RESUMO
Oxidized low density lipoprotein (ox-LDL) has been suggested to affect endothelium-dependent vascular tone through a decreased biological activity of endothelium-derived nitric oxide (NO). Oxidative inactivation of NO is regarded as an important cause of its decreased biological activity, and in this context superoxide (O(2)) is known to inactivate NO in a chemical reaction during which peroxynitrite is formed. In this study we examined the effect of ox-LDL on the intracellular NO concentration in bovine aortic endothelial cells and whether this effect is influenced by ox-LDL binding to the endothelial receptor lectin-like ox-LDL receptor-1 (LOX-1) through the formation of reactive oxygen species and in particular of O(2). ox-LDL induced a significant dose-dependent decrease in intracellular NO concentration both in basal and stimulated conditions after less than 1 min of incubation with bovine aortic endothelial cells (p < 0.01). In the same experimental conditions ox-LDL also induced O(2) generation (p < 0.001). In the presence of radical scavengers and anti-LOX-1 monoclonal antibody, O(2) formation induced by ox-LDL was reduced (p < 0.001) with a contemporary rise in intracellular NO concentration (p < 0.001). ox-LDL did not significantly modify the ability of endothelial nitric oxide synthase to metabolize l-arginine to l-citrulline. The results of this study show that one of the pathophysiological consequences of ox-LDL binding to LOX-1 may be the inactivation of NO through an increased cellular production of O(2).
Assuntos
Imidazolinas , Lipoproteínas LDL/metabolismo , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Receptores de LDL/metabolismo , Superóxidos/metabolismo , Alopurinol/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Antioxidantes/farmacologia , Aorta/metabolismo , Ácido Ascórbico/farmacologia , Aspirina/farmacologia , Bradicinina/farmacologia , Células CHO , Catecolaminas/farmacologia , Bovinos , Células Cultivadas , Cromanos/farmacologia , Cricetinae , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Sequestradores de Radicais Livres/farmacologia , Hemostáticos/farmacologia , Humanos , Camundongos , Probucol/farmacologia , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Receptores de LDL Oxidado , Receptores Depuradores Classe E , Trombina/farmacologia , Fatores de Tempo , ômega-N-Metilarginina/farmacologiaRESUMO
In this study we examined the effect of oxidized low density lipoprotein (ox-LDL) on the intracellular production of reactive oxygen species (ROS) in bovine aortic endothelial cells (BAECs) and whether this increase occurs through its binding to the endothelial receptor lectin-like ox-LDL receptor-1 (LOX-1). Furthermore, this study also aimed to ascertain whether the binding of ox-LDL to LOX-1 is associated with NF-kappaB activation. ox-LDL induced a significant dose-dependent increase in ROS production after a 30-s incubation with BAECs (p < 0.01). ROS formation was markedly reduced in BAECs incubated with anti-LOX-1 monoclonal antibody (p < 0.001), while control nonimmune IgG produced no effect. ox-LDL induced a time- and dose-dependent significant increase in ROS formation only in CHO-K1 cells stably expressing bovine LOX-1 (p < 0.001), while no increase was present in CHO-K1 cells. The activation of the transcription factor NF-kappaB in BAECs was evident after a 5-min incubation with ox-LDL and was attenuated by anti-LOX-1 monoclonal antibody. The conclusion is that one of the pathophysiological consequences of ox-LDL binding to LOX-1 may be the activation of NF-kappaB through an increased ROS production.
Assuntos
Endotélio Vascular/metabolismo , Lipoproteínas LDL/metabolismo , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de LDL/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Células CHO , Bovinos , Células Cultivadas , Cromanos/farmacologia , Cricetinae , Fluoresceínas/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Probucol/farmacologia , Ligação Proteica , Receptores de LDL Oxidado , Receptores Depuradores Classe E , Fatores de TempoRESUMO
The adhesion of monocytes to endothelium, an early event in atherosclerosis, is mediated by cell adhesion molecules. Signal-transduction pathways for these binding molecules include the translocation of the transcription factor NF-kappaB; moreover, intracellularly generated oxygen-derived free radicals (ODFR) play a major role in this process. This study evaluated the extent to which troglitazone, an oral antidiabetic agent with antioxidant properties, affects the expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin on human umbilical vein endothelial cells (HUVECs), induced by different prooxidant signals such as oxidized LDL and tumor necrosis factor-alpha (TNF-alpha). Furthermore we assessed whether the NF-kappaB activation is modulated by the antioxidative effect of troglitazone. Oxidized LDL not only caused a dose-dependent increase of ICAM-1, VCAM-1 and E-selectin (p<0.001), but also synergically increased their TNF-alpha-induced expression (p<0.001). Troglitazone reduced in a dose-dependent manner the expression of VCAM-1, ICAM-1 and E-selectin induced by different amounts of oxidized LDL (p<0.001). The addition of troglitazone to HUVECs significantly reduced the expression of ICAM-1, VCAM-1 and E-selectin induced by TNF-alpha alone or in combination with oxidized LDL (p<0.001); this reduction was paralleled by a significant fall in NF-kappaB translocation. The results suggest that troglitazone may have prevented NF-kappaB-mediated adhesion molecule expression by exerting its antioxidant effect on ODFR.
