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Cholesterol is a major lipid of the animal realm with many biological roles. It is an important component of cellular membranes and a precursor of steroid hormones and bile acids. It is particularly abundant in nervous tissues, and dysregulation of cholesterol metabolism has been associated with neurodegenerative diseases such as Alzheimer's and Huntington's diseases. Deciphering the pathophysiological mechanisms of these disorders often involves animal models such as mice and Drosophila. Accurate quantification of cholesterol levels in the chosen models is a critical point of these studies. In the present work, we compare two common methods, gas chromatography coupled to flame-ionization detection (GC/FID) and a cholesterol oxidase-based fluorometric assay to measure cholesterol in mouse brains and Drosophila heads. Cholesterol levels measured by the two methods were similar for the mouse brain, which presents a huge majority of cholesterol in its sterol profile. On the contrary, depending on the method, measured cholesterol levels were very different for Drosophila heads, which present a complex sterol profile with a minority of cholesterol. We showed that the enzyme-based assay is not specific for cholesterol and detects other sterols as well. This method is therefore not suited for cholesterol measurement in models such as Drosophila. Alternatively, chromatographic methods, such as GC/FID, offer the required specificity for cholesterol quantification. Understanding the limitations of the quantification techniques is essential for reliable interpretation of the results in cholesterol-related research.
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Colesterol , Animais , Colesterol/metabolismo , Colesterol/análise , Colesterol/sangue , Cromatografia Gasosa/métodos , Camundongos , Ensaios Enzimáticos/métodos , Drosophila melanogaster , Drosophila , Encéfalo/metabolismo , Colesterol Oxidase/metabolismo , MasculinoRESUMO
Increasing evidence supports a relationship between lipid metabolism and mental health. In particular, the biostatus of polyunsaturated fatty acids (PUFAs) correlates with some symptoms of psychiatric disorders, as well as the efficacy of pharmacological treatments. Recent findings highlight a direct association between brain PUFA levels and dopamine transmission, a major neuromodulatory system implicated in the etiology of psychiatric symptoms. However, the mechanisms underlying this relationship are still unknown. Here we demonstrate that membrane enrichment in the n-3 PUFA docosahexaenoic acid (DHA), potentiates ligand binding to the dopamine D2 receptor (D2R), suggesting that DHA acts as an allosteric modulator of this receptor. Molecular dynamics simulations confirm that DHA has a high preference for interaction with the D2R and show that membrane unsaturation selectively enhances the conformational dynamics of the receptor around its second intracellular loop. We find that membrane unsaturation spares G protein activity but potentiates the recruitment of ß-arrestin in cells. Furthermore, in vivo n-3 PUFA deficiency blunts the behavioral effects of two D2R ligands, quinpirole and aripiprazole. These results highlight the importance of membrane unsaturation for D2R activity and provide a putative mechanism for the ability of PUFAs to enhance antipsychotic efficacy.
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BACKGROUND: The vascular endothelium is markedly disrupted in sickle cell disease (SCD) and is the converging cascade of the complex pathophysiologic processes linked to sickle cell vasculopathy. Circulating endothelial activation and/or apoptotic markers may reflect this endothelial activation/damage that contributes to the pathophysiology of the SCD vascular complications. METHODS: Plasmatic levels of circulating endothelial cells (CECs), E-selectin, progenitor's endothelial cells (EPCs), and circulating extracellular vesicles (EVs) were evaluated in 50 SCD patients, 16 with vasculopathy. The association between these markers and the occurrence of disease-related microvascular injuries of the eye (retinopathy), kidney (nephropathy), and skin (chronic active ulcers) was explored. RESULTS: Among the endothelial activation markers studied, only higher plasma levels of E-selectin were found in SCD patients with vasculopathy (p = .015). Increased E-selectin levels were associated with retinopathy (p < .001) but not with nephropathy or leg ulcers. All patients, at steady state, with or without vasculopathy, did not display a high count of CEC and EPC, markers of endothelial injury and repair. We did not show any significant differences in EVs levels between vasculopathy and not vasculopathy SCD patients. CONCLUSIONS: Further studies will be required to determine whether the E-selectin could be used as an early biomarker of retinopathy sickle cell development.
