen V) did not result
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E-Modulus (kPa)
A
B
60 40 20 0
E
F
2
2
1.5
1.5
1
1
0.5
0.5
0
0 2.1 kPa 9.7 kPa 68.2 kPa
Fig. 2. Fibroblasts stained for F-actin on 9.7 kPa gels coated with collagen I (A) or hybrid collagen I/V (B and C). On a collagen I coating (A), cells are elongated and show abundant stress fibers (A, insert). On a hybrid 95/5-coating, a mixed population of spread and round cells was found (B). On an 80/20-coating (C), all cells were round and showed diffuse actin staining (C, insert). On a 0/100-coating, hardly any cells attached (not shown). Scale bars, 100 lm; scale bar inserts, 50 lm.
2.1 kPa
A 2.1 kPa 9.7 kPa 68.2 kPa
Fig. 1. (A) Elastic moduli of PA hydrogels with varying concentrations of acrylamide (Acryl) and bisacrylamide (Bis). (B–D) Representative fluorescent images of collagen I in a 100/0-coating (A), collagen V in a 0/100-coating (B) and a hybrid 80/20-coating, containing 80% collagen I and 20% collagen V (D). Note that (D) is shown at a higher magnification to visualize the homogeneous mixing of collagen I (green) and collagen V (red). The yellow dots indicate co-localization of the two collagens. Scale bars, 200 lm (A–C) and 50 lm (D). (E) Normalized fluorescence intensity of collagen I in 100/0-coatings (light grey bars) and collagen V in 0/100-coatings (dark grey bars). (F) Normalized fluorescence intensity of collagen I (light grey bars) and V (dark grey bars) in hybrid 80/20-coatings. A normalized fluorescence intensity of 1 translates to a theoretical value of 2.5 lg/cm2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
in a round cell morphology (data not shown). Finally, adding soluble collagen V to the cells did not affect cell morphology. Collagen V and substrate stiffness Twenty-four hours after plating, cells had hardly spread on the 2.1 and 9.7 kPa gels with an 80/20-coating, but were elongated on these relative soft gels with the 100/0-coating (Fig. 3A). On the stiffer 68 kPa gels, the cells were elongated regardless of the coating (100/0 or 80/20). Thus, the effect of collagen V on cell spreading appeared to be modulated by substrate stiffness. Cell shapes on the 2.1 and 9.7 kPa substrates were not significantly different from each other, but were significantly different from the 68 kPa gels and glass control (p < 0.001) (Fig 3B). Cell shape factors for the 80/20-coating were significantly larger than those for the 95/5 and 100/0-coatings (p < 0.001). Cells on glass had shapes similar to those cultured on 68 kPa gels.
9.7 kPa
68.2 kPa
% Col I/Col V (100/0)
D
(80/20)
C
B 0.8 Cell shape factor
Fluo. Intensity
5% Acr. 5% Acr. 10% Acr. 0.03% Bis 0.3% Bis 0.5% Bis
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 100/0
95/5 % Col I/Col V
80/20
Fig. 3. (A) Phase contrast images of fibroblasts on 2.1, 9.7 and 68 kPa polyacrylamide gels with a 100/0- or a hybrid 80/20-coating, 24 h after plating. Scale bars, 50 lm. (B) Cell shape factor of cells on 2.1 kPa (d), 9.7 kPa (j), 68 kPa (h) polyacrylamide gels, and on a control glass substrate (D) as a function of composition of the coating: 100/0, 95/5, and 80/20 indicate the percentages (v/v) of collagen I and V in the coating.
