RESUMEN

Angiogenesis is a pivotal process of homeostasis and tissue repair, but it also favours neovascularisation syndromes and cancer nutrition. The chemical mediation of angiogenesis is complex, involving a balance between serine proteases and their inhibitors. We addressed the mechanisms of action of a Kunitz serine protease inhibitor (KPI) on spontaneous angiogenesis, using Amblyomin-X, a KPI designed from the cDNA library of the Amblyomma cajennense tick. Amblyomin-X treatment (10-1000 ng/10 µL; each 48 h; 3 times) reduced the number of vessels in the subcutaneous dorsal tissue of male Swiss mice, as measured by intravital microscopy, haematoxylin-eosin staining, and PECAM-1 immunofluorescence labeling. Incubation of Amblyomin-X with t-End endothelial cells, a murine endothelial microvascular lineage, did not alter cell proliferation, cell-cycle phases, necrosis and apoptosis, and the production of nitric oxide and prostaglandin E2. Nevertheless, Amblyomin-X treatment reduced t-End migration and adhesion to Matrigel(®), and inhibited the VEGF-A secretion and VCAM-1 and ß3 integrin expressions by posttranscriptional pathways. Together, data herein outline novel posttranscriptional mechanisms of KPIs on endothelial cells during angiogenesis and point out the possible application of Amblyomin-X as a local inhibitor to undesired neovascularisation process.

Asunto(s)

Adhesión Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas y Péptidos Salivales/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas de Artrópodos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Dinoprostona/metabolismo , Células Endoteliales/metabolismo , Biblioteca de Genes , Masculino , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Óxido Nítrico/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas y Péptidos Salivales/genética , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética

RESUMEN

Thiazolidinediones (TZDs) are a class of drugs used for treatment of type 2 diabetes. However, the therapy with currently available TDZs (e.g. rosiglitazone) is associated with important side effects, such as edema and weight gain, suggesting that the investigation of alternative TZDs with better pharmacological properties is warranted. In this study, we investigated both anti-inflammatory and antioxidant properties of a new chemically modified TZD, the arylidene-thiazolidinedione 5-(4-methanesulfonyl-benzylidene)-3-(4-nitrobenzyl)-thiazolidine-2,4-dione (SF23), and compared the results to those obtained with rosiglitazone. We found that our SF23 displays a weaker affinity for PPARγ, up-regulating in a lower magnitude the expression of both PPARγ and CD36 compared to rosiglitazone. In lipopolysaccharide (LPS)-stimulated macrophages, SF23 decreased nitrite production and attenuated the mRNA expression of both iNOS and COX-2. These anti-inflammatory effects were comparable to those obtained with rosiglitazone. Interestingly, SF23, but not rosiglitazone, prevented LPS-induced mitochondrial membrane hyperpolarization, apoptosis, reactive oxygen species (ROS) generation, and the expression of NADPH oxidase subunits, Nox1 and Nox2. In addition, in macrophages from Nrf2⁻/⁻ mice, SF23 protected against LPSinduced cellular death and ROS production, whereas rosiglitazone was only able to protect normal Nrf2⁺/⁺ cells against oxidative injury, suggesting that, unlike rosiglitazone, the antioxidant activity of SF23 might be Nrf2-independent. Finally, in macrophages exposed to high concentrations of glucose, SF23 induced significant increases in the mRNA expression of glucose transporters, insulin receptor substrate and mitoNEET. Altogether, our data indicate that our new chemically modified TDZ displays similar anti-inflammatory properties, but superior antioxidant effects on the LPS-stimulated macrophages compared to rosiglitazone.

Asunto(s)

Antiinflamatorios/química , Antioxidantes/química , Macrófagos/efectos de los fármacos , Tiazolidinedionas/farmacología , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/química , Lipopolisacáridos/farmacología , Activación de Macrófagos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Rosiglitazona , Tiazolidinedionas/química , Tiazolidinedionas/uso terapéutico

RESUMEN

In this work we report on a study of the morphological changes of LDL induced in vitro by metallic ions (Cu(2+) and Fe(3+)). These modifications were characterized by transmission electron microscopy, nuclear magnetic resonance and the Z-scan technique. The degree of oxidative modification of LDL was determined by the TBARS and lipid hydroperoxides assays. It is shown that distinct pathways for modifying lipoproteins lead to different morphological transformations of the particles characterized by changes in size and/or shape of the resulting particles, and by the tendency to induce aggregation of the particles. There were no evidence of melting of particles promoted by oxidative processes with Cu and Fe.

