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• W2012026767 abstract "Transforming growth factor-β (TGF-β) isoforms are multifunctional cytokines that play an important role in wound healing. Transgenic mice overexpressing TGF-β in the skin under control of epidermal-specific promoters have provided models to study the effects of increased TGF-β on epidermal cell growth and cutaneous wound repair. To date, most of these studies used transgenic mice that overexpress active TGF-β in the skin by modulating the latency-associated-peptide to prevent its association with active TGF-β. The present study is the first to use transgenic mice that overexpress the natural form of latent TGF-β1 in the epidermis, driven by the keratin 14 gene promoter to investigate the effects of locally elevated TGF-β1 on the healing of partial-thickness burn wounds made on the back of the mice using a CO2 laser. Using this model, we demonstrated activation of latent TGF-β after wounding and determined the phenotypes of burn wound healing. We found that introduction of the latent TGF-β1 gene into keratinocytes markedly increases the release and activation of TGF-β after burn injury. Elevated local TGF-β significantly inhibited wound re-epithelialization in heterozygous (42% closed versus 92% in controls,P < 0.05) and homozygous (25%versus 92%, P < 0.01) animals at day 12 after wounding. Interestingly, expression of type I collagen mRNA and hydroxyproline significantly increased in the wounds of transgenic mice, probably as a result of a paracrine effect of the transgene. Transforming growth factor-β (TGF-β) isoforms are multifunctional cytokines that play an important role in wound healing. Transgenic mice overexpressing TGF-β in the skin under control of epidermal-specific promoters have provided models to study the effects of increased TGF-β on epidermal cell growth and cutaneous wound repair. To date, most of these studies used transgenic mice that overexpress active TGF-β in the skin by modulating the latency-associated-peptide to prevent its association with active TGF-β. The present study is the first to use transgenic mice that overexpress the natural form of latent TGF-β1 in the epidermis, driven by the keratin 14 gene promoter to investigate the effects of locally elevated TGF-β1 on the healing of partial-thickness burn wounds made on the back of the mice using a CO2 laser. Using this model, we demonstrated activation of latent TGF-β after wounding and determined the phenotypes of burn wound healing. We found that introduction of the latent TGF-β1 gene into keratinocytes markedly increases the release and activation of TGF-β after burn injury. Elevated local TGF-β significantly inhibited wound re-epithelialization in heterozygous (42% closed versus 92% in controls,P < 0.05) and homozygous (25%versus 92%, P < 0.01) animals at day 12 after wounding. Interestingly, expression of type I collagen mRNA and hydroxyproline significantly increased in the wounds of transgenic mice, probably as a result of a paracrine effect of the transgene. The transforming growth factor-β (TGF-β) family of growth factors are potent regulators of cell growth and differentiation and play an important role in wound healing.1Roberts AB Sporn MB Transforming growth factor-β.in: Clark RAF The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1996: 275-308Google Scholar, 2O'Kane S Ferguson MWJ Transforming growth factor βs and wound healing.Int J Biochem Cell Biol. 1997; 29: 63-78Crossref PubMed Scopus (577) Google Scholar TGF-βs exist in a number of structurally related but functionally distinct 25-kd homodimeric isoforms. In mammals, three isoforms, TGF-β1, 2, and 3, have been identified. Each isoform is synthesized as a large latent precursor that is unable to trigger signaling via high-affinity TGF-β receptors and is therefore named latent TGF-β.3Massagué J The transforming growth factor-β family.Annu Rev Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (3017) Google Scholar, 4Roberts AB Sporn MB The transforming growth factor-βs.in: Sporn MB Roberts AB Peptide Growth Factors and Their Receptors. Springer, Berlin1990: 419-472Crossref Google Scholar Latent TGF-β consists of a disulfide-linked dimer of 25-kd mature TGF-β associated with a 75-kd latency-associated-peptide (LAP). Activation of latent TGF-β, a process involving release of mature TGF-β from the latent precursor, is required for TGF-β to elicit its biological effects on the cells.5Miyazono K Ichijo H Heldin C-H Transforming growth factor-β: latent forms, binding proteins and receptors.Growth Factors. 