Focal adhesions To further analyze the stiffness-dependent effect of collagen V on cell morphology, we stained focal adhesions of fibroblast with
anti-paxillin. Cells were plated on 9.7 and 68 kPa gels coated with 100/0- or 80/20-coatings. Cells plated on 9.7 kPa gels with a 100/0coating had elongated focal adhesions (Fig. 4A), whereas the cells
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on the 80/20-coated gels showed smaller, rounder focal adhesions (Fig. 4B). In contrast, the cells on 68 kPa gels showed large elongated focal adhesions on both 100/0- and 80/20-coatings (Fig. 4C and D). Discussion Collagen V is highly expressed in developing and remodeling tissues and is believed to play an important role in processes such as tissue formation and wound healing. Until now, the function of collagen V has been studied using hard substrates rather than in the context of a soft, tissue-like environment. Using a polyacrylamide gel system, we analyzed the effects of substrate stiffness and collagen V on fibroblast cells and found that collagen V affects fibroblast morphology, attachment, focal adhesion formation and actin organization when cultured on relative soft substrates. It has been argued that rigid substrates are able to resist large actin-generated contractile forces, which promotes cell spreading and the development of abundant stress fibers and focal adhesions [20,29]. According to Yeung et al. [29], fibroblasts spread optimally on collagen -coated substrates with a stiffness of 10 kPa. This was confirmed in our experiments as cells spread and showed mature stress fiber formation and focal adhesion development on 9.7 kPa gels that were coated with collagen I (Fig. 2A). However, cells had a typical round morphology on 9.7 kPa gels coated with a mixed coating of collagen I/V. Thus, although 10 kPa may be optimal for spreading on collagen I, it does not induce cell spreading when collagen V is present. Yet, the presence of collagen V does not always prevent cell spreading, because cells spread on 68 kPa gels regardless of the coating composition (Fig. 3A). To explain these observations, it is worth considering that the interaction of fibroblasts with collagen V may disrupt the formation of actin stress fibers, as suggested by Luparello and Sirchia [30]. The disturbance of actin stress fiber formation is known to
Fig. 4. Paxillin staining of fibroblasts on 9.7 and 68 kPa polyacrylamide gels coated with a 100/0 or a 80/20-coating. Cells were stained 24 h after plating. White dotted lines indicate the contours the cell edge, whereby only a part of a complete cell is shown. Cells on 9.7 kPa with a 80/20-coating showed small focal adhesions (B), whereas cells on 100/0-coated gels had spread and showed elongated focal adhesions (A). The cells on the 68 kPa gels were spread and showed elongated focal adhesions for both 80/20- and 100/0-coatings (C and D). Scale bars, 5 lm.
have a negative effect on cell spreading [20]. On the other hand, greater substrate stiffness promotes the development of actin fibers and cell spreading. A plausible mechanistic explanation might be that the stimulating effect of greater stiffness on actin assembly competes with the disturbance of actin stress fiber formation by collagen V. This idea is supported by our data, specifically by the substrate-dependent effect of collagen V on stress fiber organization. Furthermore, a substrate stiffness of 68 kPa seems to be high enough to overcome the negative effect of collagen V on stress fiber formation and cell spreading. The present results are interesting, since in hybrid collagen I/V collagen gels (thus not the PA gel system that was coated with collagens as used in the present study), collagen V is known to modulate the structural arrangement of the ECM and hence also the stiffness. Recently we found that the addition of collagen V to collagen I gels significantly decreases the stiffness of polymerized collagen gels (Breuls, in preparation). This is intriguing in the light of the results presented here indicating that a soft environment is a prerequisite for a morphology change induced by cell binding to collagen V. This might be meaningful in the context of wound healing and tissue formation, where high levels of collagen V are found. Acknowledgments The authors acknowledge Dr. Agnes Berendsen for kindly providing the PDL fibroblasts. We are grateful to Dr. G.H. Koenderink (FOM Institute AMOLF, Amsterdam) for her help with the rheological measurements. We thank Marjolein Blaauboer for proofreading