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Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.
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Células Endoteliais , Infarto do Miocárdio , Humanos , Autofagia , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Artérias Mesentéricas/metabolismo , Infarto do Miocárdio/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Transdução de Sinais , Vasodilatação , Animais , CamundongosRESUMO
Leber's hereditary optic neuropathy (LHON) is the most common disorder due to mitochondrial DNA mutations and complex I deficiency. It is characterized by an acute vision loss, generally in young adults, with a higher penetrance in males. How complex I dysfunction induces the peculiar LHON clinical presentation remains an unanswered question. To gain an insight into this question, we carried out a non-targeted metabolomic investigation using the plasma of 18 LHON patients, during the chronic phase of the disease, comparing them to 18 healthy controls. A total of 500 metabolites were screened of which 156 were accurately detected. A supervised Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) highlighted a robust model for disease prediction with a Q2 (cum) of 55.5%, with a reliable performance during the permutation test (cross-validation analysis of variance, P-value = 5.02284e-05) and a good prediction of a test set (P = 0.05). This model highlighted 10 metabolites with variable importance in the projection (VIP) > 0.8. Univariate analyses revealed nine discriminating metabolites, six of which were the same as those found in the Orthogonal Projections to Latent Structures Discriminant Analysis model. In total, the 13 discriminating metabolites identified underlining dietary metabolites (nicotinamide, taurine, choline, 1-methylhistidine and hippurate), mitochondrial energetic substrates (acetoacetate, glutamate and fumarate) and purine metabolism (inosine). The decreased concentration of taurine and nicotinamide (vitamin B3) suggest interesting therapeutic targets, given their neuroprotective roles that have already been demonstrated for retinal ganglion cells. Our results show a reliable predictive metabolomic signature in the plasma of LHON patients and highlighted taurine and nicotinamide deficiencies.
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Mitocôndrias/genética , Niacinamida/sangue , Atrofia Óptica Hereditária de Leber/sangue , Taurina/sangue , Adolescente , Adulto , Idoso , DNA Mitocondrial/genética , Complexo I de Transporte de Elétrons/sangue , Complexo I de Transporte de Elétrons/genética , Feminino , Humanos , Masculino , Metaboloma/genética , Metabolômica , Pessoa de Meia-Idade , Mitocôndrias/patologia , Mutação/genética , Niacinamida/deficiência , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/patologia , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Taurina/deficiência , Adulto JovemRESUMO
Glaucoma is a neurodegenerative disease that affects the retinal ganglion cells (RGCs). The main risk factor is elevated intraocular pressure (IOP), but the actual cause of the disease remains unknown. Emerging evidence indicates that metabolic dysfunction plays a central role. The aim of the current study was to determine and compare the effect of universal hypoxia on the metabolomic signature in plasma samples from healthy controls (n = 10), patients with normal-tension glaucoma (NTG, n = 10), and ocular hypertension (OHT, n = 10). By subjecting humans to universal hypoxia, we aim to mimic a state in which the mitochondria in the body are universally stressed. Participants were exposed to normobaric hypoxia for two hours, followed by a 30 min recovery period in normobaric normoxia. Blood samples were collected at baseline, during hypoxia, and in recovery. Plasma samples were analyzed using a non-targeted metabolomics approach based on liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). Multivariate analyses were conducted using principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA), and univariate analysis using the Wilcoxon signed-rank test and false discovery rate (FDR) correction. Unique metabolites involved in fatty acid biosynthesis and ketone body metabolism were upregulated, while metabolites of the kynurenine pathway were downregulated in OHT patients exposed to universal hypoxia. Differential affection of metabolic pathways may explain why patients with OHT initially do not suffer or are more resilient from optic nerve degeneration. The metabolomes of NTG and OHT patients are regulated differently from control subjects and show dysregulation of metabolites important for energy production. These dysregulated processes may potentially contribute to the elevation of IOP and, ultimately, cell death of the RGCs.