Asunto(s)

Cobre/química , Hierro/química , Lipoproteínas LDL/química , Cationes/química , Espectroscopía de Resonancia Magnética , Microscopía Electrónica de Transmisión , Oxidación-Reducción , Sustancias Reactivas al Ácido Tiobarbitúrico/química

RESUMEN

The aim of this study was to determine the apoptotic pathways and mechanisms involved in electronegative LDL [LDL(-)]-induced apoptosis in RAW 264.7 macrophages and the role of Nrf2 in this process. Incubation of RAW 264.7 macrophages with LDL(-) for 24 h resulted in dose-dependent cell death. Activated caspases were shown to be involved in the apoptosis induced by LDL(-); incubation with the broad caspase inhibitor z-VAD prevented apoptosis in LDL(-)-treated cells. CD95 (Fas), CD95 ligand (FasL), CD36 and the tumor necrosis factor (TNF) ligand Tnfsf10 were overexpressed in LDL(-)-treated cells. However, Bax, Bcl-2 and Mcl-1 protein levels remained unchanged after LDL(-) treatment. LDL(-) promoted hyperpolarization of the mitochondrial membrane, elevated reactive oxygen species (ROS) production and translocation of Nrf2 to the nucleus, a process absent in cells treated with native LDL. Elicited peritoneal macrophages from Nrf2-deficient mice exhibited an elevated apoptotic response after challenge with LDL(-), together with an increase in the production of ROS in the absence of alterations in CD36 expression. These results provide evidence that CD36 expression induced by LDL(-) is Nrf2-dependent. Also, it was demonstrated that Nrf2 acts as a compensatory mechanism of LDL(-)-induced apoptosis in macrophages.

Asunto(s)

Apoptosis , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneales/patología , Factor 2 Relacionado con NF-E2/fisiología , Animales , Biomarcadores/metabolismo , Western Blotting , Antígenos CD36/genética , Antígenos CD36/metabolismo , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

In this work we report on a study of the morphological changes of LDL induced in vitro by metallic ions (Cu2+ and Fe3+). These modifications were characterized by transmission electron microscopy, nuclear magnetic resonance and the Z-scan technique. The degree of oxidative modification of LDL was determined by the TBARS and lipid hydroperoxides assays. It is shown that distinct pathways for modifying lipoproteins lead to different morphological transformations of the particles characterized by changes in size and/or shape of the resulting particles, and by the tendency to induce aggregation of the particles. There were no evidence of melting of particles promoted by oxidative processes with Cu and Fe.

Asunto(s)

Masculino , Femenino , Humanos , Lipoproteínas

RESUMEN

In order to determine the effect of antibodies against electronegative low-density lipoprotein LDL(-) on atherogenesis, five groups of LDL low receptor-deficient (LDLr-/-) mice (6 per group) were immunized with the following antibodies (100 µg each): mouse anti-LDL(-) monoclonal IgG2b, rabbit anti-LDL(-) polyclonal IgG or its Fab fragments and mouse irrelevant monoclonal IgG and non-immunized controls. Antibodies were administered intravenously one week before starting the hypercholesterolemic diet (1.25 percent cholesterol) and then every week for 21 days. The passive immunization with anti-LDL(-) monoclonal IgG2b, polyclonal antibody and its derived Fab significantly reduced the cross-sectional area of atherosclerotic lesions at the aortic root of LDLr-/- mice (28.8 ± 9.7, 67.3 ± 17.02, 56.9 ± 8.02 µm² (mean ± SD), respectively) compared to control (124.9 ± 13.2 µm²). Vascular cell adhesion molecule-1 protein expression, quantified by the KS300 image-analyzing software, on endothelium and the number of macrophages in the intima was also decreased in aortas of mice treated with anti-LDL(-) monoclonal antibody (3.5 ± 0.70 per field x 10) compared to controls (21.5 ± 3.5 per field x 10). Furthermore, immunization with the monoclonal antibody decreased the concentration of LDL(-) in blood plasma (immunized: 1.0 ± 1.4; control: 20.5 ± 3.5 RLU), the amount of cholesterol oxides in plasma (immunized: 4.7 ± 2.7; control: 15.0 ± 2.0 pg COx/mg cholesterol) and liver (immunized: 2.3 ± 1.5; control: 30.0 ± 26.0 pg COx/mg cholesterol), and the hepatic content of lipid hydroperoxides (immunized: 0.30 ± 0.020; control: 0.38 ± 0.15 ng/mg protein). In conclusion, antibodies against electronegative LDL administered intravenously may play a protective role in atherosclerosis.

Asunto(s)

Animales , Femenino , Ratones , Conejos , Anticuerpos Monoclonales/administración & dosificación , Aterosclerosis/terapia , Inmunización Pasiva/métodos , Inmunoglobulina G/administración & dosificación , Lipoproteínas LDL/administración & dosificación , Receptores de LDL/inmunología , Anticuerpos Monoclonales/inmunología , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Inmunohistoquímica , Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Peroxidación de Lípido/inmunología , Lipoproteínas LDL/inmunología , Receptores de LDL/metabolismo , Molécula 1 de Adhesión Celular Vascular/inmunología