the manuscript. This work was supported by the Dutch Program for Tissue Engineering, project BGT.6734. References [1] A. Fichard, J.P. Kleman, F. Ruggiero, Another look at collagen V and XI molecules, Matrix Biol. 14 (1995) 515–531. [2] F. Ruggiero, M.F. Champliaud, R. Garrone, M. Aumailley, Interactions between cells and collagen V molecules or single chains involve distinct mechanisms, Exp. Cell Res. 210 (1994) 215–223. [3] G. Ghersi, A.M. La Fiura, S. Minafra, Direct adhesion to type I and homotrimer collagens by breast carcinoma and embryonic epithelial cells in culture: a comparative study, Eur. J. Cell Biol. 50 (1989) 279–284. [4] N. Sakata, S. Jimi, S. Takebayashi, M.A. Marques, Type V collagen represses the attachment, spread, and growth of porcine vascular smooth muscle cells in vitro, Exp. Mol. Pathol. 56 (1992) 20–36. [5] C. Luparello, R. Schillaci, I. Pucci-Minafra, S. Minafra, Adhesion, growth and cytoskeletal characteristics of 8701-BC breast carcinoma cells cultured in the presence of type V collagen, Eur. J. Cancer 26 (1990) 231–240. [6] C. Luparello, P. Sheterline, I. Pucci-Minafra, S. Minafra, A comparison of spreading and motility behaviour of 8701-BC breast carcinoma cells on type I, I-trimer and type V collagen substrata. Evidence for a permissive effect of type I-trimer collagen on cell locomotion, J. Cell Sci. 100 (Pt 1) (1991) 179–185. [7] I. Pucci-Minafra, C. Luparello, Type V/type I collagen interactions in vitro and growth-inhibitory effect of hybrid substrates on 8701-BC carcinoma cells, J. Submicrosc. Cytol. Pathol. 23 (1991) 67–74. [8] E.H. Kerkvliet, I.C. Jansen, T. Schoenmaker, W. Beertsen, V. Everts, Collagen type I, III and V differently modulate synthesis and activation of matrix metalloproteinases by cultured rabbit periosteal fibroblasts, Matrix Biol. 22 (2003) 217–227. [9] M. Roulet, F. Ruggiero, G. Karsenty, D. LeGuellec, A comprehensive study of the spatial and temporal expression of the col5a1 gene in mouse embryos: a clue for understanding collagen V function in developing connective tissues, Cell Tissue Res. 327 (2006) 323–332. [10] B. Marian, M.W. Danner, Skin tumor promotion is associated with increased type V collagen content in the dermis, Carcinogenesis 8 (1987) 151–154. [11] H. Fischer, R. Stenling, C. Rubio, A. Lindblom, Colorectal carcinogenesis is associated with stromal expression of COL11A1 and COL5A2, Carcinogenesis 22 (2001) 875–878. [12] S.H. Barsky, C.N. Rao, G.R. Grotendorst, L.A. Liotta, Increased content of Type V Collagen in desmoplasia of human breast carcinoma, Am. J. Pathol. 108 (1982) 276–283. [13] A. Ooshima, Collagen alpha B chain: increased proportion in human atherosclerosis, Science 213 (1981) 666–668. [14] L.F. Morton, M.J. Barnes, Collagen polymorphism in the normal and diseased blood vessel wall. Investigation of collagens types I, III and V, Atherosclerosis 42 (1982) 41–51.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Collagen type V modulates fibroblast behavior dependent on substrate stiffness Roel G.M. Breuls a, Darinka D. Klumpers a, Vincent Everts b, Theo H. Smit a,* a
Department of Physics and Medical Technology, VU University Medical Center, Research Institute MOVE, Van der Boechorststraat 7, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands b
a r t i c l e
i n f o
Article history: Received 5 January 2009 Available online 23 January 2009
Keywords: Collagen V Collagen I Cell spreading Cell shape Fibroblast Actin Focal adhesions Tissue development Wound healing ECM
a b s t r a c t Collagen type V is highly expressed during tissue development and wound repair, but its exact function remains unclear. Cell binding to collagen V affects various basic cell functions and increased collagen V levels alter the structural organization and the stiffness of the ECM. We studied the combined effects of collagen V and substrate stiffness on the morphology, focal adhesion formation, and actin organization of fibroblasts. We found that a hybrid collagen I/V coating impairs fibroblast spreading on soft substrates (<10 kPa), but not on stiffer substrates (68 kPa or glass). In sharp contrast, a pure collagen I coating does not impair cell spreading on soft substrates. The impairment of cell spreading by collagen V is accompanied by diffuse actin staining patterns and small focal adhesions. These observations suggest that collagen V plays an essential role in modifying cell behavior during development and remodeling, when very soft tissues are present. Ó 2009 Elsevier Inc. All rights reserved.
