Hydration of stratum corneum

A model for the hydration behavior of human stratum corneum has been developed from measurements on in vitro samples isolated in a manner which minimized tissue treatment and trauma. Water sorption/desorption rate measurements as a function of water activity, temperature, and tissue integrity are reported. These data, together with thermodynamic data (infrared and nmr results reported earlier) are consistent with a model in which rapidly sorbed/desorbed water (ca. 0.5 mg water/mg stratum corneum) is associated with (“bound” by) the tissue, while slowly sorbed/desorbed “free” water (up to 12 mg water/mg stratum corneum) is kinetically restricted and probably intracellular in location. Both equilibrium water binding and desorption kinetic data suggest structural changes of this cellular water barrier upon hydration. Evidence for hysteresis in water sorption isotherms (reported by others) could not be reproduced.     

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.     

Beta-glucocerebrosidase activity in mammalian stratum corneum

Although previous studies have demonstrated a crucial role for the enzyme beta-glucocerebrosidase (GlcCer'ase) in the final steps of membrane structural maturation in mammalian stratum cornuem (SC) and epidermal homeostasis, the precise in vivo localization of GlcCer'ase activity and protein is not known. Here, we developed a fluorogenic in situ assay on histologic sections (zymography) to elucidate the in vivo distribution of GlcCer'ase activity, and further characterized and localized the SC GlcCer'ase activity in vitro. The zymographic technique revealed higher GlcCer'ase activity in upper stratum granulosum and SC, both in murine and human SC; activity that was both inhibited by conduritol B epoxide, a specific GlcCer'ase inhibitor, and pH-dependent; i.e., present at pH 5.2, and absent or significantly reduced at neutral pH (7.4), consistent with the known pH optimum for epidermal GlcCer'ase in vitro. Immunohistochemical staining for GlcCer'ase protein showed enhanced fluorescent signal in the outer layers of human epidermis, concentrated at the apex and margins of stratum granulosum and lower SC. Moreover, in extracts from individual epidermal layers, GlcCer'ase activity was present throughout murine epidermis, with the highest activity in the SC, peaking in the lower-to-mid-SC. The SC activity was stimulated >10-fold by sodium taurocholate, and inhibited by bromoconduritol B epoxide. Finally, isolated membrane couplets, prepared from SC sheets, also demonstrated significant GlcCer'ase activity. These data localize GlcCer'ase activity to the outer epidermis by three different techniques, and support the role of this enzyme in extracellular processing of glucosylceramides to ceramides, required for permeability barrier maturation and function.     

Carbonylation of cornified envelopes in the stratum corneum

Stratum corneum (SC), the outermost layer of the skin, is continuously exposed to oxidative stress via sunlight, lipid peroxidation, and is subsequently accompanied by oxidative modification. Previous studies have shown that major oxidative target proteins in the SC are keratins. However, it remains unclear to date whether cornified envelopes (CEs), protein envelopes of the corneocytes (cornified cells), would be oxidized. In this study, we first revealed oxidative modification of CEs using labeled hydrazide derivatives to detect carbonyl moieties. Carbonylation of CEs was confirmed by reaction with monoclonal antibodies against aldehyde-bound proteins, including anti-acrolein, anti-crotonaldehyde, anti-4-hydroxy-2-nonenal. The extent of carbonylation is stronger in CEs from the face, a sun-exposed area, than those from the inside of upper arm, an unexposed area. Carbonylation of CEs did not depend on their maturity, as evaluated by loss of involucrin antigenicity during maturation process, suggesting that CEs are carbonylated regardless of their maturation stage.     

Mechanical and failure behaviour of the stratum corneum

The load-deformation-time behaviour of heat-separated human stratum corneum was investigated using a pure shear specimen geometry. The tissue displayed non-linear load-deformation behaviour and stress relaxation, although the extensibility and amount of stress relaxation was considerably less than that shown by other soft connective tissues. Controlled failure tests were carried out after an edge cut had been made in the pure shear specimen. Sources of secondary failure, either at the free edge of the specimen, or due to the presence of inhomogeneities in the tissues, were common. Analysis of the test results suggested that the fracture surface energy of stratum corneum has a mean value of 3.6 kJ m-2 which is comparable with the tougher synthetic polymers.     

Permeability parameters of the toad isolated stratum corneum.

A technique for isolating the stratum corneum from the subjacent layers of the epithelium was developed which permits studying the stratum corneum as an isolated membrane mounted between half-chambers. The method basically consists of an osmotic shock induced by immersing a piece of skin in distilled water at 50 degrees C for 2 min. When the membrane is bathed on each surface by NaCl-Ringer's solution, its electrical resistance is 14.1 +/- 1.3 omega cm2 (n=10). This value is about 1/100 of the whole skin resistance in the presence of the same solution. The hydraulic filtration coefficient (Lp) measured by a hydrostatic pressure method, with identical solutions on each side of the membrane, is 8.8 X 10(-5) +/- 1.5 X 10(-5) cm sec-1 atm-1 (n=10) in distilled water and 9.2 X 10(-5) +/- 1.4 X 10(-5) cm sec-1 atm-1 (n=10) in NaCl-Ringer's solution. These values are not statistically different and are within the range of 1/80 to 1/120 of the whole skin Lp. The stratum corneum shows an amphoteric character when studied by KCl diffusion potentials at different pH'S. The membrane presents an isoelectric pH of 4.6 +/- 0.3 (n=10). Above the isoelectric pH the potassium transport number is higher than the chloride transport number; below it, the reverse situation is valid. Divalent cations (Ca++ or Cu++) reduce membrane ionic discrimination when the membrane is negatively charged and are ineffective when the membrane fixed charges are protonated at low pH.     

