Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 28
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
PLoS One ; 14(1): e0209573, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30608949

RESUMO

Glycosaminoglycans (GAGs), including heparan sulfates and chondroitin sulfates, are major components of the extracellular matrix. Upon interacting with heparin binding growth factors (HBGF), GAGs participate to the maintaintenance of tissue homeostasis and contribute to self-healing. Although several processes regulated by HBGF are altered in Alzheimer's disease, it is unknown whether the brain GAG capacities to bind and regulate the function of HBGF or of other heparin binding proteins, as tau, are modified in this disease. Here, we show that total sulfated GAGs from hippocampus of Alzheimer's disease have altered capacities to bind and potentiate the activities of growth factors including FGF-2, VEGF, and BDNF while their capacity to bind to tau is remarkable increased. Alterations of GAG structures and capacities to interact with and regulate the activity of heparin binding proteins might contribute to impaired tissue homeostasis in the Alzheimer's disease brain.


Assuntos
Doença de Alzheimer/metabolismo , Glicosaminoglicanos/metabolismo , Proteínas tau/fisiologia , Idoso , Idoso de 80 Anos ou mais , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Brasil , Sulfatos de Condroitina/metabolismo , Matriz Extracelular/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Hipocampo/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Lobo Temporal/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
Nat Commun ; 9(1): 3087, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30082715

RESUMO

Skeletal dysplasia with multiple dislocations are severe disorders characterized by dislocations of large joints and short stature. The majority of them have been linked to pathogenic variants in genes encoding glycosyltransferases, sulfotransferases or epimerases required for glycosaminoglycan synthesis. Using exome sequencing, we identify homozygous mutations in SLC10A7 in six individuals with skeletal dysplasia with multiple dislocations and amelogenesis imperfecta. SLC10A7 encodes a 10-transmembrane-domain transporter located at the plasma membrane. Functional studies in vitro demonstrate that SLC10A7 mutations reduce SLC10A7 protein expression. We generate a Slc10a7-/- mouse model, which displays shortened long bones, growth plate disorganization and tooth enamel anomalies, recapitulating the human phenotype. Furthermore, we identify decreased heparan sulfate levels in Slc10a7-/- mouse cartilage and patient fibroblasts. Finally, we find an abnormal N-glycoprotein electrophoretic profile in patient blood samples. Together, our findings support the involvement of SLC10A7 in glycosaminoglycan synthesis and specifically in skeletal development.

3.
Biochem J ; 475(15): 2417-2433, 2018 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-29934491

RESUMO

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.

4.
FEBS Lett ; 2018 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-29729013

RESUMO

Neurodegenerative disorders, such as Alzheimer's, Parkinson's, and prion diseases, are directly linked to the formation and accumulation of protein aggregates in the brain. These aggregates, principally made of proteins or peptides that clamp together after acquisition of ß-folded structures, also contain heparan sulfates. Several lines of evidence suggest that heparan sulfates centrally participate in the protein aggregation process. In vitro, they trigger misfolding, oligomerization, and fibrillation of amyloidogenic proteins, such as Aß, tau, α-synuclein, prion protein, etc. They participate in the stabilization of protein aggregates, protect them from proteolysis, and act as cell-surface receptors for the cellular uptake of proteopathic seeds during their spreading. This review focuses attention on the importance of heparan sulfates in protein aggregation in brain disorders including Alzheimer's, Parkinson's, and prion diseases. The presence of these sulfated polysaccharides in protein inclusions in vivo and their capacity to trigger protein aggregation in vitro strongly suggest that they might play critical roles in the neurodegenerative process. Further advances in glyco-neurobiology will improve our understanding of the molecular and cellular mechanisms leading to protein aggregation and neurodegeneration.

5.
J Lipid Res ; 59(1): 25-34, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29150495

RESUMO

The functional heterogeneity of HDL is attributed to its diverse bioactive components. We evaluated whether the vasodilatory effects of HDL differed across HDL subpopulations, reflecting their distinct molecular composition. The capacity of five major HDL subfractions to counteract the inhibitory effects of oxidized LDL on acetylcholine-induced vasodilation was tested in a rabbit aortic rings model. NO production, an essential pathway in endothelium-dependent vasorelaxation, was studied in simian vacuolating virus 40-transformed murine endothelial cells (SVECs). Small dense HDL3 subfractions displayed potent vasorelaxing activity (up to +31% vs. baseline, P < 0.05); in contrast, large light HDL2 did not induce aortic-ring relaxation when compared on a total protein basis. HDL3 particles were enriched with sphingosine-1-phosphate (S1P) (up to 3-fold vs. HDL2), with the highest content in HDL3b and -3c that concomitantly revealed the strongest vasorelaxing properties. NO generation was enhanced by HDL3c in SVECs (1.5-fold, P < 0.01), a phenomenon that was blocked by the S1P receptor antagonist, VPC 23019. S1P-enriched reconstituted HDL (rHDL) was a 1.8-fold (P < 0.01) more potent vasorelaxant than control rHDL in aortic rings. Small dense HDL3 particles displayed potent protective effects against oxidative stress-associated endothelium dysfunction, potentially reflecting their elevated content of S1P that might facilitate interaction with S1P receptors and ensuing NO generation.

