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AapoA-I, the main protein of high-density lipoprotein, plays a key role in the biogenesis and atheroprotective properties of high-density lipoprotein. We showed previously that a naturally occurring apoA-I mutation, L178P, induces major defects in protein's structural integrity and functions that may underlie the increased cardiovascular risk observed in carriers of the mutation. Here, a library of marketed drugs (956 compounds) was screened against apoA-I[L178P] to identify molecules that can stabilize the normal conformation of apoA-I. Screening was performed by the thermal shift assay in the presence of fluorescent dye SYPRO Orange. As an orthogonal assay, we monitored the change in fluorescence intensity of 8-anilinonaphthalene-1-sulfonic acid upon binding on hydrophobic sites on apoA-I. Screening identified four potential structure correctors. Subsequent analysis of the concentration-dependent effect of these compounds on secondary structure and thermodynamic stability of WT apoA-I and apoA-I[L178P] (assessed by thermal shift assay and circular dichroism spectroscopy), as well as on macrophage viability, narrowed the potential structure correctors to two, the drugs atorvastatin and bexarotene. Functional analysis showed that these two compounds can restore the defective capacity of apoA-I[L178P] to promote cholesterol removal from macrophages, an important step for atheroprotection. Computational docking suggested that both drugs target a positively charged cavity in apoA-I, formed between helix 1/2 and helix 5, and make extensive interactions that could underlie thermodynamic stabilization. Overall, our findings indicate that small molecules can correct defective apoA-I structure and function and may lead to novel therapeutic approaches for apoA-I-related dyslipidemias and increased cardiovascular risk.
Assuntos
Apolipoproteína A-I , Apolipoproteína A-I/metabolismo , Apolipoproteína A-I/química , Humanos , Animais , Camundongos , Avaliação Pré-Clínica de Medicamentos , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Paraoxonase-1 (PON1), a serum antioxidant enzyme, has been implicated in Alzheimer's disease (AD) pathogenesis that involves early oxidative damage. Corinthian currants and their components have been shown to display antioxidant and other neuroprotective effects in AD. We evaluated the effect of a Corinthian currant paste-supplemented diet (CurD), provided to 1-month-old 5xFAD mice for 1, 3, and 6 months, on PON1 activity and levels of oxidation markers in serum and the brain of mice as compared to a control diet (ConD) or glucose/fructose-matched diet (GFD). Administration of CurD for 1 month increased PON1 activity and decreased oxidized lipid levels in serum compared to ConD and GFD. Longer-term administration of CurD did not, however, affect serum PON1 activity and oxidized lipid levels. Furthermore, CurD administered for 1 and 3 months, but not for 6 months, increased PON1 activity and decreased free radical levels in the cortex of mice compared to ConD and GFD. To probe the mechanism for the increased PON1 activity in mice, we studied the effect of Corinthian currant polar phenolic extract on PON1 activity secreted by Huh-7 hepatocytes or HEK293 cells transfected with a PON1-expressing plasmid. Incubation of cells with the extract led to a dose-dependent increase of secreted PON1 activity, which was attributed to increased cellular PON1 expression. Collectively, our findings suggest that phenolics in Corinthian currants can increase the hepatic expression and activity of antioxidant enzyme PON1 and that a Corinthian currant-supplemented diet during the early stages of AD in mice reduces brain oxidative stress.
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Doença de Alzheimer , Antioxidantes , Arildialquilfosfatase , Encéfalo , Modelos Animais de Doenças , Animais , Arildialquilfosfatase/metabolismo , Arildialquilfosfatase/genética , Doença de Alzheimer/metabolismo , Camundongos , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Humanos , Camundongos Transgênicos , Estresse Oxidativo/efeitos dos fármacos , MasculinoRESUMO
OBJECTIVE: Chronic inflammatory diseases, like Systemic Lupus Erythematosus (SLE), carry an increased risk for atherosclerosis and cardiovascular events, accompanied by impairment of atheroprotective properties of high-density lipoprotein (HDL). In SLE, serum BAFF (B cell-activating factor), a cytokine implicated in disease progression, has been correlated with subclinical atherosclerosis. We investigated the impact of treatment with belimumab -an anti-BAFF monoclonal antibody- on HDL atheroprotective properties and composition in SLE patients. METHODS: Serum samples were collected from 35 SLE patients with active disease despite conventional therapy, before and after 6-month add-on treatment with belimumab, and 26 matched healthy individuals. We measured cholesterol efflux and antioxidant capacities, paraoxonase-1 activity, serum amyloid A1, myeloperoxidase and lipid peroxidation product levels of HDL. LC-MS/MS was performed to analyze the HDL lipidome. RESULTS: Following treatment with belimumab, cholesterol efflux and antioxidant capacities of HDL were significantly increased in SLE patients and restored to levels of controls. HDL-associated paraoxonase-1 activity was also increased, whereas lipid peroxidation products were decreased following treatment. HDL cholesterol efflux and antioxidant capacities correlated negatively with the disease activity. Changes were noted in the HDL lipidome of SLE patients following belimumab treatment, as well as between SLE patients and healthy individuals, and specific changes in lipid species correlated with functional parameters of HDL. CONCLUSIONS: HDL of SLE patients with active disease displays impaired atheroprotective properties accompanied by distinct lipidomic signature compared with controls. Belimumab treatment may improve the HDL atheroprotective properties and modify the HDL lipidomic signature in SLE patients, thus potentially mitigating atherosclerosis development.
