The skin: an indispensable barrier

Abstract: The skin forms an effective barrier between the organism and the environment preventing invasion of pathogens and fending off chemical and physical assaults, as well as the unregulated loss of water and solutes. In this review we provide an overview of several components of the physical barrier, explaining how barrier function is regulated and altered in dermatoses. The physical barrier is mainly localized in the stratum corneum (SC) and consists of protein‐enriched cells (corneocytes with cornified envelope and cytoskeletal elements, as well as corneodesmosomes) and lipid‐enriched intercellular domains. The nucleated epidermis also contributes to the barrier through tight, gap and adherens junctions, as well as through desmosomes and cytoskeletal elements. During epidermal differentiation lipids are synthesized in the keratinocytes and extruded into the extracellular domains, where they form extracellular lipid‐enriched layers. The cornified cell envelope, a tough protein/lipid polymer structure, resides below the cytoplasmic membrane on the exterior of the corneocytes. Ceramides A and B are covalently bound to cornified envelope proteins and form the backbone for the subsequent addition of free ceramides, free fatty acids and cholesterol in the SC. Filaggrin is cross‐linked to the cornified envelope and aggregates keratin filaments into macrofibrils. Formation and maintenance of barrier function is influenced by cytokines, 3′,5′‐cyclic adenosine monophosphate and calcium. Changes in epidermal differentiation and lipid composition lead to a disturbed skin barrier, which allows the entry of environmental allergens, immunological reaction and inflammation in atopic dermatitis. A disturbed skin barrier is important for the pathogenesis of contact dermatitis, ichthyosis, psoriasis and atopic dermatitis.     

Modulation of stratum corneum lipid composition and organization of human skin equivalents by specific medium supplements

Our in‐house human skin equivalents contain all stratum corneum (SC) barrier lipid classes, but have a reduced level of free fatty acids (FAs), of which a part is mono‐unsaturated. These differences lead to an altered SC lipid organization and thereby a reduced barrier function compared to human skin. In this study, we aimed to improve the SC FA composition and, consequently, the SC lipid organization of the Leiden epidermal model (LEM) by specific medium supplements. The standard FA mixture (consisting of palmitic, linoleic and arachidonic acids) supplemented to the medium was modified, by replacing protonated palmitic acid with deuterated palmitic acid or by the addition of deuterated arachidic acid to the mixture, to determine whether FAs are taken up from the medium and are incorporated into SC of LEM. Furthermore, supplementation of the total FA mixture or that of palmitic acid alone was increased four times to examine whether this improves the SC FA composition and lipid organization of LEM. The results demonstrate that the deuterated FAs are taken up into LEMs and are subsequently elongated and incorporated in their SC. However, a fourfold increase in palmitic acid supplementation does not change the SC FA composition or lipid organization of LEM. Increasing the concentration of the total FA mixture in the medium resulted in a decreased level of very long chain FAs and an increased level of mono‐unsaturated FAs, which lead to deteriorated SC lipid properties. These results indicate that SC lipid properties can be modulated by specific medium supplements.     

Molecular interactions of plant oil components with stratum corneum lipids correlate with clinical measures of skin barrier function

Plant‐derived oils consisting of triglycerides and small amounts of free fatty acids (FFAs) are commonly used in skincare regimens. FFAs are known to disrupt skin barrier function. The objective of this study was to mechanistically study the effects of FFAs, triglycerides and their mixtures on skin barrier function. The effects of oleic acid (OA), glyceryl trioleate (GT) and OA/GT mixtures on skin barrier were assessed in vivo through measurement of transepidermal water loss (TEWL) and fluorescein dye penetration before and after a single application. OA's effects on stratum corneum (SC) lipid order in vivo were measured with infrared spectroscopy through application of perdeuterated OA (OA‐d₃₄). Studies of the interaction of OA and GT with skin lipids included imaging the distribution of OA‐d₃₄ and GT ex vivo with IR microspectroscopy and thermodynamic analysis of mixtures in aqueous monolayers. The oil mixtures increased both TEWL and fluorescein penetration 24 h after a single application in an OA dose‐dependent manner, with the highest increase from treatment with pure OA. OA‐d₃₄ penetrated into skin and disordered SC lipids. Furthermore, the ex vivo IR imaging studies showed that OA‐d₃₄ permeated to the dermal/epidermal junction while GT remained in the SC. The monolayer experiments showed preferential interspecies interactions between OA and SC lipids, while the mixing between GT and SC lipids was not thermodynamically preferred. The FFA component of plant oils may disrupt skin barrier function. The affinity between plant oil components and SC lipids likely determines the extent of their penetration and clinically measurable effects on skin barrier functions.     