RESUMEN

BACKGROUND: Oxidative modification of low-density lipoprotein (LDL) has been demonstrated in patients with end-stage renal disease, where it is associated with oxidative stress and plays a key role in the pathogenesis of atherosclerosis. In this context, the generation of minimally oxidized LDL, also called electronegative LDL [LDL(-)], has been associated with active disease, and is a detectable sign of atherogenic tendencies. The purpose of this study was to evaluate serum LDL(-) levels and anti-LDL(-) IgG autoantibodies in end-stage renal disease patients on dialysis, comparing patients on hemodialysis (HD), peritoneal dialysis (PD) and a control group. In addition, the serum lipid profile, nutritional status, biochemical data and parameters of mineral metabolism were also evaluated. METHODS: The serum levels of LDL(-) and anti-LDL(-) IgG autoantibodies were measured in 25 patients undergoing HD and 11 patients undergoing PD at the Centro Integrado de Nefrologia, Rio de Janeiro, Brazil. Ten healthy subjects served as a control group. Serum levels of albumin, total cholesterol, triglycerides and lipoproteins were measured. Calculations of subjects' body mass index and measurements of waist circumference, triceps skin fold and arm muscle area were performed. Measurements of hematocrit, serum blood urea nitrogen, creatinine, parathyroid hormone, phosphorus and calcium were taken. RESULTS: Levels of LDL(-) were higher in HD patients (575.6 +/- 233.1 microg/ml) as compared to PD patients (223.4 +/- 117.5 microg/ml, p < 0.05), which in turn were higher than in the control group (54.9 +/- 33.3 mug/ml, p < 0.01). The anti-LDL(-) IgG autoantibodies were increased in controls (0.36 +/- 0.09 microg/ml) as compared to PD (0.28 +/- 0.12 microg/ml, p < 0.001) and HD patients (0.2 +/- 0.1 microg/ml, p < 0.001). The mean values of total cholesterol and LDL were considered high in the PD group, whereas the mean triceps skin fold was significantly lower in the HD group. CONCLUSION: Levels of LDL(-) are higher in renal patients on dialysis than in normal individuals, and are reciprocally related to IgG autoantibodies. LDL(-) may be a useful marker of oxidative stress, and this study suggests that HD patients are more susceptible to cardiovascular risk due to this condition. Moreover, autoantibodies reactive to LDL(-) may have protective effects in chronic kidney disease.

Asunto(s)

Autoanticuerpos/sangre , Inmunoglobulina G/sangre , Fallo Renal Crónico/sangre , Fallo Renal Crónico/terapia , Lipoproteínas LDL/sangre , Diálisis Peritoneal , Diálisis Renal , Adulto , Análisis de Varianza , Biomarcadores/sangre , Pesos y Medidas Corporales , Colesterol/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Fallo Renal Crónico/inmunología , Lipoproteínas LDL/inmunología , Lipoproteínas LDL/metabolismo , Masculino , Persona de Mediana Edad , Estado Nutricional , Estadísticas no Paramétricas

RESUMEN

In order to determine the effect of antibodies against electronegative low-density lipoprotein LDL(-) on atherogenesis, five groups of LDL low receptor-deficient (LDLr-/-) mice (6 per group) were immunized with the following antibodies (100 microg each): mouse anti-LDL(-) monoclonal IgG2b, rabbit anti-LDL(-) polyclonal IgG or its Fab fragments and mouse irrelevant monoclonal IgG and non-immunized controls. Antibodies were administered intravenously one week before starting the hypercholesterolemic diet (1.25% cholesterol) and then every week for 21 days. The passive immunization with anti-LDL(-) monoclonal IgG2b, polyclonal antibody and its derived Fab significantly reduced the cross-sectional area of atherosclerotic lesions at the aortic root of LDLr-/- mice (28.8 +/- 9.7, 67.3 +/- 17.02, 56.9 +/- 8.02 microm(2) (mean +/- SD), respectively) compared to control (124.9 +/- 13.2 microm(2)). Vascular cell adhesion molecule-1 protein expression, quantified by the KS300 image-analyzing software, on endothelium and the number of macrophages in the intima was also decreased in aortas of mice treated with anti-LDL(-) monoclonal antibody (3.5 +/- 0.70 per field x 10) compared to controls (21.5 +/- 3.5 per field x 10). Furthermore, immunization with the monoclonal antibody decreased the concentration of LDL(-) in blood plasma (immunized: 1.0 +/- 1.4; control: 20.5 +/- 3.5 RLU), the amount of cholesterol oxides in plasma (immunized: 4.7 +/- 2.7; control: 15.0 +/- 2.0 pg COx/mg cholesterol) and liver (immunized: 2.3 +/- 1.5; control: 30.0 +/- 26.0 pg COx/mg cholesterol), and the hepatic content of lipid hydroperoxides (immunized: 0.30 +/- 0.020; control: 0.38 +/- 0.15 ng/mg protein). In conclusion, antibodies against electronegative LDL administered intravenously may play a protective role in atherosclerosis.

Asunto(s)

Anticuerpos Monoclonales/administración & dosificación , Aterosclerosis/terapia , Inmunización Pasiva/métodos , Inmunoglobulina G/administración & dosificación , Lipoproteínas LDL/administración & dosificación , Receptores de LDL/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Femenino , Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Inmunohistoquímica , Peroxidación de Lípido/inmunología , Lipoproteínas LDL/inmunología , Ratones , Ratones Endogámicos C57BL , Conejos , Receptores de LDL/metabolismo , Molécula 1 de Adhesión Celular Vascular/inmunología

