RESUMO
Cholesterol is mostly removed from the CNS by its conversion to cerebrosterol (24(S)-hydroxycholesterol, 24(S)OH-C), which is transported to the circulation for bile formation in liver. A neurotoxic role of this oxysterol was previously demonstrated in cell culture. Here, we provide evidence that the enzyme lecithin-cholesterol acyltransferase, long known to esterify cholesterol, also produces monoesters of 24(S)OH-C. Proteoliposomes containing apolipoprotein A-I or apolipoprotein E were used to stimulate the enzyme activity and entrap the formed esters. Proteoliposomes with apolipoprotein A-I were found to be more active than those with apolipoprotein E in stimulating the production of oxysteryl esters. Cholesterol and 24(S)OH-C were found to compete for enzyme activity. High levels of haptoglobin, as those circulating during the acute inflammatory phase, inhibited 24(S)OH-C esterification. When highly neurotoxic 24(S)OH-C was treated with enzyme and proteoliposomes before incubation with differentiated SH-SY5Y cells, the neuron survival improved. The esters of 24(S)OH-C, embedded into proteoliposomes by the enzyme and isolated from unesterified 24(S)OH-C by gel filtration chromatography, did not enter the neurons in culture. These results suggest that the enzyme, in the presence of the apolipoproteins, converts 24(S)OH-C into esters restricted to the extracellular environment, thus preventing or limiting oxysterol-induced neurotoxic injuries to neurons in culture. 24-hydroxycholesterol (24(S)OH-C) is neurotoxic. The enzyme lecithin-cholesterol acyltransferase (LCAT) synthesizes monoesters of 24(S)OH-C in reaction mixtures with proteoliposomes containing phospholipids and apolipoprotein A-I or apolipoprotein E. The esters, also produced by incubation of cerebrospinal fluid only with tritiated 24(S)OH-C, are embedded into lipoproteins that do not enter neurons in culture. The enzyme activity limits the toxicity of 24-hydroxycholesterol in neuron culture.
Assuntos
Hidroxicolesteróis/antagonistas & inibidores , Hidroxicolesteróis/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Esterificação/efeitos dos fármacos , Esterificação/fisiologia , Humanos , Hidroxicolesteróis/toxicidade , Masculino , Pessoa de Meia-Idade , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/metabolismoRESUMO
Major histocompatibility complex class II mRNAs encode heterodimeric proteins involved in the presentation of exogenous antigens during an immune response. Their 3'UTRs bind a protein complex in which we identified two factors: EBP1, an ErbB3 receptor-binding protein and DRBP76, a double-stranded RNA binding nuclear protein, also known as nuclear factor 90 (NF90). Both are well-characterized regulatory factors of several mRNA molecules processing. Using either EBP1 or DRBP76/NF90-specific knockdown experiments, we established that the two proteins play a role in regulating the expression of HLA-DRA, HLA-DRB1 and HLA-DQA1 mRNAs levels. Our study represents the first indication of the existence of a functional unit that includes different transcripts involved in the adaptive immune response. We propose that the concept of 'RNA operon' may be suitable for our system in which MHCII mRNAs are modulated via interaction of their 3'UTR with same proteins.
Assuntos
Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Proteínas do Fator Nuclear 90/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Células Apresentadoras de Antígenos/imunologia , Linhagem Celular Tumoral , Citoplasma/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteínas do Fator Nuclear 90/antagonistas & inibidores , Proteínas do Fator Nuclear 90/fisiologia , Óperon , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/fisiologiaRESUMO
OBJECTIVE: In this context, our study aimed to ascertain whether the esterification of 24-hydroxycholesterol, a process heavily affected by oxidative stress, is altered in ALS. METHODS: The study examined the level of 24-hydroxycholesteryl esters in cerebrospinal fluid and plasma of 18 ALS patients by spectroscopic technique as Ultra-high performance liquid chromatography mass spectrometry (UPLC-MS). RESULTS: The level of 24-hydroxycholesteryl esters in cerebrospinal fluid was found to be lower as the brain-blood barrier was damaged. Such a level was positively correlated with the level of esters in plasma. Both cerebrospinal fluid (CSF) level and plasma level were lower in ALS patients (60.05 ± 4.24 % and 54.07 ± 20.37 % respectively) than in controls (79.51 ± 2.47 % and 80.07 ± 10.02 % respectively). CONCLUSIONS: The data suggest that the level 24-hydroxycholesteryl esters might be a new biomarker of ALS and can be measured for monitoring the disease progression.