1993; 8: 11-22Crossref PubMed Scopus (205) Google Scholar TGF-β1 is the predominant isoform in most tissues and is particularly abundant in platelets.3Massagué J The transforming growth factor-β family.Annu Rev Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (3017) Google Scholar After injury, high levels of TGF-β1 are released from degranulating platelets. However sustained levels of TGF-β in wound tissue are subsequently produced by a number of other cell types present in wound, including macrophages, keratinocytes, fibroblasts, and endothelial cells.1Roberts AB Sporn MB Transforming growth factor-β.in: Clark RAF The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1996: 275-308Google Scholar, 2O'Kane S Ferguson MWJ Transforming growth factor βs and wound healing.Int J Biochem Cell Biol. 1997; 29: 63-78Crossref PubMed Scopus (577) Google Scholar, 3Massagué J The transforming growth factor-β family.Annu Rev Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (3017) Google Scholar TGF-β acts via autocrine and paracrine mechanisms to regulate the interactions between cells and between cells and matrix in wound healing, involving inflammation, re-epithelialization, angiogenesis, and the production of extracellular matrix.1Roberts AB Sporn MB Transforming growth factor-β.in: Clark RAF The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1996: 275-308Google Scholar, 2O'Kane S Ferguson MWJ Transforming growth factor βs and wound healing.Int J Biochem Cell Biol. 1997; 29: 63-78Crossref PubMed Scopus (577) Google Scholar Application of exogenous TGF-β either locally or systemically has been found to accelerate healing, particularly in chronic or impaired wounds.6Mustoe TA Pierce GF Thomason A Gramates P Sporn MB Deuel TF Accelerated healing of incisional wounds in rats induced by transforming growth factor-β.Science. 1987; 237: 1333-1336Crossref PubMed Scopus (812) Google Scholar, 7Beck LS Deguzman L Lee WP Xu Y McFatridge LA Amento EP TGF-β1 accelerates wound healing: reversal of steroid-impaired healing in rats and rabbits.Growth Factors. 1991; 5: 295-304Crossref PubMed Scopus (107) Google Scholar However, overexpression of TGF-β1 has been implicated in various forms of fibrosis such as glomerulonephritis, liver cirrhosis, pulmonary cirrhosis,8Border WA Okuda S Languino LR Sporn MB Ruoslahti E Suppression of experimental glomerulonephritis by antiserum against transforming growth factor β1.Nature. 1990; 346: 371-374Crossref PubMed Scopus (940) Google Scholar, 9Castilla A Prieto J Fausto N Transforming growth factor β and α in chronic liver disease.N Engl J Med. 1991; 324: 933-940Crossref PubMed Scopus (645) Google Scholar, 10Broekelmann TJ Limper AH Colby TV McDonald JA Transforming growth factor β1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis.Proc Natl Acad Sci USA. 1991; 88: 6642-6646Crossref PubMed Scopus (690) Google Scholar as well as hypertrophic scar.11Tredget EE Nedelec B Scott PG Ghahary A Hypertrophic scars, keloid, and contractures. The cellular and molecular basis for therapy.Surg Clin North Am. 1997; 77: 701-730Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 12Scott PG Ghahary A Tredget EE Molecular and cellular aspects of fibrosis following thermal injury.Hand Clin. 2000; 16: 271-287PubMed Google Scholar, 13Ghahary A Shen YJ Scott PG Gong Y Tredget EE Enhanced expression of mRNA for transforming growth factor-β, type I and type III procollagen in human post-burn hypertrophic scar tissue.J Lab Clin Med. 1993; 122: 465-473PubMed Google Scholar To explore the effects of increased local TGF-β1 on skin development and wound repair, various transgenic mouse models have been established using different keratin promoters to induce the overexpression of TGF-β1 in the epidermis. To date, most investigations have been performed in transgenic mice that express the constitutively active TGF-β1 by mutation of Cys-223→Ser and Cys-225→Ser in the LAP, thus preventing its binding to mature TGF-β.14Cui W Fowlis DJ Cousins FM Duffie E Bryson