Single-cell analysis of human dermal fibroblasts isolated from a single male donor over 35 years.

The aim of this study is to examine the effects of ageing on dermal fibroblast heterogeneity based on samples obtained from the same donor. We used a dermal fibroblast lineage (named ASF-4 cell lines) isolated from the inner side of the upper arm of a healthy male donor over a 35-year period, beginning at 36 years of age. Because clonal analysis of ASF-4 cell lines demonstrated a donor age-dependent loss of proliferative capacity and acquisition of senescent traits at the single-cell level, cultured cells frozen at passage 10 at ages 36 and 72 years were subjected to single-cell RNA sequencing. Transcriptome analysis revealed an increase in senescent fibroblasts and downregulation of genes associated with extracellular matrix remodelling with ageing. In addition, two putative differentiation pathways, with one endpoint consisting of senescent fibroblasts and the other without, were speculated using a pseudo-time analysis. Knockdown of the characteristic gene of the non-senescent fibroblast cluster endpoint, EFEMP2, accelerated cellular senescence. This was also confirmed in two other normal human dermal fibroblast cell lines. The detection of a common cellular senescence-related gene from single-donor analysis is notable. This study provides new insights into the behaviour of dermal fibroblasts during skin ageing.

Development of a lip vermilion epithelium reconstruction model using keratinocytes from skin and oral mucosa.

Lip vermilion is unique and can be distinguished from the adjacent skin and oral mucosa. However, because of the lack of appropriate evaluation tools, skin and/or oral mucosa substitutes such as in vitro vermilion epithelial models have been used for lip product testing. We aimed to develop and characterize a lip vermilion epithelium reconstruction model (LVERM) using skin and oral keratinocytes. LVERM was manufactured by co-culturing primary skin and oral keratinocytes, using a device that allowed the separation of cell seeding, and created an intercalated cell-free zone, referred to as the vermilion part. After removing the device, LVERM construction was completed in 8 days, in a submerged condition. Subsequently, they were placed in an air-liquid interface for 7 days. To determine the epithelial characteristics of LVERM, keratin 2e (KRT2) and small proline-rich protein 3 (SPRR3) expression patterns were examined. The in vivo expression profiles of KRT2 and SPRR3 genes in vermilion were also examined. We found that a continuous multi-layered epithelium was generated in the LVERM that exhibited ortho- and para-keratinization in the skin and oral mucosa parts, respectively. Although an intermediate keratinization pattern was observed in the vermilion part, KRT2 and SPRR3 were co-expressed in the suprabasal layer, consistent with the expression pattern of a single vermilion epithelial model. Clustering analysis revealed that KRT2 and SPRR3 gene expression in vermilion was location-dependent within the sample. Therefore, LVERM can be used as an evaluation tool for lip products and has great importance in innovative approaches for cosmetic testing.

Distribution of Domains Formed by Lateral Packing of Intercellular Lipid in the Stratum Corneum.

Intercellular lipids fill the interstices of corneocytes and serve a barrier function. The amount of transdermal water evaporation varies depending on the packing structure of intercellular lipids, as this structure is important for maintaining barrier efficacy. This packing structure consists of a mixture of crystals (orthorhombic and hexagonal) and liquid crystals (fluid phase), and the proportion of these phases is thought to affect barrier function. However, there have been no methods to visualize the actual distribution of the domains formed by packing structure in intercellular lipids. In this study, the planar distribution of intercellular lipid structures was determined using focal plane array (FPA)-based Fourier transform (FT) IR imaging analysis of stratum corneum cell units obtained by grid stripping. The lipid composition of ceramides was revealed by electrospray ionization tandem mass spectrometry (ESI-MS/MS)-based shotgun lipidomics. The distribution of domains formed by packing structures and the lipid composition of ceramides was compared in skin with high- or low-transepidermal water loss (TEWL). The orthorhombic proportion was lower in high-TEWL skin than in low-TEWL skin. ESI-MS/MS-based shotgun lipidomics analysis showed that the alpha-hydroxyceramide content in the low- and high-TEWL groups differed regarding the distribution of fatty acid chain lengths. The evaluation of stratum corneum cell units using FPA-based FTIR imaging is an innovative technology that can visualize the distribution of domains formed by intercellular lipid-packing structures. Increased proportions of alpha-hydroxyceramide subclasses such as alpha-hydroxy-sphingosine ceramide and alpha-hydroxy-phytosphingosine ceramide were associated with a reduced proportion of the orthorhombic packing structure domain.

