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INTRODUCTION: The relationships between systemic hemodynamics and renal blood flow and renal microcirculation are poorly known in sepsis. Norepinephrine (NE) infusion may add another level of complexity. METHODS: Ventilated and anesthetized rats were submitted to various mean arterial pressure (MAP) steps by blood removal, in presence and absence of sepsis and/or NE. Renal blood flow (RBF) and blood velocity (Vm) in renal cortical capillaries (using Sidestream Dark Field Imaging) were measured. Data were analyzed using linear mixed models enabling us to display the effects of both the considered explanatory variables and their interactions. RESULTS: Positive correlations were found between MAP and RBF. Sepsis had no independent impact on RBF whereas norepinephrine decreased RBF, regardless of the presence of sepsis. The relationship between MAP and RBF was weaker above a MAP of 100 mmHg as opposed to below 100 mmHg, with RBF displaying a relative "plateau" above this threshold. Sepsis and NE impacted carotid blood flow (CBF) differently compared to RBF, demonstrating organ specificity. A positive relationship was observed between MAP and Vm. Sepsis increased Vm while nNE decreased Vm irrespective of MAP. Sepsis was associated with an increase in serum creatinine determined at the end of the experiments, which was prevented by NE infusion. CONCLUSION: In our model, sepsis at an early phase did not impact RBF over a large range of MAP. NE elicited a renal vasoconstrictive effect. Autoregulation of RBF appeared conserved in sepsis. Conversely, sepsis was associated with "hypervelocity" of blood flow in cortical peritubular capillaries reversed by NE infusion.
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Homeostase/efeitos dos fármacos , Microcirculação/efeitos dos fármacos , Norepinefrina/farmacologia , Circulação Renal/efeitos dos fármacos , Sepse/fisiopatologia , Animais , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Pressão Sanguínea , Capilares/fisiologia , Artérias Carótidas/fisiologia , Frequência Cardíaca , Córtex Renal/irrigação sanguínea , Túbulos Renais/irrigação sanguínea , Masculino , Estudos Prospectivos , Distribuição Aleatória , Ratos Wistar , VasoconstriçãoRESUMO
50% of patients with heart failure have a preserved ejection fraction (HFpEF). Numerous studies have investigated the pathophysiological mechanisms of HFpEF and have shown that endothelial dysfunction plays an important role in HFpEF. Yet no studies answered whether endothelial dysfunction could be the cause or is the consequence of HFpEF. Recently, we have shown that the endothelial overexpression of human ß 3-adrenoreceptor (Tgß 3) in rats leads to the slow development of diastolic dysfunction over ageing. The aim of the study is to decipher the involvement of endothelial dysfunction in the HFpEF development. For that, we investigated endothelial and cardiac function in 15-, 30-, and 45-week-old wild-type (WT) and Tgß 3 rats. The aortic expression of ⢠NO synthase (NOS) isoforms was evaluated by Western blot. Finally, electron paramagnetic resonance measurements were performed on aortas to evaluate ⢠NO and O2 â¢- production. Vascular reactivity was altered as early as 15 weeks of age in response to isoproterenol in Tgß 3 aortas and mesenteric arteries. NOS1 (neuronal NOS) expression was higher in the Tgß 3 aorta at 30 and 45 weeks of age (30 weeks: WT: 1.00 ± 0.21; Tgß 3: 6.08 ± 2.30; 45 weeks: WT: 1.00 ± 0.12; Tgß 3: 1.55 ± 0.17; p < 0.05). Interestingly, the endothelial NOS (NOS3) monomer form is increased in Tgß 3 rats at 45 weeks of age (ratio NOS3 dimer/NOS3 monomer; WT: 1.00 ± 0.37; Tgß 3: 0.13 ± 0.05; p < 0.05). Aortic â¢NO production was increased by NOS2 (inducible NOS) at 15 weeks of age in Tgß 3 rats (+52% vs. WT). Aortic O2 â¢- production was increased in Tgß 3 rats at 30 and 45 weeks of age (+75% and+76%, respectively, vs. WT, p < 0.05). We have shown that endothelial dysfunction and oxidative stress are present as early as 15 weeks of age and therefore conclude that endothelial dysfunction could be a cause of HFpEF development.