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Glaucoma , Doenças Neurodegenerativas , Humanos , Olho , Metaboloma , HipóxiaRESUMO
Mitochondrial complex I (CI) plays a crucial role in oxidising NADH generated by the metabolism (including photorespiration) and thereby participates in the mitochondrial electron transfer chain feeding oxidative phosphorylation that generates ATP. However, CI mutations are not lethal in plants and cause moderate phenotypes, and therefore CI mutants are instrumental to examine consequences of mitochondrial homeostasis disturbance on plant cell metabolisms and signalling. To date, the consequences of CI disruption on the lipidome have not been examined. Yet, in principle, mitochondrial dysfunction should impact on lipid synthesis through chloroplasts (via changes in photorespiration, redox homeostasis, and N metabolism) and the endoplasmic reticulum (ER) (via perturbed mitochondrion-ER crosstalk). Here, we took advantage of lipidomics technology (by LC-MS), phospholipid quantitation by 31P-NMR, and total lipid quantitation to assess the impact of CI disruption on leaf, pollen, and seed lipids using three well-characterised CI mutants: CMSII in N. sylvestris and both ndufs4 and ndufs8 in Arabidopsis. Our results show multiple changes in cellular lipids, including galactolipids (chloroplastic), sphingolipids, and ceramides (synthesised by ER), suggesting that mitochondrial homeostasis is essential for the regulation of whole cellular lipidome via specific signalling pathways. In particular, the observed modifications in phospholipid and sphingolipid/ceramide molecular species suggest that CI activity controls phosphatidic acid-mediated signalling.
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Arabidopsis , Lipidômica , Arabidopsis/genética , Arabidopsis/metabolismo , Esfingolipídeos/metabolismo , Cloroplastos/metabolismo , Ceramidas/metabolismo , Ácidos Fosfatídicos/metabolismoRESUMO
OPA1 is a dynamin GTPase implicated in mitochondrial membrane fusion. Despite its involvement in lipid remodeling, the function of OPA1 has never been analyzed by whole-cell lipidomics. We used a nontargeted, reversed-phase lipidomics approach, validated for cell cultures, to investigate OPA1-inactivated mouse embryonic fibroblasts ( Opa1 -/- MEFs). This led to the identification of a wide range of 14 different lipid subclasses comprising 212 accurately detected lipids. Multivariate and univariate statistical analyses were then carried out to assess the differences between the Opa1 -/- and Opa1 +/+ genotypes. Of the 212 lipids identified, 69 were found to discriminate between Opa1 -/- MEFs and Opa1 +/+ MEFs. Among these lipids, 34 were triglycerides, all of which were at higher levels in Opa1 -/- MEFs with fold changes ranging from 3.60 to 17.93. Cell imaging with labeled fatty acids revealed a sharp alteration of the fatty acid flux with a reduced mitochondrial uptake. The other 35 discriminating lipids included phosphatidylcholines, lysophosphatidylcholines, phosphatidylethanolamine, and sphingomyelins, mainly involved in membrane remodeling, and ceramides, gangliosides, and phosphatidylinositols, mainly involved in apoptotic cell signaling. Our results show that the inactivation of OPA1 severely affects the mitochondrial uptake of fatty acids and lipids through membrane remodeling and apoptotic cell signaling.
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Ácidos Graxos/metabolismo , Fibroblastos/enzimologia , GTP Fosfo-Hidrolases/metabolismo , Lipidômica/métodos , Triglicerídeos/metabolismo , Animais , Apoptose , Membrana Celular/metabolismo , Células Cultivadas , GTP Fosfo-Hidrolases/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismoRESUMO
Mitochondria and endoplasmic reticulum (ER) are physically and functionally connected. This close interaction, via mitochondria-associated membranes, is increasingly explored and supports the importance of studying these two organelles as a whole. Metabolomics and lipidomics are powerful approaches for the exploration of metabolic pathways that may be useful to provide deeper information on these organelles' functions, dysfunctions, and interactions. We developed a quick and simple experimental procedure for the purification of a mitochondria-ER fraction from human fibroblasts. We applied combined metabolomics and lipidomics analyses by mass spectrometry with excellent reproducibility. Seventy-two metabolites and 418 complex lipids were detected with a mean coefficient of variation around 12%, among which many were specific to the mitochondrial metabolism. Thus this strategy based on robust mitochondria-ER extraction and "omics" combination will be useful for investigating the pathophysiology of complex diseases.