Collagen V is a member of the fibrillar subfamily of collagens and is a minor component of the extracellular matrix (ECM) in connective tissue. In healthy tissue, collagen V constitutes about 1–5% of the total collagen content [1]. Although collagen V is usually buried within collagen I fibers, cells bind to collagen V when it becomes transiently available during extracellular remodeling [2,3]. In vitro studies indicate that collagen V may affect cell morphology, growth kinetics, protein synthesis and migration [1,4–8]. It was recently reported that collagen V is highly expressed in a broad variety of developing tissues [9], during inflammation and in wound healing [1]. Some diseased tissues also show increased collagen V levels compared to their healthy counterparts, both in terms of total amount of collagen V expressed and as a percentage of total collagen content [7]. For example, increased collagen V levels are found in skin and colon tumors [10–12], atherosclerotic plaques [13–15], and scar tissue [16]. These findings suggest that collagen V plays an important role in the modeling and remodeling of the ECM; however, its exact function in these processes remains unknown. Although it has long been appreciated that cells respond to different constituents of the ECM, more recently it has become clear
* Corresponding author. E-mail address: [email protected] (T.H. Smit). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.01.110
that cells are also surprisingly sensitive to small changes in stiffness of the surrounding matrix [17–21]. This is very relevant to collagen V, since increased collagen V levels appear to modify the stiffness of the ECM by changing ECM fibril organization [7,22– 25]. This is related to the role of collagen V in regulating the diameter of collagen I fibers during fibrillogenesis [23–25]. Given the biological effect of collagen V on basic cell function and its effect on the stiffness of the ECM we investigated the independent and combined effects of collagen V and substrate stiffness on the morphology, focal adhesion formation and actin organization of fibroblasts.
Materials and methods Polyacrylamide gels and protein attachment. Polyacrylamide (PA) gels with tunable stiffness were prepared according to published protocols [17] with some small modifications regarding the pH of the solutions. Briefly, stock solutions of 40% acrylamide (Acryl) and 2% N,N0 -methylene-bis-acrylamide (Bis), both from Bio-Rad Laboratories (Hercules, CA), were mixed in 10 mM Hepes pH 7.4 to obtain final concentrations of 5%/0.03%, 5%/0.3%, and 10%/0.5% Acryl/Bis (v/v%). PA gel cross-linking was induced by adding 10% ammonium persulfate and TEMED (both from Bio-Rad). For each gel, 25 ll of the acryl–bisacryl solution was pipetted into a Lab-
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TekTM Chamber SlideTM (Nalge Nunc, Rochester, NY) and covered with a 18-mm glass coverslip. After removing the coverslips, the PA gels were functionalized by covalently attaching either collagen I, collagen V, or a mixture of collagen I/V using the cross-linker sulfo-SANPAH (Pierce Biotechnology, Rockford, IL). Sulfo-SANPAH (0.5 M) was dissolved in Hepes 50 mM, added to the gels (100 ll) and treated with UV light to couple the cross-linker to the gel. Subsequently, rat tail collagen I (BD Biosciences, Bedford, MA) and/or collagen V from human placenta (Sigma, St. Louis, MO) were diluted in PBS to obtain a 0.05 mg/ml solution whereby 0.5 M NaOH was added to set the pH to 7.4. The protein solutions (100 ll) were added to the gels and incubated overnight. It was assessed that a pH of 7.4 did not adversely affect the coupling reaction but improved the homogeneity of the coatings. As a control, glass chamber slides were coated using the same coating solutions. The coatings are termed 100/0, 95/5, 80/20, and 0/100 to indicate the v/v percentages of collagen I and V in the coating solution, i.e. a 100/0-coating contains 100% collagen I and 0% collagen V. Characterization of the mechanical properties of PA gels and collagen coatings. The bulk macroscopic elastic properties (Young’s moduli) of the PA gels were quantified using a commercial rheometer (Paar Physica MCR501, Anton Paar GmbH, Graz, Austria). Gel solution (300 ll) was polymerized in the sample holder with a cone and plate geometry while monitoring storage and loss moduli at 0.5% strain and 0.5 Hz. Polymerization was