6.
Theranostics ; 8(21): 5814-5827, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30613264

RESUMO

Alteration of the extracellular matrix (ECM) is one of the major events in the pathogenesis of brain lesions following ischemic stroke. Heparan sulfate mimetics (HSm) are synthetic pharmacologically active polysaccharides that promote ECM remodeling and tissue regeneration in various types of lesions. HSm bind to growth factors, protect them from enzymatic degradation and increase their bioavailability, which promotes tissue repair. As the ECM is altered during stroke and HSm have been shown to restore the ECM, we investigated the potential of HSm4131 (also named RGTA-4131®) to protect brain tissue and promote regeneration and plasticity after a stroke. Methods: Ischemic stroke was induced in rats using transient (1 h) intraluminal middle cerebral artery occlusion (MCAo). Animals were assigned to the treatment (HSm4131; 0.1, 0.5, 1.5, or 5 mg/kg) or vehicle control (saline) groups at different times (1, 2.5 or 6 h) after MCAo. Brain damage was assessed by MRI for the acute (2 days) and chronic (14 days) phases post-occlusion. Functional deficits were evaluated with a battery of sensorimotor behavioral tests. HSm4131-99mTc biodistribution in the ischemic brain was analyzed between 5 min and 3 h following middle cerebral artery reperfusion. Heparan sulfate distribution and cellular reactions, including angiogenesis and neurogenesis, were evaluated by immunohistochemistry, and growth factor gene expression (VEGF-A, Ang-2) was quantified by RT-PCR. Results: HSm4131, administered intravenously after stroke induction, located and remained in the ischemic hemisphere. HSm4131 conferred long-lasting neuroprotection, and significantly reduced functional deficits with no alteration of physiological parameters. It also restored the ECM, and increased brain plasticity processes, i.e., angiogenesis and neurogenesis, in the affected brain hemisphere. Conclusion: HSm represent a promising ECM-based therapeutic strategy to protect and repair the brain after a stroke and favor functional recovery.


Assuntos
Isquemia Encefálica/terapia , Encéfalo/patologia , Encéfalo/fisiologia , Matriz Extracelular/metabolismo , Glucanos/administração & dosagem , Heparitina Sulfato/administração & dosagem , Acidente Vascular Cerebral/terapia , Animais , Comportamento Animal , Encéfalo/diagnóstico por imagem , Modelos Animais de Doenças , Imuno-Histoquímica , Imagem por Ressonância Magnética , Neovascularização Fisiológica , Neurogênese , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Regeneração , Resultado do Tratamento
7.
Rev. neurol. (Ed. impr.) ; 65(10): 457-468, 16 nov., 2017. tab, graf, ilus
Artigo em Espanhol | IBECS | ID: ibc-169069

RESUMO

Introducción. Numerosos trastornos neurodegenerativos se han asociado directamente a la acumulación de fibras amiloides. Estas fibras están formadas por proteínas o péptidos con conformaciones alteradas y que se agregan in vivo en asociación con polisacáridos de tipo heparán sulfatos. Objetivos. Examinar los conceptos más recientes sobre la biología de los heparán sulfatos y su papel en la agregación del péptido Abeta, de la proteína tau, de la alfa-sinucleína y de los priones, y analizar sus implicaciones en trastornos neurodegenerativos como las enfermedades de Alzheimer y de Parkinson y las enfermedades priónicas. Desarrollo. In vitro, los heparán sulfatos han desempeñado un papel importante en el proceso de oligomerización y fibrilación de proteínas o péptidos amiloidógenos, en la estabilización de estos cuerpos y su resistencia a la proteólisis, participando así en la formación de una gran variedad de fibras amiloides. Los heparán sulfatos se han relacionado también con el proceso de internalización de fibras proamiloides durante el proceso de propagación intercelular (spreading) considerado como central en la evolución de las proteinopatías, cuyo mejor ejemplo es la enfermedad de Alzheimer. Conclusión. Este trabajo sugiere que las estructuras finas de los heparán sulfatos, sus localizaciones celulares y tisulares, así como sus concentraciones locales, pueden regular los procesos de amiloidosis. Avances en la comprensión de esta área de la gliconeurobiología permitirán mejorar la comprensión de los mecanismos celulares y moleculares del proceso neurodegenerativo (AU)


