Ozone potentiates vitamin E depletion by ultraviolet radiation in the murine stratum corneum

As the outermost layer of the skin, the stratum corneum is exposed to environmental oxidants. To investigate putative synergisms of environmental oxidative stressors in stratum corneum, hairless mice were exposed to ultraviolet radiation (UV) and ozone (O(3)) alone and in combination. Whereas a significant depletion of alpha-tocopherol was observed after individual exposure to either a 0.5 minimal erythemal dose of UV or 1 ppm O(3) for 2 h, the combination did not increase the effect of UV alone. However, a dose of 0.5 ppm O(3) x 2 h, which had no effect when used alone, significantly enhanced the UV-induced depletion of vitamin E. We conclude that concomitant exposure to low doses of UV and O(3) at levels near those that humans can be exposed to causes additive oxidative stress in the stratum corneum.     

In vivo ozone exposure induces antioxidant/stress-related responses in murine lung and skin

Lung and skin are the organs directly exposed to environmental pollution. Ozone (O(3)) is a toxic, oxidant air pollutant, and exposure has been shown to induce antioxidant depletion as well as oxidation of lipids and proteins within the outermost skin layer (stratum corneum) and the lung respiratory tract lining fluids (RTLFs). To further define skin and lung responses to O(3) exposure, SKH-1 hairless mice were exposed to either 0.8 ppm of O(3) (a level occasionally reached in very polluted areas) or ambient air 6 h/day for 6 consecutive days. O(3) exposure resulted in the depletion of alpha-tocopherol in lung and plasma and induction in both skin and lung of heme oxygenase 1, cyclooxygenase 2, and proliferating cell nuclear antigen. O(3)-exposed animals showed a similar extent of upregulation of COX-2 and PCNA in lung and skin, whereas HO-1 was more responsive in skin than in lung (7-fold induction vs. 2-fold induction). In addition to these measures of response to oxidative stress, O(3) exposure led to the activation of nuclear factor kappaB measured as IkappaBalpha phosphorylation in both tissues. We conclude that in this model, O(3) at high pollutant levels is able to affect both lung and skin biology, inducing depletion of alpha-tocopherol and inducing stress-related responses in both skin epidermis and respiratory tract epithelium.     

Heterogeneous drying stresses in stratum corneum

We study the drying of stratum corneum, the skin's outermost layer and an essential barrier to mechanical and chemical stresses from the environment. Even though stratum corneum exhibits structural features across multiple length-scales, contemporary understanding of the mechanical properties of stratum corneum is based on the assumption that its thickness and composition are homogeneous. We quantify spatially resolved in-plane traction stress and deformation at the interface between a macroscopic sample of porcine stratum corneum and an adherent deformable elastomer substrate. At length-scales greater than a millimeter, the skin behaves as a homogeneous elastic material. At this scale, a linear elastic model captures the spatial distribution of traction stresses and the dependence of drying behavior on the elastic modulus of the substrate. At smaller scales, the traction stresses are strikingly heterogeneous and dominated by the heterogeneous structure of the stratum corneum.     

Using the method of homogenization to calculate the effective diffusivity of the stratum corneum with permeable corneocytes

The stratum corneum is the outermost layer of the skin, which acts as a barrier membrane against the penetration of molecules into and out of the body. It has a biphasic structure consisting of keratinized cells (corneocytes) that are embedded in a lipid matrix. The macroscopic transport properties of the stratum corneum are functions of its microstructure and the transport properties of the corneocytes and the lipid matrix, and are of considerable interest in the context of transdermal drug delivery and quantifying exposure to toxins, as well as for determining the relation of skin disorders to disruption of the stratum corneum barrier. Due to the complexity of the tissue and the difference in length scales involved in its microstructure, a direct analysis of the mass transport properties of the stratum corneum is not feasible. In this study, we undertake an approach where the macroscopic diffusion tensor of the stratum corneum is obtained through homogenization using the method of asymptotic expansions. The biphasic structure of the stratum corneum is fully accounted for by allowing the corneocytes to be permeable and considering the partitioning between the corneocytes and the lipid phases. By systematically exploring the effect of permeable corneocytes on the macroscopic transport properties of the stratum corneum, we show that solute properties such as lipophilicity and relative permeabilities in the two phases have large effects on its transdermal diffusion behavior.     

