RESUMEN
Cholesterol is a structural component of the cell and is indispensable for normal cellular function, although its excess often leads to abnormal proliferation, migration, inflammatory responses and/or cell death. To prevent cholesterol overload, ATP-binding cassette (ABC) transporters mediate cholesterol efflux from the cells to apolipoprotein A-I (apoA-I) and the apoA-I-containing high-density lipoprotein (HDL). Maintaining efficient cholesterol efflux is essential for normal cellular function. However, the role of cholesterol efflux in angiogenesis and the identity of its local regulators are poorly understood. Here we show that apoA-I binding protein (AIBP) accelerates cholesterol efflux from endothelial cells to HDL and thereby regulates angiogenesis. AIBP- and HDL-mediated cholesterol depletion reduces lipid rafts, interferes with VEGFR2 (also known as KDR) dimerization and signalling and inhibits vascular endothelial growth factor-induced angiogenesis in vitro and mouse aortic neovascularization ex vivo. Notably, Aibp, a zebrafish homologue of human AIBP, regulates the membrane lipid order in embryonic zebrafish vasculature and functions as a non-cell-autonomous regulator of angiogenesis. aibp knockdown results in dysregulated sprouting/branching angiogenesis, whereas forced Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1 (also known as Abca1a) Abcg1-deficient embryos, and cholesterol levels are increased in Aibp-deficient and Abca1 Abcg1-deficient embryos. Our findings demonstrate that secreted AIBP positively regulates cholesterol efflux from endothelial cells and that effective cholesterol efflux is critical for proper angiogenesis.
Asunto(s)
Proteínas Portadoras/metabolismo , Colesterol/metabolismo , Neovascularización Fisiológica/fisiología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico , Vasos Sanguíneos/embriología , Proteínas Portadoras/genética , Colesterol/análisis , Proteínas de Unión al ADN , Embrión no Mamífero/irrigación sanguínea , Embrión no Mamífero/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Lipoproteínas HDL/metabolismo , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Multimerización de Proteína , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/química , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Pez Cebra/embriología , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genéticaRESUMEN
Immunization with homologous malondialdehyde (MDA)-modified LDL (MDA-LDL) leads to atheroprotection in experimental models supporting the concept that a vaccine to oxidation-specific epitopes (OSEs) of oxidized LDL could limit atherogenesis. However, modification of human LDL with OSE to use as an immunogen would be impractical for generalized use. Furthermore, when MDA is used to modify LDL, a wide variety of related MDA adducts are formed, both simple and more complex. To define the relevant epitopes that would reproduce the atheroprotective effects of immunization with MDA-LDL, we sought to determine the responsible immunodominant and atheroprotective adducts. We now demonstrate that fluorescent adducts of MDA involving the condensation of two or more MDA molecules with lysine to form malondialdehyde-acetaldehyde (MAA)-type adducts generate immunodominant epitopes that lead to atheroprotective responses. We further demonstrate that a T helper (Th) 2-biased hapten-specific humoral and cellular response is sufficient, and thus, MAA-modified homologous albumin is an equally effective immunogen. We further show that such Th2-biased humoral responses per se are not atheroprotective if they do not target relevant antigens. These data demonstrate the feasibility of development of a small-molecule immunogen that could stimulate MAA-specific immune responses, which could be used to develop a vaccine approach to retard or prevent atherogenesis.