Assuntos
Esclerose Lateral Amiotrófica , Humanos , Esclerose Lateral Amiotrófica/líquido cefalorraquidiano , Ésteres , Cromatografia Líquida , Espectrometria de Massas em Tandem , BiomarcadoresRESUMO
The acute-phase protein haptoglobin (Hpt) binds apolipoprotein A-I (ApoA-I) and impairs its action on lecithin-cholesterol acyltransferase, an enzyme that plays a key role in reverse cholesterol transport. We have previously shown that an ApoA-I mimetic peptide, P2a, displaces Hpt from ApoA-I, restoring the enzyme activity in vitro. The aim of this study was to evaluate whether P2a displaces Hpt from ApoA-I in vivo and whether this event leads to anti-inflammatory activity. Mice received subplantar injections of carrageenan. Paw volume was measured before the injection and 2, 4, 6, 24, 48, 72, and 96 h thereafter. At the same time points, concentrations of HDL cholesterol (C) and cholesterol esters (CEs) were measured by high-performance liquid chromatography, and Hpt and ApoA-I plasma levels were evaluated by enzyme-linked immunosorbent assay. Western blotting analysis for nitric-oxide synthase and cyclooxygenase (COX) isoforms was also performed on paw homogenates. CEs significantly decreased in carrageenan-treated mice during edema development and negatively correlated with the Hpt/ApoA-I ratio. P2a administration significantly restored the CE/C ratio. In addition, P2a displayed an anti-inflammatory effect on the late phase of edema with a significant reduction in COX2 expression coupled to an inhibition of prostaglandin E(2) synthesis, implying that, in the presence of P2a, CE/C ratio rescue and edema inhibition were strictly related. In conclusion, the P2a effect is due to its binding to Hpt with consequent displacement of ApoA-I that exerts anti-inflammatory activity. Therefore, it is feasible to design drugs that, by enhancing the physiological endogenous protective role of ApoA-I, may be useful in inflammation-based diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Apolipoproteína A-I/farmacologia , Ésteres do Colesterol/metabolismo , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Apolipoproteína A-I/sangue , Ciclo-Oxigenase 2/genética , Dinoprostona/biossíntese , Relação Dose-Resposta a Droga , Edema/metabolismo , Esterificação , Haptoglobinas/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Fosfatidilcolina-Esterol O-Aciltransferase/fisiologiaRESUMO
Haptoglobin (Hpt) is known to capture circulating free hemoglobin (Hb) and bind apolipoprotein (Apo) A-I or E. Here, we report that Hb can be tightly bound by most of Hpt molecules (TB-Hpt, 80%), whereas loosely bound by a minor part of them (LB-Hpt, 20%). LB-Hpt amount was significantly increased (over 60%) in patients with acute coronary syndrome. LB-Hpt bound ApoA-I and ApoE less efficiently than TB-Hpt (8- and 4-fold less, respectively) and did not affect their activity of stimulating the enzyme lecithin-cholesterol acyltransferase. LB-Hpt and TB-Hpt displayed comparable levels of nitrotyrosine residues, but differences in glycan chains. Changes in LB-Hpt level might be associated with changes in Hpt functions.
