Novel double staining of the stratum corneum with fluorescent ε-poly-l-lysine and anionic dextran.

OBJECTIVE: ε-Poly-l-lysine (PLL) is a cationic polymer consisting of 25-35 l-lysine residues. Our previous study revealed that fluorescently labelled PLL can stain the stratum corneum (SC) via ionic interactions between PLL and SC constituents. In this study, to further clarify the mechanisms underlying the interaction between PLL and the SC, the staining properties of fluorescent PLL were compared with that of fluorescently labelled anionic dextran (aDex), which has approximately the same molecular weight as PLL. METHODS: SC samples were collected by non-invasive tape stripping and stained with fluorescent PLL and/or fluorescent aDex. Fluorescence images were acquired using a fluorescence microscope and then analysed. RESULTS: The SC could be stained with either fluorescent PLL or aDex, both of which were inhibited by the addition of high concentrations of salt solutions. In particular, aDex staining was inhibited at a lower salt concentration than PLL staining. Moreover, PLL staining was inhibited under acidic conditions, while aDex staining was inhibited under neutral to alkaline conditions. Double staining of SC with both fluorescent polymers produced heterogeneous staining patterns: corneocytes stained with both polymers, corneocytes stained with PLL or aDex in a mutually exclusive manner, and unstained corneocytes. Staining of SC samples from the face was more extensive than staining of SC samples from the inside of the upper arm with both polymers. In addition, pretreatment of the SC with ethanol resulted in enhanced staining with both polymers. These results suggest that double staining of SC with both polymers can provide information on the damaged SC. CONCLUSION: Staining of SC with fluorescent PLL depends on its properties of a cationic and hydrophobic polymer with appropriate molecular size, which can distinguish the damaged SC. Double staining of SC with fluorescent PLL and aDex is a novel approach to obtain information for the analysis of skin conditions.

OBJECTIF: La ε-poly-L-lysine (PLL) est un polymère cationique constitué de résidus de 25 à 35 L-lysines. Notre précédente étude a révélé que la PLL marquée par fluorescence peut colorer le stratum corneum (SC) par des interactions ioniques entre la PLL et les constituants du SC. Dans cette étude, afin de clarifier davantage les mécanismes sous-jacents à l'interaction entre la PLL et le SC, les propriétés de coloration de la PLL fluorescent ont été comparées à celles du dextran anionique (aDex) marqué par fluorescence, qui a à peu près le même poids moléculaire que la PLL. MÉTHODES: Les échantillons SC ont été prélevés par «tape stripping¼ non invasif et colorés avec de la PLL fluorescente et/ou de l'aDex fluorescent. Les images de fluorescence ont été acquises au microscope à fluorescence puis analysées. RÉSULTATS: Le SC pouvait être coloré avec de la PLL ou de l'aDex fluorescents, tous deux inhibés par l'ajout de fortes concentrations de solutions salines. En particulier, la coloration par aDex était inhibée à une concentration en sel inférieure à la coloration par PLL. En outre, la coloration de la PLL a été inhibée dans des conditions acides, tandis que la coloration de l'aDex a été inhibée dans des conditions neutres à alcalines. La double coloration de SC avec les deux polymères fluorescents a produit des modes de coloration hétérogènes: cornéocytes colorés avec les deux polymères, cornéocytes colorés avec de la PLL ou de l'aDex d'une manière mutuellement exclusive, et cornéocytes non colorés. La coloration des échantillons de SC sur le visage était plus étendue que la coloration des échantillons de SC sur la face intérieure du haut du bras avec les deux polymères. En outre, le prétraitement du SC avec de l'éthanol a entraîné une coloration améliorée avec les deux polymères. Ces résultats indiquent qu'une double coloration du CS avec les deux polymères peut fournir des informations sur le CS endommagé. CONCLUSION: La coloration du CS avec de la PLL fluorescente dépend de ses propriétés de polymère cationique et hydrophobe de taille moléculaire appropriée, ce qui permet de distinguer le CS endommagé. La double coloration de SC avec de la PLL et de l'aDex fluorescents est une nouvelle approche pour obtenir des informations pour l'analyse des affections cutanées.

Staining of stratum corneum with fluorescent ε-poly-L-lysine and its application to evaluation of skin conditions.