S Balmain A Akhurst RJ Concerted action of TGF-β1 and its type II receptor in control of epidermal homeostasis in transgenic mice.Genes Dev. 1995; 9: 945-955Crossref PubMed Scopus (90) Google Scholar, 15Cui W Fowlis DJ Bryson S Duffie E Ireland H Balmain A Akhurst RJ TGF-β1 inhibits the formation of benign skin tumors, but enhances progression to invasive spindle carcinomas in transgenic mice.Cell. 1996; 86: 531-542Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, 16Fowlis DJ Cui W Johnson SA Balmain A Akhurst RJ Altered epidermal cell growth control in vivo by inducible expression of transforming growth factor β1 in the skin of transgenic mice.Cell Growth Differ. 1996; 7: 679-687PubMed Google Scholar, 17Wang X-J Liefer KM Tsai S O'Malley BW Roop DR Development of gene-switch transgenic mice that inducibly express transforming growth factor β1 in the epidermis.Proc Natl Acad Sci USA. 1999; 96: 8483-8488Crossref PubMed Scopus (133) Google Scholar Constitutive overexpression of active TGF-β1 in the epidermis, driven by the human keratin 1 promoter, results in neonatal lethality because of developmental deficiency in skin.18Sellheyer K Bickenbach JR Rothnagel JA Bundman D Longley MA Krieg T Roche NS Roberts AB Roop DR Inhibition of skin development by overexpression of transforming growth factor β1 in the epidermis of transgenic mice.Proc Natl Acad Sci USA. 1993; 90: 5237-5241Crossref PubMed Scopus (221) Google Scholar Overexpression of TGF-β1 in the epidermis driven by the keratin 6 or keratin 10 promoters gave contradictory results, either inhibiting or stimulating keratinocyte proliferation.14Cui W Fowlis DJ Cousins FM Duffie E Bryson S Balmain A Akhurst RJ Concerted action of TGF-β1 and its type II receptor in control of epidermal homeostasis in transgenic mice.Genes Dev. 1995; 9: 945-955Crossref PubMed Scopus (90) Google Scholar, 16Fowlis DJ Cui W Johnson SA Balmain A Akhurst RJ Altered epidermal cell growth control in vivo by inducible expression of transforming growth factor β1 in the skin of transgenic mice.Cell Growth Differ. 1996; 7: 679-687PubMed Google Scholar Recently, Wang and colleagues17Wang X-J Liefer KM Tsai S O'Malley BW Roop DR Development of gene-switch transgenic mice that inducibly express transforming growth factor β1 in the epidermis.Proc Natl Acad Sci USA. 1999; 96: 8483-8488Crossref PubMed Scopus (133) Google Scholar reported a gene-switch system, in which the expression of the TGF-β1 transgene in the epidermis was controlled by topical application of an inducer. This study suggests that induction of the TGF-β1 transgene produces an inhibitory effect on keratinocyte growth in both hyperproliferative and quiescent cells. All of these studies focused on the effect of a persistent increase in active TGF-β on epidermal cell proliferation in nonwounded skin. However, phenotypes observed in these models may not necessarily reflect the physiological role of the latent form of TGF-β in wound healing. In this study, we investigated the effects of locally elevated TGF-β1 in laser-induced burn wound healing, using an established transgenic mouse model that overexpresses human latent TGF-β1 in the epidermis, driven by a keratin 14 (K14) gene promoter. We also measured the amount of total and active TGF-β in the wound tissue by the plasminogen activator inhibitor/luciferase (PAI/L) assay and the distribution of TGF-β in the wounds by immunohistochemistry. Our data suggest that injury to the skin increases the release of TGF-β1 from epidermal keratinocytes and activation of latent TGF-β. Overexpressed TGF-β1 in the epidermis, through an autocrine pathway, inhibits keratinocyte proliferation, resulting in a marked delay in wound re-epithelialization. Furthermore, overexpression of TGF-β1 in the epidermis increases the expression of type I collagen mRNA and hydroxyproline in the wound through a paracrine pathway. Transgenic mice that overexpress a latent human TGF-β1 gene in the epidermis driven by a K14 gene promoter were used in this study.19Ghahary A Tredget EE Chang L-J Scott PG Shen Q Genetically modified dermal keratinocytes express high levels of transforming growth factor-β1.J Invest Dermatol. 