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Effect of transforming growth factor-β1 antisense oligonucleotides on matrix metalloproteinases and their inhibitors in keloid fibroblasts
Otolaryngology–Head and Neck Surgery (2010) 143, 66-71
ORIGINAL RESEARCH–FACIAL PLASTIC AND RECONSTRUCTIVE SURGERY
Effect of transforming growth factor-1 antisense oligonucleotides on matrix metalloproteinases and their inhibitors in keloid fibroblasts Gregor M. Bran, MD, Ulrich R. Goessler, MD, Ariton Baftiri, Karl Hormann, MD, Frank Riedel, MD, and Haneen Sadick, MD, Mannheim, Germany Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article. ABSTRACT OBJECTIVE: To identify changes in the expression of matrixmetalloproteinases (MMPs) and their specific inhibitors tissue inhibitors of metalloproteinases (TIMPs) after targeting of transforming growth factor-1 (TGF-1) with antisense oligonucleotides. STUDY DESIGN: Cross-sectional study. SETTING: The study was performed on tissue samples from nine patients with keloid scars after otoplasty presenting to the Otolaryngology–Head and Neck Surgery Department of the University Hospital in Mannheim, Germany. SUBJECTS AND METHODS: Keloid fibroblasts and normal fibroblasts were harvested from auricular keloid scars and healthy skin regions of the same patients during resection procedure of the keloid. Cells were placed in monolayer cultures. Expression of MMPs and TIMPs were analyzed by immunohistochemistry. The effect of TGF-1 targeting using antisense oligonucleotides on the expression of both protein groups in keloid-derived fibroblasts was analyzed by enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction. RESULTS: Immunohistochemical investigation demonstrated increased expression of MMP-2, -3, -9, and -13 and TIMP-1 and -2. TGF-1 antisense therapy significantly down-regulated MMP secretion in vitro. CONCLUSION: Usage of TGF-1 antisense oligodeoxynucleotides (ODNs) may show a potential chemopreventive or therapeutic option for keloids by blocking the effect of TGF-1. Furthermore, antisense ODNs can be used as an investigative approach toward a better understanding of molecular mechanisms in keloid pathophysiology. © 2010 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved.
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carring of the skin presents a ubiquitous medical problem. The possibilities of resulting functional deficit, restriction of tissue movement, and potential risk of uncontrolled growth and adverse psychologic effects illustrate the prevailing challenge for every facial plastic surgeon. In case of a disturbance of the delicate balance of reparative processes, wound healing can be impaired drastically resulting in two pathologic extremes: chronic wounds (e.g., ulcers) or excessive scar formation (e.g., hypertrophic scars or keloids).1 A common feature of the beginning wound healing cascade is the increased activity within the extracellular matrix (ECM). Fibroblasts, and to a lesser extent keratinocytes, are the two main cell types responsible for synthesis and deposition of different collagens, elastin, glycoproteins, proteoglycans, hyaluronic acid, and growth factors into the wound ECM.2 The wound ECM forms a structural repair frame, which appears to be a key regulator of cell adhesion, migration, proliferation, and differentiation during cutaneous repair. The amount and organization of normal wound ECM are determined by a dynamic equilibrium among matrix synthesis, deposition, and degradation. The degradation of the ECM is a prerequisite for wound healing. The two main protein families that take part in this process are matrixmetalloproteinases (MMPs) and the plasminogen activation system.3 Human MMPs are a family of approximately 24 structurally related, zinc-dependent endopeptidases, which most commonly are secreted extracellularly but were also observed as membrane-associated (membrane-type MMP) or intracellularly acting enzymes.2 The activity of MMPs is inhibited by nonspecific inhibitors, e.g., ␣1-antiprotease or ␣2-macroglobulin, or more specifically by the tissue inhibitors of metalloproteinases (TIMPs). Both groups of endopeptidases are released by several different cell types found in human skin. The inhibition is regulated by a 1:1
Received December 25, 2009; revised March 3, 2010; accepted March 23, 2010.
0194-5998/$36.00 © 2010 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved. doi:10.1016/j.otohns.2010.03.029
Bran et al
Effect of transforming growth factor-1 antisense . . .
stoichiometric binding of TIMPs to the active MMPs, which thereby lose their activity.4 MMPs and TIMPs are not constitutively expressed in the skin but are induced temporarily in response to exogenous signals (e.g., cytokines, growth factors, cell-matrix interactions, and altered cell-cell interactions). It is believed that pathologic conditions represent an aberrant functioning of these regulatory mechanisms resulting in local wound milieus that favor chronic tissue turnover.4 Excessive scar tissue formation can be observed in hypertrophic scars and keloids. Hypertrophic scars follow a certain pattern of evolution, stabilization, and involution, whereas keloids do not follow this characteristic development. Keloid scars represent a dysregulated response to cutaneous wounding resulting in continuous proliferation with indefinite progression. They are characterized by exuberant, erythematous scars, which grow beyond the confines of the original wound and rarely regress over time. Besides their variable extent of elevation, they can become very painful or pruritic.1 It is well established that the transforming growth factor (TGF)  is not only a key factor in normal wound healing, but it also seems to play a role in the pathophysiology of excessive scar formation as found in keloids.1,5,6 The role of TGF- in scar formation appears to be due to chemotactic migration of mesenchymal cells into the wound; stimulation of their growth; and stimulation of expression, synthesis, and deposition of ECM. Furthermore, TGF- regulates ECM degradation by regulation of expression of MMPs and TIMPs, respectively.7 Three isoforms designated TGF-1, TGF-2, and TGF-3 exist. TGF-1 and TGF-2 are known to have profibrotic properties, and TGF-3 seems to inhibit fibrotic reactions.6 The isoforms TGF-1 and TGF2, especially TGF-1, are overexpressed in keloid fibroblasts resulting in excessive deposition of scar tissue and fibrosis.1,8 At present, in terms of keloid pathophysiology, the key question, whether excessive scar tissue formation is the result of an increased collagen synthesis or because of a reduced breakdown, or both, remains unanswered. Keloidal scarring appears to be one of the most frustrating problems in wound healing. Current treatments are empirical, unreliable, and, very often, unpredictable. The broad variety of therapeutic strategies and the concomitant frequency of recrudescence allude to the incomplete understanding of the pathogenesis of keloid formation. TGF-1 appears to be the growth factor most central to keloid pathogenesis with a stimulatory effect on MMP and TIMP expression in healthy fibroblasts.9 Because little is known, we analyzed the effect of targeting TGF-1 expression by antisense oligonucleotides on the expression of several important MMPs and TIMPs in keloid-derived fibroblasts. We chose several members of MMPs (collagenase, gelatinase, and stromelysin), and several members of the TIMP family.