Assuntos
Insuficiência Cardíaca , Doenças Vasculares , Animais , Aorta/metabolismo , Insuficiência Cardíaca/metabolismo , Humanos , Ratos , Volume Sistólico , Função Ventricular EsquerdaRESUMO
All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/Gi pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardio-vascular diseases.
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Dendroaspis , Toxinas Biológicas , Animais , Artérias/metabolismo , Colinérgicos , Dendroaspis/metabolismo , Venenos Elapídicos/química , Venenos Elapídicos/metabolismo , Venenos Elapídicos/farmacologia , Humanos , Peptídeos/farmacologia , Ratos , Receptores Muscarínicos/metabolismoRESUMO
INTRODUCTION: Hydrogen sulfide (H2S) has been shown to improve survival in rodent models of lethal hemorrhage. Conversely, other authors have reported that inhibition of endogenous H2S production improves hemodynamics and reduces organ injury after hemorrhagic shock. Since all of these data originate from unresuscitated models and/or the use of a pre-treatment design, we therefore tested the hypothesis that the H2S donor, sodium hydrosulfide (NaHS), may improve hemodynamics in resuscitated hemorrhagic shock and attenuate oxidative and nitrosative stresses. METHODS: Thirty-two rats were mechanically ventilated and instrumented to measure mean arterial pressure (MAP) and carotid blood flow (CBF). Animals were bled during 60 minutes in order to maintain MAP at 40 ± 2 mm Hg. Ten minutes prior to retransfusion of shed blood, rats randomly received either an intravenous bolus of NaHS (0.2 mg/kg) or vehicle (0.9% NaCl). At the end of the experiment (T = 300 minutes), blood, aorta and heart were harvested for Western blot (inductible Nitric Oxyde Synthase (iNOS), Nuclear factor-κB (NF-κB), phosphorylated Inhibitor κB (P-IκB), Inter-Cellular Adhesion Molecule (I-CAM), Heme oxygenase 1(HO-1), Heme oxygenase 2(HO-2), as well as nuclear respiratory factor 2 (Nrf2)). Nitric oxide (NO) and superoxide anion (O2(-)) were also measured by electron paramagnetic resonance. RESULTS: At the end of the experiment, control rats exhibited a decrease in MAP which was attenuated by NaHS (65 ± 32 versus 101 ± 17 mmHg, P < 0.05). CBF was better maintained in NaHS-treated rats (1.9 ± 1.6 versus 4.4 ± 1.9 ml/minute P < 0.05). NaHS significantly limited shock-induced metabolic acidosis. NaHS also prevented iNOS expression and NO production in the heart and aorta while significantly reducing NF-kB, P-IκB and I-CAM in the aorta. Compared to the control group, NaHS significantly increased Nrf2, HO-1 and HO-2 and limited O2(-) release in both aorta and heart (P < 0.05). CONCLUSIONS: NaHS is protective against the effects of ischemia reperfusion induced by controlled hemorrhage in rats. NaHS also improves hemodynamics in the early resuscitation phase after hemorrhagic shock, most likely as a result of attenuated oxidative stress. The use of NaHS hence appears promising in limiting the consequences of ischemia reperfusion (IR).
