Thioredoxin promotes the regeneration and binding of elastic fibre and basement membrane.

OBJECTIVE: Thioredoxin (TRX), a ubiquitous protein with strong antioxidant activity, decreases in the skin with age. A decrease in TRX is expected to induce cellular senescence, chronic inflammation, and degeneration and loss of extracellular matrix (ECM), such as collagen and elastin within the skin. In this study, we investigated the effects of TRX addition to excised skin or skin models to understand the role of TRX on cells and ECM within the skin. METHODS: To evaluate its effect on skin cells, we cultured a three-dimensional (3D) skin model in a medium containing TRX. The mRNA expression levels of proteins related to elastic and collagen fibres and the basement membrane were determined. Furthermore, 3D imaging and computational analysis were performed to evaluate the effect of TRX on the elastic fibres and extending COL VII structures in excised human skin after coculturing with TRX for 1, 4, 5 and 6 days. RESULTS: Thioredoxin application to a 3D skin model upregulated elastin, COLI and COLVII mRNA expression. Applying TRX to the excised skin increased the number of linear elastic fibres. This effect of TRX demonstrated a daily increment in a dose-dependent manner. Thioredoxin extended the fibrous structure of COL VII into the dermis, expanding its colocalization region with elastic fibres. These structural effects were confirmed using 3D imaging and computational methods. CONCLUSION: Thioredoxin elongates elastic fibres from the dermis to the basement membrane and extends the COL VII structure from the basement membrane to the dermis in excised human skin. These findings suggest the potential of TRX to protect the skin against age-related alterations such as wrinkles and sagging.

OBJECTIF: Thioredoxin (TRX), une protéine ubiquitaire dotée d'une forte activité antioxydante, diminue dans la peau avec l'âge. Une diminution de la TRX est susceptible d'induire la sénescence cellulaire, l'inflammation chronique, et la dégénérescence ainsi que la perte de la matrice extracellulaire (ECM), telle que le collagène et l'élastine de la peau. Dans cette étude, nous avons examiné les effets de l'ajout de TRX à la peau prélevée ou aux modèles de peau afin de comprendre le rôle de TRX sur les cellules et la matrice extracellulaire (ECM) de la peau. MÉTHODES: Pour évaluer son effet sur les cellules cutanées, nous avons cultivé un modèle de peau tridimensionnel (3D) dans un milieu contenant du TRX. Les niveaux d'expression de l'ARNm des protéines liées aux fibres élastiques et de collagène ainsi que de la membrane basale ont été déterminés. De plus, une imagerie 3D et une analyse informatique ont été réalisées pour évaluer l'effet de la TRX sur les fibres élastiques et les structures de COL VII étendues dans la peau humaine prélevée après une coculture avec la TRX pendant 1, 4, 5 et 6 jours. RÉSULTATS: L'application de la Thioredoxin à un modèle de peau en 3D a régulé à la hausse l'expression de l'élastine, du COLI et du COLVII au niveau de l'ARNm. L'application de TRX à la peau excisée a augmenté le nombre de fibres élastiques linéaires. Cet effet du TRX a montré une augmentation quotidienne de manière dose­dépendante. Le Thioredoxin a étendu la structure fibreuse du COL VII dans le derme, élargissant ainsi sa région de colocalisation avec les fibres élastiques. Ces effets structuraux ont été confirmés à l'aide d'imagerie 3D et de méthodes computationnelles. CONCLUSIONS: La Thioredoxin allonge les fibres élastiques du derme à la membrane basale et étend la structure de COL VII de la membrane basale au derme dans la peau humaine excisée. Ces résultats suggèrent le potentiel de la TRX pour protéger la peau contre les altérations liées à l'âge telles que les rides et le relâchement cutané.

Long Hanging Structure of Collagen VII Connects the Elastic Fibers and the Basement Membrane in Young Skin Tissue.

Aging leads to substantial structural changes in the skin. Elastic fibers maintain skin structure, but their degeneration and loss of function with age result in wrinkle formation and loss of skin elasticity. Oxytalan fiber, a type of elastic fiber, extends close to the dermal-epidermal junction (DEJ) from the back of the dermis. Oxytalan fibers are abundant in the papillary layer and contribute to skin elasticity and texture. However, to accurately understand the mechanisms of skin elasticity, the interaction between elastic fibers and DEJ should be elucidated. Here, we investigated elastic fibers and DEJ and their structural alterations with aging. Several basement membrane proteins [collagen (COL) IV, COLVII, and laminin 332], fibrous tropoelastin, and fibrillin-1 in excised human skin tissue were observed using three-dimensional imaging. Age-related alterations in COLVII, elastic fibers, and fibrillin-1 were evaluated. We found that COLVII forms long hanging structures and is co-localized with fibrous tropoelastin in young skin but not aged skin. Fibrillin-1-rich regions were observed at the tips of elastin fibers in young skin tissue, but rarely in aged skin. This co-localization of elastic fiber and COLVII may maintain skin structure, thereby preventing wrinkling and sagging. COLVII is a potential therapeutic target for skin wrinkling.

