RESUMEN
Atherosclerosis can be originated from the accumulation of modified cholesterol-rich lipoproteins in the arterial wall. The electronegative LDL, LDL(-), plays an important role in the pathogenesis of atherosclerosis once this cholesterol-rich lipoprotein can be internalized by macrophages, contributing to the formation of foam cells, and provoking an immune-inflammatory response. Herein, we engineered a nanoformulation containing highly pure surface-functionalized nanocapsules using a single-chain fragment variable (scFv) reactive to LDL(-) as a ligand and assessed whether it can affect the LDL(-) uptake by primary macrophages and the progression of atherosclerotic lesions in Ldlr -/- mice. The engineered and optimized scFv-anti-LDL(-)-MCMN-Zn nanoformulation is internalized by human and murine macrophages in vitro by different endocytosis mechanisms. Moreover, macrophages exhibited lower LDL(-) uptake and reduced mRNA and protein levels of IL1B and MCP1 induced by LDL(-) when treated with this new nanoformulation. In a mouse model of atherosclerosis employing Ldlr -/- mice, intravenous administration of scFv-anti-LDL(-)-MCMN-Zn nanoformulation inhibited atherosclerosis progression without affecting vascular permeability or inducing leukocytes-endothelium interactions. Together, these findings suggest that a scFv-anti-LDL(-)-MCMN-Zn nanoformulation holds promise to be used in future preventive and therapeutic strategies for atherosclerosis.
RESUMEN
The electronegative low-density lipoprotein, LDL (-), is an endogenously modified LDL subfraction with cytotoxic and proinflammatory actions on endothelial cells, monocytes, and macrophages contributing to the progression of atherosclerosis. In this study, epitopes of LDL (-) were mapped using a phage display library of peptides and monoclonal antibodies reactive to this modified lipoprotein. Two different peptide libraries (X6 and CX8C for 6- and 8-amino acid-long peptides, respectively) were used in the mapping. Among all tested peptides, two circular peptides, P1A3 and P2C7, were selected based on their high affinities for the monoclonal antibodies. Small-angle X-ray scattering analysis confirmed their structures as circular rings. P1A3 or P2C7 were quickly internalized by bone marrow-derived murine macrophages as shown by confocal microscopy. P2C7 increased the expression of TNFα, IL-1 ß and iNOS as well as the secretion of TNFα, CCL2, and nitric oxide by murine macrophages, similar to the responses induced by LDL (-), although less intense. In contrast, P1A3 did not show pro-inflammatory effects. We identified a mimetic epitope associated with LDL (-), the P2C7 circular peptide, that activates macrophages. Our data suggest that this conformational epitope represents an important danger-associated molecular pattern of LDL (-) that triggers proinflammatory responses.
Asunto(s)
Epítopos/metabolismo , Inflamación/metabolismo , Lipoproteínas LDL/metabolismo , Epítopos/sangre , Epítopos/aislamiento & purificación , Humanos , Lipoproteínas LDL/sangre , Lipoproteínas LDL/aislamiento & purificación , Macrófagos/metabolismo , Óxido Nítrico/análisisRESUMEN
BACKGROUND: Chronic kidney disease (CKD) patients develop metabolic acidosis when approaching stages 3 and 4, a period in which accelerated atherogenesis may ensue. Studies in vitro show that low pH may increase low-density lipoprotein (LDL) oxidation, suggesting a role for chronic metabolic acidosis in atherosclerosis. The present study attempted to evaluate the effects of conservative care using oral sodium bicarbonate (NaHCO3) supplementation on the electronegative LDL [LDL(-)], a minimally oxidized LDL, plasma levels in CKD patients. METHODS: Thirty-one CKD patients were followed by a multidisciplinary team during 15 months of care in which 1.0 mmol/kg/day oral NaHCO3 supplementation was first given in the third month. Blood samples were collected 3 months before the initiation of oral NaHCO3 supplementation (T1), at the time of the beginning of supplementation (T2), and thereafter, each 4 months (T3, T4 and T5) until month 15 of care. Blood parameters and LDL(-) were measured from these collections. RESULTS: After 12 months of conservative care, creatinine clearance (MDRD) was kept stable, and serum bicarbonate (HCO3-) increased from 20.5 ± 2.9 to 22.6 ± 1.1 mM (p < 0.003). LDL(-) plasma levels declined from 4.5 ± 3.3 to 2.1 ± 0.9 U/L (p < 0.007) after reaching mean serum HCO3- levels of 22.6 ± 1.1 mM. CONCLUSIONS: Conservative care using oral NaHCO3 supplementation was able to stabilize renal function and decrease serum levels of LDL(-), a modified proatherogenic lipoprotein, only when mean serum HCO3- levels approached 22 mM. This study constitutes evidence that alkali therapy, in addition to its beneficial effect on renal disease progression, might serve as a preventive strategy to attenuate atherogenesis in CKD patients.