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The APOE4 variant of apolipoprotein E (apoE) is the most prevalent genetic risk allele associated with late-onset Alzheimer's disease (AD). ApoE interacts with complement regulator factor H (FH), but the role of this interaction in AD pathogenesis is unknown. Here we elucidate the mechanism by which isoform-specific binding of apoE to FH alters Aß1-42-mediated neurotoxicity and clearance. Flow cytometry and transcriptomic analysis reveal that apoE and FH reduce binding of Aß1-42 to complement receptor 3 (CR3) and subsequent phagocytosis by microglia which alters expression of genes involved in AD. Moreover, FH forms complement-resistant oligomers with apoE/Aß1-42 complexes and the formation of these complexes is isoform specific with apoE2 and apoE3 showing higher affinity to FH than apoE4. These FH/apoE complexes reduce Aß1-42 oligomerization and toxicity, and colocalize with complement activator C1q deposited on Aß plaques in the brain. These findings provide an important mechanistic insight into AD pathogenesis and explain how the strongest genetic risk factor for AD predisposes for neuroinflammation in the early stages of the disease pathology.
Assuntos
Doença de Alzheimer , Apolipoproteína E4 , Humanos , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Fator H do Complemento/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doenças Neuroinflamatórias , Apolipoproteínas E/química , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Peptídeos beta-Amiloides/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
The current study focuses on the development of innovative and highly-stable curcumin (CUR)-based therapeutics by encapsulating CUR in biocompatible poly(n-butyl acrylate)-block-poly(oligo(ethylene glycol) methyl ether acrylate) (PnBA-b-POEGA) micelles. State-of-the-art methods were used to investigate the encapsulation of CUR in PnBA-b-POEGA micelles and the potential of ultrasound to enhance the release of encapsulated CUR. Dynamic light scattering (DLS), attenuated total reflection Fourier transform infrared (ATR-FTIR), and ultraviolet-visible (UV-Vis) spectroscopies confirmed the successful encapsulation of CUR within the hydrophobic domains of the copolymers, resulting in the formation of distinct and robust drug/polymer nanostructures. The exceptional stability of the CUR-loaded PnBA-b-POEGA nanocarriers over a period of 210 days was also demonstrated by proton nuclear magnetic resonance (1H-NMR) spectroscopy studies. A comprehensive 2D NMR characterization of the CUR-loaded nanocarriers authenticated the presence of CUR within the micelles, and unveiled the intricate nature of the drug-polymer intermolecular interactions. The UV-Vis results also indicated high encapsulation efficiency values for the CUR-loaded nanocarriers and revealed a significant influence of ultrasound on the release profile of CUR. The present research provides new understanding of the encapsulation and release mechanisms of CUR within biocompatible diblock copolymers and has significant implications for the advancement of safe and effective CUR-based therapeutics.
Assuntos
Antineoplásicos , Curcumina , Curcumina/química , Polímeros/química , Micelas , Antineoplásicos/química , Portadores de Fármacos/química , Polietilenoglicóis/químicaRESUMO
Three common Apolipoprotein E isoforms, ApoE2, ApoE3, and ApoE4, are key regulators of lipid homeostasis, among other functions. Apolipoprotein E can interact with amyloid proteins. The isoforms differ by one or two residues at positions 112 and 158, and possess distinct structural conformations and functions, leading to isoform-specific roles in amyloid-based neurodegenerative diseases. Over 30 different amyloid proteins have been found to share similar characteristics of structure and toxicity, suggesting a common interactome. The molecular and genetic interactions of ApoE with amyloid proteins have been extensively studied in neurodegenerative diseases, but have not yet been well connected and clarified. Here we summarize essential features of the interactions between ApoE and different amyloid proteins, identify gaps in the understanding of the interactome and propose the general interaction mechanism between ApoE isoforms and amyloid proteins. Perhaps more importantly, this review outlines what we can learn from the interactome of ApoE and amyloid proteins; that is the need to see both ApoE and amyloid proteins as a basis to understand neurodegenerative diseases.