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Anemia Falciforme , Selectina E , Doenças Retinianas , Doenças Vasculares , Humanos , Anemia Falciforme/sangue , Anemia Falciforme/complicações , Anemia Falciforme/diagnóstico , Selectina E/sangue , Células Endoteliais/patologia , Doenças Retinianas/sangue , Doenças Retinianas/etiologia , Doenças Vasculares/sangue , Doenças Vasculares/etiologiaRESUMO
Communication between neurons and glia plays a major role in nervous tissue homeostasis. It is thought to participate in tuning cholesterol metabolism to cellular demand, which is a critical issue for neuronal health. Cholesterol is a membrane lipid crucial for nervous tissue functioning, and perturbed regulation of its metabolism has been linked to several neurodegenerative disorders. In the brain, 24(S)-hydroxycholesterol (24S-OHC) is an oxysterol synthesized by neurons to eliminate cholesterol, and 24S-OHC has been shown to regulate cholesterol metabolism in astrocytes, glial cells which provide cholesterol to neurons. In the retina, 24S-OHC is also an elimination product of cholesterol produced by neurons, especially the retinal ganglion cells. However, it is not known whether Müller cells, the major macroglial cells of the retina, play the role of cholesterol provider for retinal neurons and whether they respond to 24S-OHC signaling, similarly to brain glial cells. In the present study, primary cultures of rat Müller cells were treated with 0, 0.5 or 1.5 µM 24S-OHC for 48 hours. The levels of cholesterol, precursors and oxysterols were quantified using gas chromatography coupled to flame-ionization detection or mass spectrometry. In addition, the expression of key genes related to cholesterol metabolism was analyzed using RTq-PCR. Müller cells were shown to express many genes linked to cholesterol metabolism, including genes coding for proteins implicated in cholesterol biosynthesis (HMGCR), cholesterol uptake and export via lipoproteins (LDL-R, SR-BI, ApoE and ABACA1) and regulation of cholesterol metabolism (SREBP2 and LXRß). Cholesterol and several of its precursors and oxidative products were present. CYP27A1, the main retinal enzyme implicated in cholesterol elimination via oxysterol production, was quantified at low transcript levels but neither of its two typical products were detected in Müller cells. Furthermore, our results demonstrate that 24S-OHC has a strong hypocholesterolemic effect in Müller cells, leading to cholesterol depletion (-37 % at 1.5 µM). This was mediated by a decrease in cholesterol synthesis, as illustrated by reduced levels of cholesterol precursors: desmosterol (-38 % at 1.5 µM) and lathosterol (-84 % at 1.5 µM), and strong downregulation of HMGCR gene expression (2.4 fold decrease at 1.5µM). In addition, LDL-R and SR-BI gene expression were reduced in response to 24S-OHC treatment (2 fold and 1.6 fold at 1.5 µM, respectively), suggesting diminished lipoprotein uptake by the cells. On the contrary, there was a dramatic overexpression of ABCA1 transporter (10 fold increase at 1.5 µM), probably mediating an increase in cholesterol efflux. Finally, 24S-OHC induced a small but significant upregulation of the CYP27A1 gene. These data indicate that Müller cells possess the necessary cholesterol metabolism machinery and that they are able to sharply adjust their cholesterol metabolism in response to 24S-OHC, a signal molecule of neuronal cholesterol status. This suggests that Müller cells could be major players of cholesterol homeostasis in the retina via neuron-glia crosstalk.
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Colesterol/metabolismo , Células Ependimogliais/metabolismo , Hidroxicolesteróis/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Retina/metabolismo , Animais , Células Cultivadas , Células Ependimogliais/citologia , Modelos Animais , Neuroglia/citologia , Neurônios/citologia , Ratos , Ratos Long-Evans , Retina/citologiaRESUMO
PURPOSE: Gangliosides are glycosphingolipids that are particularly abundant in the nervous system, including the retina. However, their precise role in this tissue and its pathologies remain poorly understood. The objective of the present study was to characterize the ganglioside profile of human plasma and to determine whether it is affected in age-related macular degeneration (AMD). METHODS: Eighty-three subjects were included: control subjects (n = 25), atrophic AMD patients (n = 27) and exudative AMD patients (n = 31). For each subject, gangliosides were extracted from plasma and analyzed by liquid chromatography coupled to mass spectrometry. RESULTS: GM3 appeared to be by far the major ganglioside of human plasma, associated with GD3. No specific ganglioside class was detected in the plasma of AMD patients. Fourteen molecular species of GM3 and 9 species of GD3, accounting for the variability of the ceramide moiety of the ganglioside molecule, were identified and characterized. Analyses revealed no significant differences in the proportion of these species between control, atrophic and exudative AMD patient groups. Total GM3 levels did not differ either. CONCLUSION: Although gangliosides are considered important for the retina's structure and function, it seems that circulating gangliosides are not associated with the retinal damage occurring during the course of AMD.