RESUMEN
OBJECTIVE: HIV/AIDS is generally associated with dyslipidemia and oxidative imbalance, which are caused by the infection itself and by antiretroviral therapy (ART). The flavonoids, found in cocoa and yerba mate, have antioxidant and hypolipidemic properties. The aim of this study was to evaluate the effects of the consumption of dark chocolate and mate tea on the lipid profiles of individuals with HIV/AIDS who are undergoing ART. METHODS: A randomized, double-blind, placebo-controlled crossover clinical trial was conducted with 92 patients receiving ART for ≥6 mo and with viral suppression. The participants were randomized to receive either 65 g of chocolate (with 2148 mg polyphenols) or placebo chocolate (without polyphenols) or 3 g of mate tea (with 107 mg total phenols and 84.24 mg chlorogenic acid) or placebo mate (without polyphenols) for 15 d each, separated by a washout period of 15 d. The lipid profile, including determination of electronegative low-density lipoprotein, was determined after each intervention. The data were analyzed by analysis of variance using the pkcross procedure of the Stata 11.0 software. RESULTS: Analysis of variance revealed a significant overall difference in mean high-density lipoprotein cholesterol (HDL-C) between all supplements (P = 0.047). Using the paired t test, the effect was attributed to the consumption of dark chocolate (P = 0.046). The other parameters investigated were not improved. CONCLUSIONS: The consumption of dark chocolate for 15 d improved HDL-C concentrations of individuals with HIV/AIDS undergoing ART, possibly due to the presence of fatty acids (stearic acid), polyphenols, and theobromine. This fact is important for the cardiovascular protection of these individuals.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Antirretrovirales/efectos adversos , Chocolate , Infecciones por VIH/tratamiento farmacológico , Ilex paraguariensis , Lípidos/sangre , Síndrome de Inmunodeficiencia Adquirida/sangre , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Adulto , Antirretrovirales/uso terapéutico , Antioxidantes/administración & dosificación , Bebidas , Chocolate/análisis , HDL-Colesterol/sangre , Estudios Cruzados , Método Doble Ciego , Dislipidemias/tratamiento farmacológico , Dislipidemias/etiología , Femenino , Infecciones por VIH/sangre , Infecciones por VIH/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Placebos , Extractos Vegetales/administración & dosificación , Hojas de la Planta/química , Polifenoles/administración & dosificaciónRESUMEN
Atherosclerosis is a chronic inflammatory disease responsible for the majority of cases of myocardial infarction and ischemic stroke. The electronegative low-density lipoprotein, a modified subfraction of native LDL, is pro-inflammatory and plays an important role in atherogenesis. To investigate the effects of a nanoformulation (scFv anti-LDL(-)-MCMN-Zn) containing a scFv reactive to LDL(-) on the inhibition of atherosclerosis, its toxicity was evaluated in vitro and in vivo and further it was also administered weekly to LDL receptor knockout mice. The scFv anti-LDL(-)-MCMN-Zn nanoformulation did not induce cell death in RAW 264.7 macrophages and HUVECs. The 5mg/kg dose of scFv anti-LDL(-)-MCMN-Zn did not cause any typical signs of toxicity and it was chosen for the evaluation of its atheroprotective effect in Ldlr(-/-) mice. This nanoformulation significantly decreased the atherosclerotic lesion area at the aortic sinus, compared with that in untreated mice. In addition, the Il1b mRNA expression and CD14 protein expression were downregulated in the atherosclerotic lesions at the aortic arch of Ldlr(-/-) mice treated with scFv anti-LDL(-)-MCMN-Zn. Thus, the scFv anti-LDL(-)-MCMN-Zn nanoformulation inhibited the progression of atherosclerotic lesions, indicating its potential use in a future therapeutic strategy for atherosclerosis.