Introduction. A number of neurodegenerative disorders have been linked directly to the accumulation of amyloid fibres. These fibres are made up of proteins or peptides with altered structures and which join together in vivo in association with heparan sulphate-type polysaccharides. AIMS. To examine the most recent concepts in the biology of heparan sulphates and their role in the aggregation of the peptide Abeta, of tau protein, of alpha-synuclein and of prions. The study also seeks to analyse their implications in neurodegenerative disorders such as Alzheimers and Parkinson’s disease and prion diseases. Development. In vitro, heparan sulphates have played an important role in the process of oligomerisation and fibrillation of amyloidogenic proteins or peptides, in the stabilisation of these bodies and their resistance to proteolysis, thereby participating in the formation of a wide range of amyloid fibres. Heparan sulphates have also been related to the internalisation of pro-amyloid fibres during the process of intercellular propagation (spreading), which is considered to be crucial in the development of proteinopathies, the best example of which is Alzheimers disease. Conclusion This study suggests that the fine structures of heparan sulphates, their localisation in cells and tissues, together with their local concentration, may regulate the amyloidosis processes. The advances made in the understanding of this area of glyconeurobiology will make it possible to improve the understanding of the cell and molecular mechanisms underlying the neurodegenerative process (AU)


Assuntos
Humanos , Heparitina Sulfato/farmacocinética , Amiloidose/fisiopatologia , Doença de Parkinson/fisiopatologia , Doenças Neurodegenerativas/fisiopatologia , Doenças Priônicas/fisiopatologia , Doença de Alzheimer/fisiopatologia , Agregação Patológica de Proteínas/fisiopatologia , Glicosaminoglicanos/farmacocinética , Proteínas tau/fisiologia , alfa-Sinucleína/fisiologia
8.
PLoS One ; 12(8): e0181350, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28809922

RESUMO

Engrailed 1 (En1) and 2 (En2) code for closely related homeoproteins acting as transcription factors and as signaling molecules that contribute to midbrain and hindbrain patterning, to development and maintenance of monoaminergic pathways, and to retinotectal wiring. En2 has been suggested to be an autism susceptibility gene and individuals with autism display an overexpression of this homeogene but the mechanisms remain unclear. We addressed in the present study the effect of exogenously added En2 on the morphology of hippocampal cells that normally express only low levels of Engrailed proteins. By means of RT-qPCR, we confirmed that En1 and En2 were expressed at low levels in hippocampus and hippocampal neurons, and observed a pronounced decrease in En2 expression at birth and during the first postnatal week, a period characterized by intense synaptogenesis. To address a putative effect of Engrailed in dendritogenesis or synaptogenesis, we added recombinant En1 or En2 proteins to hippocampal cell cultures. Both En1 and En2 treatment increased the complexity of the dendritic tree of glutamatergic neurons, but only En2 increased that of GABAergic cells. En1 increased the density of dendritic spines both in vitro and in vivo. En2 had similar but less pronounced effect on spine density. The number of mature synapses remained unchanged upon En1 treatment but was reduced by En2 treatment, as well as the area of post-synaptic densities. Finally, both En1 and En2 elevated mTORC1 activity and protein synthesis in hippocampal cells, suggesting that some effects of Engrailed proteins may require mRNA translation. Our results indicate that Engrailed proteins can play, even at low concentrations, an active role in the morphogenesis of hippocampal cells. Further, they emphasize the over-regulation of GABA cell morphology and the vulnerability of excitatory synapses in a pathological context of En2 overexpression.


Assuntos
Transtorno Autístico/metabolismo , Dendritos/genética , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Imunofluorescência , Hipocampo/citologia , Hipocampo/metabolismo , Proteínas de Homeodomínio/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Microscopia Confocal , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/genética , Neuroglia/citologia , Neuroglia/metabolismo , Sinapses/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Differentiation ; 93: 15-26, 2017 Jan - Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27689814

RESUMO

In vitro, extracted muscle satellite cells, called myogenic progenitor cells, can differentiate either in myotubes or preadipocytes, depending on environmental factors and the medium. Transcriptomic analyses on glycosylation genes during satellite cells differentiation into myotubes showed that 31 genes present a significant variation of expression at the early stages of murine myogenic progenitor cells (MPC) differentiation. In the present study, we analyzed the expression of 383 glycosylation related genes during murine MPC differentiation into preadipocytes and compared the data to those previously obtained during their differentiation into myotubes. Fifty-six glycosylation related genes are specifically modified in their expression during early adipogenesis. The variations correspond mainly to: a decrease of N-glycans, and of alpha (2,3) and (2,6) linked sialic acids, and to a high level of heparan sulfates. A high amount of TGF-ß1 in extracellular media during early adipogenesis was also observed. It seems that the increases of heparan sulfates and TGF-ß1 favor pre-adipogenic differentition of MPC and possibly prevent their myogenic differentiation.