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Hemodinâmica/fisiologia , Sulfeto de Hidrogênio/farmacologia , Mediadores da Inflamação/farmacologia , Estresse Oxidativo/fisiologia , Ressuscitação , Choque Hemorrágico/metabolismo , Animais , Hemodinâmica/efeitos dos fármacos , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/uso terapêutico , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/uso terapêutico , Masculino , Estresse Oxidativo/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Wistar , Ressuscitação/métodos , Choque Hemorrágico/tratamento farmacológicoRESUMO
Recombinant human activated protein C (rhAPC) is one of the treatment panels for improving vascular dysfunction in septic patients. In a previous study, we reported that rhAPC treatment in rat endotoxemia improved vascular reactivity, although the mechanisms involved are still under debate. In the present study, we hypothesized that rhAPC may improve arterial dysfunction through its nonanticoagulant properties. Ten hours after injection of LPS in mice (50 mg/kg ip), aortic rings and mesenteric arteries were isolated and incubated with or without rhAPC for 12 h. Aortic rings were mounted in a myograph, after which arterial contractility and endothelium-dependent relaxation were measured in the presence or absence of nitric oxide synthase or cyclooxygenase inhibitors. Flow (shear stress)-mediated dilation with or without the above inhibitors was also measured in mesenteric resistance arteries. Protein expression was assessed by Western blotting. Lipopolysaccharide (LPS) reduced aortic contractility to KCl and phenylephrine as well as dilation to acetylcholine. LPS also reduced flow-mediated dilation in mesenteric arteries. In rhAPC-treated aorta and mesenteric arteries, contractility and endothelial responsiveness to vasodilator drug and shear stress were improved. rhAPC treatment also improved LPS-induced endothelial dysfunction; this effect was associated with an increase in the phosphorylated form of endothelial nitric oxide synthase and protein kinase B as well as cyclooxygenase vasodilatory pathways, thus suggesting that these pathways, together with the decrease in nuclear factor-kappaB activation and inducible nitric oxide synthase expression in the vascular wall, are implicated in the endothelial effect of rhAPC. In conclusion, ex vivo application of rhAPC improves arterial contractility and endothelial dysfunction resulting from endotoxemia in mice. This finding provides important insights into the mechanism underlying rhAPC-induced improvements on arterial dysfunction during septic shock.
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Artérias/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotoxemia/tratamento farmacológico , Proteína C/farmacologia , Acetilcolina/antagonistas & inibidores , Acetilcolina/farmacologia , Animais , Aorta/efeitos dos fármacos , Western Blotting , Inibidores de Ciclo-Oxigenase/farmacologia , Endotoxemia/induzido quimicamente , Endotoxemia/patologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Lipopolissacarídeos , Masculino , Artérias Mesentéricas/fisiologia , Camundongos , NF-kappa B/biossíntese , NF-kappa B/genética , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Proteína C/uso terapêutico , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Resistência Vascular/fisiologia , Vasodilatadores/antagonistas & inibidores , Vasodilatadores/farmacologiaRESUMO
OBJECTIVE: Microparticles (MPs) are membrane vesicles with procoagulant and proinflammatory properties released during cell activation and might be potentially involved in the pathophysiology of septic shock. This study was designed to assess the effects of MPs from septic origin on the systemic hemodynamics as well as on the inflammatory, oxidative, and nitrosative stresses. DESIGN: A prospective, randomized, controlled experimental study with repeated measurements. SETTING: Investigational animal laboratory. SUBJECTS: Forty healthy rats were randomly allocated to three groups: 10 animals inoculated with MPs isolated from control rats (cMPs), 15 animals inoculated with MPs isolated from sham rats (shMPs), and 15 animals inoculated with MPs isolated from rats with peritonitis (sMPs). INTERVENTIONS: Rats were anesthetized, mechanically ventilated, and infused with the same amount of cMPs, shMPs, or sMPs. We measured the heart rate, mean arterial pressure, carotid artery, and portal vein blood flows. Hemodynamic parameters were recorded during 7 hours, and then animals were killed. Aorta and heart were harvested for further in vitro tissue analyses. MEASUREMENTS AND MAIN RESULTS: 1) The cellular origin (phenotype) but not the circulating concentration of MPs was different in septic rats, characterized by a significant increase in leukocyte-derived MPs. 2) sMPs but not cMPs or shMPs decreased mean arterial pressure without any effect on carotid artery and portal vein blood flows. 3) Rats inoculated with sMPs exhibited an increase in superoxide ion production and nuclear factor kappa B activity, overexpression of inducible nitric oxide synthase with subsequent nitric oxide overproduction and decrease in endothelial nitric oxide synthase activation. CONCLUSIONS: Rats with sepsis induced by peritonitis exhibited a specific phenotype of MPs. Inoculation of sMPs in healthy rats reproduced hemodynamic, septic inflammatory patterns, associated with oxidative and nitrosative stresses.