Assuntos
Haptoglobinas/metabolismo , Hemoglobinas/metabolismo , Síndrome Coronariana Aguda/sangue , Síndrome Coronariana Aguda/enzimologia , Apolipoproteína A-I/metabolismo , Apolipoproteínas E/metabolismo , Estudos de Casos e Controles , Haptoglobinas/farmacologia , Humanos , Lectinas/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Ligação ProteicaRESUMO
Haptoglobin is an acute phase glycoprotein, secreted by hepatocytes and other types of cells including keratinocytes. Haptoglobin has been suggested to impair the immune response, inhibit gelatinases in the extracellular matrix and promote angiogenesis, but its role in psoriasis is obscure to date. Changes in haptoglobin glycan structure were observed in several diseases. The aim of this study was to investigate whether haptoglobin displays glycan variations in psoriasis. We found that the pattern of plasma haptoglobin glycoforms, following two-dimensional electrophoresis, exhibited significant quantitative differences in spot intensities between patients and controls. Quantitative and qualitative differences in glycan mass, between patients and controls, were found by mass spectrometry of glycopeptides from tryptic digests of protein isolated from both patients and controls. The number of distinct fucosylated glycoforms of peptides NLFLNHSENATAK and MVSHHNLTTGATLINEQWLLTTAK was higher in patients than in controls, but no fucosylated glycan was detected on peptide VVLHPNYSQ-VDIGLIK in either case. The number of peptides with distinct triantennary and tetraantennary glycans was higher in patients than in controls. Abundance or structure of specific glycans, which are present in haptoglobin from patients and are different or missing in normal haptoglobin, might be associated with disease activity.
Assuntos
Haptoglobinas/química , Psoríase/metabolismo , Adulto , Sequência de Aminoácidos , Variação Genética , Glicopeptídeos/análise , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicosilação , Haptoglobinas/análise , Haptoglobinas/genética , Humanos , Masculino , Espectrometria de Massas , Dados de Sequência Molecular , Psoríase/genética , Tripsina/químicaRESUMO
Polyunsaturated fatty acids (PUFA) are fundamental building materials for cells and play crucial function as signaling molecules. When PUFA are used as substrates for non-enzymatic or enzymatic reactions and gut microbiota metabolism, they can generate electrophilic derivatives (called Reactive Lipid Species, RLS) that promptly form adducts with nucleophilic molecules. RLS participate in several signaling pathways, including the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, which is the key mechanism in the maintenance of redox, metabolic and protein homeostasis, as well as the regulation of inflammation. Recent studies have provided insights on the localization of enzymes that synthesise reactive oxygen or nitrogen species (ROS or RNS respectively) in plasma membrane compartments (raft/caveolae) which also harbour PUFA esters, from which free acid forms can be released by phospholipase A2 activity (PLA2), and the complex of Nrf2 with the inhibitory protein Kelch-like ECH-associated Protein 1(Keap1). Additional investigations have indicated that dietary PUFA insertion into specific plasma membrane microdomains may alter the lipid environment and thereby influence caveolar composition and cell signaling. Given that PUFA-originated RLS attack such a complex and promote the release of active Nrf2, it cannot be excluded that all the biochemical machinery for Nrf2 activation is present in caveolae, where it triggers the Nrf2-mediated adaptive response for rescuing or maintaining cellular redox homeostasis. Here, we specifically aimed to summarize current information with regard to the roles of dietary PUFA and RLS in Nrf2-mediated redox homeostasis, namely 1) their role as Nrf2 activators, 2) the significance of the in vivo conversion of PUFA into RLS and 3) the caveolar involvement in cell signaling for redox homeostasis.
Assuntos
Gorduras Insaturadas na Dieta/farmacologia , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos Insaturados/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Microbioma Gastrointestinal , Homeostase , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Metabolismo dos Lipídeos , Oxirredução , Proteínas Quinases/metabolismoRESUMO
Background: Traumatic caudal elbow luxation is an uncommon injury and it is rarely reported in the cat. Closed reduction is considered in early instance but open reduction and stabilization should be evaluated if the joint cannot be reduced or if gross instability persist. Case Description: This case report described two Domestic Shorthaired cats referred for monolateral forelimb non-weight bearing lameness caused by trauma. Clinical and radiographic examinations revealed a caudal elbow luxation in both patients. The cats were treated with closed reduction and the elbow joints stabilized at 40° of flexion by a transarticular external skeletal fixation for 18-22 days. The follow-up examinations at 2 months and at 3 years showed mild and moderate radiographic evidence of osteoarthritis, respectively, but good elbow function in both patients. Conclusion: This technique, for the treatment of the traumatic caudal elbow luxation, is easy and straightforward with few complications and to the authors' knowledge was not previously reported in cats.
