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Fabry disease (FD) is an X-linked lysosomal disease caused by an enzyme deficiency of alpha-galactosidase A (α-gal A). This deficiency leads to the accumulation of glycosphingolipids in lysosomes, resulting in a range of clinical symptoms. The complex pathogenesis of FD involves lysosomal dysfunction, altered autophagy, and mitochondrial abnormalities. Omics sciences, particularly transcriptomic analysis, comprehensively understand molecular mechanisms underlying diseases. This study focuses on genome-wide expression analysis in an FD human podocyte model to gain insights into the underlying mechanisms of podocyte dysfunction. Human control and GLA-edited podocytes were used. Gene expression data was generated using RNA-seq analysis, and differentially expressed genes were identified using DESeq2. Principal component analysis and Spearman correlation have explored gene expression trends. Functional enrichment and Reporter metabolite analyses were conducted to identify significantly affected metabolites and metabolic pathways. Differential expression analysis revealed 247 genes with altered expression levels in GLA-edited podocytes compared to control podocytes. Among these genes, 136 were underexpressed, and 111 were overexpressed in GLA-edited cells. Functional analysis of differentially expressed genes showed their involvement in various pathways related to oxidative stress, inflammation, fatty acid metabolism, collagen and extracellular matrix homeostasis, kidney injury, apoptosis, autophagy, and cellular stress response. The study provides insights into molecular mechanisms underlying Fabry podocyte dysfunction. Integrating transcriptomics data with genome-scale metabolic modeling further unveiled metabolic alterations in GLA-edited podocytes. This comprehensive approach contributes to a better understanding of Fabry disease and may lead to identifying new biomarkers and therapeutic targets for this rare lysosomal disorder.
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In industrialized countries, cerebral palsy affects 2.5 of preterm and term infants. At a neurochemical level, the massive release of glutamate constitutes a major process leading to excitotoxicity and neonatal brain lesions. Previous studies, conducted in the laboratory, revealed that, in (δ/δ)VEGF(A) transgenic mice, glutamate-induced brain lesions are exacerbated suggesting that VEGF(A) could play a protective action against excitotoxicity. Using a model of cultured cortical brain slices, the aim of the study was to characterize the central effects of VEGF against glutamate-induced excitotoxicity in neonates. Exposure of brain slices to glutamate induced a strong increase of necrotic cell death in the deep cortical layer VI and a decrease of apoptotic death in superficial layers II-IV. When administered alone, a 6-h treatment with VEGF(A) had no effect on both apoptotic and necrotic deaths. In contrast, VEGF(A) abolished the glutamate-induced necrosis observed in layer VI. While MEK and PI3-K inhibitors had no effect on the protective action of VEGF(A), L-NAME, a pan inhibitor of NOS, abrogated the effect of VEGF(A) and exacerbated the excitotoxic action of glutamate. Calcimetry experiments performed on brain slices revealed that VEGF(A) reduced the massive calcium influx induced by glutamate in layer VI and this effect was blocked by L-NAME. Neuroprotective effect of VEGF(A) was also blocked by LNIO and NPLA, two inhibitors of constitutive NOS, while AGH, an iNOS inhibitor, had no effect. Nitrite measurements, electron paramagnetic resonance spectroscopy and immunohistochemistry indicated that glutamate was a potent inducer of NO production via activation of nNOS in the cortical layer VI. In vivo administration of nNOS siRNA promoted excitotoxicity and mimicked the effects of L-NAME, LNIO and NPLA. A short-term glutamate treatment increased nNOS Ser1412 phosphorylation, while a long-term exposure inhibited nNOS/NR2B protein-protein interactions. Altogether, these findings indicate that, in deep cortical layers of mice neonates, glutamate stimulates nNOS activity. Contrasting with mature brain, NO production induced by high concentrations of glutamate is neuroprotective and is required for the anti-necrotic effect of VEGF(A).
