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Mitochondrial reactive oxygen species (mROS) are central to physiology. While excess mROS production has been associated with several disease states, its precise sources, regulation, and mechanism of generation in vivo remain unknown, limiting translational efforts. Here we show that in obesity, hepatic ubiquinone (Q) synthesis is impaired, which raises the QH 2 /Q ratio, driving excessive mROS production via reverse electron transport (RET) from site I Q in complex I. Using multiple complementary genetic and pharmacological models in vivo we demonstrated that RET is critical for metabolic health. In patients with steatosis, the hepatic Q biosynthetic program is also suppressed, and the QH 2 /Q ratio positively correlates with disease severity. Our data identify a highly selective mechanism for pathological mROS production in obesity, which can be targeted to protect metabolic homeostasis.
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BACKGROUND AND AIMS: Glutamine synthetase (GLUL), the sole generator of glutamine, is a metabolic nexus molecule also involved in atherosclerosis. We recently demonstrated a 2.2-fold upregulation of GLUL mRNA in stroke-causing carotid plaques when compared with plaques from asymptomatic patients. Here we compared in the same cohort GLUL mRNA expression with plaque gross morphology, and the colocalization of immunodetectable GLUL protein with histopathological changes and molecular and mechanical mediators linked to plaque development. METHODS: Endarterectomy specimens from 19 asymptomatic and 24 stroke patients were sectioned longitudinally and immunostained for GLUL, CD68, α-smooth muscle actin, iron, heme oxygenase-1 and CD163, and graded semiquantitatively in every 1 mm2. The amounts of cholesterol clefts and erythrocytes were graded. The fibrous cap thickness within each 1 mm2 area was measured. The association between the local pathological findings was analyzed by a hierarchical mixed modelling approach. RESULTS: The previously found correlation between GLUL mRNA and clinical symptomatology was supported by the increased GLUL mRNA in diseased tissue and increased local GLUL immunoreactivity in areas with multiple different atherosclerotic changes. A longer symptom-to-operation time correlated with lower GLUL mRNA (Rs = -0.423, p=0.050) but few outliers had a significantly higher GLUL mRNA levels, which persisted throughout the post-symptomatic period. Plaque ulceration associated with 1.8-fold higher GLUL mRNA (p=0.006). Macrophages were the main GLUL immunoreactive cells. GLUL immunostaining colocalized with erythrocytes, iron, CD163, and heme oxygenase-1. The correlations between local variables were consistent in both asymptomatic and stroke-causing plaques. An inverse correlation was found between the fibrous cap thickness and local GLUL immunoreactivity (p=0.012). Considerable variability in interplaque expression pattern of GLUL was present. CONCLUSIONS: Our results link connect macrophage GLUL expression with carotid plaque features characterizing plaque vulnerability.
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Aterosclerosis , Estenosis Carotídea , Endarterectomía Carotidea , Placa Aterosclerótica , Accidente Cerebrovascular , Aterosclerosis/patología , Estenosis Carotídea/patología , Fibrosis , Glutamato-Amoníaco Ligasa/metabolismo , Hemo-Oxigenasa 1/metabolismo , Humanos , Hierro/metabolismo , Macrófagos/metabolismo , Placa Aterosclerótica/metabolismo , ARN Mensajero/metabolismo , Accidente Cerebrovascular/complicacionesRESUMEN
The liberation of energy stores from adipocytes is critical to support survival in times of energy deficit; however, uncontrolled or chronic lipolysis associated with insulin resistance and/or insulin insufficiency disrupts metabolic homeostasis1,2. Coupled to lipolysis is the release of a recently identified hormone, fatty-acid-binding protein 4 (FABP4)3. Although circulating FABP4 levels have been strongly associated with cardiometabolic diseases in both preclinical models and humans4-7, no mechanism of action has yet been described8-10. Here we show that hormonal FABP4 forms a functional hormone complex with adenosine kinase (ADK) and nucleoside diphosphate kinase (NDPK) to regulate extracellular ATP and ADP levels. We identify a substantial effect of this hormone on beta cells and given the central role of beta-cell function in both the control of lipolysis and development of diabetes, postulate that hormonal FABP4 is a key regulator of an adipose-beta-cell endocrine axis. Antibody-mediated targeting of this hormone complex improves metabolic outcomes, enhances beta-cell function and preserves beta-cell integrity to prevent both type 1 and type 2 diabetes. Thus, the FABP4-ADK-NDPK complex, Fabkin, represents a previously unknown hormone and mechanism of action that integrates energy status with the function of metabolic organs, and represents a promising target against metabolic disease.