Assuntos
Gatos/cirurgia , Lesões no Cotovelo , Fixação de Fratura/veterinária , Luxações Articulares/veterinária , Coxeadura Animal/cirurgia , Amplitude de Movimento Articular , Animais , Gatos/lesões , Luxações Articulares/etiologia , Luxações Articulares/cirurgia , Resultado do TratamentoRESUMO
Haptoglobin (Hpt) binds the apolipoprotein (Apo) A-I domain, which is involved in stimulating the enzyme lecithin-cholesterol acyltransferase (LCAT) for cholesterol esterification. This binding was shown to protect ApoA-I against hydroxyl radicals, thus preventing loss of ApoA-I function in enzyme stimulation. In this study, we report that Hpt is also able to bind ApoE. The Hpt binding site on the ApoE structure was mapped by using synthetic peptides, and found homologous to the Hpt binding site of ApoA-I. Hydroxyl radicals promoted in vitro the formation of ApoE-containing adducts which were detected by immunoblotting. Hpt impaired this oxidative modification whereas albumin did not. CSF from patients with multiple sclerosis or subjects without neurodegeneration contains oxidized forms of ApoE and ApoA-I similar to those observed in vitro. CSF was analyzed for its level of ApoA-I, ApoE, Hpt, cholesteryl esters, and unesterified cholesterol. The ratio of esterified with unesterified cholesterol, assumed to reflect the LCAT activity ex vivo, did not correlate with either analyzed protein, but conversely correlated with the ratio [Hpt]/([ApoE]+[ApoA-I]). The results suggest that Hpt might save the function of ApoA-I and ApoE for cholesterol esterification, a process contributing to cholesterol elimination from the brain.
Assuntos
Apolipoproteínas E/metabolismo , Química Encefálica/fisiologia , Encéfalo/metabolismo , Líquido Cefalorraquidiano/metabolismo , Colesterol/metabolismo , Esterificação/fisiologia , Haptoglobinas/metabolismo , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Apolipoproteínas E/química , Sítios de Ligação/fisiologia , Líquido Cefalorraquidiano/química , Haptoglobinas/química , Humanos , Immunoblotting , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/fisiopatologia , Degeneração Neural/líquido cefalorraquidiano , Degeneração Neural/fisiopatologia , Neuroquímica/métodos , Ligação Proteica/fisiologiaRESUMO
Oxidative stress is caused by imbalance between the production of reactive oxygen species (ROS) and biological system ability to readily detoxify the reactive intermediates or repair the resulting damage. 2-deoxy-D-ribose (dRib) is known to induce apoptosis by provoking an oxidative stress by depleting glutathione (GSH). In this paper, we elucidate the mechanisms underlying GSH depletion in response to dRib treatment. We demonstrated that the observed GSH depletion is not only due to inhibition of synthesis, by inhibiting gamma-glutamyl-cysteine synthetase, but also due to its increased efflux, by the activity of multidrug resistance associated proteins transporters. We conclude that dRib interferes with GSH homeostasis and that likely cellular oxidative stress is a consequence of GSH depletion. Various GSH fates, such as direct oxidation, lack of synthesis or of storage, characterize different kinds of oxidative stress. In the light of our observations we conclude that dRib does not induce GSH oxidation but interferes with GSH synthesis and storage. Lack of GSH allows accumulation of ROS and cells, disarmed against oxidative insults, undergo apoptosis.
