- Email: [email protected]
Stromelysin-1-Deficient Fibroblasts Display Impaired Contractionin Vitro
Journal of Surgical Research 84, 31–34 (1999) Article ID jsre.1999.5599, available online at http://www.idealibrary.com on
Stromelysin-1-Deficient Fibroblasts Display Impaired Contraction in Vitro Kelli M. Bullard, M.D., 1 John Mudgett, Ph.D., Heinz Scheuenstuhl, B.S., Thomas K. Hunt, M.D., and Michael J. Banda, Ph.D. University of California, San Francisco, San Francisco, California 94143 Presented at the Annual Meeting of the Association for Academic Surgery, Seattle, Washington, November 18 –22, 1998
ing [1, 2]. Stromelysin-1 (MMP-3) has one of the broadest substrate specificities of the MMPs and is responsible for degrading proteoglycans, laminin, fibronectin, the nonhelical domains of collagen types IV and IX, propeptides of type I collagen, and all denatured collagens as well as proteolytic activation of procollagen [3]. Stromelysin-1 is synthesized primarily by fibroblasts [4 – 6] and has been identified in the stroma of normally healing rabbit corneal wounds [7], in stromal cells and keratinocytes of chronic ulcers [6], in burn wound fluid from human patients [8], and in basal keratinocytes of both normal and chronic, nonhealing human skin ulcers [9]. The presence of stromelysin-1 in these settings implies that it plays a key role in the process of wound healing. Targeted disruption of the stromelysin-1 gene in mice causes a delay in excisional wound healing due to a failure in the first phase of wound contraction. In normal mice, excisional wounds contract rapidly, decreasing wound area by approximately 50% per day during the first 4 days of healing. In strom-1 KO mice, however, excisional wounds lack early wound contraction, decreasing wound area by only 10 –15% per day [10]. We therefore postulated that stromelysin-1 activity plays a role in the mechanism of wound contraction. Wound fibroblasts are thought to be the functional cell initiating wound contraction and fibroblasts cultured on collagen matrices have been used as a model of wound contraction in vitro [11–13]. In this model, fibroblasts are grown on circular matrices containing predominantly type I collagen. Contractile ability is assessed by measuring changes in gel diameter or area [13]. Contractile capacity of fibroblasts from stromelysin-1-deficient mice was compared with that of fibroblasts from wild-type mice using this collagen gel model, which allowed examination of fibroblast contraction apart from other elements of wound healing.
Targeted disruption of the stromelysin-1 gene in mice causes a delay in excisional wound healing due to a failure in wound contraction. Therefore, we postulated that stromelysin-1 activity is responsible for initiating contraction. To test this hypothesis, we compared the contractile capacity of fibroblasts from stromelysin-1 knockout mice (strom-1 KO) with that of normal fibroblasts using a collagen gel contraction model. Fibroblast cultures were established from explants of skin and lung parenchyma from strom-1 KO and wild-type mice, then transferred to the surface of collagen gels. The extent of contraction was determined by measuring greatest gel diameter. Results demonstrated that (1) all fibroblasts contracted collagen gels in a uniform concentric fashion, (2) skin fibroblasts from both sets of mice exhibited greater gel contraction than did lung fibroblasts, and (3) strom-1 KO fibroblasts demonstrated significantly less contraction (21–23%) than wild-type fibroblasts. These data support the hypothesis that absence of stromelysin-1 results in defective fibroblast contraction that may contribute to delayed wound healing. © 1999 Academic Press Key Words: metalloproteinases; wound contraction; wound healing; stromelysin-1.
INTRODUCTION
Would healing occurs by a complex series of events that involves matrix deposition, remodeling, and wound contraction. Matrix metalloproteinases (MMPs) are a multigene family of enzymes that are responsible for extracellular matrix (ECM) remodeling in a wide range of physiological processes including wound heal1 To whom correspondence should be addressed at HSW-1601, Box 0570, 513 Parnassus Avenue, University of California, San Francisco, San Francisco, CA 94143– 0570. Fax: (415) 476 –2314. E-mail: [email protected].