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Micropartículas Derivadas de Células/fisiologia , Hemodinâmica , Inflamação/etiologia , Choque Séptico/patologia , Choque Séptico/fisiopatologia , Animais , Micropartículas Derivadas de Células/metabolismo , Masculino , Ratos , Ratos Wistar , Choque Séptico/metabolismoRESUMO
Lipid emulsions for parenteral nutrition are used to provide calories and essential fatty acids for patients. They have been associated with hypertriglyceridemia, hypercholesterolemia, and metabolic stress, which may promote the development of endothelial dysfunction in patients. The aim of the present study was to determine whether five different industrial lipid emulsions may affect the endothelial function of coronary arteries. Porcine coronary artery rings were incubated with lipid emulsions 0.5, 1, or 2% (v/v) for 30 min before the determination of vascular reactivity in organ chambers and the level of oxidative stress using electron paramagnetic resonance. Incubation of coronary artery rings with either Lipidem®, Medialipid® containing long- and medium-chain triacylglycerols (LCT/MCT), or SMOFlipid® containing LCT, MCT, omega-9, and -3, significantly reduced the bradykinin-induced endothelium-dependent relaxation, affecting both the nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) components, whereas, Intralipid® containing LCT (soybean oil) and ClinOleic® containing LCT (soybean and olive oil) did not have such an effect. The endothelial dysfunction induced by Lipidem® was significantly improved by indomethacin, a cyclooxygenase (COX) inhibitor, inhibitors of oxidative stress (N-acetylcysteine, superoxide dismutase, catalase) and transition metal chelating agents (neocuproine, tetrathiomolybdate, deferoxamine and L-histidine). Lipidem® significantly increased the arterial level of oxidative stress. The present findings indicate that lipid emulsions containing LCT/MCT induce endothelial dysfunction in coronary artery rings by blunting both NO- and EDH-mediated relaxations. The Lipidem®-induced endothelial dysfunction is associated with increased vascular oxidative stress and the formation of COX-derived vasoconstrictor prostanoids.
Assuntos
Bradicinina/metabolismo , Vasos Coronários/efeitos dos fármacos , Endotélio/efeitos dos fármacos , Triglicerídeos/farmacologia , Animais , Modelos Animais de Doenças , Espectroscopia de Ressonância de Spin Eletrônica , Emulsões/farmacologia , Humanos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Óleo de Soja/farmacologia , SuínosRESUMO
Endothelial senescence has been suggested to promote endothelial dysfunction in age-related vascular disorders. This study evaluated the prothrombotic properties of senescent endothelial cells (ECs) and the underlying mechanism. Serial passaging from passage (P)1 to P4 (replicative senescence) of porcine coronary artery ECs, or treatment of P1 ECs with the endothelial nitric oxide synthase (eNOS) inhibitor L-NAME (premature senescence) induced acquisition of markers of senescence including increased senescence-associated-ß-galactosidase (SA-ß-gal) activity and p53, p21, p16 expression. Approximately 55% of P3 cells were senescent with a high level oxidative stress, and decreased eNOS-derived nitric oxide (NO) formation associated with increased expression of NADPH oxidase subunits (gp91phox, p47phox), cyclooxygenase (COX)-2 but not COX-1, and a decreased eNOS expression leading to a reduced ability of ECs to inhibit platelet aggregation. P3 cells also presented increased expression and activity of tissue factor (TF), a key initiator of the coagulation cascade. Treatment of senesecent cells with a NADPH oxidase inhibitor (VAS-2870) or by a COX inhibitor (indomethacin) reduced oxidative stress, decreased TF activity and expression, and reduced the expression of gp91phox, p47phox and COX-2 and restored the ability of ECs to inhibit effectively platelet aggregation. Thus, replicative endothelial senescence promotes a prothrombotic response involving the down-regulation of the protective NO pathway and the upregulation of the NADPH oxidase- and COXs-dependent oxidative stress pathway promoting TF expression and activity.
