Overexpression of mIGF-1 in Keratinocytes Improves Wound Healing and Accelerates Hair Follicle Formation and Cycling in Mice
2008; Elsevier BV; Volume: 173; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2008.071177
ISSN1525-2191
AutoresEkaterina Semenova, Heidi Koegel, Sybille Hasse, Jennifer E. Klatte, Esfir Slonimsky, Daniel Bilbao, Ralf Paus, Sabine Werner, Nadia Rosenthal,
Tópico(s)Growth Hormone and Insulin-like Growth Factors
ResumoInsulin-like growth factor 1 (IGF-1) is an important regulator of growth, survival, and differentiation in many tissues. It is produced in several isoforms that differ in their N-terminal signal peptide and C-terminal extension peptide. The locally acting isoform of IGF-1 (mIGF-1) was previously shown to enhance the regeneration of both muscle and heart. In this study, we tested the therapeutic potential of mIGF-1 in the skin by generating a transgenic mouse model in which mIGF-1 expression is driven by the keratin 14 promoter. IGF-1 levels were unchanged in the sera of hemizygous K14/mIGF-1 transgenic animals whose growth was unaffected. A skin analysis of young animals revealed normal architecture and thickness as well as proper expression of differentiation and proliferation markers. No malignant tumors were formed. Normal homeostasis of the putative stem cell compartment was also maintained. Healing of full-thickness excisional wounds was accelerated because of increased proliferation and migration of keratinocytes, whereas inflammation, granulation tissue formation, and scarring were not obviously affected. In addition, mIGF-1 promoted late hair follicle morphogenesis and cycling. To our knowledge, this is the first work to characterize the simultaneous, stimulatory effect of IGF-1 delivery to keratinocytes on two types of regeneration processes within a single mouse model. Our analysis supports the use of mIGF-1 for skin and hair regeneration and describes a potential cell type-restricted action. Insulin-like growth factor 1 (IGF-1) is an important regulator of growth, survival, and differentiation in many tissues. It is produced in several isoforms that differ in their N-terminal signal peptide and C-terminal extension peptide. The locally acting isoform of IGF-1 (mIGF-1) was previously shown to enhance the regeneration of both muscle and heart. In this study, we tested the therapeutic potential of mIGF-1 in the skin by generating a transgenic mouse model in which mIGF-1 expression is driven by the keratin 14 promoter. IGF-1 levels were unchanged in the sera of hemizygous K14/mIGF-1 transgenic animals whose growth was unaffected. A skin analysis of young animals revealed normal architecture and thickness as well as proper expression of differentiation and proliferation markers. No malignant tumors were formed. Normal homeostasis of the putative stem cell compartment was also maintained. Healing of full-thickness excisional wounds was accelerated because of increased proliferation and migration of keratinocytes, whereas inflammation, granulation tissue formation, and scarring were not obviously affected. In addition, mIGF-1 promoted late hair follicle morphogenesis and cycling. To our knowledge, this is the first work to characterize the simultaneous, stimulatory effect of IGF-1 delivery to keratinocytes on two types of regeneration processes within a single mouse model. Our analysis supports the use of mIGF-1 for skin and hair regeneration and describes a potential cell type-restricted action. Insulin-like growth factor 1 (IGF-1) is a peptide hormone that promotes growth, survival, and differentiation of cells in various organs and tissues, including skin.1Dupont J Holzenberger M Biology of insulin-like growth factors in development.Birth Defects Res C Embryo Today. 2003; 69: 257-271Crossref PubMed Scopus (165) Google Scholar, 2Edmondson SR Thumiger SP Werther GA Wraight CJ Epidermal homeostasis: the role of the growth hormone and insulin-like growth factor systems.Endocr Rev. 2003; 24: 737-764Crossref PubMed Scopus (171) Google Scholar The importance of IGF-1 signaling in the skin is evident from the original studies with IGF-1 receptor null (Igf-1r−/−) mice, which exhibited hypotrophic skin with reduced number and size of the hair follicles.3Liu JP Baker J Perkins AS Robertson EJ Efstratiadis A Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r).Cell. 1993; 75: 59-72PubMed Scopus (2585) Google Scholar Similarly, deficiency in human growth hormone or its target IGF-1 are associated with decreased epidermal thickness and sparse hair growth.4Lange M Thulesen J Feldt-Rasmussen U Skakkebaek NE Vahl N Jorgensen JO Christiansen JS Poulsen SS Sneppen SB Juul A Skin morphological changes in growth hormone deficiency and acromegaly.Eur J Endocrinol. 