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BACKGROUND/AIMS: Impaired high-density lipoprotein (HDL) function and composition are more strongly related to cardiovascular morbidity than HDL concentration. However, it is unclear whether HDL function and composition predict ischemic stroke severity and outcome. We aimed to evaluate these associations. METHODS: We prospectively studied 199 consecutive patients who were admitted with acute ischemic stroke. The severity of stroke was evaluated at admission with the National Institutes of Health Stroke Scale (NIHSS). Severe stroke was defined as NIHSS ≥ 5. The outcome was assessed with dependency at discharge (modified Rankin scale 2-5) and in-hospital mortality. Cholesterol efflux capacity (CEC), phospholipid levels, lecithin:cholesterol acyl transferase (LCAT)-phospholipase activity, paraoxonase-1 (PON1)-arylesterase activity and serum amyloid A1 (SAA1) content of HDL were measured. RESULTS: CEC, phospholipid levels and LCAT-phospholipase activity of HDL were lower and SAA1 content of HDL was higher in patients with severe stroke. Patients who were dependent at discharge had lower CEC, PON1-arylesterase activity, phospholipid content and LCAT-phospholipase activity of HDL and higher HDL-SAA1 content. Independent predictors of dependency at discharge were the NIHSS at admission (RR 2.60, 95% CI 1.39-4.87), lipid-lowering treatment (RR 0.17, 95% CI 0.01-0.75), HDL-CEC (RR 0.21, 95% CI 0.05-0.87) and HDL-associated PON1-arylesterase activity (RR 0.95, 95% CI 0.91-0.99). In patients who died during hospitalization, phospholipids, LCAT-phospholipase and PON1-arylesterase activities of HDL were lower. CONCLUSIONS: Changes in CEC and composition of HDL appear to be associated with the severity and outcome of acute ischemic stroke and could represent biomarkers that may inform risk stratification and management strategies in these patients.
Assuntos
Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Humanos , HDL-Colesterol , Isquemia Encefálica/diagnóstico , Fosfolipídeos , Acidente Vascular Cerebral/diagnóstico , Fosfolipases , ArildialquilfosfataseRESUMO
Alzheimer's disease (AD) is associated with brain amyloid-ß (Aß) peptide accumulation and neuroinflammation. Currants, a low glycemic index dried fruit, and their components display pleiotropic neuroprotective effects in AD. We examined how diet containing 5% Corinthian currant paste (CurD) administered in 1-month-old 5xFAD mice for 1, 3, and 6 months affects Aß levels and neuroinflammation in comparison to control diet (ConD) or sugar-matched diet containing 3.5% glucose/fructose (GFD). No change in serum glucose or insulin levels was observed among the three groups. CurD administered for 3 months reduced brain Aß42 levels in male mice as compared to ConD and GFD, but after 6 months, Aß42 levels were increased in mice both on CurD and GFD compared to ConD. CurD for 3 months also reduced TNFα and IL-1ß levels in male and female mouse cortex homogenates compared to ConD and GFD. However, after 6 months, TNFα levels were increased in cortex homogenates of mice both on CurD and GFD as compared to ConD. A similar pattern was observed for TNFα-expressing cells, mostly co-expressing the microglial marker CD11b, in mouse hippocampus. IL-1ß levels were similarly increased in the brain of all groups after 6 months. Furthermore, a time dependent decrease of secreted TNFα levels was found in BV2 microglial cells treated with currant phenolic extract as compared to glucose/fructose solution. Overall, our findings suggest that a short-term currant consumption reduces neuroinflammation in 5xFAD mice as compared to sugar-matched or control diet, but longer-term intake of currant or sugar-matched diet enhances neuroinflammation.
