RESUMO
Cellular senescence is a cell fate triggered in response to stress and is characterized by stable cell-cycle arrest and a hypersecretory state. It has diverse biological roles, ranging from tissue repair to chronic disease. The development of new tools to study senescence in vivo has paved the way for uncovering its physiological and pathological roles and testing senescent cells as a therapeutic target. However, the lack of specific and broadly applicable markers makes it difficult to identify and characterize senescent cells in tissues and living organisms. To address this, we provide practical guidelines called "minimum information for cellular senescence experimentation in vivo" (MICSE). It presents an overview of senescence markers in rodent tissues, transgenic models, non-mammalian systems, human tissues, and tumors and their use in the identification and specification of senescent cells. These guidelines provide a uniform, state-of-the-art, and accessible toolset to improve our understanding of cellular senescence in vivo.
Assuntos
Senescência Celular , Humanos , Animais , Biomarcadores/metabolismo , Guias como Assunto , Neoplasias/patologiaRESUMO
Complement factor H (CFH) negatively regulates consumption of complement component 3 (C3), thereby restricting complement activation. Genetic variants in CFH predispose to chronic inflammatory disease. Here, we examined the impact of CFH on atherosclerosis development. In a mouse model of atherosclerosis, CFH deficiency limited plaque necrosis in a C3-dependent manner. Deletion of CFH in monocyte-derived inflammatory macrophages propagated uncontrolled cell-autonomous C3 consumption without downstream C5 activation and heightened efferocytotic capacity. Among leukocytes, Cfh expression was restricted to monocytes and macrophages, increased during inflammation, and coincided with the accumulation of intracellular C3. Macrophage-derived CFH was sufficient to dampen resolution of inflammation, and hematopoietic deletion of CFH in atherosclerosis-prone mice promoted lesional efferocytosis and reduced plaque size. Furthermore, we identified monocyte-derived inflammatory macrophages expressing C3 and CFH in human atherosclerotic plaques. Our findings reveal a regulatory axis wherein CFH controls intracellular C3 levels of macrophages in a cell-autonomous manner, evidencing the importance of on-site complement regulation in the pathogenesis of inflammatory diseases.
Assuntos
Aterosclerose , Complemento C3 , Animais , Humanos , Camundongos , Aterosclerose/metabolismo , Complemento C3/genética , Complemento C3/metabolismo , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Inflamação , Macrófagos/metabolismoRESUMO
Human skin equivalents (HSEs) are three-dimensional skin organ culture models raised in vitro. This review gives an overview of common techniques for setting up HSEs. The HSE consists of an artificial dermis and epidermis. 3T3-J2 murine fibroblasts, purchased human fibroblasts or freshly isolated and cultured fibroblasts, together with other components, for example, collagen type I, are used to build the scaffold. Freshly isolated and cultured keratinocytes are seeded on top. It is possible to add other cell types, for example, melanocytes, to the HSE-depending on the research question. After several days and further steps, the 3D skin can be harvested. Additionally, we show possible markers and techniques for evaluation of artificial skin. Furthermore, we provide a comparison of HSEs to human skin organ culture, a model which employs human donor skin. We outline advantages and limitations of both models and discuss future perspectives in using HSEs.
Assuntos
Pele Artificial , Pele , Humanos , Camundongos , Animais , Pele/metabolismo , Epiderme/metabolismo , Queratinócitos/metabolismo , Células Epidérmicas/metabolismo , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Células CultivadasRESUMO
Loss of cognitive function is a typical consequence of aging in humans and rodents. The extent of decline in spatial memory performance of rats, assessed by a hole-board test, reaches from unimpaired and comparable to young individuals to severely memory impaired. Recently, proteomics identified peroxiredoxin 6, an enzyme important for detoxification of oxidized phospholipids, as one of several synaptosomal proteins discriminating between aged impaired and aged unimpaired rats. In this study, we investigated several components of the epilipidome (modifications of phospholipids) of the prefrontal cortex of young, aged memory impaired (AI) and aged unimpaired (AU) rats. We observed an age-related increase in phospholipid hydroperoxides and products of phospholipid peroxidation, including reactive aldehydophospholipids. This increase went in hand with cortical lipofuscin autofluorescence. The memory impairment, however, was paralleled by additional specific changes in the aged rat brain epilipidome. There was a profound increase in phosphocholine hydroxides, and a significant decrease in phosphocholine-esterified azelaic acid. As phospholipid-esterified fatty acid hydroxides, and especially those deriving from arachidonic acid are both markers and effectors of inflammation, the findings suggest that in addition to age-related reactive oxygen species (ROS) accumulation, age-related impairment of spatial memory performance has an additional and distinct (neuro-) inflammatory component.
