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• W1969080906 abstract "Matrix metalloproteinases (MMPs) have been implicated in wound healing. To analyze the roles of MMP-9 and MMP-13 in wound healing, we generated full-thickness cutaneous wounds in MMP-9 knockout (KO), MMP-13 KO, MMP-9/13 double KO, and wild-type mice. Macroscopic wound closure was delayed in all of the KO mice, as compared with wild-type mice. The rate of re-epithelialization was significantly delayed in MMP-9 KO and MMP-13 KO mice and remarkably delayed in MMP-9/13 double KO mice, as compared with wild-type mice. Both MMP-9 and MMP-13 were expressed by the leading edges of epidermal cells in wild-type mice, and the migration of keratinocytes was suppressed by treatment with an MMP inhibitor or transfection of small interfering RNAs for MMP-9 or MMP-13, as compared with controls. The vascular density in wound granulation was significantly lower in both MMP-13 KO and MMP-9/13 double KO mice than in wild-type mice. Degradation of connective tissue growth factor in wound tissue was transiently prevented in MMP-13 KO mice. Morphometric analyses demonstrated a reduction in both wound contraction and myofibroblast formation in both MMP-13 KO and MMP-9/13 double KO mice. Proliferation and transforming growth factor-β1-induced myofibroblast differentiation of dermal fibroblasts from MMP-13 KO mice were decreased, as compared with wild-type dermal fibroblasts. These data suggest that MMP-13 plays a role in keratinocyte migration, angiogenesis, and contraction in wound healing, while MMP-9 functions in keratinocyte migration. Matrix metalloproteinases (MMPs) have been implicated in wound healing. To analyze the roles of MMP-9 and MMP-13 in wound healing, we generated full-thickness cutaneous wounds in MMP-9 knockout (KO), MMP-13 KO, MMP-9/13 double KO, and wild-type mice. Macroscopic wound closure was delayed in all of the KO mice, as compared with wild-type mice. The rate of re-epithelialization was significantly delayed in MMP-9 KO and MMP-13 KO mice and remarkably delayed in MMP-9/13 double KO mice, as compared with wild-type mice. Both MMP-9 and MMP-13 were expressed by the leading edges of epidermal cells in wild-type mice, and the migration of keratinocytes was suppressed by treatment with an MMP inhibitor or transfection of small interfering RNAs for MMP-9 or MMP-13, as compared with controls. The vascular density in wound granulation was significantly lower in both MMP-13 KO and MMP-9/13 double KO mice than in wild-type mice. Degradation of connective tissue growth factor in wound tissue was transiently prevented in MMP-13 KO mice. Morphometric analyses demonstrated a reduction in both wound contraction and myofibroblast formation in both MMP-13 KO and MMP-9/13 double KO mice. Proliferation and transforming growth factor-β1-induced myofibroblast differentiation of dermal fibroblasts from MMP-13 KO mice were decreased, as compared with wild-type dermal fibroblasts. These data suggest that MMP-13 plays a role in keratinocyte migration, angiogenesis, and contraction in wound healing, while MMP-9 functions in keratinocyte migration. Wound healing is a complex process that includes an acute inflammatory reaction, regeneration of parenchyma cells, cell migration and proliferation, angiogenesis, extracellular matrix (ECM) synthesis, contraction, and tissue remodeling.1Singer AJ Clark RA Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4611) Google Scholar, 2Cotran RS Kumar V Collins T Wound healing.in: Robbins Pathologic Basis of Disease. Sixth edition. W.B. Saunders Company, Philadelphia1999: 107-111Google Scholar, 3Li J Chen J Kirsner R Pathophysiology of acute wound healing.Clin Dermatol. 