RESUMEN
Introduction: The CC chemokine ligand 18 (CCL18) is a chemokine highly expressed in chronic inflammation in humans. Recent observations of elevated CCL18 plasma levels in patients with acute cardiovascular syndromes prompted an investigation into the role of CCL18 in the pathogenesis of human and mouse atherosclerosis. Methods and results: CCL18 was profoundly upregulated in ruptured human atherosclerotic plaque, particularly within macrophages. Repeated administration of CCL18 in Western-type diet-fed ApoE -/- mice or PCSK9mut-overexpressing wild type (WT) mice led to increased plaque burden, enriched in CD3+ T cells. In subsequent experimental and molecular modeling studies, we identified CCR6 as a functional receptor mediating CCL18 chemotaxis, intracellular Ca2+ flux, and downstream signaling in human Jurkat and mouse T cells. CCL18 failed to induce these effects in vitro in murine spleen T cells with CCR6 deficiency. The ability of CCR6 to act as CCL18 receptor was confirmed in vivo in an inflammation model, where subcutaneous CCL18 injection induced profound focal skin inflammation in WT but not in CCR6-/- mice. This inflammation featured edema and marked infiltration of various leukocyte subsets, including T cells with a Th17 signature, supporting CCR6's role as a Th17 chemotactic receptor. Notably, focal overexpression of CCL18 in plaques was associated with an increased presence of CCR6+ (T) cells. Discussion: Our studies are the first to identify the CCL18/CCR6 axis as a regulator of immune responses in advanced murine and human atherosclerosis.
Asunto(s)
Aterosclerosis , Quimiocinas CC , Receptores CCR6 , Animales , Humanos , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Ratones , Receptores CCR6/metabolismo , Receptores CCR6/genética , Quimiocinas CC/metabolismo , Quimiocinas CC/genética , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Células Jurkat , Placa Aterosclerótica/inmunología , Ratones Noqueados , Masculino , Linfocitos T/inmunología , Linfocitos T/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Femenino , Ratones Noqueados para ApoERESUMEN
Hyperlipidemia is a major risk factor for the development of atherosclerotic cardiovascular disease. Lipid-lowering drug therapies therefore still form the heart of the ongoing battle against the occurrence of cardiovascular events. However, in light of the important improvements in gene interference and editing that have been made during the last 2 decades, gene therapy-the genetic modification of cells to produce a permanent therapeutic effect-is currently employed to relief hypercholesterolemic subjects from their potential (chronic) cardiovascular disease burden. In this perspective, we review the current status regarding hepatocyte-directed base editing to treat human dyslipidemia and provide suggestions for further technological improvement.
Asunto(s)
Enfermedades Cardiovasculares , Dislipidemias , Humanos , Enfermedades Cardiovasculares/terapia , Enfermedades Cardiovasculares/tratamiento farmacológico , Edición Génica , Dislipidemias/tratamiento farmacológico , Dislipidemias/genética , Hipolipemiantes/uso terapéutico , HepatocitosRESUMEN
AIMS: (Ultra) Small superparamagnetic iron oxide nanoparticles, (U)SPIO, are widely used as magnetic resonance imaging contrast media and assumed to be safe for clinical applications in cardiovascular disease. As safety tests largely relied on normolipidaemic models, not fully representative of the clinical setting, we investigated the impact of (U)SPIOs on disease-relevant endpoints in hyperlipidaemic models of atherosclerosis. METHODS AND RESULTS: RAW264.7 foam cells, exposed in vitro to ferumoxide (dextran-coated SPIO), ferumoxtran (dextran-coated USPIO), or ferumoxytol [carboxymethyl (CM) dextran-coated USPIO] (all 1 mg Fe/mL) showed increased apoptosis and reactive oxygen species accumulation for ferumoxide and ferumoxtran, whereas ferumoxytol was tolerated well. Pro-apoptotic (TUNEL+) and pro-oxidant activity of ferumoxide (0.3 mg Fe/kg) and ferumoxtran (1 mg Fe/kg) were confirmed in plaque, spleen, and liver of hyperlipidaemic ApoE-/- (n = 9/group) and LDLR-/- (n = 9-16/group) mice that had received single IV injections compared with saline-treated controls. Again, ferumoxytol treatment (1 mg Fe/kg) failed to induce apoptosis or oxidative stress in these tissues. Concomitant antioxidant treatment (EUK-8/EUK-134) largely prevented these effects in vitro (-68%, P < 0.05) and in plaques from LDLR-/- mice (-60%, P < 0.001, n = 8/group). Repeated ferumoxtran injections of LDLR-/- mice with pre-existing atherosclerosis enhanced plaque inflammation and apoptosis but did not alter plaque size. Strikingly, carotid artery plaques of endarterectomy patients who received ferumoxtran (2.6 mg Fe/kg) before surgery (n = 9) also showed five-fold increased apoptosis (18.2 vs. 3.7%, respectively; P = 0.004) compared with controls who did not receive ferumoxtran. Mechanistically, neither coating nor particle size seemed accountable for the observed cytotoxicity of ferumoxide and ferumoxtran. CONCLUSIONS: Ferumoxide and ferumoxtran, but not ferumoxytol, induced apoptosis of lipid-laden macrophages in human and murine atherosclerosis, potentially impacting disease progression in patients with advanced atherosclerosis.