Elastin microfibril interface-located protein 1 and its catabolic enzyme, cathepsin K, regulate the age-related structure of elastic fibers in the skin.

INTRODUCTION: The elastic fiber structure becomes shorter, thicker, and curved with age. Nonetheless, the proteins and catabolic enzymes influencing the maintenance of and change in the three-dimensional (3D) structure of elastic fibers remain unknown. This study aimed to identify the proteins involved in the maintenance and degeneration of elastic fiber structures. METHODS: We performed a combined 3D structural analysis using tissue decolorization technology and mRNA abundance and comprehensive protein expression of tissue-derived cells. The relationship between the proteins was evaluated. RESULTS: Elastin microfibril interface-located protein 1 (EMILIN-1) and cathepsin K (CTSK) were implicated in structural changes in elastic fibers with aging. EMILIN-1 and CTSK levels were highly correlated and changed with age. CTSK was identified as the degrading enzyme of EMILIN-1. CTSK fragmented the otherwise linearly existing dermal elastic fiber structure, with more evident changes in oxytalan fibers. EMILIN-1 expression in fibroblasts was increased by co-culturing with keratinocytes. Furthermore, CTSK expression was increased by UV stress in keratinocytes, resulting in decreased EMILIN-1 expression. CONCLUSION: Using our new assessment strategy, we observed that EMILIN-1 and CTSK are highly linked to changes in the elastic fiber structure with aging. These results indicate that suppressing CTSK expression and increasing EMILIN-1 expression might be an effective approach to prevent elastic fiber morphological changes that lead to wrinkles and sagging. Furthermore, EMILIN-1 in the dermis increases due to interaction with the epidermis, which could provide a new target for the therapeutic care of elastic fibers (including preservation of oxytalan fibers) in epidermis-dermis interaction.

Thrombomodulin induces anti-inflammatory effects by inhibiting the rolling adhesion of leukocytes in vivo.

Thrombomodulin (TM) is an integral membrane protein expressed on the surface of vascular endothelial cells that suppresses blood coagulation. Recent studies have shown that TM exhibits anti-inflammatory effects by inhibiting leukocyte recruitment. However, the actual modes of action of TM in vivo remain unclear. Here, we describe the pharmacological effects of recombinant human soluble TM (TM alfa) on leukocyte dynamics in living mice using intravital imaging techniques. Under control conditions, neutrophils exhibited three distinct types of adhesion behavior in vessels: 1) "non-adhesion", in which cells flowed without vessel adhesion; 2) "rolling adhesion", in which cells transiently interacted with the endothelium; and 3) "tight binding", in which cells bound strongly to the endothelial cells. Compared to control conditions, local lipopolysaccharide stimulation resulted in an increased frequency of rolling adhesion that was not homogeneously distributed on vessel walls but occurred at specific endothelial sites. Under inflammatory conditions, TM alfa, particularly the D1 domain which is a lectin-like region of TM, significantly decreased the frequency of rolling adhesion, but did not influence the number of tight bindings. This was the first study to demonstrate that TM alfa exerts anti-inflammatory effects by inhibiting rolling adhesion of neutrophils to vascular endothelial cells in living mice.

Historical control data on developmental toxicity studies in rats.

Historical control data from prenatal developmental toxicity studies in rats have been used to evaluate whether toxicology outcomes were induced by exposure to a chemical or were within the range of spontaneous variation. These data are also important for monitoring animal characteristics. As a follow-up to historical control data from 1998 to 2010, this study analyzed control data from prenatal developmental studies performed in rats from 2011 to 2015. Data were collected from studies performed by 24 Japanese laboratories, including 15 pharmaceutical and chemical companies and nine contract research organizations, in Sprague-Dawley and two-sub-strains of Wistar Hannover rats. The data included maternal reproductive findings at terminal cesarean section and fetal findings, including incidences of spontaneous external, visceral, and skeletal anomalies. No noticeable differences in maternal reproductive data were observed among laboratories. The inter-laboratory variations in the incidences of fetal anomalies seemed to be due to differences in the selection of observation parameters, observation criteria, and classification of the findings, as well as to differences in terminology of fetal alterations. These historical control data may be helpful for adequate interpretation of experimental results and for evaluating the reproductive and developmental toxicities of various chemicals.