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Materials and Methods Immunohistochemistry Informed consent was obtained from all individual subjects for all procedures. The study was performed on tissue samples from nine patients with keloid scars after otoplasty. The time period for gathering of tissue was one year. Tissue specimens of keloids and control biopsy specimens of normal healthy tissue (from the same patient) were obtained during surgery, which was performed at the Department of Otolaryngology, Head and Neck Surgery, University Hospital of Mannheim. The study was approved by the Ethical Committee of the University Hospital of Mannheim, and written consent was obtained from all subjects. Samples were frozen in liquid nitrogen for MMP and TIMP identification. For in vitro analysis, dermal fibroblasts isolated from keloids and normal controls were cultured in Falcon Petri dishes (Greiner, Germany) at 37°C in a five percent CO2 fully humidified atmosphere in serum-free Fibroblast Growth Medium (PromoCell, Heidelberg, Germany) supplemented with antibiotics (Life Technologies, Inc. [Gibco BRL], Gaithersburg, MD). The immunohistochemistry for MMP-1, MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2 detection was performed using the streptavidin-biotin complex procedure. Endogenous peroxidase was blocked with 0.3 percent hydrogen peroxidase for 30 minutes. Sections were washed with phosphate-buffered saline (PBS) and incubated with normal rabbit serum in PBS for 30 minutes at room temperature to block nonspecific antibody reaction. The sections were then incubated overnight at 4°C with the primary antibody. The slides were washed in several changes of PBS. The sections were then incubated with a peroxidase-conjugated secondary antibody (DAKO, Hamburg, Germany). After being washed twice in PBS, the sections were treated with a streptavidinbiotin-peroxidase complex, and peroxidase reaction was performed using diaminobenzidine (DAB; DAKO) as chromogen. Different antibodies were diluted to the desired concentrations in PBS. Controls were carried out by omitting the primary antibody. Light microscopic investigation was performed using a Zeiss Axiophot microscope (Zeiss, Oberkochen, Germany).
Oligodeoxynucleotides Phosphorithiotated 14mer oligodeoxynucleotides (ODN) were synthesized on an Applied Biosystems 394DNA synthesizer (Applied Biosystems Inc., Foster City, CA) by means of Bcyanothylphosphoramidite chemistry to minimize degradation by endogenous nucleases. The antisense oligonucleotide (5=CGA TAG TCT TGC AG-3=) was directed against the translation start site and surrounding nucleotides of the human TGF-1 complementary DNA (cDNA). The in vitro inhibitory effect of these antisense ODNs on TGF-1 expression at both the messenger RNA (mRNA) and protein levels in human cells has been described previously.10 All experiments were performed with 3 mol/L ODNs. To determine the effect of oligonucleotides on the expression of MMP or TIMP mRNA,
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fibroblasts were plated at a density of 105 cells/microtiter well in 24-well polystyrene plates (Falcon). After 24 hours, the cells were rinsed twice with medium, and then fresh TGF-1 oligo medium containing antisense ODNs was added, followed by an incubation period of 48 hours and 72 hours.
Cytokine Immunoassay Cell culture supernatants were collected in sterile tubes and stored at ⫺20°C until used. Next, the cytokine concentrations (MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, and TIMP-2) were determined by an enzyme-linked immunosorbent assay (ELISA) technique (R&D Systems, Wiesbaden, Germany). The system used a solid-phase monoclonal antibody and an enzyme-linked polyclonal antibody raised against the recombinant cytokines. According to the manufacturer’s guidelines, each ELISA assay measured 100 m of supernatant. All analyses and calibrations were carried out in duplicate. The calibrations on each microtiter plate included recombinant human cytokine standards provided in the kit. All concentrations were documented as nanograms/milliliters. The Mann-Whitney-Wilcoxon rank sum test as a nonparametric test was used for statistical analysis. Differences were considered to be of statistical significance when P values were ⬍ 0.05. The open source environment “R” was used to perform all statistical analysis.
Reverse-Transcription Polymerase Chain Reaction To isolate the RNA from the fibroblasts grown in monolayer, the cells were directly lysed in the culture dish by addition of 1 mL RNA-Clean (RNA-Clean System; AGS,
Heidelberg, Germany). After addition of 0.2 mL chloroform per 2 mL of homogenate and centrifugation for 15 minutes at 12,000g (4°C), the supernatant was removed from the RNA precipitate. The RNA pellet was washed twice with 70 percent ethanol by vortexing and subsequent centrifugation for eight minutes at 7500g (4°C). After drying the RNA pellet, it was dissolved in diethylpyrocarbonate water. The RNA was reverse transcribed (StrataScript First-Strand Synthesis System; Stratagene, La Jolla, CA) into cDNA using random-oligonucleotide primers. MMP and TIMP mRNA levels (MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2) were measured in all cell types using reversetranscription polymerase chain reaction (MMP-CytoXpress Multiplex PCR Kit; Bio Source, San Francisco, CA) according to the manufacturer’s instruction manual. To fractionate the MPCR DNA products, the MPCR products were mixed with 6X loading buffer and separated on a two percent agarose gel containing 0.5 mg/mL ethidium bromide, visualized with ultraviolet light and recorded using a charge-couple device camera. To test the quality of the cDNA, the kit includes primers for glyceraldehyde-3-phosphate dehydrogenase. Results were obtained in two independent experiments.
Results Immunohistochemical in vitro investigation demonstrated an increased expression of MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2 in all keloid tissues in comparison with the normal human skin controls. Representative examples of expression patterns are shown in Figure 1. No expression of MMP-1 could
Figure 1 Immunohistochemical investigation of tissue samples from normal human skin control (left) and from keloid scar tissue (right). Expression patterns of MMP-2 (A) and TIMP-2 (B).
Bran et al
Effect of transforming growth factor-1 antisense . . .
