Skin Minerals: Key Roles of Inorganic Elements in Skin Physiological Functions.

As odd as it may seem at first glance, minerals, it is what we are all about…or nearly. Although life on Earth is carbon-based, several other elements present in the planet's crust are involved in and often indispensable for functioning of living organisms. Many ions are essential, and others show supportive and accessory qualities. They are operative in the skin, supporting specific processes related to the particular situation of this organ at the interface with the environment. Skin bioenergetics, redox balance, epidermal barrier function, and dermal remodeling are amongst crucial activities guided by or taking advantage of mineral elements. Skin regenerative processes and skin ageing can be positively impacted by adequate accessibility, distribution, and balance of inorganic ions.

The Fate of Epidermal Tight Junctions in the stratum corneum: Their Involvement in the Regulation of Desquamation and Phenotypic Expression of Certain Skin Conditions.

We evaluated the presence of tight junction (TJ) remnants in the stratum corneum (SC) of in vitro reconstructed human epidermis and human skin explants subjected or not to an aggressive topical treatment with beta-lipohydroxy salicylic acid (LSA) for 24 h. LSA-treated samples showed an increased presence of TJ remnants in the two lowermost layers of the SC, as quantified with standard electron microscopy. The topical aggression-induced overexpression of TJ-like cell-cell envelope fusions may influence SC functions: (1) directly, through an enhanced cohesion, and (2) indirectly, by impeding accessibility of peripheral corneodesmosomes to extracellular hydrolytic enzymes and, thus, slowing down desquamation. Observations of ichthyotic epidermis in peeling skin disease (PSD; corneodesmosin deficiency; two cases) and ichthyosis hypotrichosis sclerosing cholangitis syndrome (IHSC/NISCH; absence of claudin-1; two cases) also demonstrated increased persistence of TJ-like intercellular fusions in pathological SC and contributed to the interpretation of the diseases' pathological mechanisms.

Keratinocytes Communicate with Sensory Neurons via Synaptic-like Contacts.

OBJECTIVE: Pain, temperature, and itch are conventionally thought to be exclusively transduced by the intraepidermal nerve endings. Although recent studies have shown that epidermal keratinocytes also participate in sensory transduction, the mechanism underlying keratinocyte communication with intraepidermal nerve endings remains poorly understood. We sought to demonstrate the synaptic character of the contacts between keratinocytes and sensory neurons and their involvement in sensory communication between keratinocytes and sensory neurons. METHODS: Contacts were explored by morphological, molecular, and functional approaches in cocultures of epidermal keratinocytes and sensory neurons. To interrogate whether structures observed in vitro were also present in the human epidermis, in situ correlative light electron microscopy was performed on human skin biopsies. RESULTS: Epidermal keratinocytes dialogue with sensory neurons through en passant synaptic-like contacts. These contacts have the ultrastructural features and molecular hallmarks of chemical synaptic-like contacts: narrow intercellular cleft, keratinocyte synaptic vesicles expressing synaptophysin and synaptotagmin 1, and sensory information transmitted from keratinocytes to sensory neurons through SNARE-mediated (syntaxin1) vesicle release. INTERPRETATION: By providing selective communication between keratinocytes and sensory neurons, synaptic-like contacts are the hubs of a 2-site receptor. The permanent epidermal turnover, implying a specific en passant structure and high plasticity, may have delayed their identification, thereby contributing to the long-held concept of nerve endings passing freely between keratinocytes. The discovery of keratinocyte-sensory neuron synaptic-like contacts may call for a reassessment of basic assumptions in cutaneous sensory perception and sheds new light on the pathophysiology of pain and itch as well as the physiology of touch. ANN NEUROL 2020;88:1205-1219.

ARTICLE: Evolution of Skin Barrier Science for Healthy and Compromised Skin.