Nature versus nurture: does human skin maintain its stratum corneum lipid properties in vitro?

Human skin equivalents (HSEs) mimic human skin closely, but show differences in their stratum corneum (SC) lipid properties. The aim of this study was to determine whether isolation of primary cells, which is needed to generate HSEs, influence the SC lipid properties of HSEs. For this purpose, we expanded explants of intact full thickness human skin and isolated epidermal sheets in vitro. We investigated whether their outgrowths maintain barrier properties of human skin. The results reveal that the outgrowths and human skin have a similar morphology and expression of several differentiation markers, except for an increased expression of keratin 16 and involucrin. The outgrowths show a decreased SC fatty acid content compared with human skin. Additionally, SC lipids of the outgrowths have a predominantly hexagonal packing, whereas human skin has the dense orthorhombic packing. Furthermore, the outgrowths have lipid lamellae with a slightly reduced periodicity compared with human skin. These results demonstrate that the outgrowths do not maintain all properties observed in human skin, indicating that changes in properties of HSEs are not caused by isolation of primary cells, but by culture conditions.     

Repair of acetone- and sodium lauryl sulphate-damaged human skin barrier function using topically applied emulsions containing barrier lipids

BACKGROUND: It is generally acknowledged that well-formulated moisturizing skin care products can restore disturbed barrier function that can be assessed by transepidermal water loss (TEWL) measurements. When ceramides and/or other barrier lipids are incorporated, it is, however, not always clearly demonstrated which ingredients of the formulation exert the beneficial effects. OBJECTIVES: In this study the effects of topically applied ceramide-containing mixtures on the barrier repair of sodium lauryl sulphate (SLS)- and acetone-induced skin damage have been studied in human volunteers. TEWL and stratum corneum hydration measurements were carried out. The emulsions applied contained either a mixture of two types of ceramides, CerIII and CerIIIB (emulsion 1) or a complete mixture of ceramides III, IIIB and VI together with phytosphingosine, cholesterol and the free fatty acid linoleic acid (emulsion 2). RESULTS: After SLS damage, it was observed that barrier recovery was significantly accelerated by topical application (14 days, 2 x/d) of emulsion 2 compared with the results obtained with emulsion 1. Corneometrical results were not relevant due to the occurrence of scaly fissured skin, failing to provide a good skin/probe contact. Although no effect on TEWL could be observed, the improvement of skin hydration after acetone treatment and a single application of the emulsions, was significantly more positive for emulsion 2 than for emulsion 1. CONCLUSIONS: The investigative methods used in this study show that ceramides combined with other skin lipids can improve barrier repair after damage.     

紫外线照射对表皮屏障功能的影响

 表皮屏障在抵御外界危险因素侵害的过程中发挥重要作用。表皮屏障中各组成部分的结构及功能失调与人类的多种皮肤疾病有关。紫外线辐射等因素可引起表皮屏障功能障碍,紫外线照射使表皮屏障变得更易渗透,经皮水丢失量增加,病原体和过敏原更易进入体内,其机制可能与角质层屏障中角化包膜结构蛋白、细胞外脂质与天然保湿因子的含量降低,以及颗粒层中紧密连接屏障功能的减弱有关。近年来一些研究拓展了紫外线照射对表皮屏障的结构与功能影响的认识,本文综述紫外线照射与表皮屏障功能的影响。     

Corticosteroid-induced atrophy and barrier impairment measured by non-invasive methods in human skin

Background/aims: Atrophy is a distressing side effect of potent corticosteroids. After open application of a high potency steroid, we monitored atrophogenicity by a variety of non-invasive methods.
Methods: Volar forearms were treated twice daily for 3 or 4 weeks, with clobetasol propionate cream (Temovate^®). The following methods were used: 1) confocal microscopy, 2) transepidermal water loss (TEWL), 3) dimethyl sulfoxide whealing, 4) sodium hydroxide erosions, 5) analysis of stratum corneum lipids, and 6) B-scan ultrasound.
Results: Confocal microscopy revealed thinning of the epidermis, decreased microvasculature and decreased size of keratinocytes. Evaporimetry demonstrated transepidermal water loss. Whealing to dimethyl sulfoxide was enhanced. Sodium hydroxide erosions formed more quickly. The amount of ceramides, cholesterol, and free fatty acids was reduced. Ultrasound showed thinning of the dermis.
Conclusion: Non-invasive methods are very useful for quantifying the atrophogenicity of topical corticosteroids.     