Assuntos
Apoptose/fisiologia , Desoxirribose/metabolismo , Glutationa/metabolismo , Estresse Oxidativo/fisiologia , Animais , Western Blotting , Linhagem Celular , Cromatografia em Camada Fina , CamundongosRESUMO
24-hydroxycholesterol (24OH-C) is synthesized almost exclusively in neurons. This oxysterol is mostly present as ester form in both cerebrospinal fluid and plasma. The enzyme lecithin-cholesterol acyltransferase esterifies 24OH-C in the brain, and the level of 24OH-C esters in cerebrospinal fluid was found to be correlated with the level of 24OH-C esters in plasma. Decreased levels of 24OH-C esters levels were previously found in Alzheimer's disease and Amyotrophic Lateral Sclerosis. This finding was attributed to the inhibitory effect of oxidative stress on lecithin-cholesterol acyltransferase activity in neurodegenerative conditions. Data reported here show that the plasma level of 24OH-C esters is decreased also in Parkinson's disease. ROC analysis identified 69.0% of 24OH-C esterification as the threshold (AUCâ¯=â¯0.98) discriminating patients (Nâ¯=â¯19) from healthy subjects (Nâ¯=â¯19) with 100% specificity vs controls, 89.5% sensitivity, 94.7% accuracy, and 100% precision. The level of 24OH-C esters was not correlated with UPDRS I or UPDRS III when evaluated at the time of blood sampling. By contrast, it was negatively correlated with UPDRS I (râ¯=â¯-0.4984, pâ¯=â¯0.0299) after one year of follow up. Therefore, this level might represent a novel biomarker of neurodegeneration in Parkinson's disease. The biomarker level is here proposed as a measure to evaluate the severity of disease, as well as to monitor the progression of this pathology.
Assuntos
Hidroxicolesteróis/sangue , Doença de Parkinson/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Progressão da Doença , Ésteres/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Cholesterol (C) brain accumulation seems to play a role in the Alzheimer's disease (AD) pathogenesis. 24(S)-hydroxycholesterol (24OH-C) is the predominant metabolite of brain C and its synthesis is believed to represent a way to remove excess C from neurons. Previous studies showed that 24OH-C level is altered in patients with neurodegenerative diseases, including AD. Only one study demonstrated that 24OH-C esterification is altered in neurodegenerative diseases, i.e., amyotrophic lateral sclerosis. Herein we analyzed the level of 24OH-C esters (% 24OH-CE) in i) cerebrospinal fluid (CSF) and homologous serum of AD (nâ=â13) and controls (nâ=â8); ii) plasma from AD (nâ=â30), controls (nâ=â30), mild cognitive impairment (MCI) converting to AD (nâ=â34), and stable MCI (nâ=â40). The % 24OH-CE in CSF positively correlated with that in homologous serum and was lower in both CSF and blood from AD patients as compared to controls; moreover, the plasma value of % 24OH-CE was lower in MCI conv-AD than in non-converters. Kaplan Meier Survival curves revealed a significant anticipation of the disease onset in AD and MCI conv-AD subjects with the lowest % 24OH-CE values. In conclusion, the reduction of % 24OH-CE in AD and MCI conv-AD, as well as the anticipation of the disease in patients with the lowest % 24OH-CE, support a role of the cholesterol/lecithin-cholesterol acyltransferase axis in AD onset/progression. Thus, targeting brain cholesterol metabolism could be a valuable strategy to prevent AD associated cognitive decline.
Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Ésteres do Colesterol/sangue , Disfunção Cognitiva/sangue , Disfunção Cognitiva/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Apolipoproteína E4/genética , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Cromatografia Líquida , Disfunção Cognitiva/genética , Progressão da Doença , Diagnóstico Precoce , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Testes de Estado Mental e Demência , Pessoa de Meia-Idade , Prognóstico , Espectrometria de Massas em TandemRESUMO