BACKGROUND: ε-Poly-L-lysine (PLL) is a cationic polymer consisting of 25 to 35 L-lysine residues that adheres to the surface of skin as well as hair. However, the properties of PLL regarding its adhesion to the skin remain to be elucidated. In this study, we examined the staining of stratum corneum (SC) with fluorescence-labeled PLL and explored its relationship with skin condition. MATERIALS AND METHODS: Alexa Fluor 488-labeled PLL (AF-PLL) was reacted with tape-stripped stratum corneum (SC), and the staining properties were monitored by fluorescence microscopy. Clinical study was performed by measuring the water content of the cheek SC and transepidermal water loss (TEWL), and the tape-stripped SC was subjected to staining with AF-PLL. RESULTS: AF-PLL staining of the SC was inhibited at acidic pH or by the addition of high concentration of salt solution, suggesting the involvement of ionic interaction between PLL and the SC, at least in part. The AF-PLL staining was inhibited by unlabeled PLL or various alkyl amines, but not by L-lysine monomer. AF-PLL staining was observed inside the corneocytes as well as surrounding cornified envelope. Clinical study revealed that AF-PLL staining intensity of the SC was negatively correlated with its water content and positively correlated with its TEWL. CONCLUSION: PLL can efficiently adhere to SC and AF-PLL staining of SC can be applied to evaluate skin conditions.

Dermal Fibroblasts Internalize Phosphatidylserine-Exposed Secretory Melanosome Clusters and Apoptotic Melanocytes.

Pigmentation in the dermis is known to be caused by melanophages, defined as melanosome-laden macrophages. In this study, we show that dermal fibroblasts also have an ability to uptake melanosomes and apoptotic melanocytes. We have previously demonstrated that normal human melanocytes constantly secrete melanosome clusters from various sites of their dendrites. After adding secreted melanosome clusters collected from the culture medium of melanocytes, time-lapse imaging showed that fibroblasts actively attached to the secreted melanosome clusters and incorporated them. Annexin V staining revealed that phosphatidylserine (PtdSer), which is known as an 'eat-me' signal that triggers the internalization of apoptotic cells by macrophages, is exposed on the surface of secreted melanosome clusters. Dermal fibroblasts were able to uptake secreted melanosome clusters as did macrophages, and those fibroblasts express TIM4, a receptor for PtdSer-mediated endocytosis. Further, co-cultures of fibroblasts and melanocytes demonstrated that dermal fibroblasts internalize PtdSer-exposed apoptotic melanocytes. These results suggest that not only macrophages, but also dermal fibroblasts contribute to the collection of potentially toxic substances in the dermis, such as secreted melanosome clusters and apoptotic melanocytes, that have been occasionally observed to drop down into the dermis from the epidermis.

Placental extracts regulate melanin synthesis in normal human melanocytes with alterations of mitochondrial respiration.

Placental extracts have been widely used as skin lightening agents in the Japanese cosmetic market. Here, we show that placental extracts contain factors that can decrease or increase melanin synthesis by normal human melanocytes in vitro in possible association with mitochondrial respiration. When normal human melanocytes were treated with a whole porcine placental extract, melanin synthesis was decreased. In contrast, a porcine placental extract in which exudates and insoluble materials including lipids had been removed increased melanin synthesis. In addition, the amount of tyrosinase, the enzyme critical for melanin synthesis, changed in accordance with the alteration of melanin synthesis. Interestingly, the amount of manganese-dependent superoxide dismutase (MnSOD), a mitochondrial-resident antioxidant enzyme, was increased when melanin synthesis was decreased by the whole placental extract. Mitochondrial respiration and glycolysis also changed following treatment with the placental extracts. These results suggest that placental extracts contain factors that can increase or decrease melanin synthesis by normal human melanocytes and that mitochondrial function may be associated with the placental extract-induced regulation of melanogenesis.

Establishment of Photoaging In Vitro by Repetitive UVA Irradiation: Induction of Characteristic Markers of Senescence and its Prevention by PAPLAL with Potent Catalase Activity.

To understand a role of UVA radiation in photoaging of the skin, we established a model of photoaging cells using cultured human dermal fibroblasts. Repeated low-dose UVA radiation for 10 consecutive days induced senescence in fibroblasts, characterized with (1) increased level of senescence-associated ß-galactosidase, (2) flattened large cell shape, (3) accumulation of reactive oxygen species, (4) yellowish coloration and (5) expression of p16. These were also observed in chronologically aged fibroblasts (doubling times >20), whereas none of these were detected in young cells (doubling times <10). Collectively, we propose that fibroblasts exposed to repetitive UVA radiation may be a good model of aged cells to study the mechanism of aging and photoaging and further to search for novel agents preventing cellular senescence. In addition, H2 O2 was produced in the culture medium by a single low dose of UVA irradiation. Further, PAPLAL, a nanoparticle of platinum and palladium having potent catalase-like activity, significantly delayed the onset of H2 O2 -induced cell senescence. The present study strongly indicates that repetitive short-term UVA irradiation induces aging of cells possibly via H2 O2 and may be suppressed by potent anti-H2 O2 agents.