Skin is a complex organ comprised of multiple cell types and microstructures that work in concert to serve critical functions and support the body’s homeostasis. It is the outermost, cornified layer of our body that is primarily responsible for the permeability barrier, protecting against external aggressors and preventing water loss from within. The understanding of the organization, functionality, and underlying mechanisms of the skin barrier has evolved greatly through the years. The formation of an intact and well-maintained stratum corneum (SC), where the permeability barrier resides, relies heavily on the differentiation of epidermal keratinocytes and the synthesis, release, localization, and binding of lipids that include principally ceramides, cholesterol, and free fatty acids. The in-depth research on SC barrier, its disruption in the pathogenesis of diseases, as well as on barrier responses to environmental insults, has enabled the development of modern therapeutics and topical care routines. Among them, ceramide-containing moisturizers have clinically demonstrated the ability to support the management of skin conditions such as atopic dermatitis and psoriasis by reducing the disease severity and recurrence and improving the patients’ perception of overall skin quality and health. This review focuses on the contributions of various barrier constituents to skin barrier function in health and pathological conditions, and how topical interventions containing essential barrier lipids support barrier restoration and provide relief. J Drugs Dermatol. 20(4 Suppl):s3-9. doi:10.36849/JDD.S589A.

Five Functional Aspects of the Epidermal Barrier.

The epidermis is a living, multilayered barrier with five functional levels, including a physical, a chemical, a microbial, a neuronal, and an immune level. Altogether, this complex organ contributes to protect the host from external aggression and to preserve its integrity. In this review, we focused on the different functional aspects.

Development and characterization of Lyophilized Transparized Decellularized stroma as a replacement for living cornea in deep anterior lamellar keratoplasty.

Corneal disease is the second cause of blindness in developing countries, where the number of corneal grafts needed by far exceeds the number available. In industrialized countries, although corneas are generally available for keratoplasty, onto inflamed and vascularized host beds they are often rejected despite immune-suppression. A non-immunogenic, transparent, cytocompatible stroma is therefore required, which can be lyophilized for long-term conservation. Decellularization methods were tested on porcine corneal stromas before validation on human corneas. Decellularization and lyophilization led to opacification of the stroma, which could be reversed by soaking in 100% glycerol. Cell-depleted transparized stromas were then lyophilized (LTDC) to allow their long-term conservation and water content was measured. The ultrastructure of LTDC corneas was examined by transmission electron microscopy (TEM). Histocompatibility antigens were undetectable on LTDC stromas by antibody staining. Finally, cytocompatibility of LTDC stromas was demonstrated on an ex vivo model of anterior lamellar keratoplasty. Differential staining was used to monitor colonization of LTDC stromas by cells from the receiving cornea. Only SDS-based decellularization produced acellular porcine stromas. The lowest SDS concentration tested (0.1%) was validated on human corneas. Unlike lyophilized corneas, LTDC stromas without residual water, express no histocompatibility markers, although TEM revealed the presence of cellular debris in an ultrastructural arrangement of collagen fibers very close to that of native corneas. This structure is compatible with colonization by cells from the receiver cornea in an ex vivo lamellar graft model. Our procedure produced non-immunogenic, transparent stromas with conserved ultrastructure compatible with long-term conservation.

Changes in nano-mechanical properties of human epidermal cornified cells depending on their proximity to the skin surface.

During formation of the stratum corneum (SC) barrier, terminally differentiated keratinocytes continue their maturation process within the dead superficial epidermal layer. Morphological studies of isolated human corneocytes have revealed differences between cornified envelopes purified from the deep and superficial SC. We used atomic force microscopy to measure the mechanical properties of native human corneocytes harvested by tape-stripping from different SC depths. Various conditions of data acquisition have been tested and optimized, in order to obtain exploitable and reproducible results. Probing at 200 nN allowed us to investigate the total stiffness of the cells (at 50 nm indentation) and that of the cornified envelopes (at 10 to15 nm), and lipid envelopes (at 5 to 10 nm). The obtained data indicated statistically significant differences between the superficial (more rigid) and deep (softer) corneocytes, thus confirming the existence of depth and maturation-related morphological changes within the SC. The proposed approach can be potentially used for minimally invasive evaluation of various skin conditions such as aging, skin hydration, and pathologies linked to SC.

