Ceramides are key components of the skin's permeability barrier. In atopic dermatitis, pathological hydrolysis of ceramide precursors - glucosylceramides and sphingomyelin - into lysosphingolipids, specifically glucosylsphingosine (GS) and sphingosine-phosphorylcholine (SPC), and free fatty acids (FFAs) has been proposed to contribute to impaired skin barrier function. This study investigated whether replacing ceramides with lysosphingolipids and FFAs in skin lipid barrier models would exacerbate barrier dysfunction. When applied topically to human stratum corneum sheets, SPC and GS increased water loss, decreased electrical impedance, and slightly disordered lipid chains. In lipid models containing isolated human stratum corneum ceramides, reducing ceramides by ≥ 30% significantly increased permeability to four markers, likely due to loss of long-periodicity phase (LPP) lamellae and phase separation within the lipid matrix, as revealed by X-ray diffraction and infrared spectroscopy. However, when the missing ceramides were replaced by lysosphingolipids and FFAs, no further increase in permeability was observed. Conversely, these molecules partially mitigated the negative effects of ceramide deficiency, particularly with 5%-10% SPC, which reduced permeability even compared to control with "healthy" lipid composition. These findings suggest that while ceramide deficiency is a key factor in skin barrier dysfunction, the presence of lysosphingolipids and FFAs does not aggravate lipid structural or functional damage, but may provide partial compensation, raising further questions about the behavior of lyso(sphingo)lipids in rigid multilamellar lipid environments, such as the stratum corneum, that warrant further investigation.

• Klíčová slova
• ceramide, fatty acid, glucosylsphingosine, lipid model, lysolipid, permeability, skin barrier, sphingosine-phosphorylcholine,
• MeSH
• ceramidy * metabolismus   nedostatek   MeSH
• fosforylcholin analogy a deriváty   MeSH
• kůže * metabolismus   účinky léků   MeSH
• kyseliny mastné neesterifikované metabolismus   MeSH
• lidé MeSH
• lysofosfolipidy * metabolismus   MeSH
• permeabilita MeSH
• sfingolipidy * metabolismus   MeSH
• sfingosin analogy a deriváty   metabolismus   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ceramidy * MeSH
• fosforylcholin MeSH
• kyseliny mastné neesterifikované MeSH
• lysofosfolipidy * MeSH
• sfingolipidy * MeSH
• sfingosin MeSH
• sphingosine phosphorylcholine MeSH Prohlížeč

Oleic acid and oleyl alcohol are commonly used permeation and penetration enhancers to facilitate topical drug delivery. Here, we aimed to better understand the mechanism of their enhancing effects in terms of their interactions with the human skin barrier using diclofenac diethylamine (DIC-DEA), a nonsteroidal anti-inflammatory drug for topical pain management. Oleic acid promoted DIC-DEA permeation through ex vivo human skin more rapidly than oleyl alcohol (both applied at 0.75%) due to fluidization of stratum corneum lipids as revealed by infrared spectroscopy. After 12 h, the effect of these enhancers on DIC-DEA permeation leveled off, fluidization was no longer evident, and skin permeabilization was mainly due to the formation of fluid enhancer-rich domains. Contrary to oleyl alcohol, oleic acid adversely affected two indicators of the skin barrier integrity, transepidermal water loss and skin electrical impedance. The content of oleyl alcohol in the stratum corneum was lower than that of oleic acid (even 12 h after the enhancers were removed from the skin surface), but it caused higher DIC-DEA retention in both epidermis and dermis compared to oleic acid. The effects of oleyl alcohol and oleic acid on DIC-DEA permeation and retention in the skin were similar after a single and repeated application (4 doses every 12 h). Thus, oleyl alcohol offers several advantages over oleic acid for topical drug delivery.

