in vivo 19: 599-604 (2005) Influence of Hepatocyte Growth Factor/Scatter Factor (HGF/SF) on Fibroblast Growth Factor-2 (FGF-2) Levels in External Auditory Canal Cholesteatoma (EACC) Cell Culture RAMIN NAIM1, RAY C. CHANG2, HANEEN SADICK1 and KARL HORMANN1 1Department of Otolaryngology, Head and Neck Surgery, University Hospital Mannheim, D-68135 Mannheim, Germany; 2Department of Otolaryngology, University of Miami/Jackson Memorial Hospital, Miami, Florida, U.S.A. Abstract. Background: In previous studies, we cited angiogenesis have been identified, including fibroblast circulatory disorders and hypoxia as etiological factors for the growth factor-a (aFGF), transforming growth factor-alpha formation of external auditory canal cholesteatoma (EACC) (TGF-alpha), TGF-beta, hepatocyte growth factor/scatter resulting in angiogenesis. Here, we investigate how the factor (HGF/SF), tumor necrosis factor-alpha (TNF-alpha), angiogenic factor hepatocyte growth factor/scatter factor angiogenin and interleukin-8 (IL-8) (3, 4). (HGF/SF) influences the level of another angiogenic factor Fibroblast growth factors (FGFs) are also considered FGF-2. Materials and Methods: After 16 to 72 hours of angiogenic factors, yet the exact relationship between FGF incubation with 20ng/ml HGF/SF, levels of VEGF in the and vascular development in normal and pathological tissue HGF/SF-treated and untreated culture was analyzed. We also has long remained elusive (5). FGF-2 is a member of the investigated the influence of HGF/SF (20-80ng/ml) on the FGF family, that comprises about nine members. FGF-2 concentration of FGF-2. Results: After 16 hours of incubation stimulates smooth muscle cell growth, wound healing, tissue with HGF/SF at 20ng/ml, FGF-2 was measured at 44.19pg/ml repair, and is increased in chronic inflammation (5). FGF-2 (control: 42.24pg/ml). After 72 hours, FGF-2 was at regulates the expression of several molecules including 23.41pg/ml (control: 14.83pg/ml). After 24 hours, 40ng/ml interstitial collagenase, urokinase type plasminogen HGF/SF showed the highest concentration of FGF-2. activator (uPA), plasminogen activator inhibitor (PAI-1), Conclusion: FGF-2 levels were initially elevated after treatment uPA receptor and integrins (6). For a long time, FGF-2 was with HGF/SF, however, further incubation did not show any considered as the angiogenesis factor. This was challenged increase. We assume that HGF/SF released FGF-2 in the by the discovery of several other angiogenic factors, such as matrix but did not induce FGF-2 expression. The most the vascular endothelial growth factor (VEGF), HGF/SF effective HGF/SF concentration was 40ng/ml. and its receptor c-Met (7). HGF/SF was initially discovered as a protein secreted by Angiogenesis, the formation of new blood vessels within fibroblasts resulting in epithelial/cell mitogenesis, tissue, has been an exciting and promising new area of proliferation, morphogenesis and motogenesis (8, 9). research. During angiogenesis, endothelial cell growth, HGF/SF is a 100 kDa pleiotropic mitogenic growth factor migration and vascular tube formation are regulated by pro- and its role in establishing angiogenesis, organ regeneration, and anti-angiogenic factors, matrix-degrading proteases and development of the nervous system and tumor invasion cell–extracellular matrix interactions (1). Matrix-bound emphasizes its important role in physiological and growth factors released by proteases and/or by angiogenic pathological processes (10). HGF/SF transduces signals via factors promote different diseases by enhancing endothelial its receptor, c-Met, a transmembrane tyrosine kinase proto- migration and growth (2). Several potential regulators of oncogene (11). In the field of otolaryngology, the external auditory canal cholesteatoma (EACC) is a very rare pathological entity (12). EACC is usually found in the inferior or posterior part of the Correspondence to: Dr. med. Ramin Naim, Univ.-HNO-Klinik, bony external ear canal, and it is characterized by permanent Theodor-Kutzer-Ufer, 68135 Mannheim, Germany. Tel: +49 621 growth of the epithelial tissue (13). Establishing the 383 1600, Fax: +49 621 383 1972, e-mail: [email protected] heidelberg.de cholesteatoma, the epithelial layer is the so-called matrix, and the adjacent subepithelial tissue is called perimatrix. The Key Words: External auditory canal cholesteatoma, FGF-2, matrix contains hyperplastic epithelial tissue, which HGF/SF, culture, keratinocyte. destructively tends to grow into adjacent anatomical structures 0258-851X/2005 $2.00+.40 599 in vivo 19: 599-604 (2005) such as the bony canal and mastoid cells, in contrast to immunoassay technique. A monoclonal antibody specific for FGF-2 keratosis obturans (14). The treatment of choice remains total was pre-coated onto a microplate. Standards and samples were surgical removal of the chronic inflammatory tissue (15). In pipetted into wells and FGF-2 present was bound by the immobilized