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.

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.

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.

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.

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.

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.

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.

Mathematical model for calcium-assisted epidermal homeostasis.

Using a mathematical model of the epidermis, we propose a mechanism of epidermal homeostasis mediated by calcium dynamics. We show that calcium dynamics beneath the stratum corneum can reduce spatio-temporal fluctuations of the layered structure of the epidermis. We also demonstrate that our model can reproduce experimental results that the recovery from a barrier disruption is faster when the disrupted site is exposed to air. In particular, simulation results indicate that the recovery speed depends on the size of barrier disruption.

Abnormal Morphology of Blood Vessels in Erythematous Skin From Atopic Dermatitis Patients.

Previous studies suggest that altered peripheral blood circulation might be associated with erythema or inflammation in atopic dermatitis (AD) patients. However, the overall structure of blood vessels and capillaries in AD skin is poorly understood because most studies have involved light-microscopic observation of thin skin sections. In the present study, we compared the 3-dimensional structures of peripheral blood vessels of healthy subjects and AD patients in detail by means of 2-photon microscopy. In skin from healthy subjects, superficial vascular plexus and capillaries originating from flexous blood vessels were observed. However, skin from AD patients contained thickened, flexuous blood vessels, which might be associated with increased blood flow, in both erythematous and nonlesional areas. However, patients with lichenification did not display these morphological changes. Bifurcation of vessels was not observed in either erythematous or lichenification lesions. These results might be helpful for developing new clinical strategies to treat erythema in AD patients.

Effects of medium flow on axon growth with or without nerve growth factor.

Axon growth is a crucial process in regeneration of damaged nerves. On the other hand, elongation of nerve fibers in the epidermis has been observed in skin of atopic dermatitis patients. Thus, regulation of nerve fiber extension might be an effective strategy to accelerate nerve regeneration and/or to reduce itching in pruritus dermatosis. We previously demonstrated that neurons and epidermal keratinocytes similarly contain multiple receptors that are activated by various environmental factors, and in particular, keratinocytes are influenced by shear stress. Thus, in the present study, we evaluated the effects of micro-flow of the medium on axon growth in the presence or absence of nerve growth factor (NGF), using cultured dorsal-root-ganglion (DRG) cells. The apparatus, AXIS™, consists of two chambers connected by a set of microgrooves, through which signaling molecules and axons, but not living cells, can pass. When DRG cells were present in chamber 1, NGF was present in chamber 2, and micro-flow was directed from chamber 1 to chamber 2, axon growth was significantly increased compared with other conditions. Acceleration of axon growth in the direction of the micro-flow was also observed in the absence of NGF. These results suggest that local micro-flow might significantly influence axon growth.

Functional glycine receptor in cultured human keratinocytes.

We previously demonstrated that a series of neural receptors, those play a crucial role in nerve system, also expressed in epidermal keratinocytes. We also demonstrated that topical application of glycine on hairless mice skin after the barrier disruption also accelerated the barrier recovery and it was blocked by the strychnine. Glycine is known as one of the most important inhibitory neurotransmitters in the mammalian central nervous system. Thus, we hypothesized that glycine receptor also functionally expressed in the epidermal keratinocytes. In the present study, we first studied the expression of glycine receptor message in cultured human keratinocytes. Then we demonstrate for the first time the existence of a functional receptor with electrophysiological properties of glycine receptors in cultured human epidermal keratinocytes. Finally, we demonstrated immune-histochemical study against anti-glycine receptor subunits in human skin. Results of the present study might indicate new target of the clinical dermatology.

Coculture system of keratinocytes and dorsal-root-ganglion-derived cells for screening neurotrophic factors involved in guidance of neuronal axon growth in the skin.

The density of peripheral nerve fibres is increased in atopic dermatitis. Moreover, reduction in the fibres in a mouse model of atopic dermatitis reduces scratching behaviour. Thus, regulation of nerve fibre extension could be an effective strategy to reduce itching in pruritus dermatosis. In this study, we established a new coculture system of keratinocytes and dorsal-root-ganglion-derived cells using an apparatus, AXIS(™) , which consists of two different channels connected via a set of microgrooves, through which signalling molecules and axons, but not living cells, can pass. When we seeded keratinocytes in one chamber, extension of nerve fibres was observed from dorsal root ganglion cells seeded in the other chamber. Addition of anti-BDNF antibody in the keratinocyte-seeded chamber significantly reduced the extension. Application of Semaphorin 3A also reduced the extension by approximately 50%. We suggest that this coculture system may be useful for screening of anti-itching drugs.

Frontiers in epidermal barrier homeostasis--an approach to mathematical modelling of epidermal calcium dynamics.

