Copyright ©The Histochemical Society, Inc.

SPARC-thrombospondin-2-double-null Mice Exhibit Enhanced Cutaneous Wound Healing and Increased Fibrovascular Invasion of Subcutaneous Polyvinyl Alcohol Sponges

Pauli A. Puolakkainen, Amy D. Bradshaw, Rolf A. Brekken, May J. Reed, Themis Kyriakides, Sarah E. Funk, Michel D. Gooden, Robert B. Vernon, Thomas N. Wight, Paul Bornstein and E. Helene Sage

Hope Heart Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington (PAP,ADB,RAB,SEF,MDG,RBV, TNW,EHS); Departments of Medicine (PAP,MJR,PB) and Biochemistry (TK,PB), University of Washington, Seattle, Washington; and Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland (PAP)

Correspondence to: E. Helene Sage, PhD, Hope Heart Program, Benaroya Research Institute at Virginia Mason, 1201 9th Ave., Seattle, WA 98101. E-mail: hsage{at}benaroyaresearch.org or pauli.puolakkainen{at}hus.fi


    Summary
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 Summary
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 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Secreted protein acidic and rich in cysteine (SPARC) and thrombospondin-2 (TSP-2) are structurally unrelated matricellular proteins that have important roles in cell–extracellular matrix (ECM) interactions and tissue repair. SPARC-null mice exhibit accelerated wound closure, and TSP-2-null mice show an overall enhancement in wound healing. To assess potential compensation of one protein for the other, we examined cutaneous wound healing and fibrovascular invasion of subcutaneous sponges in SPARC-TSP-2 (ST) double-null and wild-type (WT) mice. Epidermal closure of cutaneous wounds was found to occur significantly faster in ST-double-null mice, compared with WT animals: histological analysis of dermal wound repair revealed significantly more mature phases of healing at 1, 4, 7, 10, and 14 days after wounding, and electron microscopy showed disrupted ECM at 14 days in these mice. ST-double-null dermal fibroblasts displayed accelerated migration, relative to WT fibroblasts, in a wounding assay in vitro, as well as enhanced contraction of native collagen gels. Zymography indicated that fibroblasts from ST-double-null mice also produced higher levels of matrix metalloproteinase (MMP)-2. These data are consistent with the increased fibrovascular invasion of subcutaneous sponge implants seen in the double-null mice. The generally accelerated wound healing of ST-double-null mice reflects that described for the single-null animals. Importantly, the absence of both proteins results in elevated MMP-2 levels. SPARC and TSP-2 therefore perform similar functions in the regulation of cutaneous wound healing, but fine-tuning with respect to ECM production and remodeling could account for the enhanced response seen in ST-double-null mice.

(J Histochem Cytochem 53:571–581, 2005)

Key Words: SPARC • thrombospondin • wound healing • matricellular • extracellular matrix • epidermis • dermis • angiogenesis • collagen


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
SECRETED PROTEIN ACIDIC and rich in cysteine (SPARC), thrombospondin (TSP)-1 and -2, tenascin C and X, osteopontin, and SC1/hevin are matricellular proteins that do not contribute structurally to extracellular matrix (ECM). Rather, they bind to a variety of structural proteins (e.g., collagens) and modulate the interaction of cells with the ECM (Bornstein and Sage 2002Go).

SPARC (also known as BM-40 and osteonectin) is a 32-kDa calcium-binding glycoprotein secreted by various cells, e.g., fibroblasts, endothelial cells, and platelets (Stenner et al. 1986Go; Sage et al. 1989aGo; Kasugai et al. 1991Go; Vuorio et al. 1991Go). SPARC modulates the interaction of cells with the ECM, is counteradhesive for cells from diverse sources, inhibits cell spreading, and regulates the production of several ECM proteins (Bradshaw and Sage 2001Go). Two principal functions of SPARC are modification of cell shape and inhibition of cell-cycle progression (Bradshaw and Sage 2001Go). SPARC is expressed during development and in remodeling tissues in adults (Reed et al. 1993Go; Puolakkainen et al. 1999Go; Bornstein and Sage 2002Go). Targeted disruption of the SPARC gene in mice results in a complex phenotype characterized by early cataractogenesis (Gilmour et al. 1998Go; Norose et al. 1998Go; Yan et al. 2002Go), increased amounts of subcutaneous adipose tissue (Bradshaw et al. 2003aGo), decreased amounts of collagen in the skin (Bradshaw et al. 2003bGo), and progressively severe osteopenia (Delany et al. 2000Go). The curled tails of these mice are also suggestive of altered collagen fibrillogenesis. Furthermore, enhanced fibrovascular invasion of subcutaneous polyvinyl alcohol (PVA) sponge implants (Bradshaw et al. 2001Go), enhanced growth of malignant tumors (Brekken et al. 2003Go), and reduced foreign body response (Puolakkainen et al. 2003Go) have been reported in SPARC-null mice. We have previously described the spatial and temporal distribution of SPARC during cutaneous wound healing (Reed et al. 1993Go) and during the healing of intestinal anastomoses (Puolakkainen et al. 1999Go) and have recently described accelerated cutaneous wound closure in SPARC-null mice (Bradshaw et al. 2002Go). Dermal fibroblasts from these animals displayed higher rates of migration, relative to cells from wild-type (WT) mice, in a wounding model in vitro (Bradshaw et al. 2002Go).

