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... Show moreThe 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. Show less
-aminobenzoate permeation and transepidermal water loss values as markers for barrier function, we determined that the alterations in SC lipid composition contribute to the impaired barrier... Show more-aminobenzoate permeation and transepidermal water loss values as markers for barrier function, we determined that the alterations in SC lipid composition contribute to the impaired barrier function in AD patients. By the use of biophysical techniques, we established that the largest reduction in barrier capability was observed in the model with an increased fraction of short-chain FFAs, evident by the decrease in chain packing density. Modulations in the CER subclass composition impacted the lamellar organization while having a smaller effect on the barrier function. These findings provide evidence that AD therapies normalizing the FFA composition are at least as important as normalizing CER composition. Show less
Helder, R.W.J.; Boiten, W.A.; Dijk, R. van; Gooris, G.S.; El Ghalbzouri, A.; Bouwstra, J.A. 2019
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... Show moreFull 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. Show less
Mieremet, A.; Dijk, R. van; Boiten, W.A.; Gooris, G.S.; Bouwstra, J.A.; El Ghalbzouri, A. 2019
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... Show moreHuman 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. Show less
Mieremet, A.; Boiten, W.A.; Dijk, R. van; Gooris, G.S.; Overkleeft, H.S.; Aerts, J.M.F.G.; ... ; El Ghalbzouri, A. 2019
Relative humidity (RH) levels vary continuously in vivo, although during in vitro generation of three-dimensional human skin equivalents (HSEs) these remain high (90-95%) to prevent evaporation of... Show moreRelative humidity (RH) levels vary continuously in vivo, although during in vitro generation of three-dimensional human skin equivalents (HSEs) these remain high (90-95%) to prevent evaporation of the cell-culture medium. However, skin functionality is directly influenced by environmental RH. As the barrier formation in HSEs is different, there is a need to better understand the role of cell-culture conditions during the generation of HSEs. In this study, we aim to investigate the effects of RH on epidermal morphogenesis and lipid barrier formation in HSEs. Therefore, two types of HSEs were developed at 90% or at 60% RH. Assessments were performed to determine epidermal morphogenesis by immunohistochemical analyses, ceramide composition by lipidomic analysis, and lipid organization by Fourier transform infrared spectroscopy and small-angle X-ray diffraction. We show that reduction of RH mainly affected the uppermost viable epidermal layers in the HSEs, including an enlargement of the granular cells and induction of epidermal cell activation. Neither the composition nor the organization of the lipids in the intercorneocyte space were substantially altered at reduced RH. In addition, lipid processing from glucosylceramides to ceramides was not affected by reduced RH in HSEs as shown by enzyme expression, enzyme activity, and substrate-to-product ratio. Our results demonstrate that RH directly influences epidermal morphogenesis, albeit the in vitro lipid barrier formation is comparable at 90% and 60% RH. Show less
Uche, L.E.; Gooris, G.S.; Beddoes, C.M.; Bouwstra, J.A. 2019
The intercellular lipid matrix of the stratum corneum (SC), which consist mainly of ceramides (CERs), free fatty acids and cholesterol, is fundamental to the skin barrier function. These lipids... Show moreThe intercellular lipid matrix of the stratum corneum (SC), which consist mainly of ceramides (CERs), free fatty acids and cholesterol, is fundamental to the skin barrier function. These lipids assemble into two lamellar phases, known as the long and short periodicity phases (LPP and SPP respectively). The LPP is unique in the SC and is considered important for the skin barrier function. Alterations in CER composition, as well as impaired skin barrier function, are commonly observed in diseased skin, yet the understanding of this relationship remains insufficient. In this study, we have investigated the influence of non-hydroxy and α-hydroxy sphingosine-based CERs and their phytosphingosine counterparts on the permeability and lipid organization of model membranes, which were adjusted in composition to enhance formation of the LPP. The permeability was compared by diffusion studies