View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector ORIGINAL ARTICLE Dexamethasone Induction of Keloid Regression through Effective Suppression of VEGF Expression and Keloid Fibroblast Proliferation Wen-Sheng Wu1,2, Feng-Sheng Wang1, Kuender D. Yang1, Chao-Cheng Huang3 and Yur-Ren Kuo1,2 The biological mechanism underlying steroid therapy for treating keloids remains unclear. Analytical results demonstrated that topical intra-lesional steroid injections suppress vascular endothelial growth factor (VEGF) expression in keloid tissue and induce its regression in vivo. This study investigated whether glucocorticoid (dexamethasone) downregulates VEGF expression and hinders keloid fibroblast (KF) proliferation in keloid regression. Primary KF cultures were treated with various concentrations of dexamethasone, glucocorticoid receptor (GR) antagonist (mifeprostone, RU-486), VEGF-A antibody, VEGF receptor-2 (VEGF-R2) antagonist (SU-5416), and VEGF protein. Analytical results demonstrated that dexamethasone retarded KFs proliferation. However, suppression of fibroblast proliferation by dexamethasone pre-treatment was reduced by adding exogenous VEGF protein. Dexamethasone suppressed endogenous VEGF mRNA induction, protein expressed by KFs, and angiogenesis activity detected by a tube-forming assay of human umbilical vein endothelial cells co- cultured fibroblasts. These effects were reversed by pre-treatment with RU-486, and not by pre-treatment with SU-5416. Thus, dexamethasone induces keloid regression via interaction with the GR and suppresses endogenous VEGF expression and fibroblast proliferation. However, exogenous VEGF promotes fibroblast proliferation through the GR-independent pathway. Modulation of VEGF production may comprise a valuable treatment modality for keloids. Journal of Investigative Dermatology (2006) 126, 1264–1271. doi:10.1038/sj.jid.5700274; published online 30 March 2006 INTRODUCTION The pathophysiological events resulting in keloid forma- Keloid formation following wound healing frequently occurs in tion remain unclear (Appleton et al., 1996). Research has African and Asian populations (Rockwell et al., 1989). Keloids, focused on investigating growth factors, such as vascular which are pathological scars defined as benign cutaneous endothelial growth factor (VEGF), transforming growth factor- tumors extending beyond wound margins, are distinguished by b (TGF-b), insulin growth factor (IGF), etc., that likely alter the substantial deposition of collagen in the dermis, resulting in an regeneration of the wound-healing cascade in regulating scar imbalanced production and aggregation of extracellular matrix development (Rockwell et al., 1989; Bennett and Schultz, (Diegelmann et al., 1979; Murray, 1994; Wu et al., 2004). 1993). Cytokines, such as IL-6, tumor necrosis factor-a, are also associated with keloid scar formation (McCauley et al., 1Department of Medical Research, Chang Gung Memorial Hospital at 1992; Xue et al., 2000). Excisional surgery, laser therapy, Kaohsiung, Chang Gung University, Kaohsiung, Taiwan; 2Department of radiotherapy, cryosurgery, 5-fluorouracil, and silicone sheet- Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital at ing are frequently applied therapeutic modalities for mana- 3 Kaohsiung, Chang Gung University, Kaohsiung, Taiwan and Department of ging keloids (Pollack and Goslen, 1982; Borgognoni, 2002; Pathology, Chang Gung Memorial Hospital at Kaohsiung, Chang Gung University, Kaohsiung, Taiwan Zouboulis et al., 2002; Kuo et al., 2004; Malaker et al., 2004; Presented at the 50th Anniversary Meeting of the Plastic Surgical Research Nanda and Reddy, 2004). These modalities, which amelio- Council, Toronto, Canada, May 18–21, 2005. rate or eradicate keloid scar formation, have all obtained Correspondence: Dr Yur-Ren Kuo, Department of Plastic and Reconstructive controversial outcomes for regression and arresting of keloid Surgery, Chang Gung Memorial Hospital at Kaohsiung, Chang Gung scar development (Shaffer et al., 2002). Intra-lesional University, Taiwan, 123 Ta-Pei Road, Niao-Sung Hsiang, Kaohsiung 83305, corticosteroid as a monotherapy or combined with surgery Taiwan. E-mail: [email protected] for large keloids has proven efficient for flattening and Abbreviations: GR, glucocorticoid receptor; HRP, horseradish peroxidase; HUVEC, human umbilical vein endothelial cell; IC, immunocytospin; reducing keloids (Kiil, 1977; Boyadjiev et al., 1995; Patel and IGF, insulin growth factor; IHC, immunohistochemical staining; KF, keloid Hall, 2000). However, the mechanistic role of steroids in fibroblast; PCNA, proliferating cell nuclear antigen; MTT, 3-[4,5-di- moderating keloid formation remains unclear. methylthiazole]-2,5-diphenyletetrazolium