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Doença de Alzheimer , Animais , Masculino , Feminino , Camundongos , Doença de Alzheimer/terapia , Fator de Necrose Tumoral alfa , Índice Glicêmico , Doenças Neuroinflamatórias , Camundongos Transgênicos , Peptídeos beta-Amiloides , Microglia , Modelos Animais de Doenças , Dieta , Frutose , Glucose , AçúcaresRESUMO
Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) regulates the cell-surface localization of LDL receptors in hepatocytes and is associated with LDL and lipoprotein(a) [Lp(a)] uptake, reducing blood concentrations. However, the connection between PCSK9 and HDL is unclear. Here, we investigated the association of plasma PCSK9 with HDL subpopulations and examined the effects of PCSK9 on the atheroprotective function of HDL. We examined the association of PCSK9 with HDL in apoB-depleted plasma by ELISA, native PAGE, and immunoblotting. Our analyses showed that upon apoB-depletion, total circulating PCSK9 levels were 32% of those observed in normolipidemic plasma, and only 6% of PCSK9 in the apoB-depleted plasma, including both the mature and furin-cleaved forms, was associated with HDL. We also show human recombinant PCSK9 abolished the capacity of reconstituted HDL to reduce the formation of ROS in endothelial cells, while a PCSK9-blocking antibody enhanced the capacity of human HDL (in apoB-depleted plasma) to reduce ROS formation in endothelial cells and promote endothelial cell migration. Overall, our findings suggest that PCSK9 is only minimally associated with HDL particles, but PCSK9 in apoB-depleted plasma can affect the atheroprotective properties of HDL related to preservation of endothelial function. This study contributes to the elucidation of the pathophysiological role of plasma PCSK9 and highlights further the anti-atherosclerotic effect of PCSK9 inhibition.
Assuntos
Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Humanos , Apolipoproteínas B , Células Endoteliais/metabolismo , Furina , Lipoproteína(a) , Pró-Proteína Convertases/metabolismo , Espécies Reativas de Oxigênio , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , SubtilisinasRESUMO
Nanoparticles (NPs) based on the proteolytic enzyme trypsin (TRY) were prepared by a biocompatible methodology. TRY co-assembled with the anionic polysaccharide chondroitin sulfate (CS) in complexes with well-defined distributions of radii in the range of 100-200 nm by electrostatic complexation at acidic conditions. At pH 7 the complexes were unstable and lost their monomodal size distribution which is potentially related to TRY's weak positive net surface charge and a large negative charge patch that forms at neutral pH. Thermal treatment at conditions which were not expected to interfere with TRY's proteolytic activity was used to stabilize the complexes into NPs that resisted disintegration at pH 7 taking advantage of the ability of the TRY globules to thermally aggregate. The secondary conformation of TRY within the NPs was found fairly unperturbed even after thermal treatment which is crucial for its physiological function. The CS-TRY NPs could bind and encapsulate the bioactive substances curcumin (CUR) and ß-carotene (ß-C) owing to TRY's hydrophobic domains. The CS-TRY NPs may be considered as a platform for the immobilized active enzyme and multifunctional NPs for hydrophobic bioactive compounds.
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Curcumina , Nanopartículas , Sulfatos de Condroitina/química , Curcumina/química , Portadores de Fármacos/química , Nanopartículas/química , Tamanho da Partícula , TripsinaRESUMO
INTRODUCTION: Atherosclerotic Coronary Artery Disease (ASCAD) is the leading cause of mortality worldwide. Novel therapeutic approaches aiming to improve the atheroprotective functions of High Density Lipoprotein (HDL) include the use of reconstituted HDL forms containing human apolipoprotein A-I (rHDL-apoA-I). Given the strong atheroprotective properties of apolipoprotein E3 (apoE3), rHDL-apoE3 may represent an attractive yet largely unexplored therapeutic agent. OBJECTIVE: To evaluate the atheroprotective potential of rHDL-apoE3 starting with the unbiased assessment of global transcriptome effects and focusing on endothelial cell (EC) migration as a critical process in re-endothelialization and atherosclerosis prevention. The cellular, molecular and functional effects of rHDL-apoE3 on EC migration-associated pathways were assessed, as well as the potential translatability of these findings in vivo. METHODS: Human Aortic ECs (HAEC) were treated with rHDL-apoE3 and total RNA was analyzed by whole genome microarrays. Expression and phosphorylation