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0022-4804/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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JOURNAL OF SURGICAL RESEARCH: VOL. 84, NO. 1, JUNE 1, 1999
TABLE 1
METHODS Strom-1 KO mice were generated through gene targeting by homologous recombination in murine embryonic stem cells as described previously [14] and maintained on 129Sv background. All procedures were performed in a laminar animal operating suite under aseptic conditions according to protocols approved by the University of California, San Francisco, Committee on Animal Research. Fibroblast cultures were established from explants of skin and lung parenchyma from strom-1 KO and wild-type mice and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). Collagen gels were prepared using Vitrogen type I collagen; Collagen Corp.) mixed with DMEM, phosphatebuffered saline (PBS), and 10% FBS, and pH adjusted at 4°C [15, 16]. Circular gels 20 mm in diameter were then poured into 12-well culture plates (Falcon) and incubated for 2 h at 37°C to allow polymerization of collagen. Trypsinized fibroblasts (passages 2 to 6) were then transferred to the surface of each gel (2.5 3 10 5 cells/gel) and incubated at 37°C for 24 h. Gels were rimmed and incubated at 37°C for an additional 24 h, then fixed with 10% formalin. The extent of contraction was determined by measuring the greatest diameter (mm) of each gel. Gel area was also calculated. Experiments consisted of 16 sets of skin fibroblasts (8 strom-1 KO, 8 wild type) and 8 sets of lung fibroblasts (4 strom-1 KO, 4 wild type) from 16 mice. Collagen gels without cells served as controls. Results were compared with Student’s t test (two-tailed).
RESULTS
All fibroblasts contracted collagen gels in a uniform, concentric fashion; control gels did not contract. Skin fibroblasts from both strom-1 KO mice and wild-type mice exhibited greater gel contraction than did lung fibroblasts. However, all strom-1 KO fibroblasts demonstrated less contraction than wild-type fibroblasts. Wild-type skin fibroblasts contracted gels to 45% of the original gel diameter (22% percent of original area) while strom-1 KO skin fibroblasts contracted gels to 60% of the original gel diameter (36% of original area). Similarly, wild-type lung fibroblasts contracted gels to 60% of the original gel diameter (36% of original area) versus 77% (59% of original area) for strom-1 KO lung fibroblasts. Thus, contraction measured by either gel diameter or gel area was significantly greater when wild-type fibroblasts were compared with strom-1 KO fibroblasts (P , 0.001 in all groups; Table 1, Fig. 1). DISCUSSION
Wound contraction is a fundamental event in wound healing, yet the biological process remains poorly understood. In mice, excisional dermal wounds contract more than 50% of their original size during the first 24 to 48 h after wounding, thus decreasing the volume that must be filled with granulation tissue and reducing the distance that must be traversed by keratinocytes. Our previous experiments have shown that stromelysin-1 is crucial for normal wound contraction in vivo [10]. In these experiments, stromelysin-1deficient fibroblasts contracted collagen gels to a lesser extent when compared with normal wild-type fibro-
Mean Gel Diameters and Areas after 24 h (Original Gel Diameter 5 20 mm, Original Gel area 5 314 mm 2)
Skin fibroblasts Diameter (mm) Area (mm 2) Lung fibroblasts Diameter (mm) Area (mm 2)
Control (no cells)
Wild type
Strom-1 KO
20.0 314.0
9.3 6 0.4 a 68.0 6 2.7
12.1 6 0.5* 113.0 6 4.5*
20.0 314.0
12.0 6 0.6 113.0 6 5.7
15.4 6 0.2* 186.0 6 1.9*
a 6SEM. * P , 0.001.
