Effects of the Molecular Structure of Malodor Substances and Their Masking on 1,2-Dioleoyl-sn-glycero-3-phosphocholine Molecular Layers.

Certain odors have been shown not only to cause health problems and stress but also to affect skin barrier function. Therefore, it is important to understand olfactory masking to develop effective fragrances to mask malodors. However, olfaction and olfactory masking mechanisms are not yet fully understood. To understand the mechanism of the masking effect that has been studied, the responses of several target substance (TS) molecules-1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) mixed molecular layers to odorant (OD) molecules were examined as a simple experimental model of epithelial cellular membranes injured by TS molecules. Here, we examined trans-2-nonenal, 1-nonanal, trans-2-decenal, and 1-decanal as TS molecules to clarify the effects of double bonds and hydrocarbon chain lengths on the phospholipid molecular layer. In addition, benzaldehyde and cyclohexanecarboxaldehyde were utilized as OD molecules to clarify the masking effect of the aromatic ring. Surface pressure (Π)-area (A) isotherms were measured to clarify the adsorption or desorption of TS and OD molecules on the DOPC molecular layer. In addition, Fourier transform infrared spectroscopy was performed to clarify the interactions among DOPC, TS, and OD molecules. We found that TS molecules with and without double bonds had different effects on the DOPC molecular layer and that molecules with shorter chain lengths had greater effects on the DOPC molecular layer. Furthermore, OD molecules with aromatic rings counteracted the effects of the TS molecules. On the basis of this research, not only a detailed mechanism by which odor molecules affect lipid membranes without mediating olfactory receptors is elucidated but also more effective OD molecules with masking effects are proposed.

Phospholipid Molecular Layer that Enhances Distinction of Odors Based on Artificial Sniffing.

We propose a novel odor-sensing system based on the dynamic response of phospholipid molecular layers for artificial olfaction. Organisms obtain information about their surroundings based on multidimensional information obtained from sniffing, i.e., periodic perturbations. Semiconductor- and receptor-based odor sensors have been developed previously. However, these sensors predominantly identify odors based on one-dimensional information, which limits the type of odor molecule they can identify. Therefore, the development of odor sensors that mimic the olfactory systems of living organisms is useful to overcome this limitation. In this study, we developed a novel odor-sensing system based on the dynamics of phospholipids that responds delicately to chemical substances at room temperature using multidimensional information obtained from periodic perturbations. Odor molecules are periodically supplied to the phospholipid molecular layer as an input sample. The waveform of the surface tension of the phospholipid molecular layer changes depending on the odor molecules and serves as an output. Such characteristic responses originating from the dynamics of odor molecules on the phospholipid molecular layer can be reproduced numerically. The phospholipid molecular layer amplified the information originating from the odor molecule, and the mechanism was evaluated by using surface pressure-area isotherms. This paper offers a platform for an interface-chemistry-based artificial sniffing system as an active sensor and a novel olfactory mechanism via physicochemical responses of the receptor-independent membranes of the organism.

Effect of red light on epidermal proliferation and mitochondrial activity.

BACKGROUND/PURPOSE: We previously demonstrated that irradiation with red light accelerates recovery of the epidermal water-impermeable barrier, whereas blue light delays it, and white and green light have no effect. Here, we aimed to examine in detail the effects of red and blue light in a human epidermal-equivalent model and in human skin. METHODS: We used light-emitting diodes (red light, 630 nm, 6.2 mW/cm2 ; blue light, 463 nm, 6.2 mW/cm2 ) for irradiation of an epidermal-equivalent model and human skin. Cell proliferation was evaluated by means of BrdU and Ki-67 staining, and mitochondrial activity was quantified with an extracellular flux analyzer. RESULTS: Irradiation of the epidermal-equivalent model with red light for 2 h (44.64 J/cm2 ) increased both epidermal proliferation in the basal layer and mitochondrial activity. Blue light had no effect on epidermal proliferation. Furthermore, irradiation with red light for 2 h on three consecutive days increased epidermal proliferation in human skin tissue in culture. CONCLUSION: These results suggest that red light accelerates epidermal proliferation in both an epidermal-equivalent model and human skin, and may promote epidermal homeostasis.

