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• W2034806517 abstract "The phenotype of fibroblasts repopulating experimental wounds in vivo has been shown to influence both wound healing responses and clinical outcome. Recent studies have demonstrated that the human homeobox gene PRX-2 is strongly upregulated in fibroblasts within fetal, but not adult, mesenchymal tissues during healing. Differential homeobox gene expression by fibroblasts may therefore be important in mediating the scarless healing exhibited in early fetal wounds. RNase protection analysis demonstrated that murine Prx-2 expression was involved in fetal but not adult wound healing responses in vitro. Using fibroblasts established from homozygous mutant (Prx-2-/-) and wild-type (Prx-2+/+) murine skin tissues it was demonstrated that Prx-2 affected a number of fetal fibroblastic responses believed to be important in mediating scarless healing in vivo; namely cellular proliferation, extracellular matrix reorganization, and matrix metalloproteinase 2 and hyaluronic acid production. These data demonstrate how Prx-2 may contribute to the regulation of fetal, but not adult, fibroblasts and ultimately the wound healing phenotype. This study provides further evidence for the importance of homeobox transcription factors in the regulation of scarless wound healing. A further understanding of these processes will, it is hoped, enable the targeting of specific therapies in wound healing, both to effect scarless healing and to stimulate healing in chronic, nonhealing wounds such as venous leg ulcers. The phenotype of fibroblasts repopulating experimental wounds in vivo has been shown to influence both wound healing responses and clinical outcome. Recent studies have demonstrated that the human homeobox gene PRX-2 is strongly upregulated in fibroblasts within fetal, but not adult, mesenchymal tissues during healing. Differential homeobox gene expression by fibroblasts may therefore be important in mediating the scarless healing exhibited in early fetal wounds. RNase protection analysis demonstrated that murine Prx-2 expression was involved in fetal but not adult wound healing responses in vitro. Using fibroblasts established from homozygous mutant (Prx-2-/-) and wild-type (Prx-2+/+) murine skin tissues it was demonstrated that Prx-2 affected a number of fetal fibroblastic responses believed to be important in mediating scarless healing in vivo; namely cellular proliferation, extracellular matrix reorganization, and matrix metalloproteinase 2 and hyaluronic acid production. These data demonstrate how Prx-2 may contribute to the regulation of fetal, but not adult, fibroblasts and ultimately the wound healing phenotype. This study provides further evidence for the importance of homeobox transcription factors in the regulation of scarless wound healing. A further understanding of these processes will, it is hoped, enable the targeting of specific therapies in wound healing, both to effect scarless healing and to stimulate healing in chronic, nonhealing wounds such as venous leg ulcers. conditioned medium fibroblast-populated collagen lattice fibroblast-serum containing medium hyaluronic acid matrix metalloproteinase 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide tissue inhibitor of matrix metalloproteinase extracellular matrix The healing of adult skin wounds is characterized by the production of a scar tissue. In contrast, early fetal wounds heal through a tissue regenerative process leading to the absence of scar tissue (Siebert et al., 1990Siebert J. Burd D. McCarthy J. Weinzweig J. Ehrlich H. Fetal wound healing: a biochemical study of scarless healing.Plast Reconstr Surg. 1990; 85: 495-502Crossref PubMed Scopus (91) Google Scholar;Longaker and Adzick, 1991Longaker