blasts. Because fibroblasts are involved in initiating wound contraction, this impaired ability to contract collagen gels in vitro may contribute to the impaired wound contraction we observed in vivo. Fibroblast traction on the ECM has been proposed as the mechanism underlying the first phase of wound contraction [21, 22]. Normal wound contraction requires attachment of fibroblasts to the underlying ECM, cell contraction and/or locomotion, and matrix architecture capable of transmitting force. In the collagen gel model, the underlying ECM is constant (type I collagen) and, therefore, allows fibroblast function to be studied in isolation. Fibroblast attachment to the gels involves interaction between cell surface adhesion molecules (b1 integrins) and type I collagen [23–29]. Because stromelysin-1 cleaves a wide range of matrix macromolecules, it is possible that it plays a role in integrin processing or modification of attachment sites on the underlying collagen fibrils. Fibroblast contraction in vivo also requires synthesis of an actin cable [29 –31] and an actin stress fiber network [24, 32, 33]. In a collagen gel model, depolymerization of actin filaments by cytochalasin B destroys contraction [20]. Our in vivo data suggest that an inability to adequately organize fibrillar actin may contribute to poor wound contraction in the strom-1 KO mice [10], it remains to be determined if strom-1 KO fibroblasts cultured on collagen gels produce inadequate actin networks as well. Finally, several growth factors including TGF-b1, IGF-1, and PDGF affect contraction [27, 28, 34 –36], and stromelysin-1 deficiency may result in altered processing of growth factors or growth factor receptors. Further experiments are planned to address these issues using the gel contraction model. Several lines of evidence suggest that the MMPs are involved in cell motility and contraction. Fibroblasts migrating through collagen gels are known to secrete collagenase [17]. Addition of tissue inhibitor of metalloproteinases (TIMP) decreases collagen gel contraction [18, 19]. Similarly, tumor cells that are incapable of activating MMP-2 (gelatinase) contract collagen gels
BULLARD ET AL.: STROMELYSIN-1 DEFICIENCY IMPAIRS CONTRACTION IN FIBROBLASTS
33
FIG. 1. Total area of collagen gels containing stromelysin-1 KO fibroblasts versus wild-type fibroblasts (6SEM).
poorly compared with cells capable of producing active enzyme [20]. Our data imply that stromelysin-1 may be the crucial MMP in fibroblast contraction and that absence of this proteinase in some way alters the ability of fibroblasts to contract type I collagen matrices. Because stromelysin-1 activates procollagenase, absence of stromelysin-1 may also alter the normal proteolytic cascade required for cell contraction and motility. The redundancy of the MMP system, however, may account for the fact that the defect we observed was not complete; while strom-1 KO fibroblasts contracted gels poorly compared with normal fibroblasts, they were able to contract the gels by approximately 40%. Similarly, although excisional wounds on strom-1 KO mice heal far more slowly in vivo than do normal wounds, they do eventually close [10]. We postulate that other MMPs may be capable of remodeling the ECM in the absence of stromelysin-1, albeit less efficiently, to allow contraction and healing to proceed. Gel contraction differed between lung and skin fibroblasts in both sets of mice. In both strom-1 KO and wild-type mice, dermal fibroblasts contracted more than lung fibroblasts. This functional heterogeneity among different fibroblast populations has been noted by several authors and is thought to contribute to different physiological roles played by these cells [37– 40]. However, we also observed that regardless of the tissue source of the fibroblasts, strom-1 KO fibroblasts had impaired contractile ability compared with normal fibroblasts. This observation suggests that
stromelysin-1 plays a fundamental role in fibroblast function and that the defect produced by deletion of the stromelysin-1 gene is not limited to dermal fibroblasts. It will be interesting to determine if strom-1 KO mice exhibit delayed wound healing or altered fibrosis in organs other than skin. REFERENCES 1. 2. 3. 4.
