RESUMO
The endothelium exerts many vasoprotective effects that are largely mediated by release of nitric oxide (NO). Endothelial dysfunction represents an early but reversible step in atherosclerosis and is characterized by a reduction in the bioavailability of NO. Previous studies have shown that eicosapentaenoic acid (EPA), an omega-3 fatty acid (O3FA), and statins individually improve endothelial cell function, but their effects in combination have not been tested. Through a series of in vitro experiments, this study evaluated the effects of a combined treatment of EPA and the active metabolite of atorvastatin (ATM) on endothelial cell function under conditions of oxidative stress. Specifically, the comparative and time-dependent effects of these agents on endothelial dysfunction were examined by measuring the levels of NO and peroxynitrite (ONOO-) released from human umbilical vein endothelial cells (HUVECs). The data suggest that combined treatment with EPA and ATM is beneficial to endothelial function and was unique to EPA and ATM since similar improvements could not be recapitulated by substituting another O3FA docosahexaenoic acid (DHA) or other TG-lowering agents such as fenofibrate, niacin, or gemfibrozil. Comparable beneficial effects were observed when HUVECs were pretreated with EPA and ATM before exposure to oxidative stress. Interestingly, the kinetics of EPA-based protection of endothelial function in response to oxidation were found to be significantly different than those of DHA. Lastly, the beneficial effects on endothelial function generated by combined treatment of EPA and ATM were reproduced when this study was expanded to an ex vivo model utilizing rat glomerular endothelial cells. Taken together, these findings suggest that a combined treatment of EPA and ATM can inhibit endothelial dysfunction that occurs in response to conditions such as hyperglycemia, oxidative stress, and dyslipidemia.
Assuntos
Ácido Eicosapentaenoico/farmacologia , Endotélio Vascular/fisiopatologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Óxido Nítrico/metabolismo , Animais , Atorvastatina/farmacologia , Disponibilidade Biológica , Endotélio Vascular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Cinética , Lipoproteínas LDL/farmacologia , Masculino , Ratos Endogâmicos WKYRESUMO
Cholesterol crystalline domains characterize atherosclerotic membranes, altering vascular signaling and function. Omega-3 fatty acids reduce membrane lipid peroxidation and subsequent cholesterol domain formation. We evaluated non-peroxidation-mediated effects of eicosapentaenoic acid (EPA), other TG-lowering agents, docosahexaenoic acid (DHA), and other long-chain fatty acids on membrane fluidity, bilayer width, and cholesterol domain formation in model membranes. In membranes prepared at 1.5:1 cholesterol-to-phospholipid (C/P) mole ratio (creating pre-existing domains), EPA, glycyrrhizin, arachidonic acid, and alpha linolenic acid promoted the greatest reductions in cholesterol domains (by 65.5%, 54.9%, 46.8%, and 45.2%, respectively) compared to controls; other treatments had modest effects. EPA effects on cholesterol domain formation were dose-dependent. In membranes with 1:1 C/P (predisposing domain formation), DHA, but not EPA, dose-dependently increased membrane fluidity. DHA also induced cholesterol domain formation without affecting temperature-induced changes in-bilayer unit cell periodicity relative to controls (d-space; 57Å-55Å over 15-30°C). Together, these data suggest simultaneous formation of distinct cholesterol-rich ordered domains and cholesterol-poor disordered domains in the presence of DHA. By contrast, EPA had no effect on cholesterol domain formation and produced larger d-space values relative to controls (60Å-57Å; p<0.05) over the same temperature range, suggesting a more uniform maintenance of lipid dynamics despite the presence of cholesterol. These data indicate that EPA and DHA had different effects on membrane bilayer width, membrane fluidity, and cholesterol crystalline domain formation; suggesting omega-3 fatty acids with differing chain length or unsaturation may differentially influence membrane lipid dynamics and structural organization as a result of distinct phospholipid/sterol interactions.
Assuntos
Aterosclerose/tratamento farmacológico , Colesterol/química , Ácido Eicosapentaenoico/farmacologia , Bicamadas Lipídicas/química , Fluidez de Membrana/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Relação Dose-Resposta a Droga , HumanosRESUMO
Eicosapentaenoic acid (EPA) is a triglyceride-lowering agent that reduces circulating levels of the apolipoprotein B (apoB)-containing lipoprotein particles small dense low-density lipoprotein (sdLDL), very-low-density lipoprotein (VLDL), and oxidized low-density lipoprotein (LDL). These benefits may result from the direct antioxidant effects of EPA. To investigate this potential mechanism, these particles were isolated from human plasma, preincubated with EPA in the absence or presence of atorvastatin (active) metabolite, and subjected to copper-initiated oxidation. Lipid oxidation was measured as a function of thiobarbituric acid reactive substances formation. EPA inhibited sdLDL (IC50 â¼2.0 µM) and LDL oxidation (IC50 â¼2.5 µM) in a dose-dependent manner. Greater antioxidant potency was observed for EPA in VLDL. EPA inhibition was enhanced when combined with atorvastatin metabolite at low equimolar concentrations. Other triglyceride-lowering agents (fenofibrate, niacin, and gemfibrozil) and vitamin E did not significantly affect sdLDL, LDL, or VLDL oxidation compared with vehicle-treated controls. Docosahexaenoic acid was also found to inhibit oxidation in these particles but over a shorter time period than EPA. These data support recent clinical findings and suggest that EPA has direct antioxidant benefits in various apoB-containing subfractions that are more pronounced than those of other triglyceride-lowering agents and docosahexaenoic acid.