changes of key EC migration-associated molecules were validated by qRT-PCR and Western blot analysis in primary HAEC, Human Coronary Artery ECs (HCAEC) and the human EA.hy926 EC line. The capacity of rHDL-apoE3 to stimulate EC migration was assessed by wound healing and transwell migration assays. The contribution of MEK1/2, PI3K and the transcription factor ID1 in rHDL-apoE3-induced EC migration and activation of EC migration-related effectors was assessed using specific inhibitors (PD98059: MEK1/2, LY294002: PI3K) and siRNA-mediated gene silencing, respectively. The capacity of rHDL-apoE3 to improve vascular permeability and hypercholesterolemia in vivo was tested in a mouse model of hypercholesterolemia (apoE KO mice) using Evans Blue assays and lipid/lipoprotein analysis in the serum, respectively. RESULTS: rHDL-apoE3 induced significant expression changes in 198 genes of HAEC mainly involved in re-endothelialization and atherosclerosis-associated functions. The most pronounced effect was observed for EC migration, with 42/198 genes being involved in the following EC migration-related pathways: 1) MEK/ERK, 2) PI3K/AKT/eNOS-MMP2/9, 3) RHO-GTPases, 4) integrin. rHDL-apoE3 induced changes in 24 representative transcripts of these pathways in HAEC, increasing the expression of their key proteins PIK3CG, EFNB2, ID1 and FLT1 in HCAEC and EA.hy926 cells. In addition, rHDL-apoE3 stimulated migration of HCAEC and EA.hy926 cells, and the migration was markedly attenuated in the presence of PD98059 or LY294002. rHDL-apoE3 also increased the phosphorylation of ERK1/2, AKT, eNOS and p38 MAPK in these cells, while PD98059 and LY294002 inhibited rHDL-apoE3-induced phosphorylation of ERK1/2, AKT and p38 MAPK, respectively. LY had no effect on rHDL-apoE3-mediated eNOS phosphorylation. ID1 siRNA markedly decreased EA.hy926 cell migration by inhibiting rHDL-apoE3-triggered ERK1/2 and AKT phosphorylation. Finally, administration of a single dose of rHDL-apoE3 in apoE KO mice markedly improved vascular permeability as demonstrated by the reduced concentration of Evans Blue dye in tissues such as the stomach, the tongue and the urinary bladder and ameliorated hypercholesterolemia. CONCLUSIONS: rHDL-apoE3 significantly enhanced EC migration in vitro, predominantly via overexpression of ID1 and subsequent activation of MEK1/2 and PI3K, and their downstream targets ERK1/2, AKT and p38 MAPK, respectively, and improved vascular permeability in vivo. These novel insights into the rHDL-apoE3 functions suggest a potential clinical use to promote re-endothelialization and retard development of atherosclerosis.
Assuntos
Apolipoproteína E3/farmacologia , Células Endoteliais/efeitos dos fármacos , Lipoproteínas HDL/farmacologia , Animais , Apolipoproteína E3/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/fisiologia , Humanos , Proteína 1 Inibidora de Diferenciação/antagonistas & inibidores , Proteína 1 Inibidora de Diferenciação/efeitos dos fármacos , Proteína 1 Inibidora de Diferenciação/genética , Lipoproteínas HDL/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The inverse association between plasma HDL cholesterol (HDL-C) levels and risk for cardiovascular disease (CVD) has been demonstrated by numerous epidemiological studies. However, efforts to reduce CVD risk by pharmaceutically manipulating HDL-C levels failed and refused the HDL hypothesis. HDL-C levels in the general population are highly heterogeneous and are determined by a combination of genetic and environmental factors. Insights into the causes of HDL-C heterogeneity came from the study of monogenic HDL deficiency syndromes but also from genome wide association and Μendelian randomization studies which revealed the contribution of a large number of loci to low or high HDL-C cases in the general or in restricted ethnic populations. Furthermore, HDL-C levels in the plasma are under the control of transcription factor families acting primarily in the liver including members of the hormone nuclear receptors (PPARs, LXRs, HNF-4) and forkhead box proteins (FOXO1-4) and activating transcription factors (ATFs). The effects of certain lipid lowering drugs used today are based on the modulation of the activity of specific members of these transcription factors. During the past decade, the roles of small or long non-coding RNAs acting post-transcriptionally on the expression of HDL genes have emerged and provided novel insights into HDL regulation and new opportunities for therapeutic interventions. In the present review we summarize recent progress made in the genetics and the regulation (transcriptional and post-transcriptional) of HDL metabolism.