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Apoptose/efeitos dos fármacos , Córtex Cerebral/crescimento & desenvolvimento , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Caspase 3/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Citrulina/metabolismo , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glutamato Descarboxilase/genética , Ácido Glutâmico/toxicidade , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , L-Lactato Desidrogenase/metabolismo , Camundongos , Camundongos Transgênicos , NADPH Desidrogenase/metabolismo , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , S-Nitroso-N-Acetilpenicilamina/farmacologia , Fatores de TempoRESUMO
Prenatal alcohol exposure is a major cause of neurobehavioral disabilities. MRI studies in humans have shown that alcohol is associated with white matter microstructural anomalies but these studies focused on myelin abnormalities only after birth. Only one of these studies evaluated oligodendrocyte lineage, but only for a short period during human foetal life. As data are lacking in humans and alcohol is known to impair oligodendrocyte differentiation in rodents, the present study aimed to compare by immunohistochemistry the oligodendrocyte precursor cells expressing PDGFR-α and immature premyelinating/mature oligodendrocytes expressing Olig2 in the ganglionic eminences and the frontal cortex of 14 human foetuses exposed to alcohol from 15 to 37 weeks' gestation with age-matched controls. The human brains used in this study were obtained at the time of foetal autopsies for medical termination of pregnancy, in utero or post-natal early death. Before birth, PDGFR-α expression was strongly increased in the ganglionic eminences and the cortex of all foetuses exposed to alcohol except at the earliest stage. No massive generation of Olig2 immunoreactive cells was identified in the ganglionic eminences until the end of pregnancy and the density of Olig2-positive cells within the cortex was consistently lower in foetuses exposed to alcohol than in controls. These antenatal data from humans provides further evidence of major oligodendrocyte lineage impairment at specific and key stages of brain development upon prenatal alcohol exposure including defective or delayed generation and maturation of oligodendrocyte precursors.
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Efeitos Tardios da Exposição Pré-Natal , Diferenciação Celular , Linhagem da Célula , Etanol/toxicidade , Feminino , Feto/metabolismo , Humanos , Bainha de Mielina/metabolismo , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Oligodendroglia/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismoRESUMO
Background: Vascular calcification (VC) is a cardiovascular complication associated with a high mortality rate among patients with diseases such as atherosclerosis and chronic kidney disease. During VC, vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and secrete a heterogeneous population of extracellular vesicles (EVs). Recent studies have shown involvement of EVs in the inflammation and oxidative stress observed in VC. We aimed to decipher the role and mechanism of action of macrophage-derived EVs in the propagation of inflammation and oxidative stress on VSMCs during VC. Methods: The macrophage murine cell line RAW 264.7 treated with lipopolysaccharide (LPS-EK) was used as a cellular model for inflammatory and oxidative stress. EVs secreted by these macrophages were collected by ultracentrifugation and characterized by transmission electron microscopy, cryo-electron microscopy, nanoparticle tracking analysis, and the analysis of acetylcholinesterase activity, as well as that of CD9 and CD81 protein expression by western blotting. These EVs were added to a murine VSMC cell line (MOVAS-1) under calcifying conditions (4 mM Pi-7 or 14 days) and calcification assessed by the o-cresolphthalein calcium assay. EV protein content was analyzed in a proteomic study and EV cytokine content assessed using an MSD multiplex immunoassay. Results: LPS-EK significantly decreased macrophage EV biogenesis. A 24-h treatment of VSMCs with these EVs induced both inflammatory and oxidative responses. LPS-EK-treated macrophage-derived EVs were enriched for pro-inflammatory cytokines and CAD, PAI-1, and Saa3 proteins, three molecules involved in inflammation, oxidative stress, and VC. Under calcifying conditions, these EVs significantly increase the calcification of VSMCs by increasing osteogenic markers and decreasing contractile marker expression. Conclusion: Our results show that EVs derived from LPS-EK-treated-macrophages are able to induce pro-inflammatory and pro-oxidative responses in surrounding cells, such as VSMCs, thus aggravating the VC process.
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There are numerous approaches and methodologies for assessing the identity and quantities of non-intentionally added substances (NIAS) in food contact materials (FCMs). They can give different results and it can be difficult to make meaningful comparisons. The initial approach was to attempt to prepare a prescriptive methodology but as this proved impossible; this paper develops guidelines that need to be taken into consideration when assessing NIAS. Different approaches to analysing NIAS in FCMs are reviewed and compared. The approaches for preparing the sample for analysis, recommended procedures for screening, identification, and quantification of NIAS as well as the reporting requirements are outlined. Different analytical equipment and procedures are compared. Limitations of today's capabilities are raised along with some research needs.