Assuntos
Fosfolipídeos , Fosforilcolina , Idoso , Envelhecimento/metabolismo , Animais , Encéfalo/metabolismo , Hipocampo/metabolismo , Humanos , Transtornos da Memória/metabolismo , Fosfolipídeos/metabolismo , Fosforilcolina/metabolismo , RatosRESUMO
Autophagy is a controlled mechanism of intracellular self-digestion with functions in metabolic adaptation to stress, in development, in proteostasis and in maintaining cellular homeostasis in ageing. Deletion of autophagy in epidermal keratinocytes does not prevent the formation of a functional epidermis and the permeability barrier but causes increased susceptibility to damage stress and metabolic alterations and accelerated ageing phenotypes. We here investigated how epidermal autophagy deficiency using Keratin 14 driven Atg7 deletion would affect the lipid composition of the epidermis of young and old mice. Using mass spectrometric lipidomics we found a reduction of age-related accumulation of storage lipids in the epidermis of autophagy-deficient mice, and specific changes in chain length and saturation of fatty acids in several lipid classes. Transcriptomics and immunostaining suggest that these changes are accompanied by changes in expression and localisation of lipid and fatty acid transporter proteins, most notably fatty acid binding protein 5 (FABP5) in autophagy knockouts. Thus, maintaining autophagic activity at an advanced age may be necessary to maintain epidermal lipid homeostasis in mammals.
Assuntos
Epiderme , Lipidômica , Animais , Autofagia/genética , Epiderme/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Queratina-14 , Lipídeos , Mamíferos/metabolismo , CamundongosRESUMO
To correctly assess the cleanliness of technical surfaces in a production process, corresponding online monitoring systems must provide sufficient data. A promising method for fast, large-area, and non-contact monitoring is hyperspectral imaging (HSI), which was used in this paper for the detection and quantification of organic surface contaminations. Depending on the cleaning parameter constellation, different levels of organic residues remained on the surface. Afterwards, the cleanliness was determined by the carbon content in the atom percent on the sample surfaces, characterized by XPS and AES. The HSI data and the XPS measurements were correlated, using machine learning methods, to generate a predictive model for the carbon content of the surface. The regression algorithms elastic net, random forest regression, and support vector machine regression were used. Overall, the developed method was able to quantify organic contaminations on technical surfaces. The best regression model found was a random forest model, which achieved an R2 of 0.7 and an RMSE of 7.65 At.-% C. Due to the easy-to-use measurement and the fast evaluation by machine learning, the method seems suitable for an online monitoring system. However, the results also show that further experiments are necessary to improve the quality of the prediction models.
Assuntos
Cobre , Imageamento Hiperespectral , Eletrônica , Aprendizado de Máquina , Máquina de Vetores de SuporteRESUMO
Sebaceous glands are adnexal structures, which critically contribute to skin homeostasis and the establishment of a functional epidermal barrier. Sebocytes, the main cell population found within the sebaceous glands, are highly specialized lipid-producing cells. Sebaceous gland-resembling tissue structures are also found in male rodents in the form of preputial glands. Similar to sebaceous glands, they are composed of lipid-specialized sebocytes. Due to a lack of adequate organ culture models for skin sebaceous glands and the fact that preputial glands are much larger and easier to handle, previous studies used preputial glands as a model for skin sebaceous glands. Here, we compared both types of sebocytes, using a single-cell RNA sequencing approach, to unravel potential similarities and differences between the two sebocyte populations. In spite of common gene expression patterns due to general lipid-producing properties, we found significant differences in the expression levels of genes encoding enzymes involved in the biogenesis of specialized lipid classes. Specifically, genes critically involved in the mevalonate pathway, including squalene synthase, as well as the sphingolipid salvage pathway, such as ceramide synthase, (acid) sphingomyelinase or acid and alkaline ceramidases, were significantly less expressed by preputial gland sebocytes. Together, our data revealed tissue-specific sebocyte populations, indicating major developmental, functional as well as biosynthetic differences between both glands. The use of preputial glands as a surrogate model to study skin sebaceous glands is therefore limited, and major differences between both glands need to be carefully considered before planning an experiment.