be observed within keloid fibroblast cultures or normal human skin fibroblast controls (data not shown). To quantitate cytokine secretion to the supernatant of keloid-derived fibroblasts, ELISA assay was performed after 48 hours and 72 hours, respectively. MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, and TIMP-2 were detectable in the supernatant of the keloid fibroblast cell line. In the supernatants, the treatment of keloidal fibroblasts with 3 mol/L TGF-1 antisense oligonucleotides (ODNs) for 48 hours and 72 hours resulted in significant decrease of expression of all MMPs apart from MMP-13 (MMP-1, P ⫽ 0.0012; MMP-2, P ⫽ 0.0003; MMP-3, P ⫽ 0.003; MMP-9, P ⫽ 0.0216). MMP-13 did not change expression levels either after 48 hours or after 72 hours (data not shown). As far as expression of TIMP-1 and TIMP-2 was concerned, after initial increase of expression, a relative decrease could be observed (TIMP-1, P ⫽ 0.07; TIMP-2, P ⫽ 0.401) (Fig 2). After treatment of keloid fibroblasts with TGF-1 antisense ODNs in vitro, expression of mRNA of MMP-1, MMP-2, MMP9, TIMP-1, and TIMP 2 was measured using the multiplex reverse-transcription polymerase chain reaction kit. Incubation time was 48 hours and 72 hours, respectively, containing 3 mol/L TGF-1 antisense ODNs. MMP-1 expression was increased by antisense treatment. MMP-9 mRNA expression was down-regulated after both time intervals of incuba-
Figure 2 To quantitate cytokine secretion to the supernatant of keloid-derived fibroblasts, ELISA assay was performed after 48 hours and 72 hours after treatment with medium (control) or medium containing TGF-1 antisense ODNs. Antisense treatment resulted in a significant decrease of all MMPs apart from MMP-13 (data not shown) (MMP-1, P ⫽ 0.0012; MMP-2, P ⫽ 0.0003; MMP-3, P ⫽ 0.003; MMP-9, P ⫽ 0.0216). TIMPs showed similar tendencies, although without statistical relevancy (TIMP-1, P ⫽ 0.07; TIMP-2, P ⫽ 0.401) (x-axis: relative expression ⫺ arbitrary units).
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Figure 3 Expression of mRNA of MMP-1 and MMP-9 in keloid-derived fibroblast 48 hours and 72 hours after treatment with TGF-1 antisense ODNs. c, control; as, antisense ODNs.
tion. MMP-2, TIMP-1, and TIMP-2 did not change significantly after addition of TGF-1 antisense ODNs (data not shown) (Fig 3).
Discussion Keloid scars are only observed in humans and represent a fibroproliferative aberration of wound healing, characterized by excessive deposition of large, broad, closely arranged collagen fibers, resulting in exuberant, erythematous scars, which grow beyond the confines of the original wounds and rarely regress over time. Besides their cosmetic burden for the patient, this type of scar can become very painful or pruritic.1 Two factors are generally regarded as key factors for keloid formation: genetic predisposition and skin lesion. It has been estimated that keloids most frequently occur among 15 to 20 percent of black, Hispanic, and Oriental individuals and less commonly in white individuals.11,12 Keloids usually present in individuals between 10 and 30 years of age and are less frequent at the extremes of age.12 Keloid formation tends to be regionally confined to areas such as the chest and the earlobe, whereas the hand and feet are usually spared.1 Highly qualitative reviews have already been published presenting and discussing the spectrum of therapeutic strategies and their adverse effects for keloid treatment. Common concepts include surgical management, often recommended in combination with intralesional application of corticosteroids, solitary intralesional corticosteroid injection, silicone gel sheeting, pressure therapy, radiotherapy, laser therapy, or cryotherapy. New strategies include bleomycin, interferon, intralesional 5-fluoruracil or TGF-blocking modulators.13,14 The bandwidth of therapeutic alternatives indicates the ongoing debate and reflects the limitations of pathophysiologic understanding of keloid formation. TGF- acts as a key cytokine in dermal wound healing and excessive scar tissue deposition.15 Keloid fibroblasts show a unique sensitivity to TGF-. Lee et al showed the increased expression of both profibrotic isoforms TGF-1 and TGF-2 in keloid fibroblasts relative to normal human dermal fibroblasts.16 TGF-–induced biosynthesis of ECM components occurs more rapidly and to a much larger extent, showing a greater proliferative capacity of keloid fibroblasts.5,8,14
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Dermal fibroblasts are responsible for synthesis and deposition of different components into the wound ECM.2 A deficient synthesis of products that promote matrix degradation or an excessive matrix synthesis, or both, explain the lack of scar regression in keloids.12 ECM degradation is mainly mediated by MMPs. TGF- regulates ECM degradation by regulation of expression of MMPs and TIMPs, respectively.7 Impaired wound healing is associated with an excess of MMPs.3 In keloids, the composition of the ECM is abnormal, showing elevated levels of fibronectin and proteoglycans as well as MMPs that influence the collagen architecture.11 On the search for a better understanding of wound healing processes, much effort has been put into research concerning the molecular processes of this very complex field of reparative pathways. In context of medical and scientific research, gene therapy poses a particular challenge involving several strategic mechanisms of action.9 Various models of gene therapy have been developed within the last decades. Skin appears to be an ideal tissue for this therapeutic concept: it is easily accessible, easily amenable for observation of therapeutic and adverse effects, and it is easy to transfect. Dermal fibroblasts are easily harvested and cultured, allowing in vitro investigation of gene therapy effects. The permanent interest of a better understanding of aberrant wound healing mechanisms and the mentioned advantages of the skin gave rise to the use of gene therapy in the scientific area of skin diseases, especially wound healing.10 The primary theme of antisense therapy is the inhibition of translation of a targeted protein through complementary oligonucleotides binding to target mRNA. Because growth factors control the regulation of the expression of MMPs and TIMPs, their pivotal role in wound repair has led to the development of different molecular genetic approaches that mainly focus on stimulation and enhancement or abrogation of these protein groups. Studies concerning the opposite extreme of impaired wound healing demonstrated that temporal control of the expression of distinct MMPs is associated with normalization of wound healing and ulcer repair.2,4 This prompted us to investigate the effect of TGF- antisense oligonucleotides on the expression pattern of MMPs and their specific inhibitors in cultured keloid fibroblasts. There have already been reports on keloids and MMPs. However, the literature is still sparse, with partly incongruent results. Neely et al reported significantly increased MMP-2 activity in keloids and no change in MMP-9 activity.17 Besides elevated levels of MMP-1, MMP-2, and TIMP-1, Fujiwara et al found an increase in the production of MMP-1, but no influence on MMP-2 and TIMP-1, after addition of TGF-1 antibody to cultures of keloid fibroblasts.18 Uchida et al investigated the effect of MMP and TIMP expression after addition of the MMP inhibitor tretinoin. The expression of MMP-1 and MMP-8 was significantly lower in keloid fibroblasts than in healthy controls, whereas MMP-13 expression appeared to be elevated. TIMP expression (of all 4 subtypes) was up-regulated in