2001; 145: 147-153Crossref PubMed Scopus (45) Google Scholar, 5Lurie R Ben-Amitai D Laron Z Laron syndrome (primary growth hormone insensitivity): a unique model to explore the effect of insulin-like growth factor 1 deficiency on human hair.Dermatology. 2004; 208: 314-318Crossref PubMed Scopus (32) Google Scholar Although generally recognized as a proliferation and survival factor for the skin, IGF-1 was recently also implicated in hair and skin morphogenesis.6Sadagurski M Yakar S Weingarten G Holzenberger M Rhodes CJ Breitkreutz D Leroith D Wertheimer E Insulin-like growth factor 1 receptor signaling regulates skin development and inhibits skin keratinocyte differentiation.Mol Cell Biol. 2006; 26: 2675-2687Crossref PubMed Scopus (104) Google Scholar, 7Weger N Schlake T Igf-I signalling controls the hair growth cycle and the differentiation of hair shafts.J Invest Dermatol. 2005; 125: 873-882Crossref PubMed Scopus (131) Google Scholar In the skin, its levels must be strictly controlled because overexpression of IGF-1 in proliferating and in differentiating keratinocytes resulted in hyperplasia and tumor formation.8Bol DK Kiguchi K Gimenez-Conti I Rupp T DiGiovanni J Overexpression of insulin-like growth factor-1 induces hyperplasia, dermal abnormalities, and spontaneous tumor formation in transgenic mice.Oncogene. 1997; 14: 1725-1734Crossref PubMed Scopus (126) Google Scholar, 9DiGiovanni J Bol DK Wilker E Beltran L Carbajal S Moats S Ramirez A Jorcano J Kiguchi K Constitutive expression of insulin-like growth factor-1 in epidermal basal cells of transgenic mice leads to spontaneous tumor promotion.Cancer Res. 2000; 60: 1561-1570PubMed Google Scholar In addition to its role in skin homeostasis, several studies suggest a role of IGF-1 in skin repair. Its expression is modulated during wound healing, and retarded healing has been correlated with reduced IGF-1 levels.10Gartner MH Benson JD Caldwell MD Insulin-like growth factors I and II expression in the healing wound.J Surg Res. 1992; 52: 389-394Abstract Full Text PDF PubMed Scopus (107) Google Scholar, 11Vogt PM Lehnhardt M Wagner D Jansen V Krieg M Steinau HU Determination of endogenous growth factors in human wound fluid: temporal presence and profiles of secretion.Plast Reconstr Surg. 1998; 102: 117-123Crossref PubMed Scopus (107) Google Scholar, 12Blakytny R Jude EB Martin Gibson J Boulton AJ Ferguson MW Lack of insulin-like growth factor 1 (IGF1) in the basal keratinocyte layer of diabetic skin and diabetic foot ulcers.J Pathol. 2000; 190: 589-594Crossref PubMed Scopus (129) Google Scholar, 13Brown DL Kane CD Chernausek SD Greenhalgh DG Differential expression and localization of insulin-like growth factors I and II in cutaneous wounds of diabetic and nondiabetic mice.Am J Pathol. 1997; 151: 715-724PubMed Google Scholar In vitro, IGF-1 was shown to stimulate keratinocyte proliferation and migration, as well as collagen production by fibroblasts.14Ando Y Jensen PJ Epidermal growth factor and insulin-like growth factor I enhance keratinocyte migration.J Invest Dermatol. 1993; 100: 633-639Abstract Full Text PDF PubMed Google Scholar, 15Daian T Ohtsuru A Rogounovitch T Ishihara H Hirano A Akiyama-Uchida Y Saenko V Fujii T Yamashita S Insulin-like growth factor-I enhances transforming growth factor-beta-induced extracellular matrix protein production through the P38/activating transcription factor-2 signaling pathway in keloid fibroblasts.J Invest Dermatol. 2003; 120: 956-962Crossref PubMed Scopus (57) Google Scholar, 16Granot I Halevy O Hurwitz S Pines M Growth hormone and insulin-like growth factor I regulate collagen gene expression and extracellular collagen in cultures of avian skin fibroblasts.Mol Cell Endocrinol. 