The cornified envelope-bound ceramide fraction is altered in patients with atopic dermatitis

Drug Delivery Technolog

Selectivity in cornified envelop binding of ceramides in human skin and the role of LXR inactivation on ceramide binding

The cornified lipid envelope (CLE) is a lipid monolayer covalently bound to the outside of corneocytes and is part of the stratum corneum (SC). The CLE is suggested to act as a scaffold for the unbound SC lipids. By profiling the bound CLE ceramides, a new subclass was discovered and identified as an omega-hydroxylated dihydrosphingosine (OdS) ceramide. Bound glucosylceramides were observed in superficial SC layers of healthy human skin. To investigate the relation between bound and unbound SC ceramides, the composition of both fractions was analyzed and compared. Selectivity in ceramide binding towards unsaturated ceramides and ceramides with a shorter chain length was observed. The selectivity in ceramide species bound to the cornified envelope is thought to have a physiological function in corneocyte flexibility. Next, it was examined if skin models exhibit an altered bound ceramide composition and if the composition was dependent on liver X-receptor (LXR) activation. The effects of an LXR agonist and antagonist on the bound ceramides composition of a full thickness model (FTM) were analyzed. In FTMs, a decreased amount of bound ceramides was observed compared to native human skin. Furthermore, FTMs had a bound ceramide fraction which consisted mostly of unsaturated and shorter ceramides. The LXR antagonist had a normalizing effect on the FTM bound ceramide composition. The agonist exhibited minimal effects. We show that ceramide binding is a selective process, yet, still is contingent on lipid synthesized.Drug Delivery Technolog

Contribution of Palmitic Acid to Epidermal Morphogenesis and Lipid Barrier Formation in Human Skin Equivalents

The outermost barrier layer of the skin is the stratum corneum (SC), which consists of corneocytes embedded in a lipid matrix. Biosynthesis of barrier lipids occurs de novo in the epidermis or is performed with externally derived lipids. Hence, in vitro developed human skin equivalents (HSEs) are developed with culture medium that is supplemented with free fatty acids (FFAs). Nevertheless, the lipid barrier formation in HSEs remains altered compared to native human skin (NHS). The aim of this study is to decipher the role of medium supplemented saturated FFA palmitic acid (PA) on morphogenesis and lipid barrier formation in HSEs. Therefore, HSEs were developed with 100% (25 μM), 10%, or 1% PA. In HSEs supplemented with reduced PA level, the early differentiation was delayed and epidermal activation was increased. Nevertheless, a similar SC lipid composition in all HSEs was detected. Additionally, the lipid organization was comparable for lamellar and lateral organization, irrespective of PA concentration. As compared to NHS, the level of monounsaturated lipids was increased and the FFA to ceramide ratio was drastically reduced in HSEs. This study describes the crucial role of PA in epidermal morphogenesis and elucidates the role of PA in lipid barrier formation of HSEs.Drug Delivery Technolog

Improved organotypic skin model with reduced quantity of monounsaturated ceramides by inhibiting stearoyl-CoA desaturase-1

Full thickness models (FTM) are 3D in vitro skin cultures that resemble the native human skin (NHS) to a great extent. However, the barrier function of these skin models is reduced. The skin barrier is located in the stratum corneum (SC) and consists of corneocytes embedded in a lipid matrix. In this matrix, deviations in the composition of the FTMs lipid matrix may contribute to the impaired skin barrier when compared to NHS. One of the most abundant changes in lipid composition is an increase in monounsaturated lipids for which stearoyl-CoA desaturase-1 (SCD-1) is responsible. To improve the SC lipid composition, we reduced SCD-1 activity during the generation of the FTMs. These FTMs were subsequently assessed on all major aspects, including epidermal homeostasis, lipid composition, lipid organization, and barrier functionality. We demonstrate that SCD-1 inhibition was successful and resulted in FTMs that better mimic the lipid composition of FTMs to NHS by a significant reduction in monounsaturated lipids. In conclusion, this study demonstrates an effective approach to normalize SC monounsaturated lipid concentration and may be a valuable tool in further optimizing the FTMs in future studies.Drug Delivery Technolog

Lipids as therapeutic targets for barrier repair in skin diseases

The skin is our natural barrier and lipids are a key part of this barrier. In the outer skin layer, the stratum corneum (SC), lipids form a densely organized structure dependent on the composition of these lipids. Multiple skin diseases are characterized by alterations in SC lipid composition. These alterations are related to pathological barrier defects. This thesis describes the next steps towards a treatment modifying the lipid composition and thereby restoring this barrier. We developed a novel method to quantify a key SC lipid group called ceramides. This method was applied to compare SC regeneration of skin models to healthy volunteers. Regeneration in such an ex vivo skin model proved to be a potent model for formulation development. Ensuing, a clinical study was performed to determine the mechanistic effects of a formulation on barrier repair in healthy skin. The results warranted follow up analysis of the formulation in atopic dermatitis patients. This thesis also describes a detailed analysis of the ceramide fraction that is covalently attached to the cells in the SC. It was shown that a selected group of ceramides becomes bound. Further analysis showed that this group of ceramides was also affected in atopic dermatitis patients SC. This research was financially supported by Dutch Technology Foundation TTW (grant no. 12400).Drug Delivery Technolog