M. Adzick N. The biology of fetal wound healing.Plast Reconstr Surg. 1991; 87: 788-798Crossref PubMed Scopus (159) Google Scholar). The changes that occur within these wounds are well documented and include an absence of fetal inflammation, alterations in the ratios and levels of different cytokines, changes in extracellular matrix (ECM) deposition, and the presence of phenotypically distinct populations of cells such as fibroblasts (Adzick and Lorenz, 1994Adzick N.S. Lorenz H.P. Cells, matrix, growth-factors, and the surgeon – the biology of scarless fetal wound repair.Ann Surg. 1994; 220: 10-18Crossref PubMed Scopus (223) Google Scholar). The biochemical signals that control and coordinate these highly orchestrated processes remain poorly defined, however. Fibroblasts play a pivotal and extensive role in wound healing, ranging from the synthesis of ECM to mediating its remodeling, by cytokine and metalloproteinase activity (Thomas et al., 1995Thomas D.W. O'Neill I.D. Harding K.G. Shepherd J.P. Cutaneous wound-healing – a current perspective.J Oral Maxillofac Surg. 1995; 53: 442-447Abstract Full Text PDF PubMed Scopus (65) Google Scholar). The phenotype of fibroblasts repopulating experimental wounds in vivo has been shown to influence both wound healing responses and clinical outcome (Lorenz et al., 1992Lorenz H.P. Longaker M.T. Whitby D.J. Harrison M.R. Adzick N.S. Scarless fetal skin repair is intrinsic to the fetal fibroblast.Surg Forum. 1992; 43: 694-696Google Scholar). Interestingly, it has been shown that fibroblasts from tissues that demonstrated differential wound healing responses in vivo (i.e., fetal, oral mucosal, and chronic wound tissues) exhibit differential migration, ECM reorganizational ability, and matrix metalloproteinase (MMP) production in vitro (Burd et al., 1991Burd D.A.R. Longaker M.T. Rittenberg T. Adzick N.S. Harrison M.R. Ehrlich H.P. In vitro fetal wound contraction – the effect of amniotic-fluid.Br J Plast Surg. 1991; 44: 302-305Abstract Full Text PDF PubMed Scopus (11) Google Scholar;Stephens et al., 1996Stephens P. Davies K.J. Al-Khateeb T. Shepherd J.P. Thomas D.W. A comparison of the ability of intra oral and extra oral fibroblasts to stimulate extracellular matrix reorganization in a model of wound contraction.J Dent Res. 1996; 75: 1358-1364Crossref PubMed Scopus (86) Google Scholar;Stephens et al., 2001Stephens P. Davies K.J. Occleston N. et al.Skin and oral fibroblasts exhibit phenotypic differences in extracellular matrix reorganization and matrix metalloproteinase activity.Br J Dermatol. 2001; 144: 229-237Crossref PubMed Scopus (110) Google Scholar;Al-Khateeb et al., 1997Al-Khateeb T. Stephens P. Shepherd J.P. Thomas D.W. An investigation of preferential fibroblast wound repopulation using a novel in vitro wound model.J Periodontol. 1997; 68: 1063-1069Crossref PubMed Scopus (33) Google Scholar;Ellis et al., 1997Ellis I. Banyard J. Schor S.L. Differential response of fetal and adult fibroblasts to cytokines: cell migration and hyaluronan synthesis.Development. 1997; 124: 1593-1600PubMed Google Scholar;Cook et al., 2000Cook H. Stephens P. Davies K.J. Harding K.G. Thomas D.W. Defective extracellular matrix reorganisation by chronic wound fibroblasts is associated with alterations in TIMP-1, TIMP-2 and MMP-2 activity.J Invest Dermatol. 