Assuntos
HDL-Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Fígado/metabolismo , RNA Longo não Codificante/genética , Fatores Ativadores da Transcrição/sangue , Fatores Ativadores da Transcrição/genética , HDL-Colesterol/sangue , HDL-Colesterol/genética , Fatores de Transcrição Forkhead/sangue , Fatores de Transcrição Forkhead/genética , Heterogeneidade Genética , Fatores de Risco de Doenças Cardíacas , Humanos , Lipoproteínas HDL/sangue , Lipoproteínas HDL/genética , Fígado/patologia , RNA Longo não Codificante/sangue , Receptores Citoplasmáticos e Nucleares/sangue , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
Polar phenols found in plant foods have been suggested to act protectively against pathogenic processes underlying Alzheimer's disease (AD), such as oxidative stress. The major risk factor for AD is apolipoprotein E4 (apoE4) and apoE4 forms can affect AD-related processes. It was shown previously that the hereditary apoE4 mutant apoE4[L28P], as well as the apoE4 fragment apoE4-165, induce neuronal oxidative stress. The effect of polar phenols on AD-related pathogenic functions of apoE4 forms is largely unexplored. The aim was to examine the effect of Corinthian currant polar phenolic extract and specific polar phenols resveratrol, quercetin, kaempferol and epigallocatechin gallate on AD-related functions of apoE4 forms. The polar phenolic extract and the individual compounds restored the viability of human neuroblastoma SK-N-SH cells in the presence of lipoprotein-associated apoE4[L28P] and prevented changes in cellular redox status. Furthermore, resveratrol, quercetin, kaempferol and epigallocatechin gallate prevented redox status changes induced by Aß42 uptake in SK-N-SH cells treated with lipid-free apoE4[L28P] or apoE4-165. Investigation of the molecular mechanism of action of these polar phenols showed that resveratrol prevented cellular Aß42 uptake via changes in cell membrane fluidity. Interestingly, kaempferol prevented cellular Aß42 uptake by apoE4[L28P], but not by apoE4-165, due to a modulating effect on apoE4[L28P] secondary structure and stability. The action of quercetin and epigallocatechin gallate could be attributed to free radical-scavenging or other protective activity. Overall, it is shown for the first time that natural compounds could modify the structure of apoE4 forms and ameliorate AD-related pathogenic effects of apoE4 forms.
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Doença de Alzheimer , Apolipoproteína E4 , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Peptídeos beta-Amiloides , Apolipoproteína E4/genética , Humanos , Neurônios , Fenóis/farmacologiaRESUMO
The focus of this study is the development of highly stable losartan potassium (LSR) polymeric nanocarriers. Two novel amphiphilic poly(n-butyl acrylate)-block-poly(oligo(ethylene glycol) methyl ether acrylate) (PnBA-b-POEGA) copolymers with different molecular weight (Mw) of PnBA are synthesized via reversible addition fragmentation chain transfer (RAFT) polymerization, followed by the encapsulation of LSR into both PnBA-b-POEGA micelles. Based on dynamic light scattering (DLS), the PnBA30-b-POEGA70 and PnBA27-b-POEGA73 (where the subscripts denote wt.% composition of the components) copolymers formed micelles of 10 nm and 24 nm in water. The LSR-loaded PnBA-b-POEGA nanocarriers presented increased size and greater mass nanostructures compared to empty micelles, implying the successful loading of LSR into the inner hydrophobic domains. A thorough NMR (nuclear magnetic resonance) characterization of the LSR-loaded PnBA-b-POEGA nanocarriers was conducted. Strong intermolecular interactions between the biphenyl ring and the butyl chain of LSR with the methylene signals of PnBA were evidenced by 2D-NOESY experiments. The highest hydrophobicity of the PnBA27-b-POEGA73 micelles contributed to an efficient encapsulation of LSR into the micelles exhibiting a greater value of %EE compared to PnBA30-b-POEGA70 + 50% LSR nanocarriers. Ultrasound release profiles of LSR signified that a great amount of the encapsulated LSR is strongly attached to both PnBA30-b-POEGA70 and PnBA27-b-POEGA73 micelles.