Assuntos
Adipogenia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Musculares/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Células-Tronco/citologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Heparitina Sulfato/administração & dosagem , Camundongos , Células Musculares/citologia , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Polissacarídeos/biossíntese , Células-Tronco/efeitos dos fármacos
10.
J Clin Lipidol ; 10(1): 124-33, 2016 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26892129

RESUMO

BACKGROUND: Familial hypercholesterolemia (FH) features elevated oxidative stress and accelerated atherosclerosis driven by elevated levels of atherogenic lipoproteins relative to subnormal levels of atheroprotective high-density lipoprotein (HDL). Small, dense HDL3 potently protects low-density lipoprotein (LDL) against proinflammatory oxidative damage. OBJECTIVE: To determine whether antioxidative and/or anti-inflammatory activities of HDL are defective in FH and whether such defects are corrected by LDL apheresis. METHODS: Antioxidative and antiinflammatory activities of HDL were evaluated as protection of reference LDL from oxidative stress and capacity to prevent accumulation of proinflammatory oxidised lipids, respectively. Lipid surface rigidity of HDL was assessed using a fluorescent probe. HDL components were measured by analytical approaches. Systemic oxidative stress was characterized as plasma 8-isoprostanes. RESULTS: Pre-LDL-apheresis, FH patients (n = 10) exhibited elevated systemic oxidative stress (3.3-fold, P < 0.001) vs. sex- and age-matched normolipidemic controls (n = 10). Both antioxidative and antiinflammatory activity of HDL3 were impaired (up to -91%, P < 0.01) in FH. Sphingomyelin and saturated fatty acid contents were elevated in FH HDL3, resulting in enhanced lipid surface rigidity. The surface lipid content (phospholipids, free cholesterol) was reduced in FH (up to -15%, P < 0.001), whereas content of core lipids (cholesteryl esters, triglycerides) was elevated (up to +17%, P < 0.001). Molar apolipoprotein A-I content of HDL3 was subnormal in FH. A single LDL-apheresis session partially corrected (by up to 76%) deficient HDL antiatherogenic activities, attenuated systemic oxidative stress and partially normalised both the lipid composition and surface rigidity of HDL particles. CONCLUSIONS: FH features elevated oxidative stress and deficient antioxidative and anti-inflammatory activities of small, dense HDL3; such functional deficiency is intimately linked to anomalies in lipid and protein composition, which may impair the capacity of HDL to acquire and inactivate oxidized lipids.


Assuntos
Antioxidantes/química , Antioxidantes/metabolismo , Remoção de Componentes Sanguíneos , Lipoproteínas HDL3/química , Lipoproteínas HDL3/metabolismo , Lipoproteínas LDL/sangue , Tamanho da Partícula , Adulto , Feminino , Radicais Livres/metabolismo , Humanos , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/terapia , Inflamação/metabolismo , Lipoproteínas HDL3/sangue , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo
11.
Stem Cell Res Ther ; 7: 3, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26742480

RESUMO

BACKGROUND: Expansion protocols aim at both increasing the number of umbilical cord blood (UCB) hematopoietic stem cells and progenitor cells (HSPCs) and reducing the period of neutropenia in UCB HSPC graft. Because glycosaminoglycans (GAGs) are known to be important components of the hematopoietic niche and to modulate growth factor effects, we explored the use of GAG mimetic OTR4131 to potentiate HSPC's in vitro expansion and in vivo engraftment. METHODS: UCB CD34+ cells were expanded with serum-free medium, SCF, TPO, FLT3-lig and G-CSF during 12 days in the absence or the presence of increasing OTR4131 concentrations (0-100 µg/mL). Proliferation ratio, cell viability and phenotype, functional assays, migration capacity and NOD-scid/γc(-/-) mice engraftment were assessed after expansion. RESULTS: At Day 12, ratios of cell expansion were not significantly increased by OTR4131 treatment. Better total nucleated cell viability was observed with the use of 1 µg/mL GAG mimetic compared to control (89.6 % ± 3.7 % and 79.9 % ± 3.3 %, respectively). Phenotype analysis showed a decrease of monocyte lineage in the presence of OTR4131 and HSPC migration capacity was diminished when GAG mimetic was used at 10 µg/mL (10.9 % ± 4.1 % vs. 52.9 % ± 17.9 % for control). HSPC clonogenic capacities were similar whatever the culture conditions. Finally, in vivo experiments revealed that mice successfully engrafted in all conditions, even if some differences were observed during the first month. Three months after graft, bone marrow chimerism and blood subpopulations were similar in both groups. CONCLUSIONS: UCB HSPCs ex-vivo expansion in the presence of OTR4131 is a safe approach that did not modify cell function and engraftment capacities. In our experimental conditions, the use of a GAG mimetic did not, however, allow increasing cell expansion or optimizing their in vivo engraftment.


Assuntos
Glucanos/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Animais , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Mimetismo Molecular
12.
Biomaterials ; 69: 133-47, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26283160