The human vermilion surface contains a rich amount of cholesterol sulfate than the skin.

BACKGROUND: The vermilion of the human lip presents characteristic features and undergoes aging faster than the skin. Therefore, knowledge of the vermilion surface-specific functional molecules is important to understand lip aging and formulate lip care products. Previously, we analyzed the free fatty acids distributions and showed that docosahexaenoic acid highly accumulated in the vermilion's epithelium than in the skin. OBJECTIVE: We aimed to explore the functional molecules other than the free fatty acids on the vermilion's surface. METHODS: Human lip tissues from children and tape-stripped samples from smooth and rough lips of adults were measured by desorption electrospray ionization-mass spectrometry imaging (DESI-MSI). RESULTS: DESI-MSI of children's lip sections revealed a major distribution of five phospholipid species in the viable layer, but not in the superficial area, of both the vermilion and the skin than that in the underlying tissue. Interestingly, a remarkably higher distribution of cholesterol sulfate was observed in the vermilion's superficial area compared to that in the skin in all subjects under this study. Furthermore, DESI-MSI of tape-stripped lip samples showed an overall higher accumulation of cholesterol sulfate in the stratum corneum of the rough lips than that in the smooth lips. CONCLUSION: Our study concluded that cholesterol sulfate has a characteristic distribution to the vermilion's surface and showed an association with the roughness of the lip.

Amino Acids and Their N-Acetylated Derivatives Maintain the Skin's Barrier Function.

The hydrocarbon-chain packing structure of intercellular lipids in the stratum corneum (SC) is critical to the skin's barrier function. We previously found that formation of V-shaped ceramide reduces the barrier function of skin. There are few agents, apart from ceramides and fatty acids that can improve the orthorhombic packing (Orth) ratio of the intercellular lipid packing structure. In this study, we investigated agents that directly increase the Orth ratio. We selected an intercellular lipid model consisting of ceramide, cholesterol, and palmitic acid and performed differential scanning calorimetry. We focused on natural moisturizing factor components in the SC, and therefore investigated amino acids and their derivatives. The results of our intercellular lipid model-based study indicate that N-acetyl-L-hydroxyproline (AHYP), remarkably, maintains the lamellar structure. We verified the effect of AHYP on the lamellar structure and hydrocarbon chain packing structure of intercellular lipids using time-resolved X-ray diffraction measurements of human SC. We also determined the direct physicochemical effects of AHYP on the Orth ratio of the hydrocarbon-chain packing structure. Hence, the results of our human SC study suggest that AHYP preserves skin barrier function by maintaining the hydrocarbon-chain packing structure of intercellular lipids via electrostatic repulsion. These findings will facilitate the development of skincare formulation that can maintain the skin's barrier function.

Linoleic acid and squalene are oxidized by discrete oxidation mechanisms in human sebum.

Previous studies suggest that squalene (SQ) in sebum is oxidized by a photooxidation mechanism (i.e., singlet oxygen oxidation) to create SQ hydroperoxide (SQOOH), a compound that causes adverse skin conditions. However, oxidation of other lipids in sebum, such as linoleic acid (LA), has not been fully understood. Elucidating their oxidation, especially its mechanisms, may lead to a further understanding of the relationship between sebum oxidation and skin conditions. In this study, using HPLC-MS/MS, we aimed to detect LA hydroperoxide (LAOOH) directly from sebum and identify the oxidation mechanism of LA in sebum through analysis of LAOOH isomers. We developed extraction and HPLC-MS/MS analysis conditions that can sufficiently quantify each LAOOH isomer in sebum. Using this method, LAOOH was detected in samples from healthy individuals, demonstrating the presence of LAOOH in human sebum. Moreover, isomer analysis of LAOOH and SQOOH indicated that LA and SQ are oxidized in sebum by discrete oxidation mechanisms (LA oxidized by free radical oxidation, whereas SQ oxidized by singlet oxygen oxidation). Such results may further lead to the development of mechanism-specific ways to prevent oxidation of sebum via a selection of appropriate antioxidants, ultimately leading to the promotion of skin health.

Phase Separation in Lipid Lamellae Result from Ceramide Conformations and Lateral Packing Structure.