Assuntos
Antioxidantes/farmacologia , Moléculas de Adesão Celular/biossíntese , Cromanos/farmacologia , Endotélio Vascular/metabolismo , Tiazóis/farmacologia , Tiazolidinedionas , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Selectina E/biossíntese , Endotélio Vascular/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Lipoproteínas LDL/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Troglitazona , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais , Molécula 1 de Adesão de Célula Vascular/biossíntese , Vitamina E/farmacologiaRESUMO
OBJECTIVE: Lacidipine has already been demonstrated to reduce the expression of some adhesion molecules induced by pro-oxidant signals on endothelial cells. In order to verify if this effect is a peculiarity of this molecule, or belongs to other dihydropyridinic compounds (DHPs), the activity of lacidipine was compared with that of lercanidipine, amlodipine, nimodipine and nifedipine. DESIGN AND METHODS: The compounds were incorporated in human umbilical vein endothelial cells (HUVECs) using native low-density lipoprotein as a carrier. The drug concentrations in HUVECs were measured by mass spectrometry. Human recombinant tumour necrosis factor-alpha was then incubated with HUVECs for 7 h at 37 degrees C for adhesion molecule expression. RESULTS: The cellular amount of lacidipine, lercanidipine and amlodipine was similar, while nimodipine and nifedipine were almost undetectable or undetectable, respectively. Lacidipine, at any concentration, determined a dose-dependent significant decrease of the expression of intercellular adhesion molecule-1 (ICAM-1) ICAM-1, vascular cell adhesion molecule-1 (VCAM-1) VCAM-1 and E-selectin (P < 0.01). Lercanidipine and amlodipine determined variable decreases of adhesion molecules at the intermediate and highest concentrations. Nimodipine and nifedipine determined no effect on ICAM-1, VCAM-1 and E-selectin. The lowest IC50, i.e. the concentration determining the 50% reduction of ICAM-1, VCAM-1 and E-selectin expression was obtained with lacidipine for all the adhesion molecules considered (P < 0.01). CONCLUSIONS: It is concluded that the effect of the DHPs used in this study on adhesion molecule expression is determined first by their lipophilicity and then by their intrinsic antioxidant activity.
Assuntos
Moléculas de Adesão Celular/metabolismo , Di-Hidropiridinas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , Selectina E/metabolismo , Endotélio Vascular/citologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Troglitazone, an oral antidiabetic agent with antioxidant properties, has previously been shown to increase the resistance of LDL to oxidation in vitro and in vivo in healthy volunteers. In a randomized, placebo-controlled, parallel-group study in 29 patients with NIDDM, we tested the effect of troglitazone (200 mg once daily) on the resistance of LDL to oxidation and on circulating levels of preformed lipid hydroperoxides and the adhesion molecule E-selectin. Resistance of LDL to oxidation was assessed by measuring 1) fluorescence development induced by copper treatment (lag phase), and 2) amount of thiobarbituric acid-reactive substances (TBARS) generated by incubation with umbilical vein endothelial cells. At 8 weeks, the lag phase was increased by 23% (P < 0.01 by analysis of covariance [ANCOVA]) in the patients receiving troglitazone (n = 18) compared with the group receiving placebo (n = 11). At the same time, TBARS were 3.63 +/- 0.10 nmol/l (vs. 5.32 +/- 0.10 nmol/l in the placebo group, P = 0.009), LDL hydroperoxide concentration was reduced from 1.48 +/- 0.03 to 1.19 +/- 0.03 ng/mg (no change in the placebo group, P < 0.01), and plasma E-selectin levels decreased from 56.5 +/- 2.33 to 43.7 +/- 1.77 microg/l (no change in the placebo group, P < 0.01). In NIDDM, troglitazone may slow down the development of atherosclerosis by modifying LDL-related atherogenic events.