RESUMEN

BACKGROUND: Oxidative stress plays an important role in the pathophysiology of diabetes mellitus. The aim of this study was to evaluate the formation of cholesterol oxides (ChOx) as biomarkers of oxidative stress in subjects with impaired glucose tolerance (IGT) and diabetes. METHODS: Blood plasma levels of cholesterol oxidation products were determined in the following groups: type 1 diabetes mellitus (DM1), type 2 diabetes (DM2), impaired glucose tolerance (IGT), children without diabetes (C1) and adults without diabetes (C2). The serum levels of cholest-5-ene-3alpha,7alpha-diol (7alpha-hydroxycholesterol, 7alpha-OH), cholest-5-ene-3beta,7beta-diol (7beta-hydroxycholesterol, 7beta-OH), 3beta-hydroxycholest-5-7-one (7-ketocholesterol, 7-K), 5alpha-cholestane-3beta,5,6beta-triol (cholestanetriol), 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol (cholesterol-5alpha,6alpha-epoxide,), 5,6beta-epoxy-5beta-cholestan-3beta-ol (cholesterol-5beta,6beta-epoxide) and cholest-5-eno-3beta,25-diol (25-hydroxycholesterol, 25-OH) (trivial name and abbreviations indicated in parentheses) were quantified by gas chromatography using flame ionization detection. RESULTS: The levels of total ChOx were elevated in the DM1 and DM2 groups compared to age-matched subjects without diabetes (p < 0.05). The concentrations of 7beta-hydroxycholesterol, cholesterol-alpha-epoxide and cholesterol-beta-epoxide were higher in the blood plasma of subjects in the DM2 group than in the blood plasma of subjects in the C2 and IGT groups (p < 0.05). Treatment of type 2 diabetic patients with oral hypoglycemic drugs associated with insulin resulted in lower concentrations of nitrotyrosine in the blood plasma without significant changes in the concentrations of glucose and glycated hemoglobin. Moreover, combination with statins in both treatments decreased the concentrations of ChOx. CONCLUSIONS: ChOx are suitable biomarkers of oxidative stress and may be useful in clinical studies to follow drug effects on lipid oxidative modifications in diabetic patients.

Asunto(s)

Colesterol/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Estrés Oxidativo/fisiología , Adolescente , Adulto , Anciano , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Biomarcadores , Niño , Colestanoles/sangre , Colesterol/análogos & derivados , Colesterol/sangre , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Femenino , Intolerancia a la Glucosa/sangre , Humanos , Hidroxicolesteroles/sangre , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Cetocolesteroles/sangre , Masculino , Persona de Mediana Edad

RESUMEN

Background Oxidative stress plays an important role in the pathophysiology of diabetes mellitus. The aim of this study was to evaluate the formation of cholesterol oxides (ChOx) as biomarkers of oxidative stress in subjects with impaired glucose tolerance (IGT) and diabetes. Methods Blood plasma levels of cholesterol oxidation products were determined in the following groups: type 1 diabetes mellitus (DM1), type 2 diabetes (DM2), impaired glucose tolerance (IGT), children without diabetes (C1) and adults without diabetes (C2). The serum levels of cholest-5-ene-3£/,7£/-diol (7£/-hydroxycholesterol, 7£/-OH), cholest- 5-ene-3£],7£]-diol (7£]-hydroxycholesterol, 7£]-OH), 3£]-hydroxycholest-5-7- one (7-ketocholesterol, 7-K), 5£/-cholestane-3£],5,6£]-triol (cholestanetriol), 5,6£/-epoxy-5£/-cholestan-3£/-ol (cholesterol-5£/,6£/-epoxide,), 5,6£]-epoxy- 5£]-cholestan-3£]-ol (cholesterol-5£],6£]-epoxide) and cholest-5-eno-3£],25-diol (25-hydroxycholesterol, 25-OH) (trivial name and abbreviations indicated in parentheses) were quantified by gas chromatography using flame ionization detection. Results The levels of total ChOx were elevated in the DM1 and DM2 groups compared to age-matched subjects without diabetes (p < 0.05).The concentrations of 7£]-hydroxycholesterol, cholesterol-£/-epoxide and cholesterol-£]-epoxide were higher in the blood plasma of subjects in the DM2 group than in the blood plasma of subjects in the C2 and IGT groups (p < 0.05). Treatment of type 2 diabetic patients with oral hypoglycemic drugs associated with insulin resulted in lower concentrations of nitrotyrosine in the blood plasma without significant changes in the concentrations of glucose and glycated hemoglobin. Moreover, combination with statins in both treatments decreased the concentrations of ChOx.Conclusions ChOx are suitable biomarkers of oxidative stress and may be useful in clinical studies to follow drug effects on lipid oxidative modifications in diabetic patients. Copyright ÆÉ 2006 John Wiley & Sons, Ltd.