• Klíčová slova
• diclofenac, infrared spectroscopy, lipid interactions, penetration enhancer, permeation enhancer, skin barrier, topical drug delivery,
• MeSH
• aplikace kožní MeSH
• kožní absorpce * MeSH
• kůže metabolismus   MeSH
• kyselina olejová * farmakologie   metabolismus   MeSH
• lidé MeSH
• mastné alkoholy metabolismus   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kyselina olejová * MeSH
• mastné alkoholy MeSH
• oleyl alcohol MeSH Prohlížeč

Omega-O-acyl ceramides such as 32-linoleoyloxydotriacontanoyl sphingosine (Cer[EOS]) are essential components of the lipid skin barrier, which protects our body from excessive water loss and the penetration of unwanted substances. These ceramides drive the lipid assembly to epidermal-specific long periodicity phase (LPP), structurally much different than conventional lipid bilayers. Here, we synthesized Cer[EOS] with selectively deuterated segments of the ultralong N-acyl chain or deuterated or 13C-labeled linoleic acid and studied their molecular behavior in a skin lipid model. Solid-state 2H NMR data revealed surprising molecular dynamics for the ultralong N-acyl chain of Cer[EOS] with increased isotropic motion toward the isotropic ester-bound linoleate. The sphingosine moiety of Cer[EOS] is also highly mobile at skin temperature, in stark contrast to the other LPP components, N-lignoceroyl sphingosine acyl, lignoceric acid, and cholesterol, which are predominantly rigid. The dynamics of the linoleic chain is quantitatively described by distributions of correlation times and using dynamic detector analysis. These NMR results along with neutron diffraction data suggest an LPP structure with alternating fluid (sphingosine chain-rich), rigid (acyl chain-rich), isotropic (linoleate-rich), rigid (acyl-chain rich), and fluid layers (sphingosine chain-rich). Such an arrangement of the skin barrier lipids with rigid layers separated with two different dynamic "fillings" i) agrees well with ultrastructural data, ii) satisfies the need for simultaneous rigidity (to ensure low permeability) and fluidity (to ensure elasticity, accommodate enzymes, or antimicrobial peptides), and iii) offers a straightforward way to remodel the lamellar body lipids into the final lipid barrier.

• Klíčová slova
• NMR spectroscopy, lipid assembly, lipid chain order, long periodicity phase, molecular dynamics, neutron diffraction, stratum corneum models,
• MeSH
• ceramidy chemie   MeSH
• epidermis MeSH
• kůže chemie   MeSH
• kyselina linolová * MeSH
• sfingosin analýza   MeSH
• simulace molekulární dynamiky * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ceramidy MeSH
• kyselina linolová * MeSH
• sfingosin MeSH

NRF2 is a master regulator of the cellular protection against oxidative damage in mammals and of multiple pathways relevant in the mammalian aging process. In the epidermis of the skin NRF2 contributes additionally to the formation of an antioxidant barrier to protect from environmental insults and is involved in the differentiation process of keratinocytes. In chronological aging of skin, the capacity for antioxidant responses and the ability to restore homeostasis after damage are impaired. Surprisingly, in absence of extrinsic stressors, NRF2 deficient mice do not show any obvious skin phenotype, not even at old age. We investigated the differences in chronological epidermal aging of wild type and NRF2-deficient mice to identify the changes in aged epidermis that may compensate for absence of this important transcriptional regulator. While both genotypes showed elevated epidermal senescence markers (increased Lysophospholipids, decreased LaminB1 expression), the aged NRF2 deficient mice displayed disturbed epidermal differentiation manifested in irregular keratin 10 and loricrin expression. The tail skin displayed less age-related epidermal thinning and a less pronounced decline in proliferating basal epidermal cells compared to the wildtype controls. The stratum corneum lipid composition also differed, as we observed elevated production of barrier protective linoleic acid (C18:2) and reduced abundance of longer chain saturated lignoceric acid (C24:0) among the stratum corneum fatty acids in the aged NRF2-deficient mice. Thus, despite epidermal differentiation being disturbed in aged NRF2-deficient animals in homeostasis, adaptations in keratinocyte proliferation and barrier lipid synthesis could explain the lack of a more severe phenotype.