TSP-2 is a matricellular glycoprotein that influences the formation of collagen fibrils and the interactions of cells with ECM. It is produced in many connective tissues during development and in response to injury in adult animals (Kyriakides et al. 1999bGo; Bornstein et al. 2000Go). The phenotype of TSP-2-null mice includes enhanced vascular density and angiogenesis, a bleeding disorder, increased bone growth, and abnormal collagen fibrils associated with laxity of tendons and ligaments as well as an increased fragility of skin (Kyriakides et al. 1998Go). Dermal fibroblasts from TSP-2-null mice exhibit defects in adhesion that are due to augmented levels of matrix metalloproteinase (MMP)-2 (Yang et al. 2000Go). TSP-2 is expressed during cutaneous wound healing (Kyriakides et al. 1999bGo), and TSP-2-null mice displayed accelerated excisional wound healing with irregularly organized and highly vascularized granulation tissue (Kyriakides et al. 1999bGo). No difference was found in the rate of re-epithelialization, but there was less scarring in TSP-2 mice, relative to WT animals. The fibrovascular invasion of subcutaneous PVA sponges was also increased in TSP-2-null mice (Kyriakides et al. 1999aGo).

Clearly, SPARC and TSP-2 are distinct matricellular proteins, with apparently similar functions (e.g., ECM production and/or assembly) as well as disparate ones (e.g., inhibition of angiogenesis by TSP-2). To further evaluate the function of SPARC and TSP-2 in vivo, to discriminate between their contributions, and to assess potential compensation of one protein for the other, we measured cutaneous wound healing, invasion of PVA sponges, and changes in ECM in SPARC-TSP-2 (ST) double-null mice, compared with WT animals. We report enhanced epidermal closure and dermal wound healing, as well as increased fibrovascular invasion of PVA sponges, which are associated with altered ECM in ST-double-null mice.


    Materials and Methods
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 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Analysis of Genomic DNA by Polymerase Chain Reaction (PCR)
Tail-derived DNA was digested with proteinase K (100 ng/µl) at 55C overnight, followed by precipitation in isopropanol. PCR was performed as described by Graves and Yablonka-Reuveni (2000)Go. The following primers were used. SPARC (WT allele): 5'-GATGAGGGTGGTCTGGCCCAGCCCTAGATGCCCCTCAC-3' (forward) and 5'-CACCCACACAGCTGGGGGTGATCCAGATAAGCCAAG-3' (reverse); SPARC (null allele): forward primer as above; 5'-GTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAG-3' (reverse, located in Neo insert) (Figure 1). TSP-2 (WT allele): 5'-GGTGACCACGTCAAGGACAC-3' (forward) and 5'-TGGCCACGTACATCCTGCT 3' (reverse); TSP-2 (null allele): 5'-GATCAGCAGCCTCTGTTCACATAC-3' (forward, located in Neo insert) and 5'-GGAGAAGAATTAGGGAGGCTTAGGG-3' (reverse, located in TSP-2 intron 3) (Figure 1).



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Figure 1

The SPARC and TSP-2 genes are disrupted in ST-double-null mice. PCR products of genomic DNA isolated from WT (+/+) and double-null (–/–) mouse-tail biopsies are shown. The WT allele of the TSP-2 gene was detected with primers (TS2G-A and TS2G-B) to sequences within the gene that yielded a 539-bp sequence; the disrupted allele was detected with primers to sequences within the gene (T2IN3) and the neomycin insert (447 neo) to yield a 900-bp sequence. The WT and disrupted alleles of the SPARC gene were similarly detected with primer pairs MGSPARC.FOR and MGSP.REV, and MGSPARC.FOR and NEO.REV that yield 296- and 450-bp sequences, respectively. MW markers appear on the right of each panel.