2007; 25: 9-18Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar Cutaneous wound healing by second intention is characterized by the following three continuous and overlapping processes: an inflammatory phase, a proliferative phase, and a contraction and remodeling phase.2Cotran RS Kumar V Collins T Wound healing.in: Robbins Pathologic Basis of Disease. Sixth edition. W.B. Saunders Company, Philadelphia1999: 107-111Google Scholar In the inflammatory phase, tissue injury causes the loss of cells and tissue, disruption of blood vessels, extravasation of blood constituents and infiltration of inflammatory cells, composed mainly of neutrophils and macrophages, and provides a provisional ECM for keratinocyte migration. The major events during the proliferative phase are re-epithelialization and angiogenesis, both of which require cell proliferation and migration of keratinocytes and endothelial cells, respectively. In the contraction and remodeling phase, myofibroblasts differentiated from fibroblasts play a key role in wound contraction and controlled synthesis and degradation of ECM proteins, especially collagens, leading to increased wound strength. All of these events occurring during wound healing require the collaborative efforts of many different tissues and cell types. Accumulated lines of evidence have demonstrated that members of the matrix metalloproteinase (MMP) gene family are essential to the degradation of ECM macromolecules and non-ECM molecules such as growth factors and cytokines under various pathophysiological conditions.4McCawley LJ Matrisian LM Matrix metalloproteinases: they're not just for matrix anymore!.Curr Opin Cell Biol. 2001; 13: 534-540Crossref PubMed Scopus (1092) Google Scholar, 5Overall CM McQuibban GA Clark-Lewis I Discovery of chemokine substrates for matrix metalloproteinases by exosite scanning: a new tool for degradomics.Biol Chem. 2002; 383: 1059-1066Crossref PubMed Scopus (133) Google Scholar Enhanced expression of MMPs 1, 2, 3, 8, 9, 10, 13, 14, 19, and 26 in wound tissues has been reported in experimental animals and humans.6Okada A Tomasetto C Lutz Y Bellocq JP Rio MC Basset P Expression of matrix metalloproteinases during rat skin wound healing: evidence that membrane type-1 matrix metalloproteinase is a stromal activator of pro-gelatinase A.J Cell Biol. 1997; 137: 67-77Crossref PubMed Scopus (192) Google Scholar, 7Nwomeh BC Liang HX Diegelmann RF Cohen IK Yager DR Dynamics of the matrix metalloproteinases MMP-1 and MMP-8 in acute open human dermal wounds.Wound Repair Regen. 1998; 6: 127-134Crossref PubMed Scopus (113) Google Scholar, 8Madlener M Parks WC Werner S Matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) are differentially expressed during excisional skin wound repair.Exp Cell Res. 1998; 242: 201-210Crossref PubMed Scopus (284) Google Scholar, 9Ravanti L Toriseva M Penttinen R Crombleholme T Foschi M Han J Kahari VM Expression of human collagenase-3 (MMP-13) by fetal skin fibroblasts is induced by transforming growth factor beta via p38 mitogen-activated protein kinase.FASEB J. 2001; 15: 1098-1100Crossref PubMed Scopus (67) Google Scholar, 10Hieta N Impola U Lopez-Otin C Saarialho-Kere U Kahari VM Matrix metalloproteinase-19 expression in dermal wounds and by fibroblasts in culture.J Invest Dermatol. 2003; 121: 997-1004Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 11Beare AH O'Kane S Krane SM Ferguson MW Severely impaired wound healing in the collagenase-resistant mouse.J Invest Dermatol. 2003; 120: 153-163Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 12Ahokas K Skoog T Suomela S Jeskanen L Impola U Isaka K Saarialho-Kere U Matrilysin-2 (matrix metalloproteinase-26) is upregulated in keratinocytes during wound repair and early skin carcinogenesis.J Invest Dermatol. 