Effect of allergens and irritants on levels of natural moisturizing factor and corneocyte morphology.

BACKGROUND: The irritant sodium lauryl sulfate (SLS) is known to cause a decrease in the stratum corneum level of natural moisturizing factor (NMF), which in itself is associated with changes in corneocyte surface topography. OBJECTIVE: To explore this phenomenon in allergic contact dermatitis. METHODS: Patch testing was performed on patients with previously positive patch test reactions to potassium dichromate (Cr), nickel sulfate (Ni), methylchloroisothiazolinone (MCI)/methylisothiazolinone (MI), or p-phenylenediamine. Moreover, a control (pet.) patch and an irritant (SLS) patch were applied. After 3 days, the stratum corneum from tested sites was collected, and NMF levels and corneocyte morphology, expressed as the amount of circular nanosize objects, quantified according to the Dermal Texture Index (DTI), were determined. RESULTS: Among allergens, only MCI/MI reduced NMF levels significantly, as did SLS. Furthermore, only MCI/MI caused remarkable changes at the microscopic level; the corneocytes were hexagonal-shaped with pronounced cell borders and a smoother surface. The DTI was increased after SLS exposure but not after allergen exposure. CONCLUSIONS: MCI/MI significantly decreased NMF levels, similarly to SLS. The altered corneocyte morphology suggests that skin barrier damage plays a role in the pathogenesis of MCI/MI contact allergy. The DTI seems to differentiate reactions to SLS from those to the allergens tested, as SLS was the only agent that caused a DTI increase.

Cell surface glycans in the human stratum corneum: distribution and depth-related changes.

During the formation of the stratum corneum (SC) barrier, the extracellular spaces of viable epidermis, rich in glycans, are filled with a highly organized lipid matrix and the plasma membranes of keratinocytes are replaced by cornified lipid envelopes. These structures comprise cross-linked proteins, including transmembrane glycoproteins and proteoglycans, covalently bound to a monolayer of cell surface ceramides. Little is known about the presence and distribution of glycans on the SC corneocytes despite their possible involvement in SC hydration, cohesion and desquamation. In this work, we visualized ultrastructurally and quantified the distribution of glycans on the surface of native and delipidated corneocytes. The cells were harvested at different depths of the SC, allowing us to define the relationship between the distribution of various glycans, proteoglycans and glycoproteins, and other changes occurring in SC. At the cell periphery, we found a correlation between the depth-related alterations of corneodesmosome glycoproteins and α-d-mannosyl and N-acetyl-d-glucosamine-labelling patterns. Elimination of the terminal sugars, α-linked fucose and α-(2,3) linked sialic acid, was less abrupt, but also the initial extent of their peripheral distribution was overall lower than that of concanavalin A and wheat germ agglutinin lectin-detected glycans. Diffuse labelling of heparan sulphate glycosaminoglycans disappeared completely from the outermost corneocytes, whereas that of several simple carbohydrates could be detected at all SC levels. Our results suggest that specific glycan distribution may participate in the progressive changes of SC, as it evolves from the SC compactum to the SC disjunctum, towards desquamation.

Synthesis and biological evaluation of thiophene and benzo[b]thiophene analogs of combretastatin A-4 and isocombretastatin A-4: A comparison between the linkage positions of the 3,4,5-trimethoxystyrene unit.

Combretastatin A-4 and isocombretastatin A-4 derivatives having thiophenes or benzo[b]thiophenes instead of the B ring were prepared and evaluated for their in cellulo tubulin polymerization inhibition (TPI) and antiproliferative activities. The presence of the benzo[b]thiophene ring proved to have a crucial effect as most of the thiophene derivatives, except those having one methoxy group, were inactive to inhibit tubulin polymerization into microtubules. The influence of the attachment position was also studied: benzo[b]thiophenes having iso or cis 3,4,5-trimethoxystyrenes at position 2 were 12-30-fold more active than the 3-regioisomers for the TPI activity. Some of the novel designed compounds exhibited interesting anti-proliferative effects on two different cell lines.