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Matrisian, L. M. Metalloproteinases and their inhibitors in matrix remodeling. Trends Genet. 6: 121, 1990. Woessner, J. F. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 5: 2145, 1991. Welgus, H. G. Stromelysin: Structure and function. Agents Actions, Suppl. 35: 61, 1991. Wilhelm, S. C., Collier, I. E., Kronberger, A., Eisen, A. Z., Marmer, B. L., Grant, G. A., Bauer, A. E., and Goldberg, G. I. Human skin fibroblast stromelysin: Structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells. Proc. Natl. Acad. Sci. USA 84: 6725, 1987. Welgus, H. G., Campbell, E. J., Cury, J. D., Eisen, A. D., Senior, R. M., Wilhelm, S. M., and Goldberg, G. I. Neutral metalloproteinases produced by human mononuclear phagocytes: Enzymatic profile, regulation, and expression during cellular development. J. Clin. Invest. 86: 1496, 1990. Saarialho-Kere, U. K., Pentland, A. P., Birkendal-Hansen, H., Parks, W. C., and Welgus, H. G. Distinct populations of basal keratincoytes express stromeylsin-1 and stromelysin-2 in chronic wounds. J. Clin. Invest. 94: 79, 1994. Girard, M. T., Matsubara, M., Kublin, C., Tessier, M. J., Cintron, C., and Fini, M. E. Stromal fibroblasts synthesize collagenase and stromelysin during long term tissue remodeling. J. Cell Sci. 104: 1001, 1993.
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JOURNAL OF SURGICAL RESEARCH: VOL. 84, NO. 1, JUNE 1, 1999 Young, P. K., and Grinnell, F. Metalloproteinase activation cascade after burn injury: A longitudinal analysis of the human wound environment. J. Invest. Dermatol. 103: 660, 1994. Vaalamo, M., Weckroth, M., Puolakkainen, P., Kere, J., Saarinen, P., Lauharanta, J., and Saarialho-Kere, U. K. Patterns of matrix metalloproteinase and TIMP-1 expression in chronic and normally healing human cutaneous wounds. Br. J. Dermatol. 135: 52, 1996. Bullard, K. M., Lund, L., Mudgett, J. S., Werb, Z., Mellin, T. N., Hunt, T. K., Murphy, B., Ronan J., and Banda, M. J. Impaired wound contraction in stromelysin-1 deficient mice. Ann. Surg., in press. Bell, E., Ivarsson, B., and Merrill, C. Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc. Natl. Acad. Sci. USA 76: 1274, 1979. Bellows, C. G., Melcher, A. H., and Aubin, J. E. Contraction and organization of collagen gels by cells cultured from periodontal ligament, gingiva, and bone suggest functional differences between cell types. J. Cell Sci. 50: 299, 1981. Grinnell, F. Fibroblasts, myofibroblasts, and wound contraction. J. Cell Biol. 124: 401, 1994. Mudgett, J. S., Hutchinson, N. I., Chartrain, N., Forsyth, A. J., McDonell, J., Singer, I. I., Bayna, E. K., Flanagan, J., Kawka, D., and Slein, A. F. Susceptibility of stromelysin-1 deficient mice to collagen-induced arthritis and cartilage destruction. Arthritis Rheum. 41: 110, 1997. Guidry, C., and Grinnell, F. Heparin modulates the supramolecular organization of collagen gels and inhibits gel contraction by fibroblasts. J. Cell Biol. 104: 1097, 1987. Nakagawa, S., Pawalek, P., and Grinnell, F. Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness. Exp. Cell Res. 182: 572, 1989. Lambert, C. A., Soudant, E. P., Nusgens, B. V., and Lapiere, C. M. Pretranslational regulation of extracellular matrix molecules and collagenase expression in fibroblasts by mechanical forces. Lab. Invest. 66: 444, 1992. Khaw, P. T., Occleston, N. L., Schultz, G., Grierson, I., Sherwood, M. B., and Larkin, G. Activation and suppression of fibroblast function. Eye 8: 188, 1994. Millis, A. J., Hoyle, M., McCue, H. M., and Martin, H. Differential expression of metalloproteinase and tissue inhibitor of metalloproteinase genes in aged human fibroblasts. Exp. Cell Res. 210: 373, 1992. Deryugina, E. I., Bourdon, M. A., Reisfeld, R. A., and Strongin, A. Remodeling of collagen matrix by human tumor cells requires activation and cell surface association of matrix metalloproteinse-2. Cancer Res. 58: 3743, 1998. Harris, A., Stopak, D., and Wild, P. Fibroblast traction as a mechanism for collagen morphogenesis. Nature 290: 249, 1981. Stopak, D., and Harris, A. K. Connective tissue morphogenesis by fibroblast traction. Dev. Biol. 90: 383, 1982. Gullberg, D., Tingstrom, A., Thuressen, A-C., Olsson, L., Terracio, L., Borg, T. K., and Rubin, K. Integrin-mediated collagen gel contraction is stimulated by PDGF. Exp. Cell Res. 186: 264, 1990. Seltzer, J. L., Lee, A-Y., Akers, K. T., Sudbeck, B., Southon, E., Wayner, E. A., and Eisen, A. Z. Activation of 72-kD type IV collagenase/gelatinase by normal fibroblasts in collagen lattices is mediated by integrin receptors but is not related to lattice contraction. Exp. Cell Res. 21: 365, 1994.