Intercellular lipids in the stratum corneum protect the living body from invasion by allergens and pathogens, and also suppresses water evaporation within the body. It is important to understand how differences in the microstructure of intercellular lipids arise. This microstructure is affected by lipid composition. Studies using intercellular lipid models have reported the formation of two phases with different short lamellar periodicities. However, the details of the packing structure characteristics of the two phases observed in these intercellular lipid models are unclear. Our previous report revealed that different short periodicity phases coexist in the N-(α-hydroxyoctadecanoyl)-dihydrosphingosine (CER[ADS]), cholesterol (CHOL), and palmitic acid (PA) complex model. In this study, the characteristics of the packing structure of two phases with different short lamellar periodicities, which were observed in the intercellular lipid model (CER[ADS]/CHOL/PA) that we used previously, were adjusted for models with different lipid compositions. The characteristics of the packed and lamellar structures have been determined by temperature-scanning small-angle X-ray scattering and wide-angle X-ray diffraction measurements simultaneously. These differences in lamellar structure were thought to be caused by differences in ceramides (CER) conformation between the hairpin and the V-shape type. The lamellar structure of the V-shaped CER conformation has a low orthorhombic ratio. The above results suggest that an increase in the ratio of CER with the V-shaped structure causes the lamellar structure to have low orthorhombic ratio, thereby contributing to a decrease in the bilayer's barrier function.

Assessment of skin barrier function using skin images with topological data analysis.

Recent developments of molecular biology have revealed diverse mechanisms of skin diseases, and precision medicine considering these mechanisms requires the frequent objective evaluation of skin phenotypes. Transepidermal water loss (TEWL) is commonly used for evaluating skin barrier function; however, direct measurement of TEWL is time-consuming and is not convenient for daily clinical practice. Here, we propose a new skin barrier assessment method using skin images with topological data analysis (TDA). TDA enabled efficient identification of structural features from a skin image taken by a microscope. These features reflected the regularity of the skin texture. We found a significant correlation between the topological features and TEWL. Moreover, using the features as input, we trained machine-learning models to predict TEWL and obtained good accuracy (R2 = 0.524). Our results suggest that assessment of skin barrier function by topological image analysis is promising.

A Novel Method for Visualizing Melanosome and Melanin Distribution in Human Skin Tissues.

Melanin incorporated into keratinocytes plays an important role in photoprotection; however, abnormal melanin accumulation causes hyperpigmentary disorders. To understand the mechanism behind the accumulation of excess melanin in the skin, it is essential to clarify the spatial distribution of melanosomes or melanin in the epidermis. Although several markers have been used to detect melanosomes or melanin, no suitable markers to determine the precise localization of melanin in the epidermis have been reported. In this study, we showed that melanocore-interacting Kif1c-tail (M-INK), a recently developed fluorescent probe for visualizing mature melanosomes, binds to purified melanin in vitro, and applied it for detecting melanin in human skin tissues. Frozen skin sections from different phototypes were co-stained for the hemagglutinin (HA)-tagged M-INK probe and markers of melanocytes or keratinocytes, and a wide distribution of melanin was observed in the epidermis. Analysis of the different skin phototypes indicated that the fluorescent signals of HA-M-INK correlated well with skin color. The reconstruction of three-dimensional images of epidermal sheets enabled us to observe the spatial distribution of melanin in the epidermis. Thus, the HA-M-INK probe is an ideal tool to individually visualize melanin (or melanosome) distribution in melanocytes and in keratinocytes in skin tissues.

Higher Accumulation of Docosahexaenoic Acid in the Vermilion of the Human Lip than in the Skin.

The vermilion of the human lip is a unique facial area because of certain distinguishing features from the adjacent tissues such as the white lip (skin) and oral mucosa. However, the distinction in terms of molecular distribution between the vermilion and skin has remained unexplored. Therefore, we aimed to map the human lip by mass spectrometry imaging to gain understanding of the free fatty acid distribution in the vermilion. The lip specimens trimmed off during cheiloplasty were analyzed using desorption electrospray ionization-mass spectrometry imaging. Distributions of two monounsaturated fatty acids and three polyunsaturated fatty acids were observed in the human lip tissue: palmitoleic acid (POA) and oleic acid (OA) and linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA), respectively. Although POA, OA, LA, and AA were differentially distributed across the vermilion and skin, DHA showed a higher accumulation in the epithelium of the vermilion compared to that in the skin. Our results clearly demonstrated the difference in fatty acid distributions between the vermilion and skin. The highly abundant DHA in the epithelium of the vermilion may have an antioxidant role and may thus protect the lip from aging. Our findings can provide a novel strategy for treating lip disorders.

