RESUMO
Human skin equivalents (HSEs) are 3D-cultured human skin models that mimic many aspects of native human skin (NHS). Although HSEs resemble NHS very closely, the barrier located in the stratum corneum (SC) is impaired. This is caused by an altered lipid composition in the SC of HSEs compared with NHS. One of the most pronounced changes in this lipid composition is a high level of monounsaturation. One key enzyme in this change is stearoyl-CoA desaturase-1 (SCD1), which catalyses the monounsaturation of lipids. In order to normalize the lipid composition, we aimed to target a group of nuclear receptors that are important regulators in the lipid synthesis. This group of receptors are known as the peroxisome proliferating activating receptors (PPARs). By (de)activating each isoform (PPAR-α, PPAR-δ and PPAR-γ), the PPAR isoforms may have normalizing effects on the lipid composition. In addition, another PPAR-α agonist Wy14643 was included as this supplement demonstrated normalizing effects in the lipid composition in a more recent study. After PPAR (ant)agonists supplementation, the mRNA of downstream targets, lipid synthesis genes and lipid composition were investigated. The PPAR downstream targets were activated, indicating that the supplements reached the keratinocytes to trigger their effect. However, minimal impact was observed on the lipid composition after PPAR isoform (de) activation. Only the highest concentration Wy14643 resulted in strong, but negative effects on CER composition. Although the novel tested modifications did not result in an improvement, more insight is gained on the nuclear receptors PPARs and their effects on the lipid barrier in full-thickness skin models.
Assuntos
Queratinócitos , Pele , Humanos , PPAR alfa , PPAR gama , LipídeosRESUMO
In vitro skin tissue engineering is challenging due to the manifold differences between the in vivo and in vitro conditions. Yet, three-dimensional (3D) human skin equivalents (HSEs) are able to mimic native human skin in many fundamental aspects. However, the epidermal lipid barrier formation, which is essential for the functionality of the skin barrier, remains compromised. Recently, HSEs with an improved lipid barrier formation were generated by (i) incorporating chitosan in the dermal collagen matrix, (ii) reducing the external oxygen level to 3%, and (iii) inhibiting the liver X receptor (LXR). In this study, we aimed to determine the synergic effects in full-thickness models (FTMs) with combinations of these factors as single-, double-, and triple-targeted optimization approaches. The collagen-chitosan FTM supplemented with the LXR inhibitor showed improved epidermal morphogenesis, an enhanced lipid composition, and a better lipid organization. Importantly, barrier functionality was improved in the corresponding approach. In conclusion, our leading optimization approach substantially improved the epidermal morphogenesis, barrier formation, and functionality in the FTM, which therefore better resembled native human skin.
Assuntos
Células Epidérmicas/metabolismo , Epiderme/metabolismo , Morfogênese , Pele/metabolismo , Engenharia Tecidual/métodos , Células Cultivadas , Quitosana/metabolismo , Cromatografia Líquida , Colágeno/metabolismo , Epiderme/crescimento & desenvolvimento , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Metabolismo dos Lipídeos , Lipídeos/análise , Receptores X do Fígado/metabolismo , Espectrometria de Massas , Espalhamento a Baixo Ângulo , Pele/citologia , Pele/crescimento & desenvolvimento , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
To advance drug development representative reliable skin models are indispensable. Animal skin as test model for human skin delivery is restricted as their properties greatly differ from human skin. In vitro 3D-human skin equivalents (HSEs) are valuable tools as they recapitulate important aspects of the human skin. However, HSEs still lack the full barrier functionality as observed in native human skin, resulting in suboptimal screening outcome. In this review we provide an overview of established in-house and commercially available HSEs and discuss in more detail to what extent their skin barrier biology is mimicked in vitro focusing on the lipid properties and cornified envelope. Further, we will illustrate how underlying factors, such as culture medium improvements and environmental factors affect the barrier lipids. Lastly, potential improvements in skin barrier function will be proposed aiming at a new generation of HSEs that may replace animal skin delivery studies fully.
Assuntos
Lipídeos/fisiologia , Proteínas/metabolismo , Pele Artificial , Células Cultivadas , Humanos , Permeabilidade , Proteínas/fisiologiaRESUMO
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.
Assuntos
Ceramidas/metabolismo , Inibidores Enzimáticos/farmacologia , Pele/metabolismo , Estearoil-CoA Dessaturase/antagonistas & inibidores , Adulto , Células Cultivadas , Feminino , Humanos , Técnicas de Cultura de Órgãos/métodos , Pele/citologia , Pele/efeitos dos fármacos , Pele/ultraestrutura , Estearoil-CoA Dessaturase/metabolismo , Adulto JovemRESUMO
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.