 
Animal Model
C57Bl/6 x 129SvJ SPARC-TSP-2 (ST) double-null mice and corresponding WT animals (produced from heterozygous matings) were generated from the corresponding single-null animals (Kyriakides et al. 1998Go; Bradshaw et al. 2002Go). The genotypes of the mouse colony were monitored by PCR using genome-specific primers on purified tail DNA. All mice were handled and studies were carried out according to the guidelines of the American Association for Accreditation of Laboratory Care and The Hope Heart Institute Animal Care and Use Committee. All mice were housed in a modified pathogen-free facility before and throughout the study. Twenty-eight ST-double-null and 25 WT mice (3–7 months old) were used for the experiments. For surgery, the mice were anesthetized by isoflurane inhalation (Abbott Laboratories; North Chicago, IL), and their backs were shaved and cleaned. Each animal received two excisional punch biopsies (Acu-Punch; Acuderm, Inc., Ft. Lauderdale, FL) of 5-mm diameter and two incisional (1 cm) full-thickness cutaneous wounds. Incisions were made with a scalpel and were closed with staples. All instruments were rinsed in endotoxin-free water and were sterilized prior to use. Animals were housed individually and were monitored for 4–6 hr after wounding to ensure that premature closure of the biopsy wound edges did not occur. There were no significant differences in the wound sizes at time 0. The animals were sacrificed at 1, 4, 7, 10, and 14 days after wounding.

Assessment of Wound Healing
At the time of sacrifice, the wound images were captured digitally, and the wound area and the extent of epithelial closure were calculated with computerized planimetry using Adobe Photoshop and public domain NIH-Image software. Dermal healing was assessed in hematoxylin- and eosin-stained sections of wounds with a maturity scoring system introduced by Greenhalgh et al. (1990)Go as modified by Spenny et al. (2002)Go. The amount and type of cell accumulation, the type of granulation tissue, and scarring are assessed in this model, with a score ranging from 1 to 15.

Immunohistochemical Staining
Immunolocalization of SPARC was performed as previously described (Sage et al. 1989bGo). Wound samples were immersed in Methacarn fixative (60% methanol, 30% chloroform, 10% glacial acetic acid) for 24 hr, embedded in paraffin, and cut into 5-µm sections. Sections were heated on a hotplate for 1 hr (60C), deparaffinized, and rehydrated through an ethanol gradient ending with water. Sections were incubated in 1% hydrogen peroxide for 30 min to block endogenous peroxidases, followed by blocking in Autozyme (Biomeda; Foster City, CA) at 37C for 10 min and in 25% Sea-Block (East Coast Biologics; North Berwick, ME) in 0.2% PBS-Tween for 20 min. Incubation with a goat polyclonal anti-SPARC primary antibody (10 µg/ml) (Brekken et al. 2003Go) was carried out for 1 hr at room temperature. After a 1-hr incubation with a donkey anti-goat secondary antibody (Jackson Immunoresearch Laboratories; West Grove, PA) (2.5 µg/ml) at room temperature, immune complexes were detected with 3,3'-diaminobenzidine (Sigma Chemical Co.; St Louis, MO). The sections were counterstained with hematoxylin, dehydrated with a graded series of ethanol solutions, cleared with xylene, and mounted with Permount (Fisher Scientific; Fair Lawn, NJ). Stained sections were captured digitally with a cooled CCD camera (SPOT RT Diagnostic Instruments Inc.; Sterling Heights, MI). Control sections were treated with secondary antibody only. Tissue sections of testis from ST-double-null and WT mice served as negative and positive controls, respectively.

For assessment of cell proliferation, six mice of each genotype were injected intraperitoneally (2 µg/g) on the day of sampling with 300 µg/ml of 5-bromo-2'-deoxyuridine (BrdU) (Sigma) and were sacrificed after 8 hr. Samples were prepared for immunostaining as described (Reed et al. 1996Go). The number of BrdU-positive cells/wound in the wound area was calculated from these slides.