24-Hydroxycholesterol (24OH-C) is esterified by the enzyme lecithin-cholesterol acyltransferase (LCAT) in the cerebrospinal fluid (CSF). We report here that the level of 24OH-C esters was lower in CSF of patients with amyotrophic lateral sclerosis than in healthy subjects (54% vs 68% of total 24OH-C, p=0.0005; n=8). Similarly, the level of 24OH-C esters in plasma was lower in patients than in controls (62% vs 77% of total 24OH-C; p=0.0076). The enzyme amount in CSF, as measured by densitometry of the protein band revealed by immunoblotting, was about 4-fold higher in patients than in controls (p=0.0085). As differences in the concentration of the LCAT stimulator Apolipoprotein E were not found, we hypothesized that the reduced 24OH-C esterification in CSF of patients might depend on oxidative stress. We actually found that oxidative stress reduced LCAT activity in vitro, and 24OH-C effectively stimulated the enzyme secretion from astrocytoma cells in culture. Enhanced LCAT secretion from astrocytes might represent an adaptive response to the increase of non-esterified 24OH-C percentage, aimed to avoid the accumulation of this neurotoxic compound. The low degree of 24OH-C esterification in CSF or plasma might reflect reduced activity of LCAT during neurodegeneration.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Encéfalo/enzimologia , Hidroxicolesteróis/metabolismo , Estresse Oxidativo , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Astrócitos/enzimologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Esterificação , Humanos , Hidroxicolesteróis/sangue , Hidroxicolesteróis/líquido cefalorraquidiano , Pessoa de Meia-IdadeRESUMO
Growing evidence suggests that atherogenesis is associated with inflammation or defective removal of cholesterol excess from peripheral cells. Apolipoprotein A-I [ApoA-I] activates the enzyme Lecithin-Cholesterol Acyl-Transferase to esterify cell cholesterol for transport to liver. Haptoglobin [Hpt] was previously found able to bind ApoA-I, and suggested to reduce the enzyme activation. The aim of this study was to demonstrate that enhanced levels of Hpt, as present during inflammation, are associated with low enzyme activity and increased thickness of the arterial wall. Enzyme activity and Hpt concentration were analysed in patients with rheumatoid arthritis having the same plasma levels of antioxidants (ascorbate, urate, alpha-tocopherol, retinol) or oxidation markers (nitrotyrosine, lipoperoxide) of healthy subjects. Cholesterol esterification, determined as ratio of cholesteryl esters with cholesterol in high-density lipoproteins, was lower in patients than in controls, and negatively correlated with the intima-media wall thickness of the common carotid. The ratio of Hpt with ApoA-I was negatively correlated with the enzyme activity, while positively correlated with intima-media wall thickness. The results suggest that high Hpt levels might severely impair the enzyme activity, thus contributing to cholesterol accumulation in vascular cells, and lesion formation in the endothelium.
Assuntos
Apolipoproteína A-I/fisiologia , Artrite Reumatoide/metabolismo , Colesterol/metabolismo , Haptoglobinas/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Antioxidantes/análise , Apolipoproteína A-I/sangue , Artérias Carótidas/patologia , Colesterol/sangue , Ativação Enzimática , Esterificação , Haptoglobinas/análise , Humanos , Pessoa de Meia-Idade , Oxirredução , Túnica Íntima/patologiaRESUMO
It has been demonstrated that redox homeostasis is important in the pathophysiology of several human diseases, including cardiovascular diseases. In this respect, genetic polymorphism, nutritional and environmental factors, age, lifestyle and physical activity may account for variable antioxidant defenses, which may be more or less effective at counteracting oxidative damage. Since accumulating oxidative damage may be associated with several pathologic conditions, including different cardiovascular diseases, prevention of oxidative stress appears to be a promising approach to improve such diseases. Exercise training, diets rich in antioxidants and a good control of blood glucose and lipid levels help to strengthen the physiologic antioxidant defense system, perhaps coupled to drugs capable of increasing the nitric oxide bioavailability and decreasing superoxide production. Within the next few years other therapeutic approaches will be available, such as gene therapy, which will prove to be even more effective but devoid of several important systemic side effects.