Assuntos
Metabolismo dos Lipídeos/genética , Lipídeos/genética , Glândulas Sebáceas/metabolismo , Pele/metabolismo , Transcrição Gênica/genética , Animais , Diferenciação Celular/genética , Epiderme/metabolismo , Células Epiteliais/metabolismo , Glândulas Exócrinas/metabolismo , Prepúcio do Pênis/metabolismo , Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/genéticaRESUMO
Epidermal keratinocytes undergo cornification to form the cellular building blocks of hard skin appendages such as nails and the protective layer on the surface of the skin. Cornification requires the cross-linking of structural proteins and the removal of other cellular components to form mechanically rigid and inert corneocytes. Autophagy has been proposed to contribute to this intracellular remodelling process, but its molecular targets in keratinocytes, if any, have remained elusive. Here, we deleted the essential autophagy factor Atg7 in K14-positive epithelia of mice and determined by proteomics the impact of this deletion on the abundance of individual proteins in cornified nails. The genetic suppression of autophagy in keratinocytes resulted in a significant increase in the number of proteins that survived cornification and in alterations of their abundance in the nail proteome. A broad range of enzymes and other non-structural proteins were elevated whereas the amounts of cytoskeletal proteins of the keratin and keratin-associated protein families, cytolinker proteins and desmosomal proteins were either unaltered or decreased in nails of mice lacking epithelial autophagy. Among the various types of non-cytoskeletal proteins, the subunits of the proteasome and of the TRiC/CCT chaperonin were most strongly elevated in mutant nails, indicating a particularly important role of autophagy in removing these large protein complexes during normal cornification. Taken together, the results of this study suggest that autophagy is active during nail keratinocyte cornification and its substrate specificity depends on the accessibility of proteins outside of the cytoskeleton and their presence in large complexes.
Assuntos
Autofagia/fisiologia , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Casco e Garras/fisiologia , Queratinócitos/fisiologia , Organogênese/fisiologia , Proteólise , Animais , Diferenciação Celular/genética , Epiderme/fisiologia , Espaço Intracelular/metabolismo , Queratinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Pele/metabolismoRESUMO
BACKGROUND: The main functions of the skin are to protect against environmental insults and prevent water loss, which are performed by the complex lipid- and protein matrix present in the outermost layers of the epithelium. The lipidome of these outer layers is mainly composed of ceramides, fatty acids, and cholesterol, which regulates keratinocyte differentiation and skin barrier function. SR-B1 is a multifunctional scavenger receptor that is best known for facilitating uptake of cholesterol from HDL particles in the liver, but it is also expressed in the skin. OBJECTIVE: To determine the role of SR-B1 in keratinocyte differentiation. METHODS: We investigated the relationship between SR-B1 and keratinocyte differentiation using a physiologically relevant model, organotypic skin equivalents (SEs), wherein SR-B1 was knocked down via siRNA transfection. To assess effects of SR-B1 knockdown on keratinocyte differentiation, we performed hematoxylin/eosin staining, RT-PCR, western blotting, and immunohistochemistry. We also examined the effect of SR-B1 knockdown on lipid production by performing Oil Red O staining and thin layer chromatography. RESULTS: SR-B1 knockdown resulted in decreased lipid levels in SEs, specifically ceramides, and in decreased transcript levels of LDLR, PPAR-α and PPAR-γ, which are factors involved in regulating ceramide synthesis. In addition, filaggrin levels increased in SR-B1 KD tissues, but neither keratin 14 nor keratin 10 were affected. CONCLUSION: We conclude that one of the main functions of SR-B1 in the skin is to regulate ceramide levels and thereby maintain the barrier function of the skin, resulting in the protection of cutaneous tissues from outdoor insults.
