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    Keywords: GROWTH ; IN-VITRO ; proliferation ; CELL ; Germany ; MODEL ; MODELS ; VITRO ; TOOL ; GENE-EXPRESSION ; DIFFERENTIATION ; TISSUE ; TIME ; GRAFT ; CARCINOGENESIS ; CONTRAST ; KERATINOCYTES ; SKIN ; BIOLOGY ; fibroblasts ; ACID ; ELEMENT ; STAGE ; MEMBRANE ; RATES ; LIFE-SPAN ; STABILITY ; EPIDERMAL DIFFERENTIATION ; STEM-CELLS ; keratin ; INTEGRIN ; DE-NOVO ; RECONSTRUCTION ; ARCHITECTURE ; LAYER ; BASEMENT-MEMBRANE FORMATION ; RETINOIC ACID ; MATRIX ; fibroblast ; basement membrane ; BASEMENT-MEMBRANE ; collagen ; keratins ; SKIN KERATINOCYTES ; extracellular matrix ; LIFE ; SCAFFOLD ; dermal collagen ; FULL-THICKNESS ; HUMAN-HAIR FOLLICLE ; hyaluronic acid scaffold ; KERATINOCYTE GROWTH-REGULATION ; keratinocyte-fibroblast interaction ; skin equivalents in vitro ; skin ultrastructure
    Abstract: Besides medical application as composite skin grafts, in vitro constructed skin equivalents (SEs) or organotypic co-cultures represent valuable tools for cutaneous biology. Major drawbacks of conventional models, employing collagen hydrogels as dermal equivalents (DEs), are a rather poor stability and limited life span, restricting studies to early phases of skin regeneration. Here we present an improved stabilised in vitro model actually providing the basis for skin-like homeostasis. Keratinocytes were grown on dermal equivalents (DEs) reinforced by modified hyaluronic acid fibres (Hyalograft-3D) and colonised with skin fibroblasts, producing genuine dermis-type matrix. These SEs developed a superior epidermal architecture with regular differentiation and ultrastructure, which occurred also faster than in SEs based on collagen-DEs. Critical aspects of differentiation, still unbalanced in early stages, were perfectly re-normalised, most strikingly the co-expression of keratins K1/K10 and downregulation of regeneration-associated keratins such as K16. The restriction of integrin and K15 distribution as well as keratinocyte proliferation to the basal layer underlined the restored tissue polarity, while the drop of growth rates towards physiological levels implied finally accomplishment of homeostasis. This correlated to faster basement membrane (BM) formation and ultrastructurally defined dermo-epidermal junction including abundant anchoring fibrils for strong tissue connection. Whereas the fibroblasts in the scaffold initially secreted a typical provisional regenerative matrix (fibronectin. tenascin). with time collagens of mature dermis (type I and 111) were accumulating giving rise to an in vivo-like matrix with regularly organised handles of striated collagen fibrils. In contrast to the more catabolic state in conventional DEs, the de novo reconstruction of genuine dermal tissue seemed to be a key element for maintaining prolonged normal keratinocyte proliferation (followed up to 8 wks), fulfilling the criteria of tissue-homeostasis, and possibly providing a stem cell niche
    Type of Publication: Journal article published
    PubMed ID: 15679108
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  • 3
    Keywords: EXPRESSION ; CELL ; evaluation ; Germany ; MICROSCOPY ; DISEASE ; DISTINCT ; PROTEIN ; COMPONENTS ; DIFFERENTIATION ; MARKER ; IMPACT ; SKIN ; ALPHA ; MEMBRANE ; MUTATION ; REPAIR ; COMPONENT ; ABERRATIONS ; EXTRACELLULAR-MATRIX ; MUTATIONS ; BETA ; ADHESION ; CELL-ADHESION ; HUMAN KERATINOCYTES ; INTEGRIN ; epidermis ; ultrastructure ; MATRIX ; assembly ; basement membrane ; BASEMENT-MEMBRANE ; extracellular matrix ; ARRAY ; DEFECTS ; INTEGRINS ; analysis ; methods ; JAPANESE ; DEFECT ; immunoelectron microscopy ; EXTRACELLULAR-MATRIX PROTEIN-1 ; lipoid proteinosis ; microvasculature ; aberration ; DERMO-EPIDERMAL JUNCTION ; ECM1 ; HEMIDESMOSOMES ; LAMININ ISOFORMS ; LICHEN-SCLEROSUS ; skin and oral mucosa ; VII COLLAGEN
    Abstract: Background: Excessive basement membrane (BM) deposition in skin and mucosa is characteristic for lipoid proteinosis (LP; hyalinosis cutis et mucosae), an inherited disease caused by extracellular matrix protein 1 (ECM1) mutations. According to ultrastructure there are striking differences between junctional. and microvascular BM. Objective: Distinct analysis of the junctional zone in epidermis and oral mucosa, contrasting concentric BM arrays in the microvasculature; evaluation of impact on epithelial. histogenesis and differentiation, and specifically on adhesion structures to BM (hemidesmosomes). Methods: LP-epithelia were analyzed for alterations in differentiation, BM composition and texture, and hemidesmosomal components by indirect immunofluorescence (IIF), electron microscopy (EM), and immunoelectron microscopy (ImEM). Results: Most striking was the irregular deposition of collagen IV and VII, BM-laminin, and laminin-5 at the junctional. zone, accompanied by lamellate or punctuated structures below BM (IIF), whereas integrin alpha 6 beta 4 and bullous pemphigoid antigen-1 and -2 (BPAG-1/-2) were regularly aligned. Also integrins alpha 2 beta 1 and alpha 3 beta 1 remained restricted to the epidermal basal layer, while the tissue-specific differentiation markers keratin K1/10 (mucosa, additionally K4/13) appeared delayed indicating mild hyperplasia, further confirmed by focal K6/16 expression. Ultrastructure (EM) disclosed abundance of extended basal cell protrusions and junctional aberrations like exfoliating excessive BM material. Hemidesmosomes were complete, but ImEM indicated weakened interactions between their components (BRAG-1, -2, and HD1). Confirming IIF, collagen IV and VII, and laminin-5 appeared extensively scattered, the latter two probably remaining associated. Conclusions: Subtle defects in anchorage assembly, spanning the entire BM zone, apparently compromise epithelial-matrix adhesion, which may provoke (mechanical stress-induced) erroneous BM repair. (c) 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17175139
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  • 4
    Keywords: CELLS ; EXPRESSION ; proliferation ; IN-VIVO ; PROTEIN ; PROTEINS ; COMPONENTS ; MICE ; SKIN ; DELETION ; PHENOTYPES ; ADHESION ; EGF-LIKE MOTIF ; EPITHELIAL MORPHOGENESIS ; GLOBULAR DOMAINS
    Abstract: The nidogen-laminin interaction is proposed to play a key role in basement membrane (BM) assembly. However, though there are similarities, the phenotypes in mice lacking nidogen 1 and 2 (nidogen double null) differ to those of mice lacking the nidogen binding module (gamma 1III4) of the laminin gamma 1 chain. This indicates different cell- and tissue-specific functions for nidogens and their interaction with laminin and poses the question of whether the phenotypes in nidogen double null mice are caused by the loss of the laminin-nidogen interaction or rather by other unknown nidogen functions. To investigate this, we analyzed BMs, in particular those in the skin of mice lacking the nidogen binding module. In contrast to nidogen double null mice, all skin BMs in gamma 1III4-deficient mice appeared normal. Furthermore, although nidogen 1 deposition was strongly reduced, nidogen 2 appeared unchanged. Mice with additional deletion of the laminin gamma 3 chain, which contains a gamma 1-like nidogen binding module, showed a further reduction of nidogen 1 in the dermoepidermal BM; however, this again did not affect nidogen 2. This demonstrates that in vivo only nidogen 1 deposition is critically dependent on the nidogen binding modules of the laminin gamma 1 and gamma 3 chains, whereas nidogen 2 is independently recruited either by binding to an alternative site on laminin or to other BM proteins
    Type of Publication: Journal article published
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  • 5
    Keywords: IN-VITRO ; EXTRACELLULAR-MATRIX ; organotypic cocultures ; STEM-CELLS ; SQUAMOUS-CELL CARCINOMA ; HEPARAN-SULFATE PROTEOGLYCAN ; TRANSFECTED TUMORIGENIC CLONES ; EPITHELIAL MORPHOGENESIS ; CUTIS-ET-MUCOSAE ; LAMININ-5 GAMMA-2 CHAIN
    Abstract: The epidermis functions in skin as first defense line or barrier against environmental impacts, resting on extracellular matrix (ECM) of the dermis underneath. Both compartments are connected by the basement membrane (BM), composed of a set of distinct glycoproteins and proteoglycans. Herein we are reviewing molecular aspects of BM structure, composition, and function regarding not only (i) the dermoepidermal interface but also (ii) the resident microvasculature, primarily focusing on the per se nonscaffold forming components perlecan and nidogen-1 and nidogen-2. Depletion or functional deficiencies of any BM component are lethal at some stage of development or around birth, though BM defects vary between organs and tissues. Lethality problems were overcome by developmental stage- and skin-specific gene targeting or by cell grafting and organotypic (3D) cocultures of normal or defective cells, which allows recapitulating BM formation de novo. Thus, evidence is accumulating that BM assembly and turnover rely on mechanical properties and composition of the adjacent ECM and the dynamics of molecular assembly, including further "minor" local components, nidogens largely functioning as catalysts or molecular adaptors and perlecan as bridging stabilizer. Collectively, orchestration of BM assembly, remodeling, and the role of individual players herein are determined by the developmental, tissue-specific, or functional context.