Assuntos
Senescência Celular/fisiologia , Endotélio Vascular/citologia , NADPH Oxidases/fisiologia , Estresse Oxidativo/fisiologia , Prostaglandina-Endoperóxido Sintases/fisiologia , Animais , Divisão Celular/fisiologia , Células Cultivadas , Vasos Coronários/citologia , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Humanos , Óxido Nítrico/biossíntese , Agregação Plaquetária/fisiologia , Sus scrofa , Tromboplastina/metabolismo , Trombose/enzimologia , Trombose/patologia , Regulação para Cima/fisiologiaRESUMO
INTRODUCTION: Long chain n-3 fatty acid supplementation may modulate septic shock-induced host response to pathogen-induced sepsis. The composition of lipid emulsions for parenteral nutrition however remains a real challenge in intensive care, depending on their fatty acid content. Because they have not been assessed yet, we aimed at determining the respective effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) during septic shock-induced vascular dysfunction. METHODS: In a peritonitis-induced septic shock model, rats were infused with EPA, DHA, an EPA/DHA mixture or 5% dextrose (D5) during 22 hours. From H18, rats were resuscitated and monitored during 4 hours. At H22, plasma, aorta and mesenteric resistance arteries were collected to perform ex vivo experiments. RESULTS: We have shown that septic rats needed an active resuscitation with fluid challenge and norepinephrine treatment, while SHAM rats did not. In septic rats, norepinephrine requirements were significantly decreased in DHA and EPA/DHA groups (10.6±12.0 and 3.7±8.0 µg/kg/min respectively versus 17.4±19.3 µg/kg/min in D5 group, p<0.05) and DHA infusion significantly improved contractile response to phenylephrine through nitric oxide pathway inhibition. DHA moreover significantly reduced vascular oxidative stress and nitric oxide production, phosphorylated IκB expression and vasodilative prostaglandin production. DHA also significantly decreased polyunsaturated fatty acid pro-inflammatory mediators and significantly increased several anti-inflammatory metabolites. CONCLUSIONS: DHA infusion in septic rats improved hemodynamic dysfunction through decreased vascular oxidative stress and inflammation, while EPA infusion did not have beneficial effects.
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Artérias/patologia , Ácidos Docosa-Hexaenoicos/uso terapêutico , Ácido Eicosapentaenoico/uso terapêutico , Choque Séptico/complicações , Doenças Vasculares/tratamento farmacológico , Animais , Epoprostenol/biossíntese , Masculino , Óxido Nítrico/biossíntese , Norepinefrina/administração & dosagem , Estresse Oxidativo , Ratos , Ratos Wistar , Doenças Vasculares/etiologiaRESUMO
BACKGROUND: Septic shock-induced disseminated intravascular coagulation is responsible for increased occurrence of multiple organ dysfunction and mortality. Immunothrombosis-induced coagulopathy may contribute to hypercoagulability. We aimed at determining whether recombinant human thrombomodulin (rhTM) could control exaggerated immunothrombosis by studying procoagulant responses, fibrinolysis activity borne by microvesicles (MVs) and NETosis in septic shock. METHODS: In a septic shock model after a cecal ligation and puncture-induced peritonitis (H0), rats were treated with rhTM or a placebo at H18, resuscitated and monitored during 4 h. At H22, blood was sampled to perform coagulation tests, to characterize MVs and to detect neutrophils extracellular traps (NETs). Lungs were stained with hematoxylin-eosin for inflammatory injury assessment. RESULTS: Coagulopathy was attenuated in rhTM-treated septic rats compared to placebo-treated rats, as attested by a significant decrease in procoagulant annexin A5+-MVs and plasma procoagulant activity of phospholipids and by a significant increase in antithrombin levels (84 ± 8 vs. 64 ± 6%, p < 0.05), platelet count (582 ± 157 vs. 319 ± 91 × 109/L, p < 0.05) and fibrinolysis activity borne by MVs (2.9 ± 0.26 vs. 0.48 ± 0.29 U/mL urokinase, p < 0.05). Lung histological injury score showed significantly less leukocyte infiltration. Decreased procoagulant activity and lung injury were concomitant with decreased leukocyte activation as attested by plasma leukocyte-derived MVs and NETosis reduction after rhTM treatment (neutrophil elastase/DNA: 93 ± 33 vs. 227 ± 48 and citrullinated histones H3/DNA: 96 ± 16 vs. 242 ± 180, mOD for 109 neutrophils/L, p < 0.05). CONCLUSION: Thrombomodulin limits procoagulant responses and NETosis and at least partly restores hemostasis control during immunothrombosis. Neutrophils might thus stand as a promising therapeutic target in septic shock-induced coagulopathy.