1991; 80: 1-9Crossref PubMed Scopus (27) Google Scholar, 17Haase I Evans R Pofahl R Watt FM Regulation of keratinocyte shape, migration and wound epithelialization by IGF-1- and EGF-dependent signalling pathways.J Cell Sci. 2003; 116: 3227-3238Crossref PubMed Scopus (185) Google Scholar Consequently, local administration of IGF-1 to wound sites enhanced wound closure and stimulated granulation tissue formation.18Jeschke MG Schubert T Klein D Exogenous liposomal IGF-I cDNA gene transfer leads to endogenous cellular and physiological responses in an acute wound.Am J Physiol Regul Integr Comp Physiol. 2004; 286: R958-966Crossref PubMed Scopus (26) Google Scholar, 19Jyung RW Mustoe JA Busby WH Clemmons DR Increased wound-breaking strength induced by insulin-like growth factor I in combination with insulin-like growth factor binding protein-1.Surgery. 1994; 115: 233-239PubMed Google Scholar On the other hand, increased IGF-1 receptor expression was reported in chronic wounds and in hypertrophic scars, and IGF-1 stimulation was associated with increased invasive capacity of keloid fibroblasts.20Ghahary A Shen YJ Wang R Scott PG Tredget EE Expression and localization of insulin-like growth factor-1 in normal and post-burn hypertrophic scar tissue in human.Mol Cell Biochem. 1998; 183: 1-9Crossref PubMed Scopus (33) Google Scholar, 21Yoshimoto H Ishihara H Ohtsuru A Akino K Murakami R Kuroda H Namba H Ito M Fujii T Yamashita S Overexpression of insulin-like growth factor-1 (IGF-I) receptor and the invasiveness of cultured keloid fibroblasts.Am J Pathol. 1999; 154: 883-889Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar In addition, systemic delivery of IGF-1 with the goal to improve wound healing caused hyperglycemia, electrolyte imbalance, and edema.22Bondy CA Underwood LE Clemmons DR Guler HP Bach MA Skarulis M Clinical uses of insulin-like growth factor I.Ann Intern Med. 1994; 120: 593-601Crossref PubMed Scopus (121) Google Scholar, 23Jabri N Schalch DS Schwartz SL Fischer JS Kipnes MS Radnik BJ Turman NJ Marcsisin VS Guler HP Adverse effects of recombinant human insulin-like growth factor I in obese insulin-resistant type II diabetic patients.Diabetes. 1994; 43: 369-374Crossref PubMed Google Scholar IGF-1 is also implicated in the control of hair cycling, a regenerative process that constantly occurs in the skin. In vitro, IGF-1 maintained hair follicles in a growth phase (anagen), and removal of IGF-1 led to a catagen-like regression.24Philpott MP Sanders DA Kealey T Effects of insulin and insulin-like growth factors on cultured human hair follicles: IGF-I at physiologic concentrations is an important regulator of hair follicle growth in vitro.J Invest Dermatol. 1994; 102: 857-861Abstract Full Text PDF PubMed Google Scholar Transgenic animals, in which an ultra-high-sulfur keratin gene promoter was used to target IGF-1 to the wool follicles of the sheep or hair follicles of the mouse, showed increased fleece weight and vibrissae length, respectively.25Damak S Su H Jay NP Bullock DW Improved wool production in transgenic sheep expressing insulin-like growth factor 1.Biotechnology (NY). 1996; 14: 185-188Crossref PubMed Scopus (88) Google Scholar, 26Su HY Hickford JG The PH Hill AM Frampton CM Bickerstaffe R Increased vibrissa growth in transgenic mice expressing insulin-like growth factor 1.J Invest Dermatol. 1999; 112: 245-248Crossref PubMed Scopus (33) Google Scholar Hair appeared earlier in transgenic mice overexpressing IGF-1 in the skin under control of the keratin 1 (K1) promoter.8Bol DK Kiguchi K Gimenez-Conti I Rupp T DiGiovanni J Overexpression of insulin-like growth factor-1 induces hyperplasia, dermal abnormalities, and spontaneous tumor formation in transgenic mice.Oncogene. 1997; 14: 1725-1734Crossref PubMed Scopus (126) Google Scholar In contrast, hair follicles of transgenic mice expressing IGF-1 in the skin under control of the involucrin promoter, showed a delay in anagen entry.7Weger N Schlake T Igf-I signalling controls the hair growth cycle and the differentiation of hair shafts.J Invest Dermatol. 