2000; 115: 225-233Crossref PubMed Scopus (147) Google Scholar). It therefore appears that fibroblasts within the wound environment are critical “effector” cells in the control of the repair process. The homeobox genes are a large superfamily of developmental genes that play a critical role in segment identity and pattern formation during embryogenesis (Krumlauf, 1994Krumlauf R. Hox genes in vertebrate development.Cell. 1994; 78: 191-201Abstract Full Text PDF PubMed Scopus (1712) Google Scholar). Homeobox genes contain a region of 183 nucleotides that has been conserved during evolution and encodes the 61 amino acid homeodomain. The homeobox gene products are sequence-specific DNA-binding proteins that are thought to function as transcription factors. Binding to the DNA by virtue of the helix–turn–helix motif of the homeodomain, they influence both gene activation and repression (Thali et al., 1988Thali M. Muller M.M. DeLorenzi M. Matthias P. Bienz M. Drosophila homoeotic genes encode transcriptional activators similar to mammalian OTF-2.Nature. 1988; 336: 598-601Crossref PubMed Scopus (66) Google Scholar). Regulation of homeobox gene expression involves the coordinated action of growth factors, cytokines, and hormones (Scott and Goldsmith, 1993Scott G.A. Goldsmith L.A. Homeobox genes and skin development: a review.J Invest Dermatol. 1993; 101: 3-8Abstract Full Text PDF PubMed Google Scholar) inducing auto- and allo-regulation through post-translational modifications of the homeodomain proteins and other transcription factors (Fainsod et al., 1986Fainsod A. Bogarad L.D. Ruusala T. Lubin M. Crothers D.M. Ruddle F.H. The homeo domain of a murine protein binds 5′ to its own homeo box.Proc Natl Acad Sci USA. 1986; 83: 9532-9536Crossref PubMed Scopus (37) Google Scholar;Jones et al., 1992Jones F.S. Prediger E.A. Bittner D.A. De Robertis E.M. Edelman G.M. Cell adhesion molecules as targets for Hox genes: neural cell adhesion molecule promoter activity is modulated by cotransfection with Hox-2.5 and –2.4.Proc Natl Acad Sci USA. 1992; 89: 2086-2090Crossref PubMed Scopus (192) Google Scholar). Homeobox genes are expressed strongly in an organ- and stage-specific pattern in human embryos, findings that suggest their involvement in the control of early fetal development (Mavilio et al., 1986Mavilio F. Simeone A. Giampaolo A. et al.Differential and stage-related expression in embryonic tissues of a new human homoeobox gene.Nature. 1986; 324: 664-668Crossref PubMed Scopus (116) Google Scholar). The multilayered complexity of the skin suggests that its morphogenesis involves the coordinated action of a number of genes. The expression of a number of homeobox genes in both fetal and adult skin (Bieberich et al., 1991Bieberich C.J. Ruddle F.H. Stenn K.S. Differential expression of the hox 3.1 gene in adult-mouse skin.Ann N Y Acad Sci. 1991; 642: 346-354Crossref PubMed Scopus (52) Google Scholar;Detmer et al., 1993Detmer K. Lawrence H.J. Largman C. Expression of class I homeobox genes in fetal and adult murine skin.J Invest Dermatol. 1993; 101: 517-522Abstract Full Text PDF PubMed Google Scholar;Rieger et al., 1994Rieger E. Bijl J.J. van Oostveen J.W. et al.Expression of the homeobox gene HOXC4 in keratinocytes of normal skin and epithelial skin tumors is correlated with differentiation.J Invest Dermatol. 1994; 103: 341-346Crossref PubMed Scopus (51) Google Scholar) suggests that these genes play an important role in skin development. As skin homeobox gene expression may be associated with the constant differentiation necessary to maintain the epidermis, it may therefore play an important role in the regeneration of the dermis/epidermis following cutaneous injury, where the coordinated expression of a number of genes is required to achieve successful regeneration of the skin. Interestingly, our recent studies have demonstrated that the human homeobox gene PRX-2 is strongly upregulated in scarless fetal but not scarring adult wound healing (Stelnicki et al., 1998Stelnicki E. Arbeit J. Cass D.L. Saner C. Harrison M. Largman C. Modulation of the human homeobox genes PRX-2 and HOXB13 in scarless fetal wounds.J Invest Dermatol. 