induced at room temperature. When polymerization was finished, as indicated by steady state values of the storage and loss moduli, the temperature was raised to 37 °C and the material properties were measured. The attachment of collagen I, collagen V, and hybrid collagen I/V coatings to the PA gels was examined using fluorescently labeled antibodies against collagen I and collagen V. Briefly, samples were washed with PBS and incubated with 2% glycine/1%BSA in PBS for 30 min. To stain for collagen I, the samples were incubated with 2.5 lg/ml rabbit-anti-Rat/Hu type I collagen (Abcam, Cambridge, UK) for 2 h, washed with PBS, and stained with 10 lg/ml donkey anti-rabbit Alexa 488 (Invitrogen Ltd., Paisley, UK) for 1 h. Likewise, samples were incubated with 10 lg/ml goat-anti-human type V collagen, biotin conjugated (Southern Biotech) and 25 lg/ml Streptavidin-Alexa Fluor 635 (Invitrogen) using the same incubation times. Images of the coating were taken with a confocal microscope after which the mean fluorescence intensity per image was calculated using the image processing toolbox of Matlab (The MathWorks, Natick, MA). For each gel stiffness, three gels were analyzed from which 5 images were taken at randomly chosen positions. The mean fluorescence intensity of the images from a medium gel was arbitrarily set to 1 after which other images were scaled accordingly. Cell culture. Human periodontal ligament fibroblasts were isolated as previously described [26]. Informed consent was obtained from each individual. Before use, cells were cultured in growth medium consisting of DMEM (Gibco BRL, Paisly, Schotland) + 10% FCS (HyClone, Logan, UT) + antibiotics (100 U/ml penicillin, 100 lg/ml streptomycin, and 250 ng/ml amphotericin B). Cells from passages 5–7 were trypsinized at subconfluency and plated on the PA gels at a density of 5000 cells/cm2. Confocal microscopy. Cells were visualized with a Bio-Rad MRC1000 UV Leica confocal system attached to a Leica inverted microscope (Leica Microsystems, Wetzlar, Germany). F-actin staining and paxillin staining. To stain for F-actin, cells were fixed with 4% formaldehyde and treated with 2% glycine/1% BSA in PBS for 30 min and subsequently stained with Alexa Fluor 488 phalloidin (Molecular Probes, Leiden, The Netherlands) dissolved in PBS-0.1% BSA (1:40) for 2 h at room temperature. To stain for paxillin, cells were fixed for 8 min with PHEM buffer (60 mM Pipes, 25 mM Hepes, 5 nM EGTA, 1 mM MgCl2, 3% sucrose, 0.1% Triton X-100) containing 4% formaldehyde. After washing with PBS,
samples were blocked in blocking buffer (PBS containing 5% fetal calf serum, 5% glycine, and 0.1% Triton X-100) and incubated overnight with 1:40 rabbit anti-human Phospho-Paxillin (Tyr118) antibody (Cell Signaling Technology, Inc., Danvars, MA). After 3 washing steps, cells were incubated with 1:250 dilution donkey anti-rabbit Alexa 488 (Invitrogen) in blocking buffer for 60 min. Cell shape quantification. Cell shape was quantified by determining a cell shape factor S, as defined by: S = 4pA/P2 in which A is the cell area and P the perimeter [27]. The shape factor is equal to 1 for a perfectly round cell and approaches 0 for an elongated cell. Shape factors were determined from 2D projections of single cells in a phase contrast image using custom-made software written in MatLab (The MathWorks, Natick, MA). Only solitary cells were evaluated. Statistics. Paired t-test were used to evaluate differences in fluorescence intensity of gels with different rigidities and to evaluate differences in cell shape factors for different coatings and gel rigidities. Mean values of the fluorescence intensities and cell shape factors were calculated in Matlab (The MathWorks, Natick, MA). Differences were considered significant if p < 0.05. Results Characterization of substrate stiffness and coatings Polyacrylamide (PA) hydrogels with different rigidities were coated with collagen I, collagen V, or mixtures of the two collagens. Rheometric measurements revealed that gels prepared with 5% acrylamide/0.03% bisacrylamide, 5% acrylamide/0.3% bisacrylamide and 10% acrylamide/0.5% bisacrylamide had elastic moduli of 2.1 ± 0.04, 9.7 ± 0.3 and 68 ± 0.6 kPa, respectively (mean ± SD) as shown in Fig. 1A. Each value is statistically significant from the others (p < 0.001). Immunofluorescent labeling of collagen I and V in 100/0- and 0/ 100-coatings (see methods for terminology) confirmed