2005; 125: 873-882Crossref PubMed Scopus (131) Google Scholar This difference could result from different promoter usage, but also from the use of different IGF-1 isoforms. IGF-1 is produced in multiple isoforms that differ in their amino-terminal signal peptides and carboxy-terminal extension peptide.27Adamo ML Neuenschwander S LeRoith D Roberts Jr, CT Structure, expression, and regulation of the IGF-I gene.Adv Exp Med Biol. 1993; 343: 1-11Crossref PubMed Google Scholar Previous studies28Barton ER Viral expression of insulin-like growth factor-I isoforms promotes different responses in skeletal muscle.J Appl Physiol. 2006; 100: 1778-1784Crossref PubMed Scopus (101) Google Scholar, 29Goldspink G Gene expression in skeletal muscle.Biochem Soc Trans. 2002; 30: 285-290Crossref PubMed Google Scholar and preliminary results from our laboratory suggest that the isoform determines localization and may define the mode of IGF-1 action. In muscle and heart regeneration studies using transgenic models, the outcome of regeneration depended on the isoform used.30Santini MP Tsao L Monassier L Theodoropoulos C Carter J Lara-Pezzi E Slonimsky E Salimova E Delafontaine P Song YH Bergmann M Freund C Suzuki K Rosenthal N Enhancing repair of the mammalian heart.Circ Res. 2007; 100: 1732-1740Crossref PubMed Scopus (94) Google Scholar, 31Shavlakadze T Winn N Rosenthal N Grounds MD Reconciling data from transgenic mice that overexpress IGF-I specifically in skeletal muscle.Growth Horm IGF Res. 2005; 15: 4-18Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar This stresses the importance of both IGF-1 isoform selection and the mode of delivery in preclinical regeneration studies and in therapeutic application of this growth factor. In the skin, IGF-1 is produced by cells of mesenchymal origin, such as fibroblasts of the dermis and dermal papilla, whereas its receptor is produced by both mesenchymal and epithelial cells.32Hodak E Gottlieb AB Anzilotti M Krueger JG The insulin-like growth factor 1 receptor is expressed by epithelial cells with proliferative potential in human epidermis and skin appendages: correlation of increased expression with epidermal hyperplasia.J Invest Dermatol. 1996; 106: 564-570Crossref PubMed Scopus (115) Google Scholar, 33Little JC Redwood KL Granger SP Jenkins G In vivo cytokine and receptor gene expression during the rat hair growth cycle. Analysis by semi-quantitative RT-PCR.Exp Dermatol. 1996; 5: 202-212Crossref PubMed Scopus (23) Google Scholar, 34Rudman SM Philpott MP Thomas GA Kealey T The role of IGF-I in human skin and its appendages: morphogen as well as mitogen?.J Invest Dermatol. 1997; 109: 770-777Crossref PubMed Scopus (152) Google Scholar, 35Tavakkol A Elder JT Griffiths CE Cooper KD Talwar H Fisher GJ Keane KM Foltin SK Voorhees JJ Expression of growth hormone receptor, insulin-like growth factor 1 (IGF-1) and IGF-1 receptor mRNA and proteins in human skin.J Invest Dermatol. 1992; 99: 343-349Abstract Full Text PDF PubMed Scopus (184) Google Scholar Thus, keratinocytes respond to the paracrine signal originating from the neighboring mesenchymal cells. We set out to test whether providing a localized autocrine signal to epithelial cells, including basal keratinocytes of interfollicular epidermis and outer root sheath (outer root sheath) keratinocytes of the hair follicle, can enhance wound repair and hair regeneration without causing deleterious effects, such as increased scarring, tumor formation, and systemic imbalance. To this end, we generated transgenic mice expressing a locally acting form of IGF-1 (mIGF-136Musaro A McCullagh K Paul A Houghton L Dobrowolny G Molinaro M Barton ER Sweeney HL Rosenthal N Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle.Nat Genet. 2001; 27: 195-200Crossref PubMed Scopus (890) Google Scholar) under the control of the keratin 14 promoter. These studies identify mIGF-1 as a potent stimulator of hair follicle morphogenesis and cycling, and of re-epithelialization of skin wounds. Importantly, these effects are not accompanied by spontaneous malignant tumor formation and occur in the background of normal epidermal homeostasis. The mIGF-1 rat cDNA was amplified from an MLC/mIgf-1 cassette36Musaro A McCullagh K Paul A Houghton L Dobrowolny G Molinaro M Barton ER Sweeney HL Rosenthal N Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle.Nat Genet. 