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Riikonen, T., Koivisto, L., Vihinen, P., and Heino, J. Transforming growth factor b regulates collagen gel contraction by increasing a2b1 integrin expression in osteogenic cells. J. Biol. Chem. 27: 376, 1995.
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Thiesen, S. L., Alton, M., Gadson, P. F., Patterson, E., and Rosenquist, T. H. Embryonic lineage of vascular smooth muscle cells determines responses to collagen matrices and integrin receptor expression. Exp. Cell Res. 227: 135, 1996.
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Stephens, P., Genever, P. G., Wood, E. J., and Raxworthy, J. Integrin receptor involvement in actin cable formation in an in vitro model of events associated with wound contraction. Int. J. Biochem. Cell Biol. 29: 121, 1997.
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Davis, G. E., and Camarillo, C. W. Regulation of endothelial cell morphogenesis by integrins, mechanical forces, and matrix guidance pathways. Exp. Cell Res. 216: 113, 1995.
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Eastwood, M., Porter, R., Khan, U., McGrouther, G., and Bown, R. Quantitative analysis of collagen gel contractile forces generated by dermal fibroblasts and the relationship to cell morphology. J. Cell Physiol. 166: 33, 1996.
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Allenberg, M., Weinstein, T., Li, I., and Silwerman, M. Activation of procollagenase IV by cytochalasin D and concanavalin A in cultured rat mesangial cells: Linkage to cytoskeletal reorganization. J. Am. Soc. Nephrol. 10: 760, 1994.
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Yamato, M., Adachi, E., Yamamoto, K., and Hayashi, T. Condensation of collagen fibrils to the direct vicinity of fibroblasts as a cause of gel contraction. J. Biochem. 117: 940, 1995.
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Moulin, V., Auger, F. A., O’Connor-McCourt, M., and Germaine, L. Fetal and postnatal sera differentially modulate human dermal fibroblast phenotypic and functional features in vitro. J. Cell. Physiol. 171: 1, 1997.
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Lee, Y-R., Oshita, Y., Tsuboi, R., and Ogawa, H. Combination of insulin-like growth factor (IGF)-1 and IGF-protein binding protein-1 promotes fibroblast-embedded collagen gel contraction. Endocrinology 137: 5278, 1996.
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Vernon, R. B., and Sage, H. Contraction of fibrillar type I collagen by endothelial cells: a study in vitro. J. Cell. Biochem. 60: 185, 1996.
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Dugina, V., Alexandrova, A., Chaponnier, C., Vasiliev, J., and Gabbiani, G. Rat fibroblasts cultures from various organs exhibit differences in a-smooth muscle actin expression, cytoskeletal pattern, and adhesive structure organization. Exp. Cell Res. 238: 481, 1998.