RESUMO

Tissue-engineered organs and implants hold promise for the replacement of damaged and diseased organs. However, the foreign body response (FBR) is a major obstacle that compromises the function of tissue-engineered constructs, typically causing them to fail. Two components of FBR are an inflammatory response and a lack of vascularization. To overcome these limitations, a collagen system was developed to release interleukin-6 (IL-6) siRNA and endothelial nitric oxide synthase (eNOS) pDNA in a staggered manner. Hollow collagen microspheres were assembled into a collagen sphere-in-hydrogel system that displayed a staggered release profile in vitro. This system was assessed in vivo in a subcutaneous rat model. The doses of IL-6 siRNA and eNOS pDNA were first individually optimized for their ability to reduce the volume fraction of inflammatory cells (7 days) and increase the length density of blood vessels (14 days), respectively. The identified optimal doses were combined, and the ability of the system to decrease the volume fraction of inflammatory cells and increase the length density of blood vessels was confirmed at both 7 and 14 days. Analysis of the tissue using Raman microspectroscopy revealed that in addition to changes in inflammation and angiogenesis, there were also changes in the extracellular matrix (ECM) at seven days. While changes in sulfated glycosaminoglycan (sGAG) content of the ECM were not detected, changes in the binding of sGAG of the ECM to growth factors were observed. Two growth factors tested, VEGF165 and bFGF showed increased binding to sGAG extracted from eNOS pDNA-treated samples at seven days, increasing the angiogenic potential of the ECM. Thus, we observe that changes in the tissue in terms of the balance of inflammation and angiogenesis as well changes in the activity of sGAG of the ECM occurs following dual delivery of nucleic acids from the collagen sphere-in-hydrogel system.


Assuntos
Matriz Extracelular/imunologia , Glicosaminoglicanos/imunologia , Inflamação/terapia , Interleucina-6/genética , Óxido Nítrico Sintase Tipo III/genética , Plasmídeos/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Animais , Bovinos , Colágeno/química , DNA Circular/administração & dosagem , DNA Circular/genética , DNA Circular/uso terapêutico , Feminino , Terapia Genética , Inflamação/genética , Inflamação/imunologia , Neovascularização Fisiológica , Plasmídeos/genética , Plasmídeos/uso terapêutico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Ratos Endogâmicos Lew , Engenharia Tecidual , Tecidos Suporte/química
13.
Brain ; 138(Pt 5): 1339-54, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25842390

RESUMO

Heparan sulphate (glucosamine) 3-O-sulphotransferase 2 (HS3ST2, also known as 3OST2) is an enzyme predominantly expressed in neurons wherein it generates rare 3-O-sulphated domains of unknown functions in heparan sulphates. In Alzheimer's disease, heparan sulphates accumulate at the intracellular level in disease neurons where they co-localize with the neurofibrillary pathology, while they persist at the neuronal cell membrane in normal brain. However, it is unknown whether HS3ST2 and its 3-O-sulphated heparan sulphate products are involved in the mechanisms leading to the abnormal phosphorylation of tau in Alzheimer's disease and related tauopathies. Here, we first measured the transcript levels of all human heparan sulphate sulphotransferases in hippocampus of Alzheimer's disease (n = 8; 76.8 ± 3.5 years old) and found increased expression of HS3ST2 (P < 0.001) compared with control brain (n = 8; 67.8 ± 2.9 years old). Then, to investigate whether the membrane-associated 3-O-sulphated heparan sulphates translocate to the intracellular level under pathological conditions, we used two cell models of tauopathy in neuro-differentiated SH-SY5Y cells: a tau mutation-dependent model in cells expressing human tau carrying the P301L mutation hTau(P301L), and a tau mutation-independent model in where tau hyperphosphorylation is induced by oxidative stress. Confocal microscopy, fluorescence resonance energy transfer, and western blot analyses showed that 3-O-sulphated heparan sulphates can be internalized into cells where they interact with tau, promoting its abnormal phosphorylation, but not that of p38 or NF-κB p65. We showed, in vitro, that the 3-O-sulphated heparan sulphates bind to tau, but not to GSK3B, protein kinase A or protein phosphatase 2, inducing its abnormal phosphorylation. Finally, we demonstrated in a zebrafish model of tauopathy expressing the hTau(P301L), that inhibiting hs3st2 (also known as 3ost2) expression results in a strong inhibition of the abnormally phosphorylated tau epitopes in brain and in spinal cord, leading to a complete recovery of motor neuronal axons length (n = 25; P < 0.005) and of the animal motor response to touching stimuli (n = 150; P < 0.005). Our findings indicate that HS3ST2 centrally participates to the molecular mechanisms leading the abnormal phosphorylation of tau. By interacting with tau at the intracellular level, the 3-O-sulphated heparan sulphates produced by HS3ST2 might act as molecular chaperones allowing the abnormal phosphorylation of tau. We propose HS3ST2 as a novel therapeutic target for Alzheimer's disease.