The effect of an educational program for pregnant women to prevent allergic diseases in infants: study protocol for a randomized controlled trial.

BACKGROUND: Allergic diseases in infants have dramatically increased in developed countries during the past few decades. To date, extensive research has been done on risk factors for allergies in infancy, and preventive measures against them. However, the effect of the primary approach to preventing infantile allergy is still limited. The aim of this trial is to evaluate whether prenatal education interventions, including the latest public research results on allergic diseases, prevent the onset of infant allergies. METHODS/DESIGN: We designed a randomized controlled, two-arm (standard prenatal education vs our education), parallel-group, assessor-blind trial. A sample of 120 pregnant women will be recruited at Chiba Aiyu-kai Kinen Hospital and allocation is by computer-generated randomization. Pregnant women in the intervention arm participate in the childbirth education program established by the specialist and a pediatric allergy educator. The program was developed based on evidences supporting interventions on primary prevention, which are suggested to be beneficial to infantile allergies in recent studies. The primary objective of the study is to determine whether it is possible to establish effective behaviors for allergy prevention in early infancy in the children of pregnant women who participate in an educational program developed by pediatric allergy specialists. Four months after birth, their behaviors will be compared against those of pregnant women who did not participate in the program. DISCUSSION: Allergies are common in many individuals worldwide, and can be present from babyhood through the person's lifetime. One of the strong points of this study is that it should provide pregnant women with accumulated information on preventive knowledge against allergy, that can be effective in some cases, and that women can apply a combination of these behaviors before and after pregnancy. The results of our program will be publicized to help change the behaviors of mothers, and, if the program is effective, for preventing allergies in infants, it will be disclosed worldwide as a new preventive strategy for allergy in infants. TRIAL REGISTRATION: UMIN-CTR, ID: UMIN000034730 Retrospectively registered on 1 December 2018.

Prevention of photosensitivity with action spectrum adjusted protection for erythropoietic protoporphyria.

Erythropoietic protoporphyria is a genetic disease characterized by sensitivity to sunlight caused by the accumulation of protoporphyrin IX. Photoprotection against ultraviolet A and visible light is necessary for erythropoietic porphyria patients because the absorption spectrum of protoporphyrin IX lies in both ultraviolet A and visible light region. We developed a novel index, in vitro porphyrin protection factor, based on the protoporphyrin IX absorbance spectrum. We also selected appropriate photoprotective products designed according to protoporphyrin IX absorbance. The porphyrin protection factors of a combination of make-up base with a powder as well as with a liquid foundation were significantly higher than those of a conventional sunscreen product, even at a small application dose. An in-use test carried out for 6 months showed that the efficacy of these products was 78.3%, and no adverse reactions were observed. Male subjects preferred liquid foundation, whereas all female subjects used powder foundation. The preference of the subjects could lead to the long-term use of the tested products. In conclusion, this study provided a new approach to improve photoprotection in erythropoietic protoporphyria patients.

The effects of continuous application of sunscreen on photoaged skin in Japanese elderly people - the relationship with the usage.

Since photoaging of skin is caused by chronic sun exposure, it is well-recognized that regular sunscreen use can help prevent photoaging of skin in fair-skinned people. Therefore, application of sunscreen is recommended for the prevention of photoaging in many countries. However, the relationship between UV exposure and photoaging has rarely been investigated in clinical studies in Japan. In addition, there have been almost no long-term interventional studies in Japanese people. We have previously conducted a study where Japanese actinic keratosis patients were instructed to continuously apply sunscreen. The results indicated that long-term application of sunscreen is effective in suppressing actinic keratosis progression and generation. In the present study, we investigated the effects of sunscreen on photoaged skin in 14 elderly Japanese people. Skin conditions such as water content, transepidermal water loss, the number of spots, wrinkles, and skin color tone uniformity were measured and compared before and after the study. A statistically significant difference was observed only in skin surface hydration. There were large inter-individual differences in amount of sunscreen used throughout the study. The changes in the number of spots and skin color tone uniformity during the 18 months showed good correlation with amount of sunscreen being used. These results suggest an increase in the number of spots and deterioration in skin color tone uniformity in the 18-month non-sunscreen application period, and that such skin conditions improved with increasing use of sunscreen. In this study, we suggested an inhibitory effect on photoaging symptoms such as spots and skin color tone non-uniformity, by application of the appropriate amount of sunscreen over a long period of time in Japanese people, similar to Caucasians.