1998; 111: 57-63Crossref PubMed Scopus (80) Google Scholar). This upregulation was demonstrated in the mesenchymal tissues on wounding first and second trimester fetal skin but was unaffected in adult skin wounds. Using in situ hybridization for Prx-2 it was shown that the source of this upregulation within the skin was the dermal fibroblasts. Differential homeobox gene expression was therefore theorized to be important in mediating differential fibroblast responses during wound healing. The recent availability of viable Prx-2 mutant mice (ten Berge et al., 1998ten Berge D. Brouwer A. Korving J. Martin J. Meijlink F. Prx1 and Prx2 in skeletogenesis: roles in the craniofacial region, inner ear and limbs.Development. 1998; 125: 3831-3842PubMed Google Scholar) affords the opportunity to study the role of the Prx-2 homeobox gene in the control of both fetal and adult cellular responses. We investigated how functional alteration of Prx-2 expression modulated fibroblast responses. Using RNase protection analysis we demonstrate that Prx-2 expression is involved in fetal but not adult cellular responses in vitro. Then using fibroblasts established from homozygous (Prx-2-/-) mutant and wild-type (Prx-2+/+) murine skin it was shown that Prx-2 affects a number of fibroblast cellular and biosynthetic wound healing responses that are implicated in the regulation of wound healing in vivo. These data demonstrate how Prx-2 may contribute to the regulation of fetal, but not adult, fibroblasts and demonstrate “mechanisms” by which Prx-2 expression may modulate fibroblast function in tissue repair. Mice lacking a functional Prx-2 gene were generated by homologous recombination in ES cells (ten Berge et al., 1998ten Berge D. Brouwer A. Korving J. Martin J. Meijlink F. Prx1 and Prx2 in skeletogenesis: roles in the craniofacial region, inner ear and limbs.Development. 1998; 125: 3831-3842PubMed Google Scholar). A lacZ/PGK-Hygromycin cassette was inserted into an EagI site located upstream from the homeobox, such that the lacZ-encoding sequence was in-frame with the Prx-2-coding sequence. Upon homologous recombination this allele expressed mRNA encoding the first 88 amino acids of the Prx-2 protein fused to β-galactosidase protein, in a pattern identical to that of the Prx-2 mRNA. Cultures of mouse skin fibroblasts were established from specimens of tissue obtained from fetal, time-mated (embryonic days 13.5–14.5) or adult (6 mo to 1 y) mice maintained under National Institutes of Health (U.S.A.) guidelines and in accordance with the regulations and approval of the University of California, San Francisco Committee on Animal Research. Fetal and adult mice were sacrificed by asphyxiation with CO2. Fetal skin samples were obtained from eviscerated, fetal mice that had their limbs and tail removed. Adult skin samples of 2 cm×2 cm were harvested from the backs of shaved adult mice. Fetal fibroblasts were isolated by a colony outgrowth method. The tissue was minced and placed in one well of a six-well tissue culture plate. A sterile, square coverslip was placed on top to prevent the tissue from becoming detached from the plate and 3 ml of fibroblast-serum containing medium [F-SCM: Dulbecco's modified Eagle's medium (DMEM) supplemented with L-glutamine (2 mM), antibiotics (100 U per ml penicillin G; 100 μg per ml streptomycin sulfate; 0.25 μg per ml amphotericin B), and 10% (vol/vol) fetal bovine serum (FBS); all from Life Technologies, Paisley, U.K.] was added. The cultures were maintained at 37°C in a 5% CO2 humidified atmosphere. The coverslip was removed once the cells had migrated out from the tissue. At confluency, fibroblasts were split at a ratio of 1:3. Cells were utilized for all experiments between passages 3 and 12. Adult fibroblasts were established by a single-cell suspension technique following enzymatic degradation