Assuntos
Doença de Alzheimer/metabolismo , Neurônios/metabolismo , Sulfotransferases/metabolismo , Proteínas tau/metabolismo , Animais , Comportamento Animal , Células Cultivadas , Humanos , NF-kappa B/metabolismo , Fosforilação , Tauopatias/metabolismo
14.
Anal Bioanal Chem ; 407(10): 2821-8, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25711986

RESUMO

The hyperphosphorylation of tau protein is associated with the development of the neuronal pathology of Alzheimer's disease. As most conventional methods study only particular phosphorylation sites of tau, it is necessary to develop a simple and reliable assay to determine the phosphorylation of tau at multiple sites. Capillary electrophoresis (CE)-based enzymatic assays are not yet used to monitor tau phosphorylation. The present work aims to develop CE-based assays to evaluate tau phosphorylation by the glycogen synthase kinase 3-ß (GSK3ß). A novel pre-capillary CE assay was first developed. An in-capillary CE-based enzymatic assay was also used since this approach is known to be time- and cost- effective. The enzymatic reaction was monitored by quantifying the product adenosine 5'- diphosphate (ADP). The influence of two classes of glycosaminoglycan (GAG), namely heparin and heparan sulfate, on the phosphorylation reaction was also assessed. Results obtained by both CE approaches were comparable and in excellent agreement with those reported in the literature using conventional radiometric and immunoblotting methods. In fact, CE results confirmed the inductory effect of the sulfated sugars heparin and heparan sulfate on tau hyperphosphorylation, probably because of the exposition of new sites phosphorylatable by GSK3ß. This study shows that simple (no-labeling), rapid (less than 30 min per assay), and eco-friendly (no-radioactivity) CE-based kinase assays can give insight into the abnormal phosphorylation of tau. They can be extended to screen different modulators of tau phosphorylation to highlight their function and to develop effective drugs for neurodegenerative disease treatments.


Assuntos
Eletroforese Capilar/métodos , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas tau/metabolismo , Difosfato de Adenosina/análise , Difosfato de Adenosina/metabolismo , Eletroforese Capilar/instrumentação , Glicogênio Sintase Quinase 3 beta , Heparina/química , Heparina/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Humanos , Limite de Detecção , Fosforilação
15.
Atherosclerosis ; 234(1): 162-8, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24657386

RESUMO

OBJECTIVE: Phytosterolemia is a rare autosomal recessive disorder characterized by dramatically elevated circulating levels of plant sterols (PS). Phytosterolemia is believed to be responsible for severe premature atherosclerosis. The clinical, biological and molecular genetic features of 5 patients with phytosterolemia and transient severe hypercholesterolemia challenge this hypothesis. METHODS: Our patients were referred for suspected homozygous familial hypercholesterolemia. Despite the phenotype, this diagnosis was invalidated and phytosterolemia was confirmed by the identification of mutations in the ABCG5/ABCG8 transporter complex. Plasma PS were analyzed with a mass spectrometric-gas chromatographic procedure. Vascular status was assessed with carotid ultrasonography and completed (for 4 of the 5 patients) with femoral ultrasonography; additional examinations of cardiovascular status included a stress test, determination of coronary calcium score, echocardiography, non-invasive assessment of endothelium-dependent dilatation and coronarography. RESULTS: The 5 patients displayed markedly elevated levels of both ß-sitosterol and campesterol (15-30 fold higher than normal values). However, none displayed significant signs of infraclinical premature atherosclerosis (respectively at the ages of 32, 27, 29, 11 and 11 years). All patients were characterized by very high levels of total (>450 mg/dl) and LDL-cholesterol (>350 mg/dl) at diagnosis which decreased markedly on dietary intervention alone. Treatment with cholestyramine or Ezetimibe ± atorvastatin normalized cholesterol levels, although plasma PS concentrations remained elevated. CONCLUSION: The clinical and biological characteristics of our patients, considered together with reports of cases which equally lack CVD, support the contention that the premature atherosclerosis associated with phytosterolemia in some patients may be due at least in part to mechanisms independent of elevated circulating phytosterol levels.


Assuntos
Aterosclerose/etiologia , Hipercolesterolemia/complicações , Enteropatias/complicações , Erros Inatos do Metabolismo Lipídico/complicações , Fitosteróis/efeitos adversos , Adolescente , Adulto , Fatores Etários , Feminino , Humanos , Estudos Retrospectivos , Índice de Gravidade de Doença , Adulto Jovem
16.
PLoS One ; 7(11): e49336, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23189141

RESUMO

Cholesteryl ester transfer protein (CETP) deficiency causes elevated high-density lipoprotein-cholesterol (HDL-C) levels; its impact on HDL functionality however remains elusive. We compared functional and compositional properties of HDL derived from 9 Caucasian heterozygous CETP mutation carriers (splice-site mutation in intron 7 resulting in premature truncation) with those of 9 age- and sex-matched normolipidemic family controls. As expected, HDL-C levels were increased 1.5-fold, and CETP mass and activity were decreased by -31% and -38% respectively, in carriers versus non-carriers. HDL particles from carriers were enriched in CE (up to +19%, p<0.05) and depleted of triglycerides (TG; up to -54%, p<0.01), resulting in a reduced TG/CE ratio (up to 2.5-fold, p<0.01). In parallel, the apoA-I content was increased in HDL from carriers (up to +22%, p<0.05). Both the total HDL fraction and small, dense HDL3 particles from CETP-deficient subjects displayed normal antioxidative activity by attenuating low-density lipoprotein oxidation with similar efficacy on a particle mass basis as compared to control HDL3. Consistent with these data, circulating levels of systemic biomarkers of oxidative stress (8-isoprostanes) were similar between the two groups. These findings support the contention that HDL functionality is maintained in heterozygous CETP deficiency despite modifications in lipid and protein composition.


