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Detection of lysyl oxidase gene expression in rat skin during wound healing
Poster Presentations
81
408 ClXlDASE IN THE SKIN. Noriko Yashiro, Hiroyo Miyaka Chanoki, Hiromi Kobayashi, Tomoyuki Hisa, Shall Taniyuchl, Masamitsu Ishii, Toshio Hamada and nkira ooShima*, Department of Dermatology, Osaka City University Wdlcal School, Osaka, Of Pathology, HUMAN Fushida,
LYSYL
Japan,*Department
Lysyl
oxidase initiates the cross-linking of collagen and elastin by catalyzing the formation of the lysynederived aldehyde. The activxty of lysyl oxidase in fibrotic conditions or inherited connective tissue diseases haS been reported. However, little is known about the localization of It in human Sk=". We investigated the localization of 1ySyl oxidase in normal human skin and cultured human keratlnocyte usxng indirect immunofluorescence method with monoclonal antI-human lysyl oxidase antibody. Fine fllamentous or granular posrtive immunoreactions were noted on or amonq collagen or elastic fibers. Intracellular d1strlbut1on of basal cells, endothelial cells and sweat elands also observed as well as that of fibroblasts. The cultured human keratinocytes exhibited a immunoreactlon with a filasentous cytoskeletal protein-like structure. TO rule out another possibility that certain proteins share haptens, total human keratinocytes RNAs were subjected to Northern analysrs. we detected the expression of lysyl oxidaae gene in human keratinocyto. The results suggest that lysyl oxldase would have other functions besides initiation of cross-linking in collagen and elastin.
I
I
409
406 LYSVL OXlDbSE GENE EXPRESSION IN RAT 1OUND HEALING. Hiroyo Fushlda' Ylchlo Naokl Yaekawa ' Noriko Vashlro' Hiroml liyako Chanoki' Yasamitsu Ishi Tashio Hamada and Syuzo Otarn'Dermatology and ' Blocheaistry. Osaka City Unlverslty Yedlcal School. Osaka. lapan. Ch,y” Shlota and bkira O”sh,sa Department of Pathology. “akayaaa Yed,ca, College. lakayaaa. Japan I.ysyl oxidase initlatcs the cross IInking of collage" and elastin by catalyzing the formation of the lysine-derived aldehyde. and plays an essentla, role for maturation “f collagen and therefore for wound hea,,ng. Ilthough the activity of th,s enzyme has bee" examined I" various disorders. the blologlc slgnlflcance and the function in tissues are still unknown. "e detected lysy, “xidase gene expression us,n~ Northern blot hybridization and w hybridtzatxon method I” rat skin during wound healing. Yoreover. it* lacalieation was ,nvert>aated by the ,~murlofl"orescence method. LYSY, oxndase .INI level reached a peak at 4 day and returned t" the eontro, level at 7 day after injury. In Sl," hybridization shored ~ralns corresponding to itsmRNd flbroblasts around the granulomatous tissue. It 1s s"pp*sed that these results are fundamentally important and useful for the better understand,ng of collagen metabo,,sm ,n YIVU. and further elucidation of fibrotic dlborders. DETECTION SKIN DURING Fukuda' Kobayash'i
ADHESION OF EPIDERMAL LANGERHANS CELLS TO VARIOUS EXTRACELLULAR MATRIX PROTEINS MIGHT INFLUENCE THEIR MIGRATION BEHAVIOR. Mane-Jeanne Stasuet. Yasunobu Kobavashi. Cola& m S hmut. Dermatology Research Umt. INSERM U346. CNRS SD1 6199, E. Hemot Hzpipital, Lyon, France It IS clear that ep~dermal Lagerhans cells (LC) are capable of mo”ement and of leavmg the eptdemus. Cell motihty depends on the abdity of cells to idably, attach and change shape to response to spectftc extracellular matrix (ECM) components. On the way b regtonal lymph nodes, while passmg through the baement membrane(BM), LC get m contact wth lammm (LM) and type IV collagen (Coil) at flrsl and then wth fibrooectin (FN) and ,ype I collagen I” connectwe tissue of dermw. The present study was undertaken to learn more about the adhesive interacttons between human LC and these ECM protems. For this purpose, using htghly ennched LC suspens~oos (70-908 LC), double adhesion assays were developed for the evaluation of the abibty of LC to successwely bmd to two dlfferent substrates. If bmdmg assays were first performed on Uv-ccared plates. the recovered attached LC were