Assuntos
LDL-Colesterol/metabolismo , Cromanos/farmacologia , Diabetes Mellitus Tipo 2/sangue , Selectina E/sangue , Hipoglicemiantes/farmacologia , Tiazóis/farmacologia , Tiazolidinedionas , Análise de Variância , Arteriosclerose/prevenção & controle , Cromanos/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Método Duplo-Cego , Endotélio Vascular/metabolismo , Feminino , Fluorescência , Humanos , Hipoglicemiantes/uso terapêutico , Masculino , Pessoa de Meia-Idade , Oxirredução , Peróxidos/análise , Peróxidos/sangue , Tiazóis/uso terapêutico , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Troglitazona , Vitamina E/administração & dosagemRESUMO
The present study tested the effects of ox-low density lipoprotein (LDL) on nitric oxide (NO)-dependent decrease in agonist-stimulated [Ca2+]i. The effects of ox-LDL on platelet aggregation were also evaluated. Platelets loaded with FURA 2 AM (2 micromol/litre) were incubated with NO-donors for 2-10 min to obtain a 40-50% reduction in \[Ca2+]i and with NO-donors plus ox-LDL (100 microg of protein/ml). Thrombin (0.03 U/ml) was used as an agonist. In some experiments 8-Br-cGMP (0.5-1 mmol/l) was used to investigate the NO-dependent intraplatelet signalling system. Slightly oxidized LDL was obtained by leaving native LDL in the light at room temperature for at least 7 days. Ox-LDL did not cause any increase in thrombin-induced [Ca2+] (control: 215.4 +/- 44.3 nmol/l, ox-LDL 223.4 +/- 35.3 nmol/l, M +/- SEM; n = 8) and platelet aggregation (control: 78.7 +/- 4.9% , ox-LDL: 78.9 +/- 4.2% , n = 12). Ox-LDL antagonized the effects of NO-donors on platelet [Ca2+]i (NO-donor: 137.4 +/- 22.1 nmol/l, NO + ox-LDL: 177.3 +/- 27.6 nmol/l, n = 11; P < 0.001) and platelet aggregation (NO-donor: 15.4 +/- 3.4% , NO + ox-LDL: 28.9 +/- 3.8%, n = 24; P < 0.001). Ox-LDL did not affect the inhibitory activities of 8-Br-cGMP on platelet aggregation (8-Br-cGMP: 22.0 +/- 8.5%, 8-Br-cGMP + ox-LDL: 19.3 +/- 7.8%, n = 5) and platelet [Ca2+]i . In conclusion, slightly oxidized LDL does not directly activate platelets and does not i affect the intracellular NO-dependent signalling system. The present results suggest that LDL reduces the antiplatelet activity of NO mainly by preventing its biological effects.
RESUMO
OBJECTIVE: The mechanisms by which oxidized low-density lipoprotein (ox-LDL) induces the expression of adhesion molecules on endothelial cells (HUVECs) are still not clear. The signal transduction pathways for these binding molecules include the translocation of the transcription factor NF-kB and the intracellular reactive oxygen species (ROS) are said to play a key role in this process. Aim of this study was (1) to evaluate the effect of ox-LDL on intracellular production of ROS in culture of HUVECs; (2) to evaluate if the intracellular increase of ROS induced by ox-LDL is mediated by the binding to a specific endothelial receptor; (3) to ascertain if lacidipine can decrease ox-LDL-induced ROS production in HUVECs. METHODS: Five microM Cu2+ ox-LDL were incubated with HUVECs for 5 min. 2',7'-Dichlorofluorescein (DCF) as an expression of intracellular ROS production, was measured by flow cytometry. RESULTS: ox-LDL induced a significant dose-dependent increase in DCF production (P < 0.001) through the binding to a specific receptor. The preincubation of HUVECs with radical scavengers compounds and lacidipine significantly reduced (P < 0.001) the ox-LDL-induced DCF production. CONCLUSIONS: ox-LDL increased the intracellular formation of ROS through the ligation to a specific endothelial receptor. Preincubation of HUVECs with lacidipine, a calcium antagonist with antioxidant properties, significantly reduced the intracellular ROS formation induced by ox-LDL. We propose that the effect of lacidipine on adhesion molecule expression and on NF-kB activation can be explained by its effect on intracellular ROS formation.