Asunto(s)
Aterosclerosis , Haptenos , Inmunización , Lipoproteínas LDL , Malondialdehído , Vacunas , Animales , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Aterosclerosis/prevención & control , Haptenos/química , Haptenos/inmunología , Haptenos/farmacología , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/genética , Inmunidad Humoral/efectos de los fármacos , Inmunidad Humoral/genética , Lipoproteínas LDL/química , Lipoproteínas LDL/inmunología , Lipoproteínas LDL/farmacología , Masculino , Malondialdehído/química , Malondialdehído/inmunología , Malondialdehído/farmacología , Ratones , Ratones Noqueados , Células Th2/inmunología , Células Th2/patología , Vacunas/química , Vacunas/inmunología , Vacunas/farmacologíaRESUMEN
Lipoprotein oxidation plays an important role in pathogenesis of atherosclerosis. Oxidized low density lipoprotein (OxLDL) induces profound inflammatory responses in vascular cells, such as production of monocyte chemoattractant protein-1 (MCP-1) [chemokine (C-C motif) ligand 2], a key chemokine in the initiation and progression of vascular inflammation. Here we demonstrate that OxLDL also binds MCP-1 and that the OxLDL-bound MCP-1 retains its ability to recruit monocytes. A human MCP-1 mutant in which basic amino acids Arg-18 and Lys-19 were replaced with Ala did not bind to OxLDL. The MCP-1 binding to OxLDL was inhibited by the monoclonal antibody E06, which binds oxidized phospholipids (OxPLs) in OxLDL. Because OxPLs are carried by lipoprotein(a) [Lp(a)] in human plasma, we tested to determine whether Lp(a) binds MCP-1. Recombinant wild-type but not mutant MCP-1 added to human plasma bound to Lp(a), and its binding was inhibited by E06. Lp(a) captured from human plasma contained MCP-1 and the Lp(a)-associated endogenous MCP-1 induced monocyte migration. These results demonstrate that OxLDL and Lp(a) bind MCP-1 in vitro and in vivo and that OxPLs are major determinants of the MCP-1 binding. The association of MCP-1 with OxLDL and Lp(a) may play a role in modulating monocyte trafficking during atherogenesis.
Asunto(s)
Quimiocina CCL2/metabolismo , Lipoproteína(a)/sangre , Lipoproteína(a)/metabolismo , Lipoproteínas LDL/metabolismo , Animales , Sitios de Unión , Quimiocina CCL2/sangre , Humanos , Lipoproteínas LDL/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Recombinantes/sangre , Proteínas Recombinantes/metabolismoRESUMEN
Oxidation reactions are vital parts of metabolism and signal transduction. However, they also produce reactive oxygen species, which damage lipids, proteins and DNA, generating "oxidation-specific" epitopes. In this review, we discuss the hypothesis that such common oxidation-specific epitopes are a major target of innate immunity, recognized by a variety of "pattern recognition receptors" (PRRs). By analogy with microbial "pathogen-associated molecular patterns" (PAMPs), we postulate that host-derived, oxidation-specific epitopes can be considered to represent "danger (or damage)-associated molecular patterns" (DAMPs). We also argue that oxidation-specific epitopes present on apoptotic cells and their cellular debris provided the primary evolutionary pressure for the selection of such PRRs. Furthermore, because many PAMPs on microbes share molecular identity and/or mimicry with oxidation-specific epitopes, such PAMPs provide a strong secondary selecting pressure for the same set of oxidation-specific PRRs as well. Because lipid peroxidation is ubiquitous and a major component of the inflammatory state associated with atherosclerosis, the understanding that oxidation-specific epitopes are DAMPs, and thus the target of multiple arcs of innate immunity, provides novel insights into the pathogenesis of atherosclerosis. As examples, we show that both cellular and soluble PRRs, such as CD36, toll-like receptor-4, natural antibodies, and C-reactive protein recognize common oxidation-specific DAMPs, such as oxidized phospholipids and oxidized cholesteryl esters, and mediate a variety of immune responses, from expression of proinflammatory genes to excessive intracellular lipoprotein accumulation to atheroprotective humoral immunity. These insights may lead to improved understanding of inflammation and atherogenesis and suggest new approaches to diagnosis and therapy.