The stratum corneum lipid thermotropic phase behavior.

The stratum corneum, the outermost layer of mammalian skin, is considered the least permeable skin layer to the diffusion of water and other solutes. It is generally accepted that the intercellular lipid multilayer domain is the diffusional pathway for most lipophilic solutes. Fluidization of the lipid multilayers is believed to result in the loss of barrier properties of the stratum corneum. Current investigations address the lipid thermotropic phase behavior in terms of lipid alkyl chain packing, mobility and conformational order as measured by Fourier transform infrared (FTIR) spectroscopy. A solid-solid phase transition is observed with increased alkyl chain mobility followed by a gel to liquid-crystalline phase transition near 65 degrees C. These results further elucidate the role of lipid fluidity that may contribute to the transport properties of the stratum corneum.     

Sphingolipid metabolism during epidermal barrier development in mice

In rodents, a competent skin barrier to water loss is formed within 2 or 3 days prior to birth. Acquisition of barrier function during rat gestation correlates with the formation of a stratum corneum enriched in ceramides, cholesterol, and fatty acids (Aszterbaum, M., G. K. Menon, K. R. Feingold, and M. L. Williams. 1992. Ontogeny of the epidermal barrier to water loss in the rat: correlation of function with stratum corneum structure and lipid content. PEDIATR: Res. 31: 308-317). We analyzed the formation and epidermal localization of glucosylceramides during embryonic skin barrier development in Balb/c mice. Using immunohistochemistry, epidermal glucosylceramides were hardly detectable 3 days prior to birth. After further 24 h of gestation the level of glucosylceramides was maximal and decreased with increasing gestational age. In parallel, glucosylceramides were targeted to the apical side of the outermost granular keratinocyte layer. A spectrum of five distinct epidermal ceramides was present 2 days prior to birth. With ongoing gestation the composition of the ceramide fraction changed markedly. Most importantly, the level of omega-hydroxylated acylceramides decreased paralleled by the formation of the corneocyte lipid envelope. This structure consists of omega-hydroxylated ceramides and fatty acids bound to surface proteins of the corneocytes. The covalent attachment of ceramides or glucosylceramides correlated with the maturation of the stratum corneum and might contribute to its chemical and enzymatic resistance.     

Elastic Vesicles as a Tool for Dermal and Transdermal Delivery

The main problem in delivery of drugs across the skin is the barrier function of the skin, which is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid layers, the so-called lipid lamellae. When applying drugs onto the skin, the major penetration pathway is the tortuous intercellular route along the lipid lamellae. In order to increase the number of drugs administered via the transdermal route, novel drug delivery systems have to be designed. Among these systems are iontophoresis, electroporation, microneedles, and vesicular systems.     

Structure of the skin barrier and its modulation by vesicular formulations

The natural function of the skin is to protect the body from unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. Since the lipids regions in the stratum corneum form the only continuous structure, substances applied onto the skin always have to pass these regions. For this reason the organization in the lipid domains is considered to be very important for the skin barrier function. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid phase behavior is different from that of other biological membranes. In stratum corneum crystalline phases are predominantly present, but most probably a subpopulation of lipids forms a liquid phase. Both the crystalline nature and the presence of a 13 nm lamellar phase are considered to be crucial for the skin barrier function. Since it is impossible to selectively extract individual lipid classes from the stratum corneum, the lipid organization has been studied in vitro using isolated lipid mixtures. These studies revealed that mixtures prepared with isolated stratum corneum lipids mimic to a high extent stratum corneum lipid phase behavior. This indicates that proteins do not play an important role in the stratum corneum lipid phase behavior. Furthermore, it was noticed that mixtures prepared only with ceramides and cholesterol already form the 13 nm lamellar phase. In the presence of free fatty acids the lattice density of the structure increases. In stratum corneum the ceramide fraction consists of various ceramide subclasses and the formation of the 13 nm lamellar phase is also affected by the ceramide composition. Particularly the presence of ceramide 1 is crucial. Based on these findings a molecular model has recently been proposed for the organization of the 13 nm lamellar phase, referred to as "the sandwich model", in which crystalline and liquid domains coexist. The major problem for topical drug delivery is the low diffusion rate of drugs across the stratum corneum. Therefore, several methods have been assessed to increase the permeation rate of drugs temporarily and locally. One of the approaches is the application of drugs in formulations containing vesicles. In order to unravel the mechanisms involved in increasing the drug transport across the skin, information on the effect of vesicles on drug permeation rate, the permeation pathway and perturbations of the skin ultrastructure is of importance. In the second part of this paper the possible interactions between vesicles and skin are described, focusing on differences between the effects of gel-state vesicles, liquid-state vesicles and elastic vesicles.     