the attachment and uniformity of these coatings (Fig 1B and C). Fig. 1D shows a representative image of a hybrid 80/20-coating, indicating that collagen V is homogeneously distributed and mixed with the collagen I. The fluorescence intensity of the coatings on the 2.1, 9.7 and 68 kPa gels was quantified (Fig. 1E and F), whereby a normalized value of 1 represents a theoretical value of 2.5 lg/cm2. The fluorescence intensity of the 100/0- and 0/100-coatings is uniform, regardless of the gel stiffness (Fig. 1E). The fluorescence intensities of collagen I and V in a hybrid 80/20-coating (Fig. 1F) are approximately 80% and 20% of the pure collagen I and V coatings (Fig. 1E) and again similar for the different gels stiffnesses. Noteworthy, it was previously demonstrated that attached collagen coatings in the PA gel system do not affect the gel elasticity as sensed by the cells [28]. Taken together, these results indicate that the PA gel system can be used to independently study the effects of coating composition and gel stiffness. Effect of collagen V concentration on cell morphology and F-actin staining We investigated whether collagen V affected cell morphology by plating fibroblasts onto 9.7 kPa gels coated with collagen I, collagen V, or a mixture of collagen I and V. On the 100/0-coated gels, cells were elongated and showed abundant F-actin stress fibers (Fig. 2A). The 95/5-coated gels showed a mixed population of elongated and more rounded cells (Fig. 2B), whereas almost all cells were rounded on the 80/20-coated gels (Fig. 2C). The cells on the 80/20-coated gels showed diffuse actin staining (Fig. 2C, insert). On 0/100-coated gels hardly any cells attached, suggesting that a pure collagen V coating does not allow cell attachment (data not shown). Gels coated with the same amount of collagen I as used in the 80/20-coating (but which lacked collagen V) did not result
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E-Modulus (kPa)
A
B
60 40 20 0
E
F
2
2
1.5
1.5
1
1
0.5
0.5
0
0 2.1 kPa 9.7 kPa 68.2 kPa
Fig. 2. Fibroblasts stained for F-actin on 9.7 kPa gels coated with collagen I (A) or hybrid collagen I/V (B and C). On a collagen I coating (A), cells are elongated and show abundant stress fibers (A, insert). On a hybrid 95/5-coating, a mixed population of spread and round cells was found (B). On an 80/20-coating (C), all cells were round and showed diffuse actin staining (C, insert). On a 0/100-coating, hardly any cells attached (not shown). Scale bars, 100 lm; scale bar inserts, 50 lm.
2.1 kPa
A 2.1 kPa 9.7 kPa 68.2 kPa
Fig. 1. (A) Elastic moduli of PA hydrogels with varying concentrations of acrylamide (Acryl) and bisacrylamide (Bis). (B–D) Representative fluorescent images of collagen I in a 100/0-coating (A), collagen V in a 0/100-coating (B) and a hybrid 80/20-coating, containing 80% collagen I and 20% collagen V (D). Note that (D) is shown at a higher magnification to visualize the homogeneous mixing of collagen I (green) and collagen V (red). The yellow dots indicate co-localization of the two collagens. Scale bars, 200 lm (A–C) and 50 lm (D). (E) Normalized fluorescence intensity of collagen I in 100/0-coatings (light grey bars) and collagen V in 0/100-coatings (dark grey bars). (F) Normalized fluorescence intensity of collagen I (light grey bars) and V (dark grey bars) in hybrid 80/20-coatings. A normalized fluorescence intensity of 1 translates to a theoretical value of 2.5 lg/cm2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
in a round cell morphology (data not shown). Finally, adding soluble collagen V to the cells did not affect cell morphology. Collagen V and substrate stiffness Twenty-four hours after plating, cells had hardly spread on the 2.1 and 9.7 kPa gels with an 80/20-coating, but were elongated on these relative soft gels with the 100/0-coating (Fig. 3A). On the stiffer 68 kPa gels, the cells were elongated regardless of the coating (100/0 or 80/20). Thus, the effect of collagen V on cell spreading appeared to be modulated by substrate stiffness. Cell shapes on the 2.1 and 9.7 kPa substrates were not significantly different from each other, but were significantly different from the 68 kPa gels and glass control (p < 0.001) (Fig 3B). Cell shape factors for the 80/20-coating were significantly larger than those for the 95/5 and 100/0-coatings (p < 0.001). Cells on glass had shapes similar to those cultured on 68 kPa gels.