Assuntos
Meios de Cultura/farmacologia , Receptores X do Fígado/antagonistas & inibidores , Cultura Primária de Células/métodos , Pele/efeitos dos fármacos , Sulfonamidas/farmacologia , Ceramidas/metabolismo , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/patologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ácidos Graxos não Esterificados , Humanos , Hidrocarbonetos Fluorados/farmacologia , Lipogênese/efeitos dos fármacos , Receptores X do Fígado/agonistas , Receptores X do Fígado/metabolismo , Morfogênese/efeitos dos fármacos , Pele/crescimento & desenvolvimento , Pele/metabolismo , Estearoil-CoA Dessaturase/metabolismoRESUMO
Epidermal ß-glucocerebrosidase (GBA1), an acid ß-glucosidase normally located in lysosomes, converts (glucosyl)ceramides into ceramides, which is crucial to generate an optimal barrier function of the outermost skin layer, the stratum corneum (SC). Here we report on two developed in situ methods to localize active GBA in human epidermis: i) an optimized zymography method that is less labor intensive and visualizes enzymatic activity with higher resolution than currently reported methods using either substrate 4-methylumbelliferyl-ß-D-glucopyranoside or resorufin-ß-D-glucopyranoside; and ii) a novel technique to visualize active GBA1 molecules by their specific labeling with a fluorescent activity-based probe (ABP), MDW941. The latter method pro-ved to be more robust and sensitive, provided higher resolution microscopic images, and was less prone to sample preparation effects. Moreover, in contrast to the zymography substrates that react with various ß-glucosidases, MDW941 specifically labeled GBA1. We demonstrate that active GBA1 in the epidermis is primarily located in the extracellular lipid matrix at the interface of the viable epidermis and the lower layers of the SC. With ABP-labeling, we observed reduced GBA1 activity in 3D-cultured skin models when supplemented with the reversible inhibitor, isofagomine, irrespective of GBA expression. This inhibition affected the SC ceramide composition: MS analysis revealed an inhibitor-dependent increase in the glucosylceramide:ceramide ratio.
Assuntos
Ensaios Enzimáticos , Corantes Fluorescentes/química , Glucosilceramidase/análise , Pele/enzimologia , Coloração e Rotulagem/métodos , Benzoxazinas/química , Compostos de Boro/química , Cicloexanóis/química , Compostos de Epóxi/química , Expressão Gênica , Glucosídeos/química , Glucosilceramidase/metabolismo , Humanos , Himecromona/análogos & derivados , Himecromona/química , Técnicas de Cultura de TecidosRESUMO
Terminally differentiating epidermal keratinocytes express a large number of structural and antimicrobial proteins that are involved in the physical barrier function of the stratum corneum and provide innate cutaneous host defense. Late cornified envelope (LCE) genes, located in the epidermal differentiation complex on chromosome 1, encode a family of 18 proteins of unknown function, whose expression is largely restricted to epidermis. Deletion of two members, LCE3B and LCE3C (LCE3B/C-del), is a widely-replicated psoriasis risk factor that interacts with the major psoriasis-psoriasis risk gene HLA-C*06. Here we performed quantitative trait locus analysis, utilizing RNA-seq data from human skin and found that LCE3B/C-del was associated with a markedly increased expression of LCE3A, a gene directly adjacent to LCE3B/C-del. We confirmed these findings in a 3-dimensional skin model using primary keratinocytes from LCE3B/C-del genotyped donors. Functional analysis revealed that LCE3 proteins, and LCE3A in particular, have defensin-like antimicrobial activity against a variety of bacterial taxa at low micromolar concentrations. No genotype-dependent effect was observed for the inside-out or outside-in physical skin barrier function. Our findings identify an unknown biological function for LCE3 proteins and suggest a role in epidermal host defense and LCE3B/C-del-mediated psoriasis risk.
Assuntos
Proteínas Ricas em Prolina do Estrato Córneo/genética , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Psoríase/genética , Psoríase/imunologia , Antibacterianos/imunologia , Biópsia por Agulha , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Imuno-Histoquímica , Queratinócitos , Desequilíbrio de Ligação , Masculino , Polimorfismo de Nucleotídeo Único , Psoríase/patologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Papel (figurativo)RESUMO
The effectiveness of the skin barrier underlies the outer layer of the skin: the stratum corneum (SC). However, in several skin diseases this barrier is impaired. In two inflammatory skin diseases, atopic eczema and Netherton syndrome, an increased level of monounsaturated fatty acids (MUFAs) has been observed as opposed to healthy skin. In the present study, we aimed to investigate the effect of MUFAs on the lipid organization and skin lipid barrier using an in vitro model membrane system, the stratum corneum substitute (SCS), mimicking the SC lipid composition and organization. To achieve our goal, the SCS has been prepared with increasing levels of MUFAs using various chain length. Permeation studies and trans-epidermal water loss measurements show that an increment of MUFAs reduces the lipid barrier in the SCS. The increased level of unsaturation exerts its effect by reducing the packing density in the lipid organization, while the lamellar phases are not affected. Our findings indicate that increased levels of MUFAs may contribute to the impaired skin barrier in diseased skin.