of the specimens as previously described (Stephens et al., 1996Stephens P. Davies K.J. Al-Khateeb T. Shepherd J.P. Thomas D.W. A comparison of the ability of intra oral and extra oral fibroblasts to stimulate extracellular matrix reorganization in a model of wound contraction.J Dent Res. 1996; 75: 1358-1364Crossref PubMed Scopus (86) Google Scholar). Briefly, tissue was incubated overnight with Dispase (2 mg per ml; Boehringer Mannheim, Lewes, U.K.) to separate epidermal tissue from the dermal tissue. Dermal tissue specimens were then disaggregated overnight utilizing bacterial Clostridium histolyticum A collagenase (1 mg per ml; Boehringer Mannheim). Fibroblast cultures were maintained in F-SCM. The cultures were maintained at 37°C in a 5% CO2 humidified atmosphere. At confluency, fibroblasts were split at a ratio of 1:3. Cells were utilized for all experiments between passages 3 and 12. Fetal or adult fibroblast monolayer cultures were trypsinized and utilized to construct FPCLs. The fibroblasts were re-suspended in medium (F-SCM) prepared with FBS that had been pretreated with Gelatin-A Sepharose (1 part Gelatin-A Sepharose to 5 parts FBS; Amersham Pharmacia Biotech, St. Albans, U.K.) to remove endogenous MMP-2 and MMP-9 (Azzam and Thompson, 1992Azzam H.S. Thompson E.W. Collagen-induced activation of the Mr 72,000 type-IV collagenase in normal and malignant human fibroblastoid cells.Cancer Res. 1992; 52: 4540-4544PubMed Google Scholar). Type I rat-tail collagen was extracted as described previously (Rowling et al., 1990Rowling P.J.E. Raxworthy M.J. Wood E.J. Kearney J.N. Fabrication and reorganization of dermal equivalents suitable for skin grafting after major cutaneous injury.Biomaterials. 1990; 11: 181-185Crossref PubMed Scopus (37) Google Scholar). 1×106 fibroblasts (in 0.75 ml of F-SCM, Gelatin-A Sepharose pretreated) were added to 53 mm bacteriologic grade plates containing 3 ml of 2×DMEM [40 parts 10×DMEM, 10 parts NaHCO3 (7.5% wt/vol), 4 parts L-glutamine (200 mM), 140 parts H2O, and 5 parts NaOH (1 M)], 0.75 ml of 0.1 M NaOH, 0.75 ml of Gelatin-A Sepharose pretreated FBS, and 2.25 ml of 1.7 mg per ml rat-tail type I collagen (a total lattice volume of 7.5 ml). The FPCLs were incubated at 37°C for 60 min to allow the collagen to polymerize. They were then detached from the edge of the plate and 2 ml of F-SCM (Gelatin-A Sepharose pretreated) was added. FPCLs were maintained at 37°C in a 5% CO2 humidified atmosphere. As a circular shape is retained during contraction the diameter of the contracting FPCL was measured from day 0 to day 7. The degree of cell-mediated contraction was estimated from three separate lattice diameter measurements. For each sample, experiments were performed in triplicate. On days 1, 2, 3, and 7 after initial fabrication 500 μl of CM surrounding the lattices was collected from each individual FPCL, combined, and stored at -20°C for future analysis. Five hundred microliters of F-SCM (Gelatin-A Sepharose pretreated) was added to replace that collected. FPCLs (n=3) were removed from culture, blotted on 3MM filter paper (5 min), snap-frozen in liquid nitrogen, and stored at -70°C. Total cellular RNA was isolated by a technique based on the guanidinium thiocyanate method. FPCLs were disaggregated mechanically in the presence of Ultraspec™ RNA solution (AMS Biotech, Witney, U.K.). Total RNA was then extracted with chloroform (Sigma, Poole, U.K.) and precipitated using isopropanol (Sigma). RNA was also isolated directly from monolayer cultures. RNA concentrations were determined by spectrophotometry and samples were stored at -70°C for future analysis. RNase protection analysis was undertaken as described previously (Detmer et al., 1993Detmer K. Lawrence H.J. Largman C. Expression of class I homeobox genes in fetal and adult murine skin.J Invest Dermatol. 