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-36- Matrix(1) 9/4 Room-C
EFFECTS OF MECHANICAL TENSION ON GLYCOSAMINOGLYCANS PRODUCED BY HUMAN SKIN FIBROBLASTS IN COLLAGEN LATTICE.
Masayoshi Yamanaka, Kazunori Ohnishi, Yoshiki Miyachi.
Department of Dermatology, Gunma University School of Medicine,Maebashi, Japan.
Mechanical tension to fibroblasts cultured in collagen lattice changes the cell growth and production of extracellular matrix components such as collagen. We studied the changes of glycosaminoglycans (GAGs) produced by human fibroblasts in collagen lattice. Culture in collagen lattice left attached to the dish was used as a high tension model and that detached from the dish was used as a low tension model. After 4 days culture in collagen lattices,media and lattices were collected separately and GAGs were extracted. After digestion with chondroitinase ABC or ACII, each disaccharide was quantitatively analyzed using high performance capillary electrophoresis. In the high tension model, all disaccharides, especially ŽÆDi-HA, increased in both collagen lattice and medium as compared with those in the low tension model. Mechanical tension stimulates fibroblasts to produce more GAGs.
-37- Matrix(1) 9/4 Room-C
INFLUENCE OF HYPERTHERMALLY CULTURED KERATINOCYTES ON THE FUNCTION OF DERMAL FIBROBLASTS.
Norihiro Fujimoto1, Yoshiyasu Itoh2, Shingo Tajima2, Akira Ishibashi2.
1Department of Dermatology, Self Defense Force Central Hospital, Tokyo, 2Department of Dermatology, National Defense Medical College, Saitama, Japan.
We have reported that hyperthermal therapy can prevent delayed wound healing. To know the mechanism of the therapy, we analyzed whether the conditioned media of keratinocytes cultured at different temperatures (30,34,37,39¡î) possess differential modulatory effects on mitogenic and biosynthetic phenotypes of dermal fibroblasts. The conditioned medium of the keratinocytes cultured at 39¡î showed a greater inhibitory activity for fibroblast proliferation and greater synthetic activities of collagen and glycosaminoglycans than those incubated at 30,34 or 37¡î . Transforming growth factor (TGF)¦Â1 production in skin fibroblasts was also stimulated by the conditioned medium of the keratinocytes cultured at 39¡î. The results indicate that keratinocytes cultured at hyperthermal conditions can modulate the phenotypes of dermal fibroblasts.
-38- Matrix(1) 9/4 Room-C
EXPRESSION OF FIBRILLIN-1 IN CULTURED HUMAN KERATINOCYTES.
Yoshihiro Ohnishi, Hiroyoshi Kajiya, Shingo Tajima, Akira Ishibashi.
Department of Dermatology, National Defense Medical College, Saitama, Japan.
Elastic fibers consists of two structurally different components, elastin and microfibrillar glycoproteins. Fibrillin-1 is a major constituent of microfibrillar glycoproteins. We have previously reported that cultured human keratinocytes actively express elastin and that the expression is closely related to keratinization.In the present studies, we analized whether fibrillin-1 was expressed by cultured human keratinocytes. RT- PCR assays showed the presence of fibrillin-1 mRNA in the cultured keratinocytes. When the terminal differentiation was induced by the addition of 2.0mM Ca++, the keratinized cells reacted with antifibrillin antibody. Immunohistochemical studies on skin specimen revealed that fibrillin-1 localized mostly at the granular layer of the epidermis, indicating the same localization as epidermal elastin. These results strongly suggest that elastin and fibrillin-1 are interact in the epidermis resulting in the formation of elastic fibers.
-39- Matrix(1) 9/4 Room-C
DISTRIBUTION OF TYPE XV COLLAGEN IN NORMAL SKIN AND SKIN TUMORS DETECTED BY MONOCLONAL ANTIBODY TO TYPE XV COLLAGEN.
Tomoko Fukushige1, Tamotsu Kanzaki1, Takashi Shinya2.
1Department of Dermatology, Kagoshima University Faculty of Medicine, Kagoshima, 2Fuji Chemical Industries, Biopharmaceutical Department, Ltd, Takaoka, Japan.