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Komuro, T. Re-evaluation of fibroblasts and fibroblast-like cells. Anat. Embryol. 182: 103, 1990.
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Sappino, A. P., Schurch, W., and Gabbiani, G. Differentiation repertoire of fibroblastic cells: Expression of cytoskeletal proteins as marker of phenotypic modulation. Lab. Invest. 63: 144, 1990.
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Sempowski, G. D., Borello, M. A., Blieden, T. M., Barth, R. K., and Phipps, R. P. Fibroblast heterogeneity in the healing wound. Wound Rep. Reg. 3: 120, 1995.
Stromelysin-1-Deficient Fibroblasts Display Impaired Contraction in Vitro Kelli M. Bullard, M.D., 1 John Mudgett, Ph.D., Heinz Scheuenstuhl, B.S., Thomas K. Hunt, M.D., and Michael J. Banda, Ph.D. University of California, San Francisco, San Francisco, California 94143 Presented at the Annual Meeting of the Association for Academic Surgery, Seattle, Washington, November 18 –22, 1998
ing [1, 2]. Stromelysin-1 (MMP-3) has one of the broadest substrate specificities of the MMPs and is responsible for degrading proteoglycans, laminin, fibronectin, the nonhelical domains of collagen types IV and IX, propeptides of type I collagen, and all denatured collagens as well as proteolytic activation of procollagen [3]. Stromelysin-1 is synthesized primarily by fibroblasts [4 – 6] and has been identified in the stroma of normally healing rabbit corneal wounds [7], in stromal cells and keratinocytes of chronic ulcers [6], in burn wound fluid from human patients [8], and in basal keratinocytes of both normal and chronic, nonhealing human skin ulcers [9]. The presence of stromelysin-1 in these settings implies that it plays a key role in the process of wound healing. Targeted disruption of the stromelysin-1 gene in mice causes a delay in excisional wound healing due to a failure in the first phase of wound contraction. In normal mice, excisional wounds contract rapidly, decreasing wound area by approximately 50% per day during the first 4 days of healing. In strom-1 KO mice, however, excisional wounds lack early wound contraction, decreasing wound area by only 10 –15% per day [10]. We therefore postulated that stromelysin-1 activity plays a role in the mechanism of wound contraction. Wound fibroblasts are thought to be the functional cell initiating wound contraction and fibroblasts cultured on collagen matrices have been used as a model of wound contraction in vitro [11–13]. In this model, fibroblasts are grown on circular matrices containing predominantly type I collagen. Contractile ability is assessed by measuring changes in gel diameter or area [13]. Contractile capacity of fibroblasts from stromelysin-1-deficient mice was compared with that of fibroblasts from wild-type mice using this collagen gel model, which allowed examination of fibroblast contraction apart from other elements of wound healing.
Targeted disruption of the stromelysin-1 gene in mice causes a delay in excisional wound healing due to a failure in wound contraction. Therefore, we postulated that stromelysin-1 activity is responsible for initiating contraction. To test this hypothesis, we compared the contractile capacity of fibroblasts from stromelysin-1 knockout mice (strom-1 KO) with that of normal fibroblasts using a collagen gel contraction model. Fibroblast cultures were established from explants of skin and lung parenchyma from strom-1 KO and wild-type mice, then transferred to the surface of collagen gels. The extent of contraction was determined by measuring greatest gel diameter. Results demonstrated that (1) all fibroblasts contracted collagen gels in a uniform concentric fashion, (2) skin fibroblasts from both sets of mice exhibited greater gel contraction than did lung fibroblasts, and (3) strom-1 KO fibroblasts demonstrated significantly less contraction (21–23%) than wild-type fibroblasts. These data support the hypothesis that absence of stromelysin-1 results in defective fibroblast contraction that may contribute to delayed wound healing. © 1999 Academic Press Key Words: metalloproteinases; wound contraction; wound healing; stromelysin-1.