Assuntos
Antioxidantes/farmacologia , Apolipoproteína B-100/metabolismo , Atorvastatina/farmacologia , Ácido Eicosapentaenoico/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/metabolismo , Triglicerídeos/metabolismo , Atorvastatina/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Quimioterapia Combinada , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Oxirredução , Tamanho da Partícula , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Fatores de TempoRESUMO
Lipid oxidation leads to endothelial dysfunction, inflammation, and foam cell formation during atherogenesis. Glucose also contributes to lipid oxidation and promotes pathologic changes in membrane structural organization, including the development of cholesterol crystalline domains. In this study, we tested the comparative effects of eicosapentaenoic acid (EPA), an omega-3 fatty acid indicated for the treatment of very high triglyceride (TG) levels, and other TG-lowering agents (fenofibrate, niacin, and gemfibrozil) on lipid oxidation in human low-density lipoprotein (LDL) as well as membrane lipid vesicles prepared in the presence of glucose (200 mg/dL). We also examined the antioxidant effects of EPA in combination with atorvastatin o-hydroxy (active) metabolite (ATM). Glucose-induced changes in membrane structural organization were measured using small angle x-ray scattering approaches and correlated with changes in lipid hydroperoxide (LOOH) levels. EPA was found to inhibit LDL oxidation in a dose-dependent manner (1.0-10.0 µM) and was distinguished from the other TG-lowering agents, which had no significant effect as compared to vehicle treatment alone. Similar effects were observed in membrane lipid vesicles exposed to hyperglycemic conditions. The antioxidant activity of EPA, as observed in glucose-treated vesicles, was significantly enhanced in combination with ATM. Glucose treatment produced highly-ordered, membrane-restricted, cholesterol crystalline domains, which correlated with increased LOOH levels. Of the agents tested in this study, only EPA inhibited glucose-induced cholesterol domain formation. These data demonstrate that EPA, at pharmacologic levels, inhibits hyperglycemia-induced changes in membrane lipid structural organization through a potent antioxidant mechanism associated with its distinct, physicochemical interactions with the membrane bilayer.
Assuntos
Antioxidantes/química , LDL-Colesterol/química , Ácido Eicosapentaenoico/química , Glucose/química , Lipídeos de Membrana/química , Antioxidantes/farmacologia , Atorvastatina , Ácido Eicosapentaenoico/farmacologia , Fenofibrato/química , Fenofibrato/farmacologia , Genfibrozila/química , Genfibrozila/farmacologia , Glucose/antagonistas & inibidores , Glucose/farmacologia , Ácidos Heptanoicos/química , Ácidos Heptanoicos/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Peróxidos Lipídicos/química , Membranas Artificiais , Niacina/química , Niacina/farmacologia , Pirróis/química , Pirróis/farmacologiaAssuntos
Membrana Celular/química , Colesterol/química , Espalhamento a Baixo Ângulo , Difração de Raios X/métodos , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Membrana Celular/metabolismo , Colesterol/metabolismo , Cristalização , Humanos , Cristalino/química , Cristalino/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Modelos Moleculares , Músculo Liso/química , Músculo Liso/metabolismo , Músculo Liso/patologia , Músculo Liso Vascular/química , Músculo Liso Vascular/metabolismoRESUMO
BACKGROUND: Clinical trials have shown that amlodipine reduces cardiovascular events at a rate that is not predicted by changes in brachial arterial pressure alone. These findings may be explained, in part, by the pleiotropic effects of amlodipine on endothelial cell (EC) function. In this study, we elucidated the effect of amlodipine on nitric oxide (NO) bioavailability and cytotoxic peroxynitrite (ONOO(-)) and blood pressure (BP). METHODS: Spontaneously hypertensive rats (SHRs) were treated with vehicle or amlodipine (5 mg/kg/day) for 8 weeks and compared with untreated, baseline rats. NO and ONOO(-) release from aortic and glomerular ECs were measured ex vivo using amperometric nanosensors following maximal stimulation with calcium ionophore. BP was measured using the tail-cuff method. RESULTS: As compared with baseline, vehicle treatment had reduced aortic endothelial NO release from 157 ± 11 nM to 55 ± 6 nM and increased ONOO(-) from 69 ± 7 nM to 156 ± 19 nM. The NO/ONOO(-) ratio, a comprehensive measurement of eNOS function, decreased from 2.3 ± 0.3 to 0.3 ± 0.1. Compared with vehicle, amlodipine treatment restored NO to 101 ± 3 nM, decreased ONOO(-) to 50 ± 4 nM, and increased the NO/ONOO(-) ratio to 2.0 ± 0.2, a level similar to baseline. Similar changes were observed for glomerular ECs. Mean arterial blood pressure increased from 149 ± 3 mm Hg (baseline) to 174 ± 1 mm Hg (vehicle). Amlodipine slightly, but significantly, decreased mean arterial blood pressure to 167 ± 3 mm Hg vs. vehicle treatment. CONCLUSIONS: Amlodipine increased NO bioavailability and decreased nitroxidative stress in SHRs with EC dysfunction disproportionately to BP changes. These direct, vascular effects of amlodipine on EC function may contribute to reduced risk for atherothrombotic events as observed in clinical trials.
