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.

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.

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.

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.

How does epidermal pathology interact with mental state?

The hypothesis is presented that human emotional state is influenced by epidermal pathology via the release from epidermal keratinocytes of a wide variety of chemical mediators (including neurotransmitters) that act on the brain. It has long been recognized that epidermal keratinocytes play a key role in the function of the stratum corneum as an impermeable barrier, and that skin diseases such as atopic dermatitis and psoriasis, which cause itching, sleep disturbance and concern over appearance, are associated with depression and anxiety. On the other hand, epidermal keratinocytes are known to produce and release multiple cytokines and chemical mediators in response to barrier impairment or insult, such as environmental dryness or UV radiation. Elevation of plasma cytokines is associated with depression in cancer patients. Serum levels of oxytocin and glucocorticoid have been shown to influence mental state, and a recent study showed that glucocorticoid is generated in injured epidermis. Thus, there are multiple plausible pathways through which changes in skin can affect emotional state.

In vitro formation of organized structure between keratinocytes and dorsal-root-ganglion cells.

We recently found that the morphology in a co-culture system of keratinocytes and dorsal-root ganglion-derived cells depended on the timing of seeding of the two cell types. In skin, epidermis is formed first, followed by construction of peripheral nerve structure. Therefore, we hypothesized that formation of peripheral nerve structure in the epidermis might be driven by interaction between keratinocytes and nerve cells. In the present study, we tested this idea by incubating keratinocytes and dorsal-root ganglion cells in a spatially separated manner and observing the morphological changes in the co-culture system. Extension of nerve fibre-like structures from the ganglion cells was observed, and within 3 days after seeding, many nerve fibre-like extensions penetrated into the keratinocyte cluster, subsequently forming a network that appeared to resemble the cutaneous peripheral nervous system. Our present model may be useful for studying the formation of peripheral nerve structure in the skin.

Oxytocin is expressed in epidermal keratinocytes and released upon stimulation with adenosine 5'-[γ-thio]triphosphate in vitro.

Oxytocin is a neuropeptide produced primarily in the hypothalamus and is best known for its roles in parturition and lactation. It also influences behaviour, memory and mental state. Recent studies have suggested a variety of roles for oxytocin in peripheral tissues, including skin. Here we show that oxytocin is expressed in human skin. Immunohistochemical studies showed that oxytocin and its carrier protein, neurophysin I, are predominantly localized in epidermis. RT-PCR confirmed the expression of oxytocin in both skin and cultured epidermal keratinocytes. We also show that oxytocin is released from keratinocytes after application of adenosine 5'-[γ-thio]triphosphate (ATPγS, a stable analogue of ATP) in a dose-dependent manner. The ATPγS-induced oxytocin release was inhibited by removal of extracellular calcium, or by the P2X receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). These results suggest that oxytocin is produced in human epidermal keratinocytes and is released in response to calcium influx via P2X receptors.

Ryanodine receptors are expressed in epidermal keratinocytes and associated with keratinocyte differentiation and epidermal permeability barrier homeostasis.

Ryanodine receptors (RyRs) have an important role as calcium channels in the regulation of intracellular calcium levels in the nervous system and muscle. In the present study, we investigated the expression of RyR in human epidermis. Immunohistochemical studies and reverse transcription-PCR indicated the expression of RyR type 1, 2, and 3 proteins in epidermal keratinocytes. The expression level of each RyR subtype was higher in differentiating keratinocytes than in proliferative cells. We also demonstrated the functional expression of RyR by calcium imaging. In cultured human keratinocytes, application of the RyR agonist 4-chloro-m-cresol (CMC) induced elevation of the intracellular calcium concentration, and co-application of the RyR antagonist 1,1'-diheptyl-4,4'-bipyridinium dibromide (DHBP) blocked the elevation. Application of CMC accelerated keratinocyte differentiation in vitro. On the other hand, topical application of CMC after tape-stripping of hairless mouse skin delayed barrier recovery, whereas application of an RyR antagonist, dantrolene or DHBP, accelerated the barrier recovery. These results suggest that RyR expressed in epidermal keratinocytes is associated with both differentiation of keratinocytes and epidermal barrier homeostasis.