Filaggrin breakdown products determine corneocyte conformation in patients with atopic dermatitis.

BACKGROUND: Loss-of-function (LOF) mutations in the filaggrin gene (FLG) are a well-replicated risk factor for atopic dermatitis (AD) and are known to cause an epidermal barrier defect. The nature of this barrier defect is not fully understood. Patients with AD with FLG LOF mutations are known to have more persistent disease, more severe disease, and greater risk of food allergies and eczema herpeticum. Abnormalities in corneocyte morphology have been observed in patients with AD, including prominent villus-like projections (VP); however, these ultrastructural features have not been systematically studied in patients with AD in relation to FLG genotype and acute and convalescent status. OBJECTIVE: We sought to quantitatively explore the relationship between FLG genotype, filaggrin breakdown products (natural moisturizing factor [NMF]), and corneocyte morphology in patients with AD. METHODS: We studied 15 children at first presentation of AD and after 6 weeks of standard therapy. We applied atomic force microscopy to study corneocyte conformation in patients with AD stratified by FLG status and NMF level. By using a new quantitative methodology, the number of VPs per investigated corneocyte area was assessed and expressed as the Dermal Texture Index score. Corneocytes were also labeled with an anti-corneodesmosin antibody and visualized with scanning electron microscopy. RESULTS: We found a strong correlation between NMF levels and Dermal Texture Index scores in both acute and convalescent states (respective r = -0.80 and -0.75, P < .001 and P = .002). Most, but not all, VPs showed the presence of corneodesmosin abundantly all over the cell surface in homozygous/compound heterozygous FLG patients and, to a lesser extent, in heterozygous and wild-type patients. CONCLUSIONS: NMF levels are highly correlated with corneocyte morphology in patients with AD. These corneocyte conformational changes shed further insight into the filaggrin-deficient phenotype and help explain the barrier defect in patients with AD with FLG LOF mutations.

Epidermal barrier disorders and corneodesmosome defects.

Corneodesmosomes are modified desmosomes present in the stratum corneum (SC). They are crucial for SC cohesion and, thus, constitute one of the pivotal elements of the functional protective barrier of human skin. Expression of corneodesmosomes and, notably, the process of their degradation are probably altered during several dermatoses leading to the disruption of the permeability barrier or to abnormal, often compensative, SC accumulation. These different situations are reviewed in the present paper.

Modulation of transepithelial electric resistance (TEER) in reconstructed human epidermis by excipients known to permeate intestinal tight junctions.

Several excipients are commonly used to enhance the drug absorption through simple epithelia of the digestive tract. They permeate the paracellular barrier constituted by tight junctions (TJs). We compared the effects of two excipients, sodium caprate (C10) and a self-emulsifying excipient Labrasol composed of a mixture of caprylocaproyl polyoxyl-8 glycerides, both applied to emerged reconstructed human epidermis either 'systemically', that is by addition to the culture medium, or topically. During the 'systemic' application, which produced cytoplasmic translocation of occludin and leakage of the biotin marker into the lower stratum corneum, the decrease in the trans-epithelial electrical resistance (TEER) was less abrupt with Labrasol when compared with C10, even though both excipients produced comparable final effects over time. With topical Labrasol, a significant TEER decrease was obtained with 5 times the 'systemic' concentrations. Topical application of C10 also resulted in the loss of the barrier function measured with TEER but had dramatic deleterious effects on the tissue morphology observed with light and electron microscopy. Our study demonstrates the potential value of Labrasol as an enhancer of bioavailability of molecules applied through the transcutaneous route. Our results suggest modulation of the epidermal TJs by both compounds. Even though the C10 action was at least partly due to overall cell damage and despite the fact that the decrease in TEER after topical application was apparently related to the permeabilization of the primary barrier of the stratum corneum in the first place.

High TMEM45A expression is correlated to epidermal keratinization.

