ARTICLE |
Correspondence to: E. Helene Sage, The Hope Heart Institute, 1124 Columbia St./Ste. 720, Seattle, WA 98104.
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Summary |
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Expression of SPARC (secreted protein acidic and rich in cysteine; osteonectin, BM-40), an extracellular matrix (ECM) associated protein, is coincident with matrix remodeling. To further identify the functions of SPARC in vivo, we have made excisional wounds on the dorsa of SPARC-null and wild-type mice and monitored closure over time. A significant decrease in the size of the SPARC-null wounds, in comparison to that of wild-type, was observed at Day 4 and was maximal at Day 7. Although substantial differences in the percentage of proliferating cells were not apparent in SPARC-null relative to wild-type wounds, primary cultures of SPARC-null dermal fibroblasts displayed accelerated migration, relative to wild-type fibroblasts, in wound assays in vitro. Although the expression of collagen I mRNA in wounds, as measured by in situ hybridization (ISH), was not significantly different in SPARC-null vs wild-type mice, the collagen content of unwounded skin appeared to be substantially lower in the SPARC-null animals. By hydroxyproline analysis, the concentration of collagen in SPARC-null skin was found to be half that of wild-type skin. Moreover, we found an inverse correlation between the efficiency of collagen gel contraction by dermal fibroblasts and the concentration of collagen within the gel itself. We propose that the accelerated wound closure seen in SPARC-null dermis results from its decreased collagen content, a condition contributing to enhanced contractibility. (J Histochem Cytochem 50:110, 2002)
Key Words: SPARC, matricellular, transgenic, wound healing, extracellular matrix, collagen
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Introduction |
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THE ABILITY of animals to repair cutaneous wounds is crucial for survival after injury, although advanced age and diseases such as diabetes alter efficient wound healing (
SPARC (secreted protein acidic and rich in cysteine; osteonectin, BM-40) is a matrix-associated protein that influences a variety of cellular activities in vitro (
Increases in the expression of SPARC are often observed in tissues undergoing remodeling. Induction of SPARC is associated with tumor metastasis, angiogenesis, fibrosis, and bone metabolism (
For these experiments we used mice with a targeted deletion of the SPARC gene. The phenotype of SPARC-null mice is ostensibly normal except that the mice display early-onset cataractogenesis and progressively severe osteopenia (
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Materials and Methods |
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Animals
C57BL/6 x 129SVJ SPARC-null mice were generated as described in
In Situ Hybridization
In situ hybridization (ISH) analysis was carried out as described in -[35S]dUTP (Amersham; Arlington Heights, IL). The template was a 312-bp HindIII/EcoR1 fragment from the 3 UTR of mouse
1(I) collagen in pBSK (provided by Dr. Paul Bornstein, University of Washington) (
34 x 107 cpm/µg. A riboprobe transcribed in the sense orientation was used as a control. The method of ISH was a modified protocol described by
Immunohistochemistry
Sections of wounds from wild-type and SPARC-null mice were deparaffinized and either subjected to antigen retrieval with a 20-min incubation in steaming citrate buffer (0.1 M), as recommended for the Ki-67 antibody (Dako; Carpinteria, CA), or not subjected to antigen retrieval in the case of the anti-proliferating cell nuclear antigen (PCNA) antibody (biotin-conjugated IgG; Zymed, South San Francisco, CA). Sections were blocked in 2% normal goat serum and were incubated in primary antibody for 1 hr. The Ki-67 antigen was detected with a secondary antibody conjugated to biotin, and the signals for both Ki-67 and PCNA were amplified by incubation with the ABC Elite kit (Vector Labs; Burlingame, CA). Horseradish peroxidase activity associated with the primary antibodies was visualized with 3,3'-diaminobenzidine (DAB) substrate with or without nickel enhancement. Slides were counterstained with methylene green (Ted Pella; Redding, CA). Stained sections were viewed on a Leica DMR microscope and images were captured either by Kodachrome 400 film or by digital capture with an RT spot camera (Diagnostics Instruments; Sterling Heights, MI) linked to a Macintosh G4 computer (Apple; Cupertino, CA).