• Klíčová slova
• NRF2, aging, epidermis, lipid, skin,
• MeSH
• antioxidancia * metabolismus   MeSH
• buněčná diferenciace genetika   MeSH
• epidermální buňky MeSH
• epidermis metabolismus   MeSH
• faktor 2 související s NF-E2 * genetika   metabolismus   MeSH
• keratinocyty MeSH
• myši MeSH
• ocas MeSH
• savci MeSH
• stárnutí genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antioxidancia * MeSH
• faktor 2 související s NF-E2 * MeSH

Ceramides belong to sphingolipids, an important group of cellular and extracellular lipids. Their physiological functions range from cell signaling to participation in the formation of barriers against water evaporation. In the skin, they are essential for the permeability barrier, together with free fatty acids and cholesterol. We examined the periodical structure and permeability of lipid films composed of ceramides (Cer; namely, N-lignoceroyl 6-hydroxysphingosine, CerNH24, and N-lignoceroyl sphingosine, CerNS24), lignoceric acid (LIG; 24:0), and cholesterol (Chol). X-ray diffraction experiments showed that the CerNH24-based samples form either a short lamellar phase (SLP, d ∼ 5.4 nm) or a medium lamellar phase (MLP, d = 10.63-10.78 nm) depending on the annealing conditions. The proposed molecular arrangement of the MLP based on extended Cer molecules also agreed with the relative neutron scattering length density profiles obtained from the neutron diffraction data. The presence of MLP increased the lipid film permeability to the lipophilic model permeant (indomethacin) relative to the CerNS24-based control samples and the samples that had the same lipid composition but formed an SLP. Thus, the arrangement of lipids in various nanostructures is responsive to external conditions during sample preparation. This polymorphic behavior directly affects the barrier properties, which could also be (patho)physiologically relevant.

• Publikační typ
• časopisecké články MeSH

Epidermal omega-O-acylceramides (ω-O-acylCers) are essential components of a competent skin barrier. These unusual sphingolipids with ultralong N-acyl chains contain linoleic acid esterified to the terminal hydroxyl of the N-acyl, the formation of which requires the transacylase activity of patatin-like phospholipase domain containing 1 (PNPLA1). In ichthyosis with dysfunctional PNPLA1, ω-O-acylCer levels are significantly decreased, and ω-hydroxylated Cers (ω-OHCers) accumulate. Here, we explore the role of the linoleate moiety in ω-O-acylCers in the assembly of the skin lipid barrier. Ultrastructural studies of skin samples from neonatal Pnpla1+/+ and Pnpla1-/- mice showed that the linoleate moiety in ω-O-acylCers is essential for lamellar pairing in lamellar bodies, as well as for stratum corneum lipid assembly into the long periodicity lamellar phase. To further study the molecular details of ω-O-acylCer deficiency on skin barrier lipid assembly, we built in vitro lipid models composed of major stratum corneum lipid subclasses containing either ω-O-acylCer (healthy skin model), ω-OHCer (Pnpla1-/- model), or combination of the two. X-ray diffraction, infrared spectroscopy, and permeability studies indicated that ω-OHCers could not substitute for ω-O-acylCers, although in favorable conditions, they form a medium lamellar phase with a 10.8 nm-repeat distance and permeability barrier properties similar to long periodicity lamellar phase. In the absence of ω-O-acylCers, skin lipids were prone to separation into two phases with diminished barrier properties. The models combining ω-OHCers with ω-O-acylCers indicated that accumulation of ω-OHCers does not prevent ω-O-acylCer-driven lamellar stacking. These data suggest that ω-O-acylCer supplementation may be a viable therapeutic option in patients with PNPLA1 deficiency.

