RESUMO
Annexin V (AV), a protein with anticoagulant activity, exerts antithrombotic activity by binding to phosphatidylserine (PS), inhibiting activation of serine proteases important in blood coagulation. The potential use of this protein as an anticoagulant is limited as it rapidly passes from the blood into the kidneys due to its relatively small size (36 kDa). We used recombinant DNA technology to produce a homodimer of human AV (DAV, 73 kDa), which exceeds the renal filtration threshold, and has a 6.5-hour half-life in the rat circulation. Human red blood cells with externalized PS were used to show that DAV had a higher affinity for PS-exposing cells than AV. DAV labeling sensitively identifies PS-exposing cells, was found to be a potent inhibitor of the activity of the prothombinase complexes and inhibits the ability of secretory phospholipaseA(2) to hydrolyze phospholipids of PS-exposing cells, reducing the formation of mediators of blood coagulation and reperfusion injury. DAV exerts dose-dependent antithrombotic activity in rat veins. This combination of activities suggests that DAV is a valuable probe to measure PS exposure and may be efficacious as a novel drug in a wide range of clinical situations.
Assuntos
Anexina A5/farmacologia , Anticoagulantes/farmacologia , Membrana Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Fibrinolíticos/farmacologia , Fosfatidilserinas/metabolismo , Trombose Venosa/prevenção & controle , Anemia Falciforme/sangue , Anemia Falciforme/metabolismo , Animais , Anexina A5/sangue , Anexina A5/genética , Anexina A5/metabolismo , Anticoagulantes/sangue , Anticoagulantes/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Membrana Celular/metabolismo , Dimerização , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Eritrócitos/metabolismo , Fator Va/metabolismo , Fibrinolíticos/sangue , Fibrinolíticos/metabolismo , Humanos , Masculino , Camundongos , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Trombina/metabolismo , Tromboplastina , Trombose Venosa/induzido quimicamenteRESUMO
Many compounds have been shown to prevent reperfusion injury in various animal models, although to date, translation into clinic has revealed several obstacles. Therefore, the National Heart, Lung, and Blood Institute convened a working group to discuss reasons for such failure. As a result, the concept of adequately powered, blinded, randomized studies for preclinical development of a compound has been urged. We investigated the effects of a fibrin-derived peptide Bbeta(15-42) in acute and chronic rodent models of ischemia-reperfusion at three different study centers (Universities of Dusseldorf and Vienna, TNO Biomedical Research). A total of 187 animals were used, and the peptide was compared with the free radical scavenger Tempol, CD18 antibody, alpha-C5 antibody, and the golden standard, ischemic preconditioning. We show that Bbeta(15-42) robustly and reproducibly reduced infarct size in all models of ischemia-reperfusion. Moreover, the peptide significantly reduced plasma levels of the cytokines interleukin 1beta, tumor necrosis factor alpha, and interleukin 6. In rodents, Bbeta(15-42) inhibits proinflammatory cytokine release and is cardioprotective during ischemia-reperfusion injury.
Assuntos
Fibrinogênio/química , Fibrinogênio/fisiologia , Reperfusão Miocárdica , Miocárdio/patologia , Traumatismo por Reperfusão/patologia , Animais , Antígenos CD18/química , Óxidos N-Cíclicos/farmacologia , Modelos Animais de Doenças , Sequestradores de Radicais Livres/química , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Ratos , Marcadores de SpinRESUMO
OBJECTIVE: We investigated whether the dual PPARalpha/gamma agonist tesaglitazar has anti-atherogenic effects in ApoE*3Leiden mice with reduced insulin sensitivity. METHODS AND RESULTS: ApoE*3Leiden transgenic mice were fed a high-fat (HF) insulin-resistance-inducing diet. One group received a high-cholesterol (HC) supplement (1% wt/wt; HC group). A second group received the same HC supplement along with tesaglitazar (T) 0.5 micromol/kg diet (T group). A third (control) group received a low-cholesterol (LC) supplement (0.1% wt/wt; LC group). Tesaglitazar decreased plasma cholesterol by 20% compared with the HC group; cholesterol levels were similar in the T and LC groups. Compared with the HC group, tesaglitazar caused a 92% reduction in atherosclerosis, whereas a 56% reduction was seen in the cholesterol-matched LC group. Furthermore, tesaglitazar treatment significantly reduced lesion number beyond that expected from cholesterol lowering and induced a shift to less severe lesions. Concomitantly, tesaglitazar reduced macrophage-rich and collagen areas. In addition, tesaglitazar reduced inflammatory markers, including plasma SAA levels, the number of adhering monocytes, and nuclear factor kappaB-activity in the vessel wall. CONCLUSIONS: Tesaglitazar has anti-atherosclerotic effects in the mouse model that go beyond plasma cholesterol lowering, possibly caused by a combination of altered lipoprotein profiles and anti-inflammatory vascular effects.