Assuntos
Di-Hidropiridinas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Lipoproteínas LDL/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/farmacologia , Arteriosclerose/etiologia , Arteriosclerose/prevenção & controle , Bloqueadores dos Canais de Cálcio/farmacologia , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Fluoresceínas , Corantes Fluorescentes , Humanos , Líquido Intracelular/metabolismo , Lipoproteínas LDL/metabolismo , NF-kappa B/metabolismo , OxirreduçãoRESUMO
Although elevated levels of soluble E-selectin and intercellular cell adhesion molecules-1 (ICAM-1) have been reported in non-insulin-dependent diabetes mellitus (NIDDM), it is not clear by what mechanism this elevation occurs and whether or not it is related to glycaemic control. In this study we analyse: 1) the relation of glycaemic control with the concentrations of E-selectin, vascular cell adhesion molecules-1 (VCAM-1) and ICAM-1 in NIDDM patients: 2) whether metabolic control can affect the oxidative stress (as measured by plasma hydroperoxide concentration and susceptibility of LDL to in vitro oxidation) and hence the adhesion molecule plasma concentrations. Thirty-four (19 males and 15 females) poorly controlled NIDDM patients were studied. All parameters were evaluated at the beginning of the study and after 90 days of dietary and pharmacological treatment. The treatment decreased HbA1c (p < 0.001), E-selectin (p < 0.001), plasma hydroperoxides (p < 0.003) and the susceptibility of LDL to in vitro oxidation (lag phase) (p < 0.0001). Before treatment HbA1c, lag phase and lipid hydroperoxides correlated with E-selectin plasma concentration (r = 0.51, -0.57 and 0.54, respectively, p < 0.01). There was also a correlation between HbA1c and lag phase (p < 0.01) and between HbA1c and lipid hydroperoxides (p < 0.01). In addition, the variations of HbA1c, lag phase and lipid hydroperoxide values correlated with those for E-selectin concentration after 90 days' treatment (r = 0.54, -0.64 and 0.61, respectively, p < 0.01). In multiple linear correlation analysis, however, the partial correlation coefficients of HbA1c (basal and variations) with E-selectin concentration (basal and variations) fell to non-significant values (r = 0.12 and 0.25, respectively) when LDL lag phase and plasma hydroperoxides were kept constant. The results indicate that the improvement of metabolic control in NIDDM patients is associated with a decrease of E-selectin plasma levels; they also suggest that glycaemic control per se is not directly implicated in determining E-selectin plasma concentration; glycaemic control could affect E-selectin concentration through its effect on oxidative stress.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 2/sangue , Selectina E/sangue , Lipoproteínas LDL/sangue , Estresse Oxidativo , Colesterol/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Diabetes Mellitus Tipo 2/dietoterapia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Feminino , Hemoglobinas Glicadas/análise , Humanos , Molécula 1 de Adesão Intercelular/sangue , Peróxidos Lipídicos/sangue , Masculino , Pessoa de Meia-Idade , Análise de Regressão , Triglicerídeos/sangue , Molécula 1 de Adesão de Célula Vascular/sangueRESUMO
Trolitazone is a new oral antidiabetic agent able to reduce lipid peroxidation. In this study we evaluated its effect on the susceptibility of LDL and HDL to in vitro oxidation induced by copper ions and endothelial cells. In Cu(++)-induced LDL modification, different amounts of troglitazone were added to aliquots of the same pool of plasma with subsequent ultracentrifuge separation of LDL and HDL. Differences in LDL and HDL susceptibility to in vitro oxidation with Cu(++) were studied by measuring the changes in fluorescence intensity (expressed as lag phase). LDL derived from plasma incubated with different amounts of troglitazone were also incubated with umbilical vein endothelial cells (HUVEC), the modification being monitored by LDL relative electrophoretic mobility and fluorescence. During Cu(++)- and HUVEC-induced LDL oxidation, the decay rate of vitamin E, and the potency of troglitazone as a radical scavenger in comparison with vitamin E were also studied. Troglitazone determined a significant, dose-dependent decrease in Cu(++)-induced LDL and HDL oxidation. Incubation with HUVEC was also followed by a progressive, significant decrease of LDL relative electrophoretic mobility and fluorescence intensity. During Cu(++)- and HUVEC-induced-LDL modification, troglitazone significantly reduced the rate of vitamin E decay. In this study we also demonstrated that under the same oxidative stress, troglitazone was much more potent as a radical scavenger than vitamin E. In conclusion, the results demonstrate that troglitazone can reduce LDL and HDL in vitro oxidation and that, during this process, it can protect vitamin E. In addition to ensuring blood glucose control, the drug may therefore be useful in inhibiting lipoprotein peroxidation.