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Gangliosídeos/metabolismo , Degeneração Macular/metabolismo , Retina/metabolismo , Idoso , Cromatografia Líquida , Feminino , Humanos , Degeneração Macular/diagnóstico , Masculino , Prognóstico , Retina/patologia , Estudos Retrospectivos , Índice de Gravidade de Doença , Tomografia de Coerência ÓpticaRESUMO
Expression of thioredoxin-interacting protein (TxNIP), an endogenous inhibitor of the thiol oxidoreductase thioredoxin, is augmented by high glucose (HG) and promotes oxidative stress. We previously reported that TxNIP-deficient mesangial cells showed protection from HG-induced reactive oxygen species, mitogen-activated protein kinase phosphorylation, and collagen expression. Here, we investigated the potential role of TxNIP in the pathogenesis of diabetic nephropathy (DN) in vivo. Wild-type (WT) control, TxNIP(-/-), and TxNIP(+/-) mice were rendered equally diabetic with low-dose streptozotocin. In contrast to effects in WT mice, diabetes did not increase albuminuria, proteinuria, serum cystatin C, or serum creatinine levels in TxNIP(-/-) mice. Whereas morphometric studies of kidneys revealed a thickened glomerular basement membrane and effaced podocytes in the diabetic WT mice, these changes were absent in the diabetic TxNIP(-/-) mice. Immunohistochemical analysis revealed significant increases in the levels of glomerular TGF-ß1, collagen IV, and fibrosis only in WT diabetic mice. Additionally, only WT diabetic mice showed significant increases in oxidative stress (nitrotyrosine, urinary 8-hydroxy-2-deoxy-guanosine) and inflammation (IL-1ß mRNA, F4/80 immunohistochemistry). Expression levels of Nox4-encoded mRNA and protein increased only in the diabetic WT animals. A significant loss of podocytes, assessed by Wilms' tumor 1 and nephrin staining and urinary nephrin concentration, was found in diabetic WT but not TxNIP(-/-) mice. Furthermore, in cultured human podocytes exposed to HG, TxNIP knockdown with siRNA abolished the increased mitochondrial O2 (-) generation and apoptosis. These data indicate that TxNIP has a critical role in the progression of DN and may be a promising therapeutic target.
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Proteínas de Transporte/metabolismo , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Tiorredoxinas/metabolismo , Albuminúria/etiologia , Animais , Antígenos de Diferenciação/análise , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Transporte/genética , Células Cultivadas , Colágeno Tipo IV/análise , Creatinina/sangue , Cistatina C/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Fibrose , Membrana Basal Glomerular/patologia , Glucose/farmacologia , Interleucina-1beta/genética , Glomérulos Renais/química , Glomérulos Renais/patologia , Proteínas de Membrana/análise , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Estresse Oxidativo , Oxigênio/metabolismo , Podócitos/química , Podócitos/efeitos dos fármacos , Podócitos/patologia , RNA Mensageiro/análise , Proteínas Repressoras/análise , Estreptozocina , Tiorredoxinas/genética , Fator de Crescimento Transformador beta1/análise , Proteínas WT1RESUMO
Gangliosides (GGs) make a wide family of glycosphingolipids ubiquitously expressed in mammalian tissues and particularly abundant in the brain and nervous system. They exhibit a huge diversity due to structural variations in both their oligosaccharidic chain and ceramide moiety, which represent a real analytical challenge. Since their discovery in the 1940s, methods have persistently improved until the emergence of LC/MS, which offers a high level of specificity and sensitivity and is suitable with high-throughput profiling studies. We describe here a comprehensive approach relying on various techniques and aiming at fully characterizing GGs in biological samples. First, total GG content was determined by a biochemical assay. Second, GG class composition was assessed by high-performance thin-layer chromatography followed by colorimetric revelation. Then, ceramide types of GG classes were identified, and their relative quantification was performed thanks to the development of a powerful and reliable LC/MS method. Finally, ceramides were structurally characterized, and minor and less common GG classes were identified using high-resolution MS. These methods were applied to the rat retina to provide an exhaustive description of its GG composition, giving the base for a better understanding of the precise roles of GGs in this tissue.