Elastic vesicles as a tool for dermal and transdermal delivery

The main problem in delivery of drugs across the skin is the barrier function of the skin, which is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid layers, the so-called lipid lamellae. When applying drugs onto the skin, the major penetration pathway is the tortuous intercellular route along the lipid lamellae. In order to increase the number of drugs administered via the transdermal route, novel drug delivery systems have to be designed. Among these systems are iontophoresis, electroporation, microneedles, and vesicular systems.     

Lipid mixtures prepared with well-defined synthetic ceramides closely mimic the unique stratum corneum lipid phase behavior

Lipid lamellae present in the outermost layer of the skin, the stratum corneum, form the main barrier for the diffusion of molecules through the skin. The presence of a unique 13 nm lamellar phase and its high crystallinity are characteristic for the stratum corneum lipid phase behavior. In the present study, small-angle and wide-angle X-ray diffraction were used to examine the organization in lipid mixtures prepared with a unique set of well-defined synthetic ceramides, varying from each other in head group architecture and acyl chain length. The results show that equimolar mixtures of cholesterol, free fatty acids, and synthetic ceramides (resembling the composition of pig ceramides) closely resemble the lamellar and lateral stratum corneum lipid organization, both at room and higher temperatures. Exclusion of several ceramide classes from the mixture does not affect the lipid organization. However, complete substitution of ceramide 1 (acylceramide with a sphingosine base) with ceramide 9 (acylceramide with a phytosphingosine base) reduces the formation of the long periodicity lamellar phase. This indicates that the head group architecture of acylceramides affects the lipid organization. In conclusion, lipid mixtures prepared with well-defined synthetic ceramides offer an attractive tool with which to unravel the importance of the molecular structure of individual ceramides for proper lipid organization.     

Determination of molecular conformation and permeation in skin via IR spectroscopy, microscopy, and imaging

Skin tissue, in addition to its specific use in dermal research, provides an excellent model for developing the techniques of vibrational microscopy and imaging for biomedical applications. In addition to permitting characterization of various regions of skin, the relative paucity of major biological constituents in the stratum corneum (the outermost layer of skin), permits us to image, with microscopic resolution, conformational alterations and concentration variations in both the lipid and protein components. Thus we are able to monitor the effects of exogenous materials such as models for drug delivery agents (liposomes) and permeation enhancers (DMSO) on stratum corneum lipid organization and protein structure. In addition, we are able to monitor protein conformational changes in single corneocytes. The current article demonstrates these procedures, ranging from direct univariate measures of lipid chain conformational disorder, to factor analysis which permits us to image conformational differences between liposomes that have permeated through the stratum corneum from those which have remained on the surface in a reservoir outside the skin.     

Determination of molecular conformation and permeation in skin via IR spectroscopy, microscopy, and imaging

Skin tissue, in addition to its specific use in dermal research, provides an excellent model for developing the techniques of vibrational microscopy and imaging for biomedical applications. In addition to permitting characterization of various regions of skin, the relative paucity of major biological constituents in the stratum corneum (the outermost layer of skin), permits us to image, with microscopic resolution, conformational alterations and concentration variations in both the lipid and protein components. Thus we are able to monitor the effects of exogenous materials such as models for drug delivery agents (liposomes) and permeation enhancers (DMSO) on stratum corneum lipid organization and protein structure. In addition, we are able to monitor protein conformational changes in single corneocytes. The current article demonstrates these procedures, ranging from direct univariate measures of lipid chain conformational disorder, to factor analysis which permits us to image conformational differences between liposomes that have permeated through the stratum corneum from those which have remained on the surface in a reservoir outside the skin.     