Assuntos
Arteriosclerose/metabolismo , Cromanos/farmacologia , Diabetes Mellitus/metabolismo , Hipoglicemiantes/farmacologia , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Tiazóis/farmacologia , Tiazolidinedionas , Células Cultivadas , Cobre/farmacologia , Endotélio/citologia , Humanos , Oxirredução/efeitos dos fármacos , Troglitazona , Vitamina E/metabolismoRESUMO
The oxidative modification of low density lipoprotein (LDL) and the endothelial expression of adhesion molecules are key events in the pathogenesis of atherosclerosis. In this study we evaluated the effect of oxidized LDL on the expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on human umbilical vein endothelial cells (HUVECs). The hypothesis that oxidized LDL functions as a prooxidant signal was also evaluated, by studying the effect of different radical-scavenging antioxidants on expression of adhesion molecules. LDL was oxidized by using Cu2+, HUVECs or phospholipase A2 (PLA2)/ soybean lipoxygenase (SLO), the degree of oxidation being measured as thiobarbituric acid-reactive substances (TBARS) and conjugated dienes (CD). Exposure of 200 micrograms/ml of native LDL to 1 microns Cu2+, HUVECs and to PLA2/ SLO resulted in four- to fivefold higher levels of TBARS and CD than in native LDL. Cu(2+)-(1 microM), HUVEC-, and PLA2/SLO-oxidized LDL caused a dose-dependent, significant increase of ICAM-1 and VCAM-1 (p < .01). The expression of E-selectin did not change. LDL oxidized with a 2.5 and 5 microM Cu2+ did not increase ICAM-1 and VCAM-1 significantly. Both the Cu(2+)- and HUVEC-oxidized LDL, subjected to dialysis and ultrafiltration, induced ICAM-1 and VCAM-1 expression. After incubation with the ultrafiltrate, the expression of ICAM-1 and VCAM-1 was not significantly different from that obtained with native LDL. LDL pretreated with different antioxidants (vitamin E and probucol) and subjected to oxidation by Cu2+ and HUVECs induced a significantly lower expression of ICAM-1 and VCAM-1 than nonloaded LDL (p < .01). The pretreatment of HUVECs with vitamin E and probucol significantly reduced the expression of VCAM-1 on HUVECs induced by oxidized LDL (p < .01); the effect on ICAM-1 was much less evident. In conclusion, oxidized LDL can induce the expression of different adhesion molecules on HUVECs; this induction can be prevented by pretreating either the LDL or the cells with radical-scavenging antioxidant.
Assuntos
Antioxidantes/farmacologia , Endotélio Vascular/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/biossíntese , Lipoproteínas LDL/farmacologia , Molécula 1 de Adesão de Célula Vascular/biossíntese , Células Cultivadas , Diálise , Selectina E/biossíntese , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Sequestradores de Radicais Livres/farmacologia , Radicais Livres , Humanos , Oxirredução , Espécies Reativas de Oxigênio , Ultrafiltração , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismoRESUMO
OBJECTIVE: The adhesion of monocytes to endothelium, an early event in atherosclerosis is mediated by cell adhesion molecules. Signal-transduction pathways for these binding molecules include the translocation of the transcription factor NF-kappaB; moreover, intracellularly generated oxygen-derived free radicals play a major role in this process. In this study we evaluated the extent to which lacidipine, a calcium antagonist with antioxidant properties, affects the expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin on human umbilical vein endothelial cells, induced by different pro-oxidant signals such as oxidized low density lipoprotein (LDL) and tumor necrosis factor-alpha (TNF-alpha). METHODS: We incubated 5 micromol/l Cu2+-oxidized LDL and TNF-alpha (2 ng/ml) with human umbilical vein endothelial cells for 48 and 6 h, respectively. ICAM-1, VCAM-1 and E-selectin were measured by flow cytometry. NF-kappaB was evaluated by electrophoretic mobility shift assay. RESULTS: The incubation of 5 micromol/l Cu2+-oxidized LDL not only caused a dose-dependent increase in ICAM-1, VCAM-1 and E-selectin (P < 0.001), but also synergically increased their TNF-alpha-induced expression (P < 0.001). The addition of lacidipine to human umbilical vein endothelial cells significantly reduced the expression of ICAM-1, VCAM-1 and E-selectin induced by TNF-alpha alone or with oxidized LDL (P < 0.001). The reduction in adhesion molecule expression caused by lacidipine was paralleled by a significant fall in NF-kappaB translocation. CONCLUSIONS: The results suggest that lacidipine may have prevented NF-kappaB-mediated adhesion molecule expression by exerting its effects on oxygen-derived free radicals. The results support previous observations that lacidipine may have therapeutic effects in atherosclerosis.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Moléculas de Adesão Celular/metabolismo , Di-Hidropiridinas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/metabolismo , Humanos , Oxirredução , Veias Umbilicais/citologiaRESUMO
The oxidative modification of low density lipoprotein is of importance in atherogenesis. Antioxidant supplementation has been shown, in published work, to increase low density lipoprotein resistance to oxidation in both healthy subjects and diabetic subjects; in animal studies a contemporary reduction in atherogenesis has been demonstrated. Troglitazone is a novel oral antidiabetic drug which has similarities in structure with vitamin E. The present study assessed the effect of troglitazone 400 mg twice daily for 2 weeks on the resistance of low density lipoprotein to oxidation in healthy male subjects. Ten subjects received troglitazone and ten received placebo in a randomised, placebo-controlled, parallel-group design. The lag phase (a measure of the resistance of low density lipoprotein to oxidation) was determined by measurement of fluorescence development during copper-catalysed oxidative modification of low density lipoprotein. The lag phase was increased by 27 % (p < 0.001) at week 1 and by 24% (p < 0.001) at week 2 in the troglitazone treated group compared with the placebo group. A number of variables known to influence the resistance of low density lipoprotein to oxidation were measured. They included macronutrient consumption, plasma and lipoprotein lipid profile, alpha-tocopherol, beta-carotene levels in low density lipoprotein, low density lipoprotein particle size, mono and polyunsaturated fatty acid content of low density lipoprotein and pre-formed low density lipoprotein hydroperoxide levels in low density lipoprotein. Troglitazone was associated with a significant reduction in the amount of pre-formed low density lipoprotein lipid hydroperoxides. At weeks 1 and 2, the low density lipoprotein hydroperoxide content was 17% (p < 0.05) and 18% (p < 0.05) lower in the troglitazone group compared to placebo, respectively. In summary the increase in lag phase duration in the troglitazone group appeared to be due to the compound's activity as an antioxidant and to its ability to reduce the amount of preformed low density lipoprotein lipid hydroperoxides. This antioxidant activity could provide considerable benefit to diabetic patients where atherosclerosis accounts for the majority of total mortality.