Compromising human skin in vivo and ex vivo to study skin barrier repair

Ex vivo regenerated stratum corneum (SC) after tape-stripping can be used as a model to study the barrier function of compromised skin. Yet, details about how close the regenerated SC model mimics the lipid properties (e.g. lipid composition and lipid ordering) of the in vivo situation are not known. Here, we examined using a comprehensive ceramide analysis whether human ex vivo regenerated SC showed similar lipid properties as human in vivo regenerated SC. Both in vivo and ex vivo regenerated SC had an altered ceramide subclass composition, with increased percentages of sphingosine-based subclass and decreased percentages of phytosphingosine-based subclass ceramides, a reduced mean ceramide chain length, and a higher percentage of unsaturated ceramides. Overall, regenerated SC ex vivo showed more pronounced but similar changes compared to the in vivo response. One of the purposes of these models is to use them to mimic compromised skin of inflammatory skin diseases. The altered lipid properties in regenerated SC were comparable to those observed in several inflammatory skin diseases, which makes them a valuable model for the barrier properties in inflammatory skin diseases.Drug Delivery Technolog

Combined LC/MS-platform for analysis of all major stratum corneum lipids, and the profiling of skin substitutes.

Ceramides (CERs), cholesterol, and free fatty acids (FFAs) are the main lipid classes in human stratum corneum (SC, outermost skin layer), but no studies report on the detailed analysis of these classes in a single platform. The primary aims of this study were to 1) develop an LC/MS method for (semi-)quantitative analysis of all main lipid classes present in human SC; and 2) use this method to study in detail the lipid profiles of human skin substitutes and compare them to human SC lipids. By applying two injections of 10μl, the developed method detects all major SC lipids using RPLC and negative ion mode APCI-MS for detection of FFAs, and NPLC using positive ion mode APCI-MS to analyze CERs and cholesterol. Validation showed this lipid platform to be robust, reproducible, sensitive, and fast. The method was successfully applied on ex vivo human SC, human SC obtained from tape strips and human skin substitutes (porcine SC and human skin equivalents). In conjunction with FFA profiles, clear differences in CER profiles were observed between these different SC sources. Human skin equivalents more closely mimic the lipid composition of human stratum corneum than porcine skin does, although noticeable differences are still present. These differences gave biologically relevant information on some of the enzymes that are probably involved in SC lipid processing. For future research, this provides an excellent method for (semi-)quantitative, 'high-throughput' profiling of SC lipids and can be used to advance the understanding of skin lipids and the biological processes involved

Applying a vernix caseosa based formulation accelerates skin barrier repair by modulating lipid biosynthesis

Drug Delivery Technolog

Characterization of human skin equivalents developed at body's core and surface temperatures

Human skin equivalents (HSEs) are in vitro developed three-dimensional models resembling native human skin (NHS) to a high extent. However, the epidermal lipid biosynthesis, barrier lipid composition, and organization are altered, leading to an elevated diffusion rate of therapeutic molecules. The altered lipid barrier formation in HSEs may be induced by standardized culture conditions, including a culture temperature of 37°C, which is dissimilar to skin surface temperature. Therefore, we aim to determine the influence of culture temperature during the generation of full thickness models (FTMs) on epidermal morphogenesis and lipid barrier formation. For this purpose, FTMs were developed at conventional core temperature (37°C) or lower temperatures (35°C and 33°C) and evaluated over a time period of 4 weeks. The stratum corneum (SC) lipid composition was analysed using advanced liquid chromatography coupled to mass spectrometry analysis. Our results show that SC layers accumulated at a similar rate irrespective of culture temperature. At reduced culture temperature, an increased epidermal thickness, a disorganization of the lower epidermal cell layers, a delayed early differentiation, and an enlargement of granular cells were detected. Interestingly, melanogenesis was reduced at lower temperature. The ceramide subclass profile, chain length distribution, and level of unsaturated ceramides were similar in FTMs generated at 37°C and 35°C but changed when generated at 33°C, reducing the resemblance to NHS. Herein, we report that culture temperature affects epidermal morphogenesis substantially and to a lesser extent the lipid barrier formation, highlighting the importance of optimized external parameters during reconstruction of skin.Drug Delivery Technolog

The effects of LXR agonist T0901317 and LXR antagonist GSK2033 on morphogenesis and lipid properties in full thickness skin models

Full thickness models (FTMs) are 3D-cultured human skin models that mimic many aspects of native human skin (NHS). However, their stratum corneum (SC) lipid composition differs from NHS causing a reduced skin barrier. The most pronounced differences in lipid composition are a reduction in lipid chain length and increased monounsaturated lipids. The liver-X-receptor (LXR) activates the monounsaturated lipid synthesis via stearoyl-CoA desaturase-1 (SCD-1). Therefore, the aim was to improve the SC lipid synthesis of FTMs by LXR deactivation. This was achieved by supplementing culture medium with LXR antagonist GSK2033. LXR agonist T0901317 was added for comparison. Subsequently, epidermal morphogenesis, lipid composition, lipid organization and the barrier functionality of these FTMs were assessed. We demonstrate that LXR deactivation resulted in a lipid composition with increased overall chain lengths and reduced levels of monounsaturation, whereas LXR activation increased the amount of monounsaturated lipids and led to a reduction in the overall chain length. However, these changes did not affect the barrier functionality. In conclusion, LXR deactivation led to the development of FTMs with improved lipid properties, which mimic the lipid composition of NHS more closely. These novel findings may contribute to design interventions to normalize SC lipid composition of atopic dermatitis patients.Drug Delivery Technolog