Assuntos
Anlodipino/farmacologia , Anti-Hipertensivos/farmacologia , Aorta/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Glomérulos Renais/efeitos dos fármacos , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Aorta/metabolismo , Aorta/fisiopatologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Glomérulos Renais/metabolismo , Glomérulos Renais/fisiopatologia , Masculino , Óxido Nítrico Sintase Tipo III/metabolismo , Ácido Peroxinitroso/metabolismo , Ratos , Ratos Endogâmicos SHR , Fatores de TempoRESUMO
BACKGROUND: Nebivolol is a third-generation beta-blocker used to treat hypertension. The vasodilation properties of nebivolol have been attributed to nitric oxide (NO) release. However, the kinetics and mechanism of nebivolol-stimulated bioavailable NO are not fully understood. METHODS: Using amperometric NO and peroxynitrite (ONOOâ») nanosensors, ß3-receptor (agonist: L-755,507; antagonists: SR59230A and L-748,337), ATP efflux (the mechanosensitive ATP channel blocker, gadolinium) and P2Y-receptor (agonists: ATP and 2-MeSATP; antagonist: suramin) modulators, superoxide dismutase and a NADPH oxidase inhibitor (VAS2870), we evaluated the kinetics and balance of NO and ONOOâ» stimulated by nebivolol in human umbilical vein endothelial cells (HUVECs). NO and ONOOâ» were measured with nanosensors (diameter ~ 300 nm) placed 5 ± 2 µm from the cell membrane and ATP levels were determined with a bioluminescent method. The kinetics and balance of nebivolol-stimulated NO and ONOOâ» were compared with those of ATP, 2-MeSATP, and L-755,507. RESULTS: Nebivolol stimulates endothelial NO release through ß3-receptor and ATP-dependent, P2Y-receptor activation with relatively slow kinetics (75 ± 5 nM/s) as compared to the kinetics of ATP (194 ± 10 nM/s), L-755,507 (108 ± 6 nM/s), and 2-MeSATP (105 ± 5 nM/s). The balance between cytoprotective NO and cytotoxic ONOO- was expressed as the ratio of [NO]/[ONOOâ»] concentrations. This ratio for nebivolol was 1.80 ± 0.10 and significantly higher than that for ATP (0.80 ± 0.08), L-755,507 (1.08 ± 0.08), and 2-MeSATP (1.09 ± 0.09). Nebivolol induced ATP release in a concentration-dependent manner. CONCLUSION: The two major pathways (ATP efflux/P2Y receptors and ß3 receptors) and several steps of nebivolol-induced NO and ONOOâ» stimulation are mainly responsible for the slow kinetics of NO release and low ONOOâ». The net effect of this slow kinetics of NO is reflected by a favorable high ratio of [NO]/[ONOOâ»] which may explain the beneficial effects of nebivolol in the treatment of endothelial dysfunction, hypertension, heart failure, and angiogenesis.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 3/farmacologia , Benzopiranos/farmacologia , Células Endoteliais/efeitos dos fármacos , Etanolaminas/farmacologia , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Trifosfato de Adenosina/metabolismo , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Técnicas de Cultura de Células , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Cinética , Nebivolol , Receptores Purinérgicos P2Y/metabolismo , Fatores de TempoRESUMO
PURPOSE: Naphthalene induces cataract formation through the accumulation of its reactive metabolite, 1,2-naphthoquinone (1,2-NQ), in the ocular lens. 1,2-NQ increases lens protein oxidation and disrupts fiber cell membrane function; however, the association of these effects with changes in membrane structure is not understood. The goal of this study was to determine the direct effects of 1,2-NQ on membrane lipid oxidation and structural organization. METHODS: Iodometric approaches were used to measure the effects of naphthalene and 1,2-NQ on lipid hydroperoxide (LOOH) formation in model membranes composed of cholesterol and dilinoleoylphosphatidylcholine. Membrane samples were prepared at various cholesterol-to-phospholipid mole ratios and subjected to autoxidation at 37°C for 48 hours in the absence or presence of either agent alone (0.1-5.0 µM) or in combination with vitamin E. Small-angle x-ray diffraction was used to measure the effects of naphthalene and 1,2-NQ on membrane structure before and after exposure to oxidative stress. RESULTS: 1,2-NQ increased LOOH formation by 250% (P < 0.001) and 350% (P < 0.001) at 1.0 and 5.0 µM, respectively, whereas naphthalene decreased LOOH levels by 25% (P < 0.01) and 10% (NS). The pro-oxidant effect of 1,2-NQ was inversely affected by membrane cholesterol enrichment and completely blocked by vitamin E. 1,2-NQ also increased cholesterol domain formation by 360% in membranes exposed to oxidative stress; however, no significant changes in membrane lipid organization were observed with naphthalene under the same conditions. CONCLUSIONS: These data suggest a novel mechanism for naphthalene-induced cataract, facilitated by the direct effects of 1,2-NQ on lipid peroxidation and cholesterol domain formation.