keloid fibroblasts. Addition of tretinoin caused significant down-regulation of MMP-13 and up-regulation of MMP-1 and MMP-8 at the protein level, with no change in TIMPs expression.19 Sadick et al9 demonstrated increased expression of MMP-2 and MMP-9 in tissue samples of keloids and a significant down-regulation of only MMP-9 secretion after TGF-1 antisense oligonucleotide treatment in vitro. MMP-2 synthesis seemed not to be influenced by TGF-1 antisense oligonucleotide treatment.9 Imaizumi et al20 showed expression of MMP-1, MMP-2, MMP-9, TIMP-2, and TIMP-3, but no expression of TIMP-1 in keloid fibroblasts. Expression of MMP-2, TIMP-2, and TIMP-3 was significantly higher in keloid fibroblasts than in mature scars.20 In vitro immunohistochemistry was performed and demonstrated higher expression levels of all proteins (apart from MMP-1) in keloids compared with healthy skin controls. The elevated and potentially uncontrolled state of MMPs and TIMPs appears to be a pathogenic factor in keloids. Although our in vitro findings suggest high activity of both protein groups, the specific MMP/TIMP ratios appear not to be in balance, which would explain the continuous growth of the keloid scars in clinical observation before resection. Our study demonstrated that in vitro TGF-1 antisense oligonucleotide treatment efficiently down-regulated all MMP-1, MMP-2, MMP-3, and MMP-9, but not MMP-13. The expression of TIMP-1 and TIMP-2 showed an initial increase of expression, which was followed by a relative decrease after 72-hour incubation time. A down-regulation of expression of mRNA could only be observed for MMP-9. MMP-1, MMP-2, and both investigated TIMPs did not show any down-regulation. Therefore, we suggest that synthesis of the latter proteins, including MMP-13, does not seem to be influenced by TGF-1 antisense ODNs where secretion of most MMPs (apart from MMP-13) is significantly decreased in the supernatants of keloid-derived fibroblasts. A similar effect can be seen after a prolonged incubation time for both TIMPs. Antisense ODNs may show therapeutic potential for the treatment of keloid by inhibiting collagen synthesis by blocking the effect of TGF-1 and accelerating ECM degradation by advancing the MMP/TIMP balance to physiologic ratios. At the moment, we can only speculate on the exact role of each specific MMP in keloid. It remains to be discovered which MMPs play which role in terms of keloid growth and/or infiltrative capacity into healthy adjacent skin. Furthermore, the exact regulative pathway by which TGF-1 induces transcription and expression of MMPs and their specific inhibitors need to be revealed. We used the concept of TGF-1 antisense ODNs as an investigative approach towards a better understanding of molecular mechanisms in keloid pathophysiology. Ameliorating the understanding of the regulation of MMPs and TIMPs looks promising for the development of chemopreventive therapeutic strategies, which might lead to clinical improvements in keloid therapy or prevention of keloid formation.
Bran et al
Effect of transforming growth factor-1 antisense . . .
Acknowledgment The authors thank Petra Prohaska, Department of Otorhinolaryngology, Mannheim, for her excellent technical assistance.
Author Information From the Department of Otolaryngology, Head and Neck Surgery, University Hospital of Mannheim, University of Heidelberg, Mannheim, Germany. Corresponding author: Gregor M. Bran, MD, Department of Otolaryngology, Head and Neck Surgery, University Hospital of Mannheim, TheodorKutzer-Ufer 1-3, D-68167 Mannheim, Germany. E-mail address: [email protected]. This article was presented at the 2009 AAO–HNSF Annual Meeting & OTO EXPO, San Diego, CA, October 4-7, 2009.
Author Contributions Gregor M. Bran, conception and study design, acquisition, analysis and interpretation of data, drafting and revision, final approval; Ulrich R. Goessler, conception, revision, final approval; Ariton Baftiri, acquisition and interpretation of data, drafting of the article, final approval; Karl Hormann, study design, revision, final approval; Frank Riedel, study design, analysis of data, final approval; Haneen Sadick, conception and interpretation of data, revision of article, final approval.
Disclosures Competing interests: None. Sponsorships: Department of Otolaryngology, Head and Neck Surgery, University Hospital of Mannheim, Mannheim, Germany.
References 1. Bran GM, Goessler UR, Hormann K, et al. Keloids: current concepts in pathogenesis (review). Int J Mol Med 2009;24:283–93. 2. Soo C, Shaw WW, Zhang X, et al. Differential expression of matrix metalloproteinases and their tissue-derived inhibitors in cutaneous wound repair. Plast Reconstr Surg 2000;105:638 – 47. 3. Vaalamo M, Weckroth M, Puolakkainen P, et al. Patterns of matrix metalloproteinase and TIMP-1 expression in chronic and normally healing human cutaneous wounds. Br J Dermatol 1996;135:52–9.
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4. Kähäri VM, Saarialho-Kere U. Matrix metalloproteinases in skin. Exp Dermatol 1997;6:199 –213. 5. Bock O, Mrowietz U. Keloide—Eine dermale fibroproliferative Erkrankung unbekannter Ursache. Der Hautarzt 2002;53:515–23. 6. Shah M, Foreman DM, Ferguson MW. Neutralisation of TGF-1 and TGF-2 or exogenous addition of TGF-3 to cutaneous rat wounds reduces scarring. J Cell Sci 1995;108:985–1002. 7. Ma C, Tarnuzzer RW, Chegini N. Expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases in mesothelial cells and their regulation by transforming growth factor-1. Wound Repair Regen 1999;7:477– 85. 8. Bettinger DA, Yager DR, Diegelmann RF, et al. The effect of TGF- on keloid fibroblast proliferation and collagen synthesis. Plast Recon Surg 1996;98:827–33. 9. Sadick H, Herberger A, Reidel K, et al. TGF-1 antisense therapy modulates expression of matrix metalloproteinases in keloid-derived fibroblasts. Int J Mol Med 2009;22:55– 60. 10. Hernandez A, Evers BM. Functional genomics: clinical effect and the evolving role of the surgeon. Arch Surg 1999;134:1209 –15. 11. Al-Attar A, Mess S, Thomassen JM, et al. Keloid pathogenesis and treatment. Plast Reconstr Surg 2006;117:286 –300. 12. Slemp AE, Kirschner RE. Keloids and scars: a review of keloids and scars, their pathogenesis, risk factors, and management. Curr Opin Pediatr 2006;18:396 – 402. 13. Mustoe TA, Cooter RD, Gold MH, et al. International clinical recommendations on scar management. Plast Reconstr Surg 2002; 110:560 –71. 14. Niessen FB, Spauwen PH, Schalkwijk J, et al. On the nature of hypertrophic scars and keloids: a review. Plast Reconstr Surg 1999; 104:1435–58. 15. Jagadeesan J, Bayat A. Transforming growth factor (TGF-) and keloid disease. Int J Surg 2006;5:278 – 85. 16. Lee TY, Chin GS, Kim WJ, et al. Expression of transforming growth factor  1, 2 and 3 proteins in keloids. Ann Plast Surg 1999;43:179 – 83. 17. Neely AN, Clendening CE, Gardener J, et al. Gelatinase activity in keloids and hypertrophic scars. Wound Repair Regen 1999;7:166 –71. 18. Fujiwara M, Muragaki Y, Ooshima A. Keloid-derived fibroblasts show increased secretion of factors in collagen turnover and depend on matrix metalloproteinase for migration. Br J Dermatol 2005;153:295–300. 19. Uchida G, Yoshimura K, Kitano Y, et al. Tretinoin reverses upregulation of matrix metalloproteinase-13 in human keloid-derived fibroblasts. Exp Dermatol 2003;12:35– 42. 20. Imaizumi R, Akasaka Y, Inomata N, et al. Promoted activation of matrix metalloproteinase (MMP)-2 in keloid fibroblasts and increased expression on MMP-2 in collagen bundle regions: implications for mechanisms of keloid progression. Histopathology 2009;54:722–30.