Deuterium NMR investigation of polymorphism in stratum corneum lipids

The intercellular lipid lamellae of stratum corneum constitute the major barrier to percutaneous penetration. Deuterium magnetic resonance and freeze-fracture electron microscopic investigation of hydrated lipid mixtures consisting of ceramides, cholesterol, palmitic acid and cholesteryl sulfate and approximating the stratum corneum intercellular lipid composition, revealed thermally induced polymorphism. The transition temperature of bilayer to hexagonal transition decreased as the ratio of cholesterol to ceramides in these mixtures was lowered. Lipid mixtures in which the stratum corneum ceramides were replaced by synthetic dipalmitoylphosphatidylcholine did not show any polymorphism throughout the temperature range used in the present study. The ability of the ceramide-containing samples to form hexagonal structures establishes a plausible mechanism for the assembly of the stratum corneum intercellular lamellae during the final stages of epidermal differentiation. Also, the bilayer to hexagonal phase transition of these nonpolar lipid mixtures could be used to enhance the penetration of drugs through skin.     

Cohesion and desquamation of epidermal stratum corneum.

This article attempts to provide a comprehensive review on the roles of various classes of molecules in the cohesion and desquamation of the stratum corneum. In the first part of this monograph we review the field of epidermal differentiation in vivo and vitro, describing the expression and functions of a number of key structural molecules that characterize the process. In the second part we emphasize terminal differentiation and the biogenesis of the stratum corneum. The stratum corneum is a cell layer unique to fully differentiated squamous epithelia such as skin. While it is a dead stratum, it nevertheless is in a homeostatic process of continual shedding and renewal in synchrony with basal cell replication. It is also a degradative layer containing many proteinases and glycosidases in which a variety of intracellular and intercellular macromolecules are degraded. We highlight the molecules localized within the intercorneal matrix that are most likely to play a role in cohesion and desquamation, including: glycoproteins, lipids and enzymes. Because it is difficult to study the stratum corneum and desquamation in the native tissue, we discuss a number of model systems that have been used. The stratum corneum can be dispersed into single squames in different ways; these include mechanical dispersion as well as agents such as detergents and enzymes. The solubilized molecules and the structures remaining can then be studied as to their specific roles in desquamation. Using this approach it is possible to reconstitute multilayered structures that resemble a real stratum corneum. We have shown that glycoproteins play a key role in squame reaggregation and that this process can be modulated with amino sugars in a lectin-like fashion. Cohesion and desquamation can also be studied in tissue culture. Depending on the culture system, the extent of terminal differentiation and squame accumulation varies. Yet desquamation does not normally occur. It can be induced however by the inclusion of exogenous agents such as IFN-gamma which are found in the native epidermis but are absent in vitro. Modulation of desquamation by other exogenous agents is likely to yield further knowledge of how shedding occurs in vivo. Insight has also come from studies of scaling skin disorders. The glycoprotein and lipid profiles are altered in the stratum corneum in many diseases of aberrant terminal differentiation. A number of abnormalities in the levels of cytokines and growth factors have also been reported in the lesional tissue of such diseases.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Ectoine disperses keratin and alters hydration kinetics in stratum corneum

Moisturizing compounds are commonly applied topically to human stratum corneum (SC). Many types of molecular species are employed, most commonly including humectants and occlusives. We find new evidence of keratin dispersion caused by the moisturizing compound ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid), and provide the first characterization of its impacts on the hydration kinetics and biomechanics of SC. A second compound, 2-(2-hydroxyethoxy)ethylguanidine succinate (HEG) was investigated for comparison. A suite of biomechanical and biochemical assays including FTIR, drying stress, and cellular cohesion were used. Studies were conducted on normal, lipid-extracted, and lipid plus natural moisturizing factor extracted SC. Ectoine was found to improve the dispersity and hydration of keratin bundles in corneocytes. It also decreased rates of stress development in lipid extracted SC when exposed to a dry environment by ～30% while improving stress reduction during rehydration by ～20%. Peak stresses were increased in harsh drying environments of <5% RH, but SC swelling measurements suggest that water retention was improved in ambient conditions. Further, changes up to ～4 J/m² were seen in cohesion after ectoine treatments, suggesting corneodesmosome interactions. HEG was tested and found to disperse keratin without impacting corneodesmosomes. These results indicate that keratin dispersants produce beneficial effects on SC hydration kinetics, ultimately resulting in higher SC hydration under ambient conditions.     