Assuntos
Colesterol/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos de Membrana/análise , Naftoquinonas/farmacologia , Análise de Variância , Catarata/induzido quimicamente , Humanos , Peróxidos Lipídicos/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Naftalenos/farmacologia , Naftoquinonas/efeitos adversos , Naftoquinonas/metabolismoRESUMO
We tested the hypothesis that atorvastatin active metabolite (ATM), on the basis of its distinct structural features and potent antioxidant activity, preferentially inhibits lipid oxidation in human small dense low-density lipoprotein (sdLDL) and other small lipid vesicles. LDL, sdLDL, and various subfractions were isolated from human plasma by sequential ultracentrifugation, treated with ATM, atorvastatin, pravastatin, rosuvastatin, or simvastatin and were subjected to copper-induced oxidation. Lipid oxidation was measured spectrophotometrically as a function of thiobarbituric acid reactive substances formation. Similar analyses were performed in reconstituted lipid vesicles enriched in polyunsaturated fatty acids and prepared at various sizes. ATM was found to inhibit sdLDL oxidation in a dose-dependent manner. The antioxidant effects of ATM in sdLDL were 1.5 and 4.7 times greater (P < 0.001) than those observed in large buoyant LDL and very low-density lipoprotein subfractions, respectively. ATM had similar dose- and size-dependent effects in reconstituted lipid vesicles. None of these effects were reproduced by atorvastatin (parent) or any of the other statins examined in this study. These data suggest that ATM interacts with sdLDL in a specific manner that also confers preferential resistance to oxidative stress. Such interactions may reduce sdLDL atherogenicity and improve clinical outcomes in patients with cardiovascular disease.
Assuntos
Antioxidantes/farmacologia , Ácidos Heptanoicos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lipoproteínas LDL/química , Pirróis/farmacologia , Atorvastatina , Fenômenos Químicos , Sulfato de Cobre/efeitos adversos , Sulfato de Cobre/antagonistas & inibidores , Ácidos Heptanoicos/metabolismo , Humanos , Peróxidos Lipídicos/análise , Peróxidos Lipídicos/antagonistas & inibidores , Lipoproteínas LDL/antagonistas & inibidores , Lipoproteínas LDL/isolamento & purificação , Lipoproteínas VLDL/química , Lipoproteínas VLDL/isolamento & purificação , Lipossomos/química , Concentração Osmolar , Oxidantes/efeitos adversos , Oxidantes/antagonistas & inibidores , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Pirróis/metabolismo , Ultracentrifugação , Lipossomas Unilamelares/químicaRESUMO
Most patients with diabetes also have hypertension, a risk factor associated with atherothrombotic disease and characterized by endothelial cell (EC) dysfunction and loss of nitric oxide (NO) bioavailability. Recent studies suggest a possible antihypertensive effect with dipeptidyl peptidase-4 (DPP4) inhibition; however, the underlying mechanism is not understood. In this study, we tested the effects of the DPP4 inhibitor, saxagliptin, on EC function, blood pressure, and soluble intercellular adhesion molecule 1 (sICAM-1) levels in hypertensive rats. Spontaneously hypertensive rats were treated with vehicle or saxagliptin (10 mg·kg(-1)·day(-1)) for 8 weeks. NO and peroxynitrite (ONOO(-)) release from aortic and glomerular ECs was stimulated with calcium ionophore and measured using electrochemical nanosensor technology. Changes in EC function were correlated with fasting glucose levels. Saxagliptin treatment was observed to increase aortic and glomerular NO release by 22% (P < 0.001) and 23% (P < 0.001), respectively, with comparable reductions in ONOO(-) levels; the NO/ONOO(-) ratio increased by >50% in both EC types (P < 0.001) as compared with vehicle. Saxagliptin also reduced mean arterial pressure from 170 ± 10 to 158 ± 10 mm Hg (P < 0.001) and decreased sICAM-1 levels by 37% (P < 0.01). The results of this study suggest that DPP4 inhibition reduces blood pressure and inflammation in hypertensive rats while increasing NO bioavailability.