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Contaminação de Alimentos , Embalagem de Alimentos , Alérgenos/análise , Alimentos , Contaminação de Alimentos/análiseRESUMO
(1) Background: Glioblastoma is the most common malignant brain tumor in adults. Its etiology remains unknown in most cases. Glioblastoma pathogenesis consists of a progressive infiltration of the white matter by tumoral cells leading to progressive neurological deficit, epilepsy, and/or intracranial hypertension. The mean survival is between 15 to 17 months. Given this aggressive prognosis, there is an urgent need for a better understanding of the underlying mechanisms of glioblastoma to unveil new diagnostic strategies and therapeutic targets through a deeper understanding of its biology. (2) Methods: To systematically address this issue, we performed targeted and untargeted metabolomics-based investigations on both tissue and plasma samples from patients with glioblastoma. (3) Results: This study revealed 176 differentially expressed lipids and metabolites, 148 in plasma and 28 in tissue samples. Main biochemical classes include phospholipids, acylcarnitines, sphingomyelins, and triacylglycerols. Functional analyses revealed deep metabolic remodeling in glioblastoma lipids and energy substrates, which unveils the major role of lipids in tumor progression by modulating its own environment. (4) Conclusions: Overall, our study demonstrates in situ and systemic metabolic rewiring in glioblastoma that could shed light on its underlying biological plasticity and progression to inform diagnosis and/or therapeutic strategies.
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BACKGROUND: Hemojuvelin, a critical regulator of iron homeostasis, is involved in the regulation of hepcidin expression and iron homeostasis. It is expressed both as a membrane-bound form and as a soluble one. Serum hemojuvelin can be produced by secretion following furin cleavage or by proteolytic cleavage of the membrane-bound form by matriptase 2 (TMPRSS6). These forms contribute to down-regulation of hepcidin expression upon iron deficiency or hypoxia. This study describes the development and validation of the first enzyme-linked immunosorbent assay for hemojuvelin in human serum. DESIGN AND METHODS: This assay is based on the use of a recombinant human repulsive guidance molecule-c peptide and a polyclonal antibody against hemojuvelin able to recognize the recombinant peptide and the native soluble hemojuvelin by immunoprecipitation. RESULTS: The enzyme-linked immunosorbent assay was validated and appeared to be a robust method with intra- and inter-coefficients of variance ranging from 2.6% to 15%. The assay was able to quantify hemojuvelin levels in a control population within a range from 0.88 to 1.14 mg/L. Patients with iron-refractory iron-deficiency anemia with a mutation in the TMPRSS6 gene were found to have lower levels of circulating hemojuvelin than those in healthy patients. The enzyme-linked immunosorbent assay also showed that soluble hemojuvelin levels were significantly higher in patients with anemia of chronic disease than in control individuals. CONCLUSIONS: This enzyme-linked immunosorbent assay has a good specificity and sensitivity for the quantification of soluble hemojuvelin in human serum and could be a valuable aid to understanding the physiological role of this protein.
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Anemia Ferropriva/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Ligadas por GPI/sangue , Adolescente , Adulto , Anemia/sangue , Anemia Ferropriva/tratamento farmacológico , Anemia Ferropriva/genética , Western Blotting , Criança , Pré-Escolar , Feminino , Proteína da Hemocromatose , Humanos , Ferro/metabolismo , Ferro/uso terapêutico , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mutação , Reprodutibilidade dos Testes , Serina Endopeptidases/genéticaRESUMO
Consortia of aerobic heterotrophic bacteria (AHB) have potential as sustainable microbial protein (MP) source in animal feed. A systematic screening of the nutritional value and safety of AHB biomass from full-scale activated sludge plants from 25 companies in the food sector was performed. The variable protein content (21-49%) was positively correlated with biomass-specific nitrogen loading rate and negatively with sludge retention time (SRT). Compared to the essential amino acid profile of soybean meal protein, AHB displayed an overall surplus of threonine and valine, and deficits in cysteine, histidine, lysine and phenylalanine. Histidine was positively correlated with bCOD/PO43- in the influent and valine, isoleucine and threonine with SRT. Most AHB samples were safe apropos heavy metals, polycyclic aromatic hydrocarbons and antibiotics. Some pesticides exceeded regulatory limits, necessitating mitigation. This work highlighted that the food sector can provide high-quality MP, while retrofitting existing activated sludge plants towards high-rate processes can increase AHB quality and productivity.