Assuntos
Antioxidantes/uso terapêutico , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Radicais Livres/metabolismo , Humanos , Óxido Nítrico/fisiologia , Estresse OxidativoRESUMO
Alteration in cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative disorders. Apolipoprotein E (ApoE) is the major component of brain lipoproteins supporting cholesterol transport. We previously reported that the acute-phase protein Haptoglobin (Hpt) binds ApoE, and influences its function in blood cholesterol homeostasis. Major aim of this study was to investigate whether Hpt influences the mechanisms by which cholesterol is shuttled from astrocytes to neurons. In detail it was studied Hpt effect on ApoE-dependent cholesterol efflux from astrocytes and ApoE-mediated cholesterol incorporation in neurons. We report here that Hpt impairs ApoE-mediated cholesterol uptake in human neuroblastoma cell line SH-SY5Y, and limits the toxicity of a massive concentration of cholesterol for these cells, while it does not affect cholesterol efflux from the human glioblastoma-astrocytoma cell line U-87 MG. As aging is the most important non-genetic risk factor for various neurodegenerative disorders, and our results suggest that Hpt modulates ApoE functions, we evaluated the Hpt and ApoE expression profiles in cerebral cortex and hippocampus of adolescent (2 months), adult (5 and 8 months), and middle-aged (16 months) rats. Hpt mRNA level was higher in hippocampus of 8 and 16 month-old than in 2-month old rats (p < 0.05), and Hpt concentration increased with the age from adolescence to middle-age (p < 0.001). ApoE concentration, in hippocampus, was higher (p < 0.001) in 5 month-old rats compared to 2 month but did not further change with aging. No age-related changes of Hpt (protein and mRNA) were found in the cortex. Our results suggest that aging is associated with changes, particularly in the hippocampus, in the Hpt/ApoE ratio. Age-related changes in the concentration of Hpt were also found in human cerebrospinal fluids. The age-related changes might affect neuronal function and survival in brain, and have important implications in brain pathophysiology.
RESUMO
Beta-amyloid accumulation in brain is a driving force for Alzheimer's disease pathogenesis. Apolipoprotein E (ApoE) represents a critical player in beta-amyloid homeostasis, but its role in disease progression is controversial. We previously reported that the acute-phase protein haptoglobin binds ApoE and impairs its function in cholesterol homeostasis. The major aims of this study were to characterize the binding of haptoglobin to beta-amyloid, and to evaluate whether haptoglobin affects ApoE binding to beta-amyloid. Haptoglobin is here reported to form a complex with beta-amyloid as shown by immunoblotting experiments with purified proteins, or by its immunoprecipitation in brain tissues from patients with Alzheimer's disease. The interaction between ApoE and beta-amyloid was previously shown to be crucial for limiting beta-amyloid neurotoxicity and for promoting its clearance. We demonstrate that haptoglobin, rather than impairing ApoE binding to beta-amyloid, promotes to a different extent the formation of the complex between beta-amyloid and ApoE2 or ApoE3 or ApoE4. Our data suggest that haptoglobin and ApoE functions in brain should be evaluated taking into account their mutual interaction with beta-amyloid. Hence, the risk of developing Alzheimer's disease might not only be linked to the different ApoE isoforms, but also rely on the level of critical ligands, such as haptoglobin.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Haptoglobinas/metabolismo , Adulto , Idoso , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Análise de Variância , Animais , Células CHO , Cricetulus , Ensaio de Imunoadsorção Enzimática , Feminino , Haptoglobinas/genética , Humanos , Imunoprecipitação , Masculino , Pessoa de Meia-Idade , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , TransfecçãoRESUMO
Apolipoprotein A-I and Apolipoprotein E promote different steps of reverse cholesterol transport, including lecithin-cholesterol acyltransferase stimulation. Our aim was to study the changes in the levels of Apolipoprotein A-I, Apolipoprotein E, and lecithin-cholesterol acyltransferase activity during atherosclerosis progression in rabbits. Quantitative echocardiographic parameters were analyzed in order to evaluate, for the first time, whether atherosclerosis progression in rabbit is associated to apolipoproteins changes and alteration of indices of cardiac function, such as systolic strain and strain rate of the left ventricle. Atherosclerosis was induced by feeding rabbits for 8 weeks with 2 % cholesterol diet. The HDL levels of cholesterol and cholesteryl esters were measured by HPLC. The lecithin-cholesterol acyltransferase activity was evaluated both ex vivo, as cholesteryl esters/cholesterol molar ratio, and in vitro. Apolipoproteins levels were analyzed by ELISA. The HDL levels of cholesterol and cholesteryl esters increased, during treatment, up to 3.7- and 2.5-fold, respectively, compared to control animals. The lecithin-cholesterol acyltransferase activity in vitro was halved after 4 weeks. During cholesterol treatment, Apolipoprotein A-I level significantly decreased, whereas Apolipoprotein E concentration markedly increased. The molar ratio Apolipoprotein E/Apolipoprotein A-I was negatively correlated with the enzyme activity, and positively correlated with both increases in the intima-media thickness of common carotid wall and cardiac dysfunction signs, such as systolic strain and strain rate of the left ventricle.