Asunto(s)
Epítopos/fisiología , Inmunidad Innata/fisiología , Receptores de Reconocimiento de Patrones/fisiología , Animales , Aterosclerosis/fisiopatología , Humanos , Oxidación-Reducción , Fosforilación OxidativaRESUMEN
Background The success of cardiac auscultation varies widely among medical professionals, which can lead to missed treatments for structural heart disease. Applying machine learning to cardiac auscultation could address this problem, but despite recent interest, few algorithms have been brought to clinical practice. We evaluated a novel suite of Food and Drug Administration-cleared algorithms trained via deep learning on >15 000 heart sound recordings. Methods and Results We validated the algorithms on a data set of 2375 recordings from 615 unique subjects. This data set was collected in real clinical environments using commercially available digital stethoscopes, annotated by board-certified cardiologists, and paired with echocardiograms as the gold standard. To model the algorithm in clinical practice, we compared its performance against 10 clinicians on a subset of the validation database. Our algorithm reliably detected structural murmurs with a sensitivity of 85.6% and specificity of 84.4%. When limiting the analysis to clearly audible murmurs in adults, performance improved to a sensitivity of 97.9% and specificity of 90.6%. The algorithm also reported timing within the cardiac cycle, differentiating between systolic and diastolic murmurs. Despite optimizing acoustics for the clinicians, the algorithm substantially outperformed the clinicians (average clinician accuracy, 77.9%; algorithm accuracy, 84.7%.) Conclusions The algorithms accurately identified murmurs associated with structural heart disease. Our results illustrate a marked contrast between the consistency of the algorithm and the substantial interobserver variability of clinicians. Our results suggest that adopting machine learning algorithms into clinical practice could improve the detection of structural heart disease to facilitate patient care.
Asunto(s)
Aprendizaje Profundo , Cardiopatías , Adulto , Humanos , Soplos Cardíacos/diagnóstico , Cardiopatías/diagnóstico por imagen , Auscultación Cardíaca , AlgoritmosRESUMEN
RATIONALE: Oxidized low-density lipoprotein (LDL) is an important determinant of inflammation in atherosclerotic lesions. It has also been documented that certain chronic infectious diseases, such as periodontitis and chlamydial infection, exacerbate clinical manifestations of atherosclerosis. In addition, low-level but persistent metabolic endotoxemia is often found in diabetic and obese subjects and is induced in mice fed a high-fat diet. OBJECTIVE: In this study, we examined cooperative macrophage activation by low levels of bacterial lipopolysaccharide (LPS) and by minimally oxidized LDL (mmLDL), as a model for subclinical endotoxemia-complicated atherosclerosis. METHODS AND RESULTS: We found that both in vitro and in vivo, mmLDL and LPS (Kdo2-LipidA) cooperatively activated macrophages to express proinflammatory cytokines Cxcl2 (MIP-2), Ccl3 (MIP-1alpha), and Ccl4 (MIP-1beta). Importantly, the mmLDL and LPS cooperative effects were evident at a threshold LPS concentration (1 ng/mL) at which LPS alone induced only a limited macrophage response. Analyzing microarray data with a de novo motif discovery algorithm, we found that genes transcribed by promoters containing an activator protein (AP)-1 binding site were significantly upregulated by costimulation with mmLDL and LPS. In a nuclear factor-DNA binding assay, the cooperative effect of mmLDL and LPS costimulation on c-Jun and c-Fos DNA binding, but not on p65 or p50, was dependent on mmLDL-induced activation of extracellular signal-regulated kinase (ERK) 1/2. In addition, mmLDL induced c-Jun N-terminal kinase (JNK)-dependent derepression of AP-1 by removing nuclear receptor corepressor (NCoR) from the chemokine promoters. CONCLUSIONS: The cooperative engagement of AP-1 and nuclear factor (NF)-kappaB by mmLDL and LPS may constitute a mechanism of increased transcription of inflammatory cytokines within atherosclerotic lesions.