Donor age-dependent acceleration of cellular aging by repeated ultraviolet A irradiation of human dermal fibroblasts derived from a single donor.

The relationship between cellular aging and aging of entire organisms has been studied extensively.The findings are confusing, however, and no clear relationships have been demonstrated.The conflicting data may be due to individual differences among the donors of the studied cells.It is crucial to identify the changes in cellular properties that are the result of the aging process.Here, we used human dermal fibroblast cell lines established from a single donor at different ages to assess the influence of ultraviolet A (UVA) on cellular aging. These cell lines have the same genetic background and were obtained from a restricted body region. The results indicated that cellular aging was accelerated by UVA irradiation in a donor age-dependent manner. The ratio of lifespan shortening increased with donor age. Increased donor age not only decreased cell division, but also increased the growth arrest response to UVA irradiation. The characteristics of the cultured cells reflected the age-related changes in dermal fibroblasts.

Donor age reflects the replicative lifespan of human fibroblasts in culture.

Human fibroblasts, which have a finite lifespan in cultures, have been widely used as a model system for cellular aging, and frequently used as one model of human aging. But whether cellular aging contributes to organismal aging has been controversial. To reinvestigate this question, we cultured human fibroblasts from the skin of one individual volunteer collected at different ages. Over a period of 27 years (donor age 36 years to 62 years), we obtained skin cells four times at appropriate intervals, and established eight fibroblast lines. These human fibroblasts have presented evidence for a correlation between donor age and proliferative lifespan in vitro. This result parallels the fact that telomeric DNA size cultured fibroblasts decrease with the increase in donor age. These cell lines had a normal diploid human chromosome constitution and will be useful in studies of human biology including aging.

Increased levels of a particular phosphatidylcholine species in senescent human dermal fibroblasts in vitro.

Plasma membranes are essential components of living cells, and phospholipids are major components of cellular membranes. Here, we used liquid chromatography/mass spectrometry to investigate changes in the membrane phospholipid content that occur in association with aging. Our results indicate that the levels of a particular species of phosphatidylcholine comprised of stearic acid and arachidonic acid increased with age. To determine the reason for the increased levels of this particular phosphatidylcholine, we examined the effect of highly unsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, on cellular aging. Applied arachidonic acid was incorporated into phosphatidylcholine molecules, but neither arachidonic acid nor other related unsaturated fatty acids had any effect. We conclude that increased levels of this distinctive phosphatidylcholine are a result of in vitro senescence.

Ergocalciferol promotes in vivo differentiation of keratinocytes and reduces photodamage caused by ultraviolet irradiation in hairless mice.

BACKGROUND: Ergocalciferol (VD(2)) is usually administered orally and it is metabolized to produce its biologically active metabolites in the liver and kidney. Active vitamin D is a well-known potent regulator of cell growth and differentiation. PURPOSE: Active vitamin D such as 1,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) prevents photodamage, including wrinkles and morphologic alterations. However, its clinical and cosmetic use is limited because of its potent, associated effect on calcium metabolism. We examined the efficacy of vitamin D analogues with few adverse effects for preventing skin photodamage. METHOD: Topical application of VD(2) to hairless mouse dorsal skin, and exposure to solar-simulating ultraviolet (UV) radiation at a dose of 10.8 J/cm(2) (UVA) were performed for 15 weeks, five times a week on weekdays. At the end of the final irradiation, histological and analytical studies were performed. RESULTS: Topical application of VD(2) significantly prevented wrinkle formation and abnormal accumulation of extracellular matrix components. In addition, VD(2) suppressed excessive secretion of IL-6 induced by UV irradiation in cultured human normal keratinocytes, in a dose-dependent manner. CONCLUSION: VD(2) promoted keratinocytes differentiation in the epidermis and showed diverse physiological effects, the same as the active form of VD(3). The results suggested that the suppression of skin photodamage involved the promotion of keratinocytes differentiation and suppression of IL-6 secretion induced by exposure to UV. Topical application of VD(2) may become an effective means to suppress solar UV-induced human skin damage.