2005; 124: 849-856Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 13Hartenstein B Dittrich BT Stickens D Heyer B Vu TH Teurich S Schorpp-Kistner M Werb Z Angel P Epidermal development and wound healing in matrix metalloproteinase 13-deficient mice.J Invest Dermatol. 2006; 126: 486-496Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar It is clear that MMP activity is required in wound closure by keratinocyte re-epithelialization and migration and angiogenesis, since broad-spectrum MMP inhibitors inhibit the processes.14Makela M Larjava H Pirila E Maisi P Salo T Sorsa T Uitto VJ Matrix metalloproteinase 2 (gelatinase A) is related to migration of keratinocytes.Exp Cell Res. 1999; 251: 67-78Crossref PubMed Scopus (148) Google Scholar, 15Lund LR Romer J Bugge TH Nielsen BS Frandsen TL Degen JL Stephens RW Dano K Functional overlap between two classes of matrix-degrading proteases in wound healing.EMBO J. 1999; 18: 4645-4656Crossref PubMed Scopus (226) Google Scholar, 16Burbridge MF Coge F Galizzi JP Boutin JA West DC Tucker GC The role of the matrix metalloproteinases during in vitro vessel formation.Angiogenesis. 2002; 5: 215-226Crossref PubMed Scopus (83) Google Scholar, 17Mirastschijski U Impola U Karsdal MA Saarialho-Kere U Agren MS Matrix metalloproteinase inhibitor BB-3103 unlike the serine proteinase inhibitor aprotinin abrogates epidermal healing of human skin wounds ex vivo.J Invest Dermatol. 2002; 118: 55-64Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 18Mirastschijski U Haaksma CJ Tomasek JJ Agren MS Matrix metalloproteinase inhibitor GM 6001 attenuates keratinocyte migration, contraction and myofibroblast formation in skin wounds.Exp Cell Res. 2004; 299: 465-475Crossref PubMed Scopus (117) Google Scholar However, since many MMPs are expressed by re-epithelializing keratinocytes, inflammatory cells, fibroblasts, and endothelial cells, it is uncertain which MMP species plays a central role in the process of wound healing and how these MMPs function in wounded tissues. One of the most powerful methods to directly address these questions is to analyze wound healing in MMP knockout (KO) mice. Indeed, wound healing of the skin has been studied in mice deficient for the MMP-3, MMP-8, MMP-9, MMP-13, or MMP-14 genes.13Hartenstein B Dittrich BT Stickens D Heyer B Vu TH Teurich S Schorpp-Kistner M Werb Z Angel P Epidermal development and wound healing in matrix metalloproteinase 13-deficient mice.J Invest Dermatol. 2006; 126: 486-496Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 19Bullard KM Lund L Mudgett JS Mellin TN Hunt TK Murphy B Ronan J Werb Z Banda MJ Impaired wound contraction in stromelysin-1-deficient mice.Ann Surg. 1999; 230: 260-265Crossref PubMed Scopus (188) Google Scholar, 20Gutierrez-Fernandez A Inada M Balbin M Fueyo A Pitiot AS Astudillo A Hirose K Hirata M Shapiro SD Noel A Werb Z Krane SM Lopez-Otin C Puente XS Increased inflammation delays wound healing in mice deficient in collagenase-2 (MMP-8).FASEB J. 2007; 21: 2580-2591Crossref PubMed Scopus (217) Google Scholar, 21Mohan R Chintala SK Jung JC Villar WV McCabe F Russo LA Lee Y McCarthy BE Wollenberg KR Jester JV Wang M Welgus HG Shipley JM Senior RM Fini ME Matrix metalloproteinase gelatinase B (MMP-9) coordinates and effects epithelial regeneration.J Biol Chem. 2002; 277: 2065-2072Crossref PubMed Scopus (229) Google Scholar, 22Mirastschijski U Zhou Z Rollman O Tryggvason K Agren MS Wound healing in membrane-type-1 matrix metalloproteinase-deficient mice.J Invest Dermatol. 