1998; 110: 800-805Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar The generation and characterization of this transgenic mouse strain has been described elsewhere (T. Chan and colleagues, manuscript submitted). Both heterozygous (HT) and homozygous (HM) animals were studied. Wild-type (WT) mice of the same strain were used as controls. Under metafane anesthesia, the dorsal surface of mouse was clipped, chemically depilated, and wiped with betadine and saline. Four histologically proven partial-thickness wounds (4 × 6 mm) were made in the dorsal skin using a gas-charged CO2 laser set at 10 W, and 0.2-second exposure time. The animals were allowed to recover, housed separately, and fed ad libitum until the wounds were harvested. Wounds were examined visually and the time of closure was recorded. At days 0 (within 1 hour after wounding), 6, 12, 16, and 32 after wounding, six animals from each group were sacrificed by CO2overdose and the wounds harvested using a 6-mm punch biopsy. From each animal, one wound was fixed in 4% paraformaldehyde and prepared for histology and immunocytochemistry; two wounds were collected in 4 mol/L guanidinium isothiocyanate for RNA extraction; and one wound was frozen for hydroxyproline analysis. For measurement of TGF-β in wound tissue by the PAI/L assay, three more animals were sacrificed at each time point and wound samples were processed as described below. All animal studies were conducted in compliance with Canadian Council on Animal Care guidelines and the University of Alberta Health Sciences Animal Policy and Welfare Committee regulations. Hematoxylin and eosin (H&E) staining was performed on 6-μm paraffin sections to confirm the partial-thickness wounds created by a CO2 laser and assess the phenotype of wound healing by light microscopy. From each sample at least three sections through the center of the wound were examined for re-epithelialization. The distribution of latent and active TGF-β in the skin and wounds was determined by immunohistochemistry in 6-μm paraffin sections using the avidin-biotin immunoperoxidase staining technique. Expression of latent TGF-β1 was examined using an antibody against human LAP1 (catalog no. AF-246-NA; R&D Systems, Minneapolis, MN). Active TGF-β1 in the wounds was detected using an antibody against active TGF-β1 (catalog no. AF-101-NA; R&D Systems). Sections were deparaffinized in xylene and rehydrated in graded ethanol. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in methanol (v/v) for 6 minutes at room temperature. Nonspecific protein binding was blocked with 10% normal serum from the same species as the biotinylated secondary antibody for 30 minutes. The primary antibody was applied to the slides at a final concentration of 20 μg/ml in phosphate-buffered saline (PBS) and incubated overnight in a humidified chamber at 4°C. Control sections for LAP1 were incubated with nonimmune goat IgG. The specificity of active TGF-β staining was verified by incubation of the antibody with recombinant human TGF-β1 (catalog no. 240-B; R&D Systems) before staining. After washing in PBS, sections were incubated with a biotinylated secondary antibody (1:150 dilution; Vector Laboratories, Burlingame, CA) for 40 minutes, followed by incubation with avidin-biotin complex (ABC kit; DAKO Diagnostics Canada Inc., Mississauga, Ontario, Canada) for 1 hour at room temperature. Sections were washed in PBS, then incubated for 3 minutes with 3-amino-9-ethylcarbazole for LAP1 or 0.05% (w/v) 3,3′-diaminobenzidine for active TGF-β. Sections were counterstained with hematoxylin and mounted with Permount for 3,3′-diaminobenzidine staining or AquaPerm mounting medium (Shandon, Pittsburgh, PA) for 3-amino-9-ethylcarbazole staining. Sections were viewed using a Nikon microscope and photographed using Kodak Ektachrome 200 ASA color films (Eastman-Kodak, Rochester, NY). The effect of transgene expression on keratinocyte proliferationin vivo was analyzed by 5-bromodeoxyuridine (BrdU) incorporation at day 6 after wounding as described.14Cui W Fowlis DJ Cousins FM Duffie E Bryson S Balmain A Akhurst RJ Concerted action of TGF-β1 and its type II receptor in control of epidermal homeostasis in transgenic mice.Genes Dev. 1995; 9: 945-955Crossref PubMed Scopus (90) Google Scholar Mice were injected intraperitoneally with BrdU (Sigma) solution (250 μg/g body weight in 0.9% NaCl) and killed 1 hour after injection. Wound