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Context-dependent tight regulation of collagen XVII ectodomain shedding in skin
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Abstracts / Journal of Dermatological Science 84 (2016) e1–e88
Pathogenic and non-pathogenic anti-Dsg1 monoclonal Abs (mAbs) were previously isolated as single chain variable fragments (scFvs) by phage display. However, it is unknown how each anti-Dsg1 mAb works for blister formation under polyclonal condition in vivo. In this study, we focused on the difference of a single mAb and a combination of mAbs on blister formation in the aspect of the Dsg1 distribution, desmosomal structure by using organ culture human skin injection assay. At first we created IgG form antibodies from scFv to reflect patient serum condition. A single pathogenic anti-Dsg1 IgG mAb caused subcorneal blister after 22 h injection and immunofluorescence showed IgG mAb deposited and Dsg1 localized linearly at the cell surfaces of keratinocytes. When a pathogenic and a non-pathogenic anti-Dsg1 mAbs were injected together, IgG deposits and Dsg1 localization showed an aberrant granular pattern at basal and spinous layers. This Dsg 1 clustering needed cross-linking of Dsg1 and trans-interaction blocking by a pathogenic IgG Ab. Electron microscopy revealed that a combination of pathogenic and non-pathogenic anti-Dsg1 mAbs shortened desmosome length more than a single mAb at the basal and spinous layers. In addition, dissociation assay showed a combination of pathogenic and non-pathogenic mAbs inhibited cell adhesion of keratinocytes more than a single pathogenic mAb. These findings indicated that a combination of PF mAbs induced Dsg1 clustering which may cause desmosome morphology change and promote loss of cell adhesion of keratinocytes synergistically. In conclusion, polyclonal condition of the anti-Dsg 1 Ab influences the pathogenicity in PF. http://dx.doi.org/10.1016/j.jdermsci.2016.08.087 P03-03[C08-07] Context-dependent tight regulation of collagen XVII ectodomain shedding in skin Wataru Nishie ∗ , Ken Natsuga, Kentaro Izumi, Hideyuki Ujiie, Hiroo Hata, Hiroshi Shimizu Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Pemphigoid is a common autoimmune blistering disorder in which autoAbs target transmembrane collagen XVII (COL17), a component of hemidesmosomes in basal keratinocytes. The ectodomain of COL17 can be cleaved from the cell surface within the juxtamembranous extracellular NC16A domain, and interestingly, certain autoAbs of pemphigoid patients preferentially react with the shed ectodomain. These findings suggest that COL17 ectodomain shedding generates neoepitopes on the shed form; however, the regulatory mechanism of the shedding in in vivo skin and the pathogenicity of the neoepitope-targeting antibodies are still uncertain. To address these issues, we produced rabbit Abs specifically reacting with N-terminal cleavage sites of the shed COL17 ectodomain. The Abs revealed that certain amount of human COL17 ectodomain is physiologically cleaved at Gln525 in in vivo skin. Interestingly, migrating human keratinocytes cleave COL17 at Leu524 but not at Gln525 in vitro as well as in vivo, suggesting that the cleavage of COL17 is differentially regulated in a contextdependent manner. To address the pathogenic roles of the Abs to neoepitopes on the physiological cleavage sites, the passive transfer of Abs reacting with an N-terminal cleavage site of mouse COL17 ectodomain into neonatal wild-type mice was performed. The experiment failed to induce blister formation, even though the Abs bound to the dermal-epidermal junctions, indicating that cleavage-site-specific Abs have reduced or absent pathogenicity for blister formation. This study shows the ectodomain shedding of COL17 to be a tightly regulated physiological event in in vivo
human skin that probably generates non-pathological epitopes on the cleavage sites. http://dx.doi.org/10.1016/j.jdermsci.2016.08.088 P03-04[C08-08] Biological effect of vascular endothelial growth factor-C on culture lymphatic endothelial cells Satoshi Hirakawa 1,∗ , Manami Iwasaki 1 , Akihiro Nishiguchi 2 , Michiya Matsusaki 2 , Mitsuru Akashi 2 1