2004; 123: 600-602Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar However, all of these KO mice, except for the MMP-8 KO mice, had negative results. In the MMP-8 KO mice, wound closure and re-epithelialization were inhibited mainly due to impaired infiltration of neutrophils,20Gutierrez-Fernandez A Inada M Balbin M Fueyo A Pitiot AS Astudillo A Hirose K Hirata M Shapiro SD Noel A Werb Z Krane SM Lopez-Otin C Puente XS Increased inflammation delays wound healing in mice deficient in collagenase-2 (MMP-8).FASEB J. 2007; 21: 2580-2591Crossref PubMed Scopus (217) Google Scholar which are responsible for MMP-8 production. The study suggested that the effect of MMP-8 on the wound healing is secondary to persistent inflammation at the later time point and alterations in transforming growth factor-β (TGF-β) signaling.20Gutierrez-Fernandez A Inada M Balbin M Fueyo A Pitiot AS Astudillo A Hirose K Hirata M Shapiro SD Noel A Werb Z Krane SM Lopez-Otin C Puente XS Increased inflammation delays wound healing in mice deficient in collagenase-2 (MMP-8).FASEB J. 2007; 21: 2580-2591Crossref PubMed Scopus (217) Google Scholar Keratinocytes at the leading edge of the cutaneous wound express MMP-9 and MMP-13.13Hartenstein B Dittrich BT Stickens D Heyer B Vu TH Teurich S Schorpp-Kistner M Werb Z Angel P Epidermal development and wound healing in matrix metalloproteinase 13-deficient mice.J Invest Dermatol. 2006; 126: 486-496Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 21Mohan R Chintala SK Jung JC Villar WV McCabe F Russo LA Lee Y McCarthy BE Wollenberg KR Jester JV Wang M Welgus HG Shipley JM Senior RM Fini ME Matrix metalloproteinase gelatinase B (MMP-9) coordinates and effects epithelial regeneration.J Biol Chem. 2002; 277: 2065-2072Crossref PubMed Scopus (229) Google Scholar Induction of MMP-9 at the wound site in vitro23McCawley LJ O'Brien P Hudson LG Epidermal growth factor (EGF)- and scatter factor/hepatocyte growth factor (SF/HGF)- mediated keratinocyte migration is coincident with induction of matrix metalloproteinase (MMP)-9.J Cell Physiol. 1998; 176: 255-265Crossref PubMed Scopus (181) Google Scholar and in vivo24Scott KA Arnott CH Robinson SC Moore RJ Thompson RG Marshall JF Balkwill FR TNF-alpha regulates epithelial expression of MMP-9 and integrin alphavbeta6 during tumour promotion. A role for TNF-alpha in keratinocyte migration?.Oncogene. 2004; 23: 6954-6966Crossref PubMed Scopus (83) Google Scholar promotes migration of keratinocytes probably because of its effect on detachment of basal keratinocytes from the basal membrane. Keratinocyte migration over the wound bed is known to be dependent on the attachment of keratinocytes to fibrillar type I collagen through integrins α1β1 and α2β1 and subsequent degradation of the collagen by collagenolytic MMPs.25Parks WC Matrix metalloproteinases in repair.Wound Repair Regen. 1999; 7: 423-432Crossref PubMed Scopus (330) Google Scholar, 26Beare AH Krane SM Ferguson MW Variable impairment of wound healing in the heterozygous collagenase-resistant mouse.Wound Repair Regen. 2005; 13: 27-40Crossref PubMed Scopus (6) Google Scholar These studies suggest that both MMP-9 and MMP-13 are involved in re-epithelialization in wound healing in mice, a species that lacks the gene for MMP-1.27Balbin M Fueyo A Knauper V Lopez JM Alvarez J Sanchez LM Quesada V Bordallo J Murphy G Lopez-Otin C Identification and enzymatic characterization of two diverging murine counterparts of human interstitial collagenase (MMP-1) expressed at sites of embryo implantation.J Biol Chem. 2001; 276: 10253-10262Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar However, previous studies on wound healing in MMP-9 KO and MMP-13 KO mice failed to demonstrate significant difference or showed even acceleration of wound closure and re-epithelialization, as compared with that in wild-type mice.13Hartenstein B Dittrich BT Stickens D Heyer B Vu TH Teurich S Schorpp-Kistner M Werb Z Angel P Epidermal development and wound healing in matrix metalloproteinase 13-deficient mice.J Invest Dermatol. 