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Proteínas de Unión a Ácidos Grasos , Islotes Pancreáticos , Fosfotransferasas , Adipocitos/metabolismo , Diabetes Mellitus/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Humanos , Insulina/metabolismo , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/fisiología , Lipólisis , Nucleósidos/metabolismo , Fosfotransferasas/metabolismoRESUMEN
Background: Long-term treatment with the vitamin K antagonist warfarin is widely used for the prevention of venous thrombosis and thromboembolism. However, vitamin K antagonists may promote arterial calcification, a phenomenon that has been previously studied in coronary and peripheral arteries, but not in extracranial carotid arteries. In this observational cohort study, we investigated whether warfarin treatment is associated with calcification of atherosclerotic carotid arteries. Methods: Overall, 500 consecutive patients underwent carotid endarterectomy, 82 of whom had received long-term warfarin therapy. The extent of calcification was assessed with preoperative computed tomography angiography, and both macroscopic morphological grading and microscopic histological examination of each excised carotid plaque were performed after carotid endarterectomy. Results: Compared with non-users, warfarin users had significantly more computed tomography angiography-detectable vascular calcification in the common carotid arteries (odds ratio 2.64, 95% confidence interval 1.51-4.63, P < 0.001) and even more calcification in the internal carotid arteries near the bifurcation (odds ratio 18.27, 95% confidence interval 2.53-2323, P < 0.001). Histological analysis revealed that the intramural calcified area in plaques from warfarin users was significantly larger than in plaques from non-users (95% confidence interval 3.36-13.56, P = 0.0018). Conclusions: Long-lasting warfarin anticoagulation associated with increased calcification of carotid atherosclerotic plaques, particularly in locations known to be the predilection sites of stroke-causing plaques. The clinical significance of this novel finding warrants further investigations.
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Fatty acid binding protein 4 (FABP4) is implicated in the pathogenesis of cardiometabolic disorders. Pharmacological inhibition or genetic deletion of FABP4 improves cardiometabolic health and protects against atherosclerosis in preclinical models. As cardiovascular disease (CVD) is common in type 1 diabetes, we examined the role of FABP4 in the development of complications in type 1 diabetes, focusing on a functional, low-expression variant (rs77878271) in the promoter of the FABP4 gene. For this, we assessed the risk of CVD, stroke, coronary artery disease (CAD), end-stage kidney disease, and mortality using Cox proportional hazards models for the FABP4 rs77878271 in 5,077 Finnish individuals with type 1 diabetes. The low-expression G allele of rs77878271 increased the risk of CVD, independent of confounders. Findings were tested for replication in 852 Danish and 3,678 Finnish individuals with type 1 diabetes. In the meta-analysis, each G allele increased the risk of stroke by 26% (P = 0.04), CAD by 26% (P = 0.006), and CVD by 17% (P = 0.003). In Mendelian randomization, a 1-SD unit decrease in FABP4 increased risk of CAD 2.4-fold. Hence, in contrast with the general population, among patients with type 1 diabetes the low-expression G allele of rs77878271 increased CVD risk, suggesting that genetically low FABP4 levels may be detrimental in the context of type 1 diabetes.
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Enfermedades Cardiovasculares/genética , Diabetes Mellitus Tipo 1/genética , Proteínas de Unión a Ácidos Grasos/genética , Adulto , Alelos , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/etiología , Estudios de Cohortes , Dinamarca/epidemiología , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/epidemiología , Angiopatías Diabéticas/epidemiología , Angiopatías Diabéticas/genética , Femenino , Finlandia/epidemiología , Regulación de la Expresión Génica , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Análisis de la Aleatorización Mendeliana , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Factores de RiesgoRESUMEN
Although counterregulatory hormones and mediators of the fight-or-flight responses are well defined at many levels, how energy stores per se are integrated into this system remains an enigmatic question. Recent years have seen the adipose tissue become a central focus for mediating intracellular signaling and communication through the release of a variety of bioactive lipids and substrates, as well as various adipokines. A critical integration node among these mediators and responses is controlled by FA binding protein 4 (FABP4), also known as adipocyte protein 2 (aP2), which is highly expressed in adipose tissue and functions as a lipid chaperone protein. Recently, it was demonstrated that FABP4 is a secreted hormone that has roles in maintaining glucose homeostasis, representing a key juncture facilitating communication between energy-storage systems and distant organs to respond to life-threatening situations. However, chronic engagement of FABP4 under conditions of immunometabolic stress, such as obesity, exacerbates a number of immunometabolic diseases, including diabetes, asthma, cancer, and atherosclerosis. In both preclinical mouse models and humans, levels of circulating FABP4 have been correlated with metabolic disease incidence, and reducing FABP4 levels or activity is associated with improved metabolic health. In this review, we will discuss the intriguing emerging biology of this protein, including potential therapeutic options for targeting circulating FABP4.