Primary Cell Isolation, Migration Assays, and Collagen Gel Contraction Assays
Dermal cells were isolated from the skin of wild-type and SPARC-null animals, as described in
For migration experiments, equal numbers of wild-type and SPARC-null dermal cells (as quantified by a hemacytometer) were plated in 6-well plates in growth medium and were grown to confluence. A rubber spatula was used to remove a defined area of cells and thereby create a wound in the monolayer. Uniform wounds were made in wild-type and SPARC-null culture wells and were photographed at time 0. An area of the well was designated by marks on the dish such that the same field could be followed over time. The degree of migration was monitored by photography of the same field at 24-hr intervals. Each experiment was composed of triplicate wells for both wild-type and SPARC-null cultures. Scanned images were imported into an NIH image software program and were quantified as the percent of area occupied by cells vs total area included within the image. Duplicate wells were plated in parallel to monitor proliferation of the cultured cells. Cells were removed by trypsin digestion and were quantified by a hemacytometer at 24, 48, and 72 hr, coinciding with the time of photography of the migration assays. Higher-magnification images of the cell migration front were taken to monitor the number of mitotic cells in the wound area of the dish.
Collagen gel contraction assays were carried out as described in
Hydroxyproline Analysis
Measurement of hydroxyproline content of SPARC-null and wild-type skin was carried out as described by
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Results |
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Wound Measurements and Histological Analysis
Cutaneous wound healing relies on cell migration, proliferation, and ECM remodeling to repair the injury, three processes that are influenced by the expression of SPARC (
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The rates of wound closure were monitored by measurement of the wounds at Days 4, 7, and 11 with a micrometer (Fig 2). On Day 4 the average size of the wounds in SPARC-null mice was 4.46 mm2, whereas the average of the wild-type wounds was 6.32 mm2. The differences observed on Day 4 were more pronounced on Day 7. SPARC-null wounds averaged 2.23 mm2 compared to wild-type wounds, which averaged 4.96 mm2. Measurements on Day 11 did not yield significant numbers because only one wound of six was unclosed in SPARC-null animals (three of six were unclosed and therefore measurable in wild-type animals). A significant decrease in the size of the wounds in the SPARC-null mice was observed. These data indicate an increased capacity of the null mice to close excisional wounds. In addition, the rate of closure observed at early time points implicated enhanced wound contraction in SPARC-null vs wild-type mice. Wound contraction is a significant component of dermal excisional wound healing in mice and in humans (
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The experiments shown are representative of eight separate wound-healing experiments performed in our laboratory. Although the parameters of these studies differed (the age of the mice and the size of the wound), the results were consistent. In every case, an increase in the rate of closure was observed in SPARC-null mice over that of appropriate wild-type control mice at a minimum of one time point. The trend of accelerated wound repair in the absence of SPARC has been reproducible in every experiment. Fig 1 Fig 2 Fig 3 Fig 4 represent one experiment performed with 5-mm wounds generated in 6-month-old mice.
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To determine the degree to which the absence of SPARC might affect collagen I expression in response to injury, we performed ISH analysis of wounds from wild-type and SPARC-null animals. A representative wild-type (Fig 3A) and SPARC-null wound (Fig 3B) at Day 5 is shown; arrows indicate the margins of the wound bed. We did not observe significant differences in the level or distribution of 1(I) collagen mRNA expression between wild-type and SPARC-null wounds. In addition, picrosirius red staining of Day 11 wounds showed no substantial differences in collagen deposition at the wound site of SPARC-null compared to wild-type mice (Fig 3C and Fig 3D). Collagen I was also assessed by immunohistochemistry with antibodies against the N-terminal propeptide of Type I procollagen to differentiate newly synthesized from previously-deposited collagen, as well as by immunoblotting analysis (data not shown). Although some variability among wounds was observed, the overall extent of fibrillar collagen deposition in the SPARC-null wounds did not appear to be significantly different from that in wild-type wounds. By immunohistochemistry there were also no gross differences in Type IV collagen and fibronectin between wild-type and SPARC-null wounds (data not shown). However, the collagen content of unwounded skin did appear to be decreased in SPARC-null vs wild-type skin (see below).