Assuntos
Adamantano/análogos & derivados , Pressão Sanguínea/efeitos dos fármacos , Dipeptídeos/uso terapêutico , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Hipertensão/tratamento farmacológico , Molécula 1 de Adesão Intercelular/sangue , Óxido Nítrico/metabolismo , Adamantano/administração & dosagem , Adamantano/farmacologia , Adamantano/uso terapêutico , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Dipeptídeos/administração & dosagem , Dipeptídeos/farmacologia , Inibidores da Dipeptidil Peptidase IV/administração & dosagem , Inibidores da Dipeptidil Peptidase IV/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Teste de Tolerância a Glucose , Hipertensão/enzimologia , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Insulina/sangue , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Masculino , Ácido Peroxinitroso/metabolismo , Ratos , Ratos ZuckerRESUMO
WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: ⢠Angiotensin II receptor blockers improve endothelial cell-dependent vasodilation in patients with hypertension through suppression of angiotensin II type 1 receptors but may have additional and differential effects on endothelial nitric oxide synthase (eNOS) function. WHAT THIS STUDY ADDS: ⢠The key finding from this study is that angiotensin II receptor blockers (ARBs) differentially enhanced nitric oxide (NO) release in a manner influenced by certain genetic variants of eNOS. This finding provides new insights into the effects of ARBs on endothelial cell-dependent vasodilation and eNOS function that are of high importance in vascular medicine and clinical pharmacology. AIM Angiotensin II receptor blockers (ARBs) improve endothelial cell (EC)-dependent vasodilation in patients with hypertension through suppression of angiotensin II type 1 receptors but may have additional and differential effects on endothelial nitric oxide (NO) synthase (eNOS) function. To investigate this question, we tested the effects of various ARBs on NO release in ECs from multiple donors, including those with eNOS genetic variants linked to higher cardiovascular risk. METHODS: The effects of ARBs (losartan, olmesartan, telmisartan, valsartan), at 1 µm, on NO release were measured with nanosensors in human umbilical vein ECs obtained from 18 donors. NO release was stimulated with calcium ionophore (1 µm) and its maximal concentration was correlated with eNOS variants. The eNOS variants were determined by a single nucleotide polymorphism in the promoter region (T-786C) and in the exon 7 (G894T), linked to changes in NO metabolism. RESULTS All of the ARBs caused an increase in NO release as compared with untreated samples (P < 0.01, n= 4-5 in all eNOS variants). However, maximal NO production was differentially influenced by eNOS genotype. Olmesartan increased maximal NO release by 30%, which was significantly greater (P < 0.01, n= 4-5 in all eNOS variants) than increases observed with other ARBs. CONCLUSIONS: The ARBs differentially enhanced NO release in ECs in a manner influenced by eNOS single nucleotide polymorphisms. These findings provide new insights into the effects of ARBs on EC-dependent vasodilation and eNOS function.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico/metabolismo , Polimorfismo de Nucleotídeo Único , Bloqueadores do Receptor Tipo 1 de Angiotensina II/metabolismo , Benzimidazóis/farmacologia , Benzoatos/farmacologia , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Imidazóis/farmacologia , Losartan/farmacologia , Óxido Nítrico/genética , Telmisartan , Tetrazóis/farmacologia , Veias Umbilicais/citologia , Valina/análogos & derivados , Valina/farmacologia , Valsartana , Vasodilatação/efeitos dos fármacos , Vasodilatação/genéticaRESUMO
The Smith-Lemli-Opitz syndrome (SLOS) is an inherited disorder of cholesterol synthesis caused by mutations in DHCR7 which encodes the final enzyme in the cholesterol synthesis pathway. The immediate precursor to cholesterol synthesis, 7-dehydrocholesterol (7-DHC) accumulates in the plasma and cells of SLOS patients which has led to the idea that the accumulation of abnormal sterols and/or reduction in cholesterol underlies the phenotypic abnormalities of SLOS. We tested the hypothesis that 7-DHC accumulates in membrane caveolae where it disturbs caveolar bilayer structure-function. Membrane caveolae from skin fibroblasts obtained from SLOS patients were isolated and found to accumulate 7-DHC. In caveolar-like model membranes containing 7-DHC, subtle, but complex alterations in intermolecular packing, lipid order and membrane width were observed. In addition, the BK(Ca) K(+) channel, which co-migrates with caveolin-1 in a membrane fraction enriched with cholesterol, was impaired in SLOS cells as reflected by reduced single channel conductance and a 50 mV rightward shift in the channel activation voltage. In addition, a marked decrease in BK(Ca) protein but not mRNA expression levels was seen suggesting post-translational alterations. Accompanying these changes was a reduction in caveolin-1 protein and mRNA levels, but membrane caveolar structure was not altered. These results are consistent with the hypothesis that 7-DHC accumulation in the caveolar membrane results in defective caveolar signaling. However, additional cellular alterations beyond mere changes associated with abnormal sterols in the membrane likely contribute to the pathogenesis of SLOS.