Asunto(s)
Aterosclerosis/metabolismo , Endotoxemia/metabolismo , Mediadores de Inflamación/administración & dosificación , Lipopolisacáridos/administración & dosificación , Lipoproteínas LDL/administración & dosificación , Activación de Macrófagos/fisiología , FN-kappa B/fisiología , Factor de Transcripción AP-1/fisiología , Animales , Aterosclerosis/etiología , Aterosclerosis/patología , Línea Celular , Progresión de la Enfermedad , Esquema de Medicación , Sinergismo Farmacológico , Endotoxemia/etiología , Endotoxemia/patología , Ratones , Ratones Endogámicos C57BLRESUMEN
A novel hypercholesterolemic zebrafish model has been developed to study early events of atherogenesis. This model utilizes optically transparent zebrafish larvae, fed a high cholesterol diet (HCD), to monitor processes of vascular inflammation in live animals. Because lipoprotein oxidation is an important factor in the development of atherosclerosis, in this study, we characterized the oxidized lipid milieu in HCD-fed zebrafish larvae. Using liquid chromatography-mass spectrometry, we show that feeding an HCD for only 2 weeks resulted in up to 70-fold increases in specific oxidized cholesteryl esters, identical to those present in human minimally oxidized LDL and in murine atherosclerotic lesions. The levels of oxidized phospholipids, such as 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine, and of various lysophosphatidylcholines were also significantly elevated. Moreover, lipoproteins isolated from homogenates of HCD-fed larvae induced cell spreading as well as ERK1/2, Akt, and JNK phosphorylation in murine macrophages. Removal of apoB-containing lipoproteins from the zebrafish homogenates with an anti-human LDL antibody, as well as reducing lipid hydroperoxides with ebselen, resulted in inhibition of macrophage activation. The TLR4 deficiency in murine macrophages prevented their activation with zebrafish lipoproteins. Using biotinylated homogenates of HCD-fed larvae, we demonstrated that their components bound to murine macrophages, and this binding was effectively competed by minimally oxidized LDL but not by native LDL. These data provide evidence that molecular lipid determinants of proatherogenic macrophage phenotypes are present in large quantities in hypercholesterolemic zebrafish larvae and support the use of the HCD-fed zebrafish as a valuable model to study early events of atherogenesis.
Asunto(s)
Ésteres del Colesterol/metabolismo , Colesterol en la Dieta/metabolismo , Larva/metabolismo , Macrófagos/metabolismo , Fosfolípidos/metabolismo , Pez Cebra , Animales , Línea Celular , Ésteres del Colesterol/química , Dieta , Humanos , Macrófagos/citología , Ratones , Oxidación-Reducción , Fosfolípidos/químicaRESUMEN
BACKGROUND: Percutaneous coronary intervention (PCI) outcomes for patients with significant calcification have been consistently inferior compared to patients without significant calcification. Procedural success and long-term outcomes after PCI have been worse in patients with severe coronary calcium. OBJECTIVE: A Bayesian meta-analysis of outcomes comparing rotational atherectomy (RA) with orbital atherectomy (OA) was performed. METHODS: PubMed, Embase, and Cochrane Library databases were searched through 30th November 2018 and identified 4 observational studies. RESULTS: The primary end-point, Major Adverse Cardiac Event (MACE) composing of death, MI and stroke at 1â¯year was more likely with RA (ORâ¯=â¯1.61; 95% CI: 1.11-2.33; pâ¯=â¯0.01) as compared to OA. The driver of the difference in MACE between the two groups was a statistically significant difference in mortality favoring OA (ORâ¯=â¯4.65; 95% CI: 1.36-15.87; pâ¯=â¯0.01). Peri-procedural MI, the other component of the primary end-point was 1.3 times more likely in the RA arm (ORâ¯=â¯1.35; 95% CI 0.95-1.92; p-0.09) and was not statistically different between the groups. The odds of a vascular complication were not different in the two groups (ORâ¯=â¯1.26; 95% CI: 0.73-2.17; pâ¯=â¯0.41). In an adjusted Bayesian analysis, mortality (ORâ¯=â¯3.69; 95% CI: 0.30-38.51), MACE (ORâ¯=â¯1.68; 95% CI: 0.55-5.49), MI (ORâ¯=â¯1.42; 95% CI: 0.50-4.29) and dissections/perforations (ORâ¯=â¯0.38; 95% CI: 0.10-1.38) were not different in RA and OA groups. CONCLUSION: Our study is the first published Bayesian meta-analysis comparing MACE and peri-procedural outcomes in RA compared to OA. These findings lay the foundation for a randomized comparison between the two competing technologies.