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Encéfalo/metabolismo , Ceramidas/isolamento & purificação , Gangliosídeos/isolamento & purificação , Glicoesfingolipídeos/isolamento & purificação , Animais , Química Encefálica , Ceramidas/química , Ceramidas/metabolismo , Cromatografia Líquida , Gangliosídeos/química , Gangliosídeos/metabolismo , Glicoesfingolipídeos/metabolismo , Sistema Nervoso/química , Sistema Nervoso/metabolismo , Ratos , Retina/química , Retina/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
O-GlcNAcylation on serine/threonine is a post-translational modification that controls the activity of nucleocytoplasmic proteins according to glucose availability. We previously showed that O-GlcNAcylation of FoxO1 in liver cells increases its transcriptional activity. In the present study, we evaluated the potential involvement of FoxO1 O-GlcNAcylation in the context of pancreatic ß-cell glucotoxicity. FoxO1 was O-GlcNAcylated in INS-1 832/13 ß cells and isolated rat pancreatic islets. O-GlcNAcylation of FoxO1 resulted in a 2-fold increase in its transcriptional activity toward a FoxO1 reporter gene and a 3-fold increase in the expression of the insulin-like growth factor-binding protein 1 (Igfbp1) gene at the mRNA level, resulting in IGFBP1 protein oversecretion by the cells. Of note, increased IGFBP1 in the culture medium inhibited the activity of the insulin-like growth factor 1 receptor (IGF1R)/phosphatidyl inositol 3 kinase (PI3K)/Akt pathway. We reveal in this report a novel mechanism by which O-GlcNAcylation inhibits Akt activity through an autocrine mechanism. However, although inhibition of IGFBP1 expression using siRNA restored the PI3 kinase/Akt pathway, it did not rescue INS-1 832/13 cells from high-glucose- or O-glcNAcylation-induced cell death. In contrast, FoxO1 down-regulation by siRNA led to 30 to 60% protection of INS-1 832/13 cells from death mediated by glucotoxic conditions. Therefore, whereas FoxO1 O-GlcNAcylation inhibits Akt through an IGFBP1-mediated autocrine pathway, the deleterious effects of FoxO1 O-GlcNAcylation on cell survival appeared to be independent of this pathway.
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Fatores de Transcrição Forkhead/metabolismo , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/genética , Glucose/farmacologia , Imunoprecipitação , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , RatosAssuntos
Hospitais , Imunoglobulinas Intravenosas/uso terapêutico , Doenças de von Willebrand/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Doenças de von Willebrand/sangue , Fator de von Willebrand/metabolismoRESUMO
Cholesterol is an essential component of cellular membranes, crucial for maintaining their structural and functional integrity. It is especially important for nervous tissues, including the retina, which rely on high amounts of plasma membranes for the transmission of the nervous signal. While cholesterol is by far the most abundant sterol, the retina also contains cholesterol precursors and metabolites, especially oxysterols, which are bioactive molecules. Cholesterol lack or excess is deleterious and some oxysterols are known for their effect on neuron survival. Cholesterol homeostasis must therefore be maintained. Retinal glial cells, especially Müller cells, the principal glial cells of the vertebrate retina, provide mechanical, nutritional, and metabolic support for the neighboring neurons. Several pieces of evidence indicate that Müller cells are major actors of cholesterol homeostasis in the retina, as it is known for other glial cells in the brain. This process is based on a close cooperation with neurons, and sterols can be signaling molecules participating in glia-neuron interactions. While some implication of cholesterol in age-related macular degeneration is now recognized, based on epidemiological and laboratory data, evidence for its role in glaucoma is still scarce. The association between cholesterolemia and glaucoma is controversial, but experimental data suggest that sterols could take part in the pathological processes. It has been demonstrated that Müller glial cells are implicated in the development of glaucoma through an ambivalent reactive retinal gliosis process. The early steps contribute to maintaining retinal homeostasis and favor the survival of ganglion cells, which are targeted during glaucoma. If gliosis persists, dysregulation of the neuroprotective functions, cytotoxic effects of gliotic Müller cells and disruption of glia-neuron interactions lead to an acceleration of ganglion cell death. Sterols could play a role in the glial cell response to glaucomatous injury. This represents an understudied but attractive topic to better understand glaucoma and conceive novel preventive or curative strategies. The present review describes the current knowledge on i) sterol metabolism in retinal glial cells, ii) the potential role of cholesterol in glaucoma, and iii) the possible relationships between cholesterol and oxysterols, glial cells and glaucoma. Focus is put on glia-neuron interactions.