Asunto(s)

Colesterol , Hipoglucemia , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Insulina , Lípidos , Óxidos

RESUMEN

The objective of the present study was to identify disturbances of nitric oxide radical (.NO) metabolism and the formation of cholesterol oxidation products in human essential hypertension. The concentrations of.NO derivatives (nitrite, nitrate, S-nitrosothiols and nitrotyrosine), water and lipid-soluble antioxidants and cholesterol oxides were measured in plasma of 11 patients with mild essential hypertension (H: 57.8 +/- 9.7 years; blood pressure, 148.3 +/- 24.8/90.8 +/- 10.2 mmHg) and in 11 healthy subjects (N: 48.4 +/- 7.0 years; blood pressure, 119.4 +/- 9.4/75.0 +/- 8.0 mmHg). Nitrite, nitrate and S-nitrosothiols were measured by chemiluminescence and nitrotyrosine was determined by ELISA. Antioxidants were determined by reverse-phase HPLC and cholesterol oxides by gas chromatography. Hypertensive patients had reduced endothelium-dependent vasodilation in response to reactive hyperemia (H: 9.3 and N: 15.1% increase of diameter 90 s after hyperemia), and lower levels of ascorbate (H: 29.2 +/- 26.0, N: 54.2 +/- 24.9 micro M), urate (H: 108.5 +/- 18.9, N: 156.4 +/- 26.3 micro M), beta-carotene (H: 1.1 +/- 0.8, N: 2.5 +/- 1.2 nmol/mg cholesterol), and lycopene (H: 0.4 +/- 0.2, N: 0.7 +/- 0.2 nmol/mg cholesterol), in plasma, compared to normotensive subjects. The content of 7-ketocholesterol, 5alpha-cholestane-3beta,5,6beta-triol and 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol in LDL, and the concentration of endothelin-1 (H: 0.9 +/- 0.2, N: 0.7 +/- 0.1 ng/ml) in plasma were increased in hypertensive patients. No differences were found for.NO derivatives between groups. These data suggest that an increase in cholesterol oxidation is associated with endothelium dysfunction in essential hypertension and oxidative stress, although.NO metabolite levels in plasma are not modified in the presence of elevated cholesterol oxides.

Asunto(s)

Endotelio Vascular/fisiopatología , Hipertensión/fisiopatología , Peroxidación de Lípido/fisiología , Óxido Nítrico/sangre , Estrés Oxidativo/fisiología , Vasodilatación/fisiología , Adulto , Anciano , Disponibilidad Biológica , Estudios de Casos y Controles , LDL-Colesterol/química , LDL-Colesterol/metabolismo , Cromatografía , Endotelina-1/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Hipertensión/sangre , Masculino , Persona de Mediana Edad

RESUMEN

The objective of the present study was to identify disturbances of nitric oxide radical (ANO) metabolism and the formation of cholesterol oxidation products in human essential hypertension. The concentrations ofANO derivatives (nitrite, nitrate, S-nitrosothiols and nitrotyrosine), water and lipid-soluble antioxidants and cholesterol oxides were measured in plasma of 11 patients with mild essential hypertension (H: 57.8 ± 9.7 years; blood pressure, 148.3 ± 24.8/90.8 ± 10.2 mmHg) and in 11 healthy subjects (N: 48.4 ± 7.0 years; blood pressure, 119.4 ± 9.4/75.0 ± 8.0 mmHg).Nitrite, nitrate and S-nitrosothiols were measured by chemiluminescence and nitrotyrosine was determined by ELISA. Antioxidants were determined by reverse-phase HPLC and cholesterol oxides by gas chromatography. Hypertensive patients had reduced endothelium-dependent vasodilation in response to reactive hyperemia (H: 9.3 and N: 15.1 percent increase of diameter 90 s after hyperemia), and lower levels of ascorbate (H: 29.2 ± 26.0, N: 54.2 ± 24.9 æM), urate (H: 108.5 ± 18.9, N: 156.4 ± 26.3 æM), ß-carotene (H: 1.1 ± 0.8, N: 2.5 ± 1.2 nmol/mg cholesterol), and lycopene (H: 0.4 ± 0.2, N: 0.7 ± 0.2 nmol/mg cholesterol), in plasma, compared to normotensive subjects. The content of 7-ketocholesterol, 5alpha-cholestane-3ß,5,6ß-triol and 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol in LDL, and the concentration of endothelin-1 (H: 0.9 ± 0.2, N: 0.7 ± 0.1 ng/ml) in plasma were increased in hypertensive patients. No differences were found for ANO derivatives between groups. These data suggest that an increase in cholesterol oxidation is associated with endothelium dysfunction in essential hypertension and oxidative stress, although ANO metabolite levels in plasma are not modified in the presence of elevated cholesterol oxides

Asunto(s)

Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Endotelio Vascular , Hipertensión , Peroxidación de Lípido , Óxido Nítrico , Estrés Oxidativo , Disponibilidad Biológica , Estudios de Casos y Controles , LDL-Colesterol , Cromatografía , Endotelina-1 , Ensayo de Inmunoadsorción Enzimática , Hipertensión , Vasodilatación