Asunto(s)
Aterosclerosis , Placa Aterosclerótica , Humanos , Ratones , Animales , Medios de Contraste , Dextranos/farmacología , Células Espumosas/patología , Aterosclerosis/diagnóstico por imagen , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/patología , Óxido Ferrosoférrico/farmacología , Imagen por Resonancia Magnética/métodos , Macrófagos/patología , Apoptosis , Óxidos/farmacologíaRESUMEN
While the promise of oligonucleotide therapeutics, such as (chemically modified) ASO (antisense oligonucleotides) and short interfering RNAs, is undisputed from their introduction onwards, their unfavorable pharmacokinetics and intrinsic capacity to mobilize innate immune responses, were limiting widespread clinical use. However, these major setbacks have been tackled by breakthroughs in chemistry, stability and delivery. When aiming an intervention hepatic targets, such as lipid and sugar metabolism, coagulation, not to mention cancer and virus infection, introduction of N-acetylgalactosamine aided targeting technology has advanced the field profoundly and by now a dozen of N-acetylgalactosamine therapeutics for these indications have been approved for clinical use or have progressed to clinical trial stage 2 to 3 testing. This technology, in combination with major advances in oligonucleotide stability allows safe and durable intervention in targets that were previously deemed undruggable, such as Lp(a) and PCSK9 (proprotein convertase subtilisin/kexin type 9), at high efficacy and specificity, often with as little as 2 doses per year. Their successful use even the most visionary would not have predicted 2 decades ago. Here, we will review the evolution of N-acetylgalactosamine technology. We shall outline their fundamental design principles and merits, and their application for the delivery of oligonucleotide therapeutics to the liver. Finally, we will discuss the perspectives of N-acetylgalactosamine technology and propose directions for future research in receptor targeted delivery of these gene medicines.
Asunto(s)
Acetilgalactosamina/química , Enfermedades Cardiovasculares/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Terapia Genética/métodos , Hígado/efectos de los fármacos , Oligonucleótidos/administración & dosificación , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Tratamiento con ARN de InterferenciaRESUMEN
BACKGROUND AND AIMS: There is extensive evidence from bone marrow transplantation studies that hematopoietic ATP binding cassette A1 (Abca1) is atheroprotective in low-density lipoprotein receptor (Ldlr) deficient mice. In contrast, studies using lysosyme M promoter-driven deletion of Abca1 in Ldlr deficient mice failed to show similar effects. It was hypothesized that the discrepancy between these studies might be due to the presence of Ldlr in bone marrow-derived cells in the transplantation model. In this study, we aim to determine the contribution of Ldlr to the atheroprotective effect of hematopoietic Abca1 in the murine bone marrow transplantation model. METHODS: Wild-type, Ldlr-/-, Abca1-/-, and Abca1-/-Ldlr-/- bone marrow was transplanted into hypercholesterolemic Ldlr-/- mice. RESULTS: Bone marrow Lldr deficiency did not influence the effects of Abca1 on macrophage cholesterol efflux, foam cell formation, monocytosis or plasma cholesterol. Ldlr deficiency did reduce circulating and peritoneal lymphocyte counts, albeit only in animals lacking Abca1 in bone marrow-derived cells. Importantly, the effects of Abca1 deficiency on atherosclerosis susceptibility were unaltered by the presence or absence of Ldlr. Bone marrow Ldlr deficiency did lead to marginally but consistently decreased atherosclerosis, regardless of Abca1 deficiency. Thus, Ldlr expression on bone marrow-derived cells does, to a minimal extent, influence atherosclerotic lesion development, albeit independent of Abca1. CONCLUSIONS: This study provides novel insight into the relative impact of Ldlr and Abca1 in bone marrow-derived cells on macrophage foam cell formation and atherosclerosis development in vivo. We have shown that Ldlr and Abca1 differentially and independently influence atherosclerosis development in a murine bone marrow transplantation model of atherosclerosis.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/genética , Aterosclerosis , Médula Ósea , Transportadoras de Casetes de Unión a ATP/genética , Animales , Aterosclerosis/genética , Médula Ósea/metabolismo , Colesterol , Lipoproteínas LDL/metabolismo , Ratones , Ratones Noqueados , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMEN
OBJECTIVE: We tested the hypothesis that enlarged, dysfunctional HDL (high-density lipoprotein) particles contribute to the augmented atherosclerosis susceptibility associated with SR-BI (scavenger receptor BI) deficiency in mice. Approach and Results: We eliminated the ability of HDL particles to fully mature by targeting PLTP (phospholipid transfer protein) functionality. Particle size of the HDL population was almost fully normalized in male and female SR-BI×PLTP double knockout mice. In contrast, the plasma unesterified cholesterol to cholesteryl ester ratio remained elevated. The PLTP deficiency-induced reduction in HDL size in SR-BI knockout mice resulted in a normalized aortic tissue oxidative stress status on Western-type diet. Atherosclerosis susceptibility was-however-only partially reversed in double knockout mice, which can likely be attributed to the fact that they developed a metabolic syndrome-like phenotype characterized by obesity, hypertriglyceridemia, and a reduced glucose tolerance. Mechanistic studies in chow diet-fed mice revealed that the diminished glucose tolerance was probably secondary to the exaggerated postprandial triglyceride response. The absence of PLTP did not affect LPL (lipoprotein lipase)-mediated triglyceride lipolysis but rather modified the ability of VLDL (very low-density lipoprotein)/chylomicron remnants to be cleared from the circulation by the liver through receptors other than SR-BI. As a result, livers of double knockout mice only cleared 26% of the fractional dose of [14C]cholesteryl oleate after intravenous VLDL-like particle injection. CONCLUSIONS: We have shown that disruption of PLTP-mediated HDL maturation reduces SR-BI deficiency-driven atherosclerosis susceptibility in mice despite the induction of proatherogenic metabolic complications in the double knockout mice.