TMEM45A (DERP7, DNAPTP4 or FLJ10134) gene, belonging to the TMEM family encoding predicted transmembrane proteins, is highly expressed in epidermal keratinocytes. To investigate the potential involvement of TMEM45A during the differentiation and keratinization processes, its expression has been characterized in normal human keratinocytes and the protein subcellular localization has been studied in this cell type, both in vitro and in vivo. TMEM45A expression is upregulated with differentiation, either induced by cultured keratinocyte confluence or enhanced Ca(2+) concentration in medium. In vivo, TMEM45A mRNA and protein are mostly found in the granular layer of the epidermis. TMEM45A expression is linked to keratinization, as accumulation of the protein is detected in native and reconstructed epidermis as well as in thymic Hassal bodies, but not in non-keratinized stratified epithelia. At the subcellular level, co-detection with ER and Golgi markers reveals that TM protein 45A is associated with the Golgi apparatus and more specifically with the trans-Golgi/trans-Golgi network in vitro and in granular layer in vivo. The protein is neither related to lysosomes nor transported within corneodesmosin-containing lamellar bodies. These data demonstrate a strong correlation between TMEM45A expression and epidermal keratinization, indicating the relevance of this gene in this process.

Ability of the third-generation FloTrac/Vigileo software to track changes in cardiac output in cardiac surgery patients: a polar plot approach.

OBJECTIVE: To evaluate the ability of the third-generation (3.01) of FloTrac/Vigileo monitor (Edwards Lifesciences, Irvine, CA) to follow variations in cardiac output (∆CO) using the new polar plot approach. DESIGN: Prospective interventional study. SETTING: Single hospital university study. PARTICIPANTS: Twenty-five patients referred for cardiac surgery. INTERVENTIONS: CO was measured simultaneously by 3 to 5 bolus thermodilution (COtd measurements), using a pulmonary artery catheter and by arterial pulse contour analysis, using the FloTrac/Vigileo (COvi). Data were collected at eight time points: before incision, after sternotomy, before and after protamine sulfate infusion, at the start of sternal closure, at the end of surgery, on arrival to intensive care unit, and after a standardized volume expansion with 500 mL of hetastarch 6%. MEASUREMENTS AND MAIN RESULTS: One-hundred thirty-five pairs of CO data were collected; the mean bias of all CO measurements corrected for repeated measures was 0.2 L/min with limits of agreements of -3.3 L/min and +2.9 L/min. The percentage error was 66.5%. The polar plot analysis included 71 significant ∆CO and showed a mean polar angle of -3.4 degrees with 95% polar percentage error equivalent limits of -61 to 55; 69% of analysed data points fell within the 30-degree limits and provided a correct polar concordance rate. CONCLUSIONS: Third-generation FloTrac/Vigileo software still lacks the accuracy to reliably detect changes in cardiac output (∆CO) in cardiac surgery. Improvements to FloTrac/Vigileo CO algorithm and software still are needed in this particular setting.

Hornerin is a component of the epidermal cornified cell envelopes.

A single-nucleotide polymorphism within the gene encoding hornerin (HRNR) has recently been linked with atopic dermatitis (AD) susceptibility. HRNR shares features with filaggrin, a key protein for keratinocyte differentiation, but conflicting reports have been published concerning its expression in the epidermis, and its role is still unknown. To analyze HRNR expression and function in the epidermis, anti-HRNR antibodies were produced and used in Western blot analysis and immunohistochemical, confocal, and immunoelectron microscopy analyses of human skin and of cornified cell envelopes purified from plantar stratum corneum. We also tested whether HRNR was a substrate of transglutaminases. In the epidermis, HRNR was detected at the periphery of keratohyalin granules in the upper granular layer and at the corneocyte periphery in the whole cornified layer. Detected in Western blot analysis as numerous bands, HRNR was relatively insoluble and only extracted from epidermis with urea and/or reducing agents. The presence of HRNR in the purified envelopes was confirmed by immunoelectron microscopy and by Western blot analysis after V8-protease digestion. HRNR was shown to be a substrate of transglutaminase 3. These data demonstrate that HRNR is a component of cornified cell envelopes of human epidermis. Its reduced expression in AD may contribute to the epidermal barrier defect observed in the disease.