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Retículo Endoplasmático/metabolismo , Fibroblastos/ultraestrutura , Lipídeos/análise , Metabolômica/métodos , Membranas Mitocondriais/metabolismo , Estudos Clínicos como Assunto , Humanos , Espectrometria de Massas , Reprodutibilidade dos Testes , Frações Subcelulares/ultraestruturaRESUMO
In recent years, the number of investigations based on nontargeted metabolomics has increased, although often without a thorough assessment of analytical strategies applied to acquire data. Following published guidelines for metabolomics experiments, we report a validated nontargeted metabolomics strategy with pipeline for unequivocal identification of metabolites using the MSMLS molecule library. We achieved an in-house database containing accurate m/z values, retention times, isotopic patterns, full MS, and MS/MS spectra. A UHPLC-HRMS Q-Exactive method was developed, and experimental variations were determined within and between 3 experimental days. The extraction efficiency as well as the accuracy, precision, repeatability, and linearity of the method were assessed, the method demonstrating good performances. The methodology was further blindly applied to plasma from remote ischemic pre-conditioning (RIPC) rats. Samples, previously analyzed by targeted metabolomics using completely different protocol, analytical strategy, and platform, were submitted to our analytical pipeline. A combination of multivariate and univariate statistical analyses was employed. Selection of putative biomarkers from OPLS-DA model and S-plot was combined to jack-knife confidence intervals, metabolites' VIP values, and univariate statistics. Only variables with strong model contribution and highly statistical reliability were selected as discriminated metabolites. Three biomarkers identified by the previous targeted metabolomics study were found in the current work, in addition to three novel metabolites, emphasizing the efficiency of the current methodology and its ability to identify new biomarkers of clinical interest, in a single sequence. The biomarkers were identified to level 1 according to the metabolomics standard initiative and confirmed by both RPLC and HILIC-HRMS.
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Cromatografia Líquida de Alta Pressão/métodos , Precondicionamento Isquêmico Miocárdico , Espectrometria de Massas/métodos , Metabolômica , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Bases de Dados Factuais , Humanos , Limite de Detecção , Masculino , Ratos Wistar , Reprodutibilidade dos TestesRESUMO
Leber's hereditary optic neuropathy (MIM#535000), the commonest mitochondrial DNA-related disease, is caused by mutations affecting mitochondrial complex I. The clinical expression of the disorder, usually occurring in young adults, is typically characterized by subacute, usually sequential, bilateral visual loss, resulting from the degeneration of retinal ganglion cells. As the precise action of mitochondrial DNA mutations on the overall cell metabolism in Leber's hereditary optic neuropathy is unknown, we investigated the metabolomic profile of the disease. High performance liquid chromatography coupled with tandem mass spectrometry was used to quantify 188 metabolites in fibroblasts from 16 patients with Leber's hereditary optic neuropathy and eight healthy control subjects. Latent variable-based statistical methods were used to identify discriminating metabolites. One hundred and twenty-four of the metabolites were considered to be accurately quantified. A supervised orthogonal partial least squares discriminant analysis model separating patients with Leber's hereditary optic neuropathy from control subjects showed good predictive capability (Q 2cumulated = 0.57). Thirty-eight metabolites appeared to be the most significant variables, defining a Leber's hereditary optic neuropathy metabolic signature that revealed decreased concentrations of all proteinogenic amino acids, spermidine, putrescine, isovaleryl-carnitine, propionyl-carnitine and five sphingomyelin species, together with increased concentrations of 10 phosphatidylcholine species. This signature was not reproduced by the inhibition of complex I with rotenone or piericidin A in control fibroblasts. The importance of sphingomyelins and phosphatidylcholines in the Leber's hereditary optic neuropathy signature, together with the decreased amino acid pool, suggested an involvement of the endoplasmic reticulum. This was confirmed by the significantly increased phosphorylation of PERK and eIF2α, as well as the greater expression of C/EBP homologous protein and the increased XBP1 splicing, in fibroblasts from affected patients, all these changes being reversed by the endoplasmic reticulum stress inhibitor, TUDCA (tauroursodeoxycholic acid). Thus, our metabolomic analysis reveals a pharmacologically-reversible endoplasmic reticulum stress in complex I-related Leber's hereditary optic neuropathy fibroblasts, a finding that may open up new therapeutic perspectives for the treatment of Leber's hereditary optic neuropathy with endoplasmic reticulum-targeting drugs.