Assuntos
Alcanossulfonatos/farmacologia , Apolipoproteínas E/metabolismo , Aterosclerose/patologia , Hipercolesterolemia/fisiopatologia , Resistência à Insulina , PPAR alfa/agonistas , PPAR gama/agonistas , Fenilpropionatos/farmacologia , Animais , Apolipoproteína E3 , Aterosclerose/etiologia , Aterosclerose/fisiopatologia , Biomarcadores/sangue , Vasos Sanguíneos/patologia , Adesão Celular , Colesterol/sangue , Colesterol na Dieta/administração & dosagem , Colágeno/metabolismo , Gorduras na Dieta/administração & dosagem , Feminino , Hipercolesterolemia/sangue , Hipercolesterolemia/complicações , Inflamação/sangue , Lipídeos/sangue , Lipoproteínas/sangue , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , Monócitos/patologia , NF-kappa B/metabolismoRESUMO
BACKGROUND: The prevalence of dyslipidemia and obesity resulting from excess energy intake and physical inactivity is increasing. The liver plays a pivotal role in systemic lipid homeostasis. Effective, natural dietary interventions that lower plasma lipids and promote liver health are needed. OBJECTIVE: Our goal was to determine the effect of dietary sphingolipids on plasma lipids and liver steatosis. DESIGN: APOE*3Leiden mice were fed a Western-type diet supplemented with different sphingolipids. Body cholesterol and triacylglycerol metabolism as well as hepatic lipid concentrations and lipid-related gene expression were determined. RESULTS: Dietary sphingolipids dose-dependently lowered both plasma cholesterol and triacylglycerol in APOE*3Leiden mice; 1% phytosphingosine (PS) reduced plasma cholesterol and triacylglycerol by 57% and 58%, respectively. PS decreased the absorption of dietary cholesterol and free fatty acids by 50% and 40%, respectively, whereas intestinal triacylglycerol lipolysis was not affected. PS increased hepatic VLDL-triacylglycerol production by 20%, whereas plasma lipolysis was not affected. PS increased the hepatic uptake of VLDL remnants by 60%. Hepatic messenger RNA concentrations indicated enhanced hepatic lipid synthesis and VLDL and LDL uptake. The net result of these changes was a strong decrease in plasma cholesterol and triacylglycerol. The livers of 1% PS-fed mice were less pale, 22% lighter, and contained 61% less cholesteryl ester and 56% less triacylglycerol than livers of control mice. Furthermore, markers of liver inflammation (serum amyloid A) and liver damage (alanine aminotransferase) decreased by 74% and 79%, respectively, in PS-fed mice. CONCLUSION: Sphingolipids lower plasma cholesterol and triacylglycerol and protect the liver from fat- and cholesterol-induced steatosis.