INTRODUCTION
Would healing occurs by a complex series of events that involves matrix deposition, remodeling, and wound contraction. Matrix metalloproteinases (MMPs) are a multigene family of enzymes that are responsible for extracellular matrix (ECM) remodeling in a wide range of physiological processes including wound heal1 To whom correspondence should be addressed at HSW-1601, Box 0570, 513 Parnassus Avenue, University of California, San Francisco, San Francisco, CA 94143– 0570. Fax: (415) 476 –2314. E-mail: [email protected].
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0022-4804/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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JOURNAL OF SURGICAL RESEARCH: VOL. 84, NO. 1, JUNE 1, 1999
TABLE 1
METHODS Strom-1 KO mice were generated through gene targeting by homologous recombination in murine embryonic stem cells as described previously [14] and maintained on 129Sv background. All procedures were performed in a laminar animal operating suite under aseptic conditions according to protocols approved by the University of California, San Francisco, Committee on Animal Research. Fibroblast cultures were established from explants of skin and lung parenchyma from strom-1 KO and wild-type mice and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). Collagen gels were prepared using Vitrogen type I collagen; Collagen Corp.) mixed with DMEM, phosphatebuffered saline (PBS), and 10% FBS, and pH adjusted at 4°C [15, 16]. Circular gels 20 mm in diameter were then poured into 12-well culture plates (Falcon) and incubated for 2 h at 37°C to allow polymerization of collagen. Trypsinized fibroblasts (passages 2 to 6) were then transferred to the surface of each gel (2.5 3 10 5 cells/gel) and incubated at 37°C for 24 h. Gels were rimmed and incubated at 37°C for an additional 24 h, then fixed with 10% formalin. The extent of contraction was determined by measuring the greatest diameter (mm) of each gel. Gel area was also calculated. Experiments consisted of 16 sets of skin fibroblasts (8 strom-1 KO, 8 wild type) and 8 sets of lung fibroblasts (4 strom-1 KO, 4 wild type) from 16 mice. Collagen gels without cells served as controls. Results were compared with Student’s t test (two-tailed).
RESULTS
All fibroblasts contracted collagen gels in a uniform, concentric fashion; control gels did not contract. Skin fibroblasts from both strom-1 KO mice and wild-type mice exhibited greater gel contraction than did lung fibroblasts. However, all strom-1 KO fibroblasts demonstrated less contraction than wild-type fibroblasts. Wild-type skin fibroblasts contracted gels to 45% of the original gel diameter (22% percent of original area) while strom-1 KO skin fibroblasts contracted gels to 60% of the original gel diameter (36% of original area). Similarly, wild-type lung fibroblasts contracted gels to 60% of the original gel diameter (36% of original area) versus 77% (59% of original area) for strom-1 KO lung fibroblasts. Thus, contraction measured by either gel diameter or gel area was significantly greater when wild-type fibroblasts were compared with strom-1 KO fibroblasts (P , 0.001 in all groups; Table 1, Fig. 1). DISCUSSION
Wound contraction is a fundamental event in wound healing, yet the biological process remains poorly understood. In mice, excisional dermal wounds contract more than 50% of their original size during the first 24 to 48 h after wounding, thus decreasing the volume that must be filled with granulation tissue and reducing the distance that must be traversed by keratinocytes. Our previous experiments have shown that stromelysin-1 is crucial for normal wound contraction in vivo [10]. In these experiments, stromelysin-1deficient fibroblasts contracted collagen gels to a lesser extent when compared with normal wild-type fibro-
Mean Gel Diameters and Areas after 24 h (Original Gel Diameter 5 20 mm, Original Gel area 5 314 mm 2)
Skin fibroblasts Diameter (mm) Area (mm 2) Lung fibroblasts Diameter (mm) Area (mm 2)
Control (no cells)
Wild type
Strom-1 KO
20.0 314.0
9.3 6 0.4 a 68.0 6 2.7
12.1 6 0.5* 113.0 6 4.5*
20.0 314.0
12.0 6 0.6 113.0 6 5.7
15.4 6 0.2* 186.0 6 1.9*
a 6SEM. * P , 0.001.