2006; 126: 486-496Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 21Mohan R Chintala SK Jung JC Villar WV McCabe F Russo LA Lee Y McCarthy BE Wollenberg KR Jester JV Wang M Welgus HG Shipley JM Senior RM Fini ME Matrix metalloproteinase gelatinase B (MMP-9) coordinates and effects epithelial regeneration.J Biol Chem. 2002; 277: 2065-2072Crossref PubMed Scopus (229) Google Scholar These studies did not provide the reasonable explanations for the unexpected findings except for redundancy among MMPs.13Hartenstein B Dittrich BT Stickens D Heyer B Vu TH Teurich S Schorpp-Kistner M Werb Z Angel P Epidermal development and wound healing in matrix metalloproteinase 13-deficient mice.J Invest Dermatol. 2006; 126: 486-496Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar MMP-9 and MMP-13 synergize with each other during endochondral ossification at the growth plates.28Stickens D Behonick DJ Ortega N Heyer B Hartenstein B Yu Y Fosang AJ Schorpp-Kistner M Angel P Werb Z Altered endochondral bone development in matrix metalloproteinase 13-deficient mice.Development. 2004; 131: 5883-5895Crossref PubMed Scopus (486) Google Scholar Thus, MMP-9/13 double KO mice could identify additive effects of MMP-9 and MMP-13 on wound healing, although no such studies have been reported. In the present study, we developed the MMP-9/13 double KO mice by crossing the MMP-9 KO mice with the MMP-13 KO mice, and evaluated the influence of targeted deletion of the MMP-9 and/or MMP-13 genes on healing by secondary intention by generating large skin wounds in MMP-9 KO, MMP-13 KO, MMP-9/13 double KO, and wild-type mice. Our study provides the first evidence that MMP-13 plays a key role in keratinocyte migration, angiogenesis, and contraction in wound healing, and that MMP-9 is implicated in keratinocyte migration. MMP-9 KO mice originated on the 129SvEv/CD-1 mixed background were purchased from the Jackson Laboratory29Vu TH Shipley JM Bergers G Berger JE Helms JA Hanahan D Shapiro SD Senior RM Werb Z MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes.Cell. 1998; 93: 411-422Abstract Full Text Full Text PDF PubMed Scopus (1490) Google Scholar and MMP-13 KO mice on a 129/Sv genetic background were generated by microinjection of embryonic stem cells into C57BL/6J blastocytes as described previously.30Takaishi H Kimura T Dalal S Okada Y D'Armiento J Joint diseases and matrix metalloproteinases: a role for MMP-13.Curr Pharm Biotechnol. 2008; 9: 47-54Crossref PubMed Scopus (241) Google Scholar These mice were backcrossed six times into the C57BL/6J background. MMP-9 KO and MMP-13 KO mice were obtained by breeding heterozygous mice. MMP-9/13 double KO mice were generated by interbreeding heterozygous MMP-9+/−/13+/− mice, which were produced by breeding homozygous MMP-9 KO mice with homozygous MMP-13 KO mice. Genotyping of animals was performed by PCR of DNA obtained from tail biopsies. Primers for wild-type alleles were located in exon 3 (5′-AGGCCTTCAGAAAAGCCTTC-3′) and exon 4 (5′-GCAGTTCCAAAG-3′) of the MMP-13 gene and primers for mutated alleles were located in d2EGFP (5′-GCAAGCTGACCCTGAAGTTCATCTG-3′ and 5′-CAGAAGAACGGCATCAAGGTGA-3′). Primers for wild-type alleles of the MMP-9 gene (5′-GTGGGACCATCATAACATCACA-3′ and 5′-CTCGCGGCAAGTCTTCAGAGTA-3′) and mutated alleles (5′-CTGAATGAACTGCAGGACGA-3′ and 5′-ATACTTTCTCGGCAGGAGCA-3′) were created according to the genotyping protocols from the Jackson Laboratory (Bar Harbor, Maine). MMP-9 KO, MMP-13 KO, and MMP-9/13 double KO mice exhibited a normal lifespan with sufficient fertility and did not show gross phenotype after maturation, although they had growth retardation because of defects in the growth plate during development (unpublished data for MMP-9/13 double KO mice).29Vu TH Shipley JM Bergers G Berger JE Helms JA Hanahan D Shapiro SD Senior RM Werb Z MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes.Cell. 1998; 93: 411-422Abstract Full Text Full Text PDF PubMed Scopus (1490) Google Scholar, 30Takaishi H Kimura T Dalal S Okada Y D'Armiento J Joint diseases and matrix metalloproteinases: a role for MMP-13.Curr Pharm Biotechnol. 2008; 9: 47-54Crossref PubMed Scopus (241) Google Scholar Matched control littermates of MMP-13 KO mice were used as wild-type mice for experiments. Full-thickness wounds were made with a sterile biopsy punch with a diameter of 8 mm (Keisei Medical Industrial Co., LTD) on the dorsal region of wild-type, MMP-9 KO, MMP-13 KO, and MMP-9/13 double KO mice, 9 to 11 weeks old. The wounds were left open and the animals were housed in individual cages. Wound healing was macroscopically monitored by taking digital photographs at the indicated time points. The wound areas (percentage of wound areas to initial ones) were calculated from the photographs using PhotoShop (Adobe Photoshop Element 2.0, Adobe Systems) (n = 6 per group). Mice were sacrificed by intraperitoneal injection of overdosed Nembutal, and skin with the wound was removed for further experiments. All procedures were performed according to the guidelines for the Care and Use of Laboratory Animals of Keio University School of Medicine. Excised skin specimens were fixed with Tris-buffered zinc fixative,31Beckstead JH A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues.J Histochem Cytochem. 1994; 42: 1127-1134Crossref PubMed Scopus (226) Google Scholar and paraffin sections (4 μm) were stained with H&E and Elastica-van-Gieson. For immunohistochemistry, sections were reacted with anti-CD68 antibody (1:100 dilution; FA-11; Serotec), anti-neutrophil antibody (1:100 dilution; MCA771GA; Serotec), anti-CD3 antibody (1:100 dilution; MCA1477; Serotec), anti-CD31 antibody (1:50 dilution; MEC13.3; BD Biosciences), anti-proliferating cell nuclear antigen (PCNA) antibody (1:200 dilution; PC10; DakoCytomation), anti-α-smooth muscle actin (SMA) antibody (1:200 dilution; 1A4; DakoCytomation), anti-MMP-9 antibody (1:1000 dilution; G-Tonline), or anti-MMP-13 antibody (1:100 dilution; D-17; Santa Cruz Biotechnology) after blocking endogenous peroxidase and nonspecific binding according to our methods.32Matsumura S Iwanaga S Mochizuki S Okamoto H Ogawa S Okada Y Targeted deletion or pharmacological inhibition of MMP-2 prevents cardiac rupture after myocardial infarction in mice.J Clin Invest. 2005; 115: 599-609Crossref PubMed Scopus (272) Google Scholar Then, they were incubated with peroxidase-conjugated secondary antibodies (1:200 dilution; Vector).32Matsumura S Iwanaga S Mochizuki S Okamoto H Ogawa S Okada Y Targeted deletion or pharmacological inhibition of MMP-2 prevents cardiac rupture after myocardial infarction in mice.J Clin Invest. 2005; 115: 599-609Crossref PubMed Scopus (272) Google Scholar As a control, sections were treated with non-immune IgG by replacing the first antibodies. Width of the wound and distance of the traversed epithelium were measured on H&E-stained sections, and percent re-epithelialization was calculated according to the following formula: [distance covered by epithelium]/[distance between wound bed] × 100 (n = 6 per group). Infiltration of neutrophils, macrophages, and T-lymphocytes was evaluated by counting the cells immunostained with anti-neutrophil antibody, anti-CD68 antibody, and anti-CD3 antibody, respectively (n = 3 per group).32Matsumura S Iwanaga S Mochizuki S Okamoto H Ogawa S Okada Y Targeted deletion or pharmacological inhibition of MMP-2 prevents cardiac rupture after myocardial infarction in mice.J Clin Invest. 