Distinct sets of olfactory receptors highly expressed in different human tissues evaluated by meta-transcriptome analysis: Association of OR10A6 in skin with keratinization.

Olfactory receptors (ORs) are expressed in many tissues and have multiple functions. However, most studies have focused on individual ORs. Here, we aimed to conduct a comprehensive meta-transcriptome analysis of OR gene expression in human tissues by using open-source tools to search a large, publicly available genotype-tissue expression (GTEx) data set. Analysis of RNA-seq data from GTEx revealed that OR expression patterns were tissue-dependent, and we identified distinct sets of ORs that were highly expressed in 12 tissues, involving 97 ORs in total. Among them, OR5P2, OR5P3 and OR10A6 were associated with skin. We further examined the roles of these ORs in skin by performing weighted gene correlation network analysis (WGCNA) and c3net analysis. WGCNA suggested that the three ORs are involved in epidermal differentiation and water-impermeable barrier homeostasis, and OR10A6 showed the largest gene sub-network in the c3net network. Immunocytochemical examination of human skin keratinocytes revealed a sparse expression pattern of OR10A6, suggesting that it is not uniformly distributed among all keratinocytes. An OR10A6 agonist, 3-phenylpropyl propionate (3PPP), transiently increased intracellular Ca2+ concentration and increased cornified envelope (CE) production in cultured keratinocytes. Knock-down of OR10A6 diminished the effect of 3PPP. Overall, integration of meta-transcriptome analysis and functional analysis uncovered distinct expression patterns of ORs in various human tissues, providing basic data for future studies of the biological functions of highly expressed ORs in individual tissues. Our results further suggest that expression of OR10A6 in skin is related to epidermal differentiation, and OR10A6 may be a potential target for modulation of keratinization.

Do epidermal keratinocytes have sensory and information processing systems?

It was long considered that the role of epidermal keratinocytes is solely to construct a water-impermeable protective membrane, the stratum corneum, at the uppermost layer of the skin. However, in the last two decades, it has been found that keratinocytes contain multiple sensory systems that detect environmental changes, including mechanical stimuli, sound, visible radiation, electric fields, magnetic fields, temperature and chemical stimuli, and also a variety of receptor molecules associated with olfactory or taste sensation. Moreover, neurotransmitters and their receptors that play crucial roles in the brain are functionally expressed in keratinocytes. Recent studies have demonstrated that excitation of keratinocytes can induce sensory perception in the brain. Here, we review the sensory and information processing capabilities of keratinocytes. We discuss the possibility that epidermal keratinocytes might represent the earliest stage in the development of the brain during the evolution of vertebrates.

A computational model of the epidermis with the deformable dermis and its application to skin diseases.

The skin barrier is provided by the organized multi-layer structure of epidermal cells, which is dynamically maintained by a continuous supply of cells from the basal layer. The epidermal homeostasis can be disrupted by various skin diseases, which often cause morphological changes not only in the epidermis but in the dermis. We present a three-dimensional agent-based computational model of the epidermis that takes into account the deformability of the dermis. Our model can produce a stable epidermal structure with well-organized layers. We show that its stability depends on the cell supply rate from the basal layer. Modeling the morphological change of the dermis also enables us to investigate how the stiffness of the dermis affects the structure and barrier functions of the epidermis. Besides, we show that our model can simulate the formation of a corn (clavus) by assuming hyperproliferation and rapid differentiation. We also provide experimental data for human corn, which supports the model assumptions and the simulation result.

Substrate membrane bearing close-packed array of micron-level pillars incrassates air-exposed three-dimensional epidermal equivalent model.

BACKGROUND: We showed previously that a thick three-dimensional epidermal equivalent can be constructed with passaged keratinocytes on a patterned surface. MATERIAL AND METHODS: We first carried out computer simulations of a three-dimensional epidermal equivalent model built on close-packed arrays of 10 µm, 15 µm, 20 µm, 30 µm, and 60 µm diameter pillars. Based on these predictions, we evaluated epidermal equivalents built on a series of porous plastic membranes bearing arrays of pillars 15 µm, 20 µm, 25 µm, 30 µm, and 50 µm in diameter. RESULTS: The simulations predicted that a model having near-physiological thickness would be formed on 15 ~ 30 µm pillars. In the results of in vitro study, the thickest epidermal equivalent was obtained on the 20 µm pillars. Epidermal differentiation markers, filaggrin and loricrin, were expressed at the upper layer of the epidermal equivalent model, and tight-junction proteins, claudin-1 and ZO-1, were expressed on the cell membranes. BrdU-positive cells were observed at the base and also at the top of the pillars. CONCLUSION: The results of the study suggested that mathematical modeling might be a useful tool to guide biological studies.