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DNA Mitocondrial/genética , Complexo I de Transporte de Elétrons/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Mutação/genética , Atrofia Óptica Hereditária de Leber/metabolismo , Adulto , Idoso , Células Cultivadas , Estudos de Coortes , Complexo I de Transporte de Elétrons/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Inseticidas/farmacologia , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/patologia , Piridinas/farmacologia , Rotenona/farmacologia , Adulto JovemRESUMO
Autism spectrum disorder (ASD) is a neurodevelopmental disorder with no clinical biomarker. The aims of this study were to characterize a metabolic signature of ASD and to evaluate multiplatform analytical methodologies in order to develop predictive tools for diagnosis and disease follow-up. Urine samples were analyzed using (1)H and (1)H-(13)C NMR-based approaches and LC-HRMS-based approaches (ESI+ and ESI- on HILIC and C18 chromatography columns). Data tables obtained from the six analytical modalities on a training set of 46 urine samples (22 autistic children and 24 controls) were processed by multivariate analysis (orthogonal partial least-squares discriminant analysis, OPLS-DA). The predictions from each of these OPLS-DA models were then evaluated using a prediction set of 16 samples (8 autistic children and 8 controls) and receiver operating characteristic curves. Thereafter, a data fusion block-scaling OPLS-DA model was generated from the 6 best models obtained for each modality. This fused OPLS-DA model showed an enhanced performance (R(2)Y(cum) = 0.88, Q(2)(cum) = 0.75) compared to each analytical modality model, as well as a better predictive capacity (AUC = 0.91, p-value = 0.006). Metabolites that are most significantly different between autistic and control children (p < 0.05) are indoxyl sulfate, N-α-acetyl-l-arginine, methyl guanidine, and phenylacetylglutamine. This multimodality approach has the potential to contribute to find robust biomarkers and characterize a metabolic phenotype of the ASD population.
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Transtorno do Espectro Autista/urina , Metabolômica/métodos , Adolescente , Aminoácidos/metabolismo , Transtorno do Espectro Autista/metabolismo , Biomarcadores/urina , Estudos de Casos e Controles , Criança , Pré-Escolar , Cromatografia Líquida , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Redes e Vias Metabólicas , Metaboloma , Metabolômica/estatística & dados numéricos , Análise Multivariada , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Amyotrophic lateral sclerosis (ALS) is characterized by the absence of reliable diagnostic biomarkers. The aim of the study was to (i) devise an untargeted metabolomics methodology that reliably compares cerebrospinal fluid (CSF) from ALS patients and controls by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS); (ii) ascertain a metabolic signature of ALS by use of the LC-HRMS platform; (iii) identify metabolites for use as diagnostic or pathophysiologic markers. We developed a method to analyze CSF components by UPLC coupled with a Q-Exactive mass spectrometer that uses electrospray ionization. Metabolomic profiles were created from the CSF obtained at diagnosis from ALS patients and patients with other neurological conditions. We performed multivariate analyses (OPLS-DA) and univariate analyses to assess the contribution of individual metabolites as well as compounds identified in other studies. Sixty-six CSF samples from ALS patients and 128 from controls were analyzed. Metabolome analysis correctly predicted the diagnosis of ALS in more than 80% of cases. OPLS-DA identified four features that discriminated diagnostic group (p < 0.004). Our data demonstrate that untargeted metabolomics with LC-HRMS is a robust procedure to generate a specific metabolic profile for ALS from CSF and could be an important aid to the development of biomarkers for the disease.