Asunto(s)
Aterectomía Coronaria , Enfermedad de la Arteria Coronaria/terapia , Intervención Coronaria Percutánea , Calcificación Vascular/terapia , Aterectomía Coronaria/efectos adversos , Aterectomía Coronaria/mortalidad , Teorema de Bayes , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Enfermedad de la Arteria Coronaria/mortalidad , Enfermedad de la Arteria Coronaria/fisiopatología , Humanos , Intervención Coronaria Percutánea/efectos adversos , Intervención Coronaria Percutánea/mortalidad , Factores de Riesgo , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Calcificación Vascular/diagnóstico por imagen , Calcificación Vascular/mortalidad , Calcificación Vascular/fisiopatologíaRESUMEN
BACKGROUND: Transcatheter left atrial appendage occlusion (LAAO) has become a suitable alternative to anticoagulation in patients with atrial fibrillation (AF). However, volume-outcome relationships at the individual operator level have not been studied. METHODS: Study population included 425 consecutive patients with AF undergoing LAAO from August 2015 to November 2018 by seven operators at BUMC-Phoenix. Operator volume was divided in tertiles by those with <40 cases/year (2 operators), 41-80 cases/year (3 operators) and >80 cases/year (2 operators). Patient data including comorbidities, labs, medications, procedural characteristics and outcomes were collected. The primary composite outcome was major adverse cardiac events (MACE) including mortality, stroke, bleeding and vascular complications. RESULTS: Mean age was 75⯱â¯8â¯years and 251 (59%) were males. Mean CHA2DS2-VASc score was 4.5⯱â¯1.3 points and mean HASBLED score was 3.9⯱â¯1.0 points. MACE outcome was similar in the three operator groups in both unadjusted (pâ¯=â¯0.83) and adjusted (ORâ¯=â¯0.59: 95% Confidence Interval [CI]: 0.15-2.29, pâ¯=â¯0.45) analysis. The occurrence MACE was also similar between Interventional Cardiologist (IC) and Electrophysiologist (EP) operators in an unadjusted (pâ¯=â¯0.24) and adjusted (ORâ¯=â¯0.60: 95% CI: 0.21-1.68, pâ¯=â¯0.33) analysis. The secondary outcome of technical success did not differ among the three tertiles (pâ¯=â¯0.37) and among IC & EP operators respectively (pâ¯=â¯0.24) as well. CONCLUSION: Operator experience does not affect MACE and technical success even after adjusting for comorbidities. These results suggest a lower learning curve for LAAO with high technical success achievable even by low volume operators.
Asunto(s)
Apéndice Atrial/fisiopatología , Fibrilación Atrial/terapia , Función del Atrio Izquierdo , Cateterismo Cardíaco/instrumentación , Frecuencia Cardíaca , Accidente Cerebrovascular/prevención & control , Anciano , Anciano de 80 o más Años , Fibrilación Atrial/diagnóstico , Fibrilación Atrial/mortalidad , Fibrilación Atrial/fisiopatología , Cateterismo Cardíaco/efectos adversos , Cateterismo Cardíaco/mortalidad , Competencia Clínica , Femenino , Humanos , Curva de Aprendizaje , Masculino , Diseño de Prótesis , Sistema de Registros , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/mortalidad , Factores de Tiempo , Resultado del TratamientoRESUMEN
Caveolae are specialized membrane microdomains formed as the result of local accumulation of cholesterol, glycosphingolipids, and the structural protein caveolin-1 (Cav-1). To further elucidate the role of Cav-1 in lipid homeostasis in-vivo, we analyzed fasting and post-prandial plasma from Cav-1 deficient mice on low or on high fat diet. In total plasma analysis, an increase in ceramide and hexosylceramide was observed. In cholesteryl ester (CE), we found an increased saturated+monounsaturated/polyunsaturated fatty acid ratio in fasting plasma of low fat fed Cav-1(-/-) mice with increased proportions of CE16:1, CE18:1, CE20:3, and decreased proportions of CE18:2 and CE22:6. Under high fat diet HDL-CE, free cholesterol and pre-beta-HDL were increased accompanied by a shift from slow to fast migrating alpha-HDL and expansion of apoE containing HDL. Our results demonstrate a significant role of Cav-1 in HDL-cholesterol metabolism and may reflect a variety of Cav-1 functions including modulation of ACAT activity and SR-BI function.