Effects of trans-2-nonenal and olfactory masking odorants on proliferation of human keratinocytes.

Malodorous compounds induce stress responses, mood changes, an increase of skin conductance, activation of the sympathetic nervous system and other physiological changes, and it has been suggested that sensing malodors could provide warning of danger to health. Furthermore, the human body secretes various malodorous compounds as waste products of metabolism, including trans-2-nonenal ((E)-2-nonenal), the amount of which increases with aging. In the present study, we examined the effects of some endogenous malodorous compounds ((E)-2-nonenal, nonanal, pentanal, hexanal, hexanoic acid, hexylamine and isovaleric acid) on cultured human keratinocytes. (E)-2-Nonenal decreased the viability and promoted apoptosis of cultured keratinocytes. It also reduced the thickness and the number of proliferative cells in a three-dimensional epidermal equivalent model. Co-application of masking odorants (dihydromycenol, benzaldehyde, linalool, phenethyl alcohol, benzyl acetate and anisaldehyde), but not non-masking odorants (1,8-cineol, ß-damascone, and o-t-butylcyclohexyl acetate), reduced the effect of (E)-2-nonenal on keratinocyte proliferation, and restored the thickness and number of proliferative cells in a three-dimensional epidermal equivalent model.

Polyoxyethylene/polyoxypropylene dimethyl ether (EPDME) random copolymer improves lipid structural ordering in stratum corneum of an epidermal-equivalent model as seen by two-photon microscopy.

BACKGROUND/PURPOSE: Topical application of polyoxyethylene/polyoxypropylene dimethyl ether (EPDME) random copolymer improves the barrier function of skin, whereas polyethylene glycol (PEG) and polypropylene glycol (PPG) are ineffective. The aim of this work was to examine the interaction between these polymers and lipid molecules in the stratum corneum in order to establish whether EPDME-specific changes in the structural ordering of lipids might account for the improvement of barrier function. METHODS: We used two-photon microscopy to evaluate the effects of EPDME, PEG, and PPG on the structural ordering of lipids in an epidermal-equivalent model in terms of the fluorescence changes of Laurdan, a fluorescent dye that responds to changes of membrane fluidity. The generalized polarization (GP) value, a parameter that reflects lipid ordering, was measured at various depths from the surface of the stratum corneum. RESULTS: EPDME increased the GP value to a depth of about 3 µm from the surface, indicating that lipid ordering was increased in this region, while PEG and PPG of the same molecular weight had no effect. Diffusion of Lucifer yellow into the epidermis was reduced after application of EPDME, indicating that the barrier function was improved. CONCLUSION: These results support the view that EPDME improves barrier function by increasing the ordering of lipid structures in the stratum corneum. The methodology described here could be useful for screening new compounds that would improve the structural ordering of lipids.

Comprehensive analysis of elemental distribution in human skin using laser ablation inductively coupled plasma mass spectrometry.

BACKGROUND: Multiple chemical elements play roles in skin homeostasis. The distribution of elements in skin has been studied by X-ray microanalysis methods and fluorescence microscopy using chemical indicators, but the former requires complicated sample preparation steps, while the latter is limited by the availability of suitable chemical indicators. MATERIALS AND METHODS: We applied laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to measure the distributions of thirty-eight elements in human skin. RESULTS: Among the target elements, nine (calcium: 40 Ca, 44 Ca, zinc: 64 Zn, 66 Zn, phosphorus: 31 P, potassium: 39 K, sodium: 23 Na, sulfur: 34 S, copper: 63 Cu, magnesium: 24 Mg, and iron: 56 Fe) showed distribution patterns that were consistent with previous reports, and four others (iodine: 127 I, barium: 138 Ba, strontium: 88 Sr, and molybdenum: 95 Mo) were detected for the first time in human skin. CONCLUSION: The method described here requires only slicing into sections to prepare a sample for measurement, so the elemental distributions are minimally disturbed, and comprehensive information can be obtained rapidly. The method is expected to be useful for research in a variety of fields, including skin diseases, aging, and allergenicity.