1993; 101: 517-522Abstract Full Text PDF PubMed Google Scholar). Briefly, antisense probes [a 240 nucleotide fragment of the murine cytoplasmic actin cDNA from Ambion (Austin, TX) and a 400 nucleotide fragment of the murine Prx-2 cDNA lacking the homoebox region] were synthesized by T7 polymerase (Promega, Southampton, U.K.) in the presence of 32P-labeled UTP (Amersham Pharmacia Biotech), purified through 5% (vol/vol) polyacrylamide/7 M urea gels, and eluted. Total RNA (10 μg of RNA combined from n=3 independent samples) was mixed with the test probes and hybridized overnight at 65°C. After digestion with RNase A and T1 (Promega), the protected fragments were precipitated with ethanol, separated by electrophoresis through sequencing gels, and visualized by autoradiography. Autoradiographs were scanned and analyzed using the Bio-Rad Molecular Analysis software (Bio-Rad, Hemel Hempstead, U.K.) to quantitate Prx-2 expression, which was normalized to the actin values. Positive controls involved the inclusion of a plasmid containing the Prx-2 sequence; tRNA (Promega) was used as the negative control. Fibroblast proliferation in monolayers was assayed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT; Sigma) dye-reduction assay (Mosmann, 1983Mosmann T. Rapid colormetric assay for cellular growth and survival – application to proliferation and cytotoxic assays.J Immunol Meth. 1983; 65: 55-63Crossref PubMed Scopus (43857) Google Scholar). Fibroblasts were recovered from culture by trypsinization and placed in 96-well microtiter plates at a cell density of 5×103 cells per well in F-SCM. After 24 or 72 h 25 μl of MTT (5 mg per ml) was added to each well and the plates were incubated for 4 h at 37°C. A hundred microliters of extraction buffer [10% (wt/vol) sodium dodecyl sulfate (SDS)/0.5 M dimethylformamide; Sigma] was then added to each well and the plates were incubated overnight at 37°C. The absorbance of each well was assessed using a Dynex MRX spectrophotometer (Dynex Technologies, Billinghurst, U.K.) equipped with a 550 nm filter. Cell attachment assays were performed as described previously (Aumailley et al., 1989Aumailley M. Mann K. von der Mark H. Timpl R. Cell attachment properties of collagen type VI and arg-gly-asp dependent binding to its α2(VI) and α3(VI) chains.Exp Cell Res. 1989; 181: 463-474Crossref PubMed Scopus (209) Google Scholar). Wells of a 96-well microtiter plate were incubated overnight at 4°C with a solution of either rat-tail type I collagen (40 μg per ml;Rowling et al., 1990Rowling P.J.E. Raxworthy M.J. Wood E.J. Kearney J.N. Fabrication and reorganization of dermal equivalents suitable for skin grafting after major cutaneous injury.Biomaterials. 1990; 11: 181-185Crossref PubMed Scopus (37) Google Scholar), human plasma fibronectin (20 μg per ml; Sigma), or human placental laminin (20 μg per ml; Sigma) followed by blocking of nonspecific binding by incubation with 1% (wt/vol) bovine serum albumin (Sigma) for 4 h at 4°C. Fibroblasts were trypsinized and resuspended in complete growth medium lacking FBS at a concentration of 1.5×105 cells per ml. One hundred microliters of cell suspension was added to individual wells and the plate was incubated for 60 min at 37°C in a 5% CO2 humidified atmosphere. Nonadherent cells were removed by aspiration and the remaining adherent cells were washed (phosphate-buffered saline), fixed with ethanol (70% vol/vol; 15 min), and stained with crystal violet (0.1% wt/vol; 25 min; Sigma). Excess dye was removed by washing (five times) with distilled water and the stain was solubilized using 50 μl of 0.2% (vol/vol) Triton X-100 (Sigma). The absorbance was read using a Dynex MRX spectrophotometer equipped with a 550 nm