specimens were frozen in Optimal Cutting Temperature compound. Cryosections (6 μm) were immunostained with a mouse anti-BrdU antibody (RPN202; Amersham Pharmacia Biotec, Inc., Baie d'Urté, Quebec, Canada) followed by a biotinylated goat anti-mouse secondary antibody. The immunoreaction was visualized by ABC kit and 3,3′-diaminobenzidine. The number of BrdU-labeled keratinocytes per high-power field of the epidermis from the wound edge was counted. To evaluate TGF-β activation during wound healing, we measured the levels of active and total TGF-β in cryosections of wounds by the PAI/L assay. This assay is based on the ability of TGF-β to induce plasminogen activator inhibitor-1 (PAI-1) expression in mink lung epithelial cells (MLECs) transfected with the PAI-1/luciferase construct.20Abe M Harpel JG Metz CN Nunes I Loskutoff DJ Rifkin DB An assay for transforming growth factor-β using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.Anal Biochem. 1994; 216: 276-284Crossref PubMed Scopus (678) Google Scholar Transfected MLECs were a generous gift from Dr. Daniel B. Rifkin (New York University Medical Center, New York, NY). MLECs were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc., Grand Island, NY) supplemented with 5% fetal bovine serum, antibiotic-antimycotic (100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B; Life Technologies, Inc.) and 250 μg/ml of geneticin (Life Technologies, Inc.). The cells were incubated at 37°C in an atmosphere of 5% CO2. Cells between passages 10 and 30 were used for the PAI/L assay. Wound samples were prepared from normal and transgenic mice at days 6, 12, and 16 after wounding as described.21Yang L Qiu CX Ludlow A Ferguson MWJ Brunner G Active transforming growth factor-β in wound repair: determination using a new assay.Am J Pathol. 1999; 154: 105-111Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar Briefly, four wounds, 4 × 4 mm for each, from one animal were embedded in one block of 1% sterile methyl-cellulose (Sigma Chemical Co., St. Louis, MO) and snap-frozen in liquid nitrogen. Four skin samples from the back of nonwounded normal and transgenic mice were also excised and processed as described above. The embedded tissue samples were stored at −80°C until used for analysis. When the PAI/L assay was performed, thick (24 μm) cryosections were cut and placed on sterilized 13-mm round coverslips and temporarily stored at −20°C until transferred onto MLECs. Transfected MLECs were plated into 24-well cell culture dishes (3 × 105/ml, 500 μl/well) in complete Dulbecco’s modified Eagle’s medium and incubated for 4 hours at 37°C. Then, serum-containing medium was replaced with 500 μl of Dulbecco’s modified Eagle’s medium containing 0.1% pyrogen-poor bovine serum albumin (Pierce, Rockford, IL), penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B (0.25 μg/ml). The coverslips each carrying four tissue sections from one animal were placed on MLECs with the sections facing down and incubated overnight. MLECs were lysed and luciferase activity was determined using a liquid scintillation counter (LS 6000TA; Beckman Instruments Canada Inc., Fullerton, CA) equipped with a single photon monitor. TGF-β levels were calculated by reference to a standard curve prepared with recombinant human TGF-β1. Total TGF-β in wound sections was measured after acidification.22Lawrence DA Pircher R Kryceve-Martinerie C Jullien P Normal embryo fibroblasts release transforming growth factors in a latent form.J Cell Physiol. 1984; 121: 184-188Crossref PubMed Scopus (244) Google Scholar, 23Assoian RK Sporn MB Type β transforming growth factor in human platelets: release during platelet degranulation and action on vascular smooth muscle cells.J Cell Biol. 1986; 102: 1217-1223Crossref PubMed Scopus (429) Google Scholar Coverslips carrying cryosections were placed in 24-well plates and submerged in 500 μl of Dulbecco’s modified Eagle’s medium containing 0.1% bovine serum albumin. The samples were acidified with 12.5 μl of 3 N HCl for 15 minutes at room temperature and neutralized with 35 μl of 1 mol/L HEPES/5 N NaOH (5:2, v/v). The coverslips were transferred, together with the medium, onto MLECs for TGF-β quantification. To normalize the amount of TGF-β to the wound size, each tissue specimen was trimmed to 4 × 4 mm and