Morphological and functional differences in coculture system of keratinocytes and dorsal-root-ganglion-derived cells depending on time of seeding.

Previous study indicated that in a coculture system of keratinocytes and dorsal-root-ganglion-derived (DRG) cells, mechanical stimulation of keratinocytes induced ATP-mediated calcium propagation and excitation of DRG cells. Here, we examined two different coculture systems of keratinocytes and DRG cells. In one, we seeded keratinocytes first and then seeded DRG cells on the keratinocytes. In this system, nerve fibres from DRG cells passed between keratinocytes. Mechanical stimulation of keratinocytes did not induce excitation of DRG cells. In the other, we seeded both cell types together. At first, each cell type grew separately, forming cell aggregates. Then, nerve fibres grew out from the DRG cell aggregates to keratinocyte aggregates and penetrated into them. In this system, mechanical stimulation of keratinocytes induced excitation of the nerve fibres, but the excitation was not completely blocked by apyrase, an ATP-degrading enzyme. These results suggest that coculture of keratinocytes and DRG can generate a variety of structures, depending on the seeding conditions.

Phosphodiesterase inhibitors block the acceleration of skin permeability barrier repair by red light.

We previously demonstrated that exposure to red light (550-670 nm) accelerates epidermal permeability barrier recovery after barrier disruption. Furthermore, we showed that photosensitive proteins, originally found in retina, are also expressed in epidermis. In retina, transducin and phosphodiesterase 6 play key roles in signal transmission. In this study, we evaluate the role of phosphodiesterese 6 in the acceleration by red light of epidermal permeability barrier recovery. Immunohistochemical study and reverse transcription-PCR assays confirmed the expression of both transducin and phosphodiesterase 6 in epidermal keratinocytes. Topical application of 3-isobutyl-1-methylxanthine, a non-specific phosphodiesterase inhibitor, blocked the acceleration of the barrier recovery by red light. Topical application of zaprinast, a specific inhibitor of phosphodiesterases 5 and 6, also blocked the acceleration, whereas T0156, a specific inhibitor of phosphodiesterase 5, had no effect. Red light exposure reduced the epidermal hyperplasia induced by barrier disruption under low humidity, and the effect was blocked by pretreatment with zaprinast. Our results indicate phosphodiesterase 6 is involved in the recovery-accelerating effect of red light on the disrupted epidermal permeability barrier.

Topical application of TRPM8 agonists accelerates skin permeability barrier recovery and reduces epidermal proliferation induced by barrier insult: role of cold-sensitive TRP receptors in epidermal permeability barrier homoeostasis.

TRPA1 and TRPM8 receptors are activated at low temperature (A1: below 17 degrees C and M8: below 22 degrees C). Recently, we observed that low temperature (below 22 degrees C) induced elevation of intracellular calcium in keratinocytes. Moreover, we demonstrated that topical application of TRPA1 agonists accelerated the recovery of epidermal permeability barrier function after disruption. In this study, we examined the effect of topical application of TRPM8 modulators on epidermal permeability barrier homoeostasis. Immunohistochemical study and RT-PCR confirmed the expression of TRPM8 or TRPM8-like protein in epidermal keratinocytes. Topical application of TRPM8 agonists, menthol and WS 12 accelerated barrier recovery after tape stripping. The effect of WS12 was blocked by a non-selective TRP antagonist, Ruthenium Red, and a TRPM8-specific antagonist, BTCT. Topical application of WS12 also reduced epidermal proliferation associated with barrier disruption under low humidity, and this effect was blocked by BTCT. Our results indicate that TRPM8 or a closely related protein in epidermal keratinocytes plays a role in epidermal permeability barrier homoeostasis and epidermal proliferation after barrier insult.