then plated on FW. type IV- and type I Coll~coated duhes. EachoFrhe fo,,rECM proteu,swere lhustesti ,n pars toarmlyse Ihe s"wcss~ve bmding capacmes of LC. Our resuh.s show that I, tie LC wbxh lirst attach to LM are then able to attach !n type IV Coil and vice versa. II) LC wluch adhere to LM or type IV Cdl are then able to attach LD FN and 10 lype I Coil. 1”) LC whxh f,rst adhere to FN or type Co11 preserve then abdlty IO normally arlach to type IV Co,,, ii”) !n contrast, the bmdmg capaory to LM of LC which hrst adhere 10 I%! w type Co11 IS reduced by 50%. These data mdrcate that followmg contact 10 basement membrane components, ep*dermaJ LC are able to successwely attach to ECM proteins present in the dermis whereas LC. once attached to dermis ECM protans, decrease their bmdmg capaaty to BM. espectally to lamlmn ,ntemct!ons wth the ECM env~rooment seem to play a crucial role I” the dlrccted rmgrat,o” of ep,demtal LC from the skm to prox,mal lymph n&r
OF
’
DepartmentsOf
THE EFFECT OF PLATELET-DERIVE,? GRO%IH FACTOR UPON CELLULAR INFILTRATION AND YASCULARISATION OF A CUTANEOUS COLLAGENOUS IMPLANT IN THE RAT. Peter M. Royce, Ken-ichl Ohsaki, Toshiyasu Kate and Keizaburo" Miki. Institute of Biomedical Science. Terumo R. B D. Centre, Nakai, Kanagawa, Japan. The "se of collagenous implants offers one potential treatment of full thickness skin lesions, although results may not be ideal. We have investigated the effect of the recombinant BB homodlmer of human platelet-derived growth factor (PDGF) upon wound healing following the implantation into full thickness excision wounds in the dorsal skin of rats, of a trial bovine atelocollagen sponge (collagen l:III, ca. 9:l) consisting of both fibrous and denatured collagen (9:l). stabilised by dehydrothermal crosslinking, and with an outer silicone layer. Before implantation, sponges were soaked in saline aione, or I" saline containing PDGF, in dmD"nt such that the entIre volume of liquid was absorbed Histological examination of excised sponges at 14 and 21 days postimplantation, in dose-response studies utilislng between 0-4119 PDGF per w"nd. showed an enhanced infiltration of host cells, mostly fibroblasts, and enhanced capillary formatlon. under the rnfluence of PDGF. the greatest response being observed with 4~9 of this cytokine. The proportion of inflammatory cells invading the sponge was markedly less with PDGF. althouoh d mild eosinoohilia was anparent. Desplt6 the e&anced vascularihatlon, brombheoxyurxd?ne sta?nlng demonstrated that the process still did not occur sufficiently rapidly to allow the survival of epithelial cells in split-thickness skin grafts superimposed upon sponges lacking do outer sillcone layer at the time of their implantation.
BULLOSA IDDEB). Kazoo Nomura, Dalsuke Sawamura,Atsushi Ken, Ken Harada, Hajime Nakano. Isao Hashlmoto and Jouni Ultto. Hirosakl Unlverslty School of Department of Dermatoloqy, Department of Dermatology, Medlclne, Hlrosakl. Japan,
Jefferson Medical College. Philadelphia, PA. Type "II collage" is the major collagenous component of the anchorxnq frbrils. Recent study sugqested there are two the human type "II collagen intraqenlc polymorphisms I" gene. In this study, we determined the alleleic frequencies of P"uII and Alul polymorphism in the Japanese population and attempted to establzsh the genetic lrnkaqe between DDEB and type "II collagen gene. Genom~c DNA was isolated from 40 unrelated healthy lndlvlduals and PCR was performed. The P""lI and Al"1 alleli. frequencres are 0.45 and 0.36, respectively. Next,we examined these two polymorphxsms I" a Japanese fam11y with DDEB. In this family, the affected lndlvlduals had blisters and erosions with onset shortly after birth. Electron m1croscoprc abservatlon showed the Separation ,uSt beneath the basal lamlna and the reduced number of S"ChOrilIq flbrils. PVUII and Al"1 RFLP were analyzed 1n 9 affected and 13 unaffected ,ndl"lduals. TWO -poxnt llnkaqe analysis revealed co-Segreqatlon Of P""lI and AluE poiymorphrsms in thus pedigree CZ=2.15 These data suggest that the type "II collage" 1s the candidate qene for this Japanese famrly vlth DDEB.