sectioned at 24 μm in thickness. The wound area was measured under the microscope and no significant differences were observed between sections (data not shown). Total RNA was extracted from punch biopsies of wounds at selected time points after wounding. Tissue samples were lysed in 1 ml of 4 mol/L guanidinium isothiocyanate as previously described.13Ghahary A Shen YJ Scott PG Gong Y Tredget EE Enhanced expression of mRNA for transforming growth factor-β, type I and type III procollagen in human post-burn hypertrophic scar tissue.J Lab Clin Med. 1993; 122: 465-473PubMed Google Scholar Extracted RNA was then ethanol precipitated and used for Northern analysis. Ten μg of total RNA extracted from wound tissue was loaded onto a 1% agarose gel and separated by electrophoresis. The RNA was transferred to nitrocellulose filters and baked at 80°C under vacuum for 2 hours. Filters were prehybridized in a solution containing 50% (v/v) formamide, 0.3 mol/L sodium chloride, 20 mmol/L Tris-HCl, pH 8.0, 1 mmol/L ethylenediaminetetraacetic acid, 1× Denhardt’s solution [1× = 0.02% (w/v) bovine serum albumin, Ficoll, and polyvinylpyrrolidone], 0.05% (w/v) salmon sperm DNA, and 0.005% (w/v) poly(A) for 2 to 4 hours at 45°C. Hybridization was performed in the same solution for 16 to 20 hours at 45°C using a cDNA probe specific for the pro α1(l) chain of type I procollagen (provided by Drs. G. Tromp, H. Kuivaniemi, and L. Ala-Kokko, Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Philadelphia, PA).13Ghahary A Shen YJ Scott PG Gong Y Tredget EE Enhanced expression of mRNA for transforming growth factor-β, type I and type III procollagen in human post-burn hypertrophic scar tissue.J Lab Clin Med. 1993; 122: 465-473PubMed Google Scholar The blots were subsequently rehybridized with cDNA specific for 18S ribosomal RNA (rRNA) as a control for loading. The probes were labeled with [α-32P] dCTP (DuPont Canada, Mississauga, Ontario, Canada) by nick-translation. Filters were initially washed at room temperature with 2× standard saline citrate (1× = 0.15 mpl/L sodium chloride, 0.015 mol/L sodium citrate) and 0.1% sodium dodecyl sulfate for 30 minutes and then for 20 minutes at 65°C in 0.2× standard saline citrate and 0.1% sodium dodecyl sulfate. Autoradiography was performed by exposing Kodak X-Omat film to the nitrocellulose filters at −70°C in the presence of an enhancing screen. The cDNA probe for 18S ribosomal RNA was obtained from the American Type Culture Collection (Rockville, MD). The content of collagen in wound tissue was determined by mass spectrometric analysis for 4-hydroxyproline.24Tredget EE Falk N Scott PG Hogg AM Burke J Determination of 4-hydroxyproline in collagen by gas chromatography/mass spectrometry.Anal Biochem. 1990; 190: 259-265Crossref PubMed Scopus (36) Google Scholar Wound samples taken from normal and transgenic mice at days 6, 12, and 16 after wounding were freeze-dried. Internal standard (N-methyl-l-proline) and 6 N HCl solution was added to wound tissue, and each sample was then hydrolyzed overnight at 115°C. The O-butyl ester derivatives were prepared with 10% BF2-butanol for 30 minutes at 120°C after drying the hydrolysate. Liquid chromatography (column: Eclipse XDB-C18)/mass spectrometry analysis was performed on a Hewlett-Packard (series 1100, Atlanta, GA) mass selective detector monitoring the ions m/z 188. The data on wound re-epithelialization were analyzed using Fisher’s exact test. The levels of active/total TGF-β, the number of BrdU-labeled keratinocytes and relative expression of type I collagen mRNA and hydroxyproline were compared using a one-way analysis of variance. For all comparisons P < 0.05 was considered to be significant. All data are expressed as mean values ± SEM. To verify expression of the transgene at the protein level, immunostaining for human latent TGF-β1 was performed in nonwounded skin and wounds using an antibody against human LAP1. In nonwounded skin, a strong staining of latent TGF-β1 was seen in basal keratinocytes of both HT and HM mice (Figure 1A). The staining pattern was mainly intracellular in distribution. In normal skin of WT mice, immunostaining of latent TGF-β was visible in the epidermis but the intensity of staining was markedly less than that in transgenic mouse skin (Figure 1A). At day 