SPARC-thrombospondin-2-double-null Mice Exhibit Enhanced Cutaneous Wound Healing and Increased Fibrovascular Invasion of Subcutaneous Polyvinyl Alcohol Sponges
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
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Summary |
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(J Histochem Cytochem 53:571581, 2005)
Key Words: SPARC thrombospondin wound healing matricellular extracellular matrix epidermis dermis angiogenesis collagen
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Introduction |
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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. 1986; Sage et al. 1989a
; Kasugai et al. 1991
; Vuorio et al. 1991
). 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 2001
). Two principal functions of SPARC are modification of cell shape and inhibition of cell-cycle progression (Bradshaw and Sage 2001
). SPARC is expressed during development and in remodeling tissues in adults (Reed et al. 1993
; Puolakkainen et al. 1999
; Bornstein and Sage 2002
). Targeted disruption of the SPARC gene in mice results in a complex phenotype characterized by early cataractogenesis (Gilmour et al. 1998
; Norose et al. 1998
; Yan et al. 2002
), increased amounts of subcutaneous adipose tissue (Bradshaw et al. 2003a
), decreased amounts of collagen in the skin (Bradshaw et al. 2003b
), and progressively severe osteopenia (Delany et al. 2000
). 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. 2001
), enhanced growth of malignant tumors (Brekken et al. 2003
), and reduced foreign body response (Puolakkainen et al. 2003
) 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. 1993
) and during the healing of intestinal anastomoses (Puolakkainen et al. 1999
) and have recently described accelerated cutaneous wound closure in SPARC-null mice (Bradshaw et al. 2002
). 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. 2002
).
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. 1999b; Bornstein et al. 2000
). 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. 1998
). 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. 2000
). TSP-2 is expressed during cutaneous wound healing (Kyriakides et al. 1999b
), and TSP-2-null mice displayed accelerated excisional wound healing with irregularly organized and highly vascularized granulation tissue (Kyriakides et al. 1999b
). 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. 1999a
).
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.
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Materials and Methods |
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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) as modified by Spenny et al. (2002)
. 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. 1989b). 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. 2003
) 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. 1996). 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 2000) 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)
.
Electron Microscopy
Samples were immersed in Karnovsky's fixative and were processed for transmission electron microscopy according to established methods (Wight et al. 1997).
Isolation and Culture of Dermal Fibroblasts
Dermal cells were isolated from the skin as described (Bradshaw et al. 2002). 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 46 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. 2002
). All experiments were performed with cells at early passage (P1P4), 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). 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. 2002). 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. 2002). 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. 2002
).
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Results |
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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. 2002). In contrast, no differences in wound re-epithelialization were found between TSP-2-null and WT mice (Kyriakides et al. 1999b
).
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Cell Proliferation in ST-double-null vs WT Wounds
Both SPARC and TSP-2 affect the cell cycle (Brekken and Sage 2000; Armstrong et al. 2002
). 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. 2003b, Puolakkainen et al. 2003
), and irregular collagen fibers arranged in a basket-weave fashion were reported in wounds from TSP-2-null mice (Kyriakides et al. 1999b
). Thus, changes in ECM composition are seemingly correlated with alterations in wound repair in the double-null mice.
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Discussion |
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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. 1999b) 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. 2002
) (Table 1).
Greenhalgh et al. (1990) 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. 2002
) 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. 1999b
). In addition, TSP-2-null wounds manifested a higher cellularity and healed with less scarring than control wounds (Kyriakides et al. 1999b
) (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. 2002) (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. 1993; Puolakkainen et al. 1999
) 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. 1999b
). 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. 2002
).
Altered ECM is characteristic of mice with targeted disruptions of genes for several matricellular proteins (Bornstein and Sage 2002). By hydroxyproline analysis, the concentration of collagen in SPARC-null skin was found to be half that of WT skin (Bradshaw et al. 2002
). 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. 2003b
; Puolakkainen et al. 2003
). The wound beds of TSP-2-null mice also contained disorganized collagen fibers (Kyriakides et al. 1999b
). 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 1994). 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. 2002
) and diminished gel contraction (Kyriakides et al. 1999b
), 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. 2002), and Kyriakides et al. (2001)
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.
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Acknowledgments |
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The authors thank Eileen Neligan for assistance with the manuscript.