Department of Dermatology, Hamamatsu University School of Medicine, Japan 2 Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Japan Vascular endothelial growth factors (VEGFs) promote new lymphatic vessel growth in mammalian development and pathological condition. Among the VEGF family members, VEGF-C plays a key role in promoting lymphangiogenesis. Recent studies showed that experimental animal models reveal the potent effect of VEGF-C in several pathological conditions such as tumor progression. Therefore, we initially isolated cultured LEC from human skin to show the molecular mechanism of VEGF-C-mediated lymphangiogenesis. However, cultured LEC showed a response to highly- concentrated recombinant VEGF-C in mono-layered culture condition. Therefore, we decided to generate a novel three-dimensional culture system that gives rise to the biological effect of recombinant VEGF-C to cultured LEC. Initially, cultured LEC were seeded on the fibroblasts. Thereafter, those cells were incubated for 48 h in the presence or absence of recombinant VEGF-C. Our three dimensional tissues showed that cultured LEC develop highly organized networks in the presence of VEGF-C at a physiological concentration as compared with controls. Therefore, we next assessed the biological effect of recombinant VEGF-A by the three dimensional tissue analysis. Cultured LEC markedly developed capillary-sized vascular networks in the presence of VEGF-A, whereas these networks were highly condensed and enlarged, demonstrating a pathological condition. Therefore, our results indicate, for the first time, that physiological lymphatic vessel growth is induced by VEGF-C in the three dimensional tissue, and that our novel culture system may clarify the biological mechanism of lymphangiogenesis in physiological and pathological condition. http://dx.doi.org/10.1016/j.jdermsci.2016.08.089 P03-05 Inhibitory action against fibrosis in systemic sclerosis by apelin Yoko Yokoyama ∗ , Akihiko Uchiyama, Kazuya Yamada, Sachiko Ogino, Osamu Ishikawa, Sei-ichiro Motegi Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan The secreted protein apelin and its receptor APJ signaling mainly regulates angiogenesis and cardiovascular functions. Recently, it has been identified that apelin: APJ signaling also regulates cardiac and arterial fibrosis, and is associated with the pathogenesis of pulmonary arterial hypertension. However, the mechanisms and roles of apelin in skin fibrosis in systemic sclerosis (SSc) have not been well characterized. The aim of this study was to elucidate the
Abstracts / Journal of Dermatological Science 84 (2016) e1–e88
Pathogenic and non-pathogenic anti-Dsg1 monoclonal Abs (mAbs) were previously isolated as single chain variable fragments (scFvs) by phage display. However, it is unknown how each anti-Dsg1 mAb works for blister formation under polyclonal condition in vivo. In this study, we focused on the difference of a single mAb and a combination of mAbs on blister formation in the aspect of the Dsg1 distribution, desmosomal structure by using organ culture human skin injection assay. At first we created IgG form antibodies from scFv to reflect patient serum condition. A single pathogenic anti-Dsg1 IgG mAb caused subcorneal blister after 22 h injection and immunofluorescence showed IgG mAb deposited and Dsg1 localized linearly at the cell surfaces of keratinocytes. When a pathogenic and a non-pathogenic anti-Dsg1 mAbs were injected together, IgG deposits and Dsg1 localization showed an aberrant granular pattern at basal and spinous layers. This Dsg 1 clustering needed cross-linking of Dsg1 and trans-interaction blocking by a pathogenic IgG Ab. Electron microscopy revealed that a combination of pathogenic and non-pathogenic anti-Dsg1 mAbs shortened desmosome length more than a single mAb at the basal and spinous layers. In addition, dissociation assay showed a combination of pathogenic and non-pathogenic mAbs inhibited cell adhesion of keratinocytes more than a single pathogenic mAb. These findings indicated that a combination of PF mAbs induced Dsg1 clustering which may cause desmosome morphology change and promote loss of cell adhesion of keratinocytes synergistically. In conclusion, polyclonal condition of the anti-Dsg 1 Ab influences the pathogenicity in PF. http://dx.doi.org/10.1016/j.jdermsci.2016.08.087 P03-03[C08-07] Context-dependent tight regulation of collagen XVII ectodomain shedding in skin Wataru Nishie ∗ , Ken Natsuga, Kentaro Izumi, Hideyuki Ujiie, Hiroo Hata, Hiroshi Shimizu Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Pemphigoid is a common autoimmune blistering disorder in which autoAbs target transmembrane collagen XVII (COL17), a