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Proteínas de Unión a Ácidos Grasos/metabolismo , Animales , Metabolismo Energético , Proteínas de Unión a Ácidos Grasos/sangre , Humanos , RatonesRESUMEN
INTRODUCTION: Every fifth ischemic stroke is caused by thromboembolism originating from an atherosclerotic carotid artery plaque. While prevention is the most cost-effective stroke therapy, antiplatelet and cholesterol-lowering drugs have a ceiling effect in their efficacy. Therefore, discovery of novel pathophysiologic targets are needed to improve the primary and secondary prevention of stroke. This article provides a detailed study design and protocol of HeCES2, an observational prospective cohort study with the objective to investigate the pathophysiology of carotid atherosclerosis. MATERIALS AND METHODS: Recruitment and carotid endarterectomies of the study patients with carotid atherosclerosis were performed from October 2012 to September 2015. After brain and carotid artery imaging, endarterectomised carotid plaques (CPs) and blood samples were collected from 500 patients for detailed biochemical and molecular analyses. Findings to date: We developed a morphological grading for macroscopic characteristics within CPs. The dominant macroscopic CP characteristics were: smoothness 62%, ulceration 61%, intraplaque hemorrhage 60%, atheromatous gruel 59%, luminal coral-type calcification 34%, abundant (44%) and moderate (39%) intramural calcification, and symptom-causing "hot spot" area 53%. Future plans: By combining clinically oriented and basic biomedical research, this large-scale study attempts to untangle the pathophysiological perplexities of human carotid atherosclerosis. Key Messages This article is a rationale and design of the HeCES2 study that is an observational prospective cohort study with the objective to investigate the pathophysiology of carotid atherosclerosis. The HeCES2 study strives to develop diagnostic algorithms including radiologic imaging to identify carotid atherosclerosis patients who warrant surgical treatment. In addition, the study aims at finding out new tools for clinical risk stratification as well as novel molecular targets for drug development.
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Infarto Encefálico/prevención & control , Arterias Carótidas/patología , Estenosis Carotídea/patología , Endarterectomía Carotidea , Placa Aterosclerótica/patología , Anciano , Anciano de 80 o más Años , Encéfalo/irrigación sanguínea , Encéfalo/diagnóstico por imagen , Infarto Encefálico/sangre , Infarto Encefálico/patología , Arterias Carótidas/diagnóstico por imagen , Arterias Carótidas/cirugía , Estenosis Carotídea/sangre , Estenosis Carotídea/complicaciones , Estenosis Carotídea/cirugía , Angiografía por Tomografía Computarizada , Estudios Transversales , Femenino , Finlandia , Humanos , Estudios Longitudinales , Angiografía por Resonancia Magnética , Masculino , Persona de Mediana Edad , Placa Aterosclerótica/sangre , Placa Aterosclerótica/complicaciones , Placa Aterosclerótica/cirugía , Estudios Prospectivos , Proyectos de Investigación , Medición de RiesgoRESUMEN
Lipid accumulation is a key characteristic of advancing atherosclerotic lesions. Herein, we analyzed the ultrastructure of the accumulated lipids in endarterectomized human carotid atherosclerotic plaques using three-dimensional (3D) electron microscopy, a method never used in this context before. 3D electron microscopy revealed intracellular lipid droplets and extracellular lipoprotein particles. Most of the particles were aggregated, and some connected to needle-shaped or sheet-like cholesterol crystals. Proteomic analysis of isolated extracellular lipoprotein particles revealed that apolipoprotein B is their main protein component, indicating their origin from low-density lipoprotein, intermediate-density lipoprotein, very-low-density lipoprotein, lipoprotein (a), or chylomicron remnants. The particles also contained small exchangeable apolipoproteins, complement components, and immunoglobulins. Lipidomic analysis revealed differences between plasma lipoproteins and the particles, thereby indicating involvement of lipolytic enzymes in their generation. Incubation of human monocyte-derived macrophages with the isolated extracellular lipoprotein particles or with plasma lipoproteins that had been lipolytically modified in vitro induced intracellular lipid accumulation and triggered inflammasome activation in them. Taken together, extracellular lipids accumulate in human carotid plaques as distinct 3D structures that include aggregated and fused lipoprotein particles and cholesterol crystals. The particles originate from plasma lipoproteins, show signs of lipolytic modifications, and associate with cholesterol crystals. By inducing intracellular cholesterol accumulation (ie, foam cell formation) and inflammasome activation, the extracellular lipoprotein particles may actively enhance atherogenesis.
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Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/metabolismo , Mediadores de Inflamación/metabolismo , Metabolismo de los Lípidos/fisiología , Arterias Carótidas/ultraestructura , Enfermedades de las Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/cirugía , Células Cultivadas , Colesterol/metabolismo , Endarterectomía Carotidea , Espacio Extracelular/metabolismo , Humanos , Imagenología Tridimensional/métodos , Inflamasomas/metabolismo , Lipólisis/fisiología , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Microscopía Electrónica de Transmisión/métodosRESUMEN
Cholesterol is a critical nutrient requiring tight constraint in the endoplasmic reticulum (ER) due to its uniquely challenging biophysical properties. While the mechanisms by which the ER defends against cholesterol insufficiency are well described, it remains unclear how the ER senses and effectively defends against cholesterol excess. Here, we identify the ER-bound transcription factor nuclear factor erythroid 2 related factor-1, Nrf1/Nfe2L1, as a critical mediator of this process. We show that Nrf1 directly binds to and specifically senses cholesterol in the ER through a defined domain and that cholesterol regulates Nrf1 turnover, processing, localization, and activity. In Nrf1 deficiency, in vivo cholesterol challenges induce massive hepatic cholesterol accumulation and damage, which is rescued by replacing Nrf1 exogenously. This Nrf1-mediated mechanism involves the suppression of CD36-driven inflammatory signaling and derepression of liver X receptor activity. These findings reveal Nrf1 as a guardian of cholesterol homeostasis and a core component of adaptive responses to excess cellular cholesterol.