Assuntos
Apolipoproteína A-I/sangue , Apolipoproteínas E/sangue , Aterosclerose/enzimologia , Ésteres do Colesterol/sangue , Fosfatidilcolina-Esterol O-Aciltransferase/sangue , Animais , Aorta Torácica/patologia , Aterosclerose/sangue , Aterosclerose/patologia , Aterosclerose/fisiopatologia , HDL-Colesterol/sangue , Progressão da Doença , Esterificação , Masculino , Miocárdio/patologia , Placa Aterosclerótica/patologia , Coelhos , Volume Sistólico , Túnica Íntima/patologia , Função VentricularRESUMO
Improved diagnosis of psoriasis, by new biomarkers, is required for evaluating the progression rate of the disease and the response to treatment. Haptoglobin (Hpt), a glycoprotein secreted by hepatocytes and other types of cells including keratinocytes, was found with glycan changes in psoriasis and other diseases. We previously reported that Hpt isolated from plasma of psoriatic patients is more fucosylated than Hpt of healthy subjects. The aim of this study was to compare the glycosylation pattern of Hpt isolated from skin scales or plasma of patients with psoriasis with that of Hpt from cornified epidermal layer or plasma of healthy subjects. High performance liquid chromatography analysis of the glycans isolated from the protein backbone revealed that glycan patterns from skin and plasma of patients were similar, and mostly displayed quantitative rather than qualitative differences from normal pattern. Biotin-labeled lectins were used to evaluate quantitative differences in the glycoforms of Hpt from plasma and psoriatic skin scales. Hpt from skin and plasma of patients showed more fucosylated and branched glycans than Hpt from plasma of healthy subjects. Tryptic glycopeptides of Hpt were also analyzed by mass spectrometry, and a decreased amount of sialylated glycan chains was found in glycopeptides of skin Hpt, as compared with Hpt from plasma. High levels of glycans with fucosylated and tetra-antennary chains were detected on the peptide NLFLNHSENATAK from Hpt of psoriatic patients. Our data demonstrate that specific changes in glycan structures of Hpt, such as enhanced glycan branching and fucose content, are associated with psoriasis, and that differences between circulating and skin Hpt do exist. A lower extent of glycan fucosylation and branching was found in Hpt from plasma of patients in disease remission. Altered glycoforms might reflect changes of Hpt function in the skin, and could be used as markers of the disease.
Assuntos
Haptoglobinas/metabolismo , Psoríase/metabolismo , Adulto , Sequência de Aminoácidos , Biomarcadores/sangue , Biomarcadores/metabolismo , Cromatografia Líquida de Alta Pressão , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicosilação , Haptoglobinas/química , Humanos , Lectinas/química , Lectinas/metabolismo , Espectrometria de Massas , Polissacarídeos/sangue , Polissacarídeos/metabolismoRESUMO
Haptoglobin (Hpt) binds apolipoprotein A-I (ApoA-I), and impairs its stimulation of lecithin:cholesterol acyltransferase (LCAT). LCAT plays a major role in reverse cholesterol transport (RCT). Apolipoprotein E (ApoE), like ApoA-I, promotes different steps of RCT, including LCAT stimulation. ApoE contains amino acid sequences that are homologous with the ApoA-I region bound by Hpt and are involved in the interaction with LCAT. Therefore, Hpt was expected to also bind ApoE, and inhibit the ApoE stimulatory effect on LCAT. Western blotting and ELISA experiments demonstrated that the Hpt beta-subunit binds ApoE. The affinity of Hpt for ApoE was higher than that for ApoA-I. High ratios of Hpt with either apolipoprotein, such as those associated with the acute phase of inflammation, inhibited, in vitro, the stimulatory effect of ApoE on the cholesterol esterification activity of LCAT. Hpt also impaired human hepatoblastoma-derived cell uptake of [(3)H]cholesterol from proteoliposomes containing ApoE or ApoA-I. We suggest that the interaction between Hpt and ApoE represents a mechanism by which inflammation affects atherosclerosis progression. Hpt might influence ApoE function in processes other than RCT.