Assuntos
Colesterol/sangue , Fígado Gorduroso/prevenção & controle , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Esfingolipídeos/administração & dosagem , Triglicerídeos/sangue , Animais , Apolipoproteína E3 , Apolipoproteínas E/genética , Colesterol na Dieta/farmacocinética , Relação Dose-Resposta a Droga , Ácidos Graxos não Esterificados/farmacocinética , Fezes/química , Feminino , Expressão Gênica , Absorção Intestinal/efeitos dos fármacos , Metabolismo dos Lipídeos/fisiologia , Lipólise/efeitos dos fármacos , Lipólise/fisiologia , Lipoproteínas VLDL/química , Lipoproteínas VLDL/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Camundongos , Camundongos Transgênicos , RNA/metabolismo , Distribuição Aleatória , Esfingolipídeos/farmacologiaRESUMO
BACKGROUND: Statins can exert anti-inflammatory antiatherosclerotic effects through an anti-inflammatory action, independent of lowering cholesterol. We addressed the question whether the anti-inflammatory activities of statins can reduce atherosclerosis beyond the reduction achieved by cholesterol lowering per se. METHODS AND RESULTS: Two groups of 20 female APOE*3-Leiden mice received either a high-cholesterol diet (HC) or a high-cholesterol diet supplemented with 0.005% (wt/wt) rosuvastatin (HC+R). The HC diet alone resulted in a plasma cholesterol concentration of 18.9+/-1.4 mmol/L, and administration of rosuvastatin lowered plasma cholesterol to 14.1+/-0.7 mmol/L. In a separate low-cholesterol (LC) control group, the dietary cholesterol intake was reduced, which resulted in plasma cholesterol levels that were comparable to the HC+R group (13.4+/-0.8 mmol/L). Atherosclerosis in the aortic root area was quantified after 24 weeks. As compared with the HC group, the LC group had a 62% (P<0.001) reduction in cross-sectional lesion area. When compared with the LC group, the HC+R group showed a further decrease in cross-sectional lesion area (80%, P<0.001), size of individual lesions (63%, P<0.05), lesion number (58%, P<0.001), monocyte adherence (24%, P<0.05), and macrophage-containing area (60%, P<0.001). Furthermore, rosuvastatin specifically suppressed the expression of the inflammation parameters MCP-1 and TNF-alpha in the vessel wall and lowered plasma concentrations of serum amyloid A and fibrinogen, independent of its cholesterol-lowering effect. CONCLUSIONS: Rosuvastatin reduces atherosclerosis beyond and independent of the reduction achieved by cholesterol lowering alone. This additional beneficial effect of rosuvastatin may be explained, at least partly, by its anti-inflammatory activity.
Assuntos
Anti-Inflamatórios/uso terapêutico , Apolipoproteínas E/genética , Arteriosclerose/tratamento farmacológico , Fluorbenzenos/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Pirimidinas , Sulfonamidas , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Apolipoproteína E3 , Arteriosclerose/sangue , Arteriosclerose/imunologia , Arteriosclerose/patologia , Adesão Celular , Colesterol/sangue , Citocinas/metabolismo , Feminino , Lipídeos/sangue , Lipoproteínas/sangue , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Monócitos/imunologia , Rosuvastatina CálcicaRESUMO
In 328 type 2 diabetic patients followed for 9.0 years (mean), we investigated whether endothelial dysfunction and chronic inflammation (estimated from plasma markers) can explain the association between (micro)albuminuria and mortality. Of the patients, 113 died. Mortality was increased in patients with baseline microalbuminuria or macroalbuminuria (odds ratios as compared with normoalbuminuria, 1.78 [P < 0.05] and 2.86 [P < 0.01]) and in patients with soluble vascular cell adhesion molecule 1 in the third tertile and C-reactive protein in the second and third tertiles (odds ratios as compared with the first tertile, 2.05 [ P < 0.01], and 1.80 [P < 0.05] and 2.92 [ P < 0.01]). These associations were mutually independent. The mean yearly change in urinary albumin excretion was 9.4%; in von Willebrand factor, 8.1%; in tissue-type plasminogen activator, 2.8%; in soluble vascular cell adhesion molecule 1, 5.2%; in soluble E-selectin, -2.3%; in C-reactive protein, 3.8%; and in fibrinogen, 2.3%. The longitudinal development of urinary albumin excretion was significantly and independently determined by baseline levels of and the longitudinal development of BMI, systolic blood pressure, serum creatinine, glycated hemoglobin and plasma von Willebrand factor (baseline only), soluble E-selectin (baseline only), tissue-type plasminogen activator, C-reactive protein, and fibrinogen. The longitudinal developments of markers of endothelial function and inflammation were interrelated. In type 2 diabetes, increased urinary albumin excretion, endothelial dysfunction, and chronic inflammation are interrelated processes that develop in parallel, progress with time, and are strongly and independently associated with risk of death.