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Footnotes |
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Literature Cited |
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Agah A, Kyriakides TR, Lawler J, Bornstein P (2002) The lack of thrombospondin-1 (TSP 1) dictates the course of wound healing in double-TSP1/TSP2-null mice. Am J Pathol 161:831839
Armstrong LC, Bjorkblom B, Hankenson KD, Siadak AW, Stiles CE, Bornstein P (2002) Thrombospondin 2 inhibits microvascular endothelial cell proliferation by a caspase-independent mechanism. Mol Biol Cell 13:18931905
Bornstein P, Armstrong LC, Hankenson KD, Kyriakides TR, Yang Z (2000) Thrombospondin 2, a matricellular protein with diverse functions. Matrix Biol 19:557568[CrossRef][Medline]
Bornstein P, Sage EH (2002) Matricellular proteins: extracellular modulators of cell function. Curr Opin Cell Biol 14:608616[CrossRef][Medline]
Bradshaw AD, Graves DC, Motamed K, Sage EH (2003a) SPARC-null mice exhibit increased adiposity without significant differences in overall body weight. Proc Natl Acad Sci USA 100:60456050
Bradshaw AD, Puolakkainen P, Dasgupta J, Davidson JM, Wight TN, Sage EH (2003b) SPARC-null mice display abnormalities in the dermis characterized by decreased collagen fibril diameter and reduced tensile strength. J Invest Dermatol 120:949955[CrossRef][Medline]
Bradshaw AD, Reed MJ, Carbon JG, Pinney E, Brekken RA, Sage EH (2001) Increased fibrovascular invasion of subcutaneous polyvinyl alcohol sponges in SPARC-null mice. Wound Rep Reg 9:522530[CrossRef]
Bradshaw AD, Reed MJ, Sage EH (2002) SPARC-null mice exhibit accelerated cutaneous wound closure. J Histochem Cytochem 50:110
Bradshaw AD, Sage EH (2001) SPARC, a matricellular protein that functions in cellular differentiation and tissue response to injury. J Clin Invest 107:10491054
Brekken RA, Puolakkainen P, Graves DC, Workman G, Lubkin SR, Sage EH (2003) Enhanced growth of tumors in SPARC null mice is associated with changes in the ECM. J Clin Invest 111:487495
Brekken RA, Sage EH (2000) SPARC, a matricellular protein: at the crossroads of cell-matrix communication. Matrix Biol 19:569580[CrossRef][Medline]
Delany AM, Amling M, Priemel M, Howe C, Baron R, Canalis E (2000) Osteopenia and decreased bone formation in osteonectin-deficient mice. J Clin Invest 105:915923
Gilmour DT, Lyon GJ, Carlton MBL, Sanes JR, Cunningham JM, Anderson JR, Hogan BLM, et al. (1998) Mice deficient for the secreted glycoprotein SPARC/osteonectin/BM-40 develop normally but show severe age-onset cataract formation and disruption of the lens. EMBO J 17:18601870
Graves DC, Yablonka-Reuveni Z (2000) Vascular smooth muscle cells spontaneously adopt a skeletal muscle phenotype: a unique Myf5()/MyoD(+) myogenic program. J Histochem Cytochem 48:11731193
Greenhalgh DG, Sprugel KH, Murray MJ, Ross R (1990) PDGF and FGF stimulate wound healing in the genetically diabetic mouse. Am J Pathol 136:12351246[Abstract]
Grinnell F (1994) Fibroblasts, myofibroblasts and wound contraction. J Cell Biol 124:401404[CrossRef][Medline]
Kasugai S, Todescan R, Nagata T, Yao K-L, Butler WT, Sodek J (1991) Expression of bone matrix proteins associated with mineralized tissue formation by adult rat bone marrow cells in vitro: inductive effects of dexamethasone on the osteoblastic phenotype. J Cell Physiol 147:111117[CrossRef][Medline]
Koike T, Vernon RB, Hamner MA, Sadoun E, Reed MJ (2002) MT1-MMP, but not secreted MMPs, influences the migration of human microvascular endothelial cells in 3-dimensional collagen gels. J Cell Biochem 86:748758[CrossRef][Medline]
Kyriakides TR, Leach KJ, Hoffman AS, Ratner BD, Bornstein P (1999a) Mice that lack the angiogenesis inhibitor, thrombospondin 2, mount an altered foreign body reaction characterized by increased vascularity. Proc Natl Acad Sci USA 96:44494454