bromide; TGF-b, transforming Glucocorticoid dexamethasone has numerous effects growth factor-b; VEGF, vascular endothelial growth factor; VEGF-R, vascular endothelial growth factor receptor during wound-healing sequences. In vitro studies have Received 11 March 2005; revised 27 January 2006; accepted 28 January demonstrated that glucocorticoid dexamethasone suppresses 2006; published online 30 March 2006 fibroblast proliferation of TGF-b (Slavin et al., 1994; Carroll 1264 Journal of Investigative Dermatology (2006), Volume 126 & 2006 The Society for Investigative Dermatology W-S Wu et al. Dexamethasone-Induced Keloid Regression et al., 2002). Recently, speculation based on experimental a bc data hypothesized that excess production of VEGF is crucial to keloid tissue formation (Steinbrech et al., 1999; Gira et al., 2004). However, few experimental studies have investi- gated the relationship between VEGF expression and dexa- methasone. Pre-treatment Post-treatment Negative control VEGF, a proangiogenic cytokine, has been implicated as d e f crucial to normal and pathological wound healing (Le et al., PCNA 2004). As an angiogenic peptide composed a variety of isoforms, VEGF is also a vascular permeability factor that promotes neo-vascularization and cell growth (Sayah et al., g hi 1999; Carmeliet and Jain, 2000). The most abundant of the four VEGF isoforms, VEGF-A, is typically utilized in VEGF biological angiogenic studies (Ferrara et al., 1992; Thomas, 1996). Recently, Gira et al. (2004) indicated that VEGF production is abundant in the underlying dermis of keloids. In Figure 1. Histological changes in keloid tissues before and following steroid vitro studies have indicated that VEGF is expressed at higher treatment. (a–c) Patient with keloid formation produced by of ear piercing. In levels in keloid-derived fibroblasts than in normal skin histological hematoxylin and eosin staining, the untreated keloid tissues fibroblasts (Steinbrech et al., 1999; Gira et al., 2004). indicated hyperkeratosis, thickness of the epidermis and dermis layers, and Therefore, VEGF likely plays a significant role in keloid overproduction of excess dense collagen fibers in the extracellular matrix. formation by changing the extracellular matrix. Scale bar is (b)10mmand(c)50mm. (d–f) The IHC staining with an HRP- diaminobenzidine staining kit indicated substantial PCNA expression (brown In the authors’ clinical experience, patients with keloids color in cell nuclei; arrow) in keloid before (d) intra-lesional steroid demonstrated substantial regression following steroid (triam- (triamcinolone) injection. Expression of PCNA in keloid biopsy tissue was cinolone) injections. In this study, keloid tissues were retarded after multiple steroid injections at 4-week intervals. (g–h) The IHC obtained before and after steroid injections and analyzed. staining results indicated VEGF expression (brown color; arrow) in keloid For in vitro study, human keloid fibroblasts (KFs) were tissue before (g) intra-lesional steroid treatment. Expression of VEGF in keloid obtained as a cell line. Proliferating cell nuclear antigen tissue was suppressed after steroid injections. Scale bar ¼ 50 mm. (PCNA) and VEGF expressions in keloid tissues were determined utilizing immunohistochemical staining (IHC). The mRNA expressions of growth factors in keloids, such as Dexamethasone suppresses KF proliferation, but not when VEGF, IGF-1, TGF-b, and VEGF-R2, were determined using in exogenous VEGF is added situ hybridization, and quantitative real-time PCR. This study The effect of dexamethasone on cellular proliferation of KFs investigated whether steroids (e.g. dexamethasone) can in vitro was detected using the MTT assay. Proliferation effectively downregulate VEGF expression and suppress KF activities of KFs compared with that in the control group were À7 proliferation under keloid regression. Cell proliferation of KFs clearly suppressed by 10 M of dexamethasone treatment was assessed with a 3-[4,5-dimethylthiazole]-2,5-diphenyle- after culturing for 96 hours (Figure 3a). Adding an optimal tetrazolium bromide (MTT) assay and by quantifying PCNA dosage of exogenous VEGF protein alone without dexa- expression. Protein and mRNA expressions of VEGF were methasone treatment significantly enhanced KF proliferation demonstrated in KFs before and after experimental treatments. compared with that in the control group (without treatment) (Figure 3b). The VEGF monoclonal antibody treatment group mimicked dexamethasone-suppressed cell proliferation. Add- RESULTS ing exogenous VEGF protein to the culture medium following VEGF involved in the steroid-induced keloid tissue regression dexamethasone pre-treatment for 24 hours did not