    Type of Publication: Journal article published
    PubMed ID: 23586018
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  • 6
    Keywords: EXPRESSION ; COMBINATION ; GENE ; PROTEIN ; PROTEINS ; DIFFERENTIATION ; COMPLEX ; COMPLEXES ; FAMILY ; DOMAIN ; chromosome ; ACID ; intermediate filaments ; INTERMEDIATE-FILAMENTS ; CALCIUM ; epidermis ; CORNIFIED CELL-ENVELOPE ; BARRIER FUNCTION ; TERMINAL DIFFERENTIATION ; HUMAN SKIN ; intermediate filament ; keratinocyte ; HAIR FOLLICLE ; AMINO-ACID ; hair ; GENE FAMILY ; SWITZERLAND ; STRUCTURAL PROTEINS ; ENVELOPE ; fused gene ; HUMAN-CHROMOSOME 1Q21 ; KERATINOCYTE TRANSGLUTAMINASE ; LORICRIN ; lq21 ; PROFILAGGRIN ; repetin
    Abstract: The human repetin gene is a member of the "fused" gene family and localized in the epidermal differentiation complex on chromosome 1q21. The "fused" gene family comprises profilaggrin, trichohyalin, repetin, hornerin, the profilaggrin-related protein and a protein encoded by c1orf10. Functionally, these proteins are associated with keratin intermediate filaments and partially crosslinked to the cell envelope (CE). Here, we report the isolation and characterization of the human repetin gene and of its protein product. The repetin protein of 784 amino acids contains EF (a structure resembling the E helix-calcium-binding loop-F helix domain of parvalbumin) hands of the S100 type and internal tandem repeats typical for CE precursor proteins, a combination which is characteristic for "fused" proteins. Repetin expression is scattered in the normal epidermis but strong in the acrosyringium, the inner hair root sheat and in the filiform papilli of the tongue. Ultrastructurally, repetin is a component of cytoplasmic non-membrane "keratohyalin" F-granules in the stratum granulosum of normal epidermis, similar to profilaggrin. Finally, we show that EF hands are functional and reversibly bind Ca2+. Our results indicate that repetin is indeed a member of the fused gene family similar to the prototypical members profilaggrin and trichohyalin
    Type of Publication: Journal article published
    PubMed ID: 15854042
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  • 7
    Keywords: EXPRESSION ; BLOOD ; Germany ; PROTEIN ; DIFFERENTIATION ; MARKER ; EXTRACELLULAR-MATRIX ; MUTATIONS ; HUMAN KERATINOCYTES ; MORPHOLOGY ; INTEGRIN ; INVOLVEMENT ; TUMOR ANGIOGENESIS ; ultrastructure ; MATRIX ; VESICLES ; extracellular matrix ; laminin ; HISTOLOGY ; function ; LOSSES ; JAPANESE ; POLARITY ; immunoelectron microscopy ; HUMAN-SKIN ; microvascular density ; basement membrane components ; EXTRACELLULAR-MATRIX PROTEIN-1 ; GENE ECM1 ; lipoid proteinosis ; microvasculature ; PERLECAN
    Abstract: Background: In lipoid proteinosis (LP) vascular anomalies represent severe functional defects caused by excessive deposition of basement membrane (BM)-like matrix, particularly around small subepithelial blood vessels. Objective: Correlation of microvascular anomalies in morphology and ultrastructure with extracellular matrix composition and cell interactions for elucidating vascular involvement in LP-pathophysiology. Methods: Biopsies from non-related LP-patients were analyzed by indirect immunofluorescence (IIF), electron microscopy (EM), and immune-EM (ImEM). Results: In LP-skin and mucosa the thickened vessel walls stained strongly for the BM-components type IV collagen, laminin, perlecan, and nidogen (IIF). Integrin (alpha 6 beta 4 was regularly collocated with endothelial surface markers such as PECAM (CD31). Ultrastructure (EM) revealed highly ordered matrix deposits around microvessels, with frequently collapsed lumina, functionally compensated by increased vascular density (histology, IIF). Pericytes were trapped between these concentric BM-layers at varying distances towards the periphery (EM), contrasting their regularly close endothelial apposition. Periodic type IV collagen patterns (ImEM) corroborated the multiple BM-leaflet structure and the lack of a common 'fused' endothelial-pericyte BM, seen normally. Presumptive secretory vesicles, abundant in both cell types, implied an equal contribution to BM-synthesis, but also indicated partial loss of endothelial polarity. Conclusions: In LP thickened vessel walls, composed of multiple BM, profoundly alter microvascular properties, also by interference with endothelial-pericyte interactions. The increased microvascular density reflects compensatory restoration for disabled function. Most remarkable was the exaggerated secretory activity (also at luminal surfaces) underlining the regulatory key role of extracellular matrix protein 1 (ECM1; mutated in LP) in export or turnover of all major BM-components. (c) 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16497486