Assuntos
Diferenciação Celular/fisiologia , Homeostase , Queratinócitos/citologia , Metabolismo dos Lipídeos , Receptores Depuradores Classe B/fisiologia , Pele/metabolismo , Células Cultivadas , Proteínas Filagrinas , Regulação da Expressão Gênica , Humanos , Metabolismo dos Lipídeos/genética , Receptores Depuradores Classe B/genética , Pele/citologiaRESUMO
We have reported recently that inactivation of the essential autophagy-related gene 7 (Atg7) in keratinocytes has little or no impact on morphology and function of the epidermal barrier in experimental animals. When these mice aged, mutant males, (Atg7 ΔKC), developed an oily coat. As the keratin 14 promoter driven cre/LoxP system inactivates floxed Atg7 in all keratin 14 (K14) expressing cells, including sebocytes, we investigated whether the oily hair phenotype was the consequence of changes in function of the skin sebaceous glands. Using an antibody to the GFP-LC3 fusion protein, autophagosomes were detected at the border of sebocyte disintegration in control but not in mutant animals, suggesting that autophagy was (a) active in normal sebaceous glands and (b) was inactivated in the mutant mice. Detailed analysis established that dorsal sebaceous glands were about twice as large in all Atg7 ΔKC mice compared to those of controls (Atg7 F/F), and their rate of sebocyte proliferation was increased. In addition, male mutant mice yielded twice as much lipid per unit hair as age-matched controls. Analysis of sebum lipids by thin layer chromatography revealed a 40% reduction in the proportion of free fatty acids (FFA) and cholesterol, and a 5-fold increase in the proportion of fatty acid methyl esters (FAME). In addition, the most common diester wax species (58-60 carbon atoms) were increased, while shorter species (54-55 carbon atoms) were under-represented in mutant sebum. Our data show that autophagy contributes to sebaceous gland function and to the control of sebum composition.
Assuntos
Proteína 7 Relacionada à Autofagia/genética , Autofagia/genética , Glândulas Sebáceas/patologia , Glândulas Sebáceas/fisiopatologia , Sebo/química , Animais , Autofagossomos , Proliferação de Células/genética , Colesterol/análise , Ácidos Graxos não Esterificados/análise , Cabelo , Masculino , Camundongos , Fenótipo , Ceras/análiseRESUMO
BACKGROUND: Several activities are attributed to antimicrobial peptides (AMPs), including bacterial killing, leucocyte recruitment and angiogenesis. Despite promises of advanced cellular therapies for treatment of diabetic foot ulcer, it is currently accepted that paracrine factors rather than cellular components are causative for the observed effects. Whether AMPs are present in the mononuclear cell (MNC) secretome (MNC-sec) of white blood cells that are beneficial in experimental wound healing is not known. MATERIALS AND METHODS: Antimicrobial activity of the secretomes of nonirradiated (MNC-sec) and γ-irradiated MNCs (MNC-sec rad) was analysed by microdilution assay. AMPs were determined by quantitative real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Whether human MNC-sec rad causes AMP secretion in vivo was examined in an experimental rat model. Image flow cytometry was used to determine the type of cell death induced in MNCs after exposure to γ-radiation. RESULTS: The antimicrobial activity assay revealed a bactericidal activity of MNC-sec rad and to a lesser degree also of MNC-sec. Image flow cytometry showed that γ-irradiation of MNCs induced early apoptosis followed mainly by necroptosis. RT-PCR and ELISA revealed a high abundance of different AMPs in the secretome of MNCs. In addition, human MNC-sec elicited an increase in de novo endogenous AMP production in rats in vivo. CONCLUSION: We provide evidence that the secretome of MNCs has direct and indirect positive effects on the immune defence system, including augmentation of antibacterial properties. Our data further suggest that necroptosis could play a key role for the release of paracrine factors and the therapeutic action of MNC-sec rad.