Assuntos
Albuminúria/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Endotélio Vascular/fisiopatologia , Inflamação/fisiopatologia , Biomarcadores/sangue , Pressão Sanguínea , Proteína C-Reativa/análise , Colesterol/sangue , HDL-Colesterol/sangue , Creatinina/sangue , Diabetes Mellitus Tipo 2/mortalidade , Selectina E/urina , Feminino , Hemoglobinas Glicadas/análise , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Valor Preditivo dos Testes , Valores de Referência , Fatores de Risco , Fumar , Ativador de Plasminogênio Tecidual/urina , Molécula 1 de Adesão de Célula Vascular/sangue , Fator de von Willebrand/urinaRESUMO
OBJECTIVE: To investigate the short-term effects of HMR 3339, a novel selective estrogen receptor modulator, on markers of coagulation and fibrinolysis. STUDY DESIGN: In a multicenter, 14-week, randomized, placebo-controlled, double-blind, dose-ranging study, healthy postmenopausal women received daily placebo (n = 22), HMR 3339 2.5 mg (n = 25), HMR 3339 10 mg (n = 24), HMR 3339 50 mg (n = 24), or raloxifene 60 mg (n = 23). Statistical analysis was performed using standard parametric tests. RESULTS: After 12 weeks, compared with placebo, HMR 3339 50 mg induced the largest mean percentage changes in antithrombin (-14.6%, P < .001), protein C (-12.9%, P = .029), and fibrinogen (-26.3%, P = .001). Decreases were observed in the HMR 3339 2.5 mg group, compared with placebo, in tissue-type plasminogen activator (-55.0%, P = .026 after 4 weeks), plasmin-alpha2-antiplasmin complex (-85%, P = .031 and -63.3%, P = .008, respectively, after 4 and 12 weeks), and D-dimer (-91.4%, P = .018 after 12 weeks). Compared with placebo, raloxifene 60 mg decreased total protein S (-8.2%, P = .009) after 4 weeks and antithrombin (-6.0%, P = .034) and fibrinogen (-18.1%, P = .007) after 12 weeks. CONCLUSION: HMR 3339 and raloxifene decreased fibrinogen levels. In the low dosage, HMR 3339 showed potentially beneficial effects on some markers of fibrinolysis. Both drugs impaired the anticoagulatory potential.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Estradiol/análogos & derivados , Fibrinólise/efeitos dos fármacos , Pós-Menopausa , Cloridrato de Raloxifeno/farmacologia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Biomarcadores/sangue , Relação Dose-Resposta a Droga , Método Duplo-Cego , Estradiol/farmacologia , Feminino , Humanos , Pessoa de Meia-Idade , Fatores de TempoRESUMO
OBJECTIVE: To look for evidence of a fibrinolytic insufficiency as a cause of adhesion formation. DESIGN: Retrospective and prospective study. SETTING: University medical center. PATIENT(S): Retrospective study: 50 patients undergoing laparoscopy, divided into patients with and without endometriosis. Prospective study: 18 patients undergoing infertility surgery involving a second-look laparoscopy. INTERVENTION(S): During all surgical procedures, adhesions were scored, and peritoneal fluid and plasma were collected. MAIN OUTCOME MEASURE(S): Parameters of the fibrinolytic system were measured to establish a possible relation with the presence and formation of adhesions. RESULT(S): In patients with endometriosis and adhesions, significantly higher peritoneal fluid concentrations were found for plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), and plasminogen, compared with patients with endometriosis but without adhesions. In the prospective study, initial peritoneal PAI-1 concentrations correlated significantly with the extent of adhesion formation (r(s) = 0.49) and adhesion-improvement scores (r(s) = -0.52). Also, the change in concentration of tPA and fibrinogen from the initial surgical procedure to the second-look laparoscopy correlated significantly with adhesion-improvement scores (DeltatPA: r(s)= 0.50; Deltafibrinogen: r(s) = -0.64). CONCLUSION(S): This first prospective study in humans adds further weight to the hypothesis that adhesions are caused by an insufficiency in peritoneal fibrinolytic activity. Plasminogen activator inhibitor-1 is a potential marker for the identification of patients at risk for developing adhesions.