Asunto(s)
Caveolina 1/metabolismo , Grasas de la Dieta/metabolismo , Ayuno/metabolismo , Metabolismo de los Lípidos/fisiología , Lípidos/sangre , Lipoproteínas/sangre , Periodo Posprandial/fisiología , Animales , Caveolina 1/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Elevated cholesterol levels are clearly independently associated with adverse cardiovascular events. Another class of lipid particles, triglycerides, is also abundant in the human body and has been found in atherosclerotic plaques. Recent observational studies have demonstrated an association between elevated triglyceride levels and increased risk for future cardiovascular events. With this knowledge and the discovery of effective agents to lower triglyceride levels, the management of triglycerides is currently undergoing a renaissance. Unfortunately, no randomized, controlled clinical trials have been completed to date, proving that lowering triglycerides will reduce cardiovascular events. In this review we highlight some of the evidence that led to this stage and discuss the current data on pharmacologic intervention of triglyceride levels and the effect on clinical outcomes. Lastly, we want to give the reader insight on what the most recent lipid guidelines state about clinical triglyceride management, mention new pharmacological agents, and highlight the clinical evidence for safe and effective lowering of triglycerides levels with life style modification.
Asunto(s)
Enfermedades Cardiovasculares/prevención & control , Dislipidemias/sangre , Dislipidemias/tratamiento farmacológico , Hipolipemiantes/uso terapéutico , Triglicéridos/sangre , Biomarcadores/sangre , Enfermedades Cardiovasculares/etiología , Dislipidemias/complicaciones , Humanos , Medición de Riesgo , Factores de Riesgo , Conducta de Reducción del Riesgo , Resultado del TratamientoRESUMEN
OBJECTIVE: We report the clinical outcomes of the "single-operator" technique, whereby the operator both advances the rotational atherectomy (RA) device and keeps the distal wire in place. BACKGROUND: Severely calcified lesions are associated with increased ischemic complications during percutaneous coronary intervention. RA, which utilizes a differential cutting mechanism of action for plaque modification, is a valuable treatment option for patients with severely calcified vessels prior to stent implantation. Reasons that may explain the underutilization include lack of operator experience and the availability of a skilled assistant to maintain wire position while the operator advances the device to the lesion. Loss of wire position can lead to increased procedural and fluoroscopic times. METHODS: In a prospective single-center study, a total of 67 consecutive patients underwent RA from July 2012 to June 2015. The primary endpoint was successful delivery of the RA device to the lesion without losing wire position with procedural success. RESULTS: The primary endpoint was met in 100% of the patients. The 30-day major adverse cardiac and cerebrovascular event rate was 6.0%, all due to non-fatal myocardial infarction. There was no cardiac death, target lesion revascularization, stroke, stent thrombosis, perforation, or flow-limiting dissection. Five patients had slow flow, but resolved with intracoronary vasodilator therapy and achieved TIMI grade 3 flow. CONCLUSION: RA can be performed successfully without a skilled assistant to maintain wire position during advancement of the burr, and the absence of an assistant should therefore not eliminate the performance of RA.