2001; 27: 195-200Crossref PubMed Scopus (890) Google Scholar with primers containing BamHI sites: the forward primer was 5′-CGGGATCCCTGTTTCCTGTCTACAGTGTC-3′ and the reverse primer was 5′-CGGGATCCCTCGGGAGGCTCCTCCTAC-3′. The polymerase chain reaction (PCR) product was analyzed by sequencing and cloned into the BamHI site located downstream of the β-globin intron in the pG3Z.K14 cassette (kindly provided by Dr. Elaine Fuchs, Rockefeller University, New York, NY).37Vassar R Rosenberg M Ross S Tyner A Fuchs E Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice.Proc Natl Acad Sci USA. 1989; 86: 1563-1567Crossref PubMed Scopus (298) Google Scholar The pG3Z.K14 cassette includes a 2-kb AvaI fragment of the human K14 promoter followed by a 700-bp β-globin intron and 500 bp of the human K14 polyadenylation signal (poly A). The transgene fragment was excised using KpnI upstream of the K14 promoter and HindIII downstream of the K14 poly A. The fragment was gel-purified and microinjected into male pronuclei of FVB zygotes. Founders were crossed with wild-type FVB animals and positive progeny were maintained in a hemizygous state. Genotyping, using tail genomic DNA as a template, was performed by PCR using standard conditions and two sets of PCR primers. The first set is located within the human K14 poly A sequence: 5′-GTGTGGACACAGATCCCAC-3′ and 5′-GGAGACACCACATATGACC-3′. The second set is located within exons 3 and 4 of the rat IGF-1 sequence: 5′-TTCCTGTCTACAGTGTCTGTG-3′ and 5′-GAGCTGACTTTGTAGGCTTCA-3′. Animals were housed in a clean, temperature-controlled mouse facility on a 12-hour light/dark cycle, and standard diet was provided. All mouse procedures were approved by the European Molecular Biology Laboratory Monterotondo Ethical Committee (Monterotondo, Italy) and were in accordance with national and European regulations. Eight- to ten-week-old sex-matched male and female animals (unless otherwise specified) were used for the study. Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA). For Northern blot analysis, 15 μg of total RNA for each sample were blotted and hybridized using standard conditions. In situ hybridization was performed as previously described.38Neubuser A Koseki H Balling R Characterization and developmental expression of Pax9, a paired-box-containing gene related to Pax1.Dev Biol. 1995; 170: 701-716Crossref PubMed Scopus (240) Google Scholar A probe corresponding to the complete rat mIGF-1 cDNA described above was used for both Northern blotting and in situ hybridization. To determine circulating IGF-1 levels, the OCTEIA rat/mouse IGF-1 immunoenzymometric assay for the quantitative determination of IGF-1 in rat and mouse serum was used according to the manufacturer's instructions (IDS Limited, Frankfurt, Germany). For histological analysis, upper back skin was isolated, fixed overnight in 4% paraformaldehyde in phosphate-buffered saline (PBS), and embedded in paraffin. Dewaxed sections (7 μm) were stained using hematoxylin and eosin (H&E) and photographed using a Leica DC 500 camera (Leica Microsystems, Wetzlar, Germany). Mice were injected intraperitoneally with BrdU (250 mg/kg BrdU in 0.9% NaCl) and sacrificed 2 hours after injection. Back skin and wound sections were fixed overnight in acetic ethanol (1% acetic acid/95% EtOH) and incubated with a horseradish peroxidase-conjugated monoclonal antibody directed against BrdU (Roche, Basel, Switzerland). BrdU-positive cells were visualized using 3,3-diaminobenzidine substrate (Sigma, St. Louis, MO). Counterstaining was performed with H&E. Upper back skin sections fixed in 4% buffered formalin were stained with the anti-Ki-67 antibody (Novocastra, Newcastle upon Tyne, UK). All Ki-67+ cells and the total number of matrix keratinocytes below the Auber's line were counted in morphogenesis stage 8 on day 8 after birth and in anagen on day 28 after birth. Seven to thirteen individual hair follicles derived from three different mice per group were counted (mean ± SEM, *P < 0.05), analyzed by Mann-Whitney test for unpaired samples (GraphPad Prism; GraphPad Software Inc., San Diego, CA). All Ki-67+ cells of the outer root sheath in the visual field above the hair bulb were counted at a magnification of ×200 in morphogenesis stage 8 on day 8 after birth and in anagen on day 28 after birth. Twelve individual HFs derived from three different mice per group were counted (mean ± SEM, *P < 0.05 for day 8, *P < 0.0001 for day 28, analyzed by Mann-Whitney test for unpaired samples). Migration assay was performed using transwell migration chambers (6.5 mm diameter, 8-μm pore size) (Costar; Corning, Lowell, MA) in 24-well plates. In all of the experiments, cell adhesion was achieved by precoating the transwell with vitrogen 100 collagen (Cohesion Technologies, Palo Alto, CA) and fibronectin (Invitrogen). Primary keratinocytes were isolated from 3-day-old pups as described.39Montanez E Piwko-Czuchra A Bauer M Li S Yurchenco P Fassler R Analysis of integrin functions in peri-implantation embryos, hematopoietic system, and skin.Methods Enzymol. 