Assuntos
Esclerose Lateral Amiotrófica/líquido cefalorraquidiano , Metaboloma , Doenças do Sistema Nervoso Periférico/líquido cefalorraquidiano , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/líquido cefalorraquidiano , Adulto , Idoso , Esclerose Lateral Amiotrófica/diagnóstico , Análise de Variância , Biomarcadores/líquido cefalorraquidiano , Estudos de Casos e Controles , Cromatografia Líquida , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doenças do Sistema Nervoso Periférico/diagnóstico , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/diagnóstico , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Metabolomics is a powerful data-driven tool for in-depth biological phenotyping that could help identify the specific metabolic profile of cryptogenic strokes, for which no precise cause has been identified. We performed a targeted quantitative metabolomics study in West African patients who had recently suffered an ischemic stroke, which was either cryptogenic (n = 40) or had a clearly identified cause (n = 39), compared to a healthy control group (n = 40). Four hundred fifty-six metabolites were accurately measured. Multivariate analyses failed to reveal any metabolic profile discriminating between cryptogenic ischemic strokes and those with an identified cause but did show superimposable metabolic profiles in both groups, which were clearly distinct from those of healthy controls. The blood concentrations of 234 metabolites were significantly affected in stroke patients compared to controls after the Benjamini-Hochberg correction. Increased methionine sulfoxide and homocysteine concentrations, as well as an overall increase in saturation of fatty acids, were indicative of acute oxidative stress. This signature also showed alterations in energetic metabolism, cell membrane integrity, monocarbon metabolism, and neurotransmission, with reduced concentrations of several metabolites known to be neuroprotective. Overall, our results show that cryptogenic strokes are not pathophysiologically distinct from ischemic strokes of established origin, and that stroke leads to intense metabolic remodeling with marked oxidative and energetic stresses.
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(1) Background: The development of mitochondrial medicine has been severely impeded by a lack of effective therapies. (2) Methods: To better understand Mitochondrial Encephalopathy Lactic Acidosis Syndrome Stroke-like episodes (MELAS) syndrome, neuronal cybrid cells carrying different mutation loads of the m.3243A > G mitochondrial DNA variant were analysed using a multi-omic approach. (3) Results: Specific metabolomic signatures revealed that the glutamate pathway was significantly increased in MELAS cells with a direct correlation between glutamate concentration and the m.3243A > G heteroplasmy level. Transcriptomic analysis in mutant cells further revealed alterations in specific gene clusters, including those of the glutamate, gamma-aminobutyric acid pathways, and tricarboxylic acid (TCA) cycle. These results were supported by post-mortem brain tissue analysis from a MELAS patient, confirming the glutamate dysregulation. Exposure of MELAS cells to ketone bodies significantly reduced the glutamate level and improved mitochondrial functions, reducing the accumulation of several intermediate metabolites of the TCA cycle and alleviating the NADH-redox imbalance. (4) Conclusions: Thus, a multi-omic integrated approach to MELAS cells revealed glutamate as a promising disease biomarker, while also indicating that a ketogenic diet should be tested in MELAS patients.
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BACKGROUND: Male factor is incriminated in approximately 50% of cases of infertility. The metabolomic approach has recently been used in the assessment of sperm quality and male fertility. MATERIALS AND METHODS: We analyzed the metabolomic signatures of the seminal plasma in 20 men with severe oligoasthenospermia (prewash total motile sperm count < 5.106 ) (SOA) and compared it to 20 men with normal semen parameters, with a standardized approach of targeted and quantitative metabolomics using high-performance liquid chromatography, coupled with tandem mass spectrometry, and the Biocrates Absolute IDQ p180 kit. RESULTS: Among the 188 metabolites analyzed, 110 were accurately measured in the seminal plasma. A robust model discriminating the two populations (Q2(cum) = 55.2%) was obtained by OPLS-DA (orthogonal partial least-squares discriminant analysis), based on the drop in concentrations of 37 metabolites with a VIP (variable important for projection) greater than 1. Overall, in men with SOA, there was a significant decrease in: 17 phosphatidylcholines and four sphingomyelins; acylcarnitines, with free L-carnitine being the most discriminating metabolite; polyunsaturated fatty acids; six amino acids (glutamate, aspartate, methionine, tryptophan, proline, and alanine); and four biogenic amines (spermine, spermidine, serotonin, and alpha-aminoadipate). DISCUSSION: Our signature includes several metabolic changes with different impacts on the sperm quality: a change in phospholipid composition and the saturation of their fatty acids that is potentially linked to the deterioration of sperm membranes; a carnitine deficiency that can negatively impact the energy production via fatty acid oxidation and oxidative phosphorylation; and a decreased level of amino acids and biogenic amines that can lead to dysregulated metabolic and signaling pathways. CONCLUSION: We provide a global overview of the metabolic defects contributing to the structural and functional alteration of spermatozoa in severe oligoasthenospermia. These findings offer new insights into the pathophysiology of male factor infertility that could help to develop future specific treatments.