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Colesterol/metabolismo , Retículo Endoplásmico/metabolismo , Hígado/metabolismo , Factor Nuclear 1 de Respiración/metabolismo , Animales , Antígenos CD36/metabolismo , Hígado Graso/metabolismo , Regulación de la Expresión Génica , Homeostasis , Humanos , Hígado/citología , Ratones , Transcripción GenéticaRESUMEN
BACKGROUND AND PURPOSE: Haptoglobin (Hp) is an acute phase plasma protein protecting tissues from oxidative damage. It exists in 2 variant alleles (hp1/hp2) giving rise to 3 protein isoforms with different biochemical properties and efficiency to limit oxidative stress. We previously found that hp2 variant is associated with stroke risk in the patients with carotid stenosis and the risk of ischemic cardiovascular events in a general population cohort. This study examined the hypothesis that Hp genotype is associated with general cardiovascular risk in patients with stroke. METHODS: Hp was genotyped in SAM study (Helsinki Stroke Aging Memory, n=378). A total of 1426 individuals ascertained from a nationally representative cross-sectional health survey served as population controls. RESULTS: Hp genotype frequencies were 15.6% (hp1-1), 44.2% (hp1-2), and 40.2% (hp2-2) in patients with stroke. During a mean of 7.5-year follow-up after first-ever stroke, hp2 carriers had a substantially higher rate of cardiac deaths (24.5% versus 8.5%; P=0.006) and a trend toward more fatal strokes (23.5% versus 13.6%; P=0.122). The combined risk of ischemic cardiovascular deaths was 2.4-fold higher among hp2 carriers (95% confidence interval, 1.28-4.43) after adjustment for major cardiovascular risk factors. CONCLUSIONS: Hp2 allele is associated with premature ischemic cardiovascular deaths after first-ever ischemic stroke.
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Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/mortalidad , Haptoglobinas/genética , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/mortalidad , Anciano , Anciano de 80 o más Años , Estudios Transversales , Femenino , Finlandia/epidemiología , Estudios de Seguimiento , Genotipo , Humanos , Masculino , Persona de Mediana Edad , SobrevivientesRESUMEN
BACKGROUND: Atherosclerosis affects several vascular trees systemically and though surgical plaque removal diminishes the risk of stroke in patients with carotid stenosis, they still face a risk of other atherothrombotic complications like myocardial infarction and premature death. AIMS AND/OR HYPOTHESIS: This study was designed to reveal the long-term risk of death and atherothrombotic events following carotid endarterectomy. METHODS: Eighty-nine previously (1997-2000) endarterectomized carotid patients (56-92 years) were followed up to 15·2 years. Causes of death, cardiovascular events (stroke, transient ischemic attack, acute myocardial infarction), comorbidities, and medications were recorded and analyzed by Cox regression analysis. Four population controls and four controls with coronary disease (n = 712) were selected for each case from a population cohort for age- and gender-matched analysis. RESULTS: At the end of follow-up, 41 (44·6%) patients had died and 48 were alive. Ten patients (24,4%) died due to acute myocardial infarction and one (2,4%) due to stroke. Nineteen (21%) patients had an acute myocardial infarction, 12 (13%) had a stroke, 13 (15%) had a transient ischemic attack, and 5 (6%) had other atherothrombotic events. The risk of death was 5·7-fold in diabetics (P < 0·001) and 3·9-fold in smokers (P < 0·001). Patients who did not use statins had 5·0-fold, and irregular users 3·3-fold risk of death compared with active users (P = 0·005 and P = 0·001, respectively). The major factors associated with acute myocardial infarction were diabetes (6·0-fold risk, P = 0·004), bilateral carotid disease (3·5-fold risk, P = 0·014), and lack of statin use (4·4-fold risk, P = 0·038). Compared with population controls, carotid patients had a 4·4-fold risk of acute myocardial infarction (P = 0·002). CONCLUSIONS: Endarterectomized carotid patients have a high risk of acute myocardial infarction and death, and need an intensified cardiovascular disease-risk-lowering treatment. Although asymptomatic, the evaluation of prognostically significant myocardial ischemia should be considered in these high-risk patients. Eventually, a clinical trial is needed to address whether carotid patients would benefit from early intervention.