Assuntos
Anti-Infecciosos/farmacocinética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Leucócitos Mononucleares/metabolismo , Adolescente , Adulto , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Apoptose/efeitos da radiação , Morte Celular , Raios gama , Humanos , Leucócitos Mononucleares/fisiologia , Masculino , Ratos Sprague-Dawley , Adulto JovemRESUMO
Senescent cells accumulate during ageing in various tissues and contribute to organismal ageing. However, factors that are involved in the induction of senescence in vivo are still not well understood. SNEV(P) (rp19/) (PSO) (4) is a multifaceted protein, known to be involved in DNA damage repair and senescence, albeit only in vitro. In this study, we used heterozygous SNEV(+/-) mice (SNEV-knockout results in early embryonic lethality) and wild-type littermate controls as a model to elucidate the role of SNEV(P) (rp19/) (PSO) (4) in DNA damage repair and senescence in vivo. We performed PUVA treatment as model system for potently inducing cellular senescence, consisting of 8-methoxypsoralen in combination with UVA on mouse skin to induce DNA damage and premature skin ageing. We show that SNEV(P) (rp19/) (PSO) (4) expression decreases during organismal ageing, while p16, a marker of ageing in vivo, increases. In response to PUVA treatment, we observed in the skin of both SNEV(P) (rp19/) (PSO) (4) and wild-type mice an increase in γ-H2AX levels, a DNA damage marker. In old SNEV(P) (rp19/) (PSO) (4) mice, this increase is accompanied by reduced epidermis thickening and increase in p16 and collagenase levels. Thus, the DNA damage response occurring in the mouse skin upon PUVA treatment is dependent on SNEV(P) (rp19/) (PSO) (4) expression and lower levels of SNEV(P) (rp19/) (PSO) (4) , as in old SNEV(+/-) mice, result in increase in cellular senescence and acceleration of premature skin ageing.
Assuntos
Colagenases/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Terapia PUVA/métodos , Fatores de Processamento de RNA/genética , Envelhecimento da Pele/fisiologia , Pele/metabolismo , Senilidade Prematura , Animais , Senescência Celular , Colágeno/metabolismo , Dano ao DNA , Epiderme/metabolismo , Feminino , Genótipo , Heterozigoto , Histonas/metabolismo , Masculino , Metoxaleno/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Processamento de RNA/metabolismoRESUMO
Reverse transcription polymerase chain reaction (qRT-PCR) has become a mainstay in many areas of skin research. To enable quantitative analysis, it is necessary to analyse expression of reference genes (RGs) for normalization of target gene expression. The selection of reliable RGs therefore has an important impact on the experimental outcome. In this study, we aimed to identify and validate the best suited RGs for qRT-PCR in human primary keratinocytes (KCs) over a broad range of experimental conditions using the novel bioinformatics tool 'RefGenes', which is based on a manually curated database of published microarray data. Expression of 6 RGs identified by RefGenes software and 12 commonly used RGs were validated by qRT-PCR. We assessed whether these 18 markers fulfilled the requirements for a valid RG by the comprehensive ranking of four bioinformatics tools and the coefficient of variation (CV). In an overall ranking, we found GUSB to be the most stably expressed RG, whereas the expression values of the commonly used RGs, GAPDH and B2M were significantly affected by varying experimental conditions. Our results identify RefGenes as a powerful tool for the identification of valid RGs and suggest GUSB as the most reliable RG for KCs.
Assuntos
Expressão Gênica , Glucuronidase/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Queratinócitos , Microglobulina beta-2/genética , Actinas/genética , Biomarcadores , Biomarcadores Tumorais/genética , Diferenciação Celular , Biologia Computacional/métodos , Bases de Dados Genéticas , Complexo II de Transporte de Elétrons/genética , Humanos , Integrina alfa6/genética , Queratina-5/genética , Lectinas/genética , Proteínas de Neoplasias/genética , Fosfoglicerato Quinase/genética , Psoríase/genética , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/genética , Proteína A7 Ligante de Cálcio S100 , Proteínas S100/genética , Software , Receptores Toll-Like/genética , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Sebaceous glands drive acne, however, their role in other inflammatory skin diseases remains unclear. To shed light on their potential contribution to disease development, we investigated the spatial transcriptome of sebaceous glands in psoriasis and atopic dermatitis patients across lesional and non-lesional human skin samples. Both atopic dermatitis and psoriasis sebaceous glands expressed genes encoding key proteins for lipid metabolism and transport such as ALOX15B, APOC1, FABP7, FADS1/2, FASN, PPARG, and RARRES1. Also, inflammation-related SAA1 was identified as a common spatially variable gene. In atopic dermatitis, genes mainly related to lipid metabolism (e.g. ACAD8, FADS6, or EBP) as well as disease-specific genes, i.e., Th2 inflammation-related lipid-regulating HSD3B1 were differentially expressed. On the contrary, in psoriasis, more inflammation-related spatially variable genes (e.g. SERPINF1, FKBP5, IFIT1/3, DDX58) were identified. Other psoriasis-specific enriched pathways included lipid metabolism (e.g. ACOT4, S1PR3), keratinization (e.g. LCE5A, KRT5/7/16), neutrophil degranulation, and antimicrobial peptides (e.g. LTF, DEFB4A, S100A7-9). In conclusion, our results show that sebaceous glands contribute to skin homeostasis with a cell type-specific lipid metabolism, which is influenced by the inflammatory microenvironment. These findings further support that sebaceous glands are not bystanders in inflammatory skin diseases, but can actively and differentially modulate inflammation in a disease-specific manner.