; 8=0.00).
81
408 ClXlDASE IN THE SKIN. Noriko Yashiro, Hiroyo Miyaka Chanoki, Hiromi Kobayashi, Tomoyuki Hisa, Shall Taniyuchl, Masamitsu Ishii, Toshio Hamada and nkira ooShima*, Department of Dermatology, Osaka City University Wdlcal School, Osaka, Of Pathology, HUMAN Fushida,
LYSYL
Japan,*Department
Lysyl
oxidase initiates the cross-linking of collagen and elastin by catalyzing the formation of the lysynederived aldehyde. The activxty of lysyl oxidase in fibrotic conditions or inherited connective tissue diseases haS been reported. However, little is known about the localization of It in human Sk=". We investigated the localization of 1ySyl oxidase in normal human skin and cultured human keratlnocyte usxng indirect immunofluorescence method with monoclonal antI-human lysyl oxidase antibody. Fine fllamentous or granular posrtive immunoreactions were noted on or amonq collagen or elastic fibers. Intracellular d1strlbut1on of basal cells, endothelial cells and sweat elands also observed as well as that of fibroblasts. The cultured human keratinocytes exhibited a immunoreactlon with a filasentous cytoskeletal protein-like structure. TO rule out another possibility that certain proteins share haptens, total human keratinocytes RNAs were subjected to Northern analysrs. we detected the expression of lysyl oxidaae gene in human keratinocyto. The results suggest that lysyl oxldase would have other functions besides initiation of cross-linking in collagen and elastin.
I
I
409
406 LYSVL OXlDbSE GENE EXPRESSION IN RAT 1OUND HEALING. Hiroyo Fushlda' Ylchlo Naokl Yaekawa ' Noriko Vashlro' Hiroml liyako Chanoki' Yasamitsu Ishi Tashio Hamada and Syuzo Otarn'Dermatology and ' Blocheaistry. Osaka City Unlverslty Yedlcal School. Osaka. lapan. Ch,y” Shlota and bkira O”sh,sa Department of Pathology. “akayaaa Yed,ca, College. lakayaaa. Japan I.ysyl oxidase initlatcs the cross IInking of collage" and elastin by catalyzing the formation of the lysine-derived aldehyde. and plays an essentla, role for maturation “f collagen and therefore for wound hea,,ng. Ilthough the activity of th,s enzyme has bee" examined I" various disorders. the blologlc slgnlflcance and the function in tissues are still unknown. "e detected lysy, “xidase gene expression us,n~ Northern blot hybridization and w hybridtzatxon method I” rat skin during wound healing. Yoreover. it* lacalieation was ,nvert>aated by the ,~murlofl"orescence method. LYSY, oxndase .INI level reached a peak at 4 day and returned t" the eontro, level at 7 day after injury. In Sl," hybridization shored ~ralns corresponding to itsmRNd flbroblasts around the granulomatous tissue. It 1s s"pp*sed that these results are fundamentally important and useful for the better understand,ng of collagen metabo,,sm ,n YIVU. and further elucidation of fibrotic dlborders. DETECTION SKIN DURING Fukuda' Kobayash'i
ADHESION OF EPIDERMAL LANGERHANS CELLS TO VARIOUS EXTRACELLULAR MATRIX PROTEINS MIGHT INFLUENCE THEIR MIGRATION BEHAVIOR. Mane-Jeanne Stasuet. Yasunobu Kobavashi. Cola& m S hmut. Dermatology Research Umt. INSERM U346. CNRS SD1 6199, E. Hemot Hzpipital, Lyon, France It IS clear that ep~dermal Lagerhans cells (LC) are capable of mo”ement and of leavmg the eptdemus. Cell motihty depends on the abdity of cells to idably, attach and change shape to response to spectftc extracellular matrix (ECM) components. On the way b regtonal lymph nodes, while passmg through the baement membrane(BM), LC get m contact wth lammm (LM) and type IV collagen (Coil) at flrsl and then wth fibrooectin (FN) and ,ype I collagen I” connectwe tissue of dermw. The present study was undertaken to learn more about the adhesive interacttons between human LC and these ECM protems. For this purpose, using htghly ennched LC suspens~oos (70-908 LC), double adhesion assays were developed for the evaluation of the abibty of LC to successwely bmd to two dlfferent substrates. If bmdmg assays were first performed on Uv-ccared plates. the recovered attached LC were then plated on FW. type IV- and type I Coll~coated duhes. EachoFrhe fo,,rECM