Andrej Kováčik

Different Phase Behavior and Packing of Ceramides with Long (C16) and Very Long (C24) Acyls in Model Membranes: Infrared Spectroscopy Using Deuterated Lipids

Ceramides (Cer) are the central molecules in sphingolipid metabolism that participate in cellular signaling and also prevent excessive water loss by the skin. Previous studies showed that sphingosine-based Cer with a long 16C chain (CerNS16) and very long 24C-chain ceramides (CerNS24) differ in their biological actions. Increased levels of long CerNS16 at the expense of the very long CerNS24 have been found in atopic dermatitis patients, and this change correlated with the skin barrier properties. To probe the membrane behavior of the long CerNS16 and the very long chain CerNS24, we studied their interactions with fatty acids and cholesterol in model stratum corneum membranes using infrared spectroscopy. Using Cer with deuterated acyls and/or deuterated fatty acids, we showed differences in lipid mixing, packing, and thermotropic phase behavior between long and very long Cer. These differences were observed in the presence of lignoceric acid or a heterogeneous fatty acid mixture (C16-C24), in the presence or absence of cholesterol sulfate, and at 5-95% humidity. In these membranes, very long CerNS24 prefers an extended (splayed-chain) conformation in which the fatty acid is associated with the very long Cer chain. In contrast, the shorter CerNS16 and fatty acids are mostly phase separated.

Stimulation of PPARα normalizes the skin lipid ratio and improves the skin barrier of normal and filaggrin deficient reconstructed skin

BACKGROUND Therapeutic options for atopic dermatitis mostly address the symptoms but causal therapies are still missing. Peroxisome proliferator activated receptor (PPAR) agonists exert beneficial effects in patients suffering this disease, whereas the stimulation of PPARα and γ seemed most promising. OBJECTIVES To elucidate the effects of the PPARα specific agonist WY14643, the PPARγ agonist ciglitazone, and the dual PPARα+γ agonist docosahexaenoic acid (DHA) on the homeostasis and barrier function of filaggrin deficient skin. METHODS The effects of the PPAR agonists on skin differentiation were evaluated via qPCR, Western blot, histological or immunofluorescence staining. Skin lipid organization was determined by ATR-FTIR and lipid composition was analyzed by HPTLC. Ultimately, the skin barrier function was assessed by skin absorption studies using the radioactively labeled compound testosterone. RESULTS Significant upregulation of filaggrin after DHA and WY14643 supplementation, but no effect of ciglitazone, on protein and mRNA level was detected. DHA and WY14643, but not ciglitazone, normalized the molar ratio of the main skin barrier lipids to 1:1:1 (free fatty acids:ceramides:cholesterol). Furthermore, DHA and WY14643 supplementation normalized the skin lipid profile in filaggrin deficient skin, but only WY14643 significantly improved the skin barrier function. CONCLUSION Supplementation particularly with the PPARα agonist WY14643 improved the homeostasis and barrier function of filaggrin deficient skin models by normalization of the free fatty acid profile underlining the potential of PPAR agonists for the treatment of filaggrin-associated skin diseases.

The Chemistry and Biology of 6‐Hydroxyceramide, the Youngest Member of the Human Sphingolipid Family

Sphingolipids are crucial for the life of the cell. In land‐dwelling mammals, they are equally important outside the cell—in the extracellular space of the skin barrier—because they prevent loss of water. Although a large body of research has elucidated many of the functions of sphingolipids, their extensive structural diversity remains intriguing. A new class of sphingolipids based on 6‐hydroxylated sphingosine has recently been identified in human skin. Abnormal levels of these 6‐hydroxylated ceramides have repeatedly been observed in atopic dermatitis; however, neither the biosynthesis nor the roles of these unique ceramide subclasses have been established in the human body. In this Minireview, we summarize the current knowledge of 6‐hydroxyceramides, including their discovery, structure, stereochemistry, occurrence in healthy and diseased human epidermis, and synthetic approaches to 6‐hydroxysphingosine and related ceramides.