Assuntos
Antioxidantes/farmacologia , Cromanos/farmacologia , Hipoglicemiantes/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Lipoproteínas LDL/efeitos dos fármacos , Tiazóis/farmacologia , Tiazolidinedionas , Administração Oral , Adulto , Antioxidantes/administração & dosagem , Cromanos/administração & dosagem , Humanos , Hipoglicemiantes/administração & dosagem , Peróxidos Lipídicos/sangue , Masculino , Valores de Referência , Tiazóis/administração & dosagem , Fatores de Tempo , Triglicerídeos/sangue , TroglitazonaRESUMO
In this study we evaluated the time course and mechanism of low density lipoprotein (LDL) oxidation induced by human umbilical vein endothelial cells (HUVECs), cell-free medium (CFM) and Cu2+. After incubating LDL (200 micrograms/ml) with HUVECs, CFM and Cu2+ (concentration adjusted to obtain the same degree of LDL modification as with HUVECs), the extent of LDL lipid peroxidation and apoprotein B modification was monitored at different times from 0 to 24 h. This involved evaluating the time course of LDL conjugated diene, peroxide, malonyldialdehyde (MDA), fluorescence, relative electrophoretic mobility (REM), vitamin E and monounsaturated and polyunsaturated fatty acids. After incubation with HUVECs, the LDL REM was significantly higher than that obtained in CFM (p < 0.01). When balanced for the same degree of LDL modification as obtained with HUVECs, Cu2+ gave a REM similar to that obtained with HUVECs. At the different times of incubation there was no statistical difference between conjugated diene and peroxide values after incubation with HUVECs and with CFM. The values obtained with Cu2+ were significantly higher than those obtained with HUVECs and CFM (p < 0.01). MDA and LDL fluorescence were significantly higher after exposure to HUVECs than to CFM (p < 0.01), values being similar to those obtained with Cu2+. There was no statistical difference between the values of LDL oleic, linoleic, arachidonic and eicosapentaenoic acids after incubation with HUVECs and CFM. Eicosatetraynoic acid (ETYA), a lipoxygenase inhibitor, determined dose-dependent reduction of MDA formation induced by the incubation of LDL with HUVECs; it did not affect LDL conjugated diene. ETYA did not have any effect on the MDA derived from LDL after incubation with Cu2+ or CFM. The results of this study demonstrate that, unlike Cu2+, the contribution of HUVECs to LDL modification does not involve only lipid peroxidation of the lipoprotein; it also includes intracellular radical and non-radical processes.
Assuntos
Cobre/farmacologia , Endotélio Vascular/fisiologia , Lipoproteínas LDL/metabolismo , Ácido 5,8,11,14-Eicosatetrainoico/farmacologia , Ácido Araquidônico/metabolismo , Células Cultivadas , Meios de Cultura , Ácido Eicosapentaenoico/metabolismo , Eletroforese , Endotélio Vascular/citologia , Ácidos Graxos Insaturados/metabolismo , Fluorescência , Guanidinas/farmacologia , Humanos , Cinética , Peroxidação de Lipídeos , Peróxidos Lipídicos/metabolismo , Malondialdeído/metabolismo , Ácido Oleico/metabolismo , Veias Umbilicais , Vitamina E/metabolismoRESUMO
The authors interviewed 76.2% of the pediatricians from the Pediatric Department-School of Medical Sciences-Campinas University-UNICAMP, to analyse the difficulties of this professional group in the evaluation of the domestic violence against children. From the group of professionals, 86.7% had already attended a situation involving child abuse; the therapeutic aspects were referred in 44.4% as most important, and in 46%, the difficulties in the diagnostic aspects were referred. They conclude that, despite the experiences with the theme, a lot more could be done and studied.