Glutathione Counteracts the Effects of Japanese Cedar (Cryptomeria japonica) Pollen Allergen Cry j1.

Japanese cedar (Cryptomeria japonica) pollen allergen Cry j1 increases the intracellular calcium concentration in human keratinocytes, and also impairs the epidermal barrier function. Here, we show that reduced glutathione (GSH) blocks both thrombin activation and the Cry j1-induced intracellular calcium elevation in cultured human keratinocytes, and also prevents the Cry j1-induced decrease of barrier function in ex vivo human skin.

Live imaging of alterations in cellular morphology and organelles during cornification using an epidermal equivalent model.

The stratum corneum plays a crucial role in epidermal barrier function. Various changes occur in granular cells at the uppermost stratum granulosum during cornification. To understand the temporal details of this process, we visualized the cell shape and organelles of cornifying keratinocytes in a living human epidermal equivalent model. Three-dimensional time-lapse imaging with a two-photon microscope revealed that the granular cells did not simply flatten but first temporarily expanded in thickness just before flattening during cornification. Moreover, before expansion, intracellular vesicles abruptly stopped moving, and mitochondria were depolarized. When mitochondrial morphology and quantity were assessed, granular cells with fewer, mostly punctate mitochondria tended to transition to corneocytes. Several minutes after flattening, DNA leakage from the nucleus was visualized. We also observed extension of the cell-flattening time induced by the suppression of filaggrin expression. Overall, we successfully visualized the time-course of cornification, which describes temporal relationships between alterations in the transition from granular cells to corneocytes.

Can simple physicochemical studies predict the effects of molecules on epidermal water-impermeable barrier function?

Improvement of the water-impermeable barrier function of skin is clinically important, because barrier abnormality is associated with various skin diseases, such as psoriasis or atopic dermatitis. We have shown that topical application of fatty acids, sex hormones, hexoses, polyols and polymers influences barrier homeostasis, but the effects are highly dependent on even small variations of molecular structure. Moreover, the effects appear within one hour after application and thus are likely to be non-genomic (physicochemical) phenomena. Secretion of lipids from lamellar bodies into the intercellular space between stratum granulosum and stratum corneum is a crucial step in epidermal water-impermeable barrier homeostasis, especially at the early stage of barrier recovery after damage, and phase transition of the lipid lamellar structure in the epidermis is an important part of this process. Therefore, we evaluated the effects of the above molecules on the physicochemical properties of phospholipid monolayers and liposomes as models of the lamellar body membrane and cell membrane. Molecules that influenced the barrier recovery process also altered the stability of liposomes and the air-water surface pressure of phospholipid monolayers. Studies using attenuated total reflection Fourier-transform infrared spectroscopy (ATR FT-IR), differential scanning calorimetry (DSC) and 13 C nuclear magnetic resonance (NMR) spectrometry suggested that molecules influencing barrier recovery interact specifically with phospholipids. The idea that molecules interacting with phospholipids may influence barrier homeostasis should open up new approaches to the treatment of a variety of skin diseases.

Red light-promoted skin barrier recovery: Spatiotemporal evaluation by transepidermal potential.

The light-promoted recovery of epidermal barrier of skin was evaluated by the associated recovery of transepidermal potential (TEP), the potential difference between the surface and dermis of skin, by using porcine skin samples. An accelerated recovery of TEP was observed by irradiation of red light with the irradiance of 40 mW/cm2 and a duration of > 10 min. The influence of the light stimulation to the surroundings (~ 20 mm) was also observed. The irradiations of blue and purple lights were ineffective in accelerating the barrier recovery. These characteristics of the light stimulation would be useful for the design of effective and safe phototherapy devices for skin. The present study proves that the TEP can serve as a spatiotemporal indicator of the epidermal barrier function.

Modulation of lipid fluidity likely contributes to the fructose/xylitol-induced acceleration of epidermal permeability barrier recovery.