9.7 kPa
68.2 kPa
% Col I/Col V (100/0)
D
(80/20)
C
B 0.8 Cell shape factor
Fluo. Intensity
5% Acr. 5% Acr. 10% Acr. 0.03% Bis 0.3% Bis 0.5% Bis
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 100/0
95/5 % Col I/Col V
80/20
Fig. 3. (A) Phase contrast images of fibroblasts on 2.1, 9.7 and 68 kPa polyacrylamide gels with a 100/0- or a hybrid 80/20-coating, 24 h after plating. Scale bars, 50 lm. (B) Cell shape factor of cells on 2.1 kPa (d), 9.7 kPa (j), 68 kPa (h) polyacrylamide gels, and on a control glass substrate (D) as a function of composition of the coating: 100/0, 95/5, and 80/20 indicate the percentages (v/v) of collagen I and V in the coating.
Focal adhesions To further analyze the stiffness-dependent effect of collagen V on cell morphology, we stained focal adhesions of fibroblast with
anti-paxillin. Cells were plated on 9.7 and 68 kPa gels coated with 100/0- or 80/20-coatings. Cells plated on 9.7 kPa gels with a 100/0coating had elongated focal adhesions (Fig. 4A), whereas the cells
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on the 80/20-coated gels showed smaller, rounder focal adhesions (Fig. 4B). In contrast, the cells on 68 kPa gels showed large elongated focal adhesions on both 100/0- and 80/20-coatings (Fig. 4C and D). Discussion Collagen V is highly expressed in developing and remodeling tissues and is believed to play an important role in processes such as tissue formation and wound healing. Until now, the function of collagen V has been studied using hard substrates rather than in the context of a soft, tissue-like environment. Using a polyacrylamide gel system, we analyzed the effects of substrate stiffness and collagen V on fibroblast cells and found that collagen V affects fibroblast morphology, attachment, focal adhesion formation and actin organization when cultured on relative soft substrates. It has been argued that rigid substrates are able to resist large actin-generated contractile forces, which promotes cell spreading and the development of abundant stress fibers and focal adhesions [20,29]. According to Yeung et al. [29], fibroblasts spread optimally on collagen -coated substrates with a stiffness of 10 kPa. This was confirmed in our experiments as cells spread and showed mature stress fiber formation and focal adhesion development on 9.7 kPa gels that were coated with collagen I (Fig. 2A). However, cells had a typical round morphology on 9.7 kPa gels coated with a mixed coating of collagen I/V. Thus, although 10 kPa may be optimal for spreading on collagen I, it does not induce cell spreading when collagen V is present. Yet, the presence of collagen V does not always prevent cell spreading, because cells spread on 68 kPa gels regardless of the coating composition (Fig. 3A). To explain these observations, it is worth considering that the interaction of fibroblasts with collagen V may disrupt the formation of actin stress fibers, as suggested by Luparello and Sirchia [30]. The disturbance of actin stress fiber formation is known to
Fig. 4. Paxillin staining of fibroblasts on 9.7 and 68 kPa polyacrylamide gels coated with a 100/0 or a 80/20-coating. Cells were stained 24 h after plating. White dotted lines indicate the contours the cell edge, whereby only a part of a complete cell is shown. Cells on 9.7 kPa with a 80/20-coating showed small focal adhesions (B), whereas cells on 100/0-coated gels had spread and showed elongated focal adhesions (A). The cells on the 68 kPa gels were spread and showed elongated focal adhesions for both 80/20- and 100/0-coatings (C and D). Scale bars, 5 lm.