• Klíčová slova
• PNPLA1 deficiency, Skin, acylceramides, barrier function, ceramides, linoleic acid, lipids, model membranes, sphingolipids, stratum corneum,
• MeSH
• acyltransferasy MeSH
• ceramidy * chemie   MeSH
• epidermis MeSH
• ichtyóza MeSH
• kůže * MeSH
• kyselina linolová MeSH
• lipasa MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acyltransferasy MeSH
• ceramidy * MeSH
• kyselina linolová MeSH
• lipasa MeSH
• PNPLA1 protein, mouse MeSH Prohlížeč

Ceramides (Cers) with ultralong (∼32-carbon) chains and ω-esterified linoleic acid, composing a subclass called omega-O-acylceramides (acylCers), are indispensable components of the skin barrier. Normal barriers typically contain acylCer concentrations of ∼10 mol%; diminished concentrations, along with altered or missing long periodicity lamellar phase (LPP), and increased permeability accompany an array of skin disorders, including atopic dermatitis, psoriasis, and ichthyoses. We developed model membranes to investigate the effects of the acylCer structure and concentration on skin lipid organization and permeability. The model membrane systems contained six to nine Cer subclasses as well as fatty acids, cholesterol, and cholesterol sulfate; acylCer content-namely, acylCers containing sphingosine (Cer EOS), dihydrosphingosine (Cer EOdS), and phytosphingosine (Cer EOP) ranged from zero to 30 mol%. Systems with normal physiologic concentrations of acylCer mixture mimicked the permeability and nanostructure of human skin lipids (with regard to LPP, chain order, and lateral packing). The models also showed that the sphingoid base in acylCer significantly affects the membrane architecture and permeability and that Cer EOP, notably, is a weaker barrier component than Cer EOS and Cer EOdS. Membranes with diminished or missing acylCers displayed some of the hallmarks of diseased skin lipid barriers (i.e., lack of LPP, less ordered lipids, less orthorhombic chain packing, and increased permeability). These results could inform the rational design of new and improved strategies for the barrier-targeted treatment of skin diseases.

• Klíčová slova
• acylceramide, ceramides, disease models, epidermis, extracellular matrix, membrane nanostructure, membranes/model, permeability, sphingolipids,
• MeSH
• ceramidy analýza   metabolismus   MeSH
• kožní nemoci metabolismus   MeSH
• kůže chemie   metabolismus   MeSH
• lidé MeSH
• membránové lipidy chemie   metabolismus   MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ceramidy MeSH
• membránové lipidy MeSH

Membrane models of the stratum corneum (SC) lipid barrier, either healthy or affected by recessive X-linked ichthyosis, constructed from ceramide [Cer; nonhydroxyacyl sphingosine N-tetracosanoyl-d-erythro-sphingosine (CerNS24) alone or with omega-O-acylceramide N-(32-linoleyloxy)dotriacontanoyl-d-erythro-sphingosine (CerEOS)], FFAs(C16-24), cholesterol (Chol), and sodium cholesteryl sulfate (CholS) were investigated. X-ray diffraction (XRD) revealed a previously unreported polymorphism of the membranes. In the absence of CerEOS, the membranes formed a short lamellar phase (SLP; the repeat distance d = 5.3 nm), a medium lamellar phase (MLP; d = 10.6 nm), or very long lamellar phases (VLLP; d = 15.9 and 21.2 nm). An increased CholS-to-Chol ratio modulated the membrane polymorphism, although the CholS phase separated at ≥ 7 weight% (of total lipids). The presence of CerEOS led to the stable long lamellar phase (LLP) with d = 12.2 nm and prevented VLLP formation. Our XRD results agree well with recently published cryo-electron microscopy data for vitreous skin sections, while also revealing new structures. Thus, lamellar phases with long repeat distances (MLP and VLLP) may be formed in the absence of omega-O-acylceramide, whereas these ultralong Cer species likely stabilize the final SC lipid architecture of LLP by riveting the adjacent lipid layers.