using ethyl-p-aminobenzoate as a model drug, and the lipid organization was characterized by X-ray diffraction and infrared spectroscopy. Both the sphingosine- and phytosphingosine-based CER models formed the LPP, while the latter exhibited a longer LPP repeat distance. The ethyl-p-aminobenzoate flux across the sphingosine-based CER models was higher when compared to the phytosphingosine counterparts, contrary to the fact that the α-hydroxy phytosphingosine-based CER model had the lowest chain packing density. The unanticipated low permeability of the α-hydroxy phytosphingosine-based model is probably associated with a stronger headgroup hydrogen bonding network. Our findings indicate that the increased level of sphingosine-based CERs at the expense of phytosphingosine-based CERs, as observed in the diseased skin, may contribute to the barrier function impairment. Show less
Cardoso, R.M.; Creemers, E.; Absalah, S.; Gooris, G.S.; Hoekstra, M.; Eck, M. van; Bouwstra, J.A. 2019
Long-term exposure to hypercholesterolemia induces the development of skin xanthoma's characterized by the accumulation of lipid-laden foam cells in humans and in mice. Early skin changes in... Show moreLong-term exposure to hypercholesterolemia induces the development of skin xanthoma's characterized by the accumulation of lipid-laden foam cells in humans and in mice. Early skin changes in response to hypercholesterolemia are however unknown. In this study, we investigated the skin lipid composition and associated barrier function in young adult low-density lipoprotein receptor knockout (LDLR−/−) and apolipoprotein E knockout (APOE−/−) mice, two commonly used hypercholesterolemic mouse models characterized by the accumulation of apolipoprotein B containing lipoproteins. No effects were observed on cholesterol content in the epidermis in LDLR−/− mice nor in the more extremely hypercholesterolemic APOE−/− mice. Interestingly, the free fatty acids in the APOE−/− epidermis shifted towards shorter and unsaturated chains. Genes involved in the synthesis of cholesterol and fatty acids were downregulated in APOE−/− skin suggesting a compensation for the higher influx of plasma lipids, most probably as cholesteryl esters. Importantly, in vivo transepidermal water loss and permeability studies with murine lipid model membranes revealed that the lipid composition of the APOE−/− skin resulted in a reduced skin barrier function. In conclusion, severe hypercholesterolemia associated with increased apolipoprotein B containing lipoproteins affects the epidermal lipid composition and its protective barrier. Show less
Berkers, M.A.J.; Visscher, D.; Gooris, G.S.; Bouwstra, J.A. 2018
The extracellular lipid matrix in the skin's outermost layer, the stratum corneum, is crucial for the skin barrier. The matrix is composed of ceramides (CERs), cholesterol (CHOL) and free fatty... Show moreThe extracellular lipid matrix in the skin's outermost layer, the stratum corneum, is crucial for the skin barrier. The matrix is composed of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) and involves two lamellar phases: the short periodicity phase (SPP) and the long periodicity phase (LPP). To understand the skin barrier thoroughly, information about the molecular arrangement in the unit cell of these lamellar phases is paramount. Previously we examined the molecular arrangement in the unit cell of the SPP. Furthermore X-ray and neutron diffraction revealed a trilayer arrangement of lipids within the unit cell of the LPP [D. Groen et al., Biophysical Journal, 97, 2242-2249, 2009]. In the present study, we used neutron diffraction to obtain more details about the location of lipid (sub)classes in the unit cell of the LPP. The diffraction pattern revealed at least 8 diffraction orders of the LPP with a repeating unit of 129.6±0.5Å. To determine the location of lipid sub(classes) in the unit cell, samples were examined with either only protiated lipids or selectively deuterated lipids. The diffraction data obtained by means of D2O/H2O contrast variation together with a gradual replacement of one particular CER, the acyl CER, by its partly deuterated counterpart, were used to construct the scattering length density profiles. The acyl chain of the acyl CER subclass is located at a position of ~21.4±0.2Å from the unit cell centre of the LPP. The position and orientation of CHOL in the LPP unit cell were determined using tail and head-group deuterated forms of the sterol. CHOL is located with its head-group positioned ~26±0.2Å from the unit cell centre. This allows the formation of a hydrogen bond with the ester group of the acyl CER located in close proximity. Based on the positions of the deuterated moieties of the acyl CER, CHOL and the previously determined location of two other lipid subclasses [E.H. Mojumdar et al., Biophysical Journal, 108, 2670-2679, 2015], a molecular model is proposed for the unit cell of the LPP. In this model CHOL is located in the two outer layers of the LPP, while CER EOS is linking the two outer layers with the central lipid layers. Finally the two other lipid subclasses are predominantly located in the central layer of the LPP. Show less