Assuntos
Metaboloma/fisiologia , Oligospermia/metabolismo , Análise do Sêmen , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/patologia , Adulto , Aminas/análise , Aminoácidos/análise , Carnitina/análogos & derivados , Carnitina/análise , Ácidos Graxos Insaturados/análise , Humanos , Masculino , Metabolômica/métodos , Fosfatidilcolinas/análise , Estudos Prospectivos , Sêmen/citologia , Esfingomielinas/análiseRESUMO
Glaucoma is an age related disease characterized by the progressive loss of retinal ganglion cells, which are the neurons that transduce the visual information from the retina to the brain. It is the leading cause of irreversible blindness worldwide. To gain further insights into primary open-angle glaucoma (POAG) pathophysiology, we performed a non-targeted metabolomics analysis on the plasma from POAG patients (n = 34) and age- and sex-matched controls (n = 30). We investigated the differential signature of POAG plasma compared to controls, using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). A data mining strategy, combining a filtering method with threshold criterion, a wrapper method with iterative selection, and an embedded method with penalization constraint, was used. These strategies are most often used separately in metabolomics studies, with each of them having their own limitations. We opted for a synergistic approach as a mean to unravel the most relevant metabolomics signature. We identified a set of nine metabolites, namely: nicotinamide, hypoxanthine, xanthine, and 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline with decreased concentrations and N-acetyl-L-Leucine, arginine, RAC-glycerol 1-myristate, 1-oleoyl-RAC-glycerol, cystathionine with increased concentrations in POAG; the modification of nicotinamide, N-acetyl-L-Leucine, and arginine concentrations being the most discriminant. Our findings open up therapeutic perspectives for the diagnosis and treatment of POAG.
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In established tumors, tumor-associated macrophages (TAM) orchestrate nonresolving cancer-related inflammation and produce mediators favoring tumor growth, metastasis, and angiogenesis. However, the factors conferring inflammatory and protumor properties on human macrophages remain largely unknown. Most solid tumors have high lactate content. We therefore analyzed the impact of lactate on human monocyte differentiation. We report that prolonged lactic acidosis induces the differentiation of monocytes into macrophages with a phenotype including protumor and inflammatory characteristics. These cells produce tumor growth factors, inflammatory cytokines, and chemokines as well as low amounts of IL10. These effects of lactate require its metabolism and are associated with hypoxia-inducible factor-1α stabilization. The expression of some lactate-induced genes is dependent on autocrine M-CSF consumption. Finally, TAMs with protumor and inflammatory characteristics (VEGFhigh CXCL8+ IL1ß+) are found in solid ovarian tumors. These results show that tumor-derived lactate links the protumor features of TAMs with their inflammatory properties. Treatments that reduce tumor glycolysis or tumor-associated acidosis may help combat cancer.
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Acidose Láctica/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Inflamação/imunologia , Inflamação/patologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/imunologia , Neoplasias Ovarianas/patologia , Acidose Láctica/patologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Citocinas/metabolismo , Feminino , Humanos , Inflamação/etiologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Neoplasias Ovarianas/etiologia , Neoplasias Ovarianas/metabolismo , Fenótipo , Células Tumorais CultivadasRESUMO
Pathogenic variants of OPA1, which encodes a dynamin GTPase involved in mitochondrial fusion, are responsible for a spectrum of neurological disorders sharing optic nerve atrophy and visual impairment. To gain insight on OPA1 neuronal specificity, we performed targeted metabolomics on rat cortical neurons with OPA1 expression inhibited by RNA interference. Of the 103 metabolites accurately measured, univariate analysis including the Benjamini-Hochberg correction revealed 6 significantly different metabolites in OPA1 down-regulated neurons, with aspartate being the most significant (p < 0.001). Supervised multivariate analysis by OPLS-DA yielded a model with good predictive capability (Q2cum = 0.65) and a low risk of over-fitting (permQ2 = -0.16, CV-ANOVA p-value 0.036). Amongst the 46 metabolites contributing the most to the metabolic signature were aspartate, glutamate and threonine, which all decreased in OPA1 down-regulated neurons, and lysine, 4 sphingomyelins, 4 lysophosphatidylcholines and 32 phosphatidylcholines which were increased. The phospholipid signature may reflect intracellular membrane remodeling due to loss of mitochondrial fusion and/or lipid droplet accumulation. Aspartate and glutamate deficiency, also found in the plasma of OPA1 patients, is likely the consequence of respiratory chain deficiency, whereas the glutamate decrease could contribute to the synaptic dysfunction that we previously identified in this model.