Assuntos
Cavéolas/metabolismo , Desidrocolesteróis/metabolismo , Fibroblastos/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Síndrome de Smith-Lemli-Opitz/metabolismo , Caveolina 1/metabolismo , Células Cultivadas , Desidrocolesteróis/química , Genótipo , Humanos , Immunoblotting , Membranas Artificiais , Microscopia Eletrônica , Estrutura Molecular , Pele/citologia , Esteróis/metabolismo , Difração de Raios XRESUMO
AIM: Endothelial cell (EC) dysfunction contributes to insulin resistance in diabetes and is characterized by reduced nitric oxide (NO) release, increased nitroxidative stress and enhanced inflammation. The purpose of this study was to test the effect of improved postprandial glucose control on EC function in insulin-resistant rats as compared to fasting glucose (FG) changes. METHODS: Obese Zucker rats were treated with 10 mg/kg/day saxagliptin, a dipeptidyl peptidase-4 (DPP4) inhibitor, for 4 or 8 weeks and compared to lean rats. NO and peroxynitrite (ONOO(-)) release from aortic and glomerular ECs was measured ex vivo using amperometric approaches and correlated with FG, postprandial glucose, insulin, soluble CD40 (sCD40) and L-citrulline levels. RESULTS: Saxagliptin treatment improved NO production and reduced ONOO(-) release prior to any observed changes in FG levels. In untreated obese animals, NO release from aortic and glomerular ECs decreased by 22% and 31%, respectively, while ONOO(-) release increased by 26% and 40%. Saxagliptin increased aortic and glomerular NO release by 18% and 31%, respectively, with comparable reductions in ONOO(-) levels; the NO/ONOO(-) ratio, an indicator of NO synthase coupling, increased by >40%. Improved glycemic control was further associated with a reduction in sCD40 levels by more than ten-fold (from 300 ± 206 to 22 ± 22 pg/mL, p < 0.001). CONCLUSION: These findings indicate that enhanced glycemic control with DPP4 inhibition improved NO release and reduced inflammation in a manner not predicted by FG changes alone.
Assuntos
Adamantano/análogos & derivados , Glicemia/análise , Antígenos CD40/metabolismo , Dipeptídeos/farmacologia , Hipoglicemiantes/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Obesidade/sangue , Adamantano/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Colesterol/sangue , Citrulina/sangue , Teste de Tolerância a Glucose , Insulina/sangue , Obesidade/enzimologia , Obesidade/metabolismo , Ratos , Ratos Zucker , Triglicerídeos/sangueRESUMO
A number of structural and functional mechanisms have been identified in the pathogenesis of hypertensive vascular disease, each of which requires effective therapy to reduce global cardiovascular risk. Hypertension, together with other cardiovascular risk factors, promotes endothelial dysfunction as evidenced by decreased nitric oxide (NO) release and reduced vascular responsiveness to normal vasodilatory stimuli. In addition, the mechanical forces inherent in hypertension activate neurohormonal mechanisms, including the renin-angiotensin system, which modulate vessel wall structure and function. Antihypertensive drugs may have class-specific hemodynamic and physiologic effects that attenuate these vascular disease processes. Pharmacologic approaches that enhance endothelial NO bioavailability have been shown to restore vasodilation while reducing clinical events. These agents improve NO bioavailability by increasing endogenous production through enzymatic mechanisms or by promoting the direct release of NO by its redox congeners in a spontaneous fashion. In this article, we review the basic mechanisms of endothelial dysfunction along with the use and comparative therapeutic benefits of various pharmacologic interventions, with particular emphasis on antihypertensive agents.
Assuntos
Anti-Hipertensivos/uso terapêutico , Hipertensão/tratamento farmacológico , Óxido Nítrico/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/uso terapêutico , Anti-Hipertensivos/farmacologia , Disponibilidade Biológica , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Hemodinâmica , Humanos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Resistência à Insulina , Óxido Nítrico/biossíntese , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Fosfodiesterase/uso terapêutico , Sistema Renina-Angiotensina/efeitos dos fármacos , Fatores de RiscoRESUMO
BACKGROUND: Cardiovascular disease remains the leading cause of morbidity and premature mortality in most industrialized countries as well as in developing nations. A pro-oxidative state appears to promote and/or exacerbate vascular disease complications. Furthermore, a state of low-grade chronic inflammation can promote increased oxidative stress and lead to endothelial cell and platelet dysfunction ultimately contributing to thrombogenesis. OBJECTIVES: In this study, the effect of a proprietary astaxanthin prodrug (CDX-085) on thrombus formation was investigated using a mouse model of arterial thrombosis. The influence of free astaxanthin, the active drug of CDX-085, on human endothelial cells and rat platelets was evaluated to investigate potential mechanisms of action. METHODS AND RESULTS: Oral administration of CDX-085 (0.4% in chow, approximately 500 mg/kg/day) to 6-8 week old C57BL/6 male mice for 14 days resulted in significant levels of free astaxanthin in the plasma, liver, heart and platelets. When compared to control mice, the CDX-085 fed group exhibited significant increases in basal arterial blood flow and significant delays in occlusive thrombus formation following the onset of vascular endothelial injury. Primary human umbilical vein endothelial cells (HUVECs) and platelets isolated from Wistar-Kyoto rats treated with free astaxanthin demonstrated significantly increased levels of released nitric oxide (NO) and significantly decreased peroxynitrite (ONOO-) levels. CONCLUSION: Observations of increased NO and decreased ONOO- levels in endothelial cells and platelets support a potential mechanism of action for astaxanthin (CDX-085 active drug). These studies support the potential of CDX-085 and its metabolite astaxanthin in the treatment or prevention of thrombotic cardiovascular complications.