The collagen family of proteins consists of 19 types encoded by 33genes. One of the more recently discovered collagen is the ¦Á1 chain of type XV. We produced a polypeptide of a noncollagenous part of ¦Á1 chain and monoclonal antibodies were made by a hybridoma technique using the polypeptide as an antigen. We immunohistochemically investigated the localization of type XV collagen in normal skin and various skin tumors to elucidate the possible function of collagen XV. Type XV exhibited a surprisingly restricted and strong association with epidermal, vascular, neural,and mesenclymal basement membrane zones. Type XV collagen was associated with benign skin tumors but less than in normal counterparts and much diminished in malignant tumors. This suggests that type XV collagen may have a close association with type IV collagen producing lamina densa of basement membranes and the amount is related with cellular function.
-40- Matrix(1) 9/4 Room-C
PREFERENTIAL EXPRESSION OF a2 AND a4 CHAINS OF TYPE iv COLLAGEN IN THE DEVELOPING HUMAN FETAL SKIN.
Nobuhiko Tanaka1, Shingo Tajima1, Akira Ishibashi1, Yoshikazu Sado2, Yoshifumi Ninomiya3.
1Department of Dermatology, National Defense Medical College, Saitama, 2Division of Immunology, Shigei Medical Research Institute. 3Department of Molecular Biology and Biochemistry, Okayama University Medical School, Okayama, Japan.
Localization of ¦Á1-¦Á6 chains of type ª¤ collagen in the developing skin was studied by use of chain-specific monoclonal antibodies. On 10-weeks of gestation, the antibody for ¦Á2 and ¦Á4 chains stained the base- ment membranes of dermal-epidermal junction (DEJ), and no staining was observed with the other (¦Á1,¦Á3,¦Á5 and ¦Á6) chain antibodies. On 20-weeks of gestation, the antibodies showed essentially similar staining patterns to those of adult skin; the antibodies for ¦Á1 and ¦Á2 chain stained the basement membranes of both DEJ and capillaries, the antibodies for ¦Á5 and ¦Á6 chain stained only the basement membrane of DEJ, and no staining was observed with ¦Á3 and ¦Á4 chain antibodies. The results indicate that the expression of ¦Á2 and ¦Á4 chains occured preferentially in the early phase of skin development and that ¦Á4 chain disappeared with further development. Chain-specific expression of ¦Á2 and ¦Á4 chains during early phase of development may suggest their potential role in basement membrane formation.
-41- Matrix(1) 9/4 Room-C
EXOGENOUS LAMININ 5 CAN PROMOTE THE ASSEMBLY OF LAMINA DENSA IN THE ORGANOTYPIC CULTURE OF SKIN.
Makoto Tsunenaga1,Eijirou Adachi2, Satoshi Amano1, Yasuhisa Nakayama1, Robert E. Burgeson3, Toshio Nishiyama1.
1Shiseido Research Center, 2Department of Anatomy, Kitasato University, Japan. 3MGH/Harvard Cutaneous Biology Research Center, Boston, MA.
Although major components of the lamina densa were revealed along the epidermal-dermal junction in the skin model, the lamina densa, as the supramolecular strucuture, could rarely be observed. Therefore, we examined whether exogenous laminin-5 can promote the assembly of lamian densa in the skin model or not, because the concentration of the components might be too low to form the lamina densa. Human keratinocytes were seeded onto the surface of the gels of collagen I contracted by human skin fibroblasts and lifted at the air-liquid interface. The keratinocytes formed the stratified squamous epithelium within 7 days and was continued to culture for additional 7 days under supplementing laminin 5 at the concentrations of 0, 1, 5 and 20 mg/ml. A lot of patches of the lamina densa with or without microfibrils were observed along the epidermal-dermal junction and also in secretory granules, approximately 0.6 mm in diameter, in basal keratinocytes. Morphometric analysis suggest that the total length of the lamina densa along the epidermal-dermal junction increased depending on the concentration of laminin 5 supplemented. Immuno-electron microscopy revealed that collagen IV, laminin 1 and 5, and fibrillin 1 were localized on the lamina densa, and on microfibrils, respectively. Based on these obserbations, exogenous laminin 5 can promote the formation of the lamina densa and microfibrils in the skin model.