Phytosphingosine, sphingosine and dihydrosphingosine ceramides in model skin lipid membranes: permeability and biophysics

Ceramides based on phytosphingosine, sphingosine and dihydrosphingosine are essential constituents of the skin lipid barrier that protects the body from excessive water loss. The roles of the individual ceramide subclasses in regulating skin permeability and the reasons for C4-hydroxylation of these sphingolipids are not completely understood. We investigated the chain length-dependent effects of dihydroceramides, sphingosine ceramides (with C4-unsaturation) and phytoceramides (with C4-hydroxyl) on the permeability, lipid organization and thermotropic behavior of model stratum corneum lipid membranes composed of ceramide/lignoceric acid/cholesterol/cholesteryl sulfate. Phytoceramides with very long C24 acyl chains increased the permeability of the model lipid membranes compared to dihydroceramides or sphingosine ceramides with the same chain lengths. Either unsaturation or C4-hydroxylation of dihydroceramides induced chain length-dependent increases in membrane permeability. Infrared spectroscopy showed that C4-hydroxylation of the sphingoid base decreased the relative ratio of orthorhombic chain packing in the membrane and lowered the miscibility of C24 phytoceramide with lignoceric acid. The phase separation in phytoceramide membranes was confirmed by X-ray diffraction. In contrast, phytoceramides formed strong hydrogen bonds and highly thermostable domains. Thus, the large heterogeneity in ceramide structures and in their aggregation mechanisms may confer resistance towards the heterogeneous external stressors that are constantly faced by the skin barrier.

Scalable Synthesis of Human Ultralong Chain Ceramides

Ceramides with ultralong chains (≥30 carbons), also known as acylceramides, play a critical role in the survival of mammals on dry land. An efficient and scalable synthesis of four major classes of ultralong human skin ceramides is reported. The key approach involves the use of a succinimidyl ester that acts as a protective group, helps overcome the extremely low solubility, and simultaneously activates the fatty acid for its clean and high-yielding attachment to a sphingoid base.

Synthesis of 6-hydroxyceramide using ruthenium-catalyzed hydrosilylation–protodesilylation. Unexpected formation of a long periodicity lamellar phase in skin lipid membranes

The synthesis of a ceramide with a 6-hydroxysphingosine base, a unique component of the human epidermal barrier, is reported. The key step involves a mild and selective trans-reduction of a triple bond using [Cp*Ru(CH3CN)3]PF6-catalyzed hydrosilylation followed by protodesilylation. The oxidation of sphingosine-based ceramide to 6-hydroxyceramide is also described. X-Ray powder diffraction on the model skin lipid membranes showed that 6-hydroxyceramide promotes the formation of a lamellar phase with 10.6 nm periodicity, which might explain why keratinocytes hydroxylate some ceramides at carbon 6.

Effects of 6-Hydroxyceramides on the Thermotropic Phase Behavior and Permeability of Model Skin Lipid Membranes

Ceramides (Cer) based on 6-hydroxysphingosine are important components of the human skin barrier, the stratum corneum. Although diminished concentrations of 6-hydroxyCer have been detected in skin diseases such as atopic dermatitis, our knowledge on these unusual sphingolipids, which have only been found in the skin, is limited. In this work, we investigate the biophysical behavior of N-lignoceroyl-6-hydroxysphingosine (Cer NH) in multilamellar lipid membranes composed of Cer/free fatty acids (FFAs) (C16-C24)/cholesterol/cholesteryl sulfate. To probe the Cer structure-activity relationships, we compared Cer NH membranes with membranes containing Cer with sphingosine (Cer NS), dihydrosphingosine, and phytosphingosine (Cer NP), all with the same acyl chain length (C24). Compared with Cer NS, 6-hydroxylation of Cer not only increased membrane water loss and permeability in a lipophilic model compound but also dramatically increased the membrane opposition to electrical current, which is proportional to the flux of ions. Infrared spectroscopy revealed that Cer hydroxylation (in either Cer NH or Cer NP) increased the main transition temperature of the membrane but prevented good Cer mixing with FFAs. X-ray powder diffraction showed not only lamellar phases with shorter periodicity upon Cer hydroxylation but also the formation of an unusually long periodicity phase (d = 10.6 nm) in Cer NH-containing membranes. Thus, 6-hydroxyCer behaves differently from sphingosine- and phytosphingosine-based Cer. In particular, the ability to form a long-periodicity lamellar phase and highly limited permeability to ions indicate the manner in which 6-hydroxylated Cer contribute to the skin barrier function.