6 after skin injury, staining for latent TGF-β1 was increased in the suprabasal keratinocytes in WT mice (Figure 1B). At this time point, latent TGF-β staining was especially strong and present throughout the epidermis adjacent to the wounds of transgenic mice (Figure 1B). Similar differences in the intensity and localization of latent TGF-β staining were also observed between wounds in transgenic and WT mice on day 12 (data not shown). Because activation of latent TGF-β is a key point in the regulation of TGF-β action, we evaluated active TGF-β in the skin and wounds by immunohistochemistry. In nonwounded skin, no active TGF-β1 was detectable (data not shown). After wounding, marked differences in the intensity and localization of TGF-β staining were observed between wounds in transgenic and WT mice. In day 6 wounds, active TGF-β was found in the migrating epithelial sheet in both WT and transgenic mice (Figure 2). In wounds in WT mice, active TGF-β1 was present mainly in the suprabasal keratinocytes, whereas, in wounds in transgenic mice, TGF-β1 was distributed throughout the epidermis. The same staining patterns were also observed in day 12 wounds (data not shown). The specificity of TGF-β staining was confirmed by the fact that 100 μl of antibody (20 μg/ml) was completely neutralized by incubation with 200 μg of recombinant human TGF-β overnight at 4°C (data not shown). To further quantitate the amount of total and active TGF-β in the wounds, we further analyzed samples taken from WT and transgenic mice before and after injury using the PAI/L assay. The levels of total TGF-β were significantly higher in nonwounded skin and wounds of transgenic mice compared with WT controls. In WT mice, the amount of total TGF-β was twofold to threefold higher in wounds than in normal skin at days 6, 12, and 16 after wounding, whereas this increase was significantly higher in wounds of HT and HM mice at these time points (Figure 3A). However, there were no significant differences in the amount of active TGF-β in nonwounded skin for transgenic and WT mice. After injury, the level of active TGF-β markedly increased in all wounds, however, the increase was more apparent in wounds of transgenic mice compared with controls. At day 6, there was a very significant increase in active TGF-β in HT (15.9 ± 1.1 pg/section, P < 0.01,n = 12) and HM (18.8 ± 2.9 pg/section,P < 0.01, n = 12) mice, compared to normal controls (4.5 ± 1.7 pg/section). Although the levels of active TGF-β in the wounds of transgenic mice remained higher than those in WT wounds at days 12 and 16, the differences were not statistically significant (Figure 3B). First, we determined the time course and morphological changes of partial-thickness burn wounds created by a CO2laser in WT mice by visual observation and light microscopy. Histologically, the epidermis on the wound surface and collagen in the papillary dermis were destroyed soon after wounding (within 1 hour) with a thick layer of fatty tissue remaining under the dermis. There were no immediately visible changes in the panniculus (Figure 4A). At day 6, the wounds remained open and were characterized by a large number of infiltrated inflammatory cells. The keratinocyte layers at the wound edges were becoming thicker and the panniculus was no longer present at the wound sites (Figure 4B). Re-epithelialization was complete in majority of the wounds at day 12, at which time high cellularity was still obvious and some new collagen was seen within the wounds (Figure 4C). By day 16, the wound areas became smaller, with dense granulation tissue present (Figure 4D). By day 32, cellularity was markedly reduced and scar tissue was seen at the wound site (data not shown). Next, we examined the extent of wound closure and re-epithelialization in transgenic mice (48 wounds in each" @default.
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• W2012026767 date "2001-12-01" @default.
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• W2012026767 title "Healing of Burn Wounds in Transgenic Mice Overexpressing Transforming Growth Factor-β1 in the Epidermis" @default.
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• W2012026767 doi "https://doi.org/10.1016/s0002-9440(10)63066-0" @default.
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