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Estenosis Carotídea/complicaciones , Estenosis Carotídea/mortalidad , Enfermedad de la Arteria Coronaria/complicaciones , Enfermedad de la Arteria Coronaria/mortalidad , Anciano , Anciano de 80 o más Años , Estenosis Carotídea/cirugía , Comorbilidad , Endarterectomía Carotidea , Femenino , Finlandia/epidemiología , Estudios de Seguimiento , Hospitales Universitarios , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Infarto del Miocardio/complicaciones , Infarto del Miocardio/mortalidad , Prevalencia , Índice de Severidad de la Enfermedad , Análisis de SupervivenciaRESUMEN
The gelatinase enzymes, matrix metalloproteinases -2 and -9, are central mediators of blood-brain barrier disruption, actively studied in experimental models of neurological disease. Staining with in situ zymography (ISZ) allows visualization of gelatinase activity directly in brain tissue sections. However, quantifying microvascular gelatinase activity from ISZ-images is challenging and time consuming, as surrounding cell types often show significant confounding activity. We describe validation and performance of a workflow for automated image analysis of cerebromicrovascular gelatinase activity, now released for open-access use. In comparison to manual analysis, the automated workflow showed superior accuracy, was faster to execute and allows for more detailed analysis of heterogeneity in the microvasculature. We further suggest recommendations for quantifying and reporting this type of activity in experimental studies, focusing on ischemic stroke.
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Encéfalo/irrigación sanguínea , Gelatinasas/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Inmunohistoquímica/métodos , Infarto de la Arteria Cerebral Media/enzimología , Microscopía Fluorescente/métodos , Microvasos/enzimología , Flujo de Trabajo , Animales , Automatización de Laboratorios , Modelos Animales de Enfermedad , Masculino , Ratas Wistar , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Fatty acid-binding protein 4 (FABP4 or aP2 in mice) has been identified as a key regulator of core aspects of cardiometabolic disorders, including lipotoxic endoplasmic reticulum stress in macrophages. A functional promoter polymorphism (rs77878271) of human FABP4 gene has been described resulting in reduced FABP4 transcription. METHODS AND RESULTS: We investigated the effects of this low-expression variant of FABP4 on cardiovascular morbidity and carotid atherosclerosis on a population level (n=7491) and in patient cohorts representing endarterectomized patients with advanced carotid atherosclerosis (n=92) and myocardial infarction (n=3432). We found that the low-expression variant was associated with decreased total cholesterol levels (P=0.006) with the largest reduction in variant allele homozygotes. Obese variant allele carriers also showed reduced carotid intima-media thickness (P=0.010) and lower prevalence of carotid plaques (P=0.060). Consistently, the variant allele homozygotes showed 8-fold lower odds for myocardial infarction (P=0.019; odds ratio, 0.12; 95% confidence interval, 0.003-0.801). Within the carotid plaques, the variant allele was associated with a 3.8-fold reduction in FABP4 transcription (P=0.049) and 2.7-fold reduction in apoptosis (activated caspase 3; P=0.043). Furthermore, the variant allele was enriched to patients with asymptomatic carotid stenosis (P=0.038). High FABP4 expression in the carotid plaques was associated with lipid accumulation, intraplaque hemorrhages, plaque ulcerations, and phosphoactivated endoplasmic reticulum stress markers. CONCLUSIONS: Our results reveal FABP4 rs77878271 as a novel variant affecting serum total cholesterol levels and cardiovascular risk. A therapeutic regimen reducing FABP4 expression within the atherosclerotic plaque may promote lesion stability through modulation of endoplasmic reticulum stress signaling, and attenuation of apoptosis, lipid burden, and inflammation.
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Proteínas de Unión a Ácidos Grasos/genética , Placa Aterosclerótica/genética , Anciano , Alelos , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/genética , Arterias Carótidas/patología , Endarterectomía , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Ensayo de Inmunoadsorción Enzimática , Femenino , Finlandia , Variación Genética , Genotipo , Homocigoto , Humanos , Inflamación/sangre , Lípidos/sangre , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Placa Aterosclerótica/sangre , Polimorfismo Genético , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Thrombolysis with tissue plasminogen activator (tPA) traditionally demands baseline imaging to rule out intracerebral hemorrhage (ICH), which causes delays in treatment. Preventing possible adverse effects of tPA on ICH would allow rapid on-site thrombolysis in patients with presumed acute ischemic stroke, reducing onset-to-treatment times. We examined how intravenous tPA alters ICH evolution during an extended follow-up, and how mast cell stabilization affects this process. Intracerebral hemorrhage was induced in rats by collagenase injection. Rats received either saline (n=10), tPA (n=13), tPA+low-dose cromoglycate (n=10), or tPA+high-dose cromoglycate (n=10). Magnetic resonance imaging was performed at 24, 48, and 72 hours after ICH induction, together with neurologic evaluations. During 72 hours of follow-up, tPA administration did not significantly increase hematoma volume (mean±s.d. 83.5±14.3 versus 66.7±14.7 µL; P=0.256) or hemispheric expansion (14.5±5.0 versus 11.5±5.0%; P=0.457) compared with saline. However, tPA-treated animals had worse neurologic outcomes (P<0.05), and mortality (8/13 versus 3/10). Combining tPA with high-dose cromoglycate mitigated hemispheric expansion (7.4±1.7 versus 14.5±5.0%; P=0.01), improved neurologic outcome (P<0.001) and decreased mortality (1/10; P<0.05) compared with tPA alone. Our results suggest tPA increases neurologic deficit in ICH, an effect that was abolished by concomitant mast cell stabilization. Further studies are needed to establish the clinical relevance of these findings.