Asunto(s)
Aterectomía Coronaria/instrumentación , Enfermedad de la Arteria Coronaria/cirugía , Intervención Coronaria Percutánea/instrumentación , Angiografía Coronaria , Enfermedad de la Arteria Coronaria/diagnóstico , Diseño de Equipo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Factores de Tiempo , Resultado del TratamientoAsunto(s)
Oclusión Coronaria/terapia , Alta del Paciente , Intervención Coronaria Percutánea , Anciano , Arizona , Enfermedad Crónica , Oclusión Coronaria/diagnóstico por imagen , Oclusión Coronaria/mortalidad , Estudios de Factibilidad , Femenino , Humanos , Tiempo de Internación , Masculino , Persona de Mediana Edad , Intervención Coronaria Percutánea/efectos adversos , Intervención Coronaria Percutánea/mortalidad , Estudios Retrospectivos , Factores de Riesgo , Factores de Tiempo , Resultado del TratamientoRESUMEN
Oxidative modification of low-density lipoprotein (LDL) turns it into an endogenous ligand recognized by pattern-recognition receptors. We have demonstrated that minimally oxidized LDL (mmLDL) binds to CD14 and mediates TLR4/MD-2-dependent responses in macrophages, many of which are MyD88-independent. We have also demonstrated that the mmLDL activation leads to recruitment of spleen tyrosine kinase (Syk) to TLR4 and TLR4 and Syk phosphorylation. In this study, we produced a macrophage-specific Syk knockout mouse and used primary Syk(-/-) macrophages in our studies. We demonstrated that Syk mediated phosphorylation of ERK1/2 and JNK, which in turn phosphorylated c-Fos and c-Jun, respectively, as assessed by an in vitro kinase assay. c-Jun phosphorylation was also mediated by IKKε. c-Jun and c-Fos bound to consensus DNA sites and thereby completed an AP-1 transcriptional complex and induced expression of CXCL2 and IL-6. These results suggest that Syk plays a key role in TLR4-mediated macrophage responses to host-generated ligands, like mmLDL, with subsequent activation of an AP-1 transcription program.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/fisiología , Receptores de Lipopolisacáridos/metabolismo , Lipoproteínas LDL/metabolismo , Proteínas Tirosina Quinasas/fisiología , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Animales , Células de la Médula Ósea/citología , Células CHO , Quimiocina CXCL2/metabolismo , Cricetinae , Humanos , Quinasa I-kappa B/metabolismo , Interleucina-6/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Quinasa Syk , Receptor Toll-Like 4/metabolismoRESUMEN
Spleen tyrosine kinase (SYK) is the best known for its involvement in immune receptor signalling, mediated by binding of SYK tandem Src-homology 2 domains to tandem phosphotyrosine in immunoreceptor tyrosine-based activation motifs (ITAMs). ITAM adaptors or ITAM-containing receptor tails mediate signalling from B- and T-cell receptors, Fc receptors and many C-type lectins, including dectin-1. Recent data point to constitutive binding of SYK to the cytoplasmic domain of toll-like receptor-4 (TLR4). This SYK-TLR4 binding increases upon TLR4 dimerization and phosphorylation, and SYK plays a prominent role in TLR4 signalling in response to LPS in neutrophils and monocytes. SYK also plays an important role in TLR4-mediated macrophage responses to minimally oxidized low-density lipoprotein (mmLDL), which is a form of oxidized LDL relevant to development of human atherosclerosis. Interestingly, mmLDL-induced effects in macrophages, which occur via TLR4, are predominantly MyD88 independent. This unmasks the role of the SYK branch of TLR4 signalling, which mediates modest cytokine release via activation of AP-1 transcription and robust reactive oxygen species generation and cytoskeletal rearrangements. The latter results in extensive membrane ruffling and macropinocytosis, leading to lipoprotein uptake and foam cell formation, a hallmark of atherosclerotic lesions. Because inhibitors of SYK activity, such as fostamatinib, are in advanced clinical trials for rheumatoid arthritis and other autoimmune diseases, understanding the role of SYK in signalling via TLR4 is of immediate importance. This signalling pathway seems to be particularly important in TLR4 activation by host-derived, damage-associated molecular pattern ligands, such as mmLDL, relevant to development of atherosclerosis and other chronic inflammatory diseases.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/inmunología , Lipoproteínas LDL/inmunología , Proteínas Tirosina Quinasas/inmunología , Receptor Toll-Like 4/inmunología , Animales , Humanos , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Transducción de Señal , Quinasa SykRESUMEN