Calcium ion propagation in cultured keratinocytes and other cells in skin in response to hydraulic pressure stimulation.

We have previously suggested that a variety of environmental factors might be first sensed by epidermal keratinocytes, which represent the frontier of the body. To further examine this idea, in the present study, we examined the intracellular calcium responses of cultured keratinocytes to external hydraulic pressure. First, we compared the responses of undifferentiated and differentiated keratinocytes with those of fibroblasts, vascular endothelial cells (VEC), and lymphatic endothelial cells. Elevation of intracellular calcium was observed after application of pressure to keratinocytes, fibroblasts, and VEC. The calcium propagation extended over a larger area and continued for a longer period of time in differentiated keratinocytes, as compared with the other cells. The response of the keratinocytes was dramatically reduced when the cells were incubated in medium without calcium. Application of a non-selective transient receptor potential (TRP) channel blocker also attenuated the calcium response. These results suggest that differentiated keratinocytes are sensitive to external pressure and that TRP might be involved in the mechanism of their response.

Glycolic acid induces keratinocyte proliferation in a skin equivalent model via TRPV1 activation.

BACKGROUND: Glycolic acid (GA) is the most commonly used alpha-hydroxy acid (AHA) for dermatologic applications, and is considered as a versatile superficial peeling agent for facial rejuvenation. Its therapeutic effect includes acceleration of epidermal turnover without apparent inflammation, and its action is pH-dependent. However, little is known about the molecular mechanism of GA-induced peeling. OBJECTIVE: To investigate the effects of topical application of GA on cell proliferation using a skin equivalent model and to examine the molecular mechanisms of GA-induced peeling. METHODS: GA solution was applied on the surface of a skin equivalent model, and cell proliferation was measured by means of BrdU-incorporation and immunohistochemical methods. Release of chemical mediators such as ATP into the medium was examined. The effects of antagonists of ion channels were also analyzed. RESULTS: At 24h after GA application, BrdU-incorporation into basal keratinocytes was significantly increased. Induction of keratinocyte proliferation was pH-dependent, and was inhibited by antagonists of TRPV1, an acid-sensitive ion channel. Furthermore, transient ATP release was detected in the culture medium after GA stimulation, and this was also suppressed by TRPV1 antagonists. CONCLUSION: These results suggest that one of the mechanisms of GA-induced epidermal proliferation is a growth response of basal keratinocytes to the local elevation of H(+)-ion concentration by infiltrated GA. This response is mediated by TRPV1 activation and ATP release. Activation of P2 receptors by the released ATP may also be involved.

Neuronal nitric oxide synthase in epidermis is involved in cutaneous circulatory response to mechanical stimulation.

The source of nitric oxide (NO) in the cutaneous circulation remains controversial. We hypothesized that epidermis might generate NO in response to mechanical stimulation. In hairless mouse (HR-1) skin organ culture, mechanical stimulation resulted in NO release, which declined within 30 minutes after cessation. A similar NO release occurred in a reconstructed skin model containing only keratinocytes and fibroblasts and was suppressed after detachment of the epidermal layer. Moreover, the stimulation-induced NO release was significantly lower in skin organ culture from neuronal NO synthase knockout (nNOS-KO) mice, compared with wild-type (WT) mice. Mechanical stimulation of skin organ cultures from HR-1, nNOS-KO, endothelial NOS-KO (eNOS-KO), and WT mice caused an enlargement of cutaneous lymphatic vessels. The enlargement was significantly lower after detachment of the epidermal layer than in normal skin samples and was significantly lower for nNOS-KO than for WT mice. Skin blood flow in nNOS-KO mice after stimulation was significantly lower than in WT mice. eNOS-KO mice also showed lower responses than WT mice, and the difference was similar to that in the case of nNOS-KO mice. These results are consistent with the idea that NO generated by epidermal nNOS has a significant role in the cutaneous circulatory response to mechanical stimulation.