RESUMEN

As modificaçöes oxidativas da lipoproteína de baixa densidade (LDL) säo consideradas um fator importante para o desenvolvimento da aterosclerose. Estas modificaçöes ocorrem in vivo, originando uma sub-traçäo denominada de LDL, eletronegativa (LDL-). O monitoramento clínico da LDL- é de extrema importância, mas estava sendo limitado pela dificuldade para detecçäo desta partícula em fluídos biológicos. Neste estudo desenvolveu-se novas metodologias para detectar a LDL- no plasma, utilizando-se um anticorpo monoclonal anti-LDL- humana (3D1036) e avaliar a resposta imune humoral relacionada à LDL-. A LDL- plasmática foi analisada através de um ELISA com detecçäo por quimioluminescência com boa sensibilidade (<1,0µg/mL) e precisäo (CVintra=6,44 ñ 1,15 porcento e CVinter=8,59 ñ 3,42 porcento). As análises dos auto-anticorpos anti-LDL- evidenciaram a presença de uma resposta imune específica para LDL- em humanos e em coelhos. A determinaçäo da LDL-, abre novas perspectivas para o monitoramento das modificaçöes oxidativas endógenas da LDL em estudos clínicos e de intervençäo que utilizam um elevado número de amostras. Além disto, a detecçäo dos auto-anticorpos anti-LDL- demonstra o potencial imunogênico desta partícula. Portanto, a detecçäo da LDL- e dos auto-anticorpos anti-LDL- abre novas perspectivas para o monitoramento dos fatores de risco para a aterosclerose vinculados às reaçöes oxidativas

Asunto(s)

Humanos , Conejos , Anticuerpos Monoclonales , Arteriosclerosis , Autoanticuerpos , Lipoproteínas LDL/farmacología , Plasma , Ensayo de Inmunoadsorción Enzimática , Biomarcadores/sangre

RESUMEN

Lipid and acute-phase protein alterations have been described in various infection diseases, and they have been recorded during the early stages of HIV infection. Lipid and acute-phase protein profiles also have been correlated with cellular immunological abnormalities. To document these correlations during HIV infection, we studied 75 HIV-infected patients and 26 HIV-negative controls. Patients were classified according to the criteria proposed by the Walter Reed Army Institute: as WR-1 (CD4 lymphocytes, 1154 +/- 268/mm3), WR-2 (CD4, 793 +/- 348/mm3) and WR3/4 (CD4, 287+/-75 mm3). Triglycerides, total cholesterol and HDL-cholesterol concentrations were measured by enzymatic methods. Immunoglobulins (IgA and IgG) and acute-phase proteins (haptoglobin, alpha1-acid glycoprotein, C-reactive protein and transferrin) were determined by immunonephelometry. Haptoglobin levels were significantly increased in HIV-positive patients and correlated with the progression of HIV-infection (control

Asunto(s)

Proteínas de Fase Aguda/metabolismo , Infecciones por VIH/sangre , VIH-1 , Lipoproteínas/sangre , Adulto , Proteína C-Reactiva/metabolismo , Recuento de Linfocito CD4 , Estudios de Casos y Controles , Colesterol/sangre , HDL-Colesterol/sangre , Femenino , Haptoglobinas/metabolismo , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Masculino , Orosomucoide/metabolismo , Transferrina/metabolismo , Triglicéridos/sangre

RESUMEN

Free radicals superoxide (O(2)(-)) and nitric oxide (*NO) are generated by blood vessels and can rapidly react to produce a peroxynitrite anion (ONOO(-)), a powerful oxidant that modifies lipoproteins making them more atherogenic. The aim of this study was to investigate the effect of peroxynitrite-induced modifications on beta-very-low-density lipoprotein (beta-VLDL) as to its biodistribution and plasma clearance rate, as well as the uptake of these particles by THP-1 cells. After being injected into New Zealand White rabbits, the peroxynitrite-modified beta-VLDL (99mTc-per-beta-VLDL) was cleared from circulation faster than the native beta-VLDL (99mTc-nat-beta-VLDL) in both normocholesterolemic rabbits (NC) and in hypercholesterolemic rabbits (HC). In HC rabbits, the fractional clearance of 99mTc-labeled beta-VLDL was significantly lower than in NC rabbits. The in vivo studies showed that accumulation of 99mTc-labeled beta-VLDL, expressed per gram of tissue, followed the decreasing order: kidney > liver > spleen > adrenal gland >or= lung > aortic arch > heart >or= abdominal aorta > thoracic aorta > psoas muscle. The high accumulation in the kidneys suggests the processing of 99mTc-labeled apolipoproteins by receptors present in kidney cells. The accumulation of 99mTc-nat-beta-VLDL in the whole organ was the following: liver > kidney > heart > spleen > adrenal gland > aorta in HC and NC rabbits. The uptake of 99mTc-per-beta-VLDL by the spleen was greater than the uptake by the heart in both groups. The in vitro studies showed that the uptake of 99mTc-per-beta-VLDL by THP-1 cells was higher than that of 99mTc-nat-beta-VLDL. These results show that peroxynitrite-modified beta-VLDL is rapidly removed from plasma and accumulates in several tissues, mainly in the liver and kidney. This may be particularly important in hypercholesterolemic situations that could favor the accumulation of native and peroxynitrite-modified beta-VLDL in several tissues.

