RESUMEN
BACKGROUND: Macromolecules in skin cells are damaged when exposed to environmental stressors, leading to disrupted cellular function and homeostasis. While epidermal turnover can eliminate some of this damage, autophagy can rapidly remove these defective components. Niacinamide (Nam) is known to induce autophagy and optimizing formulations to maximize this response could provide improved homeostasis in stressed skin. OBJECTIVE: To determine (i) whether Nam can induce autophagy related 5 (ATG5), an autophagy marker, in human keratinocytes and (ii) whether optimized low pH Nam formulations can enhance the response in 3D skin models. METHODS: Human keratinocytes treated with Nam were evaluated for autophagosome accumulation and induction of ATG5 by gene expression, immunoblotting and immune-fluorescence microscopy. 3D skin equivalents were topically treated with Nam formulations at pH 5.8 and 3.8. Gene expression profiling and immunoblot analysis of ATG5 were performed. RESULTS: Nam treatment of keratinocytes led to an accumulation of autophagosomes with a maximal signal at 48 h. Gene expression of ATG5 was induced by Nam, and immunoblots stained for ATG5 showed a significant increase after 6 h of treatment. Gene expression profiling of 3D epidermal skin equivalents treated with Nam at pH 3.8 showed stronger induction of autophagy-related genes, including ATG5, compared with pH 5.8 formulas. Enrichment for gene ontology terms on autophagy showed an increased linkage with Nam formulas at pH 3.8. CONCLUSIONS: We found that Nam induces autophagosome accumulation and ATG5 levels in keratinocytes. We also discovered that a Nam formulation at pH 3.8 can further increase levels of ATG5 in 3D skin models when compared to Nam at pH 5.8. These data support that Nam can induce autophagy in keratinocytes and formulations at pH 3.8 can enhance the impact. We hypothesize that optimized formulations at pH 3.8 can improve skin ageing appearance via autophagy induction.
Asunto(s)
Proteína 5 Relacionada con la Autofagia , Autofagia , Queratinocitos , Niacinamida , Proteína 5 Relacionada con la Autofagia/genética , Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Queratinocitos/metabolismoRESUMEN
BACKGROUND: Transition of hair shaft keratinocytes from actively respiring, nucleated cells to structural cells devoid of nucleus and cytoplasm is key to hair production. This form of cell 'death', or cornification, requires cellular organelle removal to allow the cytoplasm to become packed with keratin filament bundles that further require cross-linking to create a strong hair fibre. Although these processes are well described in epidermal keratinocytes, there is a lack of understanding of such mechanisms, specifically in the hair follicle. OBJECTIVES: To gain insights into cornification mechanisms within the hair follicle and thus improve our understanding of normal hair physiology. METHODS: Scalp biopsies and hair-pluck samples were obtained from healthy human donors and analysed microscopically after immunohistochemical staining. RESULTS: A focal point of respiratory activity was evident in keratogenous zone cells within the hair shaft, which also exhibited nuclear damage. Nuclear degradation occurred via both caspase-dependent and caspase-independent pathways. Conversely, mitophagy was driven by Bnip3L and restricted to the boundary of the keratogenous zone at Adamson's Fringe. CONCLUSIONS: We propose a model of stepwise living-dead transition within the first 1 mm of hair formation, whereby fully functional, nucleated cells first consolidate required functions by degrading nuclear DNA, yet continue to respire and provide the source of reactive oxygen species required for keratin cross-linking. Finally, as the cells become packed with keratin bundles, Bnip3L expression triggers mitophagy to rid the cells of the last remaining 'living' characteristic, thus completing the march from 'living' to 'dead' within the hair follicle.
Asunto(s)
Cabello/crecimiento & desarrollo , Queratinocitos/citología , Orgánulos/ultraestructura , Adolescente , Adulto , Anciano , Apoptosis/fisiología , Autofagia/fisiología , Muerte Celular/fisiología , Diferenciación Celular , Núcleo Celular/ultraestructura , Reactivos de Enlaces Cruzados/metabolismo , Femenino , Cabello/citología , Cabello/ultraestructura , Folículo Piloso/citología , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/ultraestructura , Voluntarios Sanos , Humanos , Queratinocitos/ultraestructura , Queratinas/metabolismo , Microscopía Confocal , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Mitocondrias/ultraestructura , Oxidación-Reducción , Estrés Oxidativo/fisiología , Cuero Cabelludo/citología , Cuero Cabelludo/crecimiento & desarrollo , Cuero Cabelludo/ultraestructura , Adulto JovenRESUMEN
There is an urgent need to validate in vitro human skin models for use in safety testing. An important component of validation is characterizing the metabolizing capacity of these models. We report comparison of the expression of 139 genes encoding xenobiotic metabolizing enzymes in the EpiDerm model and human skin. In microarray analysis, the expression of 87% of the genes was consistent between the EpiDerm model and human skin indicating the presence of similar metabolic pathways suggesting commonality in function. Analysis of EpiDerm models constructed from four donors showed highly comparable expression of xenobiotic metabolizing genes demonstrating reproducibility of the model. Overall, the expression of Phase II enzymes appeared to be more pronounced in human skin and the EpiDerm model than that of Phase I enzymes, consistent with the role of skin in detoxification of xenobiotics. Though the basal expression of CYPs in particular was low in EpiDerm, significant induction of CYP1A1/1B1 activity was observed following treatment with 3-methylcholanthrene. These results indicate that the xenobiotic metabolizing capacity of the EpiDerm model appears to be representative of human skin. Models such as EpiDerm provide a valuable in vitro approach for evaluation of metabolism and toxicity of cutaneous exposures to xenobiotics.
Asunto(s)
Epidermis/metabolismo , Expresión Génica/efectos de los fármacos , Modelos Biológicos , Piel/metabolismo , Xenobióticos/metabolismo , Adolescente , Biotransformación , Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Inducción Enzimática/efectos de los fármacos , Epidermis/efectos de los fármacos , Epidermis/enzimología , Femenino , Glucuronosiltransferasa/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Técnicas In Vitro , Inactivación Metabólica , Análisis de Secuencia por Matrices de Oligonucleótidos , Piel/efectos de los fármacos , Piel/enzimología , Xenobióticos/toxicidad , Adulto JovenRESUMEN
This study reports the concentration of collagen and its hydroxypyridinoline crosslinks, collagen fibril organization in the dorsal aortas, and systolic blood pressure during the progression of atherosclerosis in Japanese quail selected for cholesterol-induced atherosclerosis. The quail were placed on either a control or 0.5% cholesterol-added diet at approximately 16 weeks of age. The concentration of total collagen did not change in the control arteries during the course of the study, whereas at 5 and 10 weeks of cholesterol feeding, collagen levels decreased in the cholesterol-fed birds. Hydroxypyridinoline concentration increased during the duration of the study in the cholesterol-fed birds and by 15 and 20 weeks of cholesterol feeding, levels were significantly increased over those observed in the control arteries. Transmission electron microscopy showed changes in the organization of collagen fibrils. Increased systolic blood pressure was noted beginning at 10 weeks of cholesterol feeding, which is suggestive of other systemic changes induced by hypercholesterolemia. These results demonstrated remodeling of the collagen component of the dorsal aorta extracellular matrix during the progression of atherosclerosis and are suggestive of other systemic cardiovascular system changes.
Asunto(s)
Aorta/metabolismo , Enfermedades de las Aves/metabolismo , Colágeno/metabolismo , Enfermedad de la Arteria Coronaria/veterinaria , Coturnix , Animales , Aorta/ultraestructura , Presión Sanguínea , Colesterol , Colágeno/análisis , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Microscopía Electrónica , Piridinas/análisisRESUMEN
The temporal and spatial distribution, and relative levels of the proteoglycan decorin and collagen type I were examined during the progression of atherosclerosis in the dorsal aortas of Japanese quail selected for cholesterol induced atherosclerosis. The quail were placed on either a control or 0.5% added cholesterol diet at approximately 16 weeks of age. Dorsal aortas were collected at 1- or 2-week intervals over a 15-week period after initiating cholesterol feeding. Biochemical analysis for decorin and collagen type I showed that both increased in the cholesterol-fed birds compared to control-fed birds beginning at 9 weeks and continued through the duration of the study. Through immunohistochemical staining for decorin and collagen type I, the spatial localization of decorin and collagen type I in control and less severe plaques in cholesterol-fed birds was most prominent in the arterial adventitia. However, in severe atherosclerotic plaques, decorin was localized in foam cell regions and collagen type I was found surrounding the foam cell regions where decorin accumulated. These results demonstrated a localization of decorin in the core of the atherosclerotic plaque foam cell region with collagen type I being located on the plaque surface.