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Reatores Biológicos , Esgotos , Animais , Bactérias Aeróbias , Bebidas , Processos Heterotróficos , Eliminação de Resíduos LíquidosRESUMO
The contamination of foods with mineral oil hydrocarbons (MOH) is a serious concern, requiring in most cases tedious mitigation measures that span across the whole food supply chain. A major issue today is the significant variability of the results generated by laboratories. This study was therefore designed to achieve a deeper insight into the analytical procedures used by commercial laboratories, identifying possible gaps and suggesting improvements that will enhance the reliability of the MOH data, an important prerequisite for risk assessment. In total six different food matrices, i.e. infant formula (IF), cocoa butter, cocoa powder, biscuits, fruit-based baby food containing biscuit and roast and ground coffee were subjected to comparative inter-laboratory studies, as well as one vegetable oil analysed within the frame of a professionally conducted proficiency test. The results indicate that on some matrices with possibly low amounts of MOH contamination, the current methodologies cannot reliably conclude whether or not a food sample is indeed contaminated with mineral oils (<10 mg/kg food). Urgently needed are: (i) an aligned and fully validated sample preparation strategy tested on a range of different food matrices; (ii) a confirmation of positive flame ionisation detection (FID) results by confirmatory methods such as mass spectrometry - in line with the CEN Standard and the Joint Research Centre (JRC) Guidance Document, (iii) a more detailed root-cause analysis in the reports of laboratories through the use of mineral oil markers, and (iv) a fully validated official method for the concerned foods with a limit of application <10 mg/kg food.
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Análise de Alimentos , Contaminação de Alimentos/análise , Hidrocarbonetos/análise , Óleo Mineral/análise , Chocolate/análise , Café/química , Gorduras na Dieta/análise , Farinha/análise , Análise de Alimentos/normas , Frutas/química , Humanos , Lactente , Fórmulas Infantis/química , Reprodutibilidade dos TestesRESUMO
Alcohol affects multiple neurotransmitter systems, notably the GABAergic system and has been recognised for a long time as particularly damaging during critical stages of brain development. Nevertheless, data from the literature are most often derived from animal or in vitro models. In order to study the production, migration and cortical density disturbances of GABAergic interneurons upon prenatal alcohol exposure, we performed immunohistochemical studies by means of the proliferation marker Ki67, GABA and calretinin antibodies in the frontal cortical plate of 17 foetal and infant brains antenatally exposed to alcohol, aged 15 weeks' gestation to 22 postnatal months and in the ganglionic eminences and the subventricular zone of the dorsal telencephalon until their regression, i.e., 34 weeks' gestation. Results were compared with those obtained in 17 control brains aged 14 weeks of gestation to 35 postnatal months. We also focused on interneuron vascular migration along the cortical microvessels by confocal microscopy with double immunolabellings using Glut1, GABA and calretinin. Semi-quantitative and quantitative analyses of GABAergic and calretininergic interneuron density allowed us to identify an insufficient and delayed production of GABAergic interneurons in the ganglionic eminences during the two first trimesters of the pregnancy and a delayed incorporation into the laminar structures of the frontal cortex. Moreover, a mispositioning of GABAergic and calretininergic interneurons persisted throughout the foetal life, these cells being located in the deep layers instead of the superficial layers II and III. Moreover, vascular migration of calretininergic interneurons within the cortical plate was impaired, as reflected by low numbers of interneurons observed close to the cortical perforating vessel walls that may in part explain their abnormal intracortical distribution. Our results are globally concordant with those previously obtained in mouse models, in which alcohol has been shown to induce an interneuronopathy by affecting interneuron density and positioning within the cortical plate, and which could account for the neurological disabilities observed in children with foetal alcohol disorder spectrum.
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Consumo de Bebidas Alcoólicas , Encéfalo/embriologia , Calbindina 2/metabolismo , Transtornos do Espectro Alcoólico Fetal/metabolismo , Feto/embriologia , Interneurônios/metabolismo , Antígeno Ki-67/metabolismo , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Ácido gama-Aminobutírico/metabolismo , Alcoolismo , Consumo Excessivo de Bebidas Alcoólicas , Encéfalo/metabolismo , Encéfalo/patologia , Estudos de Casos e Controles , Movimento Celular , Feminino , Transtornos do Espectro Alcoólico Fetal/patologia , Feto/metabolismo , Feto/patologia , Lobo Frontal/embriologia , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/patologia , Humanos , Lactente , Recém-Nascido , Interneurônios/patologia , Masculino , Gravidez , Complicações na Gravidez , Segundo Trimestre da Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Telencéfalo/embriologia , Telencéfalo/metabolismo , Telencéfalo/patologiaRESUMO
Background: Fabry disease (FD) is an X-linked progressive lysosomal disease (LD) due to glycosphingolipid metabolism impairment. Currently, plasmatic globotriaosylsphingosine (LysoGb3) is used for disease diagnosis and monitoring. However, this biomarker is inconstantly increased in mild forms and in some female patients. Materials and Methods: We applied a targeted proteomic approach to explore disease-related biological patterns that might explain the disease pathophysiology. Forty proteins, involved mainly in inflammatory and angiogenesis processes, were assessed in 69 plasma samples retrieved from the French Fabry cohort (FFABRY) and from 83 healthy subjects. For predictive performance assessment, we also included other LD samples (Gaucher, Pompe and Niemann Pick C). Results: The study yielded four discriminant proteins that include three angiogenesis proteins (fibroblast growth factor 2 (FGF2), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor C (VEGFC)) and one cytokine interleukin 7 (IL-7). A clear elevation of FGF2 and IL-7 concentrations was observed in FD compared to other LD samples. No correlation was observed between these proteins and globotriaosylsphingosine (LysoGb3). A significant correlation exists between IL-7 and residual enzyme activity in a non-classical phenotype. This highlights the orthogonal biological information yielded by these proteins that might help in stratifying Fabry patients. Conclusion: This work highlights the potential of using proteomics approaches in exploring FD and enhancing FD diagnosis and therapeutic monitoring performances.