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The olfactory mucosa (OM) and olfactory bulb (OB) are neuronal tissues that contribute to the early processing of olfactory information. They contain significant amounts of n-3 and n-6 polyunsaturated fatty acids (PUFAs), which are crucial for neuronal tissue development. In this study, we evaluated the impact of feeding mice diets that are either deficient in α-linolenic acid (ALA) or supplemented with n-3 long-chain PUFAs from gestation to adolescence on the phospholipid and ganglioside composition of these tissues. Both diets modified the levels of some phospholipid classes, notably the phosphatidylserine and phosphatidylethanolamine levels. In addition, the low-ALA diet enriched n-6 PUFAs in the main phospholipid classes of both tissues, while the diet supplemented with n-3 PUFAs enhanced the n-3 PUFA-containing phospholipid species level, mainly in OM. The diets also modulated the levels and profiles of several ganglioside classes in OM and OB. These modifications may have repercussions on the olfactory sensitivity.
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Ácidos Graxos Ômega-3 , Fosfolipídeos , Gravidez , Feminino , Camundongos , Animais , Gangliosídeos , Desmame , Dieta , Ácidos Graxos Ômega-6RESUMO
Only a few clusters of invasive pneumococcal disease have been described globally in children, and most of these cases occurred before pneumococcal vaccination implementation. Two unusual cases of pneumococcal meningitis, occurring in the same daycare center over a 3-day period, were reported. Both cerebrospinal fluid (CSF) were sent to the National reference center for pneumococci. In addition, we decided to perform a pneumococcal carriage study on all children and staff of the daycare center to analyze the pneumococcal serotypes circulating in this DCC and to discuss an antibiotic chemoprophylaxis. CSF culture was positive for pneumococcus, and serotype 25A was identified by latex agglutination. The second case had negative CSF culture, but CSF antigen test and gene amplification results were positive for Streptococcus pneumoniae. Serotype 12F was identified by using molecular biology. The absence of correlation between these strains was confirmed by multi-locus sequence typing. In the carriage study, we included 29 children (median age 1.9 years, interquartile range 1.4-2.5) and 10 adults. Among the children, 24 carried Streptococcus pneumoniae (83%). The main serotypes isolated were 23A for 6 children and 25A for 5 children; serotypes were non-typeable for 3 children. Only 1 of 10 adults tested carried Streptococcus pneumoniae (serotype 12F). Despite this temporo-spatial pattern, the cases were unrelated and not due to carriage of a particular serotype. No specific action has been taken for the other children attending this DCC, and no other case of bacterial meningitis occurred.
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Alterations of cholesterol metabolism have been described for many neurodegenerative pathologies, such as Alzheimer's disease in the brain and age-related macular degeneration in the retina. Recent evidence suggests that glaucoma, which is characterized by the progressive death of retinal ganglion cells, could also be associated with disruption of cholesterol homeostasis. In the present study we characterized cholesterol metabolism in a rat model of laser-induced intraocular hypertension, the main risk factor for glaucoma. Sterol levels were measured using gas-chromatography and cholesterol-related gene expression using quantitative RT-PCR at various time-points. As early as 18 hours after the laser procedure, genes implicated in cholesterol biosynthesis and uptake were upregulated (+49% and +100% for HMG-CoA reductase and LDLR genes respectively, vs. naive eyes) while genes involved in efflux were downregulated (-26% and -37% for ApoE and CYP27A1 genes, respectively). Cholesterol and precursor levels were consecutively elevated 3 days post-laser (+14%, +40% and +194% for cholesterol, desmosterol and lathosterol, respectively). Interestingly, counter-regulatory mechanisms were transcriptionally activated following these initial dysregulations, which were associated with the restoration of retinal cholesterol homeostasis, favorable to ganglion cell viability, one month after the laser-induced ocular hypertension. In conclusion, we report here for the first time that ocular hypertension is associated with transient major dynamic changes in retinal cholesterol metabolism.