Reverse Transcriptase PCR (RT-PCR)
RNA was isolated from wound tissue with Tri Reagent (Molecular Research Center, Inc.; Cincinnati, OH) according to the manufacturer's instructions. PCR was performed as described (Graves and Yablonka-Reuveni 2000Go) with the primer sequences (sense/anti-sense) for SPARC, rpS6, transforming growth factor (TGF)-ß, vascular endothelial growth factor (VEGF), MMP-2, collagen I, and collagen VI, as described by Brekken et al. (2003)Go.

Electron Microscopy
Samples were immersed in Karnovsky's fixative and were processed for transmission electron microscopy according to established methods (Wight et al. 1997Go).

Isolation and Culture of Dermal Fibroblasts
Dermal cells were isolated from the skin as described (Bradshaw et al. 2002Go). In brief, pieces of shaved skin were incubated in 0.25% trypsin (Sigma) in Solution A (10 mM glucose, 3 mM KCl, 130 mM NaCl, 1 mM Na2HPO4/7H2O, 30 mM Hepes, pH 7.4) overnight at 4C to separate the epidermis from the dermis. The dermal pieces were incubated in 0.25% Clostridium histolyticum collagenase (Worthington Enzymes; Freehold, NJ) at 37C for 4–6 hr. The digested dermis was triturated and plated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (Gibco-BRL; Gaithersburg, MD), 500 U/ml penicillin G, 500 U/ml streptomycin sulfate, and 2.5 µg/ml fungizone (growth medium). The majority of cells in these primary cultures were dermal fibroblasts (Bradshaw et al. 2002Go). All experiments were performed with cells at early passage (P1–P4), before cellular senescence or transformation.

In Vitro Wounding Model for Cell Migration
For migration studies, equal numbers of ST-double-null and WT dermal fibroblasts were plated in six-well plates in growth medium and were grown to confluence. A rubber spatula was used to remove a defined area of cells, i.e., to create a wound in the monolayer. An area of each well was designated by marks on the dish such that the same field could be followed over time. The degree of cell migration was monitored by photography of the same field at 24-hr intervals (24, 48, and 72 hr after wounding). Scanned images were imported into NIH-Image and were quantified as the percent of area occupied by cells vs the total area of the original wound.

Collagen Gel Contraction Assays
Assays of cell-mediated contraction of native, fibrillar type-I collagen gels were performed according to Vernon and Gooden (2002)Go. The disk-shaped, 12.7-mm diameter gels, which were polymerized from 0.5 mg/ml rat tail collagen (BD Biosciences; Bedford, MA), contained ST-double-null or WT dermal fibroblasts at three different concentrations. (6, 12, and 24 x 103 cells/400 µl gel). The gels were cultured for 20 hr at 37C in DMEM/10% fetal calf serum to permit contraction and subsequently were fixed with neutral-buffered formalin (Sigma), immersed in deionized water, and viewed under darkfield illumination with a stereomicroscope equipped with a CCD (SPOT) camera. Areas of gels were measured from digital images with NIH-Image.

Zymography
Equivalent numbers of ST-double-null and WT dermal fibroblasts were cultured in DMEM/1% fetal calf serum. The culture media were conditioned for 3 days, after which zymography was performed as described (Koike et al. 2002Go). Control medium was defined as that which had no contact with the cells.

Fibrovascular Invasion of PVA sponges
Prior to implantation, circular PVA sponges of uniform size (10-mm diameter, clinical PVA sponges grade 3; M-PACT Worldwide Management Co., Eudora, KS) were treated as previously described (Bradshaw et al. 2002Go). The sponges were implanted into five ST-double-null and four WT mice (three sponges per animal), sampled at 14 days after implantation, processed for histology, and stained with hematoxylin and eosin. For quantification of invasion, microscopic images were captured with a CCD (SPOT) camera, imported into NIH-Image, and the area of fibrovascular invasion calculated as a percentage of the total area of the sponge (Bradshaw et al. 2002Go).


    Results
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 Materials and Methods
 Results
 Discussion
 Literature Cited
 
ST-double-null Mice
ST-double-null mice appeared grossly normal. However, they did have kinked tails and early cataract formation similar to SPARC-null mice. Previously, it has been shown that SPARC-null mice do not produce SPARC protein (Bradshaw et al. 2003bGo) and that TSP-2-null mice do not produce TSP-2 (Kyriakides et al. 1998Go). Confirmation of the disruption of both genes in the ST-double-null mice is shown in Figure 1.