Barrier recovery rate varies time-dependently in human skin

The recovery in cutaneous barrier functions, assessed in terms of transepidermal water loss, 1 h after tape stripping of volar forearm skin in human volunteers, was investigated at different times over the 24 h day. The barrier recovery rate was significantly lower between 20:00 h and 23:00 h than that at other time points. The skin surface temperature and the basal transepidermal water loss reached their highest values at about 03:00 h (33.6 degrees C and 0.30 mg cm-2 h-1), while the cortisol level in the saliva was highest at 09:00 h (7.8 pmol mL-1). These results suggest significant time-dependent variation in cutaneous barrier repair independent of changes in skin temperature and cortisol level.     

Modulators of the endocannabinoid system influence skin barrier repair,epidermal proliferation,differentiation and inflammation in a mouse model

Endocannabinoids (ECs) are important regulators of cell signalling. Cannabinoid receptors are involved in keratinocyte proliferation/differentiation. Elevation of the endogenous cannabinoid tone leads to strong anti‐inflammatory effects. Here, we explored the influence of endocannabinoid system (ECS) modulators on skin permeability barrier repair, epidermal proliferation, differentiation and inflammation in hairless mice. We used WOBE440, a selective fatty acid amide hydrolase (FAAH) inhibitor, WOL067‐531, an inhibitor of endocannabinoid reuptake with no relevant FAAH activity, which both signal via cannabinoid receptor‐1 and cannabinoid receptor‐2 (CB‐1R and CB‐2R) and compared them to WOBE15 which signals via CB‐2R. Barrier disruption and skin irritation were induced by tape stripping or by sodium dodecyl sulphate (SDS) patch testing. Immediately after barrier disruption, 30 μL of 0.5% WOBE440, WOL067‐531 and WOBE15 solutions or the vehicle was applied topically. Barrier repair was monitored by transepidermal water loss at 1.5, 3, 5 and 7 hours. We found that barrier repair was significantly delayed by WOL067‐531. A tendency for a delay was noticed for WOBE440, whereas for WOBE15, no effect was observed. Immunohistology showed that the tape‐stripping‐induced increase in epidermal proliferation and filaggrin expression was significantly reduced by topical applications of WOL067‐531 and WOBE440, but not by WOBE15. Also, the SDS‐induced inflammation, as determined by the number of inflammatory cells, was reduced by WOL067‐531 and WOBE440. In summary, we showed that WOL067‐531 exhibits a significant effect on skin barrier repair, epidermal proliferation/differentiation and inflammation.     

Non‐invasive assessment of tryptophan fluorescence and confocal microscopy provide information on skin barrier repair dynamics beyond TEWL

The stratum corneum (SC) serves a primary function of skin barrier and understanding the kinetics of SC formation may provide great insight for skin diagnosis and evaluation of therapies. Besides trans‐epidermal water loss (TEWL), few methods have been characterized to assess skin barrier non‐invasively in vivo, particularly for dynamic measurements on the same specimen over time. The objective of this study was to characterize alternative non‐invasive methods to evaluate the dynamic processes involved in the recovery of normal human SC after total removal. TEWL, tryptophan fluorescence and reflectance confocal microscopy (RCM) were used to determine skin barrier function, cell turnover and epidermal morphology over a period of 10 days after total removal of the SC by tape stripping. The results show a biphasic recovery of TEWL over time, which contrasted with a linear increase of 2.3 μm/day in SC thickness. Tryptophan assessment of cell turnover also demonstrated a biphasic pattern attaining a maximum three to four times the levels of the control site 3 days after injury that slowly returned to baseline and displayed great correlation (R² > 0.95) to viable epidermis thickness that also achieved a maximum about 3 days after injury with an approximate increase of 55%. When plotting the change of TEWL versus SC thickness, a single exponential function is observed [Δ‐TEWL = 55 exp (?0.157×)] which contrasts with other proposed models. These methods were able to present rates for SC recovery processes beyond skin barrier (TEWL) that may provide new insights on kinetics of barrier formation for evaluation of skin conditions and treatments.     

Fluorescence induction of protoporphyrin IX by a new 5‐aminolevulinic acid nanoemulsion used for photodynamic therapy in a full‐thickness ex vivo skin model