Skin viscoelasticity studied in vitro by microprobe-based techniques

Time-dependent deformation of porcine skin was studied in vitro using specialized microprobe instruments. The deformation behavior of stratum corneum, dermis, and whole skin is examined in the context of results of creep strain, elastic stiffness, and viscoelastic constants obtained in terms of the hold time, loading/unloading rate, and maximum indentation depth (load). Skin time-dependent deformation is significantly influenced by dermis viscoelasticity up to a critical indentation depth (load) beyond which it is controlled by the outermost hard epidermis, particularly stratum corneum. Skin viscoelastic behavior under constant load (creep) and constant displacement (stress relaxation) is interpreted in the light of phenomenological observations and experimental trends.     

The skin barrier in healthy and diseased state

The primary function of the skin is to protect the body for unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid regions. As most drugs applied onto the skin permeate along the lipid domains, the lipid organization is considered to be very important for the skin barrier function. It is for this reason that the lipid organization has been investigated quite extensively. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid organization is different from that of other biological membranes. In stratum corneum, two lamellar phases are present with repeat distances of approximately 6 and 13 nm. Moreover the lipids in the lamellar phases form predominantly crystalline lateral phases, but most probably a subpopulation of lipids forms a liquid phase. Diseased skin is often characterized by a reduced barrier function and an altered lipid composition and organization. In order to understand the aberrant lipid organization in diseased skin, information on the relation between lipid composition and organization is crucial. However, due to its complexity and inter-individual variability, the use of native stratum corneum does not allow detailed systematic studies. To circumvent this problem, mixtures prepared with stratum corneum lipids can be used. In this paper first the lipid organization in stratum corneum of normal and diseased skin is described. Then the role the various lipid classes play in stratum corneum lipid organization and barrier function has been discussed. Finally, the information on the role various lipid classes play in lipid phase behavior has been used to interpret the changes in lipid organization and barrier properties of diseased skin.     

The skin barrier in healthy and diseased state

The primary function of the skin is to protect the body for unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid regions. As most drugs applied onto the skin permeate along the lipid domains, the lipid organization is considered to be very important for the skin barrier function. It is for this reason that the lipid organization has been investigated quite extensively. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid organization is different from that of other biological membranes. In stratum corneum, two lamellar phases are present with repeat distances of approximately 6 and 13 nm. Moreover the lipids in the lamellar phases form predominantly crystalline lateral phases, but most probably a subpopulation of lipids forms a liquid phase. Diseased skin is often characterized by a reduced barrier function and an altered lipid composition and organization. In order to understand the aberrant lipid organization in diseased skin, information on the relation between lipid composition and organization is crucial. However, due to its complexity and inter-individual variability, the use of native stratum corneum does not allow detailed systematic studies. To circumvent this problem, mixtures prepared with stratum corneum lipids can be used. In this paper first the lipid organization in stratum corneum of normal and diseased skin is described. Then the role the various lipid classes play in stratum corneum lipid organization and barrier function has been discussed. Finally, the information on the role various lipid classes play in lipid phase behavior has been used to interpret the changes in lipid organization and barrier properties of diseased skin.     

Cigarette smoke affects keratinocytes SRB1 expression and localization via H2O2 production and HNE protein adducts formation

Scavenger Receptor B1 (SR-B1), also known as HDL receptor, is involved in cellular cholesterol uptake. Stratum corneum (SC), the outermost layer of the skin, is composed of more than 25% cholesterol. Several reports support the view that alteration of SC lipid composition may be the cause of impaired barrier function which gives rise to several skin diseases. For this reason the regulation of the genes involved in cholesterol uptake is of extreme significance for skin health. Being the first shield against external insults, the skin is exposed to several noxious substances and among these is cigarette smoke (CS), which has been recently associated with various skin pathologies. In this study we first have shown the presence of SR-B1 in murine and human skin tissue and then by using immunoblotting, immunoprecipitation, RT-PCR, and confocal microscopy we have demonstrated the translocation and the subsequent lost of SR-B1 in human keratinocytes (cell culture model) after CS exposure is driven by hydrogen peroxide (H(2)O(2)) that derives not only from the CS gas phase but mainly from the activation of cellular NADPH oxidase (NOX). This effect was reversed when the cells were pretreated with NOX inhibitors or catalase. Furthermore, CS caused the formation of SR-B1-aldheydes adducts (acrolein and 4-hydroxy-2-nonenal) and the increase of its ubiquitination, which could be one of the causes of SR-B1 loss. In conclusion, exposure to CS, through the production of H(2)O(2), induced post-translational modifications of SR-B1 with the consequence lost of the receptor and this may contribute to the skin physiology alteration as a consequence of the variation of cholesterol uptake.     

Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient

Two-photon fluorescence lifetime imaging is used to identify microdomains (1-25 microm) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), whose lifetime tau (pH 4.5, tau = 2.75 ns; pH 8.5, tau = 3.90 ns) is pH dependent over the pH range of the stratum corneum (pH 4.5 to pH 7.2). Hairless mice (SKH1-hrBR) are used as a model for human skin. Images (< or =50 microm x 50 microm) are acquired every 1.7 microm from the stratum corneum surface to the first viable layer (stratum granulosum). Acidic microdomains (average pH 6.0) of variable size (~1 microm in diameter with variable length) are detected within the extracellular matrix of the stratum corneum, whereas the intracellular space of the corneocytes in mid-stratum corneum (25 microm diameter) approaches neutrality (average pH 7.0). The surface is acidic. The average pH of the stratum corneum increases with depth because of a decrease in the ratio of acidic to neutral regions within the stratum corneum. The data definitively show that the stratum corneum acid mantle results from the presence of aqueous acidic pockets within the lipid-rich extracellular matrix.     

The mechanical properties of stratum corneum. I. The effect of water and ambient temperature on the tensile properties of newborn rat stratum corneum.

The tensile properties of the outermost layer of skin of neonatal rats, the stratum corneum, were investigated at a constant strain rate as a function of moisture content and ambient test temperature. The results show that the mechnical behavior of this membrane, whose primary constituent is the fibrous protein keratin, can be significantly altered by variations in both the sorbed water content and ambient temperature. In particular, a brittle to ductile transition was observed at 25 degrees C once the hydration level exceeded 70% relative humidity. Similarly, an identical phenomenon was detected at temperatures beyond 40 degrees C for specimens whose equilibrium moisture concentrations were maintained at 10 g H2 O/100 g dry protein. Differential scanning calrimetry measurements showed the presence of a molecular relaxation process which migrated from 42 degrees C at 40% relative humidity to --18 degrees C at 95% relative humidity. It is postulated that this relaxation process, possibly corresponding to the glass transition of the fibrous protein component of stratum corneum, is primarily responsible for the observed behavior.     

The mechanical properties of stratum corneum: I. The effect of water and ambient temperature on the tensile properties of newborn rat stratum corneum

The tensile properties of the outermost layer of skin of neonatal rats, the stratum corneum, were investigated at a constant strain rate as a function of moisture content and ambient test temperature. The results show that the mechanical behavior of this membrane, whose primary constituent is the fibrous protein keratin, can be significantly altered by variations in both the sorbed water content and ambient temperature. In particular, a brittle to ductile transition was observed at 25°C once the hydration level exceeded 70% relative humidity. Similarly, an identical phenomenon moisture concentrations were maintained at 10 g H₂O/100 g dry protein. Differential scanning calorimetry measurements showed the presence of a molecular relaxation process which migrated from 42°C at 40% relative humidity to −18°C at 95% relative humidity. It is postulated that this relaxation process, possibly corresponding to the glass transition of the fibrous protein component of stratum corneum, is primarily respnsible for the observed behavior.     

The phase behaviour of skin lipid mixtures based on synthetic ceramides

The lipid lamellae present in the outermost layer of the skin, the stratum corneum (SC), form the main barrier for diffusion of molecules across the skin. The main lipid classes in SC are cholesterol (CHOL), free fatty acids (FFA) and at least nine classes of ceramides (CER), referred to as CER1 to CER9. In the present study the phase behaviour of four synthetic CER, either single or mixed with CHOL or CHOL and FFA, has been studied using small and wide angle X-ray diffraction. The lipid mixtures showed complex phase behaviour with coexistence of several phases. The results further revealed that the presence of synthetic CER1 as well as a proper composition of the other CER in the mixture were crucial for the formation of a phase with a long periodicity, characteristic for SC lipid phase behaviour. Only a mixture containing synthetic CER1 and CER3, CHOL and FFA showed similar phase behaviour to that of SC.