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Glaucoma , Hipertensão Ocular , Animais , Colesterol/metabolismo , Modelos Animais de Doenças , Glaucoma/metabolismo , Hipertensão Ocular/metabolismo , Ratos , Retina/patologia , Células Ganglionares da Retina/patologiaRESUMO
Gangliosides (GG) are glycosphingolipids, composed of a ceramide moiety (fatty acid and long chain base) linked to an oligosaccharide chain containing one (or more) molecule of sialic acid. After lipid extraction from biological matrices, quantification of GG by liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI/MS) can be impacted by ion suppression effects due to co-elution with more abundant lipids in the matrix. In this study, a simple, rapid and efficient method to purify GG from biological samples by Phree columns is proposed. This approach proved to be useful in eliminating phospholipids (PL) from the matrix and thus increasing the signal of GG classes and molecular species in rat brain samples during LC-ESI/MS analysis.
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Cromatografia Líquida/métodos , Gangliosídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Química Encefálica , Masculino , Ratos , Ratos WistarRESUMO
Reward-processing impairment is a common symptomatic dimension of several psychiatric disorders. However, whether the underlying pathological mechanisms are common is unknown. Herein, we asked if the decrease in the n-3 polyunsaturated fatty acid (PUFA) lipid species, consistently described in these pathologies, could underlie reward-processing deficits. We show that reduced n-3 PUFA biostatus in mice leads to selective motivational impairments. Electrophysiological recordings revealed increased collateral inhibition of dopamine D2 receptor-expressing medium spiny neurons (D2-MSNs) onto dopamine D1 receptor-expressing MSNs in the nucleus accumbens, a main brain region for the modulation of motivation. Strikingly, transgenically preventing n-3 PUFA deficiency selectively in D2-expressing neurons normalizes MSN collateral inhibition and enhances motivation. These results constitute the first demonstration of a causal link between a behavioral deficit and n-3 PUFA decrease in a discrete neuronal population and suggest that lower n-3 PUFA biostatus in psychopathologies could participate in the etiology of reward-related symptoms.
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Ácidos Graxos Ômega-3/deficiência , Motivação , Neurônios , Núcleo Accumbens , Receptores de Dopamina D2/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Núcleo Accumbens/metabolismo , Núcleo Accumbens/patologiaRESUMO
The peripheral olfactory tissue (OT) plays a primordial role in the detection and transduction of olfactory information. Recent proteomic and transcriptomic studies have provided valuable insight into proteins and RNAs expressed in this tissue. Paradoxically, there is little information regarding the lipid composition of mammalian OT. To delve further into this issue, using a set of complementary state-of-the-art techniques, we carried out a comprehensive analysis of OT lipid composition in rats and mice fed with standard diets. The results showed that phospholipids are largely predominant, the major classes being phosphatidylcholine and phosphatidylethanolamine. Two types of plasmalogens, plasmenyl-choline and plasmenyl-ethanolamine, as well as gangliosides were also detected. With the exception of sphingomyelin, substantial levels of n-3 polyunsaturated fatty acids, mainly docosahexaenoic acid (22:6n-3; DHA), were found in the different phospholipid classes. These findings demonstrate that the rodent OT shares several features in common with other neural tissues, such as the brain and retina.