Assuntos
Proteínas de Transferência de Ésteres de Colesterol/deficiência , Proteínas de Transferência de Ésteres de Colesterol/genética , Heterozigoto , Lipoproteínas HDL/metabolismo , Adulto , Idoso , Antioxidantes/química , Antioxidantes/metabolismo , Feminino , Humanos , Lipoproteínas HDL/sangue , Lipoproteínas HDL/química , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo
17.
Biochem Biophys Res Commun ; 414(3): 587-91, 2011 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-21982770

RESUMO

The conversion of the endogenous cellular prion protein to an abnormally folded isoform is a hallmark of transmissible spongiform encephalopathies. It occurs when a misfolded prion protein contacts the cellular PrP. Among the molecular partners suggested to be involved in the misfolding process, the glycosaminoglycans seem to be good candidates. The present study was aimed to examine a possible link between PrP conversion efficiency and transcript level of Chst8 gene that encodes the carbohydrate N-acetylgalactosamine 4-O-sulfotransferase 8. Mov cells expressing ovine PrP were transfected with shRNA directed against Chst8 transcripts. Resulting clones were characterized for their Chst8 and Prnp transcript levels, and for their content in sulfated glycosaminoglycans, more particularly sulfated chondroitins. Unexpectedly, the decreased amount of Chst8 transcript induced an increase of the chondroitin sulfate percentage among total GAGs, with an increased amount of 4-O-sulfation of GalNAc residues. Upon to infection by a sheep prion, a slight amount of PrP(Sc) was observed, which rapidly disappeared upon subpassaging. Together, these findings indicate that the Chst8 transcript level affects the glycosaminoglycan environment of the cellular prion protein, and as a consequence its ability for conversion into PrP(Sc).


Assuntos
Regulação Enzimológica da Expressão Gênica , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Sulfotransferases/genética , Animais , Linhagem Celular , Glicosaminoglicanos/metabolismo , Camundongos , Proteínas PrPC/genética , Proteínas PrPSc/genética , Ovinos , Transcrição Genética
18.
Am J Cardiovasc Drugs ; 11(5): 317-25, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21699274

RESUMO

BACKGROUND AND OBJECTIVE: High-density lipoproteins (HDLs) exert multiple antiatherogenic activities including protection of low-density lipoproteins (LDLs) from oxidative stress. Beneficial effects of calcium channel blockers on cardiovascular disease may in part be related to the reduction of oxidative stress, potentially enhancing the antioxidative activity (AOX) of HDLs. This study aimed to assess the effect of 1 month's treatment with amlodipine on HDL AOX in hypertensive subjects. METHODS: This was a prospective trial of amlodipine 10 mg/day administered for 1 month in primary-care patients with hypertension (n = 28), 46% of whom were obese and 57% of whom displayed the metabolic syndrome. The main outcome measure was HDL AOX, assessed as the capacity of small, dense HDL3c particles to attenuate LDL oxidation induced in vitro by an azo initiator (AAPH). RESULTS: Mean (± SD) systolic (SBP) and diastolic (DBP) BP were reduced by amlodipine by 22.1 mmHg (± 13.2) and 10.4 mmHg (± 7.5), respectively (p < 0.001). Body mass index, waist circumference, and plasma levels of triglycerides, cholesterol, and fasting blood glucose did not change significantly. Amlodipine treatment did not modify HDL3c AOX in the whole study population; changes in AOX were, however, positively correlated with SBP (r = 0.37, p = 0.05 for maximal diene concentration; r = 0.34, p = 0.08 for LDL oxidation rate). When the population was divided into two subgroups according to the BP response to amlodipine (change in SBP below or above the median), HDL3c AOX was significantly improved in hyper-responders (BP-lowering response >22/10 mmHg) as compared with hypo-responders (BP-lowering response <22/10 mmHg: mean [± SD] change in the LDL oxidation rate in the presence of HDL3c, -6.8% [± 11.2] vs +1.9% [± 5.2], respectively, p = 0.04; maximal diene concentration, -8.6% [± 13.0] vs +1.9% [± 8.2], respectively, p < 0.05). By contrast, neither plasma concentrations of oxidized LDL, a marker of systemic oxidative stress, nor the chemical composition of HDL3c were modified between the subgroups. CONCLUSIONS: In hypertensive patients, amlodipine treatment enhanced HDL AOX in subjects who had a BP reduction that exceeded the median response. This effect appears to be secondary to the hypotensive effect, rather than to the direct antioxidant properties, of the drug.