ORIGINAL RESEARCH–FACIAL PLASTIC AND RECONSTRUCTIVE SURGERY
Effect of transforming growth factor-1 antisense oligonucleotides on matrix metalloproteinases and their inhibitors in keloid fibroblasts Gregor M. Bran, MD, Ulrich R. Goessler, MD, Ariton Baftiri, Karl Hormann, MD, Frank Riedel, MD, and Haneen Sadick, MD, Mannheim, Germany Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article. ABSTRACT OBJECTIVE: To identify changes in the expression of matrixmetalloproteinases (MMPs) and their specific inhibitors tissue inhibitors of metalloproteinases (TIMPs) after targeting of transforming growth factor-1 (TGF-1) with antisense oligonucleotides. STUDY DESIGN: Cross-sectional study. SETTING: The study was performed on tissue samples from nine patients with keloid scars after otoplasty presenting to the Otolaryngology–Head and Neck Surgery Department of the University Hospital in Mannheim, Germany. SUBJECTS AND METHODS: Keloid fibroblasts and normal fibroblasts were harvested from auricular keloid scars and healthy skin regions of the same patients during resection procedure of the keloid. Cells were placed in monolayer cultures. Expression of MMPs and TIMPs were analyzed by immunohistochemistry. The effect of TGF-1 targeting using antisense oligonucleotides on the expression of both protein groups in keloid-derived fibroblasts was analyzed by enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction. RESULTS: Immunohistochemical investigation demonstrated increased expression of MMP-2, -3, -9, and -13 and TIMP-1 and -2. TGF-1 antisense therapy significantly down-regulated MMP secretion in vitro. CONCLUSION: Usage of TGF-1 antisense oligodeoxynucleotides (ODNs) may show a potential chemopreventive or therapeutic option for keloids by blocking the effect of TGF-1. Furthermore, antisense ODNs can be used as an investigative approach toward a better understanding of molecular mechanisms in keloid pathophysiology. © 2010 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved.
S
carring of the skin presents a ubiquitous medical problem. The possibilities of resulting functional deficit, restriction of tissue movement, and potential risk of uncontrolled growth and adverse psychologic effects illustrate the prevailing challenge for every facial plastic surgeon. In case of a disturbance of the delicate balance of reparative processes, wound healing can be impaired drastically resulting in two pathologic extremes: chronic wounds (e.g., ulcers) or excessive scar formation (e.g., hypertrophic scars or keloids).1 A common feature of the beginning wound healing cascade is the increased activity within the extracellular matrix (ECM). Fibroblasts, and to a lesser extent keratinocytes, are the two main cell types responsible for synthesis and deposition of different collagens, elastin, glycoproteins, proteoglycans, hyaluronic acid, and growth factors into the wound ECM.2 The wound ECM forms a structural repair frame, which appears to be a key regulator of cell adhesion, migration, proliferation, and differentiation during cutaneous repair. The amount and organization of normal wound ECM are determined by a dynamic equilibrium among matrix synthesis, deposition, and degradation. The degradation of the ECM is a prerequisite for wound healing. The two main protein families that take part in this process are matrixmetalloproteinases (MMPs) and the plasminogen activation system.3 Human MMPs are a family of approximately 24 structurally related, zinc-dependent endopeptidases, which most commonly are secreted extracellularly but were also observed as membrane-associated (membrane-type MMP) or intracellularly acting enzymes.2 The activity of MMPs is inhibited by nonspecific inhibitors, e.g., ␣1-antiprotease or ␣2-macroglobulin, or more specifically by the tissue inhibitors of metalloproteinases (TIMPs). Both groups of endopeptidases are released by several different cell types found in human skin. The inhibition is regulated by a 1:1
Received December 25, 2009; revised March 3, 2010; accepted March 23, 2010.
0194-5998/$36.00 © 2010 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved. doi:10.1016/j.otohns.2010.03.029
Bran et al
Effect of transforming growth factor-1 antisense . . .
stoichiometric binding of TIMPs to the active MMPs, which thereby lose their activity.4 MMPs and TIMPs are not constitutively expressed in the skin but are induced temporarily in response to exogenous signals (e.g., cytokines, growth factors, cell-matrix interactions, and altered cell-cell interactions). It is believed that pathologic conditions represent an aberrant functioning of these regulatory mechanisms resulting in local wound milieus that favor chronic tissue turnover.4 Excessive scar tissue formation can be observed in hypertrophic scars and keloids. Hypertrophic scars follow a certain pattern of evolution, stabilization, and involution, whereas keloids do not follow this characteristic development. Keloid scars represent a dysregulated response to cutaneous wounding resulting in continuous proliferation with indefinite progression. They are characterized by exuberant, erythematous scars, which grow beyond the confines of the original wound and rarely regress over time. Besides their variable extent of elevation, they can become very painful or pruritic.1 It is well established that the transforming growth factor (TGF)  is not only a key factor in normal wound healing, but it also seems to play a role in the pathophysiology of excessive scar formation as found in keloids.1,5,6 The role of TGF- in scar formation appears to be due to chemotactic migration of mesenchymal cells into the wound; stimulation of their growth; and stimulation of expression, synthesis, and deposition of ECM. Furthermore, TGF- regulates ECM degradation by regulation of expression of MMPs and TIMPs, respectively.7 Three isoforms designated TGF-1, TGF-2, and TGF-3 exist. TGF-1 and TGF-2 are known to have profibrotic properties, and TGF-3 seems to inhibit fibrotic reactions.6 The isoforms TGF-1 and TGF2, especially TGF-1, are overexpressed in keloid fibroblasts resulting in excessive deposition of scar tissue and fibrosis.1,8 At present, in terms of keloid pathophysiology, the key question, whether excessive scar tissue formation is the result of an increased collagen synthesis or because of a reduced breakdown, or both, remains unanswered. Keloidal scarring appears to be one of the most frustrating problems in wound healing. Current treatments are empirical, unreliable, and, very often, unpredictable. The broad variety of therapeutic strategies and the concomitant frequency of recrudescence allude to the incomplete understanding of the pathogenesis of keloid formation. TGF-1 appears to be the growth factor most central to keloid pathogenesis with a stimulatory effect on MMP and TIMP expression in healthy fibroblasts.9 Because little is known, we analyzed the effect of targeting TGF-1 expression by antisense oligonucleotides on the expression of several important MMPs and TIMPs in keloid-derived fibroblasts. We chose several members of MMPs (collagenase, gelatinase, and stromelysin), and several members of the TIMP family.
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Materials and Methods Immunohistochemistry Informed consent was obtained from all individual subjects for all procedures. The study was performed on tissue samples from nine patients with keloid scars after otoplasty. The time period for gathering of tissue was one year. Tissue specimens of keloids and control biopsy specimens of normal healthy tissue (from the same patient) were obtained during surgery, which was performed at the Department of Otolaryngology, Head and Neck Surgery, University Hospital of Mannheim. The study was approved by the Ethical Committee of the University Hospital of Mannheim, and written consent was obtained from all subjects. Samples were frozen in liquid nitrogen for MMP and TIMP identification. For in vitro analysis, dermal fibroblasts isolated from keloids and normal controls were cultured in Falcon Petri dishes (Greiner, Germany) at 37°C in a five percent CO2 fully humidified atmosphere in serum-free Fibroblast Growth Medium (PromoCell, Heidelberg, Germany) supplemented with antibiotics (Life Technologies, Inc. [Gibco BRL], Gaithersburg, MD). The immunohistochemistry for MMP-1, MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2 detection was performed using the streptavidin-biotin complex procedure. Endogenous peroxidase was blocked with 0.3 percent hydrogen peroxidase for 30 minutes. Sections were washed with phosphate-buffered saline (PBS) and incubated with normal rabbit serum in PBS for 30 minutes at room temperature to block nonspecific antibody reaction. The sections were then incubated overnight at 4°C with the primary antibody. The slides were washed in several changes of PBS. The sections were then incubated with a peroxidase-conjugated secondary antibody (DAKO, Hamburg, Germany). After being washed twice in PBS, the sections were treated with a streptavidinbiotin-peroxidase complex, and peroxidase reaction was performed using diaminobenzidine (DAB; DAKO) as chromogen. Different antibodies were diluted to the desired concentrations in PBS. Controls were carried out by omitting the primary antibody. Light microscopic investigation was performed using a Zeiss Axiophot microscope (Zeiss, Oberkochen, Germany).
Oligodeoxynucleotides Phosphorithiotated 14mer oligodeoxynucleotides (ODN) were synthesized on an Applied Biosystems 394DNA synthesizer (Applied Biosystems Inc., Foster City, CA) by means of Bcyanothylphosphoramidite chemistry to minimize degradation by endogenous nucleases. The antisense oligonucleotide (5=CGA TAG TCT TGC AG-3=) was directed against the translation start site and surrounding nucleotides of the human TGF-1 complementary DNA (cDNA). The in vitro inhibitory effect of these antisense ODNs on TGF-1 expression at both the messenger RNA (mRNA) and protein levels in human cells has been described previously.10 All experiments were performed with 3 mol/L ODNs. To determine the effect of oligonucleotides on the expression of MMP or TIMP mRNA,
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fibroblasts were plated at a density of 105 cells/microtiter well in 24-well polystyrene plates (Falcon). After 24 hours, the cells were rinsed twice with medium, and then fresh TGF-1 oligo medium containing antisense ODNs was added, followed by an incubation period of 48 hours and 72 hours.
Cytokine Immunoassay Cell culture supernatants were collected in sterile tubes and stored at ⫺20°C until used. Next, the cytokine concentrations (MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, and TIMP-2) were determined by an enzyme-linked immunosorbent assay (ELISA) technique (R&D Systems, Wiesbaden, Germany). The system used a solid-phase monoclonal antibody and an enzyme-linked polyclonal antibody raised against the recombinant cytokines. According to the manufacturer’s guidelines, each ELISA assay measured 100 m of supernatant. All analyses and calibrations were carried out in duplicate. The calibrations on each microtiter plate included recombinant human cytokine standards provided in the kit. All concentrations were documented as nanograms/milliliters. The Mann-Whitney-Wilcoxon rank sum test as a nonparametric test was used for statistical analysis. Differences were considered to be of statistical significance when P values were ⬍ 0.05. The open source environment “R” was used to perform all statistical analysis.
Reverse-Transcription Polymerase Chain Reaction To isolate the RNA from the fibroblasts grown in monolayer, the cells were directly lysed in the culture dish by addition of 1 mL RNA-Clean (RNA-Clean System; AGS,
Heidelberg, Germany). After addition of 0.2 mL chloroform per 2 mL of homogenate and centrifugation for 15 minutes at 12,000g (4°C), the supernatant was removed from the RNA precipitate. The RNA pellet was washed twice with 70 percent ethanol by vortexing and subsequent centrifugation for eight minutes at 7500g (4°C). After drying the RNA pellet, it was dissolved in diethylpyrocarbonate water. The RNA was reverse transcribed (StrataScript First-Strand Synthesis System; Stratagene, La Jolla, CA) into cDNA using random-oligonucleotide primers. MMP and TIMP mRNA levels (MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2) were measured in all cell types using reversetranscription polymerase chain reaction (MMP-CytoXpress Multiplex PCR Kit; Bio Source, San Francisco, CA) according to the manufacturer’s instruction manual. To fractionate the MPCR DNA products, the MPCR products were mixed with 6X loading buffer and separated on a two percent agarose gel containing 0.5 mg/mL ethidium bromide, visualized with ultraviolet light and recorded using a charge-couple device camera. To test the quality of the cDNA, the kit includes primers for glyceraldehyde-3-phosphate dehydrogenase. Results were obtained in two independent experiments.
Results Immunohistochemical in vitro investigation demonstrated an increased expression of MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2 in all keloid tissues in comparison with the normal human skin controls. Representative examples of expression patterns are shown in Figure 1. No expression of MMP-1 could
Figure 1 Immunohistochemical investigation of tissue samples from normal human skin control (left) and from keloid scar tissue (right). Expression patterns of MMP-2 (A) and TIMP-2 (B).
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be observed within keloid fibroblast cultures or normal human skin fibroblast controls (data not shown). To quantitate cytokine secretion to the supernatant of keloid-derived fibroblasts, ELISA assay was performed after 48 hours and 72 hours, respectively. MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, and TIMP-2 were detectable in the supernatant of the keloid fibroblast cell line. In the supernatants, the treatment of keloidal fibroblasts with 3 mol/L TGF-1 antisense oligonucleotides (ODNs) for 48 hours and 72 hours resulted in significant decrease of expression of all MMPs apart from MMP-13 (MMP-1, P ⫽ 0.0012; MMP-2, P ⫽ 0.0003; MMP-3, P ⫽ 0.003; MMP-9, P ⫽ 0.0216). MMP-13 did not change expression levels either after 48 hours or after 72 hours (data not shown). As far as expression of TIMP-1 and TIMP-2 was concerned, after initial increase of expression, a relative decrease could be observed (TIMP-1, P ⫽ 0.07; TIMP-2, P ⫽ 0.401) (Fig 2). After treatment of keloid fibroblasts with TGF-1 antisense ODNs in vitro, expression of mRNA of MMP-1, MMP-2, MMP9, TIMP-1, and TIMP 2 was measured using the multiplex reverse-transcription polymerase chain reaction kit. Incubation time was 48 hours and 72 hours, respectively, containing 3 mol/L TGF-1 antisense ODNs. MMP-1 expression was increased by antisense treatment. MMP-9 mRNA expression was down-regulated after both time intervals of incuba-
Figure 2 To quantitate cytokine secretion to the supernatant of keloid-derived fibroblasts, ELISA assay was performed after 48 hours and 72 hours after treatment with medium (control) or medium containing TGF-1 antisense ODNs. Antisense treatment resulted in a significant decrease of all MMPs apart from MMP-13 (data not shown) (MMP-1, P ⫽ 0.0012; MMP-2, P ⫽ 0.0003; MMP-3, P ⫽ 0.003; MMP-9, P ⫽ 0.0216). TIMPs showed similar tendencies, although without statistical relevancy (TIMP-1, P ⫽ 0.07; TIMP-2, P ⫽ 0.401) (x-axis: relative expression ⫺ arbitrary units).
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Figure 3 Expression of mRNA of MMP-1 and MMP-9 in keloid-derived fibroblast 48 hours and 72 hours after treatment with TGF-1 antisense ODNs. c, control; as, antisense ODNs.
tion. MMP-2, TIMP-1, and TIMP-2 did not change significantly after addition of TGF-1 antisense ODNs (data not shown) (Fig 3).
Discussion Keloid scars are only observed in humans and represent a fibroproliferative aberration of wound healing, characterized by excessive deposition of large, broad, closely arranged collagen fibers, resulting in exuberant, erythematous scars, which grow beyond the confines of the original wounds and rarely regress over time. Besides their cosmetic burden for the patient, this type of scar can become very painful or pruritic.1 Two factors are generally regarded as key factors for keloid formation: genetic predisposition and skin lesion. It has been estimated that keloids most frequently occur among 15 to 20 percent of black, Hispanic, and Oriental individuals and less commonly in white individuals.11,12 Keloids usually present in individuals between 10 and 30 years of age and are less frequent at the extremes of age.12 Keloid formation tends to be regionally confined to areas such as the chest and the earlobe, whereas the hand and feet are usually spared.1 Highly qualitative reviews have already been published presenting and discussing the spectrum of therapeutic strategies and their adverse effects for keloid treatment. Common concepts include surgical management, often recommended in combination with intralesional application of corticosteroids, solitary intralesional corticosteroid injection, silicone gel sheeting, pressure therapy, radiotherapy, laser therapy, or cryotherapy. New strategies include bleomycin, interferon, intralesional 5-fluoruracil or TGF-blocking modulators.13,14 The bandwidth of therapeutic alternatives indicates the ongoing debate and reflects the limitations of pathophysiologic understanding of keloid formation. TGF- acts as a key cytokine in dermal wound healing and excessive scar tissue deposition.15 Keloid fibroblasts show a unique sensitivity to TGF-. Lee et al showed the increased expression of both profibrotic isoforms TGF-1 and TGF-2 in keloid fibroblasts relative to normal human dermal fibroblasts.16 TGF-–induced biosynthesis of ECM components occurs more rapidly and to a much larger extent, showing a greater proliferative capacity of keloid fibroblasts.5,8,14
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Dermal fibroblasts are responsible for synthesis and deposition of different components into the wound ECM.2 A deficient synthesis of products that promote matrix degradation or an excessive matrix synthesis, or both, explain the lack of scar regression in keloids.12 ECM degradation is mainly mediated by MMPs. TGF- regulates ECM degradation by regulation of expression of MMPs and TIMPs, respectively.7 Impaired wound healing is associated with an excess of MMPs.3 In keloids, the composition of the ECM is abnormal, showing elevated levels of fibronectin and proteoglycans as well as MMPs that influence the collagen architecture.11 On the search for a better understanding of wound healing processes, much effort has been put into research concerning the molecular processes of this very complex field of reparative pathways. In context of medical and scientific research, gene therapy poses a particular challenge involving several strategic mechanisms of action.9 Various models of gene therapy have been developed within the last decades. Skin appears to be an ideal tissue for this therapeutic concept: it is easily accessible, easily amenable for observation of therapeutic and adverse effects, and it is easy to transfect. Dermal fibroblasts are easily harvested and cultured, allowing in vitro investigation of gene therapy effects. The permanent interest of a better understanding of aberrant wound healing mechanisms and the mentioned advantages of the skin gave rise to the use of gene therapy in the scientific area of skin diseases, especially wound healing.10 The primary theme of antisense therapy is the inhibition of translation of a targeted protein through complementary oligonucleotides binding to target mRNA. Because growth factors control the regulation of the expression of MMPs and TIMPs, their pivotal role in wound repair has led to the development of different molecular genetic approaches that mainly focus on stimulation and enhancement or abrogation of these protein groups. Studies concerning the opposite extreme of impaired wound healing demonstrated that temporal control of the expression of distinct MMPs is associated with normalization of wound healing and ulcer repair.2,4 This prompted us to investigate the effect of TGF- antisense oligonucleotides on the expression pattern of MMPs and their specific inhibitors in cultured keloid fibroblasts. There have already been reports on keloids and MMPs. However, the literature is still sparse, with partly incongruent results. Neely et al reported significantly increased MMP-2 activity in keloids and no change in MMP-9 activity.17 Besides elevated levels of MMP-1, MMP-2, and TIMP-1, Fujiwara et al found an increase in the production of MMP-1, but no influence on MMP-2 and TIMP-1, after addition of TGF-1 antibody to cultures of keloid fibroblasts.18 Uchida et al investigated the effect of MMP and TIMP expression after addition of the MMP inhibitor tretinoin. The expression of MMP-1 and MMP-8 was significantly lower in keloid fibroblasts than in healthy controls, whereas MMP-13 expression appeared to be elevated. TIMP expression (of all 4 subtypes) was up-regulated in