Proliferation in SPARC-null vs Wild-type Wounds
SPARC interferes with the cell-cycle progression induced by a variety of growth factors in vitro (
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Migration of Dermal Fibroblasts In Vitro
Because cell migration is another critical component of cutaneous wound repair, we asked whether there were differences in migration rates between primary wild-type and SPARC-null dermal cells. Wild-type and SPARC-null cells were plated at equal densities and were allowed to reach confluence. A wound of uniform size was made across the monolayer with a soft rubber spatula to avoid excessive injury to the remaining cells, and a designated field was photographed at time 0. The same field was photographed at 24 and 48 hr after wounding. The images were scanned for quantification of cell invasion, as shown in Table 2. The experiment was repeated four separate times with primary isolates from three different preparations, with similar results. The SPARC-null dermal cells consistently showed an enhanced capacity to invade the abraded area on the dish, compared to wild-type cells. To assess whether differences in the rates of proliferation affected invasion, we plated parallel cultures to monitor cell division in two separate experiments. At each migration time point, the cells were detached with trypsin and were counted by a hemacytometer. It was anticipated that SPARC-null cells would divide more rapidly in vitro relative to wild-type cells (
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Collagen Composition of SPARC-null Skin
Although differences in the migration of SPARC-null vs wild-type cells were apparent, the marked rate of wound closure observed at early time points in the SPARC-null mice was suggestive of differences in wound contraction. In addition, preliminary observations of SPARC-null skin implied differences in the collagenous ECM of the dermis compared to that of wild-type. For example, SPARC-null skin appeared to be more prone to tearing than wild-type skin. In addition, collagenase treatments performed during primary dermal fibroblast isolation (see Materials and Methods) revealed an increased sensitivity of the SPARC-null dermis to digestion. Whereas wild-type dermis typically required 45 hr for complete dissolution of the tissue by collagenase, parallel samples of SPARC-null dermis required 23 hr under identical conditions (unpublished experiments).
To quantify potential differences in collagen composition, we performed hydroxyproline analyses on SPARC-null and wild-type skins. As shown in Fig 4, SPARC-null skin showed an average hydroxyproline content of 8.7 µg/mg tissue, whereas wild-type skin yielded a value of 17.6 µg/mg tissue. The level of hydroxyprolyl post-translational modification of collagen chains did not appear to be significantly different between SPARC-null and wild-type mice, as assessed by SDS-PAGE analysis of collagen extracted from skin and tails (data not shown). We conclude that the skin of SPARC-null mice has a substantially reduced collagen concentration in comparison to that of wild-type mice. Because wound healing in mice results in part from enhanced contraction of the dermal matrix, the accelerated rate of closure observed in SPARC-null animals could be a consequence of the decreased content of dermal collagen relative to that of wild-type mice, or due to an increased contractile capacity of dermal fibroblasts in the absence of SPARC.
Collagen Gel Contraction Assays
To determine the capacity of SPARC-null dermal cells to contract collagen gels, SPARC-null and wild-type fibroblasts were plated in gels of increasing collagen concentration. As shown in Fig 5, SPARC-null fibroblasts did not exhibit an increased capacity for collagen gel contraction in comparison to wild-type cells. An equal number of SPARC-null (Fig 5, diamonds) or wild-type dermal fibroblasts (Fig 5, circles) contracted a collagen gel of 0.5 mg/ml to an area of 19.6 mm2, whereas an area of 75 mm2 was observed with a collagen gel of 1.25 mg/ml. Therefore, there was an inverse correlation between the degree of contraction and the concentration of the collagen gel for both cell genotypes. We have observed in some preparations of fibroblasts a decrease in the capacity of SPARC-null cells to contract collagen gels in comparison to wild-type cells. This phenomenon has been variable. More importantly, SPARC-null cells consistently showed an inverse correlation between the capacity of the cells to contract collagen gels and the concentration of collagen within the gel itself. Because SPARC-null fibroblasts did not exhibit an increased capacity for collagen gel contraction in comparison to wild-type cells, the increased wound closure observed in the absence of SPARC most likely results from the altered collagenous ECM of the dermis. Given that SPARC-null skin contained approximately half the amount of collagen (as measured by hydroxyproline analysis) as that of wild-type skin, we propose that the skin of SPARC-null mice is more susceptible to contraction than that of wild-type. This property could account, at least in part, for the enhanced rate of wound closure observed in SPARC-null vs wild-type mice.