Permeability Barrier and Microstructure of Skin Lipid Membrane Models of Impaired Glucosylceramide Processing

Ceramide (Cer) release from glucosylceramides (GlcCer) is critical for the formation of the skin permeability barrier. Changes in β-glucocerebrosidase (GlcCer’ase) activity lead to diminished Cer, GlcCer accumulation and structural defects in SC lipid lamellae; however, the molecular basis for this impairment is not clear. We investigated impaired GlcCer-to-Cer processing in human Cer membranes to determine the physicochemical properties responsible for the barrier defects. Minor impairment (5–25%) of the Cer generation from GlcCer decreased the permeability of the model membrane to four markers and altered the membrane microstructure (studied by X-ray powder diffraction and infrared spectroscopy), in agreement with the effects of topical GlcCer in human skin. At these concentrations, the accumulation of GlcCer was a stronger contributor to this disturbance than the lack of human Cer. However, replacement of 50–100% human Cer by GlcCer led to the formation of a new lamellar phase and the maintenance of a rather good barrier to the four studied permeability markers. These findings suggest that the major cause of the impaired water permeability barrier in complete GlcCer’ase deficiency is not the accumulation of free GlcCer but other factors, possibly the retention of GlcCer bound in the corneocyte lipid envelope.

Influence of a Novel Dimeric Ceramide Molecule on the Nanostructure and Thermotropic Phase Behavior of a Stratum Corneum Model Mixture

The stratum corneum (SC) is the outermost layer of the skin and is composed of a multilayered assembly of mostly ceramids (Cer), free fatty acids, cholesterol (Chol), and cholesterol sulfate (Chol-S). Because of the tight packing of these lipids, the SC features unique barrier properties defending the skin from environmental influences. Under pathological conditions, where the skin barrier function is compromised, topical application of molecules that rigidify the SC may lead to a restored barrier function. To this end, molecules are required that incorporate into the SC and bring back the original rigidity of the skin barrier. Here, we investigated the influence of a novel dimeric ceramide (dim-Cer) molecule designed to feature a long, rigid hydrocarbon chain ideally suited to forming an orthorhombic lipid phase. The influence of this molecules on the thermotropic phase behavior of a SC mixture consisting of Cer[AP18] (55 wt %), cholesterol (Chol, 25 wt %), steric acid (SA, 15 wt %), and cholesterol sulfate (Chol-S, 5 wt %) was studied using a combination of neutron diffraction and 2H NMR spectroscopy. These methods provide detailed insights into the packing properties of the lipids in the SC model mixture. Dim-Cer remains in an all-trans state of the membrane-spanning lipid chain at all investigated temperatures, but the influence on the phase behavior of the other lipids in the mixture is marginal. Biophysical experiments are complemented by permeability measurements in model membranes and human skin. The latter, however, indicates that dim-Cer only partially provides the desired effect on membrane permeability, necessitating further optimization of its structure for medical applications.

Ceramides in the skin barrier

Abstract The skin barrier, which is essential for human survival on dry land, is located in the uppermost skin layer, the stratum corneum. The stratum corneum consists of corneocytes surrounded by multilamellar lipid membranes that prevent excessive water loss from the body and entrance of undesired substances from the environment. To ensure this protective function, the composition and organization of the lipid membranes is highly specialized. The major skin barrier lipids are ceramides, fatty acids and cholesterol in an approximately equimolar ratio. With hundreds of molecular species of ceramide, skin barrier lipids are a highly complex mixture that complicate the investigation of its behaviour. In this minireview, the structures of the major skin barrier lipids, formation of the stratum corneum lipid membranes and their molecular organization are described.