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Hemorragia Cerebral/tratamiento farmacológico , Fibrinolíticos/uso terapéutico , Mastocitos/efectos de los fármacos , Activador de Tejido Plasminógeno/uso terapéutico , Animales , Conducta Animal/efectos de los fármacos , Hemorragia Cerebral/patología , Modelos Animales de Enfermedad , Fibrinolíticos/administración & dosificación , Hematoma/patología , Hematoma/prevención & control , Inyecciones Intravenosas , Estimación de Kaplan-Meier , Imagen por Resonancia Magnética , Masculino , Mastocitos/patología , Microcirculación/fisiología , Ratas , Ratas Sprague-Dawley , Activador de Tejido Plasminógeno/administración & dosificaciónRESUMEN
OBJECTIVE: Intraplaque hemorrhages (IPH) may predispose to unstable atherosclerotic disease and its atherothrombotic complications, ischemic stroke and coronary syndromes. However, the discriminative value of IPH has been limited in histological and imaging studies suggesting that confounding factors modulate the response to IPH. We studied whether common variants of haptoglobin (Hp), which facilitates the removal of free hemoglobin and protects tissues from heme-iron induced oxidative damage, would modify the inflammatory response to IPH and the risk of unstable carotid stenosis (CS) and major cardiovascular diseases. METHODS: We genotyped Hp polymorphism in 91 patients with a high-grade CS from Helsinki Carotid Endarterectomy Study (HeCES) and in 1417 individuals from Health 2000, a Finnish epidemiological cross-sectional health survey, and determined heme oxygenase-1 (HO1) expression in relation to Hp genotypes in carotid plaques. RESULTS: In the Health 2000 cohort, Hp genotype frequencies were 0.143 (hp1-1), 0.486 (hp1-2) and 0.371 (hp2-2) consistent with Hardy-Weinberg equilibrium and those reported from other Caucasian populations. Among patients with unstable CS, the frequency of hp2-2 genotype was higher than in the control population (0.516 vs. 0.371, P = 0.025). Hp genotypes correlated with HO1 expression in the plaque (r = 0.47, P = 0.027). In the Health 2000 cohort, hp2 allele was associated with an increased risk of major cardiovascular diseases (ischemic stroke, TIA, myocardial infarction, coronary heart disease) with an adjusted OR of 1.46 (95% CI 1.03-2.06). CONCLUSION: Common haptoglobin variants modulate the inflammatory response to IPH and associate with the risk of unstable carotid stenosis and major ischemic cardiovascular events.
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Enfermedades Cardiovasculares/genética , Arterias Carótidas/patología , Estenosis Carotídea/genética , Haptoglobinas/genética , Placa Aterosclerótica/genética , Polimorfismo Genético , Anciano , Alelos , Enfermedades Cardiovasculares/sangre , Estenosis Carotídea/sangre , Estudios de Cohortes , Estudios Transversales , Endarterectomía Carotidea , Femenino , Genotipo , Haptoglobinas/metabolismo , Hemo-Oxigenasa 1/metabolismo , Humanos , Inflamación , Masculino , Persona de Mediana Edad , Placa Aterosclerótica/sangre , Riesgo , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/genéticaRESUMEN
BACKGROUND AND PURPOSE: Perivascularly positioned cerebral mast cells (MC) have been shown to participate in acute blood-brain barrier disruption and expansive brain edema following experimental transient cerebral ischemia. However, the underlying molecular mechanisms remain unknown. Because proteolytic gelatinase enzymes, matrix metalloproteinases (MMP)-2 and MMP-9, are thought to have a central role in compromising the integrity of the blood-brain barrier following ischemia, we examined whether cerebral MCs influence gelatinase activity in ischemic cerebral microvasculature. METHODS: Rats underwent 60 minutes of middle cerebral artery occlusion followed by 3-hour reperfusion, and were treated with a MC-stabilizing (cromoglycate), or MC-degranulating (compound 48/80) agent, or vehicle. Genetically manipulated, MC-deficient WsRc(Ws/Ws) rats and their wild-type littermates (WT) underwent the same procedures. Cerebral edema and extravasation of Evans blue albumin were measured. Gelatinase activity was visualized by in situ zymography and was quantified with computerized high-throughput image and data analysis. RESULTS: Activated MCs showed secretion of gelatinase-positive granules. Genetic MC deficiency decreased global gelatinase-active area (-69%, compared with WT; P<0.001) and the mean gelatinase activity of the ischemic microvasculature (-57% compared with WT; P=0.002). MC stabilization with cromoglycate decreased the percentage of microvessels with high gelatinase activity (-36% compared with saline; P<0.05). Compound 48/80 showed increased area of in situ zymography activity in the ischemic lesion (+55% compared with saline; P<0.001). Microvascular gelatinase activity correlated with brain swelling (r=0.84; P<0.001; and r=0.61; P=0.02). CONCLUSIONS: Our data demonstrate that cerebral MCs participate in regulation of acute microvascular gelatinase activation and consequent blood-brain barrier disruption following transient cerebral ischemia.