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Naturally occurring point mutations in apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), may affect plasma HDL-cholesterol levels and cardiovascular risk. Here, we evaluated the effect of human apoA-I mutations L144R (associated with low HDL-cholesterol), L178P (associated with low HDL-cholesterol and increased cardiovascular risk) and A164S (associated with increased cardiovascular risk and mortality without low HDL-cholesterol) on the structural integrity and functions of lipid-free and lipoprotein-associated apoA-I in an effort to explain the phenotypes of subjects carrying these mutations. All three mutants, in lipid-free form, presented structural and thermodynamic aberrations, with apoA-I[L178P] presenting the greatest thermodynamic destabilization. Additionally, apoA-I[L178P] displayed reduced ABCA1-mediated cholesterol efflux capacity. When in reconstituted HDL (rHDL), apoA-I[L144R] and apoA-I[L178P] were more thermodynamically destabilized compared to wild-type apoA-I, both displayed reduced SR-BI-mediated cholesterol efflux capacity and apoA-I[L144R] showed severe LCAT activation defect. ApoA-I[A164S] was thermodynamically unaffected when in rHDL, but exhibited a series of functional defects. Specifically, it had reduced ABCG1-mediated cholesterol and 7-ketocholesterol efflux capacity, failed to reduce ROS formation in endothelial cells and had reduced capacity to induce endothelial cell migration. Mechanistically, the latter was due to decreased capacity of rHDL-apoA-I[A164S] to activate Akt kinase possibly by interacting with endothelial LOX-1 receptor. The impaired capacity of rHDL-apoA-I[A164S] to preserve endothelial function may be related to the increased cardiovascular risk for this mutation. Overall, our structure-function analysis of L144R, A164S and L178P apoA-I mutants provides insights on how HDL-cholesterol levels and/or atheroprotective properties of apoA-I/HDL are impaired in carriers of these mutations.
Assuntos
Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Apolipoproteína A-I/genética , Doenças Cardiovasculares/genética , HDL-Colesterol/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteína A-I/metabolismo , Apolipoproteína A-I/ultraestrutura , Doenças Cardiovasculares/patologia , Movimento Celular/genética , HDL-Colesterol/metabolismo , HDL-Colesterol/ultraestrutura , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Fatores de Risco de Doenças Cardíacas , Humanos , Cetocolesteróis/genética , Cetocolesteróis/metabolismo , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/ultraestrutura , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/ultraestrutura , Mutação/genética , Receptores Depuradores Classe E/genética , Receptores Depuradores Classe E/metabolismo , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Drug encapsulation into amphiphilic block copolymer micelles aims to increase drug solubility and minimize drug degradation upon administration, avoid undesirable side effects and ameliorate drug bioavailability. Drug encapsulation methodologies including thin-film hydration method and organic cosolvent method are described in this chapter. Often, it is desirable to determine the most efficient solubilization protocol leading to functional drug delivery nanovehicles in each case. The encapsulation of curcumin into PEO-b-PPO-b-PEO (Pluronic F-127) polymeric micelles through thin-film hydration method presents the most promising results. Indomethacin can be loaded successfully into the hydrophobic cores of PEO-b-PCL amphiphilic block copolymer micelles following both encapsulation protocols.
Assuntos
Curcumina/química , Portadores de Fármacos/química , Micelas , Polietilenoglicóis/química , Propilenoglicóis/química , Curcumina/uso terapêutico , Portadores de Fármacos/uso terapêutico , Interações Hidrofóbicas e Hidrofílicas , Polietilenoglicóis/uso terapêutico , Propilenoglicóis/uso terapêutico , SolubilidadeRESUMO
High-Density Lipoprotein cholesterol (HDL-C) levels do not correlate well with Coronary Artery Disease (CAD) risk, while HDL functionality affects atherogenesis and is a better prognostic marker for CAD. Often, the extreme HDL-C levels have a multigenic origin. Here, we searched for single-nucleotide polymorphisms (SNPs) in ten genes of HDL metabolism in a Greek cohort with very low (<10th percentile, n = 13) or very high (>90th percentile, n = 21) HDL-C. We also evaluated the association between HDL-C levels, HDL functionality (anti-oxidant capacity) and CAD in the subjects of this cohort. Individuals with low HDL-C levels had higher triglyceride levels, lower apoA-I levels, decreased HDL anti-oxidant capacity and higher incidence of CAD compared with individuals with control or high HDL-C levels. With next generation sequencing we identified 18 exonic SNPs in 6 genes of HDL metabolism and for selected amino acid changes we performed computer-aided structural analysis and modeling. A previously uncharacterized rare apolipoprotein A-IV variant, apoA-IV [V336M], present in a subject with low HDL-C (14 mg/dL) and CAD, was expressed in recombinant form and structurally and functionally characterized. ApoA-IV [V336M] had similar α-helical content to WT apoA-IV but displayed a small thermodynamic stabilization by chemical unfolding analysis. ApoA-IV [V336M] was able to associate with phospholipids but presented reduced kinetics compared to WT apoA-IV. Overall, we identified a rare apoA-IV variant in a subject with low HDL levels and CAD with altered biophysical and phospholipid binding properties and showed that subjects with very low HDL-C presented with HDL dysfunction and higher incidence of CAD in a Greek cohort.