antibody. After washing away unbound substances, an previous immunohistochemical studies, we showed that the enzyme-linked polyclonal antibody specific for FGF-2 was added growth factors VEGF, FGF-2, HGF/FS and the receptor to the wells. Following a wash to remove unbound antibody- c-Met are up-regulated in EACC (16). Sudhoff et al. reported enzyme reagent, a substrate solution was added to the wells and that angiogenesis is a pivotal factor for the formation of middle color developed in proportion to the amount of FGF-2 bound in ear cholesteatoma (17). They showed an altered expression the initial step. The color development was stopped and the and distribution of VEGF, FGF-2, TGF-· and TGF-‚1 in intensity of the color measured. middle ear cholesteatoma in relation to middle ear mucosa and The cells were grown in 96-well plates (Part 890218) with 12 strips of 8 walls coated with a mouse monoclonal antibody against auditory meatal skin. Further, Adamczyk et al. showed, by FGF-2. After 16, 24, 48 and 72 hours of incubation with 20 ng/ml immunohistochemistry, that EACC has enhanced expression HGF/SF, the expression of the FGF-2 protein in the supernants of of TGF-·, another potent angiogenic factor (18). the HGF/SF-treated and untreated culture cell lines was analyzed. Many authors reported that growth of several tumors is Concurrently, cultured EACC were incubated with different levels induced by HGF/SF, c-Met, VEGF and FGF-2 (5, 10, 11). of HGF/SF (20-80 ng/ml) for 24 hours. The assay recognized all The aim of this study was to explore the effect of HGF/SF different forms of FGF-2 (Figure 1a). on the matrix tissue of EACC with respect to the Western blot analysis. For Western blot analysis, the protein extracts concentration of FGF-2. To the best of our knowledge, we of the cholesteatoma cell culture were separated in a Tris-glycine are the first to grow EACC in vitro by removing gel (Invitrogen, Carlsbad, CA, USA) and transferred onto a mesenchymal tissue including fibroblasts from purified polyvinylidene difluoride membrane. Western blot analysis was endothelial cell culture (19). We present data that HGF/SF performed, as described by Zhang (20). influences the FGF-2 concentration, which might be important in the pathogenesis of EACC. Statistical analysis. The statistical analysis was calculated according to Bortz et al. (21). This method enabled the calculation of the p-value (p<0.05) for our small collective and data. Materials and Methods Results Tissue collection, isolation and culture of human skin keratinocytes and cholesteatoma culture. All EACC cell cultures were used at passage 3. Incubation up to 72 hours with 20 ng HGF/SF. After 16 hours All cells were obtained from 5 patients undergoing reconstructive of incubation with HGF/SF, FGF-2 was measured at 44.19 ear canal surgery at the Department of Otorhinolaryngology of the University of Mannheim, Germany. (Prior to surgery, written pg/ml. In untreated EACC (control), the level of FGF-2 was consent was obtained from all patients to take tissue samples of the 42.24 pg/ml. After 24 hours, the treated EACC culture resected cholesteatoma. This study was approved by the Ethics showed 46.36 pg/ml, while the control was 36.52 pg/ml. committee of the Faculty of Clinical Medicine, Mannheim, After 48 hours, the difference between the treated EACC University of Heidelberg, Germany). After removal of connective and control was less than 13% (24.79 pg/ml and 28.42 pg/ml, tissue, skin and cholesteatoma samples were cut into small pieces respectively). Cells incubated for 72 hours with HGF/SF had and incubated in a trypsin solution (0.25% trypsin in phosphate- 23.41 pg/ml FGF-2, whereas in untreated EACC culture buffered saline, PBS) overnight at 4ÆC. For primary culture of keratinocytes and cholesteatoma cells, the suspension of epidermal 14.83 pg/ml was measured. The proliferation assay, based cells was added onto mitomycin-treated (23.9 ÌM) human fibroblast on determination of total protein (Bradford), showed a slow monolayers and cultured in FAD2-medium (Dulbecco’s Modified growth of the EACC culture (311 Ìg/ml to 414 Ìg/ml). Eagle Medium and Ham’s F12 in a 3:1 ratio supplemented with fetal Comparing total protein content and the fraction of FGF-2 calf serum, adenine, insulin, triiodothyronin hydrocortisone, at different intervals of time, the concentration of FGF-2 epidermal growth factor, cholera toxin and penicillin/streptomycin decreased in untreated EACC; however, HGF/SF-incubated at 37ÆC in a 10% CO atmosphere). On reaching subconfluency, the 2 EACC culture showed an increase of FGF-2 protein feeder layer was removed by incubation with 0.02% ethylenediamine tetraacetic acid (EDTA) in PBS for 4 min at 37ÆC and the concentration as a fraction of total protein content. There cholesteatoma cells were further cultured in keratinocyte growth was a decrease of FGF-2 after 48 hours, which stagnated up medium (KGM, Clonetics, San Diego, CA, USA) without serum.