Asunto(s)
Barrera Hematoencefálica/fisiopatología , Isquemia Encefálica/fisiopatología , Gelatinasas/metabolismo , Mastocitos/enzimología , Accidente Cerebrovascular/fisiopatología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/enzimología , Isquemia Encefálica/enzimología , Corteza Cerebral , Cromolin Sódico/farmacología , Masculino , Mastocitos/efectos de los fármacos , Ratas , Ratas Wistar , Accidente Cerebrovascular/enzimología , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
Atherosclerotic carotid stenosis is an important risk factor for stroke. Carotid plaques (CPs) causing stroke may present a distinct type of molecular pathology compared with transient ischemic attack (TIA)-associated or asymptomatic plaques. We compared the gene expression profiles of CPs from stroke patients (n = 12) and asymptomatic patients (n = 9), both with similar risk factors and severity of carotid stenosis (>70%). Sixty probes showed over 1.5-fold expression difference at 5% false discovery rate. Functional clustering showed enrichment of genes in 51 GO categories and seven pathways, the most significant of which relate to extracellular-matrix interaction, PPAR gamma signaling, scavanger receptor activity, and lysosomal activity. Differential expression of ten genes was confirmed in an extended replication group (n = 43), where the most significant expression differences were found in CD36 (2.1-fold change, p = 0.005), CD163 (1.7-fold change, p = 0.007) and FABP4 (2.2-fold change, p = 0.015). These include four genes not previously linked to plaque destabilization: GLUL (2.2-fold change, p = 0.016), FUCA1 (2.2-fold change, p = 0.025), IL1RN (1.6-fold change, p = 0.034), and S100A8 (2.5-fold change, p = 0.047). Strong correlations were found to plaque ulceration, plaque hemorrhage, and markers of apoptosis and proliferation (activated caspase 3, TUNEL, and Ki67). Protein expression of these genes was confirmed by immunohistochemistry and was found in the atheromatous areas of CPs critical for plaque destabilization. This study presents a comprehensive transcriptional analysis of stroke-associated CPs and demonstrates a significant transcriptome difference between stroke-associated and asymptomatic CPs. Follow-up studies on the identified genes are needed to define whether they could be used as biomarkers of symptomatic CPs or have a role in plaque destabilization.
Asunto(s)
Estenosis Carotídea/complicaciones , Estenosis Carotídea/genética , Perfilación de la Expresión Génica , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/genética , Anciano , Estenosis Carotídea/patología , Análisis por Conglomerados , Sondas de ADN/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reproducibilidad de los Resultados , Accidente Cerebrovascular/patologíaRESUMEN
Paracoccus denitrificans is a soil bacterium which can respire aerobically and also denitrify if oxygen is absent. Both processes are highly dependent on copper enzymes and copper is therefore likely to be an essential trace element for the bacterium. If copper is not easily available, a copper-acquisition mechanism would be highly beneficial. In this paper, we have addressed the question of whether Paracoccus secretes a copper-acquisition compound functionally analogous to that found in some methanotrophs. Bacteria were grown both in copper-containing and copper-deficient denitrification media, cells were removed by centrifugation and the supernatant was analysed using chromatography and spectroscopy. Bacterial growth yield in the absence of copper was 70-80% of that in the copper-containing medium. A notable difference between the two culture conditions was that spent copper-deficient medium was pigmented, whereas the copper-containing medium was not. Spectrophotometry indicated that a red compound with an absorption maximum at 405 nm was produced under copper-limited conditions. In addition to the strong 405 nm maximum, the visible spectrum of the purified red molecule had weaker maxima at 535 nm and 570 nm, features typical of metallated tetrapyrroles. Mass spectrometry showed that the purified pigment had a molecular mass of 716.18. Moreover, the fine structure of the mass spectrum suggested the presence of zinc and was consistent with the chemical formula of C(36)H(36)N(4)O(8)Zn. The presence of zinc was also demonstrated using inductively coupled plasma atomic emission spectroscopy. Fragmentation analysis with mass spectrometry showed the release of consecutive 59 Da fragments, assignable to four -CH(2)-COOH moieties. Thin layer chromatography as well as NMR analysis of the C-13/N-15 labelled red pigment suggested that it is predominantly zinc coproporphyrin III with a minor fraction of metal-free coproporphyrin III. We propose that in a copper-poor environment P. denitrificans secretes coproporphyrin III for copper chelation and subsequent uptake of the bound copper into the cell. Consistent with this idea, cell yields of copper-deficient cultures grown in the presence of 1 microM copper-coproporphyrin III were 90-95% of the yields of cultures grown in the normal copper-containing media. Coproporphyrin III may work as a copper-acquisition compound in P. denitrificans.
Asunto(s)
Cobre/metabolismo , Coproporfirinas/metabolismo , Paracoccus denitrificans/metabolismo , Zinc/metabolismo , Cromatografía en Capa Delgada , Coproporfirinas/aislamiento & purificación , Desnitrificación , Espectroscopía de Resonancia Magnética , Oxígeno/metabolismo , Paracoccus denitrificans/crecimiento & desarrollo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
BACKGROUND: CD36 is a macrophage scavenger receptor mediating the uptake of modified lipoproteins, whereas the ABCA1 transporter counteracts this effect by mediating cellular lipid efflux. Based on a DNA microarray, we previously found that the CD36 and ABCA1 genes were overexpressed in symptom-causing carotid plaques (CP) compared with nonsymptom-causing CP. To evaluate their role in CP destabilization, we conducted detailed immunohistochemical studies on the localization of lipids, CD36 and ABCA1 proteins, extravasated red blood cells, and atheromatous/necrotic tissue. METHODS: Ninety-two high-grade (>70%) stenosing CP obtained from carotid endarterectomy were Oil-red-O-stained for evaluation of neutral lipids. Subgroups of nonsymptom-causing and symptom-causing CP (n=42) were further analyzed by immunostaining adjacent histological sections against CD36 and ABCA1 and examining them microscopically. RESULTS: When compared with nonsymptom-causing CP, the amount of extracellular lipid and the expression of CD36 protein were elevated in symptom-causing CP, but no difference was found in ABCA1 expression. These observations were also confirmed when ulcerated and nonulcerated CP were compared. In ulcerated CP, CD36 protein expression was higher than that of ABCA1, and the opposite was true in nonulcerated CP. CD36 colocalized with extravasated red blood cells and atheromatous or necrotic areas in the various types of CP. CONCLUSIONS: Our results suggest that an imbalance between lipid influx (CD36) and efflux (ABCA1) favors lipid accumulation in macrophages of ulcerated CP, thus contributing to plaque destabilization. Furthermore, colocalization of CD36 protein with red blood cells suggests that intraplaque hemorrhages may contribute to the lipid load and thus the stability of CP.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/sangre , Antígenos CD36/sangre , Estenosis Carotídea/metabolismo , Estenosis Carotídea/fisiopatología , Metabolismo de los Lípidos/fisiología , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/fisiopatología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/análisis , Anciano , Biomarcadores/análisis , Biomarcadores/sangre , Antígenos CD36/análisis , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Arterias Carótidas/fisiopatología , Estenosis Carotídea/complicaciones , Estenosis Carotídea/patología , Eritrocitos/metabolismo , Eritrocitos/patología , Femenino , Humanos , Inmunohistoquímica , Trastornos del Metabolismo de los Lípidos/etiología , Trastornos del Metabolismo de los Lípidos/metabolismo , Trastornos del Metabolismo de los Lípidos/fisiopatología , Masculino , Persona de Mediana Edad , Accidente Cerebrovascular/etiología , Túnica Íntima/metabolismo , Túnica Íntima/patología , Túnica Íntima/fisiopatología , Regulación hacia Arriba/fisiologíaRESUMEN
Apoptotic cell death contributes to neuronal loss in the penumbral region of brain infarction. Activated caspase-3 (ACA-3) cleaves proteins including poly(ADP-ribose) polymerase-1 (PARP-1) important in DNA repair, thus promoting apoptosis. Overactivation of PARP-1 depletes NAD(+) and ATP, resulting in necrosis. These cell death phenomena have been investigated mostly in experimental animals. We studied an autopsy cohort of 13 fatal ischemic stroke cases (symptoms 15 h to 18 days) and 2 controls by immunohistochemical techniques. The number of PARP-1 immunoreactive neurons was highest in the periinfarct area. Nuclear PARP-1 correlated with increasing neuronal necrosis (P = 0.013). Cytoplasmic PARP-1 correlated with TUNEL in periinfarct and core areas (P = 0.01). Cytoplasmic cleaved PARP-1 was inversely correlated with increasing necrotic damage (P = 0.001). PAR-polymers were detected in neurons confirming enzymatic activity of PARP-1. Cytoplasmic ACA-3 correlated with death receptor Fas (r (s) = 0.48; P = 0.005). In conclusion, the confirmation of the same pathways of cell death than previously described in experimental animal models encourages neuroprotective treatments acting on these mediators also in human stroke.