Assuntos
Fibrinólise , Aderências Teciduais/etiologia , Adulto , Líquido Ascítico/química , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Humanos , Laparoscopia , Inibidor 1 de Ativador de Plasminogênio/análise , Estudos Prospectivos , Estudos Retrospectivos , Ativador de Plasminogênio Tecidual/análiseRESUMO
An elevated urinary albumin excretion rate (UAER) is associated with an increased risk of cardiovascular mortality, but the pathophysiological mechanism underlying this association is poorly understood. To investigate the role of endothelial dysfunction, leukocyte adhesion, and low-grade inflammation (1) in the development of elevated UAER (study I) and (2) in linking elevated UAER with risk of cardiovascular mortality (study II), we performed a prospective study in an age-, sex-, and glucose tolerance- stratified sample of a population-based cohort aged 50 to 75 years. High levels of von Willebrand factor, soluble vascular cell adhesion molecule-1 (sVCAM-1), and C-reactive protein (CRP) were used as markers of endothelial dysfunction, leukocyte adhesion, and low-grade inflammation, respectively. For study I, subjects who had normal UAER at baseline (n=316 subjects, 66 with type 2 diabetes) were reexamined after a mean follow-up of 6.1 years. The development of elevated UAER was defined as a mean albumin-to-creatinine ratio >2.0 mg/mmol at follow-up. Age-, sex-, and glucose tolerance- adjusted logistic regression analyses showed the development of elevated UAER to be significantly associated with levels of sVCAM-1 and CRP (odds ratio 1.14 [95% CI 1.02 to 1.27] per 10% increase of sVCAM-1 and odds ratio 1.17 [95% CI 1.04 to 1.32] per 50% increase of CRP). The results were not materially different after additional adjustment for hypertension, body mass index, cardiovascular disease, and creatinine clearance or stratification by the presence of diabetes. For study II, the vital status of all subjects (n= 575) was determined after a mean follow-up of 6.6 years. Eighty-one of 575 subjects died (30 died of cardiovascular disease). The presence of elevated UAER at baseline was associated with a 4.1-fold (1.94 to 8.73) increased risk of cardiovascular death after adjustment for age, sex, and glucose tolerance status. Adjustment for levels of von Willebrand factor, sVCAM-1, or CRP did not materially affect the results, nor did additional adjustment for the presence of hypertension, retinopathy, and cardiovascular disease and for levels of homocysteine, triglycerides, and high density lipoprotein cholesterol. Leukocyte adhesion (sVCAM-1) and low-grade inflammation (CRP) are determinants of the development of elevated UAER. However, these determinants do not explain the association between elevated UAER and cardiovascular mortality.
Assuntos
Albuminúria/mortalidade , Doenças Cardiovasculares/mortalidade , Idoso , Albuminúria/sangue , Albuminúria/complicações , Biomarcadores/sangue , Biomarcadores/urina , Proteína C-Reativa/metabolismo , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/complicações , Adesão Celular , Estudos de Coortes , Creatinina/urina , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Inflamação/sangue , Masculino , Pessoa de Meia-Idade , Razão de Chances , Estudos Prospectivos , Análise de Regressão , Molécula 1 de Adesão de Célula Vascular/sangue , Fator de von Willebrand/metabolismoRESUMO
This study was designed to investigate the potential antiatherosclerotic effects of the calcium antagonist amlodipine as compared with the HMG-CoA reductase inhibitor atorvastatin and the combination of both in ApoE*3-Leiden transgenic mice. Four groups of 15 ApoE*3-Leiden mice were put on a high-cholesterol diet. One group received 0.002% (wt/wt) amlodipine in the diet, which had no effect on plasma cholesterol levels. Another group received 0.01% (wt/wt) atorvastatin, resulting in a decrease of plasma cholesterol by 50% by a reduction in very low density lipoprotein production. The combination group received both amlodipine and atorvastatin. After 28 weeks, atherosclerosis in the aortic root was quantified. Treatment with amlodipine had no significant effect on atherosclerotic lesion area, whereas atorvastatin markedly reduced atherosclerosis by 77% compared with the control group. Atorvastatin also reduced inflammation markers. The combination of amlodipine and atorvastatin tended to reduce lesion area by 61% compared with the atorvastatin-only group; however, this effect did not reach statistical significance. Amlodipine treatment significantly reduced calcification in the lesions, whereas atorvastatin alone had no effect. The combination of amlodipine and atorvastatin resulted in a near absence of calcium deposits in the lesions. This study demonstrates that amlodipine treatment alone does not significantly reduce atherosclerotic lesion development. Atorvastatin was shown to have strong antiatherosclerotic effects, and cotreatment with amlodipine may potentiate the antiatherosclerotic effect of atorvastatin.