Synthesis and biological evaluation of novel heterocyclic derivatives of combretastatin A-4.

A novel series of combretastatin A-4 heterocyclic analogues was prepared by replacement of the B ring with indole, benzofurane or benzothiophene, attached at the C2 position. These compounds were evaluated for their abilities to inhibit tubulin assembly: derivative cis3b, having a benzothiophene, showed an activity similar to those of colchicine or deoxypodophyllotoxine. The antiproliferative and antimitotic properties of cis3b against keratinocyte cancer cell lines were also evaluated and the intracellular organization of microtubules in the cells after treatment with both stereoisomers of 3b was also determined, using confocal microscopy.

Human epidermal desmosome-enriched tissue fractions for analytical and prospective studies.

Here, we report a method, adapted to the human epidermis, allowing isolation of desmosomes in small tissue fractions. The methods previously developed for animal skin did not work efficiently with human tissue. Enrichment of desmosomes was performed by the association of two incubation steps in acidic solutions containing detergent NP-40 at two different concentrations followed by a sonication step. The suspension was centrifuged twice: first to remove the heavy cell fragments and then at 16000 g on a discontinuous sucrose gradient. A desmosome-enriched fraction (DsF) was collected at the 30-50% sucrose interface. We demonstrate by immunoelectron microscopy and by western blotting that the central part of the desmosome structure is preserved as well as the antigenicity of its components. Our approach, allowing a significant enrichment of the cell fractions containing desmosomes, can be used to immunize animals and create new antibodies directed against desmosomal components. Using this strategy, new and so far poorly studied molecules incorporated into the desmosome cores could be targeted more easily.

Compartmentalization of the human stratum corneum by persistent tight junction-like structures.

Several tight junction (TJ) proteins were detected in the living layers of adult human epidermis, and TJ-like membrane ridges were observed at the top of the stratum granulosum (SG) in freeze-fracture studies. We applied standard and immunoelectron microscopy to look for TJ-derived structures in the stratum corneum (SC) of human adult epidermis and in cornified envelopes purified from the plantar SC. Besides confirming claudin-1 labelling in the proximity of SG desmosomes, we also observed immunolocalization near corneodesmosomes in the lower SC. In addition, TJ proteins were consistently detected in the purified cornified envelopes. Lateral but not horizontal walls of the corneocytes showed frequent points of molecular fusion between lipid envelopes. These structural associations were very frequently localized at the top of the lateral corneocyte membranes, thus sealing the extremities of lateral intercorneocyte spaces. We propose that TJ-like structures persist in the SC and contribute to the reinforcement of lateral contacts and to the formation of membrane interdigitations between corneocytes. Their presence could contribute to subdivision of the extracellular spaces of SC into consecutive individualized compartments. Intercellular lipids, enzymes and other (glyco)protein content could thus evolve in the keratinized epidermal layer at different paces, as preprogrammed in the underlying living cells and influenced by the environment, e.g. humidity. Such situation might explain differences in the degradation rates between the 'peripheral' and the 'non-peripheral' corneodesmosomes observed during physiological desquamation, as previously suggested by us and others.

Development and in vitro assay of oxidative stress modifying formulations for wound healing promotion.

Often presented as metabolism byproducts, reactive oxygen species are linked to detrimental effects such as chronic wound, mutagenesis, cancer and skin ageing. However, recent in vitro and in vivo observations suggest that ROS, and mainly hydrogen peroxide, interfere with cell signaling acting like second messenger and inducing adaptive responses. This is particularly observed in skin wound healing where cells are exposed to H2O2 following injury. In this study, we developed and characterized an innovative formulation producing H2O2 at low concentrations, in order to mimic physiological inflammation phase. Then, this pro-oxidative formulation (CAM-GOx) was assayed in vitro on keratinocytes cell culture, compared to the blank formulation (CAM) and the anti-oxidative formulation (CAM-CAT) to assess whether oxidative stress was implied or not in cellular responses.