Asunto(s)
Aterosclerosis/prevención & control , Lipoproteínas LDL/inmunología , Receptores de LDL/fisiología , Anticuerpos de Cadena Única/inmunología , Animales , Línea Celular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de LDL/genéticaRESUMEN
Experimental studies on atherosclerosis are crucial for investigating its pathophysiology, defining new therapeutic targets, and developing new drugs and diagnostic tools. Thus, many imaging markers have been developed and introduced in experimental studies. The main advantage of these new tools is that they allow the noninvasive diagnosis of atherosclerotic vascular disease. Here, we describe the cloning, expression, purification, and stabilization of a chimeric protein specifically designed to probe cells and tissues for the presence of LDL(-), a relevant marker of atherosclerosis. The DNA sequence that encodes the anti-LDL(-) scFv, previously obtained from a hybridoma secreting an anti-LDL(-) monoclonal antibody, was inserted into the bacterial vector pET-28a(+) in tandem with a DNA sequence encoding GFP. The recombinant protein was expressed in high yields in E. coli as inclusion bodies. The applicability of GFP-scFv was assessed by ELISA, which determined its affinity for LDL(-) and confocal microscopy, that showed macrophage uptake of the protein along with LDL(-). In conclusion, our data suggest that the anti-LDL(-) GFP-scFv chimeric protein could be useful in studies on atherogenesis as well as for developing diagnostic tools for atherosclerosis.
Asunto(s)
Biomarcadores/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Lipoproteínas LDL/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/metabolismo , Animales , Aterosclerosis/metabolismo , Línea Celular Tumoral , Endocitosis , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/farmacocinética , Ratones , Imagen Molecular/métodos , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacocinética , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/farmacocinéticaRESUMEN
The in vivo modified forms of low-density lipoprotein (LDL) are important for the formation of foam cells and as mediators of the immuno-inflammatory process involved in the progression of atherosclerosis. Electronegative LDL, LDL(-), is a LDL subfraction with pro-inflammatory properties that is present in human blood. To investigate possible atheroprotective effects, an anti-LDL(-) single-chain variable fragment (scFv) was expressed in the methylotrophic yeast Pichia pastoris and its activity was evaluated in vitro against macrophages and in experimental atherosclerosis in Ldlr(-/-) mice. The recombinant 2C7 scFv was produced in a yield of 9.5 mg of protein/L. The specificity and affinity of purified 2C7 scFv against LDL(-) was confirmed by ELISA. To assess the activity of 2C7 scFv on foam cell formation, RAW 264.7 macrophages were exposed to LDL(-) in the presence or absence of 2C7 scFv. The 2C7 scFv inhibited the uptake of LDL(-) by macrophages in a dose-dependent manner, and internalization of LDL(-) by these cells was found to be mediated by the CD36 and CD14 receptor. In addition, compared with untreated cells, lipid accumulation in macrophages was decreased, and the expression of Cd36, Tlr-4 and Cox-2 was downregulated in macrophages treated with 2C7 scFv. Importantly, compared with untreated mice, the treatment of Ldlr(-/-) mice with 2C7 scFv decreased the atherosclerotic lesion area at the aortic sinus. In conclusion, our data show that 2C7 scFv inhibits foam cell formation and atherosclerotic plaque development by modulating the expression of genes relevant to atherogenesis. These results encourage further use of this antibody fragment in the development of new therapeutic strategies that neutralize the pro-atherogenic effects of LDL(-).