2005; 115: 599-609Crossref PubMed Scopus (272) Google Scholar To analyze keratinocyte proliferative activity in migration tongue (mitosis zone), PCNA-positive cell index (percentage of positive cells to whole keratinocytes) was calculated by counting the immunoreactive cells and total keratinocytes in the mitosis zone (n = 3 per group). The vascular density in the granulation tissue of six random fields was determined by counting numbers of CD31-positive vessels per square millimeter (n = 5 for the samples on days 0, 3, 5, 7 and 10 and n = 3 for those on days 14 and 28).33Komiya K Enomoto H Inoki I Okazaki S Fujita Y Ikeda E Ohuchi E Matsumoto H Toyama Y Okada Y Expression of ADAM15 in rheumatoid synovium: up-regulation by vascular endothelial growth factor and possible implications for angiogenesis.Arthritis Res Ther. 2005; 7: R1158-R1173Crossref PubMed Scopus (39) Google Scholar Total RNA was extracted from skin wounds in wild-type, MMP-9 KO, MMP-13 KO, and MMP-9/13 double KO mice, and cDNA was prepared from 1 μg of total RNA with SuperScript II reverse transcriptase (Life Technologies Inc.) according to our methods.34Mitsui Y Mochizuki S Kodama T Shimoda M Ohtsuka T Shiomi T Chijiiwa M Ikeda T Kitajima M Okada Y ADAM28 is overexpressed in human breast carcinomas: implications for carcinoma cell proliferation through cleavage of insulin-like growth factor binding protein-3.Cancer Res. 2006; 66: 9913-9920Crossref PubMed Scopus (109) Google Scholar Reaction products were subjected to RT-PCR analysis at 28, 30, and 25 cycles for the expression of MMP-9, MMP-13, and β-actin, respectively. Sequences of the primers were as follows: for MMP-9 5′-GCGCCACCACAGCCAACTATG-3′ (forward), 5′-TGGATGCCGTCTATGTCGTCTTTA-3′ (reverse); for MMP-13 5′-GGTCCCAAACGAACTTAACTTACA-3′ (forward), 5′-CCTTGAACGTCATCAGGAAGC-3′ (reverse); for β-actin 5′-TTCTACAATGAGCTGCGTGTGGC-3′ (forward), 5′-CTCATAGCTCTTCTCCAGGGAGGA-3′ (reverse). The nucleotide sequence of the amplified fragments was confirmed by cycle sequencing using a DYEnamic ET dye terminator cycle sequencing kit (MegaBACE; Amersham Pharmacia Biotech) and MegaBACE 1000 DNA sequencer (Amersham Pharmacia Biotech).34Mitsui Y Mochizuki S Kodama T Shimoda M Ohtsuka T Shiomi T Chijiiwa M Ikeda T Kitajima M Okada Y ADAM28 is overexpressed in human breast carcinomas: implications for carcinoma cell proliferation through cleavage of insulin-like growth factor binding protein-3.Cancer Res. 2006; 66: 9913-9920Crossref PubMed Scopus (109) Google Scholar Mouse keratinocytes (PAM212 cells; a gift from Dr. Takashi Hashimoto, in Department of Dermatology, Kurume University)35Chaturvedi V Sitailo LA Qin JZ Bodner B Denning MF Curry J Zhang W Brash D Nickoloff BJ Knockdown of p53 levels in human keratinocytes accelerates Mcl-1 and Bcl-x(L) reduction thereby enhancing UV-light induced apoptosis.Oncogene. 2005; 24: 5299-5312Crossref PubMed Scopus (41) Google Scholar were grown to confluence on type I collagen-coated 6-well plates (BD Biosciences) in Spinner’s modified minimum essential medium (Gibco) containing 10% fetal bovine serum, 1% antibiotic solution, 2 mmol/L l-glutamine and 100 μmol/L CaCl2 and then scratch-wounded with a blue pipette tip according to the previous methods.36Daniel RJ Groves RW Increased migration of murine keratinocytes under hypoxia is mediated by induction of urokinase plasminogen activator.J Invest Dermatol. 