Wound Healing Is Accelerated in ST-double-null Mice
Cell migration, proliferation, and ECM remodeling are critical components of cutaneous wound healing. Epidermal closure, as reflected by a decreased wound area, occurred faster in ST-double-null mice, relative to WT animals. This difference between ST-double-null and WT mice was statistically significant (p<0.01) at 1-, 4-, 7-, and 10-day time points after wounding (Figures 2A and 2B). Some of the wounds in the WT mice remained open 14 days after wounding, whereas all the wounds in the ST-double-null mice were epithelialized in less than 10 days. In comparison, by day 11, five of six SPARC-null wounds showed no visible scab or opening, whereas only three of six WT wounds exhibited complete closure (Bradshaw et al. 2002Go). In contrast, no differences in wound re-epithelialization were found between TSP-2-null and WT mice (Kyriakides et al. 1999bGo).



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Figure 2

Epidermal closure is enhanced in ST-double-null mice. (A) Stereomicroscopic image of excisional wounds at 1, 4, and 10 days after wounding. Part of the wounds in WT mice remained non-epithelialized at 10 days. Bar = 2 mm. (B) The areas (mm2 ± SD) of the wounds were measured by NIH-Image software. The wound area was significantly smaller in ST-double-null mice (gray bars), relative to WT animals (white bars), an indication of enhanced epidermal closure. *p<0.01.

 
Wound healing was accelerated in both incisional (Figure 3A) and excisional (Figure 3B) wounds in ST-double-null mice at all time points, in comparison to wounds in WT mice. Because cell accumulation, development and maturation of granulation tissue, and scarring occurred earlier, the maturity score was higher in the ST-double-null animals. The difference was significant (p<0.01) at 4, 7, and 10 days (Figure 3C). Wound maturity scoring produced comparable results from incisional (data not shown) and excisional wounds (Figure 3C).



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Figure 3

Dermal wound healing is accelerated in ST-double-null mice. Hematoxylin- and eosin-stained sections of representative incisional (A) and excisional (B) wounds at 1, 4, 7, 10, and 14 days after wounding indicate more mature and advanced wound healing in ST-double-null mice, as reflected by cell accumulation (short arrows), development of granulation tissue (arrowheads), and scarring (long arrows). Asterisks indicate edges of wounds. Magnification: x2.5–10. (C) Dermal wound healing was analyzed according to a modified Greenhalgh classification of wound maturity (score from 1 to 15) (mean ± SD). A significantly enhanced dermal wound maturity score was found in ST-double-null mice (gray bars), relative to WT animals (white bars), at all time points but 14 days. *p<0.01.

 
SPARC Is Present in Healing Wounds
Because critical components of cutaneous wound healing are influenced by the expression of SPARC, we confirmed its appearance in healing wounds of WT animals. Immunostaining for SPARC revealed increased expression in the wound area at 4 and 7 days after wounding, in comparison to uninjured dermis. SPARC was located intracellularly in fibroblasts and endothelial cells of the wound area (data not shown) (Reed et al. 1993Go). No staining for SPARC was seen in the wounds or skin from ST-double-null mice. RT-PCR revealed SPARC mRNA in the wounds of WT animals at all time points, with highest levels at days 4 and 7 after wounding (data not shown). The expression pattern of TSP-2 in healing wounds has previously been reported (Kyriakides et al. 1999bGo). At day 3, TSP-2 was detected in only a small number of fibroblast-like cells in the wound area, and no ECM-associated expression was observed. In 7-, 10-, and 14-day wounds, TSP-2 was prominent in both the cells and ECM within the wound area (Kyriakides et al. 1999bGo). Thus, both SPARC and TSP-2 are expressed in a temporal fashion that is relevant to healing cutaneous wounds of WT animals.

Cell Proliferation in ST-double-null vs WT Wounds
Both SPARC and TSP-2 affect the cell cycle (Brekken and Sage 2000Go; Armstrong et al. 2002Go). We therefore asked whether an increase in cell proliferation in vivo contributed to the accelerated wound repair in ST-double-null mice. We found no significant difference in the amount of cell proliferation assessed by BrdU incorporation between wounds from ST-double-null and WT mice. The mean number (±SD) of BrdU-positive cells in wounds from ST-double-null mice at 4 days was 105 ± 40 and at 7 days was 131 ± 7. Corresponding values for WT mice were 97 ± 9 and 104 ± 37, respectively. These results indicate that altered rates of cell proliferation are not responsible for the accelerated wound repair at 4 days.