Assuntos
Apolipoproteínas A/genética , HDL-Colesterol/metabolismo , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/metabolismo , Lipoproteínas HDL/metabolismo , Adulto , Apolipoproteínas A/química , Arildialquilfosfatase/metabolismo , Estudos de Coortes , Feminino , Grécia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Polimorfismo de Nucleotídeo ÚnicoRESUMO
We report on the preparation of drug nanocarriers by encapsulating losartan potassium (LSR) into amphiphilic block copolymer micelles, utilizing the biocompatible/biodegradable poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL) diblock copolymer. The PEO-b-PCL micelles and LSR-loaded PEO-b-PCL nanocarriers were prepared by organic solvent evaporation method (OSEM). Light scattering and nuclear magnetic resonance (NMR) provide information on micelle structure and polymer-drug interactions. According to dynamic light scattering (DLS) analysis, the PEO-b-PCL micelles and LSR-loaded PEO-b-PCL nanocarriers formed nanostructures in the range of 17-26 nm in aqueous milieu. Attenuated total reflection Fourier transform infrared (ATR-FTIR) and ultraviolet-visible (UV-Vis) measurements confirmed the presence of LSR in the polymeric drug solutions. NMR results proved the successful encapsulation of LSR into the PEO-b-PCL micelles by analyzing the drug-micelles intermolecular interactions. Specifically, 2D-NOESY experiments clearly evidenced the intermolecular interactions between the biphenyl ring and butyl chain of LSR structure with the methylene signals of PCL. Additionally, NMR studies as a function of temperature demonstrated an unexpected, enhanced proton mobility of the PEO-b-PCL micellar core in D2O solutions, probably caused by the melting of the PCL hydrophobic core.
RESUMO
We report on the preparation of novel and multifunctional hybrid spherical-shaped nanostructures involving a double-hydrophilic block copolymer, namely the neutral cationic poly[oligo(ethylene glycol) methacrylate]-b-poly[(vinyl benzyl trimethylammonium chloride)] (POEGMA-b-PVBTMAC) diblock copolymer, initially complexed with hydrophilic anionic magnetic nanoparticles (MNPs), and subsequently, with short deoxyribonucleic acid (113 bases DNA). The POEGMA-b-PVBTMAC copolymer, the copolymer/MNPs and the copolymer/MNPs/DNA tricomponent hybrid electrostatic complexes were studied by dynamic/electrophoretic light scattering (DLS/ELS) and cryogenic transmission electron microscopy (cryo-TEM) techniques for the determination of their structure and solution properties. The MNPs were complexed efficiently with the oppositely charged diblock chains, leading to well-defined hybrid organic-inorganic spherical-shaped nanostructures. A significant aggregation tendency of the MNPs is noticed in cryo-TEM measurements after the electrostatic complexation of DNA, implying an accumulation of the DNA macromolecules on the surface of the hybrid tricomponent complexes. Magnetophoretic experiments verified that the MNPs maintain their magnetic properties after the complexation initially with the copolymer, and subsequently, within the block polyelectrolyte/MNPs/DNA nanostructures.
RESUMO
Eighteen 3-aryl-5-substituted-coumarins-six 5-acetyloxy-derivatives, six 5-hydroxy-derivatives, and six 5-geranyloxy-derivatives-were synthesized, structurally characterized and their antioxidant activity, lipoxygenase inhibitory ability, as well as their cytotoxic activity against human neuroblastoma SK-N-SH and HeLa adenocarcinoma cell lines were evaluated. The 5-acetyloxy-compounds 3a-3f were found to be the best cytotoxic agents among all the compounds studied. The bromo-substituted coumarins 3a and 3b were remarkably active against HeLa cell line showing IC50 1.8 and 6.1 µM, respectively. Coumarin 5e possessing a geranyloxy-chain on position 5 of the coumarin scaffold presented dual bioactivity, while 5-geranyloxy-coumarin 5f was the most competent soybean lipoxygenase inhibitor of this series (IC50 10 µM). As shown by in silico docking studies, the studied molecules present allosteric interactions with soybean lipoxygenases.