2002; 119: 1304-1309Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar The keratinocytes were allowed to heal the wounds in the presence and absence of BB94 (British Biotech) in the medium containing 100 μmol/L hydroxyurea (Sigma-Aldrich), an inhibitor of cell proliferation. The marked areas of the wound were photographed, and cell migration areas were determined using the public domain Scion Image program. A pool of small interfering (si)RNAs for MMP-9 or MMP-13 and control non-silencing oligonucleotide (AATTCTCGGAACGTGTCAXGT) were purchased from Dharmacon SMART Pool and QIAGEN, Inc, respectively. Transfection of these siRNAs to keratinocytes was performed using the Human Keratinocyte Nucleofetor Kit (Amaxa Inc.). As a positive control, green fluorescent protein vector siRNA was transfected in a similar way. Specific silencing of the MMP-9 and MMP-13 genes was examined by RT-PCR and immunoblotting analyses. Wound tissues were obtained from wild-type, MMP-9 KO, MMP-13 KO, and MMP-9/13 double KO mice. Proteins were separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting using anti-connective tissue growth factor (CTGF) antibody (1:1000 dilution)37Hashimoto G Inoki I Fujii Y Aoki T Ikeda E Okada Y Matrix metalloproteinases cleave connective tissue growth factor and reactivate angiogenic activity of vascular endothelial growth factor 165.J Biol Chem. 2002; 277: 36288-36295Crossref PubMed Scopus (302) Google Scholar or anti-β-actin antibody (1:1000 dilution; AC-74; Sigma-Aldrich). Degradation of CTGF in the wound tissues was monitored by densitometrical analysis of the bands of intact CTGF and its degradation fragments using Scion Image. The expression of vascular endothelial growth factor (VEGF) was also monitored by RT-PCR and immunoblotting according to the previous methods.38Claffey KP Wilkison WO Spiegelman BM Vascular endothelial growth factor. Regulation by cell differentiation and activated second messenger pathways.J Biol Chem. 1992; 267: 16317-16322Abstract Full Text PDF PubMed Google Scholar, 39Vandervelde S van Luyn MJ Rozenbaum MH Petersen AH Tio RA Harmsen MC Stem cell-related cardiac gene expression early after murine myocardial infarction.Cardiovasc Res. 2007; 73: 783-793Crossref PubMed Scopus (62) Google Scholar When the wounds of each group were completely re-epithelialized, the height of dermis (HD) in the center of the wounds and the width of middle dermis (WD) in the wounds were determined on Elastica-van-Gieson-stained sections according to the previous methods40Akasaka Y Ono I Yamashita T Jimbow K Ishii T Basic fibroblast growth factor promotes apoptosis and suppresses granulation tissue formation in acute incisional wounds.J Pathol. 2004; 203: 710-720Crossref PubMed Scopus (76) Google Scholar (n = 3 per group). Myofibroblasts were identified by immunostaining of α-SMA in the granulation tissue, and percentage of the area with myofibroblasts to total granulation area was determined by planimetric image analysis using PhotoShop (Adobe Photoshop Element 2.0, Adobe Systems) according to the following formula: [α-SMA-positive cell area]/[dermal area] × 100 (n = 3 per group). Fibroblasts were cultured from dermal explants of adult wild-type, MMP-9 KO, MMP-13 KO, or MMP-9/13 double KO mice in Dulbecco’s Modified Eagle’s medium containing 10% fetal bovine serum. Mitogenic activity of fibroblasts was measured by 5-bromo-2′-deoxy-uridine (BrdU) Labeling and Detection Kit III (Roche Molecular Biochemicals).34Mitsui Y Mochizuki S Kodama T Shimoda M Ohtsuka T Shiomi T Chijiiwa M Ikeda T Kitajima M Okada Y ADAM28 is overexpressed i" @default.
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• W1969080906 title "MMP-13 Plays a Role in Keratinocyte Migration, Angiogenesis, and Contraction in Mouse Skin Wound Healing" @default.
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