blasts. Because fibroblasts are involved in initiating wound contraction, this impaired ability to contract collagen gels in vitro may contribute to the impaired wound contraction we observed in vivo. Fibroblast traction on the ECM has been proposed as the mechanism underlying the first phase of wound contraction [21, 22]. Normal wound contraction requires attachment of fibroblasts to the underlying ECM, cell contraction and/or locomotion, and matrix architecture capable of transmitting force. In the collagen gel model, the underlying ECM is constant (type I collagen) and, therefore, allows fibroblast function to be studied in isolation. Fibroblast attachment to the gels involves interaction between cell surface adhesion molecules (b1 integrins) and type I collagen [23–29]. Because stromelysin-1 cleaves a wide range of matrix macromolecules, it is possible that it plays a role in integrin processing or modification of attachment sites on the underlying collagen fibrils. Fibroblast contraction in vivo also requires synthesis of an actin cable [29 –31] and an actin stress fiber network [24, 32, 33]. In a collagen gel model, depolymerization of actin filaments by cytochalasin B destroys contraction [20]. Our in vivo data suggest that an inability to adequately organize fibrillar actin may contribute to poor wound contraction in the strom-1 KO mice [10], it remains to be determined if strom-1 KO fibroblasts cultured on collagen gels produce inadequate actin networks as well. Finally, several growth factors including TGF-b1, IGF-1, and PDGF affect contraction [27, 28, 34 –36], and stromelysin-1 deficiency may result in altered processing of growth factors or growth factor receptors. Further experiments are planned to address these issues using the gel contraction model. Several lines of evidence suggest that the MMPs are involved in cell motility and contraction. Fibroblasts migrating through collagen gels are known to secrete collagenase [17]. Addition of tissue inhibitor of metalloproteinases (TIMP) decreases collagen gel contraction [18, 19]. Similarly, tumor cells that are incapable of activating MMP-2 (gelatinase) contract collagen gels
BULLARD ET AL.: STROMELYSIN-1 DEFICIENCY IMPAIRS CONTRACTION IN FIBROBLASTS
33
FIG. 1. Total area of collagen gels containing stromelysin-1 KO fibroblasts versus wild-type fibroblasts (6SEM).
poorly compared with cells capable of producing active enzyme [20]. Our data imply that stromelysin-1 may be the crucial MMP in fibroblast contraction and that absence of this proteinase in some way alters the ability of fibroblasts to contract type I collagen matrices. Because stromelysin-1 activates procollagenase, absence of stromelysin-1 may also alter the normal proteolytic cascade required for cell contraction and motility. The redundancy of the MMP system, however, may account for the fact that the defect we observed was not complete; while strom-1 KO fibroblasts contracted gels poorly compared with normal fibroblasts, they were able to contract the gels by approximately 40%. Similarly, although excisional wounds on strom-1 KO mice heal far more slowly in vivo than do normal wounds, they do eventually close [10]. We postulate that other MMPs may be capable of remodeling the ECM in the absence of stromelysin-1, albeit less efficiently, to allow contraction and healing to proceed. Gel contraction differed between lung and skin fibroblasts in both sets of mice. In both strom-1 KO and wild-type mice, dermal fibroblasts contracted more than lung fibroblasts. This functional heterogeneity among different fibroblast populations has been noted by several authors and is thought to contribute to different physiological roles played by these cells [37– 40]. However, we also observed that regardless of the tissue source of the fibroblasts, strom-1 KO fibroblasts had impaired contractile ability compared with normal fibroblasts. This observation suggests that
stromelysin-1 plays a fundamental role in fibroblast function and that the defect produced by deletion of the stromelysin-1 gene is not limited to dermal fibroblasts. It will be interesting to determine if strom-1 KO mice exhibit delayed wound healing or altered fibrosis in organs other than skin. REFERENCES 1. 2. 3. 4.
5.
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