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Aqueous solutions of two important quaternary ammonium compounds--16-BAC (benzyl-dimethyl-hexadecylammonium-chloride) and 18-BAC (benzyl-dimethyl-stearylammonium-chloride)--were treated by the ozonation and photo-Fenton processes at different ozone doses and hydrogen peroxide concentrations, respectively. During the photo-Fenton experiments, two different types of lamps were used--a UV mercury vapor medium pressure lamp and a xenon lamp, which simulates solar radiation. The total organic carbon removal was monitored to follow the mineralization of the surfactants. According to the experimental results, after 90 minutes of treatment, the photo-Fenton process achieved up to 80% of mineralization when the UV lamp was used. The efficiency of the photo-Fenton with the xenon lamp was lower. The ozonation process reached, at most, 50% mineralization at the used conditions (ozone dose = 7.57 g/h).
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Peróxido de Hidrogênio , Ferro , Ozônio , Compostos de Amônio Quaternário/química , Purificação da Água/métodos , Compostos de Amônio Quaternário/efeitos da radiação , Tensoativos , Raios Ultravioleta , Eliminação de Resíduos Líquidos , Poluentes Químicos da Água/químicaRESUMO
Microalgal biomass production is a resource-efficient answer to the exponentially increasing demand for protein, yet variability in biomass quality is largely unexplored. Nutritional value and safety were determined for Chlorella and Spirulina biomass from different producers, production batches and the same production batch. Chlorella presented a similar protein content (47⯱â¯8%) compared to Spirulina (48⯱â¯4%). However, protein quality, expressed as essential amino acid index, and digestibility were lower for Chlorella (1.1⯱â¯0.1 and 51⯱â¯9%, respectively) compared to Spirulina (1.3⯱â¯0.1 and 61⯱â¯4%, respectively). Generally, variability was lower between batches and within a batch. Heavy metals, pesticides, mycotoxins, antibiotics and nitrate did not violate regulatory limits, while polycyclic aromatic hydrocarbon levels exceeded the norm for some samples, indicating the need for continuous monitoring. This first systematic screening of commercial microalgal biomass revealed a high nutritional variability, necessitating further optimization of cultivation and post-processing conditions. Based on price and quality, Spirulina was preferred above Chlorella.
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Chlorella/metabolismo , Spirulina/metabolismo , Biomassa , Microalgas/metabolismo , Valor NutritivoRESUMO
Most children with in utero alcohol exposure do not exhibit all features of fetal alcohol syndrome (FAS), and a challenge for clinicians is to make an early diagnosis of fetal alcohol spectrum disorders (FASD) to avoid lost opportunities for care. In brain, correct neurodevelopment requires proper angiogenesis. Since alcohol alters brain angiogenesis and the placenta is a major source of angiogenic factors, we hypothesized that it is involved in alcohol-induced brain vascular defects. In mouse, using in vivo repression and overexpression of PLGF, we investigated the contribution of placenta on fetal brain angiogenesis. In human, we performed a comparative molecular and morphological analysis of brain/placenta angiogenesis in alcohol-exposed fetuses. Results showed that prenatal alcohol exposure impairs placental angiogenesis, reduces PLGF levels and consequently alters fetal brain vasculature. Placental repression of PLGF altered brain VEGF-R1 expression and mimicked alcohol-induced vascular defects in the cortex. Over-expression of placental PGF rescued alcohol effects on fetal brain vessels. In human, alcohol exposure disrupted both placental and brain angiogenesis. PLGF expression was strongly decreased and angiogenesis defects observed in the fetal brain markedly correlated with placental vascular impairments. Placental PGF disruption impairs brain angiogenesis and likely predicts brain disabilities after in utero alcohol exposure. PLGF assay at birth could contribute to the early diagnosis of FASD.