Assuntos
Fibrinolíticos/uso terapêutico , Pró-Fármacos/uso terapêutico , Trombose/tratamento farmacológico , Administração Oral , Animais , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Fibrinolíticos/administração & dosagem , Fibrinolíticos/farmacocinética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacocinética , Ratos , Ratos Wistar , Trombose/fisiopatologia , Vasodilatadores/administração & dosagem , Vasodilatadores/farmacocinética , Vasodilatadores/uso terapêutico , Xantofilas/administração & dosagem , Xantofilas/farmacocinética , Xantofilas/uso terapêuticoRESUMO
BACKGROUND: Endothelial cell (EC) dysfunction contributes to hypertension and mechanisms of atherosclerosis. Agents that improve EC function may provide vascular protection, especially in patients with multiple risk factors. In this study, we examined the effects of beta(1)-selective antagonists, nebivolol and metoprolol, on vascular and renal EC function in spontaneously hypertensive (SH) rats with diabetes. METHODS: Male SH rats were treated with streptozotocin (STZ) to induce type 2 diabetes, followed by treatment with nebivolol or metoprolol at 2 mg/kg/day (vs. vehicle). After 4 weeks, aortic and glomerular ECs were isolated, stimulated with calcium ionophore (CaI), and assayed for nitric oxide (NO), and peroxynitrite (ONOO(-)) release using amperometric approaches. RESULTS: Glucose and mean blood pressure (BP) levels were significantly elevated in diabetic SH rats. In aortic ECs isolated from diabetic SH rats, NO production decreased by 20% whereas ONOO(-) increased by 16%, an effect linked to NAD(P)H oxidase and endothelial NO synthase (eNOS) uncoupling. Nebivolol treatment reduced glucose and BP levels and restored aortic EC function in diabetic SH rats, as indicated by a 30% increase and 23% decrease in NO and ONOO(-) levels, respectively. The NO/ONOO(-) ratio increased by more than twofold with nebivolol treatment in aortic and glomerular ECs. Despite similar reductions in glucose and mean BP levels, metoprolol had a smaller effect on the NO/ONOO(-) ratio in glomerular ECs but no effect in aortic ECs. CONCLUSIONS: Vascular and renal NO was significantly reduced in diabetic hypertensive rats and correlated with metabolic changes. Nebivolol reversed these effects in a manner consistent with enhanced endothelial function.
Assuntos
Antagonistas Adrenérgicos beta/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Benzopiranos/uso terapêutico , Diabetes Mellitus Experimental/metabolismo , Angiopatias Diabéticas/metabolismo , Etanolaminas/uso terapêutico , Hipertensão/metabolismo , Metoprolol/uso terapêutico , Óxido Nítrico/metabolismo , Animais , Artérias/metabolismo , Glicemia/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Angiopatias Diabéticas/tratamento farmacológico , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Hipertensão/complicações , Hipertensão/tratamento farmacológico , Rim/metabolismo , Masculino , Nebivolol , Ácido Peroxinitroso/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Oxidative damage to vascular cell membrane phospholipids causes physicochemical changes in membrane structure and lipid organization, contributing to atherogenesis. Oxidative stress combined with hyperglycemia has been shown to further increase the risk of vascular and metabolic diseases. In this study, the effects of glucose on oxidative stress-induced cholesterol domain formation were tested in model membranes containing polyunsaturated fatty acids and physiologic levels of cholesterol. Membrane structural changes, including cholesterol domain formation, were characterized by small angle X-ray scattering (SAXS) analysis and correlated with spectrophotometrically-determined lipid hydroperoxide levels. Glucose treatment resulted in a concentration-dependent increase in lipid hydroperoxide formation, which correlated with the formation of highly-ordered cholesterol crystalline domains (unit cell periodicity of 34 A) as well as a decrease in overall membrane bilayer width. The effect of glucose on lipid peroxidation was further enhanced by increased levels of cholesterol. Treatment with free radical-scavenging agents inhibited the biochemical and structural effects of glucose, even at elevated cholesterol levels. These data demonstrate that glucose promotes changes in membrane organization, including cholesterol crystal formation, through lipid peroxidation.