Uchiyama, M.; Oguri, M.; Mojumdar, A.; Gooris, G.S.; Bouwstra, J.A. 2016
The lipid matrix in the stratum corneum (SC) plays an important role in the barrier function of the skin. The main lipid classes in this lipid matrix are ceramides (CERs), cholesterol (CHOL) and... Show moreThe lipid matrix in the stratum corneum (SC) plays an important role in the barrier function of the skin. The main lipid classes in this lipid matrix are ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The aim of this study was to determine whether a variation in CER subclass composition and chain length distribution of FFAs affect the permeability of this matrix. To examine this, we make use of lipid model membranes, referred to as stratum corneum substitute (SCS). We prepared SCS containing i) single CER subclass with either a single FFA or a mixture of FFAs and CHOL, or ii) a mixture of various CER subclasses with either a single FFA or a mixture of FFAs and CHOL. In vitro permeation studies were performed using ethyl-p-aminobenzoic acid (E-PABA) as a model drug. The flux of E-PABA across the SCS containing the mixture of FFAs was higher than that across the SCS containing a single FA with a chain length of 24 C atoms (FA C24), while the E-PABA flux was not effected by the CER composition. To select the underlying factors for the changes in permeability, the SCSs were examined by Fourier transform infrared spectroscopy (FTIR) and Small angle X-ray scattering (SAXS). All lipid models demonstrated a similar phase behavior. However, when focusing on the conformational ordering of the individual FFA chains, the shorter chain FFA (with a chain length of 16, 18 or 20 C atoms forming only 11m/m% of the total FFA level) had a higher conformational disordering, while the conformational ordering of the chains of the CER and FA C24 and FA C22 hardly did not change irrespective of the composition of the SCS. In conclusion, the conformational mobility of the short chain FFAs present only at low levels in the model SC lipid membranes has a great impact on the permeability of E-PABA. Show less
Our in-house human skin equivalents contain all stratum corneum (SC) barrier lipid classes, but have a reduced level of free fatty acids (FAs), of which a part is mono-unsaturated. These... Show moreOur in-house human skin equivalents contain all stratum corneum (SC) barrier lipid classes, but have a reduced level of free fatty acids (FAs), of which a part is mono-unsaturated. These differences lead to an altered SC lipid organization and thereby a reduced barrier function compared to human skin. In this study, we aimed to improve the SC FA composition and, consequently, the SC lipid organization of the Leiden epidermal model (LEM) by specific medium supplements. The standard FA mixture (consisting of palmitic, linoleic and arachidonic acids) supplemented to the medium was modified, by replacing protonated palmitic acid with deuterated palmitic acid or by the addition of deuterated arachidic acid to the mixture, to determine whether FAs are taken up from the medium and are incorporated into SC of LEM. Furthermore, supplementation of the total FA mixture or that of palmitic acid alone was increased four times to examine whether this improves the SC FA composition and lipid organization of LEM. The results demonstrate that the deuterated FAs are taken up into LEMs and are subsequently elongated and incorporated in their SC. However, a fourfold increase in palmitic acid supplementation does not change the SC FA composition or lipid organization of LEM. Increasing the concentration of the total FA mixture in the medium resulted in a decreased level of very long chain FAs and an increased level of mono-unsaturated FAs, which lead to deteriorated SC lipid properties. These results indicate that SC lipid properties can be modulated by specific medium supplements. Show less