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Córtex Cerebral/patologia , GTP Fosfo-Hidrolases/deficiência , Neurônios/patologia , Atrofia Óptica Autossômica Dominante/patologia , Animais , Ácido Aspártico/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Modelos Animais de Doenças , Regulação para Baixo , Embrião de Mamíferos , Feminino , GTP Fosfo-Hidrolases/genética , Ácido Glutâmico/metabolismo , Humanos , Metabolômica , Atrofia Óptica Autossômica Dominante/genética , Fosfolipídeos/metabolismo , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , RatosRESUMO
Amyotrophic lateral sclerosis (ALS) is characterized by a wide metabolic remodeling, as shown by recent metabolomics and lipidomics studies performed in samples from patient cohorts and experimental animal models. Here, we explored the metabolome and lipidome of fibroblasts from sporadic ALS patients (n = 13) comparatively to age- and sex-matched controls (n = 11), and the subcellular fraction containing the mitochondria and endoplasmic reticulum (mito-ER), given that mitochondrial dysfunctions and ER stress are important features of ALS patho-mechanisms. We also assessed the mitochondrial oxidative respiration and the mitochondrial genomic (mtDNA) sequence, although without yielding significant differences. Compared to controls, ALS fibroblasts did not exhibit a mitochondrial respiration defect nor an increased proportion of mitochondrial DNA mutations. In addition, non-targeted metabolomics and lipidomics analyses identified 124 and 127 metabolites, and 328 and 220 lipids in whole cells and the mito-ER fractions, respectively, along with partial least-squares-discriminant analysis (PLS-DA) models being systematically highly predictive of the disease. The most discriminant metabolomic features were the alteration of purine, pyrimidine, and energetic metabolisms, suggestive of oxidative stress and of pro-inflammatory status. The most important lipidomic feature in the mito-ER fraction was the disturbance of phosphatidylcholine PC (36:4p) levels, which we had previously reported in the cerebrospinal fluid of ALS patients and in the brain from an ALS mouse model. Thus, our results reveal that fibroblasts from sporadic ALS patients share common metabolic remodeling, consistent with other metabolic studies performed in ALS, opening perspectives for further exploration in this cellular model in ALS.
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Esclerose Lateral Amiotrófica/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Metabolômica , Mitocôndrias/metabolismo , Fosfolipídeos , Purinas/metabolismo , Pirimidinas/metabolismo , DNA Mitocondrial/genética , Metabolismo Energético , Feminino , Humanos , Metabolismo dos Lipídeos , Masculino , Pessoa de Meia-Idade , Fosforilação Oxidativa , Fosfolipídeos/metabolismoRESUMO
Purpose: To investigate the plasma concentration of nicotinamide in primary open-angle glaucoma (POAG). Methods: Plasma of 34 POAG individuals was compared to that of 30 age- and sex-matched controls using a semiquantitative method based on liquid chromatography coupled to high-resolution mass spectrometry. Subsequently, an independent quantitative method, based on liquid chromatography coupled to mass spectrometry, was used to assess nicotinamide concentration in the plasma from the same initial cohort and from a replicative cohort of 20 POAG individuals and 15 controls. Results: Using the semiquantitative method, the plasma nicotinamide concentration was significantly lower in the initial cohort of POAG individuals compared to controls and further confirmed in the same cohort, using the targeted quantitative method, with mean concentrations of 0.14 µM (median: 0.12 µM; range, 0.06-0.28 µM) in the POAG group (-30%; P = 0.022) and 0.19 µM (median: 0.18 µM; range, 0.08-0.47 µM) in the control group. The quantitative dosage also disclosed a significantly lower plasma nicotinamide concentration (-33%; P = 0.011) in the replicative cohort with mean concentrations of 0.14 µM (median: 0.14 µM; range, 0.09-0.25 µM) in the POAG group, and 0.19 µM (median: 0.21 µM; range, 0.09-0.26 µM) in the control group. Conclusions: Glaucoma is associated with lower plasmatic nicotinamide levels, compared to controls, suggesting that nicotinamide supplementation might become a future therapeutic strategy. Further studies are needed, in larger cohorts, to confirm these preliminary findings.