OBJECTIVES: This study sought to evaluate the in vivo magnetic resonance imaging (MRI) efficacy of manganese [Mn(II)] molecular imaging probes targeted to oxidation-specific epitopes (OSE). BACKGROUND: OSE are critical in the initiation, progression, and destabilization of atherosclerotic plaques. Gadolinium [Gd(III)]-based MRI agents can be associated with systemic toxicity. Mn is an endogenous, biocompatible, paramagnetic metal ion that has poor MR efficacy when chelated, but strong efficacy when released within cells. METHODS: Multimodal Mn micelles were generated to contain rhodamine for confocal microscopy and conjugated with either the murine monoclonal IgG antibody MDA2 targeted to malondialdehyde (MDA)-lysine epitopes or the human single-chain Fv antibody fragment IK17 targeted to MDA-like epitopes ("targeted micelles"). Micelle formulations were characterized in vitro and in vivo, and their MR efficacy (9.4-T) evaluated in apolipoprotein-deficient (apoE(-/-)) and low-density lipoprotein receptor negative (LDLR(-/-)) mice (0.05 mmol Mn/kg dose) (total of 120 mice for all experiments). In vivo competitive inhibition studies were performed to evaluate target specificity. Untargeted, MDA2-Gd, and IK17-Gd micelles (0.075 mmol Gd/kg) were included as controls. RESULTS: In vitro studies demonstrated that targeted Mn micelles accumulate in macrophages when pre-exposed to MDA-LDL with â¼10× increase in longitudinal relativity. Following intravenous injection, strong MR signal enhancement was observed 48 to 72 h after administration of targeted Mn micelles, with colocalization within intraplaque macrophages. Co-injection of free MDA2 with the MDA2-Mn micelles resulted in full suppression of MR signal in the arterial wall, confirming target specificity. Similar MR efficacy was noted in apoE(-/-) and LDLR(-/-) mice with aortic atherosclerosis. No significant differences in MR efficacy were noted between targeted Mn and Gd micelles. CONCLUSIONS: This study demonstrates that biocompatible multimodal Mn-based molecular imaging probes detect OSE within atherosclerotic plaques and may facilitate clinical translation of noninvasive imaging of human atherosclerosis.
Asunto(s)
Materiales Biocompatibles , Lipoproteínas/química , Espectroscopía de Resonancia Magnética/métodos , Manganeso , Placa Aterosclerótica/metabolismo , Animales , Modelos Animales de Enfermedad , Epítopos , Ratones , Ratones Noqueados , Micelas , Oxidación-Reducción , Placa Aterosclerótica/diagnóstico , Reproducibilidad de los ResultadosRESUMEN
Glycerophospholipid and sphingolipid species and their bioactive metabolites are important regulators of lipoprotein and cell function. The aim of the study was to develop a method for lipid species profiling of separated lipoprotein classes. Human serum lipoproteins VLDL, LDL, and HDL of 21 healthy fasting blood donors were separated by fast performance liquid chromatography (FPLC) from 50 microl serum. Subsequently, phosphatidylcholine (PC), lysophosphatidylcholine, sphingomyelin (SM), ceramide (CER), phosphatidylethanolamine (PE), PE-based plasmalogen (PE-pl), cholesterol, and cholesteryl ester (CE) content of the separated lipoproteins was quantified by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Analysis of FPLC fractions with PAGE demonstrated that albumin partially coelutes with HDL fractions. However, analysis of an HDL deficient serum (Tangier disease) showed that only lysophosphatidylcholine, but none of the other lipids analyzed, exhibited a significant coelution with the albumin containing fractions. Approximately 60% of lipoprotein CER were found in LDL fractions and 60% of PC, PE, and plasmalogens in HDL fractions. VLDL, LDL, and HDL displayed characteristic lipid class and species pattern. The developed method provides a detailed lipid class and species composition of lipoprotein fractions and may serve as a valuable tool to identify alterations of lipoprotein lipid species profiles in disease with a reasonable experimental effort.