Assuntos
Dermatite Atópica , Psoríase , Humanos , Dermatite Atópica/genética , Glândulas Sebáceas , Metabolismo dos Lipídeos/genética , Inflamação/genética , Psoríase/genética , Perfilação da Expressão Gênica , Transcriptoma , Proteínas de MembranaRESUMO
Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.
Assuntos
Autofagia/imunologia , Queratina-5/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Timo/imunologia , Animais , Autoimunidade , Autofagia/genética , Proteína 5 Relacionada à Autofagia , Peso Corporal/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Epiteliais/citologia , Células Epiteliais/imunologia , Deleção de Genes , Marcação de Genes , Queratina-15 , Queratina-5/genética , Camundongos , Camundongos Transgênicos , Timo/citologia , Aumento de Peso/genéticaRESUMO
Oxidized phospholipids (OxPLs) are pleiotropic lipid mediators known to induce proangiogenic and proinflammatory cellular effects that are increasingly recognized to be involved in a number of physiologic and pathologic processes in the retina. Immunohistochemical studies have detected OxPLs in retinal structures, such as retinal pigment epithelium (RPE) or photoreceptor cells. This study analyzed whether OxPLs could play a role in upregulation of VEGF, which is a cause of pathological neovascularization characteristic of eye diseases such as age-related macular degeneration. We confirmed accumulation of OxPLs in the eye using reversed-phase liquid chromatography coupled to mass spectrometry. Multiple species of oxidized phosphatidylcholines (OxPCs) were detected in human vitreous, including biologically active fragmented species POVPC, PGPC, PONPC and PAzPC. In in vitro experiments human fetal RPE and primary RPE cells were stimulated with OxPLs. Primary RPE cells were transfected with small interfering RNAs targeting ATF4. mRNA levels of VEGF in fetal and primary RPE cells were determined by real-time quantitative PCR. VEGF protein concentrations were measured in culture medium by ELISA. We found that OxPCs and other classes of OxPLs upregulated the expression of VEGF in fetal and primary RPE cells, which critically depended on ATF4. In addition, upregulation of VEGF in primary RPE cells was blocked by a chemical inhibitor of protein kinase CK2 known to suppress induction of ATF4 and VEGF by OxPLs. Our data show that different species of OxPLs, which are present in the human eye are capable of stimulating expression of VEGF in fetal and primary RPE cells via ATF4-dependent mechanisms.
Assuntos
Fator 4 Ativador da Transcrição/genética , Caseína Quinase II/genética , Fosfolipídeos/metabolismo , RNA Mensageiro/genética , Epitélio Pigmentado da Retina/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Fator 4 Ativador da Transcrição/biossíntese , Western Blotting , Caseína Quinase II/biossíntese , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Espectrometria de Massas , Oxirredução , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/patologia , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
PURPOSE: Stage II posterior tibial tendon dysfunction (PTTD) can be treated by flexor digitorum longus (FDL) tendon transfer and medial displacement calcaneal osteotomy (MDCO). Numerous authors have studied the clinical and radiographic results of this procedure. However, little is known about the kinematic changes. Therefore, the purpose of this study was to assess plantar-pressure distribution in these patients. METHODS: Seventy-three patients with PTTD stage II underwent FDL tendon transfer and MDCO. Plantar pressure distribution and American Orthopaedic Foot and Ankle Society (AOFAS) score were assessed 48 months after surgery. Pedobarographic parameters included lateral and medial force index of the gait line, peak pressure (PP), maximum force (MF), contact area (CA), contact time (CT) and force-time integral (FTI). RESULTS: In the lesser-toe region, PP, MF, CT, FTI and CA were reduced and MF in the forefoot region was increased. These changes were statistically significant. We found statistically significant correlations between AOFAS score and loading parameters of the medial midfoot. CONCLUSIONS: Study results reveal that FDL tendon transfer and MDCO leads to impaired function of the lesser toes during the stance phase. However, there seems to be a compensating increased load in the forefoot region.