Assuntos
Colesterol/química , Glucose/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Anlodipino/farmacologia , Atorvastatina , Membrana Celular/efeitos dos fármacos , Glucose/antagonistas & inibidores , Ácidos Heptanoicos/farmacologia , Lipídeos de Membrana/química , Estresse Oxidativo , Fosfolipídeos/química , Pirróis/farmacologia , Difração de Raios XRESUMO
Several classes of antihypertensive drugs have been shown to improve vascular function through mechanisms other than reducing blood pressure (BP) alone. Certain dihydropyridine calcium channel blockers (CCBs) and inhibitors of the renin-angiotensin system (RAS) increase nitric oxide (NO) bioavailability and decrease oxidative stress, thereby improving endothelial activity and vascular function. Pulse wave analyses have shown that these agents reduce the impact of pressure wave reflections on central systolic BP (SBP), consistent with a decrease in arterial stiffness. The complementary vascular mechanisms of these drug classes suggest that combination therapy may be effective for improving clinical outcomes. In animal model studies, combination calcium channel/RAS blockade has been shown to be more effective in improving endothelial dysfunction than treatment with drugs from either class alone. Furthermore, results from recent clinical trials suggest a greater reduction in central aortic SBP, pulse pressure, and cardiovascular events with calcium channel/RAS blockade vs. beta-blocker/diuretic therapy. These studies support the potential benefit of combination calcium channel and RAS blockade in the prevention and treatment of cardiovascular disease.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Bloqueadores dos Canais de Cálcio/uso terapêutico , Hipertensão/tratamento farmacológico , Sistema Renina-Angiotensina/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Humanos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Estresse Oxidativo/fisiologia , Resultado do Tratamento , Resistência Vascular/efeitos dos fármacosRESUMO
Carotenoids are naturally occurring organic pigments that are believed to have therapeutic benefit in treating cardiovascular disease (CVD) because of their antioxidant properties. However, prospective randomized trials have failed to demonstrate a consistent benefit for the carotenoid beta-carotene in patients at risk for CVD. The basis for this apparent paradox is not well understood but may be attributed to the distinct antioxidant properties of various carotenoids resulting from their structure-dependent physicochemical interactions with biologic membranes. To test this hypothesis, we measured the effects of astaxanthin, zeaxanthin, lutein, beta-carotene, and lycopene on lipid peroxidation using model membranes enriched with polyunsaturated fatty acids. The correlative effects of these compounds on membrane structure were determined using small-angle x-ray diffraction approaches. The nonpolar carotenoids, lycopene and beta-carotene, disordered the membrane bilayer and stimulated membrane lipid peroxidation (>85% increase in lipid hydroperoxide levels), whereas astaxanthin (a polar carotenoid) preserved membrane structure and exhibited significant antioxidant activity (>40% decrease in lipid hydroperoxide levels). These results suggest that the antioxidant potential of carotenoids is dependent on their distinct membrane lipid interactions. This relation of structure and function may explain the differences in biologic activity reported for various carotenoids, with important therapeutic implications.
Assuntos
Doenças Cardiovasculares/prevenção & controle , Carotenoides/metabolismo , Carotenoides/farmacologia , Doenças Cardiovasculares/metabolismo , Carotenoides/química , Carotenoides/uso terapêutico , Endotélio Vascular/efeitos dos fármacos , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Luteína/farmacologia , Licopeno , Estresse Oxidativo/efeitos dos fármacos , Xantofilas/farmacologia , Zeaxantinas , beta Caroteno/farmacologiaRESUMO
OBJECTIVES: This study was designed to determine the predictive value of lipid hydroperoxide (LOOH) levels for adverse cardiovascular outcomes in patients with stable coronary artery disease (CAD). BACKGROUND: Oxidative modification of circulating lipids contributes to inflammation and endothelial dysfunction, which are hallmark features of atherosclerosis. A serum biomarker of oxidation is LOOH, which is a primary product of fatty acid peroxidation. METHODS: Serum LOOH levels were measured and correlated with clinical events over a 3-year period in 634 patients with angiographic evidence of CAD. RESULTS: Baseline LOOH levels in the highest quartile were associated with hazard ratios of 3.24 (95% confidence interval [CI] 1.86 to 5.65; p = 0.0001) for nonfatal vascular events (n = 149), 1.80 (95% CI 1.13 to 2.88; p = 0.014) for major vascular procedures (n = 139), and 2.23 (95% CI 1.44 to 3.44; p = 0.0003) for all vascular events and procedures. Baseline LOOH levels correlated with serum levels of soluble intercellular adhesion molecule-1 (p = 0.001) and thiobarbituric acid reactive substances (p = 0.001) as well as the mean percent change in stenosis for large segments >50% stenosed (p = 0.048). A multivariate proportional hazards model, adjusted for traditional risk factors and inflammatory markers, showed an independent effect of LOOH on nonfatal vascular events, vascular procedures, and all events or procedures. Amlodipine treatment was associated with reduced cardiovascular events and changes in LOOH levels compared with placebo. CONCLUSIONS: Elevated LOOH levels were predictive of nonfatal vascular events and procedures in patients with stable CAD, independent of traditional risk factors and inflammatory markers.