We previously showed that topical application of hexoses such as fructose accelerates barrier recovery after disruption. We also showed that various hexoses and polyols interact with phospholipid and alter the phase transition temperature. Thus, we hypothesized that the improvement of barrier recovery by hexoses and polyols might be related to the interaction with phospholipid. Here, we tested this idea by examining the effects of xylitol (a component of some skin-care products) and fructose on lipid dynamics in an epidermal-equivalent model at the single-cell level by means of two-photon microscopy after staining with Laurdan, a fluorescent dye sensitive to the physical properties of its membrane environment. First, we confirmed that topical application of xylitol aqueous solution on tape-stripped human skin accelerated barrier recovery. Then, we examined changes of lipid fluidity in the epidermal-equivalent model after application of water or an aqueous solution of xylitol or fructose. Application of xylitol and/or fructose increased the lipid fluidity in the uppermost part of the stratum granulosum layer, compared to treatment with water alone, and accelerated the exocytosis of lamellar bodies to the intercellular domain between stratum corneum and stratum granulosum. Our results support the idea that the improvement of epidermal barrier homeostasis upon topical application of xylitol or fructose is due to increased lipid fluidity in the uppermost layer of the stratum granulosum, which enables accelerated release of lipid from the stratum granulosum, thereby improving the lamellar structure and accelerating epidermal permeability barrier recovery.

Mathematical-model-guided development of full-thickness epidermal equivalent.

Epidermal equivalents prepared with passaged keratinocytes are typically 10-20 µm thick, whereas intact human epidermis is up to 100 µm thick. Our established mathematical model of epidermal homeostasis predicted that the undulatory pattern of the papillary layer beneath the epidermis is a key determinant of epidermal thickness. Here, we tested this prediction by seeding human keratinocytes on polyester textiles with various fiber-structural patterns in culture dishes exposed to air, aiming to develop a more physiologically realistic epidermal model using passaged keratinocytes. Textile substrate with fiber thickness and inter-fiber distance matching the computer predictions afforded a three-dimensional epidermal-equivalent model with thick stratum corneum and intercellular lamellar lipid structure. The basal layer structure was similar to that of human papillary layer. Cells located around the textile fibers were proliferating, as indicated by BrdU and YAP (Yes-associated protein) staining and expression of melanoma-associated chondroitin sulfate proteoglycan. Filaggrin, loricrin, claudin 1 and ZO-1 were all appropriately expressed. Silencing of transcriptional coactivator YAP with siRNA disturbed construction of the three-dimensional structure. Measurement of trans-epidermal water loss (TEWL) indicated that the model has excellent barrier function. Our results support the idea that mathematical modeling of complex biological processes can have predictive ability and practical value.

Real-time imaging of human epidermal calcium dynamics in response to point laser stimulation.

BACKGROUND: Changes of epidermal calcium ion concentration are involved in regulation of barrier homeostasis and keratinocyte differentiation. Moreover, intracellular calcium dynamics might play a role in skin sensation. But, although calcium dynamics of cultured keratinocytes in response to mechanical stresses has been well studied, calcium propagation in stimulated human epidermis is still poorly understood. OBJECTIVE: The aim of this study was to demonstrate a novel method for real-time measurement of calcium dynamics in response to point stimulation of human epidermis at the single-cell level. METHODS: We examined calcium propagation in cross-sectional samples of living human epidermis ex vivo, as well as in cultured human keratinocytes, by means of two-photon microscopy after stimulating cells in stratum granulosum with the emission laser of a two-photon microscope. RESULTS: Cells in different epidermal layers showed different responses, and those in stratum basale showed the greatest elevation of intracellular calcium. Calcium propagation in epidermis was inhibited in the presence of apyrase (which degrades adenosine triphosphate; ATP) or gap-junction blockers. In cultured keratinocytes, on the other hand, calcium propagated in a simple concentric wave-like manner from the stimulation site, and propagation was strongly suppressed by apyrase. CONCLUSION: Our results suggested that ATP and gap junctions play important roles in calcium propagation induced by point laser stimulation of the uppermost layer of epidermis. Our method should be broadly useful to study calcium dynamics, epidermal physiological mechanisms, and mechanisms of skin sensation at the single-cell level.