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Ácidos Graxos/análise , Lipídeos/análise , Mucosa Olfatória/química , Animais , Cromatografia Líquida , Gangliosídeos/análise , Gangliosídeos/química , Lipídeos/química , Masculino , Camundongos Endogâmicos C57BL , Fosfolipídeos/análise , Fosfolipídeos/química , Plasmalogênios/análise , Plasmalogênios/química , Ratos Wistar , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
BACKGROUND/OBJECTIVES: Sickle cell disease (SCD) complications mostly result from vascular dysfunction, concerning systemic microvasculature and cerebral large vessels. The aim of this cohort study was to identify potential circulating biomarkers predictive for further vascular event occurrence in pediatric SCD. METHODS: We consecutively enrolled 108 children with SCD at steady state, aged 3-18 years old (median 9.8 years). Hematology, coagulation, hemolysis, endothelial, platelet and vascular activation parameters were recorded at inclusion. Neurovascular and systemic vascular events were prospectively recorded during a mean follow-up period of 27 months. RESULTS: Patients at steady state displayed significantly higher hemolysis and platelet activation markers, higher leukocyte, CD34+ hematopoietic stem cell and microvesicle counts, and a pro-coagulant profile compared to controls matched for age and ethnicity. Circulating endothelial cell or nucleosome level did not differ. During the follow-up period, 36 patients had at least one neurovascular (n = 12) or systemic vascular event (n = 25). In a multivariate model, high CD34+ cell count was the best predictor for the occurrence of a vascular event (OR 1.2 for 1000 cell/mL increase, 95% CI [1.049-1.4], p = 0.013, sensitivity 53%, specificity 84% for a threshold of 8675 cells/mL). CONCLUSION: CD34+ cell count at steady state is a promising biomarker of further vascular event in children with SCD.
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Anemia Falciforme/sangue , Antígenos CD34/sangue , Vasos Sanguíneos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Adolescente , Anemia Falciforme/patologia , Biomarcadores/sangue , Plaquetas/metabolismo , Vasos Sanguíneos/patologia , Micropartículas Derivadas de Células/genética , Cerebelo/irrigação sanguínea , Criança , Pré-Escolar , Feminino , Células-Tronco Hematopoéticas/patologia , Humanos , MasculinoRESUMO
Glaucoma is a progressive and irreversible blinding neuropathy that is characterized by the loss of retinal ganglion cells (RGCs). Muller Glial Cell (MGC) activation is induced in retinal gliosis. MGCs are the most numerous glial cells in the retina and one of their roles is to sustain cholesterol homeostasis. 24S-hydroxycholesterol (24S-OHC) is one of the form of cholesterol elimination from the retina and is overexpressed during glaucoma. The objective of this study was to determine whether 24S-OHC triggers MGC membrane dynamics involving lipid rafts and contributes to gliosis at early and late time points. A proteomic analysis was carried out by nanoLC-MS/MS in raft and non-raft fractions from MGCs after treatment with 24S-OHC (10µM). The expression of structural and functional proteins was further analyzed by Western-blotting, as well as the levels of GM3 ganglioside by LC-MS. Cholesterol, sphingomyelin, saturated fatty acids and ganglioside GM3 are enriched in the rafts fractions in MGCs. Caveolin-1, flotillin-1, connexin-30 and -43 are localized in the MGCs rafts. Proteins implicated in adhesion or oxidative stress pathways in raft fractions were up and down-regulated by the treatment. Our data showed that 24S-OHC induced early changes in protein distribution in raft microdomains; however, further studies are needed to better characterize the surrounded mechanisms.
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Membrana Celular/efeitos dos fármacos , Colesterol/metabolismo , Células Ependimogliais/citologia , Glaucoma/metabolismo , Hidroxicolesteróis/farmacologia , Animais , Membrana Celular/metabolismo , Células Cultivadas , Ratos , Ratos Long-EvansRESUMO
Advanced glycation end products (AGEs) are involved in the development of microvascular complications, including alterations of retinal pericyte and renal mesangial cell growth occurring during diabetic retinopathy and diabetic nephropathy, respectively. Because gangliosides are implicated in the regulation of cell proliferation, we hypothesized that AGEs could exert cellular effects in part by modulating ganglioside levels. Results of the present study indicate that AGEs caused an inhibition of both bovine retinal pericyte (BRP) and rat renal mesangial cell (RMC) proliferation, associated with an increase of a-series gangliosides consecutive to GM3 synthase activity increase and GD3 synthase activity inhibition. Similar modifications were also found in the renal cortex of diabetic db/db mice compared with controls. Treatment of BRP and RMC with exogenous a-series gangliosides decreased proliferation and blockade of a-series gangliosides with specific antibodies partially protecting the two cell types from the AGE-induced proliferation decrease. Further, inhibition of GM3 synthase using specific SiRNA partially reversed the AGE effects on mesangial cell proliferation. These results suggest that a-series gangliosides are mediators of the adverse AGE effects on BRP and RMC proliferation. They also raise the hypothesis of common mechanisms involved in the development of diabetic retinopathy and diabetic nephropathy.