Assuntos
Anlodipino/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Antioxidantes/análise , Pressão Sanguínea/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/uso terapêutico , Hipertensão/tratamento farmacológico , Lipoproteínas HDL3/sangue , Idoso , Anlodipino/farmacocinética , Anti-Hipertensivos/farmacocinética , Antioxidantes/química , Índice de Massa Corporal , Bloqueadores dos Canais de Cálcio/farmacocinética , Feminino , Humanos , Hipertensão/sangue , Hipertensão/etiologia , Lipoproteínas HDL3/química , Lipoproteínas LDL/análise , Lipoproteínas LDL/sangue , Lipoproteínas LDL/química , Masculino , Síndrome Metabólica/fisiopatologia , Pessoa de Meia-Idade , Obesidade/fisiopatologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos
19.
J Cell Mol Med ; 14(3): 608-20, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19243471

RESUMO

Plasma high-density lipoproteins (HDLs) protect endothelial cells against apoptosis induced by oxidized low-density lipoprotein (oxLDL). The specific component(s) of HDLs implicated in such cytoprotection remain(s) to be identified. Human microvascular endothelial cells (HMEC-1) were incubated with mildly oxLDL in the presence or absence of each of five physicochemically distinct HDL subpopulations fractionated from normolipidemic human plasma (n= 7) by isopycnic density gradient ultracentrifugation. All HDL subfractions protected HMEC-1 against oxLDL-induced primary apoptosis as revealed by nucleic acid staining, annexin V binding, quantitative DNA fragmentation, inhibition of caspase-3 activity and reduction of cytoplasmic release of cytochrome c and apoptosis-inducing factor. Small, dense HDL 3c displayed twofold superior intrinsic cytoprotective activity (as determined by mitochondrial dehydrogenase activity) relative to large, light HDL 2b on a per particle basis (P < 0.05). Equally, all HDL subfractions attenuated intracellular generation of reactive oxygen species (ROS); such anti-oxidative activity diminished from HDL 3c to HDL 2b. The HDL protein moiety, in which apolipoprotein A-I (apoA-I) predominated, accounted for approximately 70% of HDL anti-apoptotic activity. Furthermore, HDL reconstituted with apoA-I, cholesterol and phospholipid potently protected HMEC-1 from apoptosis. By contrast, modification of the content of sphingosine-1-phosphate in HDL did not significantly alter cytoprotection. We conclude that small, dense, lipid-poor HDL 3 potently protects endothelial cells from primary apoptosis and intracellular ROS generation induced by mildly oxLDL, and that apoA-I is pivotal to such protection.


Assuntos
Apoptose/efeitos dos fármacos , Lipoproteínas HDL3/farmacologia , Lipoproteínas LDL/farmacologia , Apolipoproteína A-I/sangue , Apolipoproteína A-I/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Citocromos c/metabolismo , Células Endoteliais/citologia , Humanos , Immunoblotting , Lipoproteínas HDL3/sangue , Lisofosfolipídeos/sangue , Lisofosfolipídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Esfingosina/análogos & derivados , Esfingosina/sangue , Esfingosina/farmacologia
20.
Arterioscler Thromb Vasc Biol ; 29(12): 2169-75, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19762782

RESUMO

OBJECTIVE: Small dense HDL3 particles of defined lipidome and proteome potently protect atherogenic LDL against free radical-induced oxidation; the molecular determinants of such antioxidative activity in these atheroprotective, antiinflammatory particles remain indeterminate. METHODS AND RESULTS: Formation of redox-active phosphatidylcholine hydroperoxides (PCOOH) and redox-inactive phosphatidylcholine hydroxides (PCOH) was initiated in LDL by free radical-induced oxidation. Human HDL3 inactivated LDL-derived PCOOH (-62%, P<0.01) and enhanced accumulation of PCOH (2.1-fold, P<0.05); in parallel, HDL3 accumulated minor amounts of PCOOH. Enzyme-deficient reconstituted dense HDL potently inactivated PCOOH (-43%, P<0.01). HDL3-mediated reduction of PCOOH to PCOH occurred concomitantly with oxidation of methionine residues in HDL3-apolipoprotein AI (apoAI). Preoxidation of methionine residues by chloramine T markedly attenuated PCOOH inactivation (-35%); by contrast, inhibition of HDL3-associated enzymes was without effect. PCOOH transfer rates from oxidized LDL to phospholipid liposomes progressively decreased with increment in the rigidity of the phospholipid monolayer. CONCLUSIONS: The redox status of apoAI and surface lipid rigidity represent major determinants of the potent HDL3-mediated protection of LDL against free radical-induced oxidation. Initial transfer of PCOOH to HDL3 is modulated by the surface rigidity of HDL3 particles with subsequent reduction of PCOOH to PCOH by methionine residues of apoAI.


Assuntos
Apolipoproteína A-I/metabolismo , Aterosclerose/etiologia , Inflamação/etiologia , Peróxidos Lipídicos/metabolismo , Lipoproteínas HDL3/metabolismo , Lipoproteínas LDL/metabolismo , Fosfolipídeos/metabolismo , Apolipoproteína A-I/química , Aterosclerose/metabolismo , Ésteres do Colesterol/metabolismo , Radicais Livres/metabolismo , Humanos , Técnicas In Vitro , Inflamação/metabolismo , Cinética , Metionina/química , Modelos Biológicos , Oxirredução
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA