RESUMEN
Nitric oxide (NO) donors are commonly used for the prevention and treatment of ischemic heart disease. Besides their effects on the heart, NO donors may also prevent hypoxic brain damage and exert beneficial effects on atherosclerosis by favoring features of plaque stability. We recently described that apolipoprotein E (ApoE) deficient mice with a mutation in the fibrillin-1 (Fbn1) gene (ApoE-/-Fbn1C1039G+/-) develop accelerated atherosclerosis, plaque rupture, myocardial infarction, cerebral hypoxia and sudden death. In the present study, we evaluated the effects of chronic treatment with the NO donor molsidomine on atherosclerotic plaque stability, cardiac function, neurological symptoms and survival in the ApoE-/-Fbn1C1039G+/- mouse model. Female ApoE-/-Fbn1C1039G+/- mice were fed a Western diet (WD). After 8â¯weeks of WD, the mice were divided into two groups receiving either molsidomine via the drinking water (1â¯mg/kg/day; nâ¯=â¯34) or tap water (control; nâ¯=â¯36) until 25â¯weeks of WD. Survival tended to increase after molsidomine treatment (68% vs. 58% in controls). Importantly, atherosclerotic plaques of molsidomine-treated mice had a thicker fibrous cap (11.1⯱â¯1.2 vs. 8.1⯱â¯0.7⯵m) and showed an increased occurrence of plaque macrocalcifications (30% vs. 0%), indicative of a more stable phenotype. Molsidomine also improved cardiac function, as fractional shortening was increased (40⯱â¯2% vs. 27⯱â¯2%) combined with a decreased end diastolic (3.1⯱â¯0.2 vs. 3.9⯱â¯0.2â¯mm) and end systolic diameter (1.9⯱â¯0.1 vs. 2.9⯱â¯0.2â¯mm). Furthermore, perivascular fibrosis (23⯱â¯2 vs. 30⯱â¯2%) and the occurrence of myocardial infarctions (12% vs. 36%) was significantly reduced. Track width, a measure of the animal's hind limb base of support and representative of hypoxic brain damage, was also normalized as a result of molsidomine treatment (2.54⯱â¯0.04 vs. 2.91⯱â¯0.09â¯cm in controls). These findings demonstrate that the NO donor molsidomine improves cardiac function, reduces neurological symptoms and enhances atherosclerotic plaque stability.
Asunto(s)
Aterosclerosis/tratamiento farmacológico , Molsidomina/farmacología , Actividad Motora/efectos de los fármacos , Infarto del Miocardio/prevención & control , Miocardio/patología , Donantes de Óxido Nítrico/farmacología , Placa Aterosclerótica , Animales , Aterosclerosis/complicaciones , Aterosclerosis/genética , Aterosclerosis/patología , Modelos Animales de Enfermedad , Femenino , Fibrilina-1/genética , Fibrosis , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/fisiopatología , Hipertrofia Ventricular Izquierda/prevención & control , Ratones Noqueados para ApoE , Mutación , Contracción Miocárdica/efectos de los fármacos , Infarto del Miocardio/etiología , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Necrosis , Rotura Espontánea , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacosRESUMEN
OBJECTIVE: The MEDCOR trial is a double-blind, randomized study aiming at demonstrating the superiority of molsidomine (direct NO donor) over placebo, used as add-on treatments, on improving endothelial function (EF) after 12 months, in stable angina patients undergoing percutaneous coronary intervention. METHODS: EF was assessed by peripheral vasodilator response (i.e. Endoscore) using arterial tonometry and by several biomarkers, in terms of changes versus baseline after a one-year treatment. RESULTS: The change in Endoscore was +75 ± 130% in placebo group and +39 ± 145% in molsidomine group (p = 0.143). There was a decrease in sICAM-1 with molsidomine (-6%) and an increase with placebo (+6%). The MPO activity/antigen ratio slightly increased with placebo (+9%) and strongly decreased with molsidomine (-42%) (p = 0.020). CONCLUSION: The MEDCOR trial was not able to demonstrate significant differences between molsidomine and placebo for all parameters, except the MPO activity/antigen ratio which significantly decreased with molsidomine (p = 0.020 versus placebo).
Asunto(s)
Angina Estable/terapia , Enfermedad de la Arteria Coronaria/terapia , Endotelio Vascular/efectos de los fármacos , Molsidomina/uso terapéutico , Donantes de Óxido Nítrico/uso terapéutico , Intervención Coronaria Percutánea , Vasodilatación/efectos de los fármacos , Vasodilatadores/uso terapéutico , Anciano , Angina Estable/sangre , Angina Estable/diagnóstico , Angina Estable/fisiopatología , Bélgica , Biomarcadores/sangre , Terapia Combinada , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/diagnóstico , Enfermedad de la Arteria Coronaria/fisiopatología , Método Doble Ciego , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Femenino , Humanos , Molécula 1 de Adhesión Intercelular/sangre , Masculino , Manometría , Persona de Mediana Edad , Molsidomina/efectos adversos , Donantes de Óxido Nítrico/efectos adversos , Intervención Coronaria Percutánea/efectos adversos , Peroxidasa/sangre , Factores de Tiempo , Resultado del Tratamiento , Vasodilatadores/efectos adversosRESUMEN
AIMS: There is a need for animal models of plaque rupture. We previously reported that elastin fragmentation, due to a mutation (C1039G(+/-)) in the fibrillin-1 (Fbn1) gene, promotes atherogenesis and a highly unstable plaque phenotype in apolipoprotein E deficient (ApoE(-/-)) mice on a Western-type diet (WD). Here, we investigated whether plaque rupture occurred in ApoE(-/-)Fbn1(C1039G+/-) mice and was associated with myocardial infarction, stroke, and sudden death. METHODS AND RESULTS: Female ApoE(-/-)Fbn1(C1039G+/-) and ApoE(-/-) mice were fed a WD for up to 35 weeks. Compared to ApoE(-/-) mice, plaques of ApoE(-/-)Fbn1(C1039G+/-) mice showed a threefold increase in necrotic core size, augmented T-cell infiltration, a decreased collagen I content (70 ± 10%), extensive neovascularization, intraplaque haemorrhage, and a significant increase in matrix metalloproteinase-2, -9, -12, and -13 expression or activity. Plaque rupture was observed in 70% of ascending aortas and in 50% of brachiocephalic arteries of ApoE(-/-)Fbn1(C1039G+/-) mice. In ApoE(-/-) mice, plaque rupture was not seen in ascending aortas and only in 10% of brachiocephalic arteries. Seventy percent of ApoE(-/-)Fbn1(C1039G+/-) mice died suddenly, whereas all ApoE(-/-) mice survived. ApoE(-/-)Fbn1(C1039G+/-) mice showed coronary plaques and myocardial infarction (75% of mice). Furthermore, they displayed head tilt, disorientation, and motor disturbances (66% of cases), disturbed cerebral blood flow (73% of cases; MR angiograms) and brain hypoxia (64% of cases), indicative of stroke. CONCLUSIONS: Elastin fragmentation plays a key role in plaque destabilization and rupture. ApoE(-/-)Fbn1(C1039G+/-) mice represent a unique model of acute plaque rupture with human-like complications.
Asunto(s)
Muerte Súbita/etiología , Elastina/metabolismo , Infarto del Miocardio/etiología , Placa Aterosclerótica/etiología , Accidente Cerebrovascular/etiología , Animales , Aorta , Apolipoproteínas E/deficiencia , Biomarcadores/metabolismo , Tronco Braquiocefálico , Cardiomegalia/etiología , Cardiomegalia/fisiopatología , Arteria Carótida Común , Circulación Cerebrovascular/fisiología , Dieta Occidental , Modelos Animales de Enfermedad , Femenino , Fibrilina-1 , Fibrilinas , Hemorragia/etiología , Hipoxia Encefálica/etiología , Hipoxia Encefálica/fisiopatología , Ratones , Proteínas de Microfilamentos/deficiencia , Microvasos , Infarto del Miocardio/fisiopatología , Neovascularización Patológica/etiología , Neovascularización Patológica/fisiopatología , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/fisiopatología , Placa Aterosclerótica/fisiopatología , Rotura Espontánea/etiología , Rotura Espontánea/fisiopatología , Disfunción Ventricular Izquierda/etiología , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
The effects of molsidomine (a direct nitric oxide donor) on the endothelial dysfunction have never been evaluated using reactive hyperemia peripheral arterial tonometry (RH-PAT). The objective of the MEDCOR double-blind trial will be to demonstrate the superiority of molsidomine (Coruno® 16 mg, once daily) over placebo, on improving the endothelial function (Endoscore by RH-PAT) after 12 months of treatment in stable angina patients undergoing elective percutaneous coronary intervention (PCI). Study design will take care of the real-life situation, in which patients are being offered PCI and stent placement (drug-eluting or bare metal), but also gold standard medical therapy (beta-blockers, statins, angiotensin-converting enzyme inhibitors (ACEIs), and/or calcium antagonists). Demonstrating clinical and statistical superiority of the study drug over placebo will be a real challenge. Therefore, a sequential approach has been designed with a pilot phase aiming at recruiting 50 patients. Upon evaluation of the results by an independent data steering committee, a larger sample size phase will eventually be considered.
Asunto(s)
Angina Estable/terapia , Vasos Coronarios/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Molsidomina/uso terapéutico , Donantes de Óxido Nítrico/uso terapéutico , Intervención Coronaria Percutánea , Proyectos de Investigación , Angina Estable/diagnóstico , Angina Estable/fisiopatología , Bélgica , Protocolos Clínicos , Vasos Coronarios/fisiopatología , Método Doble Ciego , Quimioterapia Combinada , Endotelio Vascular/fisiopatología , Humanos , Intervención Coronaria Percutánea/efectos adversos , Intervención Coronaria Percutánea/instrumentación , Proyectos Piloto , Stents , Factores de Tiempo , Resultado del TratamientoRESUMEN
OBJECTIVE: Recent evidence suggests that amyloid precursor protein (APP) is overexpressed in atherosclerosis-prone regions of mouse aorta. We therefore investigated in the present study whether APP has a role in the progression and composition of atherosclerotic plaques. METHODS AND RESULTS: Apolipoprotein E-deficient (apoE(-/-)) mice were crossbred with animals lacking APP (APP(-/-)). After 16 weeks on a Western-type diet, apoE(-/-) and APP(-/-)/apoE(-/-) mice showed similar cholesterol levels. However, atherosclerotic plaque size was significantly reduced in the distal thoracic aorta (90% reduction) and abdominal aorta (75% reduction) of APP(-/-)/apoE(-/-) mice as compared to apoE(-/-). Plaques at the level of the aortic valves were not different in size, but showed a more stable phenotype in APP(-/-)/apoE(-/-) mice, as indicated by a reduced macrophage content, an increased amount of collagen and a thicker fibrous cap. CONCLUSION: Our findings provide evidence that lack of APP attenuates atherogenesis and leads to plaque stability.
Asunto(s)
Precursor de Proteína beta-Amiloide/deficiencia , Enfermedades de la Aorta/prevención & control , Apolipoproteínas E/deficiencia , Aterosclerosis/prevención & control , Placa Aterosclerótica/prevención & control , Precursor de Proteína beta-Amiloide/genética , Animales , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aorta Torácica/metabolismo , Aorta Torácica/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Colesterol/sangre , Colágeno/metabolismo , Modelos Animales de Enfermedad , Fibrosis , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Rotura EspontáneaRESUMEN
Because macrophages play an important role in the destabilization of atherosclerotic plaques, and smooth muscle cells (SMCs) contribute to plaque stability, selective clearance of macrophages in atherosclerotic plaques is a promising strategy for plaque stabilization. In the present study, we investigated the effects of fluvastatin, simvastatin, lovastatin, and pravastatin on the viability of macrophages and SMCs. All statins, except pravastatin, induced cell death of J774A.1 macrophages after 24 hours, albeit with different sensitivity. The viability of rabbit aortic SMCs was hardly affected. Fluvastatin-induced macrophage cell death was characterized as apoptosis and could be reversed by isoprenoid intermediates of the mevalonate pathway. Peritoneal macrophages from male or female mice were much more resistant to statin-induced cell death. The high sensitivity of J774A.1 macrophages to statin-induced cell death was related to the strong 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in these cells. Macrophage and SMC content of rabbit atherosclerotic plaques was not affected after in vitro treatment with fluvastatin or lovastatin for 3 days. In conclusion, fluvastatin, simvastatin, and lovastatin, but not pravastatin, can selectively induce apoptosis in J774A.1 macrophages, but not in SMCs, primary macrophages or rabbit atherosclerotic plaques. This effect was related to the degree of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in the different cell types.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Macrófagos/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Animales , Aorta/citología , Aorta/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Femenino , Hidroximetilglutaril-CoA Reductasas/efectos de los fármacos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Macrófagos/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/metabolismo , ConejosRESUMEN
AIM: Transglutaminase 2 (TG2) is important for the deposition and stability of the extracellular matrix via effects on cross-linking of matrix proteins and transforming growth factor beta (TGFbeta) activity. The purpose of this study was to investigate the effect of TG2 deficiency on the composi- tion of atherosclerotic plaques. METHODS: Apolipoprotein E (ApoE)(-/-) mice were crossbred with TG2(-/-) mice to obtain ApoE(-/-)TG2(-/-) mice. ApoE(-/-) and ApoE(-/-)TG2(-/-) mice were fed a Western-type diet for 16 or 30 weeks to determine the effect of TG2 deficiency on early and advanced atherosclerosis, respectively. RESULTS: Atherosclerotic plaques of ApoE(-/-)TG2(-/-) mice showed decreased cross-linking of matrix proteins, as well as decreased nuclear staining for phospho-Smad2/-Smad3, indicative of decreased TGFbeta activity. Compared to ApoE(-/-) mice, plaque area was decreased by 45 and 48% in ApoE(-/-)TG2(-/-) mice after 16 and 30 weeks, respectively. Sirius red staining showed a significant decrease in collagen content in early and advanced atherosclerotic plaques of ApoE(-/-)TG2(-/-) mice. Furthermore, there was a significant increase in macrophages in advanced atherosclerotic plaques of ApoE(-/-)TG2(-/-) mice. CONCLUSION: TG2 deficiency resulted in a decreased collagen content and increased inflammation, which are features of a more unstable plaque.
Asunto(s)
Apolipoproteínas E/deficiencia , Aterosclerosis/enzimología , Proteínas de Unión al GTP/deficiencia , Inflamación/enzimología , Transglutaminasas/deficiencia , Animales , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/prevención & control , Colágeno/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibrosis , Proteínas de Unión al GTP/genética , Inflamación/genética , Inflamación/patología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Glutamina Gamma Glutamiltransferasa 2 , Rotura , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismo , Transglutaminasas/genéticaRESUMEN
The ubiquitin-proteasome system is involved in the development and progression of atherosclerosis. The aim of this study was to investigate whether plaque composition is affected by proteasome function. In vitro, the potent and selective proteasome inhibitor bortezomib induced apoptosis in both cultured smooth muscle cells (SMCs) and activated macrophages. This effect was associated with increased expression of C/EBP homologous protein and cleavage of caspase-12, indicative of endoplasmic reticulum stress. The sensitivity to the proapoptotic effects of proteasome inhibition correlated with the protein synthesis rate. Proteasome inhibition in explanted atherosclerotic plaques of ApoE-deficient mice resulted in a significant decrease in SMCs and macrophages, indicating that both cell types in the atherosclerotic plaque were susceptible to the proapoptotic effects of proteasome inhibition. In vivo proteasome inhibition in ApoE-deficient mice did not affect plaque size or composition of early atherosclerotic plaques, but resulted in a significant decrease in collagen content as well as a significant enlargement of the necrotic core in advanced atherosclerotic plaques. In conclusion, our results indicate that an impaired proteasome function promotes features of a more rupture-prone plaque phenotype.
Asunto(s)
Aterosclerosis/metabolismo , Ácidos Borónicos/farmacología , Macrófagos/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Pirazinas/farmacología , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Apoptosis/efectos de los fármacos , Bortezomib , Células Cultivadas , Femenino , Ratones , Ratones Noqueados , Fenotipo , Inhibidores de Proteasoma , Conejos , Rotura EspontáneaRESUMEN
Rupture-prone and ruptured plaques are characterized by the presence of large numbers of macrophages. N1177 is a contrast agent consisting of iodinated nanoparticles that are selectively phagocytosed by macrophages. The aim of this study was to investigate the effect of N1177 on the CT attenuation of rupture-prone and ruptured plaques in rabbits. In addition, we examined in vitro whether uptake of N1177 occurred without cytotoxic or pro-inflammatory effects on macrophages. In vitro, the viability of J774 macrophages was not affected by treatment with N1177. Moreover, N1177 had no effect on the phagocytic capacity or cytokine production of macrophages. For the in vivo experiments, 6 New Zealand White rabbits were fed a cholesterol-supplemented diet for 12-15 months, resulting in the development of large atherosclerotic plaques that resembled rupture-prone plaques in humans. In three rabbits, mechanical plaque rupture was induced by retrograde pullback of an embolic protection device. N1177 had no effect on the median density of rupture-prone plaques [35 HU (range 3-85) before injection vs. 32 HU (range 1-93) 2 h after injection of N1177; P > 0.05]. However, after induction of mechanical plaque rupture, the median density of the atherosclerotic plaques increased from 40 HU (range 6-86) before injection to 74 HU (range 14-111) 2 h after injection of N1177 (P < 0.001). Using time-of-flight static secondary ion mass spectrometry, the presence of N1177 nanoparticles was demonstrated in macrophage-rich areas of ruptured plaques, but not of non-ruptured plaques. In conclusion, our results show that N1177 is a contrast agent that can identify ruptured atherosclerotic plaques.
Asunto(s)
Aterosclerosis/diagnóstico por imagen , Medios de Contraste , Yodo , Macrófagos/efectos de los fármacos , Nanopartículas , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Medios de Contraste/efectos adversos , Ratones , Conejos , Rotura Espontánea/diagnóstico por imagen , Tomografía Computarizada por Rayos XRESUMEN
INTRODUCTION: Steatosis, without fibrosis, may lead to changes in liver blood flow, which are poorly understood, and to date have not been correlated to portal pressure and related haemodynamics. AIMS: To study the temporal relation between progressive steatosis, portal pressure, systemic haemodynamics, vascular responsiveness, mesenteric and portal blood flow in methionine-choline-deficient diet (MCDD)-fed rats. METHODS: Male Wistar rats fed the MCDD were examined at week (w) 0-1-2-3-4-5-6-7-8, respectively, including systemic haemodynamics and portal pressure. At w0-4-8, in vivo blood flow was measured in the portal vein and the superior mesenteric artery. Dose-response curves to phenylephrine (PE) were established in abdominal aortic rings. RESULTS: Histology showed 100% steatosis from w3 on. Fibrosis was absent. Significant inflammation was nearly absent upon w4. Portal pressure slightly increased at w2, reached a maximum at w4 [9.4 +/- 0.3 vs 2.9 +/- 0.6 mmHg at w0 (P=0.003)] and remained stable upon w8. Mean arterial blood pressure (MABP) decreased from w2 on [98.7 +/- 5.7 mmHg on w4 compared with 123.8 +/- 1.8 on w0 (P=0.002)]. Portal flow increased from 1.85 +/- 0.11 to 3.07 +/- 0.44 ml/min/100 g on w0 and w8 respectively (P=0.039). Mesenteric artery flow increased from 3.40 +/- 0.26 to 4.56 +/- 0.30 ml/min/100 g on w0 and w8 respectively (P=0.043). Vascular responsiveness to PE gradually decreased from 138 +/- 3% on w0 to 110 +/- 5% on w4 (P=0.013). CONCLUSION: Steatohepatitis induces significant portal hypertension (PHT) in the absence of fibrosis, associated with an increase in mesenteric arterial and portal venous flow, arterial hyporesponsiveness to vasoconstrictors and a decrease in MABP, indicating the presence of splanchnic vasodilation and hyperdynamic circulation. These alterations resemble those seen in cirrhotic PHT.
Asunto(s)
Hígado Graso/fisiopatología , Hemodinámica , Hipertensión Portal/etiología , Circulación Hepática , Presión Portal , Circulación Esplácnica , Vasodilatación , Animales , Aorta , Velocidad del Flujo Sanguíneo , Citocinas/sangre , Relación Dosis-Respuesta a Droga , Hígado Graso/complicaciones , Hígado Graso/patología , Técnicas In Vitro , Hígado/metabolismo , Hígado/patología , Masculino , Peroxidasa/metabolismo , Fenilefrina/farmacología , Ratas , Ratas Wistar , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacologíaRESUMEN
BACKGROUND: Arterial stiffness has been associated with an increased cardiovascular risk. The aim of this study was to investigate the interaction between arterial stiffness and atherosclerosis. METHODS AND RESULTS: Mice with a mutation C1039G+/-) in the fibrillin-1 gene leading to fragmentation of the elastic fibers were crossbred with apolipoprotein E-deficient (ApoE-/-) mice. Subsequently, ApoE-/- and ApoE-/-C1039G+/- mice were fed a Western-type diet for 10 or 20 weeks. Our results show that the interaction between arterial stiffness and atherosclerosis is bidirectional. On the one hand, arterial stiffness in ApoE-/-C1039G+/- mice increased more rapidly in the presence of atherosclerotic plaques. On the other hand, arterial stiffness promoted the development of larger and more unstable plaques in ApoE-/-C1039G+/- mice. The plaque area at the aortic root was increased 1.5- and 2.1-fold in ApoE-/-C1039G+/- mice after 10 and 20 weeks of Western-type diet, respectively. After 10 weeks of Western-type diet, plaques of ApoE-/-C1039G+/- mice showed increased apoptosis of smooth muscle cells, which was associated with a decrease in collagen content, an enlargement of the necrotic core, and an increase in macrophages. After 20 weeks of Western-type diet, the number of buried fibrous caps was increased in atherosclerotic lesions of ApoE-/-C1039G+/- mice, not only at the level of the aortic valves but also in the brachiocephalic artery and in the upper, middle, and lower thoracic aorta. Furthermore, acute plaque rupture was observed. CONCLUSIONS: These results indicate that fragmentation of the elastic fibers leads to increased vascular stiffness, which promotes features of multifocal plaque instability.
Asunto(s)
Apolipoproteínas E/deficiencia , Aterosclerosis/genética , Aterosclerosis/fisiopatología , Proteínas de Microfilamentos/fisiología , Animales , Aorta Torácica/metabolismo , Aorta Torácica/fisiopatología , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Femenino , Fibrilina-1 , Fibrilinas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genéticaRESUMEN
Unstable atherosclerotic plaques are characterized by a thin fibrous cap that contains few smooth muscle cells (SMCs) and numerous foam cells of macrophage origin. Previously we and others demonstrated that macrophages disappear from atherosclerotic plaques after dietary lipid lowering. However, it remains unclear whether loss of macrophages after lipid lowering occurs via increased apoptosis, decreased macrophage replication and/or recruitment, or via a combination of both. Rabbits were fed a diet supplemented with cholesterol (0.3%) for 24 weeks followed by a normal diet for 4, 12, or 24 weeks. After 24 weeks of cholesterol supplement, plaques showed apoptosis in both macrophages and SMCs, as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling. Cell replication (Ki-67 immunolabeling) was predominantly present in macrophages. After 24 weeks of cholesterol withdrawal, the thickness and areas of the plaques were unchanged. Nevertheless, plaques showed a considerable loss of macrophages. This event was associated with a reduced immunoreactivity for vascular cell adhesion molecule-1 (VCAM-1) in the endothelial cells starting 4 weeks after cholesterol withdrawal. Apoptosis did not increase after lipid lowering but showed a steady decline. Apart from decreased VCAM-1 expression, a strong decrease in Ki-67 immunolabeling was observed after 12 weeks of cholesterol withdrawal. Our findings suggest that loss of macrophages in atherosclerotic plaques after dietary lipid lowering is not related to induction of macrophage apoptosis but mainly a consequence of impaired monocyte recruitment followed by decreased macrophage replication. This information is essential for understanding the effects of aggressive lipid lowering on plaque stability.
Asunto(s)
Apoptosis , Aterosclerosis/patología , Colesterol en la Dieta/administración & dosificación , Macrófagos/patología , Actinas/análisis , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Aterosclerosis/sangre , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Colesterol en la Dieta/sangre , LDL-Colesterol/sangre , Células Endoteliales/metabolismo , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Antígeno Ki-67/análisis , Lípidos/administración & dosificación , Lípidos/sangre , Macrófagos/metabolismo , Masculino , Músculo Liso/química , Conejos , Triglicéridos/sangre , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
AIMS: Most acute coronary syndromes are caused by plaque rupture. The risk of plaque rupture is related to plaque composition. The purpose of this study was to validate VH-IVUS for in vivo plaque characterisation. METHODS AND RESULTS: Six rabbits were fed a cholesterol-supplemented diet for 12 to 18 months. Thereafter, VH-IVUS imaging of the aorta was performed. After sacrifice, the VH-IVUS images were matched to the corresponding histological cross sections. A total of 260 atherosclerotic plaques were analysed. VH-IVUS had a high sensitivity, specificity and positive predictive value for the detection of non-calcified thin cap fibroatheroma (88%, 96%, 87%, respectively) and calcified thin cap fibroatheroma (95%, 99%, 93%, respectively). These values were respectively 82%, 94%, 85% for non-calcified fibroatheroma and 78%, 98%, 84% for calcified fibroatheroma. The lowest values were obtained for pathological intimal thickening (74%, 92%, 70%, respectively). For all plaque types, VH-IVUS had a kappa-value of 0.79. Linear regression analysis and Bland-Altman plots showed a strong correlation between VH-IVUS and histology for fibrous tissue, fibrofatty tissue, necrotic calcified tissue and confluent necrotic core. CONCLUSIONS: VH-IVUS showed a good accuracy for in vivo plaque characterisation and is a promising technique for the detection of the vulnerable plaque.
Asunto(s)
Enfermedades de la Aorta/diagnóstico por imagen , Aterosclerosis/diagnóstico por imagen , Calcinosis/diagnóstico por imagen , Modelos Animales de Enfermedad , Ultrasonografía Intervencional , Interfaz Usuario-Computador , Animales , Enfermedades de la Aorta/etiología , Aterosclerosis/etiología , Calcinosis/etiología , Colesterol en la Dieta , Fibrosis , Necrosis , Valor Predictivo de las Pruebas , Conejos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Because macrophages play a major role in atherosclerotic plaque destabilization, selective removal of macrophages represents a promising approach to stabilize plaques. We showed recently that the protein synthesis inhibitor cycloheximide, in contrast to puromycin, selectively depleted macrophages in rabbit atherosclerotic plaques without affecting smooth muscle cells (SMCs). The mechanism of action of these two translation inhibitors is dissimilar and could account for the differential effects on SMC viability. It is not known whether selective depletion of macrophages is confined to cycloheximide or whether it can also be achieved with translation inhibitors that have a similar mechanism of action. Therefore, in the present study, we investigated the effect of anisomycin, a translation inhibitor with a mechanism of action similar to cycloheximide, on macrophage and SMC viability. In vitro, anisomycin induced apoptosis of macrophages in a concentration-dependent manner, whereas SMCs were only affected at higher concentrations. In vivo, anisomycin selectively decreased the macrophage content of rabbit atherosclerotic plaques through apoptosis. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole] prevented anisomycin-induced macrophage death, without affecting SMC viability. SB202190 decreased anisomycin-induced p38 MAPK phosphorylation, did not alter c-Jun NH(2)-terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. The latter effect was abolished by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene ethanolate], although the prevention of anisomycin-induced macrophage death by SB202190 remained unchanged. The JNK phosphorylation inhibitor SP600125 did not affect anisomycin-induced macrophage or SMC death. In conclusion, anisomycin selectively decreased the macrophage content in rabbit atherosclerotic plaques, indicating that this effect is not confined to cycloheximide. p38 MAPK, but not ERK1/2 or JNK, plays a major role in anisomycin-induced macrophage death.
Asunto(s)
Anisomicina/farmacología , Apoptosis/efectos de los fármacos , Estenosis Carotídea/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Anisomicina/uso terapéutico , Antracenos/farmacología , Aorta/citología , Butadienos/farmacología , Arterias Carótidas/citología , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/metabolismo , Estenosis Carotídea/patología , Línea Celular Tumoral , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Imidazoles/farmacología , Macrófagos/citología , Macrófagos Alveolares/citología , Macrófagos Alveolares/efectos de los fármacos , Ratones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Nitrilos/farmacología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Conejos , Túnica Íntima/citología , Túnica Íntima/efectos de los fármacos , Túnica Íntima/metabolismo , Túnica Media/citología , Túnica Media/efectos de los fármacos , Túnica Media/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Phagocytosis represents a mechanism used by macrophages to remove pathogens and cellular debris. Recent evidence suggests that phagocytosis is stimulated under specific conditions of stress, such as extracellular pressure and hypoxia. In the present study, we show that amino acid or glucose deprivation caused an increase in the phagocytosis of heat-inactivated Escherichia coli and Staphylococcus aureus by macrophages, but not the uptake of platelets, apoptotic cells or beads. Increased phagocytosis of bacteria could be blocked by phagocytosis inhibitors and was found to be dependent on p38 mitogen-activated protein kinase activity and scavenger receptor A. Although nutrient deprivation is a strong stimulus of autophagy, autophagosome formation was not critical for the uptake of bacteria because phagocytic clearance was not inhibited after down-regulation of the autophagy essential gene Atg7. Moreover, enhanced uptake of bacteria should not be considered as a general stress response because phagocytosis of bacteria was not stimulated after exposure of macrophages to the genotoxic agent camptothecin, heat (40 degrees C) or thapsigargin-induced endoplasmic reticulum stress. Overall, the results obtained in the present study indicate that nutrient deprivation can stimulate macrophages to fight bacterial infections.
Asunto(s)
Escherichia coli/metabolismo , Macrófagos/microbiología , Fagocitosis/fisiología , Staphylococcus aureus/metabolismo , Animales , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Receptores Depuradores/metabolismoRESUMEN
The lack of exposure to helminth infections, as a result of improved living standards and medical conditions, may have contributed to the increased incidence of IBD in the developed world. Epidemiological, experimental, and clinical data sustain the idea that helminths could provide protection against IBD. Studies investigating the underlying mechanisms by which helminths might induce such protection have revealed the importance of regulatory pathways, for example, regulatory T-cells. Further investigation on how helminths influence both innate and adaptive immune reactions will shed more light on the complex pathways used by helminths to regulate the hosts immune system. Although therapy with living helminths appears to be effective in several immunological diseases, the disadvantages of a treatment based on living parasites are explicit. Therefore, the identification and characterization of helminth-derived immunomodulatory molecules that contribute to the protective effect could lead to new therapeutic approaches in IBD and other immune diseases.
Asunto(s)
Proteínas del Helminto/uso terapéutico , Helmintiasis/inmunología , Helmintos/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/terapia , Linfocitos T Reguladores/inmunología , Animales , Helmintiasis/parasitología , Helmintos/metabolismo , Humanos , Inmunidad Mucosa , Lactante , Recién Nacido , RatonesRESUMEN
Recent evidence indicates that the protein synthesis inhibitor cycloheximide triggers selective macrophage death in rabbit atheroma-like lesions without affecting smooth muscle cells (SMCs) or the endothelium, thereby favoring a stable plaque phenotype. In this study, we report that puromycin, a protein synthesis inhibitor with a different mode of action but with similar ability to inhibit de novo protein synthesis, did not reveal plaque-stabilizing effects. The macrophage and the SMC content readily decreased in puromycin-treated atheroma-like lesions in rabbit carotid arteries. Moreover, puromycin induced apoptosis in macrophages and SMCs in vitro. Puromycin-treated SMCs showed signs of endoplasmic reticulum (ER) stress, as demonstrated by CCAAT/enhancer-binding protein homologous protein (CHOP) protein expression, splicing of X-box-binding protein 1 mRNA, and phosphorylation of eukaryotic translation initiation factor 2alpha. The ER stress inducer thapsigargin up-regulated CHOP protein expression in SMCs without affecting their viability, indicating that ER stress not necessarily results in cell death. Puromycin, but not thapsigargin, activated the ER stress-related caspase-12. Treatment of SMCs with a combination of cycloheximide and puromycin inhibited ER stress and partially improved SMC viability. In addition, puromycin, but not cycloheximide or thapsigargin, induced intracellular accumulation of polyubiquitinated proteins in SMCs, whereas the proteasome function was not affected. Taken together, puromycin, in contrast to cycloheximide, induces SMC apoptosis, thereby favoring an unstable plaque phenotype. SMC death upon puromycin treatment could only be partially prevented by cycloheximide, which completely blocked ER stress. However, other or additional mechanisms, such as increased polyubiquitination of proteins, might be involved in puromycin-induced SMC death.
Asunto(s)
Aterosclerosis/patología , Cicloheximida/farmacología , Macrófagos/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Puromicina/farmacología , Animales , Apoptosis , Aterosclerosis/metabolismo , Arterias Carótidas/efectos de los fármacos , Caspasa 12/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN , Proteínas de Unión al ADN/genética , Grasas de la Dieta/administración & dosificación , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patología , Técnicas In Vitro , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Proteínas Nucleares/genética , ARN Mensajero/metabolismo , Conejos , Factores de Transcripción del Factor Regulador X , Factores de TranscripciónRESUMEN
AIMS: Rupture-prone atherosclerotic plaques show an elevated temperature, but a molecular explanation for this phenomenon is unknown. Here, we investigated whether mitochondrial uncoupling protein 2 (UCP2) could be involved because this protein is a macrophage homologue of thermogenin in brown fat tissue. METHODS AND RESULTS: Immunohistochemistry, western blotting, and real-time quantitative polymerase chain reaction were used to detect UCP2 expression in human and rabbit atherosclerotic plaques. Temperature was measured in plaques with thermography catheters and in cultured cells with precision thermometers. UCP2 was abundantly expressed in subendothelial macrophages of atherosclerotic plaques but not in deeper layers of the plaque. Ex vivo temperature measurements in atherosclerotic rabbit thoracic aorta demonstrated a correlation between local plaque temperature, total macrophage mass, and UCP2 expression. In vitro, chemical uncoupling of macrophages with sodium cyanide resulted in heat production (DeltaT = 0.13 +/- 0.04 degrees C vs. controls). Also, overexpression of UCP2 in cultured cells led to a similar increase in temperature. CONCLUSION: Our findings provide evidence that temperature heterogeneity in atherosclerotic plaques is at least in part attributed to UCP2 expression in macrophages. The heat generated might be used to detect unstable, macrophage-rich, atherosclerotic plaques via thermography.
Asunto(s)
Aterosclerosis/fisiopatología , Canales Iónicos/fisiología , Proteínas Mitocondriales/fisiología , Termogénesis , Animales , Células Cultivadas , Humanos , Canales Iónicos/genética , Macrófagos/metabolismo , Masculino , Ratones , Proteínas Mitocondriales/genética , ARN Mensajero/análisis , Conejos , Termografía , Proteína Desacopladora 2RESUMEN
Acetylcholine (ACh)-induced relaxation declines in apolipoprotein E-deficient (apoE-/-) mouse aortas, but only after atherosclerotic plaque formation. This study investigated intracellular calcium concentrations [Ca2+]i and changes in phenylephrine-induced contractions as index of baseline nitric oxide (NO) bioavailability before plaque development. Isometric contractions of thoracic aorta rings of young (4 months) apoE-/- and C57BL/6J (WT) mice were evoked by phenylephrine (3x10(-9)-3x10(-5) M) in the presence and absence of endothelial cells (ECs) or NO synthase (NOS) inhibitors. [Ca2+]i (Fura-2 AM) and endothelium-dependent relaxation were measured at baseline and after ACh stimulation. Segments of apoE-/- mice were significantly more sensitive and developed more tension than WT segments in response to phenylephrine. The differences disappeared after NOS inhibition or EC removal or upon increasing [Ca2+]i in apoE-/- strips with 10(-6) M cyclopiazonic acid or 10(-7) M Ca2+-ionophore A23187. Expression of endothelial NOS (eNOS) mRNA was similar in apoE-/- and WT aorta segments. Basal [Ca2+]i was significantly lower in apoE-/- than in WT strips. Relaxation by ACh (3x10(-9)-10(-5) M) was time- and dose-dependently related to [Ca2+]i, but neither ACh-induced relaxation nor Ca2+ mobilization were diminished in apoE-/- strips. In conclusion, basal, but not ACh-induced NO bioavailability, was compromised in lesion-free aorta of apoE-/- mice. Decreased basal NO bioavailability was not related to lower eNOS expression, but most likely related to lower basal [Ca2+]i. These findings further point to important differences between basal and stimulated eNOS activity.
Asunto(s)
Apolipoproteínas E/genética , Aterosclerosis/fisiopatología , Endotelio Vascular/fisiología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Acetilcolina/farmacología , Animales , Aorta Torácica/enzimología , Aorta Torácica/patología , Aorta Torácica/fisiología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Calcio/metabolismo , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Femenino , Contracción Isométrica/efectos de los fármacos , Contracción Isométrica/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo III , Fenilefrina/farmacología , ARN Mensajero/metabolismo , Vasoconstrictores/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiología , Vasodilatadores/farmacologíaRESUMEN
Efficient phagocytosis of cells undergoing apoptosis by macrophages is important to prevent immunological responses and development of chronic inflammatory disorders such as systemic lupus erythematosus, cystic fibrosis and atherosclerosis. To study phagocytosis of apoptotic cells (AC) by macrophages in tissue, we validated different apoptosis markers (DNA fragmentation, caspase-3 activation and cleavage of its substrate poly(ADP-ribose)polymerase-1) in combination with macrophage immunostaining. Human tonsils were used as a model because they show a high apoptosis frequency under physiological conditions as well as efficient phagocytosis of AC by macrophages. On the other hand, advanced human atherosclerotic plaques were examined since plaques show severely impaired phagocytosis of AC. Our results demonstrate that the presence of non-phagocytized terminal deoxynucleotidyl transferase end labelling (TUNEL)-positive AC represents a suitable marker of poor phagocytosis by macrophages in situ. Other markers for apoptosis, such as cleavage of caspase-3 or PARP-1, should not be used to assess phagocytosis efficiency, because activation of the caspase cascade and cleavage of their substrates can occur in AC when they have not yet been phagocytized by macrophages.