Kyriakides TR, Tam JWY, Bornstein P (1999b) Accelerated wound healing in mice with disruption of the thrombospondin 2 gene. J Invest Dermatol 113:782787[CrossRef][Medline]
Kyriakides TR, Zhu YH, Smith LT, Bain SD, Yang Z, Lin MT, Danielson KG, et al. (1998) Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis. J Cell Biol 140:419430
Kyriakides TR, Zhu YH, Yang Z, Huynh G, Bornstein P (2001) Altered extracellular matrix remodeling in sponge granulomas of thrombospondin 2-null mice. Am J Pathol 159:12551262
Liaw L, Birk D, Ballas CB, Whitsitt JS, Davidson JM, Hogan BLM (1998) Altered wound healing in mice lacking a functional osteopontin gene (ssp1). J Clin Invest 101:14681478
Mackie EJ, Halfter W, Liverani D (1988) Induction of tenascin in healing wounds. J Cell Biol 107:27572767[Abstract]
Norose K, Clark JI, Syed NA, Basu A, Heberkatz ES, Sage EH, Howe CC (1998) SPARC deficiency leads to early-onset cataractogenesis. Invest Ophthalmol Vis Sci 39:26742680[Abstract]
Puolakkainen P, Bradshaw A, Kyriakides TR, Reed M, Brekken R, Wight T, Bornstein P, et al. (2003) Compromised production of extracellular matrix in mice lacking Secreted Protein, Acidic and Rich in Cysteine (SPARC) leads to a reduced foreign body reaction to implanted biomaterials. Am J Pathol 162:627635
Puolakkainen P, Reed M, Vento P, Sage EH, Kiviluoto T, Kivilaakso E (1999) Expression of SPARC in healing intestinal anastomosis and short bowel syndrome in rats. Dig Dis Sci 44:15541564[CrossRef][Medline]
Reed MJ, Penn PE, Li Y, Birnbaum R, Vernon RB, Johnson TS, Pendergrass WR, et al. (1996) Enhanced cell proliferation and biosynthesis mediate improved wound repair in refed, caloric-restricted mice. Mech Ageing Dev 89:2143[CrossRef][Medline]
Reed MJ, Puolakkainen P, Lane TF, Dickerson D, Bornstein P, Sage EH (1993) Differential expression of SPARC and thrombospondin 1 in wound repair: Immunolocalization and in situ hybridization. J Histochem Cytochem 41:14671477
Sage EH, Decker J, Funk S, Chow M (1989a) SPARC: a Ca2+-binding extracellular protein associated with endothelial cell injury and proliferation. J Mol Cell Cardiol 21:1322[CrossRef][Medline]
Sage EH, Vernon RB, Decker J, Funk S, Iruela-Arispe ML (1989b) Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice. J Histochem Cytochem 37:819824[Abstract]
Spenny ML, Muangman P, Sullivan SR, Bunnett NW, Ansel JC, Olerud JE, Gibran NS (2002) Neutral endopeptidase inhibition in diabetic wound repair. Wound Repair Regen 10:295301[CrossRef][Medline]
Stenner DD, Tracy RP, Riggs BL, Mann KG (1986) Human platelets contain and secrete osteonectin, a major protein of mineralized bone. Proc Natl Acad Sci USA 83:68926896
Vernon RB, Gooden MD (2002) An improved method for the collagen gel contraction assay. In Vitro Cell Dev Biol 38:97101[CrossRef]
Vuorio T, Kahari V-M, Black C, Vuorio E (1991) Expression of osteonectin, decorin, and transforming growth factor beta 1 genes in fibroblast cultures from patients with systemic sclerosis and morphea. J Rheumatol 18:247253[Medline]
Wight TN, Lara S, Riessen R, Le Baron R, Isner J (1997) Selective deposits of versican in the extracellular matrix of restenotic lesions from human peripheral arteries. Am J Pathol 151:963973[Abstract]
Yan Q, Clark JI, Wight T, Sage EH (2002) Alterations in the lens capsule contribute to cataractogenesis in SPARC-null mice. J Cell Sci 115:27472756
Yang Z, Kyriakides TR, Bornstein P (2000) Matricellular proteins as modulators of cell-matrix interactions: adhesive defect in thrombospondin-2-null fibroblasts is a consequence of increased levels of matrix metalloproteinase-2. Mol Biol Cell 11:33533364