ECM Is Altered in ST-double-null Mice
Because a lack of SPARC or TSP-2 results in changes in the organization and composition of ECM, we asked whether such changes could contribute to the accelerated wound healing in ST-double-null mice. Electron microscopy revealed that tissue from the 14-day wounds of WT animals contained collagen fibrils of variable size. Moreover, the fibrils were arranged in parallel and formed the anticipated, well-organized architecture of the ECM (Figures 4A and 4B). In contrast, collagen fibrils in wound tissue from ST-double-null mice were small and rather uniform in size, and the organization of the ECM was loose and disrupted (Figures 4C and 4D). Previously, small collagen fibrils of uniform size were described in the skin and in foreign body capsules of SPARC-null mice (Bradshaw et al. 2003bGo, Puolakkainen et al. 2003Go), and irregular collagen fibers arranged in a basket-weave fashion were reported in wounds from TSP-2-null mice (Kyriakides et al. 1999bGo). Thus, changes in ECM composition are seemingly correlated with alterations in wound repair in the double-null mice.



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Figure 4

ECM is altered in ST-double-null mice. (A) Transmission electron microscopy of wound tissue from day 14 wounds of WT animals showed collagen fibrils of variable size (arrows). (B) The collagen fibrils (e.g., arrow) in WT wounds were parallel and formed a densely packed, well-organized dermal ECM. (C) In contrast, collagen fibrils (arrows) in the wound tissue from ST-double-null mice were small and uniform in size. (D) The organization of the collagen fibrils (e.g., arrow) in ST-double-null ECM was loose and disrupted. Original magnifications: x50,000 (A,C) and x30,000 (B,D).

 
Dermal Cell Migration Is Accelerated in ST-double-null Mice
We also asked whether there were differences in rates of migration between primary WT and ST-double-null dermal cells. Confluent dermal cell monolayers were wounded, after which cell migration was analyzed as a function of time. The denuded area of the monolayer was covered faster by cells from ST-double-null mice, a result indicating that the fibroblasts from the skin of ST-double-null mice migrated significantly (p<0.01) more rapidly than those from WT animals (Figure 5). Because wound healing is complex and relies in part on cell migration, further studies were performed in vivo on PVA sponge implants to determine the effect of combined SPARC and TSP-2 deficiency. Fibrovascular invasion of the sponges, determined as the percentage of the total area of the sponge devoted to cells, ECM, and vasculature, was increased significantly (p<0.01) in ST-double-null mice, relative to WT animals (Figure 6). No significant differences between genotypes were found in the morphology of the tissue invading the sponge implants. The finding of enhanced fibrovascular invasion was consistent with that seen in SPARC-null and TSP-2-null mice (Bradshaw et al. 2001Go; Kyriakides et al. 2001Go).



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Figure 5

Enhanced migration of dermal fibroblasts from ST-double-null mice. Dermal fibroblasts were isolated and cultured until confluence. Wounds were created in the cell monolayers, and cell migration into the denuded area was analyzed as the percentage of area covered by the cells as a function of time. Fibroblasts from the skin of ST-double-null mice (gray bars) migrated significantly faster than those from WT animals (white bars) at 24 and 48 hr. *p<0.01.

 


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Figure 6

Fibrovascular invasion of PVA sponges was increased in ST-double-null mice. PVA sponges were implanted subcutaneously into ST-double-null and WT mice and were evaluated histologically at 14 days after implantation. Fibrovascular invasion of the sponges was determined as the percentage of the total area of the sponge devoted to cells, vasculature, and ECM. The invasion was increased significantly (*p<0.01) in ST-double-null relative to WT mice.

 
One possibility for the enhanced migration of ST-double-null over WT cells could be an increased secretion of MMPs. We examined levels of MMP2 because augmented levels of this proteinase have been reported in cells from TSP-2-null mice (Yang et al. 2000Go). Equivalent numbers of ST-double-null and WT dermal fibroblasts were cultured in DMEM/1% fetal calf serum. The supernate was conditioned for 3 days, after which zymography was performed. Cells from ST-double-null mice produced more MMP-2 than cells from their WT counterparts (Figure 7). No difference was found in the expression of transforming growth factor beta (TGFbeta), vascular endothelial cell growth factor (VEGF), collagen I, or collagen VI mRNA between wounds from ST-double-null and WT mice (data not shown). Levels of TGFbeta mRNA were maximal at 4 and 7 days, whereas those of VEGF were constant at different time points.