filter. Fibroblast proliferation in the collagen lattices was assessed using “scaled-down” FPCLs (containing 2×104 cells in a total lattice volume of 400 μl) fabricated in 1% (wt/vol) bovine serum albumin coated (to render them nonadherent) 24-well plates. Fibroblasts were recovered from five combined FPCLs by enzymatic degradation as previously described (Stephens et al., 1996Stephens P. Davies K.J. Al-Khateeb T. Shepherd J.P. Thomas D.W. A comparison of the ability of intra oral and extra oral fibroblasts to stimulate extracellular matrix reorganization in a model of wound contraction.J Dent Res. 1996; 75: 1358-1364Crossref PubMed Scopus (86) Google Scholar). Briefly lattices were solubilized by incubation for 60 min at 37°C with 200 μl of phosphate buffered saline containing 2 mg per ml collagenase. Following incubation for 20 min at 37°C with 100 μl of 0.05% (wt/vol) Trypsin/0.53 mM ethylenediamine tetraacetic acid (Life Technologies) cells were recovered by centrifugation and viable cell numbers were determined by direct counting in a Neubauer hemocytometer using a 0.02% (wt/vol) Trypan Blue solution (Sigma). To determine the relative amounts of pro-enzyme and active MMP species produced by the fibroblasts in the FPCL systems gelatin zymography was employed. Equal volumes (15 μl) of CM were electrophoresed through gelatin (2 mg per ml; Sigma) containing 12% (vol/vol) SDS-polyacrylamide gels. SDS was removed by soaking the gels in 2.5% (vol/vol) Triton X-100 (60 min at room temperature) and MMPs were activated by incubation in activation buffer (25 mM Tris pH 7.6, 5 mM CaCl2, 25 mM NaCl, 5% wt/vol Brij 35; Sigma) overnight at 37°C. Gels were stained with a Coomassie Blue solution (0.05% wt/vol Coomassie Blue, 12% vol/vol acetic acid, 54% vol/vol methanol; Sigma) and destained (5% vol/vol methanol, 7.5% vol/vol acetic acid), and images of the gels were captured with a Bio-Rad gel documentation system. MMPs were identified by the appearance of clear bands and by calculation of relative molecular weights. Quantitation of the MMP bands was undertaken using the Molecular Analyst software. Controls involved running normal, nonconditioned F-SCM to establish that any observed gelatinolytic activity was not due to the presence of endogenous MMPs within the F-SCM. Reverse zymography was employed to determine the relative amounts of the tissue inhibitors of matrix metalloproteinase (TIMP) species produced by the fibroblasts in the FPCL systems. Reverse zymography was performed in a similar manner to zymography except purified MMP-2 (30 μg per ml;Coughlan et al., 1998Coughlan A.R. Robertson D.H.L. Burke R. Beynon R.J. Carter S.D. Isolation and identification of canine matrix metalloproteinase-2 (MMP-2).Vet J. 1998; 155: 231-237Crossref PubMed Scopus (25) Google Scholar) was incorporated into a 14% (vol/vol) SDS-polyacrylamide gel along with 1 mg per ml gelatin. TIMPs were identified by the appearance of dark bands and by calculation of relative molecular weights. Quantitation of the TIMP bands was undertaken using the Molecular Analyst software. Controls involved running normal, nonconditioned F-SCM. CM samples were initially visually assessed by adding 200 μl of dimethylmethylene blue reagent [1,9-dimethylmethylene blue (16 μg per μl; Sigma) in 40 mM glycine–HCl, 42 mM NaCl; pH 3.0] to 40 μl of each of the samples to be assayed (Farndale and Barrett, 1986Farndale R.W.A. Barrett A.J. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue.Biochim Biophys Acta. 1986; 883: 173-177Crossref PubMed Scopus (2832) Google Scholar). If a pink color was observed dilutions were carried out to bring the samples into the blue-purple range (the linear range of the assay). Forty microliters of the appropriately diluted samples to be tested were added to a 96-well plate in triplicate and the plate was read at 525 nm on a Dynex MRX microplate reader. Dimethylmethylene blue reagent was then added (200 μl per well) and the plate was immediately re-read at 525 nm. The sulfated glycosaminoglycan content of each CM sample was calculated by reference to a standard curve (10–40 μg per ml chondroitin sulfate C; Sigma). Eight hundred microliters of concentrated sulfuric acid (containing 120 mM sodium tetraborate; Sigma) was added to 160 μl of each CM sample and incubated for 1 h at 80°C (van den Hoogen et al., 1998van den Hoogen B.M. van Weeren P.R. Lopes-Cardozo M. van Golde L.M. Barneveld A. van de Lest C.H. A microtiter plate assay for the determination of uronic acids.Anal Biochem. 1998; 257: 107-111Crossref PubMed Scopus (125) Google Scholar). After cooling to room temperature 40 μl of each sample was added to a 96-well plate in triplicate. Background absorbance was measured at 540 nm on a Dynex MRX microplate reader and 40 μl of m-hydroxy diphenyl reagent [100 μl of m-hydroxy diphenyl in dimethyl sulfoxide (100 mg per ml) mixed with 4.9 ml of 80% (vol/vol) sulfuric acid just before use; Sigma] was added to each sample. After a 15 min incubation at room temperature, the absorbance was re-read at 540 nm. The uronic acid content of each CM sample was calculated by reference to a standard curve (0–1000 μg per ml uronic acid; Sigma). Relative HA size was determined by gel filtration chromatography on a Sepharose CL-4B (Pharmacia, Uppsala, Sweden) column (Meyer and Stern, 1994Meyer L.J.M. Stern R. Age-dependent changes of hyaluronan in human skin.J Invest Dermatol. 1994; 102: 385-389Abstract Full Text PDF PubMed Google Scholar). Sepharose CL-4B beads were suspended in chromatography buffer (50 mM Tris–HCl, 150 mM NaCl; pH 8.0) and degassed for 30 min. A column (1 cm inner diameter) with a 35 ml bed volume of Sepharose CL-4B beads was then poured and the beads were packed by passing three column volumes of chromatography buffer through the column. CM samples were freeze dried overnight (-70°C) and resuspended in 2 ml of chromatography buffer. Each sample was loaded onto the column and 1 ml fractions collected. Between samples the column was washed with 150 ml of chromatography buffer. The relative HA levels in each fraction were determined using the uronic acid assay. Assessment of collagen production by the fibroblast populations was undertaken utilizing the cold hydoxyproline method. 5×105 fibroblasts (in F-SCM) were seeded into each well of a 12-well tissue culture plate and cultured to confluence. F-SCM was replaced with serum-free medium and the cells were cultured for a further 24 h. Cells were then cultured in (i) serum-free medium, (ii) serum-free medium containing human recombinant transforming growth factor β1 (TGF-β1) (10 ng per ml; CHEMICON, Chandlers Ford, U.K.), or (iii) serum-containing medium (F-SCM) for 72 h. Medium was supplemented with 50 μg per ml of ascorbic acid (Sigma) and 0.2 mM proline (Sigma) with the ascorbic acid replenished after every 24 h. After 72 h the medium and cells from three wells were combined and used for analysis of collagen production; a fourth well was used for cell counting. Proteins in the cell/medium suspension were precipitated by the addition of ice-cold absolute ethanol (Sigma) to a final concentration of 67% (vol/vol) and storage at 4°C overnight. Precipitated protein wa" @default.
• W2034806517 created "2016-06-24" @default.
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• W2034806517 date "2003-01-01" @default.
• W2034806517 modified "2023-09-25" @default.
• W2034806517 title "Deletion of the Homeobox Gene PRX-2 Affects Fetal but Not Adult Fibroblast Wound Healing Responses" @default.
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