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Encéfalo/efeitos dos fármacos , Transtornos do Espectro Alcoólico Fetal/metabolismo , Fator de Crescimento Placentário/metabolismo , Placenta/efeitos dos fármacos , Animais , Encéfalo/irrigação sanguínea , Encéfalo/embriologia , Encéfalo/patologia , Modelos Animais de Doenças , Etanol/toxicidade , Feminino , Humanos , Camundongos , Neovascularização Patológica/induzido quimicamente , Neovascularização Patológica/embriologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Placenta/irrigação sanguínea , Placenta/metabolismo , Placenta/patologia , Fator de Crescimento Placentário/genética , Gravidez , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Brain developmental lesions are a devastating consequence of prenatal alcohol exposure (PAE). We recently showed that PAE affects cortical vascular development with major effects on angiogenesis and endothelial cell survival. The underlying molecular mechanisms of these effects remain poorly understood. This study aimed at characterizing the ethanol exposure impact on the autophagic process in brain microvessels in human fetuses with fetal alcohol syndrome (FAS) and in a PAE mouse model. Our results indicate that PAE induces an increase of autophagic vacuole number in human fetal and neonatal mouse brain cortical microvessels. Subsequently, ex vivo studies using green fluorescent protein (GFP)-LC3 mouse microvessel preparations revealed that ethanol treatment alters autophagy in endothelial cells. Primary cultures of mouse brain microvascular endothelial cells were used to characterize the underlying molecular mechanisms. LC3 and p62 protein levels were significantly increased in endothelial cells treated with 50 mM ethanol. The increase of autophagic vacuole number may be due to excessive autophagosome formation associated with the partial inhibition of the mammalian target of rapamycin pathway upon ethanol exposure. In addition, the progression from autophagosomes to autolysosomes, which was monitored using autophagic flux inhibitors and mRFP-EGFP vector, showed a decrease in the autolysosome number. Besides, a decrease in the Rab7 protein level was observed that may underlie the impairment of autophagosome-lysosome fusion. In addition, our results showed that ethanol-induced cell death is likely to be mediated by decreased mitochondrial integrity and release of apoptosis-inducing factor. Interestingly, incubation of cultured cells with rapamycin prevented ethanol effects on autophagic flux, ethanol-induced cell death and vascular plasticity. Taken together, these results are consistent with autophagy dysregulation in cortical microvessels upon ethanol exposure, which could contribute to the defects in angiogenesis observed in patients with FAS. Moreover, our results suggest that rapamycin represents a potential therapeutic strategy to reduce PAE-related brain developmental disorders.
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Autofagia/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Etanol/efeitos adversos , Microvasos/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/patologia , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Transtornos do Espectro Alcoólico Fetal/metabolismo , Transtornos do Espectro Alcoólico Fetal/patologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Microvasos/metabolismo , Microvasos/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Modelos Animais , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sirolimo/farmacologiaRESUMO
The pathogenesis of ischemic coronary events involves degradation of the extracellular matrix in atherosclerotic lesions. The cysteine protease inhibitor cystatin-C may be involved in this phenomenon. The association of plasma cystatin-C with the incidence of myocardial infarction-coronary death and angina, was examined in a nested case-control (two controls per case) design within the prospective cohort study (Prospective Epidemiological Study of Myocardial Infarction (PRIME Study)) which included 9,758 men aged 50-59 years who were free of coronary heart disease (CHD) on entry and followed for a 5-year period. Three hundred and thirteen participants suffered myocardial infarction or coronary death (n = 159) or angina pectoris (n = 154) during follow-up. Cystatin-C was positively correlated with body mass index (BMI), low-density lipoprotein (LDL)-cholesterol, triglycerides and several inflammatory markers such as fibrinogen (r = 0.18), C-reactive protein (CRP) (r = 0.24), interleukin-6 (= 0.20), tumor necrosis factor-alpha (TNFalpha) (r = 0.27) and two TNFalpha receptors: TNFR1A (r = 0.43) and TNFR1B (r = 0.41); and negatively with high-density lipoprotein (HDL)-cholesterol (r = -0.25). After adjustment for traditional risk factors (age, diabetes, smoking, hypertension, BMI, triglycerides, LDL- and HDL-cholesterol), cystatin-C was significantly associated with the occurrence of the first ischemic coronary event. However, this association was no longer significant when CRP was included in the analysis. A decrease in glomerular filtration rate did not explain higher cystatin-C in cases than in controls. Cystatin-C appears to participate in the inflammatory phenomenon observed in the atherosclerotic process. Cystatin-C is not a more predictive risk marker of CHD than CRP or interleukin-6, but could be useful in detecting moderate chronic renal disease.