2007; 426: 239-289Crossref PubMed Scopus (23) Google Scholar After 2 days in culture, keratinocytes were trypsinized and seeded at 1 × 104 cells in 200 μl of Dulbecco's modified Eagle's medium per upper chamber. Six hundred μl of Dulbecco's modified Eagle's medium or of Dulbecco's modified Eagle's medium supplemented with 1% chelated serum were added to the lower chamber. Cells were allowed to migrate for 4 hours after which nonmigrated cells were removed from the top of the filter. The filter was stained in 20% methanol/0.1% crystal violet, mounted, and the whole filter was photographed using a Leica DMRx microscope (with ×5 objective) and a Leica DFC290 digital camera. Keratinocytes from three to four wild-type and transgenic animals were pooled and each assay was performed in triplicates. The total number of migrated cells was counted for each filter. The data presented are a mean of triplicate experiments ± SD. Statistical analysis was performed using the unpaired t-test with GraphPad Prism software. The migration experiment was repeated with three different litters. Wounds were generated as described above. Wound samples, including clot and 2 mm of the surrounding skin were collected 24 hours after wounding. Seven to fourteen wounds from five wild-type and five transgenic animals were split into three groups per genotype and thus a total of six groups were subjected to Affymetrix array analysis (Affymetrix, Santa Clara, CA). Gene expression in wound skin samples was determined using Mouse Genome 430 2.0 arrays (Affymetrix). Each sample was hybridized to an individual microarray chip. Target preparation, hybridization, and scanning were conducted according to the Affymetrix GeneChip Expression technical manual by Gene Core facility (EMBL, Heidelberg, Germany). Data files were analyzed using GeneSpring 7.3 software (Silicon Genetics, Redwood City, CA). Briefly, Affymetrix data were imported into GeneSpring software and normalized by using the default normalization methods recommended by the software. Lists of differentially expressed transcripts were generated by using a one-way analysis of variance parametric test and a P value cutoff of 0.05. Additional stringent filtering criteria were applied to comparative analyzes, including filter on flags, volcano plot, presence of the signal in three of three replicates. Venn diagrams were generated to identify differentially regulated transcripts. Additionally, the gene list of cytokines, chemokines, and growth factors containing GenBank ID and the fold expression change was generated manually. Seven-μm sections from the middle of the acidic ethanol-fixed wounds or from back skin were deparaffinized, rehydrated, rinsed in PBS, and incubated overnight at 4°C with the primary antibodies diluted in PBS containing 1% bovine serum albumin and 0.01% Nonidet P-40. After three 10-minute washes with PBS/0.1% Tween 20, the sections were incubated for 1 hour at room temperature with the Cy2- or Cy3-coupled secondary antibodies (Jackson ImmunoResearch Laboratory Inc., West Grove, PA), washed, mounted with Mowiol (Hoechst, Frankfurt, Germany), and analyzed with a Zeiss Axioplan fluorescence microscope (Zeiss, Oberkochen, Germany). The following antibodies were used: a mouse monoclonal antibody directed against keratin 10 (1:100; DAKO, Glostrup, Denmark), rabbit polyclonal antibodies directed against keratin 14 (1:5000; Babco, Richmond, CA), keratin 6 (1:1000, Babco), and loricrin (1:250; Covance, Denver, PA). Back skin from wild-type and transgenic 47 dpp male animals was used for the analysis as described previously.40Blanpain C Lowry WE Geoghegan A Polak L Fuchs E Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche.Cell. 