• Klíčová slova
• X-ray crystallography, ceramide, cholesterol, cholesteryl sulfate, extracellular matrix, membranes/model, skin, skin barrier,
• MeSH
• biologické modely * MeSH
• elektronová kryomikroskopie MeSH
• ichtyóza vázaná na chromozom X genetika   metabolismus   patologie   MeSH
• kůže chemie   metabolismus   patologie   MeSH
• lidé MeSH
• membránové lipidy chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové lipidy MeSH

Preclinical studies frequently lack predictive value for human conditions. Human cell-based disease models that reflect patient heterogeneity may reduce the high failure rates of preclinical research. Herein, we investigated the impact of primary cell age and body region on skin homeostasis, epidermal differentiation, and drug uptake. Fibroblasts derived from the breast skin of female 20- to 30-year-olds or 60- to 70-year-olds and fibroblasts from juvenile foreskin (<10 years old) were compared in cell monolayers and in reconstructed human skin (RHS). RHS containing aged fibroblasts differed from its juvenile and adult counterparts, especially in terms of the dermal extracellular matrix composition and interleukin-6 levels. The site from which the fibroblasts were derived appeared to alter fibroblast-keratinocyte crosstalk by affecting, among other things, the levels of granulocyte-macrophage colony-stimulating factor. Consequently, the epidermal expression of filaggrin and e-cadherin was increased in RHS containing breast skin fibroblasts, as were lipid levels in the stratum corneum. In conclusion, the region of the body from which fibroblasts are derived appears to affect the epidermal differentiation of RHS, while the age of the fibroblast donors determines the expression of proteins involved in wound healing. Emulating patient heterogeneity in preclinical studies might improve the treatment of age-related skin conditions.

• MeSH
• buněčná diferenciace MeSH
• dospělí MeSH
• epidermální buňky cytologie   metabolismus   MeSH
• fibroblasty metabolismus   patologie   MeSH
• filagriny MeSH
• hojení ran MeSH
• homeostáza MeSH
• kultivované buňky MeSH
• kůže anatomie a histologie   cytologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• předkožka cytologie   MeSH
• primární buněčná kultura MeSH
• prsy anatomie a histologie   cytologie   MeSH
• senioři MeSH
• stárnutí buněk fyziologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Aging depicts one of the major challenges in pharmacology owing to its complexity and heterogeneity. Thereby, advanced glycated end-products modify extracellular matrix proteins, but the consequences on the skin barrier function remain heavily understudied. Herein, we utilized transmission electron microscopy for the ultrastructural analysis of ribose-induced glycated reconstructed human skin (RHS). Molecular and functional insights substantiated the ultrastructural characterization and proved the relevance of glycated RHS beyond skin aging. In particular, electron microscopy mapped the accumulation and altered spatial orientation of fibrils and filaments in the dermal compartment of glycated RHS. Moreover, the epidermal basement membrane appeared thicker in glycated than in non-glycated RHS, but electron microscopy identified longitudinal clusters of the finest collagen fibrils instead of real thickening. The stratum granulosum contained more cell layers, the morphology of keratohyalin granules decidedly differed, and the stratum corneum lipid order increased in ribose-induced glycated RHS, while the skin barrier function was almost not affected. In conclusion, dermal advanced glycated end-products markedly changed the epidermal morphology, underlining the importance of matrix⁻cell interactions. The phenotype of ribose-induced glycated RHS emulated aged skin in the dermis, while the two to three times increased thickness of the stratum granulosum resembled poorer cornification.

• Klíčová slova
• advanced glycated end products, aging, diabetes, electron microscopy, nanomedicine, reconstructed human skin, ribose, skin absorption,
• MeSH
• bazální membrána účinky léků   ultrastruktura   MeSH
• buněčná diferenciace účinky léků   MeSH
• epidermis účinky léků   ultrastruktura   MeSH
• fibroblasty účinky léků   ultrastruktura   MeSH
• keratinocyty účinky léků   ultrastruktura   MeSH
• kůže účinky léků   ultrastruktura   MeSH
• lidé MeSH
• produkty pokročilé glykace metabolismus   MeSH
• ribosa farmakologie   MeSH
• škára účinky léků   ultrastruktura   MeSH
• stárnutí kůže účinky léků   MeSH
• transmisní elektronová mikroskopie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• produkty pokročilé glykace MeSH
• ribosa MeSH