Asunto(s)

Lipoproteínas VLDL/farmacocinética , Animales , Masculino , Tasa de Depuración Metabólica , Ácido Peroxinitroso , Conejos , Tecnecio/farmacocinética , Distribución Tisular

RESUMEN

The relationship between free radical and scavenger enzymes has been found in the epileptic phenomena and reactive oxygen species have been implicated in seizure-induced neurodegeneration. Using the epilepsy model obtained by systemic administration of pilocarpine (PILO) in rats, we investigated the superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities as well as the hydroperoxide (HPx) concentration in the hippocampus of rats during status epilepticus (SE), silent and chronic periods. The enzyme activities as well as the HPx concentration were measured using spectrophotometric methods and the results compared to values obtained from saline-treated animals. The SOD activity decreased after long-lasting SE period and during the chronic phase. In addition, HPx levels increased in same periods whereas the GPx activity increased only in the hippocampus of animals submitted to 1 h of SE. Animals presenting partial seizures, those submitted to 5 h of SE and animals from the silent period (seizure free) showed normal levels of SOD, GPx and HPx. These results show a direct evidence of lipid peroxidation during seizure activity that could be responsible for neuronal damage in the hippocampus of rats, during the establishment of PILO model of epilepsy.

Asunto(s)

Epilepsia/metabolismo , Glutatión Peroxidasa/metabolismo , Hipocampo/metabolismo , Peróxido de Hidrógeno/metabolismo , Superóxido Dismutasa/metabolismo , Animales , Enfermedad Crónica , Epilepsia/inducido químicamente , Masculino , Agonistas Muscarínicos , Concentración Osmolar , Pilocarpina , Ratas , Ratas Wistar , Estado Epiléptico/inducido químicamente , Estado Epiléptico/metabolismo

RESUMEN

BACKGROUND/AIMS: Nutrients able to modify the susceptibility of lipoproteins to oxidation and/or reduce the cholesterol levels of blood plasma are important for prevention and/or treatment of atherosclerosis. The influence of animal and vegetable proteins on hypercholesterolemia and atherogenesis has been studied, concerning the mechanisms able to modify the digestion, absorption and bioavailability of lipids. In this study, the influence of casein and soy protein isolate on lipoprotein oxidation and atherosclerosis progression was investigated in cholesterol-fed rabbits. METHODS: During 2 months, 20 New Zealand rabbits were fed with diets containing 1% cholesterol and 27% casein or 27% soy protein isolate. Blood samples were collected at baseline, 15, 30, 45 and 60 days of feeding. RESULTS: Casein feeding contributed to increasing cholesterol and triglyceride concentrations, lipoprotein oxidation and the area of aorta atherosclerotic lesions. In contrast, the soy protein isolate reduced, when compared to casein, the concentrations of cholesterol and lipid peroxides of beta-VLDL and LDL fractions during the experimental time course, as well as the area of atherosclerotic lesions at the end of the study. CONCLUSION: Soy protein isolate, in comparison with casein, promoted a decrease of lipid peroxides, cholesterol and triglyceride content of atherogenic lipoproteins (beta-VLDL and LDL), which had beneficial effects over atherosclerosis progression in cholesterol-fed rabbits.

Asunto(s)

Arteriosclerosis/sangre , Caseínas/farmacología , Hiperlipidemias/sangre , Lipoproteínas/metabolismo , Proteínas de Soja/farmacología , Animales , Arteriosclerosis/patología , Caseínas/administración & dosificación , Colesterol/sangre , Colesterol en la Dieta/administración & dosificación , Progresión de la Enfermedad , Hiperlipidemias/patología , Peróxidos Lipídicos/sangre , Lipoproteínas/sangre , Masculino , Oxidación-Reducción , Conejos , Proteínas de Soja/administración & dosificación , Factores de Tiempo , Triglicéridos/sangre

RESUMEN

Nitric oxide (NO) reacts with thiol-containing biomolecules to form S-nitrosothiols (RSNOs). RSNOs are considered as NO reservoirs as they generate NO by homolytic cleavage. Ceruloplasmin has recently been suggested to have a potent catalytic activity towards RSNO production. Considering that NO activity is impaired in hypercholesterolemia and that RSNOs may act as important NO donors, we investigated the relation between concentrations of ceruloplasmin and RSNOs in plasma of hypercholesterolemic (HC) patients compared to normolipidemic (N) controls. Concentrations of ceruloplasmin (0.36 +/- 0.07 x 0.49 +/- 0.11 mg/dl, N x HC), nitrate (19.10 +/- 12.03 x 40.19 +/- 18.70 microM, N x HC), RSNOs (0.25 +/- 0.20 x 0.54 +/- 0.26 microM, N x HC), nitrated LDL (19.51 +/- 6.98 x 35.29 +/- 17.57 nM nitro-BSA equivalents, N x HC), and cholesteryl ester-derived hydroxy/hydroperoxides (CEOOH, 0.19 +/- 0.06 x 1.46 +/- 0.97 microM) were increased in plasma of HC as compared to N. No difference was found for nitrite levels between the two groups (1.01 +/- 0.53 x 1.02 +/- 0.33 microM, N x HC). The concentrations of RSNOs, nitrate, and nitrated LDL were positively correlated to those of total cholesterol, LDL cholesterol, and apoB. Ceruloplasmin levels were directly correlated to apoB and apoE concentrations. Data suggest that: (i) ceruloplasmin may have a role in the enhancement of RSNOs found in hypercholesterolemia; (ii) the lower NO bioactivity associated with hypercholesterolemia is not related to a RSNOs paucity or a defective NO release from RSNOs; and (iii) the increased nitrotyrosine levels found in hypercholesterolemia indicate that superoxide radicals contribute to inactivation of NO, directly generated by NO synthase or originated by RSNO decomposition.