RESUMO
BACKGROUND: Immunonutrition in sepsis, including n-3 poly-unsaturated fatty acids (PUFAs) or L-arginine supplementation, is a controversial issue that has yielded a great number of studies for the last thirty-five years, and the conclusions regarding the quantity and quality of this support in patients are deceiving. The aim of the present experimental study is to investigate the effects of a pretreatment with enteral nutrition enriched with n-3 PUFAs or L-arginine on vascular dysfunctions, inflammation and oxidative stress during septic shock in rats. DESIGN: Rats were fed with enteral Peptamen® HN (HN group), Peptamen® AF containing n-3 PUFAs (AF group) or Peptamen® AF enriched with L-arginine (AFA group). On day 4, peritonitis by cecal ligation and puncture (CLP) was performed. Rats were resuscitated (H18) once septic shock was established. After a 4-hour resuscitation, vessels and organs were harvested to assess inflammation, superoxide anion, nitric oxide and prostacyclin levels. Ex-vivo vascular reactivity was also performed. RESULTS: Compared to CLP-AF or CLP-HN groups, 47.6% of CLP-AFA rats died before the beginning of hemodynamic measurements (vs. 8.0% and 20.0% respectively, p<0.05). AF and AFA rats required significantly increased norepinephrine infusion rates to reach the mean arterial pressure objective, compared to CLP-HN rats. Both CLP-AF and CLP-AFA reduced mesenteric resistance arterial contractility, decreased vascular oxidative stress, but increased NF-κB (0.40±0.15 in CLP-AF and 0.69±0.06 in CLP-AFA vs. 0.09±0.03 in SHAM rats and 0.30±0.06 in CLP-HN, ß-actin ratio, p<0.05) and pIκB expression (0.60±0.03 in CLP-AF and 0.94±0.15 in CLP-AFA vs. 0.04±0.01 in SHAM rats and 0.56±0.07 in CLP-HN, ß-actin ratio, p<0.05), nitric oxide and prostacyclin production in septic rats. CONCLUSIONS: Although n-3 PUFAs or L-arginine supplementation exhibited an antioxidant effect, it worsened the septic shock-induced vascular dysfunction. Furthermore, mortality was higher after L-arginine supplementation.
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Peritonite/tratamento farmacológico , Choque Séptico/tratamento farmacológico , Animais , Arginina/uso terapêutico , Ácidos Graxos Ômega-3/uso terapêutico , Masculino , Peritonite/mortalidade , Ratos , Ratos Wistar , Sepse/tratamento farmacológico , Sepse/mortalidade , Choque Séptico/mortalidadeRESUMO
INTRODUCTION: Enriching the diet with Omega-3 for several weeks improves myocardial resistance to ischemia-reperfusion (IR) in rats. However, patients with myocardial infarction requiring an emergency reperfusion cannot be pretreated with such a diet. The objective of our study was to describe the effects of an intravenous Omega-3 bolus before reperfusion in a rat model of myocardial IR. METHODS: In a rat model of acute myocardial IR, an intravenous Omega-3 bolus (EPA:DHA 6:1), associated or not with iodinated contrast media, was administered after a 30-min ischemia, before reperfusion. Hemodynamic parameters were assessed. Circulating procoagulant microparticles were phenotyped. Vascular and heart inflammation, superoxide anion, and nitric oxide were measured. Ex vivo vascular reactivity was performed with a pharmacological selective inhibitor of inductible nitric oxide synthase. Cardiac troponin I (cTn-I) plasma levels were measured. RESULTS: Compared with untreated IR rats, an Omega-3 bolus before reperfusion significantly decreased the IR syndrome, improving mean arterial pressure (114â±â9 vs. 61â±â17 mmHg 4 h after reperfusion, Pâ<â0.05) and carotid blood flow, and decreasing plasma cTn-I levels after revascularization. These beneficial effects may be due to improved ex vivo mesenteric resistance artery sensitivity to phenylephrine, endothelial protection assessed by decreased endothelial CD54 microparticle release (9.1â±â2.5 vs. 4.8â±â2.0ânM Eq PhtdSer, Pâ<â0.05) and reduced vascular inflammation and oxidative stress. CONCLUSIONS: In this rat model of myocardial IR, an intravenous Omega-3 bolus before reperfusion decreases IR-induced vascular failure and shock. These results open therapeutic perspectives as far as myocardial reperfusion process is concerned that deserve further explorations in humans.