proteu,swere lhustesti ,n pars toarmlyse Ihe s"wcss~ve bmding capacmes of LC. Our resuh.s show that I, tie LC wbxh lirst attach to LM are then able to attach !n type IV Coil and vice versa. II) LC wluch adhere to LM or type IV Cdl are then able to attach LD FN and 10 lype I Coil. 1”) LC whxh f,rst adhere to FN or type Co11 preserve then abdlty IO normally arlach to type IV Co,,, ii”) !n contrast, the bmdmg capaory to LM of LC which hrst adhere 10 I%! w type Co11 IS reduced by 50%. These data mdrcate that followmg contact 10 basement membrane components, ep*dermaJ LC are able to successwely attach to ECM proteins present in the dermis whereas LC. once attached to dermis ECM protans, decrease their bmdmg capaaty to BM. espectally to lamlmn ,ntemct!ons wth the ECM env~rooment seem to play a crucial role I” the dlrccted rmgrat,o” of ep,demtal LC from the skm to prox,mal lymph n&r
OF
’
DepartmentsOf
THE EFFECT OF PLATELET-DERIVE,? GRO%IH FACTOR UPON CELLULAR INFILTRATION AND YASCULARISATION OF A CUTANEOUS COLLAGENOUS IMPLANT IN THE RAT. Peter M. Royce, Ken-ichl Ohsaki, Toshiyasu Kate and Keizaburo" Miki. Institute of Biomedical Science. Terumo R. B D. Centre, Nakai, Kanagawa, Japan. The "se of collagenous implants offers one potential treatment of full thickness skin lesions, although results may not be ideal. We have investigated the effect of the recombinant BB homodlmer of human platelet-derived growth factor (PDGF) upon wound healing following the implantation into full thickness excision wounds in the dorsal skin of rats, of a trial bovine atelocollagen sponge (collagen l:III, ca. 9:l) consisting of both fibrous and denatured collagen (9:l). stabilised by dehydrothermal crosslinking, and with an outer silicone layer. Before implantation, sponges were soaked in saline aione, or I" saline containing PDGF, in dmD"nt such that the entIre volume of liquid was absorbed Histological examination of excised sponges at 14 and 21 days postimplantation, in dose-response studies utilislng between 0-4119 PDGF per w"nd. showed an enhanced infiltration of host cells, mostly fibroblasts, and enhanced capillary formatlon. under the rnfluence of PDGF. the greatest response being observed with 4~9 of this cytokine. The proportion of inflammatory cells invading the sponge was markedly less with PDGF. althouoh d mild eosinoohilia was anparent. Desplt6 the e&anced vascularihatlon, brombheoxyurxd?ne sta?nlng demonstrated that the process still did not occur sufficiently rapidly to allow the survival of epithelial cells in split-thickness skin grafts superimposed upon sponges lacking do outer sillcone layer at the time of their implantation.
BULLOSA IDDEB). Kazoo Nomura, Dalsuke Sawamura,Atsushi Ken, Ken Harada, Hajime Nakano. Isao Hashlmoto and Jouni Ultto. Hirosakl Unlverslty School of Department of Dermatoloqy, Department of Dermatology, Medlclne, Hlrosakl. Japan,
Jefferson Medical College. Philadelphia, PA. Type "II collage" is the major collagenous component of the anchorxnq frbrils. Recent study sugqested there are two the human type "II collagen intraqenlc polymorphisms I" gene. In this study, we determined the alleleic frequencies of P"uII and Alul polymorphism in the Japanese population and attempted to establzsh the genetic lrnkaqe between DDEB and type "II collagen gene. Genom~c DNA was isolated from 40 unrelated healthy lndlvlduals and PCR was performed. The P""lI and Al"1 alleli. frequencres are 0.45 and 0.36, respectively. Next,we examined these two polymorphxsms I" a Japanese fam11y with DDEB. In this family, the affected lndlvlduals had blisters and erosions with onset shortly after birth. Electron m1croscoprc abservatlon showed the Separation ,uSt beneath the basal lamlna and the reduced number of S"ChOrilIq flbrils. PVUII and Al"1 RFLP were analyzed 1n 9 affected and 13 unaffected ,ndl"lduals. TWO -poxnt llnkaqe analysis revealed co-Segreqatlon Of P""lI and AluE poiymorphrsms in thus pedigree CZ=2.15 These data suggest that the type "II collage" 1s the candidate qene for this Japanese famrly vlth DDEB.
; 8=0.00).