RESUMO
Oxidized low-density lipoproteins (LDL) play an important role in the pathogenesis of atherosclerosis. An increased sensitivity of red blood cell membranes to lipid peroxidation has been previously demonstrated in patients with chronic renal failure, suggesting that the antioxidant defence of lipoproteins might be impaired. Fish oil supplementation has been proposed in dialysis patients, but it is still unclear if the positive effects of fish oil depend only on its polyunsaturated fatty acid content or on other factors, such as the usually added antioxidants. Moreover, the increased concentration of highly peroxidable n-3 polyunsaturated fatty acids induced by fish oil in LDL particles could favour LDL oxidation and possibly the development of atherosclerosis. The present study was designed to evaluate the susceptibility of LDL to in vitro oxidation (lag phase) and the rate of lipid peroxidation (propagation phase) by fluorescence development during copper exposure in 14 hemodialysis patients. A further aim was to compare the effects on lipid metabolism and LDL oxidation of fish oil supplementation (20 ml containing vitamin E 20 IU as antioxidant) for 30 days and of vitamin E administration (50 IU) for another 30 days. The length of the lag phase and vitamin E concentration were significantly reduced (p < 0.01) in hemodialysis patients and increased significantly (p < 0.01) after administration of both fish oil and vitamin E. Fish oil supplementation also reduced plasma lipids significantly (p < 0.01) and increased the propagation phase (p < 0.01). Our results demonstrate that the susceptibility of LDL to oxidation is enhanced in hemodialysis patients, suggesting a possible relationship between excessive LDL peroxidation and accelerated atherosclerosis. The increased susceptibility of LDL to in vitro oxidation can be explained, at least partially, by a reduced LDL vitamin E concentration. Since fish oil increased the lag phase to the same extent as vitamin E supplementation, the positive effect of fish oil could be partly explained by its antioxidant content.
Assuntos
Arteriosclerose/prevenção & controle , Membrana Eritrocítica/metabolismo , Óleos de Peixe/administração & dosagem , Falência Renal Crônica/terapia , Lipoproteínas LDL/sangue , Vitamina E/administração & dosagem , Arteriosclerose/sangue , Arteriosclerose/etiologia , Dieta , Membrana Eritrocítica/efeitos dos fármacos , Feminino , Humanos , Falência Renal Crônica/sangue , Falência Renal Crônica/complicações , Masculino , Pessoa de Meia-Idade , Oxirredução , Diálise Renal , Fatores de RiscoRESUMO
The adhesion of leucocytes to the endothelium, an early step in atherogenesis, is mediated by cell adhesion molecules. In this study we evaluated the concentration of soluble adhesion molecules in patients with insulin-dependent (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) and studied its relation to glycaemic control. Soluble adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) were measured in 31 diabetic patients (18 with IDDM and 13 with NIDDM), 20 hyperlipoproteinaemic patients (10 with type IIa and 10 with type IIb) and 20 healthy subjects. Increased E-selectin concentrations were found in the patients with IDDM and NIDDM and in the hyperlipoproteinaemic patients when compared to the control subjects (p < 0.01 for all the groups). ICAM-1 was found to be elevated only in the patients with NIDDM (p < 0.01). No significant differences in VCAM-1 concentration were found in the different groups of subjects. The concentration of plasma E-selectin was positively correlated with the glycated haemoglobin (r = 0.54, p < 0.01) in patients with IDDM and NIDDM. In the same patients E-selectin was not related to the concentrations of plasma lipids in spite of the fact that it was found to be elevated in hyperlipoproteinaemic subjects. The results though preliminary suggest that in diabetic patients the concentration of soluble adhesion molecules and especially of E-selectin may be related to metabolic control.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 2/sangue , Selectina E/sangue , Hemoglobinas Glicadas/análise , Hiperlipoproteinemia Tipo II/sangue , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Humanos , Molécula 1 de Adesão Intercelular/sangue , Masculino , Pessoa de Meia-Idade , Valores de Referência , Análise de Regressão , Molécula 1 de Adesão de Célula Vascular/sangueRESUMO
The authors report the case of a 50-day-old baby who was brought to the Pediatric Emergency Room of the "Hospital de Clinicas da UNICAMP" with seizures. At the examination she presented without external signs of trauma, hypoactive and with generalized seizures. Numerous hemorrhages were found in the ocular fundi. CT scan showed interhemisferic hemorrhage and brain swelling. The child was admitted to the Pediatric Intensive Care Unit, kept under controlled ventilation and hydantal. Although these measures, she died three days later. Since the beginning there was suspicion of child abuse. One day after the admission the father told that he had shaken the baby because she was crying too much. The present paper discuss social and epidemiological aspects, and about the difficulties in the diagnosis of this syndrome that sometimes may be fatal, as in this case.