have a negative effect on cell spreading [20]. On the other hand, greater substrate stiffness promotes the development of actin fibers and cell spreading. A plausible mechanistic explanation might be that the stimulating effect of greater stiffness on actin assembly competes with the disturbance of actin stress fiber formation by collagen V. This idea is supported by our data, specifically by the substrate-dependent effect of collagen V on stress fiber organization. Furthermore, a substrate stiffness of 68 kPa seems to be high enough to overcome the negative effect of collagen V on stress fiber formation and cell spreading. The present results are interesting, since in hybrid collagen I/V collagen gels (thus not the PA gel system that was coated with collagens as used in the present study), collagen V is known to modulate the structural arrangement of the ECM and hence also the stiffness. Recently we found that the addition of collagen V to collagen I gels significantly decreases the stiffness of polymerized collagen gels (Breuls, in preparation). This is intriguing in the light of the results presented here indicating that a soft environment is a prerequisite for a morphology change induced by cell binding to collagen V. This might be meaningful in the context of wound healing and tissue formation, where high levels of collagen V are found. Acknowledgments The authors acknowledge Dr. Agnes Berendsen for kindly providing the PDL fibroblasts. We are grateful to Dr. G.H. Koenderink (FOM Institute AMOLF, Amsterdam) for her help with the rheological measurements. We thank Marjolein Blaauboer for proofreading the manuscript. This work was supported by the Dutch Program for Tissue Engineering, project BGT.6734. References [1] A. Fichard, J.P. Kleman, F. Ruggiero, Another look at collagen V and XI molecules, Matrix Biol. 14 (1995) 515–531. [2] F. Ruggiero, M.F. Champliaud, R. Garrone, M. Aumailley, Interactions between cells and collagen V molecules or single chains involve distinct mechanisms, Exp. Cell Res. 210 (1994) 215–223. [3] G. Ghersi, A.M. La Fiura, S. Minafra, Direct adhesion to type I and homotrimer collagens by breast carcinoma and embryonic epithelial cells in culture: a comparative study, Eur. J. Cell Biol. 50 (1989) 279–284. [4] N. Sakata, S. Jimi, S. Takebayashi, M.A. Marques, Type V collagen represses the attachment, spread, and growth of porcine vascular smooth muscle cells in vitro, Exp. Mol. Pathol. 56 (1992) 20–36. [5] C. Luparello, R. Schillaci, I. Pucci-Minafra, S. Minafra, Adhesion, growth and cytoskeletal characteristics of 8701-BC breast carcinoma cells cultured in the presence of type V collagen, Eur. J. Cancer 26 (1990) 231–240. [6] C. Luparello, P. Sheterline, I. Pucci-Minafra, S. Minafra, A comparison of spreading and motility behaviour of 8701-BC breast carcinoma cells on type I, I-trimer and type V collagen substrata. Evidence for a permissive effect of type I-trimer collagen on cell locomotion, J. Cell Sci. 100 (Pt 1) (1991) 179–185. [7] I. Pucci-Minafra, C. Luparello, Type V/type I collagen interactions in vitro and growth-inhibitory effect of hybrid substrates on 8701-BC carcinoma cells, J. Submicrosc. Cytol. Pathol. 23 (1991) 67–74. [8] E.H. Kerkvliet, I.C. Jansen, T. Schoenmaker, W. Beertsen, V. Everts, Collagen type I, III and V differently modulate synthesis and activation of matrix metalloproteinases by cultured rabbit periosteal fibroblasts, Matrix Biol. 22 (2003) 217–227. [9] M. Roulet, F. Ruggiero, G. Karsenty, D. LeGuellec, A comprehensive study of the spatial and temporal expression of the col5a1 gene in mouse embryos: a clue for understanding collagen V function in developing connective tissues, Cell Tissue Res. 327 (2006) 323–332. [10] B. Marian, M.W. Danner, Skin tumor promotion is associated with increased type V collagen content in the dermis, Carcinogenesis 8 (1987) 151–154. [11] H. Fischer, R. Stenling, C. Rubio, A. Lindblom, Colorectal carcinogenesis is associated with stromal expression of COL11A1 and COL5A2, Carcinogenesis 22 (2001) 875–878. [12] S.H. Barsky, C.N. Rao, G.R. Grotendorst, L.A. Liotta, Increased content of Type V Collagen in desmoplasia of human breast carcinoma, Am. J. Pathol. 108 (1982) 276–283. [13] A. Ooshima, Collagen alpha B chain: increased proportion in human atherosclerosis, Science 213 (1981) 666–668. [14] L.F. Morton, M.J. Barnes, Collagen polymorphism in the normal and diseased blood vessel wall. Investigation of collagens types I, III and V, Atherosclerosis 42 (1982) 41–51.
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