Assuntos
Ácidos Docosa-Hexaenoicos/uso terapêutico , Ácido Eicosapentaenoico/uso terapêutico , Infarto do Miocárdio/sangue , Infarto do Miocárdio/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Animais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácido Eicosapentaenoico/administração & dosagem , Hemodinâmica/efeitos dos fármacos , Injeções Intravenosas , Masculino , Óxido Nítrico Sintase/sangue , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Troponina I/sangueRESUMO
AIMS: Omega-3 fatty acid products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have vasoprotective effects, in part, by stimulating the endothelial formation of nitric oxide (NO). This study determined the role of the EPA:DHA ratio and amount, and characterized the mechanism leading to endothelial NO synthase (eNOS) activation. METHODS AND RESULTS: EPA:DHA 6â¶1 and 9â¶1 caused significantly greater endothelium-dependent relaxations in porcine coronary artery rings than EPA:DHA 3â¶1, 1â¶1, 1â¶3, 1â¶6, 1â¶9, EPA and DHA alone, and EPA:DHA 6â¶1 with a reduced EPA + DHA amount, which were inhibited by an eNOS inhibitor. Relaxations to EPA:DHA 6â¶1 were insensitive to cyclooxygenase inhibition, and reduced by inhibitors of either oxidative stress, Src kinase, PI3-kinase, p38 MAPK, MEK, or JNK. EPA:DHA 6â¶1 induced phosphorylation of Src, Akt, p38 MAPK, ERK, JNK and eNOS; these effects were inhibited by MnTMPyP. EPA:DHA 6â¶1 induced the endothelial formation of ROS in coronary artery sections as assessed by dihydroethidium, and of superoxide anions and hydrogen peroxide in cultured endothelial cells as assessed by electron spin resonance with the spin probe CMH, and the Amplex Red based assay, respectively. CONCLUSION: Omega-3 fatty acids cause endothelium-dependent NO-mediated relaxations in coronary artery rings, which are dependent on the EPA:DHA ratio and amount, and involve an intracellular activation of the redox-sensitive PI3-kinase/Akt and MAPKs pathways to activate eNOS.
Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinases da Família src/metabolismo , Animais , Western Blotting , Células Cultivadas , Espectroscopia de Ressonância de Spin Eletrônica , Ácidos Graxos Ômega-3/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Óxido Nítrico Sintase Tipo III/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos , Vasodilatação/efeitos dos fármacos , Quinases da Família src/genéticaRESUMO
BACKGROUND AND AIMS: Lipid emulsions for parenteral nutrition interfere with immunity and may alter the cell plasma membrane and microparticle release, thus modulating their biological effects. Our aim was to evaluate the effect of two lipid emulsions for parenteral nutrition containing either a mixture of long- and medium-chain triglycerides (LCTs and MCTs) or LCTs only, to assess their role on microparticle release and acute inflammation during septic shock in rats. METHODS AND RESULTS: Septic rats (cecal ligation and puncture) and sham rats were infused with 5% dextrose or a lipid emulsion during 22 h. After 18 h, rats were resuscitated during 4 h and hemodynamic parameters monitored. Circulating microparticles and their phenotype were measured by prothrombinase assay; heart and aorta were collected for Western blotting and electron paramagnetic resonance measurements. No significant effect of lipid emulsions was observed in sham rats. In septic rats, norepinephrine requirements were increased in MCT/LCT-infused rats compared with 5% dextrose- or LCT-infused rats (2.7 ± 0.2 vs. 1.9 ± 0.8 and 1.2 ± 0.3 µg/kg per minute, respectively; P < 0.05) with increased procoagulant microparticle generation (38.6 ± 5.8 vs. 18.8 ± 3.1 and 19.2 ± 3.0 nM equivalent phosphatidylserine [Eq PhtdSer]; P < 0.05), leukocyte- (17.4 ± 3.5 vs. 7.7 ± 1.8 and 6.0 ± 1.1 nM Eq PhtdSer; P < 0.05), platelet- (13.9 ± 2.5 vs. 4.4 ± 0.7 and 5.4 ± 1.3 nM Eq PhtdSer; P < 0.05), and endothelial-derived microparticles (16.9 ± 3.6 vs. 6.4 ± 1.4 and 5.6 ± 0.8 nM Eq PhtdSer; P < 0.05). The mixture of MCTs/LCTs significantly increased cardiac and vascular nitric oxide and superoxide anion production, phosphorylated IκB, and cyclooxygenase 2 expression compared with the lipid emulsion containing only LCTs. CONCLUSIONS: Compared with 5% dextrose, MCT/LCT supplementation during septic shock in rats induced deleterious effects with increased inflammation and cell activation, associated to vascular hyporeactivity. During septic shock, LCT supplementation seemed to be neutral compared with 5% dextrose infusion.
Assuntos
Membrana Celular/metabolismo , Peritonite/fisiopatologia , Choque Séptico/fisiopatologia , Triglicerídeos/efeitos adversos , Animais , Aorta/metabolismo , Coagulantes/química , Espectroscopia de Ressonância de Spin Eletrônica , Emulsões/química , Glucose/química , Hemodinâmica , Inflamação , Lipídeos/química , Masculino , Microesferas , Miocárdio/metabolismo , Óxido Nítrico/química , Soluções de Nutrição Parenteral/química , Fosforilação , Ratos , Ratos Wistar , Choque Séptico/induzido quimicamente , Choque Séptico/metabolismo , Superóxidos/química , Fatores de Tempo , Triglicerídeos/químicaRESUMO
Circulating microparticles play a pro-inflammatory and procoagulant detrimental role in the vascular dysfunction of septic shock. It was the objective of this study to investigate mechanisms by which a pharmacological modulation of microparticles could affect vascular dysfunction in a rat model of septic shock. Septic or sham rats were treated by activated protein C (aPC) and resuscitated during 4 hours. Their microparticles were harvested and inoculated to another set of healthy recipient rats. Haemodynamic parameters were monitored, circulating total procoagulant microparticles assessed by prothrombinase assay, and their cell origin characterised. Mesenteric resistance arteries, aorta and heart were harvested for western blotting analysis. We found that a) the amount and phenotype of circulating microparticles were altered in septic rats with an enhanced endothelial, leucocyte and platelet contribution; b) aPC treatment significantly reduced the generation of leucocyte microparticles and norepinephrine requirements to reach the mean arterial pressure target in septic rats; c) Microparticles from untreated septic rats, but not from aPC-treated ones, significantly reduced the healthy recipients' mean arterial pressure; d) Microparticle thromboxane content and aPC activity were significantly increased in aPC-treated septic rats. In inoculated naïve recipients, microparticles from aPC-treated septic rats prompted reduced NF-κB and cyclooxygenase-2 arterial activation, blunted the generation of pro-inflammatory iNOS and secondarily increased platelet and endothelial microparticles. In conclusion, in this septic shock model, increased circulating levels of procoagulant microparticles led to negative haemodynamic outcomes. Pharmacological treatment by aPC modified the cell origin and levels of circulating microparticles, thereby limiting vascular inflammation and favouring haemodynamic improvement.