Assuntos
Lesões Encefálicas , Maus-Tratos Infantis/diagnóstico , Lesões Encefálicas/diagnóstico por imagem , Evolução Fatal , Feminino , Humanos , Lactente , Tomografia Computadorizada por Raios XRESUMO
It has been suggested that the oxidative modification of low-density lipoprotein (LDL) plays a major role in atherogenesis. We evaluated the oxidative resistance to copper-induced oxidative changes of LDL derived from patients affected by type IIa hyperlipoproteinemia compared with healthy subjects and faced the question of the importance of the antioxidants and polyunsaturated fatty acids (PUFAs) contained in LDL in determining its variability. LDL isolated from the plasmas of 25 subjects affected by familial hypercholesterolemia and 15 control subjects was oxidatively modified with Cu2+ in vitro, and the differences in LDL susceptibilities (lag and propagation phases) to lipid peroxidation were studied by measuring the changes in fluorescence intensity. LDL alpha-tocopherol and PUFAs were also measured. The lag phase was significantly lower and the propagation phase significantly higher in the type IIa patients than in control subjects (p < 0.01). The linoleic and arachidonic acids, expressed as percentage of total LDL fatty acids, were significantly higher in type IIa patients than in the control subjects (p < 0.01). There was a positive significant correlation between the LDL cholesterol and the linoleic and arachidonic acids as percentage of total LDL fatty acids (p < 0.01). Both linoleic and arachidonic acids turned out to be negatively correlated with the lag phase and positively with the propagation phase (p < 0.01). The concentration of LDL alpha-tocopherol was similar in the two groups. Therefore, type IIa patients have a greater susceptibility to LDL oxidation than control subjects. This may be due to a relative higher concentration of linoleic and arachidonic acids in LDL derived from patients with familial hypercholesterolemia.
Assuntos
Hipercolesterolemia/metabolismo , Lipoproteínas LDL/metabolismo , Adulto , Antropometria , Antioxidantes/metabolismo , Ácido Araquidônico/sangue , Ácido Araquidônico/metabolismo , LDL-Colesterol/sangue , LDL-Colesterol/metabolismo , Cobre/farmacologia , Ácidos Graxos Insaturados/sangue , Ácidos Graxos Insaturados/metabolismo , Feminino , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/genética , Ácido Linoleico , Ácidos Linoleicos/sangue , Ácidos Linoleicos/metabolismo , Peroxidação de Lipídeos , Lipoproteínas LDL/sangue , Masculino , Pessoa de Meia-Idade , Oxirredução , Fatores de Tempo , Vitamina E/sangue , Vitamina E/metabolismoRESUMO
The determination of lipid hydroperoxides in plasma and lipoproteins recently reached a clinical relevance in disorders such as atherosclerosis, where oxidative reactions have been suggested to play a fundamental pathogenetic role. The peroxide content of lipoproteins is usually measured after ultracentrifugation and extraction. During this procedure, some peroxides might decompose causing a too low recovery. To screen this possibility, the disappearance, in the presence of human plasma, of hydroperoxides of linoleic acid and Cu-oxidized low density lipoprotein (LDL) have been investigated, using both a iodometric titration and an enzymatic assay. While only in the presence of GSH plasma decomposes linoleic acid hydroperoxides quite rapidly, peroxides in Cu-oxidized LDL were stable both in presence as well as in absence of GSH. This indicated that lipid hydroperoxides are stable in plasma and that peroxides of Cu-oxidized LDL are not substrate for the glutathione-dependent peroxidase activity in plasma. The relevant decrease of the iodometric titre of LDL peroxides observed in the presence of elevated amounts of plasma was shown to be artifactual, since some compounds extracted from plasma do react with iodine generated by peroxides. Whole plasma itself, indeed, has been shown to reduce back to I- appreciable amount of free iodine.
Assuntos
Cobre/farmacologia , Ácidos Linoleicos/sangue , Peróxidos Lipídicos/sangue , Lipoproteínas LDL/metabolismo , Glutationa/farmacologia , Humanos , Cinética , Lipoproteínas LDL/efeitos dos fármacos , Oxirredução , Peroxidases/sangue , Valores de Referência , Especificidade por Substrato , Fatores de TempoRESUMO
A cross-sectional study was carried out to estimate prevalence of child abuse with battering among patients under age 14 for alleged disciplinary purposes. The reference population were children attending pediatric out-patient services at the University Hospital in Campinas, State of São Paulo, Brazil, a city of one million inhabitants with universal coverage in terms of accessing health care in the unit under study. Interviews were carried out with 130 parents or guardians in a randomly selected group according to order of attendance, and the prevalence of child abuse was estimated at 10.8%, considering as positive cases those where parents referred child battering under age one year or where there was evidence of bodily lesion over age one year. A validation study for the experimental questionnaire is proposed in order to allow for future estimation of population prevalence or incidence. The use of qualitative methods is also recommended as a means of gathering detailed data on socio-psychological determination for the supposedly disciplinary aggression with child abuse.