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Figure 7

Production of MMP-2 by dermal fibroblasts. Equivalent numbers of ST-double-null and WT dermal fibroblasts were cultured in DMEM/1% fetal calf serum. The culture media were conditioned for 3 days, after which zymography was performed. Control lane contained an equivalent amount of medium not conditioned by cells. The representative zymogram indicates that cells from ST-double-null mice produced higher amounts of MMP-2 (arrow) than cells from their WT counterparts. The band migrating at ~90 kDa represents MMP-9 (arrowhead). Molecular mass standards x 10–3 in kDa, on right.

 
Fibroblasts from ST-double-null Animals Exhibit Enhanced Contraction of Native Collagen Gels
The capacity of dermal fibroblasts to remodel ECM was tested in a collagen gel contraction assay. Over a fourfold range of cell concentration, fibroblasts from ST-double-null mice contracted collagen gels to a greater extent than did fibroblasts from WT mice (Figure 8). This result was distinct from observations of collagen gel contraction by single-null SPARC or TSP-2 dermal fibroblasts. Relative to WT, contraction of collagen gels by SPARC-null fibroblasts was similar (Bradshaw et al. 2002Go), whereas gel contraction by TSP-2-null fibroblasts was diminished (Kyriakides et al. 1999bGo).



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Figure 8

ST-double-null dermal fibroblasts exhibit enhanced contraction of native collagen gels. Over a fourfold range of cell concentration, fibroblasts from ST-double-null mice (open circles) contracted the gels to a greater extent than fibroblasts from WT mice (closed circles). Values are mean ± SD (n=4 replicates).

 
A summary of the data relating to wound healing in ST-double-null, SPARC-null, and TSP-2-null mice is shown in Table 1.


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Table 1

Characteristics of SPARC-null, TSP-2-null, and ST-double-null mice in healing dermal excisional wounds

 

    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
A number of matricellular proteins including SPARC, TSP-1 and -2, osteopontin, and tenascin C (Mackie et al. 1988Go; Reed et al. 1993Go; Liaw et al. 1998Go) show increased expression in response to injury, such as cutaneous wound healing. Previously, the role of SPARC and TSP-2 in wound healing has been studied in mice with a targeted disruption of each gene: SPARC-null mice exhibited accelerated epidermal closure, and TSP-2-null mice showed an enhanced dermal wound healing (Table 1).

Here we report significantly enhanced epithelial closure in mice lacking gene function for both SPARC and TSP-2. That no difference in the re-epithelialization rate was detected in TSP-2-null mice (Kyriakides et al. 1999bGo) indicates that SPARC functions in vivo more specifically in epidermal migration and closure than TSP-2. This result is consistent with our finding that dermal fibroblasts cultured from ST-double-null mice showed enhanced migration, relative to fibroblasts from WT mice in vitro. Accelerated migration relative to WT controls was also seen in dermal fibroblasts from SPARC-null mice (Bradshaw et al. 2002Go) (Table 1).

Greenhalgh et al. (1990)Go introduced a scoring system to assess wound repair and to quantify the maturity of the wound as a function of time. This score allows comparison of wounds, e.g., after treatment or between genotypes. We have used a simplified scoring method that excludes the epithelial component (Spenny et al. 2002Go) and found significantly more mature phases of both excisional and incisional wound healing at 1, 4, 7, 10, and 14 days after wounding in ST-double-null mice. This result is in accord with a previous study that showed an irregularly organized but highly vascularized granulation tissue in TSP-2-null mice (Kyriakides et al. 1999bGo). In addition, TSP-2-null wounds manifested a higher cellularity and healed with less scarring than control wounds (Kyriakides et al. 1999bGo) (Table 1). We conclude that both SPARC and TSP-2 affect the dermal component of cutaneous wound healing by their modulation of angiogenesis and by the production and assembly of ECM.

Given the capacity of SPARC to retard cell-cycle progression in vitro, we asked whether differences in cell proliferation might account for the increased healing of ST-double-null mice. There was no significant difference in cell proliferation assessed by BrdU incorporation between wounds from ST-double-null and WT mice. This finding is consistent with data from SPARC-null mice, in which cell proliferation was excluded as a mechanism that could account for the accelerated wound healing in these animals (Bradshaw et al. 2002Go) (Table 1).