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Doença das Coronárias/sangue , Cistatinas/sangue , Inibidores de Cisteína Proteinase/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Estudos de Coortes , Doença das Coronárias/diagnóstico , Doença das Coronárias/mortalidade , Cistatina C , Inibidores Enzimáticos/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Infarto do Miocárdio/mortalidade , Fatores de RiscoRESUMO
Phosphonates are widely used as chelating agents, e.g., in water cooling systems, in bleaching baths or as scale inhibitors in deflocculation agents. They are considered to be difficult to degrade and produce aminomethylphosphonic acid (AMPA) as a metabolite. As the fate of phosphonates in the environment is not very well known the present work aims at simulating the time dependent photodegradation of four selected phosphonates: nitrilotris-methylenephosphonic acid (NTMP), ethylenediamine-tetra-methylenephosphonic acid (EDTMP), diethylenetriaminepenta-methylenephosphonic acid (DTPMP) and hexaethylenediamine-tetra-methylenephosphonic acid (HDTMP), at concentrations of 1 mg/l (i.e. 3.2 microM NTMP, 2.3 microM EDTMP, 1.7 microM DTPMP and 2.0 microM HDTMP) irradiated by a middle pressure mercury lamp emitting between 190 and 600 nm. The influence of iron under different pH ranges (3, 5-6 and 10) are tested. The degradation of phosphonates is measured by the release of orthophosphates (PO4-P) and aminomethylphosphonic acid (AMPA). This study shows that phosphonates are substances that undergo UV light conversion, which is enhanced in the presence of iron. The half-life without iron is between 15 and 35 min at pH 3, between 10 and 35 min at pH 5-6 and between 50 and 75 min at pH 10. The half-life in the presence of 3.6 microM iron is between 5 and 10 min at pH 3, between 5 and 15 min at pH 5-6 and between 35 and 60 min at pH 10. The individual substances do not significantly influence the reaction rates whereas the presence of iron and the pH have significant effects. The total conversion of phosphonates after 90 min is 75-100% for pH values of 3 and 5-6 and 55-75% for a pH of 10 dependent on the presence of iron. In the environment longer degradation times are to be expected since natural light is weaker by a factor between 125 and 300 in the UVB, a factor between 3 and 8 in the UVA and of the same intensity in the visible range than the light in our study. Although orthophosphates are the major products, AMPA is also shown to be a by-product of the photodegradation of phosphonates that is later converted into orthophosphate.
Assuntos
Quelantes/química , Organofosfonatos/química , Água/química , Catálise , Quelantes/análise , Concentração de Íons de Hidrogênio , Ferro/química , Organofosfonatos/análise , Fotólise , Raios UltravioletaRESUMO
In neonates, excitotoxicity is a major process involved in hypoxic-ischemic brain lesions, and several research groups have suggested the use of NMDA antagonists for neuroprotection. However, despite their clinical interest, there is more and more evidence suggesting that, in the immature brain, these molecules exert deleterious actions on migrating GABAergic interneurons by suppressing glutamatergic trophic inputs. Consequently, preventing the side effects of NMDA antagonists would be therapeutically useful. Because macroautophagy is involved in the adaptive response to trophic deprivation, the aim of the present study was to investigate the impact of autophagy modulators on the MK801-induced death of immature GABAergic interneurons and to characterize the crosstalk between autophagic and apoptotic mechanisms in this cell type. Ex vivo, using cortical slices from NMRI and Gad67-GFP mice, we show that blockade of the NMDA receptor results in an accumulation of autophagosomes due to the disruption of the autophagic flux. This effect precedes the activation of the mitochondrial apoptotic pathway, and the degeneration of immature GABAergic neurons present in developing cortical layers II-IV and is prevented by 3-MA, an autophagy inhibitor. In contrast, modulators of autophagy (3-MA, rapamycin) do not interfere with the anti-excitotoxic and neuroprotective effect of MK801 observed in deep layers V and VI. In vivo, 3-MA blocks the rapid increase in caspase-3 cleavage induced by the blockade of NMDA receptors and prevents the resulting long-term decrease in Gad67-GFP neurons in layers II-IV. Together, these data suggest that, in the developing cortex, the suppression of glutamatergic inputs through NMDA receptor inhibition results in the impairment of the autophagic flux and the subsequent switch to apoptotic death of immature GABAergic interneurons. The concomitant inhibition of autophagy prevents this pro-apoptotic action of the NMDA blocker and favors the long-term rescue of GABAergic interneurons without interfering with its neuroprotective actions. The use of autophagy modulators in the developing brain would create new opportunities to prevent the side effects of NMDA antagonists used for neuroprotection or anesthesia.