component of hemidesmosomes in basal keratinocytes. The ectodomain of COL17 can be cleaved from the cell surface within the juxtamembranous extracellular NC16A domain, and interestingly, certain autoAbs of pemphigoid patients preferentially react with the shed ectodomain. These findings suggest that COL17 ectodomain shedding generates neoepitopes on the shed form; however, the regulatory mechanism of the shedding in in vivo skin and the pathogenicity of the neoepitope-targeting antibodies are still uncertain. To address these issues, we produced rabbit Abs specifically reacting with N-terminal cleavage sites of the shed COL17 ectodomain. The Abs revealed that certain amount of human COL17 ectodomain is physiologically cleaved at Gln525 in in vivo skin. Interestingly, migrating human keratinocytes cleave COL17 at Leu524 but not at Gln525 in vitro as well as in vivo, suggesting that the cleavage of COL17 is differentially regulated in a contextdependent manner. To address the pathogenic roles of the Abs to neoepitopes on the physiological cleavage sites, the passive transfer of Abs reacting with an N-terminal cleavage site of mouse COL17 ectodomain into neonatal wild-type mice was performed. The experiment failed to induce blister formation, even though the Abs bound to the dermal-epidermal junctions, indicating that cleavage-site-specific Abs have reduced or absent pathogenicity for blister formation. This study shows the ectodomain shedding of COL17 to be a tightly regulated physiological event in in vivo
human skin that probably generates non-pathological epitopes on the cleavage sites. http://dx.doi.org/10.1016/j.jdermsci.2016.08.088 P03-04[C08-08] Biological effect of vascular endothelial growth factor-C on culture lymphatic endothelial cells Satoshi Hirakawa 1,∗ , Manami Iwasaki 1 , Akihiro Nishiguchi 2 , Michiya Matsusaki 2 , Mitsuru Akashi 2 1
Department of Dermatology, Hamamatsu University School of Medicine, Japan 2 Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Japan Vascular endothelial growth factors (VEGFs) promote new lymphatic vessel growth in mammalian development and pathological condition. Among the VEGF family members, VEGF-C plays a key role in promoting lymphangiogenesis. Recent studies showed that experimental animal models reveal the potent effect of VEGF-C in several pathological conditions such as tumor progression. Therefore, we initially isolated cultured LEC from human skin to show the molecular mechanism of VEGF-C-mediated lymphangiogenesis. However, cultured LEC showed a response to highly- concentrated recombinant VEGF-C in mono-layered culture condition. Therefore, we decided to generate a novel three-dimensional culture system that gives rise to the biological effect of recombinant VEGF-C to cultured LEC. Initially, cultured LEC were seeded on the fibroblasts. Thereafter, those cells were incubated for 48 h in the presence or absence of recombinant VEGF-C. Our three dimensional tissues showed that cultured LEC develop highly organized networks in the presence of VEGF-C at a physiological concentration as compared with controls. Therefore, we next assessed the biological effect of recombinant VEGF-A by the three dimensional tissue analysis. Cultured LEC markedly developed capillary-sized vascular networks in the presence of VEGF-A, whereas these networks were highly condensed and enlarged, demonstrating a pathological condition. Therefore, our results indicate, for the first time, that physiological lymphatic vessel growth is induced by VEGF-C in the three dimensional tissue, and that our novel culture system may clarify the biological mechanism of lymphangiogenesis in physiological and pathological condition. http://dx.doi.org/10.1016/j.jdermsci.2016.08.089 P03-05 Inhibitory action against fibrosis in systemic sclerosis by apelin Yoko Yokoyama ∗ , Akihiko Uchiyama, Kazuya Yamada, Sachiko Ogino, Osamu Ishikawa, Sei-ichiro Motegi Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan The secreted protein apelin and its receptor APJ signaling mainly regulates angiogenesis and cardiovascular functions. Recently, it has been identified that apelin: APJ signaling also regulates cardiac and arterial fibrosis, and is associated with the pathogenesis of pulmonary arterial hypertension. However, the mechanisms and roles of apelin in skin fibrosis in systemic sclerosis (SSc) have not been well characterized. The aim of this study was to elucidate the