keloid fibroblasts. Addition of tretinoin caused significant down-regulation of MMP-13 and up-regulation of MMP-1 and MMP-8 at the protein level, with no change in TIMPs expression.19 Sadick et al9 demonstrated increased expression of MMP-2 and MMP-9 in tissue samples of keloids and a significant down-regulation of only MMP-9 secretion after TGF-1 antisense oligonucleotide treatment in vitro. MMP-2 synthesis seemed not to be influenced by TGF-1 antisense oligonucleotide treatment.9 Imaizumi et al20 showed expression of MMP-1, MMP-2, MMP-9, TIMP-2, and TIMP-3, but no expression of TIMP-1 in keloid fibroblasts. Expression of MMP-2, TIMP-2, and TIMP-3 was significantly higher in keloid fibroblasts than in mature scars.20 In vitro immunohistochemistry was performed and demonstrated higher expression levels of all proteins (apart from MMP-1) in keloids compared with healthy skin controls. The elevated and potentially uncontrolled state of MMPs and TIMPs appears to be a pathogenic factor in keloids. Although our in vitro findings suggest high activity of both protein groups, the specific MMP/TIMP ratios appear not to be in balance, which would explain the continuous growth of the keloid scars in clinical observation before resection. Our study demonstrated that in vitro TGF-1 antisense oligonucleotide treatment efficiently down-regulated all MMP-1, MMP-2, MMP-3, and MMP-9, but not MMP-13. The expression of TIMP-1 and TIMP-2 showed an initial increase of expression, which was followed by a relative decrease after 72-hour incubation time. A down-regulation of expression of mRNA could only be observed for MMP-9. MMP-1, MMP-2, and both investigated TIMPs did not show any down-regulation. Therefore, we suggest that synthesis of the latter proteins, including MMP-13, does not seem to be influenced by TGF-1 antisense ODNs where secretion of most MMPs (apart from MMP-13) is significantly decreased in the supernatants of keloid-derived fibroblasts. A similar effect can be seen after a prolonged incubation time for both TIMPs. Antisense ODNs may show therapeutic potential for the treatment of keloid by inhibiting collagen synthesis by blocking the effect of TGF-1 and accelerating ECM degradation by advancing the MMP/TIMP balance to physiologic ratios. At the moment, we can only speculate on the exact role of each specific MMP in keloid. It remains to be discovered which MMPs play which role in terms of keloid growth and/or infiltrative capacity into healthy adjacent skin. Furthermore, the exact regulative pathway by which TGF-1 induces transcription and expression of MMPs and their specific inhibitors need to be revealed. We used the concept of TGF-1 antisense ODNs as an investigative approach towards a better understanding of molecular mechanisms in keloid pathophysiology. Ameliorating the understanding of the regulation of MMPs and TIMPs looks promising for the development of chemopreventive therapeutic strategies, which might lead to clinical improvements in keloid therapy or prevention of keloid formation.
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Acknowledgment The authors thank Petra Prohaska, Department of Otorhinolaryngology, Mannheim, for her excellent technical assistance.
Author Information From the Department of Otolaryngology, Head and Neck Surgery, University Hospital of Mannheim, University of Heidelberg, Mannheim, Germany. Corresponding author: Gregor M. Bran, MD, Department of Otolaryngology, Head and Neck Surgery, University Hospital of Mannheim, TheodorKutzer-Ufer 1-3, D-68167 Mannheim, Germany. E-mail address: [email protected]. This article was presented at the 2009 AAO–HNSF Annual Meeting & OTO EXPO, San Diego, CA, October 4-7, 2009.
Author Contributions Gregor M. Bran, conception and study design, acquisition, analysis and interpretation of data, drafting and revision, final approval; Ulrich R. Goessler, conception, revision, final approval; Ariton Baftiri, acquisition and interpretation of data, drafting of the article, final approval; Karl Hormann, study design, revision, final approval; Frank Riedel, study design, analysis of data, final approval; Haneen Sadick, conception and interpretation of data, revision of article, final approval.
Disclosures Competing interests: None. Sponsorships: Department of Otolaryngology, Head and Neck Surgery, University Hospital of Mannheim, Mannheim, Germany.
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4. Kähäri VM, Saarialho-Kere U. Matrix metalloproteinases in skin. Exp Dermatol 1997;6:199 –213. 5. Bock O, Mrowietz U. Keloide—Eine dermale fibroproliferative Erkrankung unbekannter Ursache. Der Hautarzt 2002;53:515–23. 6. Shah M, Foreman DM, Ferguson MW. Neutralisation of TGF-1 and TGF-2 or exogenous addition of TGF-3 to cutaneous rat wounds reduces scarring. J Cell Sci 1995;108:985–1002. 7. Ma C, Tarnuzzer RW, Chegini N. Expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases in mesothelial cells and their regulation by transforming growth factor-1. Wound Repair Regen 1999;7:477– 85. 8. Bettinger DA, Yager DR, Diegelmann RF, et al. The effect of TGF- on keloid fibroblast proliferation and collagen synthesis. Plast Recon Surg 1996;98:827–33. 9. Sadick H, Herberger A, Reidel K, et al. TGF-1 antisense therapy modulates expression of matrix metalloproteinases in keloid-derived fibroblasts. Int J Mol Med 2009;22:55– 60. 10. Hernandez A, Evers BM. Functional genomics: clinical effect and the evolving role of the surgeon. Arch Surg 1999;134:1209 –15. 11. Al-Attar A, Mess S, Thomassen JM, et al. Keloid pathogenesis and treatment. Plast Reconstr Surg 2006;117:286 –300. 12. Slemp AE, Kirschner RE. Keloids and scars: a review of keloids and scars, their pathogenesis, risk factors, and management. Curr Opin Pediatr 2006;18:396 – 402. 13. Mustoe TA, Cooter RD, Gold MH, et al. International clinical recommendations on scar management. Plast Reconstr Surg 2002; 110:560 –71. 14. Niessen FB, Spauwen PH, Schalkwijk J, et al. On the nature of hypertrophic scars and keloids: a review. Plast Reconstr Surg 1999; 104:1435–58. 15. Jagadeesan J, Bayat A. Transforming growth factor (TGF-) and keloid disease. Int J Surg 2006;5:278 – 85. 16. Lee TY, Chin GS, Kim WJ, et al. Expression of transforming growth factor  1, 2 and 3 proteins in keloids. Ann Plast Surg 1999;43:179 – 83. 17. Neely AN, Clendening CE, Gardener J, et al. Gelatinase activity in keloids and hypertrophic scars. Wound Repair Regen 1999;7:166 –71. 18. Fujiwara M, Muragaki Y, Ooshima A. Keloid-derived fibroblasts show increased secretion of factors in collagen turnover and depend on matrix metalloproteinase for migration. Br J Dermatol 2005;153:295–300. 19. Uchida G, Yoshimura K, Kitano Y, et al. Tretinoin reverses upregulation of matrix metalloproteinase-13 in human keloid-derived fibroblasts. Exp Dermatol 2003;12:35– 42. 20. Imaizumi R, Akasaka Y, Inomata N, et al. Promoted activation of matrix metalloproteinase (MMP)-2 in keloid fibroblasts and increased expression on MMP-2 in collagen bundle regions: implications for mechanisms of keloid progression. Histopathology 2009;54:722–30.