Assuntos
Autofagia/genética , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Neurônios GABAérgicos/fisiologia , Glutamato Descarboxilase/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Animais Recém-Nascidos , Autofagia/efeitos dos fármacos , Caspase 3/metabolismo , Córtex Cerebral/efeitos dos fármacos , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Neurônios GABAérgicos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Glutamato Descarboxilase/genética , Imunossupressores/farmacologia , Técnicas In Vitro , L-Lactato Desidrogenase/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Modelos Biológicos , Fatores de TempoRESUMO
In human neonates, immature GABAergic interneurons are markedly affected by an excitotoxic insult. While in adults the interest of cell transplantation has been demonstrated in several neurological disorders, few data are available regarding the immature brain. The low survival rate constitutes a strong limitation in the capacity of transplanted neurons to integrate the host tissue. Because i) autophagy is an adaptive process to energetic/nutrient deprivation essential for cell survival and ii) literature describes cross-talks between autophagy and apoptosis, we hypothesized that regulation of autophagy would represent an original strategy to favor long-term survival of GABAergic precursors grafted in the immature neocortex. Morphological, neurochemical, and functional data showed that in control conditions, few grafted Gad67-GFP precursors survived. The first hours following transplantation were a critical period with intense apoptosis. Experiments performed on E15.5 ganglionic eminences revealed that Gad67-GFP precursors were highly sensitive to autophagy. Rapamycin and 3-MA impacted on LC3 cleavage, LC3II translocation, and autophagosome formation. Quantification of Bax, mitochondrial integrity, caspase-3 cleavage, and caspase-3 immunolocalization and activity showed that 3-MA induced a significant decrease of Gad67-GFP precursor apoptosis. In vivo, 3-MA induced, within the first 24 h, a diffuse LC3 pattern of grafted Gad67-GFP precursors, an increase of precursors with neurites, a reduction of the density of caspase-3 immunoreactive cells. A twofold increase in the survival rate occurred 15 days after the graft. Surviving neurons were localized in the cortical layers II-IV, which were still immature when the transplantation was done. Altogether, these data indicate that inhibition of autophagy represents an original strategy to allow GABAergic interneurons to overpass the first critical hours following transplantation and to increase their long-term survival in mice neonates.
Assuntos
Adenina/análogos & derivados , Células-Tronco Embrionárias/transplante , Neurônios GABAérgicos/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Neocórtex/citologia , Células-Tronco Neurais/transplante , Adenina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transplante de Células/métodos , Feminino , Neurônios GABAérgicos/citologia , Glutamato Descarboxilase/biossíntese , Proteínas de Fluorescência Verde/biossíntese , Interneurônios/citologia , Interneurônios/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neocórtex/efeitos dos fármacos , Neocórtex/cirurgia , Gravidez , Proteínas Recombinantes de Fusão/biossíntese , Sirolimo/farmacologiaRESUMO
In the intestine, NF-κB is the main transcription factor involved in the anti-inflammatory effect of glutamine and we previously demonstrated that glutamine via its conversion to glutamate diminished the p65 protein content in Caco-2/TC7 cell nuclei without affecting the stimulating effect of IL-1ß on NF-κB [21]. However, the molecular mechanism by which glutamine acts is not established. We therefore tried to identify such a mechanism. Our results demonstrate that glutamine decreased the intracellular NF-κB through the nuclear ubiquitin-proteasome pathway requiring therefore the nuclear translocation of the factor. Indeed, time-course study revealed that glutamine induced an increase in the nuclear p65 content within the first 15 min of culture, the p65 nuclear and cytosolic content decreasing gradually thereafter to reach 50 % of the control value after 60 min. This translocation was initiated by the phosphorylation of IκBα by the IKKß subunit inducing its degradation and the p65 translocation. In parallel, glutamine activated the IKKα subunit which in turn phosphorylates p65 at Ser 536 which was responsible for p65 degradation by the nuclear proteasome. We also demonstrate that p38 MAPK lies between glutamine and the NF-κB pathway. In conclusion, this study identified for the first time the signaling pathway by which glutamine may protect against inflammatory conditions.