Intact epidermal barrier function is crucial for survival and is associated with the presence of gradients of both calcium ion concentration and electric potential. Although many molecules, including ion channels and pumps, are known to contribute to maintenance of these gradients, the mechanisms involved in epidermal calcium ion dynamics have not been clarified. We have established that a variety of neurotransmitters and their receptors, originally found in the brain, are expressed in keratinocytes and are also associated with barrier homeostasis. Moreover, keratinocytes and neurons show some similarities of electrochemical behaviour. As mathematical modelling and computer simulation have been employed to understand electrochemical phenomena in brain science, we considered that a similar approach might be applicable to describe the dynamics of epidermal electrochemical phenomena associated with barrier homeostasis. Such methodology would also be potentially useful to address a number of difficult problems in clinical dermatology, such as ageing and itching. Although this work is at a very early stage, in this essay, we discuss the background to our approach and we present some preliminary results of simulation of barrier recovery.

Dynamics of intracellular calcium in cultured human keratinocytes after localized cell damage.

Recovery of cultured keratinocytes after scratch damage is considered to be a wound-healing model. In this study, we observed changes in intracellular calcium concentration ([Ca(2+) ]i ) in cultured human keratinocytes after scratch damage. Immediately after scratch damage, a wave of increased [Ca(2+) ]i radiated outward from the damaged area and then disappeared gradually. But, [Ca(2+) ]i remained elevated in a peripheral layer of cells around the damaged area for several minutes. This layer did not appear in calcium-free medium. When the culture was switched to calcium-free medium for 30 min immediately after scratch damage, then switched back to standard (Ca(2+) -containing) medium, the recovery ratio after 24 h was approximately 25% lower than that of the culture in standard medium throughout. We speculate that delineation of damage sites by a layer of cells with increased [Ca(2+) ]i might be part of a signalling pathway that appropriately directs the wound-healing process in epidermis.

Low environmental humidity induces synthesis and release of cortisol in an epidermal organotypic culture system.

Dry environmental conditions induce a variety of skin pathologies and a recent report indicating that cortisol synthesis in epidermis was increased during wound healing led us to hypothesize that environmental dryness might induce increased cortisol secretion in epidermis. Therefore, we incubated a skin equivalent model under dry (relative humidity: less than 10%) and humid (relative humidity: approximately 100%) conditions for 48 hours and evaluated cortisol secretion and mRNA levels of cortisol-synthesizing enzyme (steroid 11ß-hydroxylase, CYP11B1) and IL-1ß. Cortisol secretion was increased threefold, and CYP11B1 and IL-1ß mRNAs were increased 38-fold and sixfold, respectively, in the dry condition versus the humid condition. Occlusion with a water-impermeable plastic membrane partially blocked the increases in cortisol secretion and CYP11B1 and IL-1ß mRNA expression in the dry condition. Thus, environmental dryness might induce increased cortisol secretion in epidermis of diseased skin characterized by epidermal barrier dysfunction, potentially influencing mental state and systemic physiology.

External negative electric potential accelerates exocytosis of lamellar bodies in human skin ex vivo.

Exocytosis of lamellar bodies at the uppermost nucleated layer of the epidermis is a crucial process for epidermal permeability barrier homoeostasis. We have previously suggested that skin surface electric potential might be associated with barrier homoeostasis. Thus, we hypothesized that the potential might drive exocytosis of lamellar bodies. In this study, we tested this idea by applying negative electric potential (-0.5 V) to human skin samples ex vivo for 2 h and observing the ultrastructure of the uppermost layer. The secretion of lamellar bodies was accelerated in the potential-applied skin, compared to that in untreated control skin. Multiphoton observation indicated that extracellular lipid domains were more extensive in treated skin than in control skin. Moreover, the calcium ion gradient was greater at the uppermost layer of the epidermis of treated skin, compared to that in control skin. These results indicate that electric potential may regulate lamellar body secretion in healthy human skin.

Distinct intracellular calcium responses of individual cultured human keratinocytes to air pressure changes.

BACKGROUND: We previously showed that application of hydraulic pressure to cultured human keratinocytes induced elevation of intracellular calcium concentration ([Ca(2+) ]i ), but the absolute value of the pressure could not be determined. PURPOSE: To evaluate the effect of the absolute value of pressure on keratinocytes and other skin cells. METHODS: In the present work, we examined the effect of changes in absolute pressure level by observing the [Ca(2+) ]i responses of cultured human keratinocytes and other cells cultured at the bottom of a hermetically sealed plastic flask as the air pressure in the flask was increased gradually, held stable, and then decreased abruptly, using the Ca(2+) -indicator fura-2. RESULTS: We found that the [Ca(2+) ]i of differentiated keratinocytes was changed significantly in each phase, whereas undifferentiated keratinocytes and other cells derived from skin or dorsal root ganglion showed no response. Removal of calcium from the medium blocked the increase in [Ca(2+) ]i in differentiated keratinocytes. The [Ca(2+) ]i responses of individual differentiated keratinocytes in the increasing, stable and decreasing phases of pressure change varied from cell to cell. The threshold of air-pressure increase from the original level for inducing [Ca(2+) ]i response was 5 - 20 hPa. CONCLUSION: These results suggest that epidermal keratinocytes might contain a sensory system that detects changes of external pressure on the skin.

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.