The presence of SPARC in the wound area, with maximal levels at days 4 and 7 after wounding, supports our previous conclusions (Reed et al. 1993Go; Puolakkainen et al. 1999Go) and speaks to a potentially significant function for this protein during the phase of granulation tissue formation of cutaneous wound healing. TSP-2 has previously been shown to be present in both early and late wounds, either associated with cells or more widely distributed in cells and ECM (Kyriakides et al. 1999bGo). This temporal expression is important because the protein that is produced first may determine the course of the wound-healing process (see Table 1), as was observed in a study on TSP-1- and TSP-2-null mice (Agah et al. 2002Go).

Altered ECM is characteristic of mice with targeted disruptions of genes for several matricellular proteins (Bornstein and Sage 2002Go). By hydroxyproline analysis, the concentration of collagen in SPARC-null skin was found to be half that of WT skin (Bradshaw et al. 2002Go). Electron microsopy studies have revealed that the collagen fibrils are smaller and more uniform in size in SPARC-null mice, relative to their WT counterparts (Bradshaw et al. 2003bGo; Puolakkainen et al. 2003Go). The wound beds of TSP-2-null mice also contained disorganized collagen fibers (Kyriakides et al. 1999bGo). Given these findings, we asked whether altered ECM might account for the enhanced wound healing observed in ST-double-null mice. By electron microscopy, we found that the collagen fibers in day-14 wounds of ST-double-null mice were small and uniform in diameter, whereas those from WT mice were larger and more variable in size. In WT animals the collagen fibrils were parallel and constituted a well-organized architecture. In contrast, in ST-double-null mice, a severely disrupted ECM was found.

Collagen gel contraction has been proposed as a model in vitro for wound contraction in vivo that results in part from the contractile action of myofibroblasts within the wound bed (Grinnell 1994Go). It is noteworthy, therefore, that the contraction of gels in vitro by ST-double-null dermal fibroblasts was increased relative to WT fibroblasts, a result in accordance with the increased rate of wound closure in ST-double-null mice. The mechanism by which concomitant ablation of SPARC and TSP-2 enhances the capacity of fibroblasts to remodel collagen in vitro is unclear, particularly because single-null SPARC and TSP-2 fibroblasts exhibited, respectively, no difference in collagen gel contraction (Bradshaw et al. 2002Go) and diminished gel contraction (Kyriakides et al. 1999bGo), relative to WT fibroblasts. It is possible that the enhanced contractile action of fibroblasts characteristic of the ST-double-null phenotype is, in part, a consequence of synthesis of an abnormal pericellular ECM in association with elevated levels of MMP-2.

Fibrovascular invasion of PVA sponges was increased in SPARC-null mice (Bradshaw et al. 2002Go), and Kyriakides et al. (2001)Go reported increased angiogenesis and fibrotic encapsulation of sponges in TSP-2-null mice. Therefore, we were not surprised to find a significantly increased fibrovascular invasion of PVA sponges in ST-double-null animals, relative to WT mice. The fibrovascular invasion of PVA sponges has been shown to reflect angiogenic responses. Thus, part of the enhanced wound repair in ST-double-null mice might be due to increased angiogenesis during the healing process.

In summary, our findings indicate that the wound-healing phenotype of ST-double-null mice represents collectively the characteristics of the single-null animals. Lack of SPARC seems to dominate epithelial closure, whereas lack of TSP-2 dominates dermal wound healing. Both lead to altered ECM in the wound tissue. We propose that the enhanced cutaneous wound healing seen in ST-double-null mice results from accelerated epidermal closure and alterations in the ECM. Collectively, and consistent with earlier reports, these findings indicate that SPARC and TSP-2 have important roles in epidermal wound healing and in the repair of the dermis in response to injury.


    Acknowledgments
 
This work was supported in part by grants from the National Institutes of Health (F32 HL-10352 to RAB, K01 AR-0022200 to ADB, R01 AR-45418 to PB, and R01 GM-40711 and R01 HL-59574 to EHS), from the National Science Foundation to the University of Washington Engineered Biomaterials (EEC9529161), from The Gilbertson Foundation to The Hope Heart Program, and from the Helsinki University Central Hospital Research Funds (EV0, Finland) (to PAP).

The authors thank Eileen Neligan for assistance with the manuscript.


    Footnotes
 
Received for publication May 26, 2004; accepted September 29, 2004


    Literature Cited
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
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