Please cite this paper as: Fluorescence induction of protoporphyrin IX by a new 5‐aminolevulinic acid nanoemulsion used for photodynamic therapy in a full‐thickness ex vivo skin model. Experimental Dermatology 2010; 19 : e302–e305. Abstract: An ex vivo porcine skin model was utilized to analyse the penetration of 5‐aminolevulinic acid (5‐ALA) contained in a nanoemulsion‐based formulation BF‐200 ALA (10% 5‐ALA‐hydrochloride) versus 16% aminolevulinate methyl ester‐hydrochloride in a commercially cream (MAL cream) by fluorescence microscopy of their common metabolite protoporphyrin IX (PpIX) after 3, 5, 8 and 12 h. Fluorescence signals of PpIX in pig skin treated with BF‐200 ALA were stronger than those for MAL cream. At 8 and 12 h, the PpIX fluorescence signals were 4.8‐ and 5.0‐fold higher than those measured after MAL cream application. Fluorescence signals of PpIX after application of BF‐200 ALA were detected in deeper tissue layers of the epidermis than after application of MAL cream (97.2 ± 5.7 μm for BF‐200 ALA vs 42.0 ± 4.2 μm for MAL cream). These data implicate that BF‐200 ALA in photodynamic therapy might lead to a superior therapeutically effect of intraepidermal (in situ) squamous cell carcinomas.     

International guidelines for the in vivo assessment of skin properties in non‐clinical settings: Part 2. transepidermal water loss and skin hydration

Background

There is an emerging perspective that it is not sufficient to just assess skin exposure to physical and chemical stressors in workplaces, but that it is also important to assess the condition, i.e. skin barrier function of the exposed skin at the time of exposure. The workplace environment, representing a non‐clinical environment, can be highly variable and difficult to control, thereby presenting unique measurement challenges not typically encountered in clinical settings.

Methods

An expert working group convened a workshop as part of the 5th International Conference on Occupational and Environmental Exposure of Skin to Chemicals (OEESC) to develop basic guidelines and best practices (based on existing clinical guidelines, published data, and own experiences) for the in vivo measurement of transepidermal water loss (TEWL) and skin hydration in non‐clinical settings with specific reference to the workplace as a worst‐case scenario.

Results

Key elements of these guidelines are: (i) to minimize or recognize, to the extent feasible, the influences of relevant endogenous‐, exogenous‐, environmental‐ and measurement/instrumentation‐related factors; (ii) to measure TEWL with a closed‐chamber type instrument; (iii) report results as a difference or percent change (rather than absolute values); and (iv) accurately report any notable deviations from this guidelines.

Conclusion

It is anticipated that these guidelines will promote consistent data reporting, which will facilitate inter‐comparison of study results.     

Topically applied fatty acids are elongated before incorporation in the stratum corneum lipid matrix in compromised skin

In several skin diseases, both the lipid composition and organization in the stratum corneum (SC) are altered which contributes to the impaired skin barrier function in patients. One of the approaches for skin barrier repair is treatment with topical formulations to normalize SC lipid composition and organization. Vernix caseosa (VC), a white cheesy cream on the skin during gestational delivery, has shown to enhance skin barrier repair. In this study, we examined how a fatty acid (FA) containing formulation mimicking the lipid composition of VC interacts with the lipid matrix in the SC. The formulation was applied on ex vivo human skin after SC removal. Subsequently, the ex vivo human skin generated SC during culture. The effect of FA containing formulations on the lipid organization and composition in the regenerated SC was analysed by Fourier transform infrared (FTIR) spectroscopy and liquid chromatography mass spectroscopy (LC/MS), respectively. FTIR results demonstrate that the FAs are intercalated in the lipid matrix of the regenerated SC and partition in the same lattice with the endogenous SC lipids, thereby enhancing the fraction of lipids forming an orthorhombic (very dense) packing in the SC. LC/MS data show that the topically applied FAs are elongated before intercalation in the lipid matrix and are thus involved in the lipid biosynthesis in the skin.     

A topical heparinoid‐containing product improves epidermal permeability barrier homeostasis in mice

Because of the importance of epidermal functions, including stratum corneum hydration and maintenance of permeability barrier homeostasis, in the pathogenesis of a variety of cutaneous and systemic disorders, a wide range of products has been developed to improve epidermal functions. However, the underlying mechanisms whereby certain products, including heparinoid‐containing product, are far little understood. In the present study, we assessed the impact of a heparinoid‐containing product, Hirudoid^® cream, on epidermal permeability barrier function and expression levels of a panel of epidermal mRNA related to the formation/maintenance of the permeability barrier in mouse skin. Our results showed that while the baseline levels of transepidermal water rates remained unchanged, treatment with Hirudoid^® cream twice daily for 7 days significantly accelerated permeability barrier recovery and increased stratum corneum hydration. In parallel, expression levels of epidermal mRNA for certain differentiation marker‐related proteins, lipid synthetic enzymes, keratinocyte proliferation and antimicrobial peptides also increased significantly. Together, these results provide the underlying mechanisms by which topical Hirudoid^® cream improves epidermal permeability barrier and antimicrobial function. Because of its benefits for epidermal functions, heparinoid‐containing product could be more useful in the management of skin conditions, characterized by abnormal permeability barrier and antimicrobial function.