Asunto(s)

Ceruloplasmina/metabolismo , Hipercolesterolemia/sangre , Mercaptoetanol , Compuestos Nitrosos/sangre , S-Nitrosotioles , Tirosina/análogos & derivados , Apolipoproteínas B/sangre , Ácido Ascórbico/sangre , Catálisis , Colesterol/sangre , Ésteres del Colesterol/sangre , LDL-Colesterol/sangre , Humanos , Peróxido de Hidrógeno/sangre , Lipoproteínas LDL/sangre , Nitratos/sangre , Nitritos/sangre , Tirosina/sangre , Ácido Úrico/sangre , Vitamina E/sangre

RESUMEN

The incidence of atherosclerosis can be modified by diet, and plant-derived proteins have a beneficial effect, but the underlying mechanisms remain unclear. It has been suggested that oxidized LDL (oxLDL) and autoantibodies against oxLDL are important in the development of atherosclerosis. We analyzed these factors in rabbits fed a nonpurified diet supplemented with high cholesterol (10.0 g/kg) containing either 270.0 g/kg casein (CAS, n = 10) or 270.0 g/kg soy protein isolate (SPI, n = 10) for 2 mo. Plasma and purified serum LDL from rabbits were analyzed at d 0, 15, 30, 45 and 60 of treatment, and the size of atherosclerotic lesions was evaluated at d 60 of treatment. CAS-fed rabbits had significantly higher plasma cholesterol at d 15-45 and LDL cholesterol levels at d 15 and 30. Levels of trilinolein and phosphatidylcholine hydroperoxides were higher in the LDL fraction of rabbits fed CAS than in those fed SPI. Also, CAS-fed rabbits had higher levels of highly oxidized LDL [monoclonal antibody (mAb) 24-reactive oxLDL] in plasma at d 60, whereas SPI-fed rabbits had higher levels of minimally oxidized LDL (mAb 28-reactive oxLDL) at d 45. These results were consistent with the earlier formation of anti-oxLDL antibodies and the presence of a larger area of atherosclerotic lesion in rabbits fed the CAS diet. These data indicate the importance of both the type of dietary protein used in the induction of atherosclerosis and the relevance of immunologic mechanisms in addition to biochemical and physiologic factors in the pathogenesis of atherosclerosis.

Asunto(s)

Arteriosclerosis/inducido químicamente , Caseínas/farmacología , Colesterol en la Dieta/efectos adversos , Lipoproteínas/metabolismo , Proteínas de Soja/uso terapéutico , Análisis de Varianza , Animales , Arteriosclerosis/metabolismo , Arteriosclerosis/prevención & control , Autoanticuerpos/biosíntesis , Caseínas/administración & dosificación , Colesterol en la Dieta/administración & dosificación , LDL-Colesterol/sangre , Dieta , Ensayo de Inmunoadsorción Enzimática , Masculino , Oxidación-Reducción/efectos de los fármacos , Conejos , Proteínas de Soja/administración & dosificación

RESUMEN

Information is scarce on the prevalence of hepatitis B (HBV) and hepatitis C (HCV) among voluntary blood donors and patients infected with the human immunodeficiency virus (HIV) in Florianópolis, Brazil. A total of 2,678 serum samples from 2,583 blood donors and 95 HIV-infected patients, collected between April, 1994, and March, 1995, were examined for markers of HBV and HCV. All the samples were analyzed to detect HBV and HCV markers (HBsAg, anti-HBc, and anti-HCV). Hepatitis B and C prevalence among the studied blood donors reached 9.3% and 1.0%, respectively; 0.7% being seropositive for HBsAg and 9.2% for anti-HBc. It was also verified that 0.1% of blood donors were seropositive for HBsAg alone, 8.6% seropositive for the anti-HBc alone, and 0.6% presented a positive reaction for both of the HBV markers studied. Among HIV-infected patients, prevalence of 69.5% and 54.7% for hepatitis B and hepatitis C, respectively, were observed. Of these patients, 18.9% were seropositive for HBsAg, and 66.3% for the anti-HBc. The prevalence of a reaction for HBsAg alone, and for anti-HBc alone was 3.1% and 50.5%, respectively, for HIV-infected patients, whereas 15.8% were seropositive for both of the studied markers. HBV and HCV coinfection was 0.1% in blood donors, and 40% of those patients tested seropositive for HIV. Results show prevalence of HBV and HCV infection to be significantly greater among HIV-infected patients than among blood donors. These observations confirm the high frequency of HIV-infected patients exposure to these other viruses.

Asunto(s)

Donantes de Sangre/estadística & datos numéricos , Infecciones por VIH/complicaciones , VIH-1 , Hepatitis B/epidemiología , Hepatitis C/epidemiología , Brasil/epidemiología , Femenino , Infecciones por VIH/virología , Hepatitis B/inmunología , Anticuerpos contra la Hepatitis B/sangre , Hepatitis C/inmunología , Anticuerpos contra la Hepatitis C/sangre , Humanos , Masculino , Estudios Seroepidemiológicos