Assuntos
Disfunção do Tendão Tibial Posterior/fisiopatologia , Disfunção do Tendão Tibial Posterior/cirurgia , Adulto , Idoso , Fenômenos Biomecânicos , Calcâneo/cirurgia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteotomia , Pressão , Transferência TendinosaRESUMO
Atopic dermatitis (AD) is a complex disease characterized by chronic recurring eczema and pruritus. In addition, patients with AD display increased cutaneous and systemic levels of oxidative damage markers, whose source remains elusive. In this study, we investigated oxidative and mitochondrial stress in AD epidermis. The levels of superoxide dismutase 2 and hydrogen peroxide are augmented in the mitochondria of flaky tail (ft/ft) mouse keratinocytes, which is associated with the inhibition of the glutathione system and catalase. Furthermore, reduced levels of glutathione peroxidase 4 are associated with accumulation of malondialdehyde, 4-hydroxy-2-nonenal, and oxidized phosphatidylcholines in ft/ft epidermis. Cytochrome c is markedly increased in ft/ft epidermis, hence showing mitochondrial stress. Topical application of MitoQ, which is a mitochondrial-targeting antioxidant, to ft/ft mouse skin reduced damage to macromolecules and inflammation and restored epidermal homeostasis. Absence of alteration in the expression of superoxide dismutase 2, catalase, and glutathione peroxidase 4 and limited lipid peroxidation as well as oxidized phosphatidylcholines in the epidermis of Flg-/- mice suggest that FLG deficiency marginally contributes to oxidative stress in ft/ft epidermis. Increased superoxide dismutase 2, lipid peroxidation, and cytochrome c in the epidermis of patients with AD, associated with reduced antioxidant response in primary AD keratinocytes, corroborate mitochondrial dysfunction and lack of cellular adjustment to oxidative stress in AD epidermis.
Assuntos
Dermatite Atópica , Eczema , Humanos , Camundongos , Animais , Dermatite Atópica/metabolismo , Catalase/genética , Catalase/metabolismo , Peróxido de Hidrogênio/metabolismo , Antioxidantes , Citocromos c/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Estresse Oxidativo , Mitocôndrias/metabolismoRESUMO
Linoleic acid (LA) is an essential omega-6 polyunsaturated fatty acid (PUFA) derived from the diet. Sebocytes, whose primary role is to moisturise the skin, process free fatty acids (FFAs) to produce the lipid-rich sebum. Importantly, like other sebum components such as palmitic acid (PA), LA and its derivative arachidonic acid (AA) are known to modulate sebocyte functions. Given the different roles of PA, LA and AA in skin biology, the aim of this study was to assess the specificity of sebocytes for LA and to dissect the different roles of LA and AA in regulating sebocyte functions. Using RNA sequencing, we confirmed that gene expression changes in LA-treated sebocytes were largely distinct from those induced by PA. LA, but not AA, regulated the expression of genes related to cholesterol biosynthesis, androgen and nuclear receptor signalling, keratinisation, lipid homeostasis and differentiation. In contrast, a set of mostly down-regulated genes involved in lipid metabolism and immune functions overlapped in LA- and AA-treated sebocytes. Lipidomic analyses revealed that the changes in the lipid profile of LA-treated sebocytes were more pronounced than those of AA-treated sebocytes, suggesting that LA may serve not only as a precursor of AA but also as a potent regulator of sebaceous lipogenesis, which may not only influence the gene expression profile but also have further specific biological relevance. In conclusion, we have shown that sebocytes are able to respond selectively to different lipid stimuli and that LA-induced effects can be both AA-dependent and independent. Our findings allow for the consideration of LA application in the therapy of sebaceous gland-associated inflammatory skin diseases such as acne, where lipid modulation and selective targeting of AA metabolism are potential treatment options.