Fatty acids in normal and pathological stratum corneum of humans

Fatty acids of triacylglycerols and free fatty acids have been studied in normal and hyperkeratotic human plantar stratum corneum. The results emphasize a decrease of long-chain fatty acids (greater than C18) in the hyperkeratotic tissues. Possible explanation for these findings are discussed in relation to the fatty acids metabolism in living epidermis.     

Interaction between corneocytes and stratum corneum lipid liposomes in vitro

Small unilamellar vesicles were made from a mixture of epidermal ceramides (45%), cholesterol (35%), free fatty acids (15%) and cholesteryl sulfate (5%). Isolated corneocytes prepared from pig epidermis were added to the liposomes and the interaction between corneocytes and liposomes was studied by (1) thin-section electron microscopy and (2) monitoring the release of aqueous contents of the vesicles by following the fluorescence intensity of carboxyfluorescein entrapped in the vesicles. The vesicles adsorbed readily onto the corneocytes and slowly transformed into lamellar sheets. Enhanced fluorescence intensity indicated a corneocyte-induced membrane fusion process that resulted in the release of aqueous contents of the vesicles. The results suggest a cohesive role for the corneocyte cell envelope, which consists of a monomolecular layer of lipids covalently bound to the outside of a cross-linked protein envelope. This may be one of the major factors in the reassembly of extruded membranous disks into lamellar sheets which occurs during the final stages of epidermal differentiation.     

Immunohistochemical analysis of stratum corneum components in oral squamous epithelia

The generation of a stratum corneum in squamous epithelia involves marked changes in morphology and in the expression of cell products. We have examined the expression of some of the components involved in this process in oral squamous epithelia with different terminal differentiation patterns by use of immunofluorescent techniques. Involucrin and transglutaminase are involved in formation of cornified envelopes consistently seen in the stratum corneum. Both components were present in keratinized oral epithelia (palatal epithelium and hyperkeratinized buccal epithelium). The nonkeratinized normal buccal epithelium stained positive as well. Filaggrin, a protein derived from a precursor present in keratohyalin granules, is proposed to aggregate keratin filaments in the cornified layer. Although the staining differed markedly in quantity, this component was likewise detected in both keratinized and nonkeratinized epithelia. The staining patterns for different keratin polypeptides, however, showed qualitative differences between the different epithelia. Thus, it seems that the keratin composition shows differentiation-specific characteristics, whereas the presence of other important components needed to generate a stratum corneum is not as closely related to the terminal differentiation pattern of oral epithelia.     

DNase 2 is the main DNA-degrading enzyme of the stratum corneum

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA.     

New arrangement of proteins and lipids in the stratum corneum cornified envelope

A new arrangement of proteins and lipids of stratum corneum (SC) cornified envelope (CE) is proposed. The chemical analysis of CE revealed the presence of free fatty acids (FFA), ceramides (Cer), and important percentages of glutamic acid/glutamine (Glx) and serine (Ser) residues. The molecular structure of these components suggests the existence of covalent links not only between Cer and Glx but also between FFA and Ser. The protein distribution of extracellular surface of CE, i.e., the proteins that could be involved in the bonds with lipids, was studied using post- and pre-embedding immunolabeling electron microscopy. Some loricrin (protein rich in Ser) was detected in the outermost part of the CE protein layer. The external arrangement of some domains of this protein may give rise to form linkages with FFA, yielding further insight into the CE arrangement in which Cer-Glx bonds and FFA-Ser bonds would be involved. Although the importance of fatty acids in the cohesion and barrier function of SC has been widely demonstrated, their role could be associated not only to the presence of these lipids in the intercellular lamellae but also in the CE, in the same way that Cer.     

New arrangement of proteins and lipids in the stratum corneum cornified envelope

A new arrangement of proteins and lipids of stratum corneum (SC) cornified envelope (CE) is proposed. The chemical analysis of CE revealed the presence of free fatty acids (FFA), ceramides (Cer), and important percentages of glutamic acid/glutamine (Glx) and serine (Ser) residues. The molecular structure of these components suggests the existence of covalent links not only between Cer and Glx but also between FFA and Ser. The protein distribution of extracellular surface of CE, i.e., the proteins that could be involved in the bonds with lipids, was studied using post- and pre-embedding immunolabeling electron microscopy. Some loricrin (protein rich in Ser) was detected in the outermost part of the CE protein layer. The external arrangement of some domains of this protein may give rise to form linkages with FFA, yielding further insight into the CE arrangement in which Cer-Glx bonds and FFA-Ser bonds would be involved. Although the importance of fatty acids in the cohesion and barrier function of SC has been widely demonstrated, their role could be associated not only to the presence of these lipids in the intercellular lamellae but also in the CE, in the same way that Cer.     

Determination of polyethylene glycols of different molecular weight in the stratum corneum

We developed a sensitive method for determination of polyethylene glycols (PEGs) of different molecular weight (MW) in the human stratum corneum (SC) obtained by tape stripping. The analysis is based on derivatization with pentafluoropropionic anhydride (PFPA) and gas chromatography-electron capture detection (GC-ECD). The identification and quantification of PEGs was done using individual oligomers. The method showed to be suitable for studying permeability in normal and impaired skin with respect to MW in the range of 150-600 Da.