2004; 118: 635-648Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar Primary antibodies used for flow cytometric analysis were anti-α6 integrin (CD49f) directly coupled to phycoerythrin and anti-CD34 directly coupled to fluorescein isothiocyanate (BD Pharmingen, Franklin Lakes, NJ). Dead cells were stained with 7-AAD (Sigma) and excluded from the analysis. Analysis was performed in a three laser standard configuration FACS Aria (BD Biosciences, San Jose, Ca). Data analysis was performed using FACS Diva (BD Biosciences) and FlowJo software (Tree Star, Inc., Ashland, OR). Compensation was performed manually after acquisition of 110,000 events. Gating of the putative stem cell populations was performed as indicated in figure legend. Four full-thickness excisional wounds, 5 mm in diameter, were made on either side of the dorsal midline of 8- to 10-week-old mice by excising skin and panniculus carnosus. Wounds were left uncovered and harvested 5, 14, and 21 days after injury. Mice were housed individually during the healing period. For histological analysis the complete wounds including 2 mm of the epithelial margins were isolated, bisected, fixed overnight in 4% paraformaldehyde in PBS (for Masson trichrome analysis) or in acetic ethanol (for immunofluorescence and BrdU staining), and embedded in paraffin. Sections (7 μm) from the middle of the wound were stained using the Masson's trichrome procedure as described by the manufacturer (Sigma) and photographed using a Leica DC 500 camera (Leica Microsystems). Morphometric measurements of the wounds were performed on both BrdU- and Masson's trichrome-stained sections using the OpenLab software (Improvision Ltd., Basel, Switzerland). Wound-healing experiments (excisional and incisional wounds; see below) were performed with permission from the local veterinary authorities of Zurich, Switzerland. Statistical analysis was performed using the unpaired t-test (given the variances were normally distributed) included in the GraphPad Prism4 software package. Mice were anesthetized and the dorsal region was shaved and treated with a depilatory agent (Pilca Perfect; Stafford-Miller Continental, Oevel, Belgium). Four full-thickness incisions (1 cm) were made at two anterior and two posterior dorsal sites, and the skin margins were closed with strips of wound plaster (Fixomull stretch; Beiersdorf, Hamburg, Germany). Mice were sacrificed on day 5 after wounding, and bursting strength of the wounds was determined in situ using the BTC-2000 system (SRLI Technologies, Nashville, TN) according to the manufacturer's protocol for the nonhuman disruptive linear incision analysis. Hair was plucked from five wild-type and five transgenic 28 dpp male animals and at least 200 single hair shafts per animal were analyzed to determine the percentage of each hair type. Hair shafts were analyzed using a Leica MZ12 stereo microscope, photographed using a Leica DC 500 camera, and hair shaft length was measured using MetaMorph7 software (Molecular Devices Corp., Sunnyvale, CA). Skin samples of K14/mIGF-1 mice and their wild-type littermates were harvested from the back at 1, 8, 17, 28, and 49 days post partum (dpp). This allows to reliably assess differences in early morphogenesis (dpp 1), late morphogenesis (dpp 8), as well as the entry into hair follicle cycling (dpp 17, first catagen), and in subsequent hair cycling activity (dpp 28, first anagen; dpp 49, second telogen).41Muller-Rover S Handjiski B van der Veen C Eichmuller S Foitzik K McKay IA Stenn KS Paus R A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.J Invest Dermatol. 2001; 117: 3-15Crossref PubMed Google Scholar, 42Paus R Muller-Rover S Van Der Veen C Maurer M Eichmuller S Ling G Hofmann U Foitzik K Mecklenburg L Handjiski B A comprehensive guide for the recognition and classification of distinct stages of hair follicle morphogenesis.J Invest Dermatol. 1999; 113: 523-532Crossref PubMed Scopus (453) Google Scholar For cryosectioning, skin samples were embedded as described elsewhere42Paus R Muller-Rover S Van Der Veen C Maurer M Eichmuller S Ling G Hofmann U Foitzik K Mecklenburg L Handjiski B A comprehensive guide for the recognition and classification of distinct stages of hair follicle
Referência(s)