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Discussion |
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The function(s) of SPARC, a matricellular protein with several apparent effects in vitro, has been elusive in vivo. Recent evidence from SPARC-null mice indicates that this protein is required for lens transparency and maintenance of bone mass. We now report that the absence of SPARC in mice leads to accelerated dermal wound repair. SPARC-null mice were able to close excisional wounds more rapidly than wild-type counterparts. Given the capacity of SPARC to retard cell cycle progression in culture, we sought to determine whether differences in cell proliferation might account for the increased healing observed in SPARC-null vs wild-type mice. The percentage of proliferating cells at Days 4 and 7 did not appear to differ significantly in SPARC-null vs wild-type wounds, as determined by quantification of PCNA- and Ki-67-immunoreactive cells. However, we can not exclude the possibility that differences in the rate of proliferation of individual cells present in the wound bed might be accelerated in SPARC-null mice, i.e., the absence of SPARC might render cells more sensitive to mitogenic stimuli and thus shorten the time required for cell-cycle traverse at early times after injury. Nevertheless, a substantial increase in the profile of dividing cells was not apparent in SPARC-null wounds and therefore is most likely not the principal mechanism that leads to accelerated wound closure in these mice.
Consistent differences in the deposition of certain ECM components (fibrillar collagen, Type IV collagen, and fibronectin) at the wound site were not detected between SPARC-null and wild-type wounds, although some variability among wounds was observed. However, the collagen composition of the uninjured dermal ECM in SPARC-null mice is significantly reduced in comparison to wild-type mice. Hydroxyproline analysis confirmed that SPARC-null skin contains 50% less collagen than age-matched wild-type skin. However, abundant production of newly synthesized collagen in the wound bed was not substantially reduced in the absence of SPARC. Therefore, we propose that collagen production in response to wounding is more robust than during routine maintenance of the dermal collagen layer, most likely due to the presence of multiple collagen-stimulating factors in the wound. Alternatively, SPARC might be involved in the incorporation and/or stabilization of collagen into the more mature fibers of remodeled, structured ECM rather than the expression and secretion of collagen I per se. In this case, alterations in collagen expression and accumulation would not be substantial in the days and weeks after injury but would become more apparent at later time points during scar formation. Experiments are under way to address these possibilities.
The reduced collagen content of the SPARC-null dermis appears to render the skin intrinsically more susceptible to cell contraction than that of wild-type mice. Collagen gel contraction assays demonstrated a linear relationship between the concentration of collagen gels and their propensity for contraction by dermal fibroblasts. In general, collagen gel contraction by SPARC-null vs wild-type dermal fibroblasts was not increased. Therefore, the increased wound contraction was most likely due not to an increased cellular capacity for contraction in the absence of SPARC but instead to an altered ECM. Interestingly, the tight-skin mouse (Tsk) exhibits delayed wound contraction in response to excisional wounding (
The capacity of SPARC to influence the amount of collagen in the uninjured dermis was somewhat unexpected. Although SPARC binds to a number of different collagens, including Types I, III, IV, and V, all of which are represented in the dermal or epidermal ECM, a prior function of SPARC in collagen accumulation has not been described (
The activity of MMPs is critical for both collagen gel contraction and wound contraction (
Although the composition of the ECM in the absence of SPARC undoubtedly influences the process of accelerated closure, the effect of SPARC on growth factor activity might also contribute to the increase in the rate of wound repair in SPARC-null mice. The capacity of SPARC to bind to and diminish the activity of growth factors, such as PDGF and VEGF, and to associate with the ECM make it a prime candidate for a modulator of cell behavior in response to injury. The absence of SPARC might allow increased migratory and/or contractile activity mediated by growth factors and would thereby accelerate wound repair (
The concept that matrix-associated proteins might modulate cell behavior in response to injury is gaining support with the publication of recent studies involving transgenic animals. A number of matricellular proteins show increased expression in response to injury: thrombospondins 1 and 2, osteopontin, and tenascin C (
In conclusion, cutaneous wound healing provides an excellent milieu for the study of factors that influence cell interaction with ECM. For repair of damage to the skin, the matrix must be degraded, rearranged, and eventually reconstructed to restore a functional barrier for the organism. We have found evidence that the absence of SPARC expression enhances wound closure in mice. Future experiments will provide valuable insights into the molecular mechanisms governing SPARC activity in the skin and in wound repair.
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Acknowledgments |
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Supported by National Institutes of Health grants GM 40711 and HL 59574 (EHS), AG 15837 (MJR), and DK 07467 (ADB), by National Science Foundation EE 04-150 (EHS), and by a Beeson Scholar Award (MJR).
We are indebted to Emmett Pinney for the hydroxyproline analysis. In addition, we would like to thank Juliet G. Carbon for valuable assistance with the mouse colony, and Dr Pauli Puolakkainen and the members of the Sage Laboratory for helpful discussions and suggestions.
Received for publication July 18, 2001; accepted October 10, 2001.
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