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  • 8
    Keywords: IN-VITRO ; CELL ; Germany ; IN-VIVO ; MICROSCOPY ; MODEL ; MODELS ; VITRO ; VIVO ; MICE ; TISSUES ; SKIN ; DYNAMICS ; BIOLOGY ; FORM ; GLYCOPROTEIN ; MEMBRANE ; REPAIR ; EXTRACELLULAR-MATRIX ; HUMAN KERATINOCYTES ; organotypic cocultures ; SQUAMOUS-CELL CARCINOMA ; RETINOIC ACID ; ultrastructure ; MATRIX ; review ; assembly ; basement membrane ; BASEMENT-MEMBRANE ; EGF-LIKE MOTIF ; MOUSE NIDOGEN-2 ; PROTEOGLYCANS ; USA ; DERMO-EPIDERMAL JUNCTION ; Genetic ; Extracellular ; CUTIS-ET-MUCOSAE ; EPIDERMAL STEM-CELLS ; Micro-vasculature ; Molecular assembly ; Molecular composition ; Nidogen function ; NIDOGEN-BINDING ; Organotypic co-culture
    Abstract: The view of extracellular matrix (ECM) has evolved from a merely scaffolding and space filling tissue element to an interface actively controlling cellular activities and tissue functions. A highly specialized form of ECM is the basement membrane (BM), an ubiquitous sheet-like polymeric structure composed of a set of distinct glycoproteins and proteoglycans. In this review we are largely focusing on function and assembly of BM in skin (1) at the dermo-epidermal interface and (2) in the resident micro-vasculature. The role of the non-polymeric components perlecan and particularly nidogen is exemplified by reviewing experiments based on genetic approaches and adequate experimental skin models in vivo and in vitro. While in mice total deficiency of one of these components is eventually developmentally lethal, the severity of the defects varies drastically between tissues and also the skin models recapitulating BM formation in vitro. There is accumulating evidence that this relies on the mechanical properties, the molecular composition of the BM, the adjacent ECM or connective tissue, the dynamics of molecular assembly, and 'minor' tissue-specific modifier or adapter components. Though the role of nidogen or perlecan is still remaining a controversial issue, the statements 'being essential for BM/or not' should be consequently referred to the developmental, tissue, and functional (e.g., repair) context
    Type of Publication: Journal article published
    PubMed ID: 19333614
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    Keywords: CELLS ; EXPRESSION ; GROWTH ; IN-VITRO ; INVASION ; carcinoma ; SITES ; GENE-EXPRESSION ; HYBRIDIZATION ; PROTEIN ; DIFFERENTIATION ; KERATINOCYTES ; fibroblasts ; TUMOR PROGRESSION ; EPITHELIAL-CELLS ; vimentin ; HACAT-RAS ; ALPHA-6-BETA-4 INTEGRIN ; atypical keratins ; BASEMENT- MEMBRANE ; CYTOKERATIN EXPRESSION ; malignant human keratinocytes ; mouse grafts ; stromal reaction ; TRANSFECTED TUMORIGENIC CLONES
    Abstract: Accumulating evidence indicates a decisive role for the adjacent stroma in tumour growth and dissemination. However, it is not clear how far altered differentiation such as expression of aberrant keratins and vimentin, common in invasive human carcinomas, may reflect intrinsic cell properties or a response to the tumour environment. We have addressed this by transplanting benign and malignant human HaCaT-ras keratinocytes, seeded on collagen matrix, onto nude mice. Initially, epithelia derived from benign and malignant cells, being separated from host stroma by collagen, were poorly organized and exhibited the same differentiation markers, as identified by immunofluorescence and in situ hybridization. Epidermal basal and suprabasal keratins were expressed persistently even upon contact with newly formed stroma and malignant cell invasion. In contrast, non-epidermal keratins (K4/K13, K8/18, K19), which were similarly synthesized by benign and malignant cells in culture and in early transplants, were differentially regulated with increasing stromal vicinity. While both proteins and mRNAs were downregulated in benign epithelia, the malignant, invasive tumour cells continuously expressed these non-epidermal keratins throughout (K19), suprabasally (K4/13) or at invasive sites (K8/18). Furthermore, the mesenchymal protein vimentin was expressed de novo in invasive areas confronting tumour stroma. Thus, atypical tissue markers, similarly synthesized in isolated cells in vitro, are downregulated in benign but maintained and upregulated in malignant epithelia. This is presumably caused by the neighbouring stroma being permanently activated by malignant epithelia. Copyright (C) 2003 John Wiley Sons, Ltd
    Type of Publication: Journal article published
    PubMed ID: 12845625
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