Asunto(s)
Aterosclerosis/prevención & control , HDL-Colesterol/sangre , Metabolismo Energético , Hígado/metabolismo , Síndrome Metabólico/sangre , Proteínas de Transferencia de Fosfolípidos/deficiencia , Receptores Depuradores de Clase B/deficiencia , Animales , Aorta/metabolismo , Aorta/patología , Aterosclerosis/sangre , Aterosclerosis/genética , Aterosclerosis/patología , Ésteres del Colesterol/administración & dosificación , Ésteres del Colesterol/sangre , Modelos Animales de Enfermedad , Femenino , Intolerancia a la Glucosa/sangre , Intolerancia a la Glucosa/genética , Hipertrigliceridemia/sangre , Hipertrigliceridemia/genética , Masculino , Síndrome Metabólico/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/sangre , Obesidad/genética , Proteínas de Transferencia de Fosfolípidos/genética , Placa Aterosclerótica , Receptores Depuradores de Clase B/genéticaRESUMEN
The anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) plays an important role in survival and differentiation of leukocytes, more specifically of neutrophils. Here, we investigated the impact of myeloid Mcl-1 deletion in atherosclerosis. Western type diet fed LDL receptor-deficient mice were transplanted with either wild-type (WT) or LysMCre Mcl-1fl/fl (Mcl-1-/-) bone marrow. Mcl-1 myeloid deletion resulted in enhanced apoptosis and lipid accumulation in atherosclerotic plaques. In vitro, Mcl-1 deficient macrophages also showed increased lipid accumulation, resulting in increased sensitivity to lipid-induced cell death. However, plaque size, necrotic core and macrophage content were similar in Mcl-1-/- compared to WT mice, most likely due to decreased circulating and plaque-residing neutrophils. Interestingly, Mcl-1-/- peritoneal foam cells formed up to 45% more multinucleated giant cells (MGCs) in vitro compared to WT, which concurred with an increased MGC presence in atherosclerotic lesions of Mcl-1-/- mice. Moreover, analysis of human unstable atherosclerotic lesions also revealed a significant inverse correlation between MGC lesion content and Mcl-1 gene expression, coinciding with the mouse data. Taken together, these findings suggest that myeloid Mcl-1 deletion leads to a more apoptotic, lipid and MGC-enriched phenotype. These potentially pro-atherogenic effects are however counteracted by neutropenia in circulation and plaque.
Asunto(s)
Apoptosis , Células Gigantes/citología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Células 3T3 , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Diferenciación Celular , Eliminación de Gen , Humanos , Inmunohistoquímica , Lípidos/química , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Neutrófilos/metabolismo , Fenotipo , Placa Aterosclerótica/metabolismoRESUMEN
BACKGROUND: Membrane-exposed sulfatides are proposed to contribute to P-selectin-dependent platelet aggregation. Here, we demonstrated that P-selectin-mediated platelet aggregation on a collagen-coated surface under flow indeed depended on sulfatides and that this interaction differed considerably from the interaction of P-selectin with P-selectin Glycoprotein Ligand-1 (PSGL-1), which underlies leukocyte-endothelium adhesion. METHODS AND RESULTS: Upon platelet activation, sulfatides were translocated to the platelet surface to form focal hot-spots. Interestingly, P-selectin was observed to exclusively interact with liposomes with a sulfatide density higher than 21% (w/w), indicating that the binding profile of P-selectin for sulfatide-rich liposomes was dependent on sulfatide density. Sulfatide-liposome binding to P-selectin and sulfatide/P-selectin-dependent platelet aggregation was blunted by peptide antagonists, carrying the EWVDV motif within N-terminal extensions, such as CDVEWVDVSC (half maximal inhibitory concentration IC50 = 0.2 µM), but not by the EWVDV core motif itself (IC50 > 1000 µM), albeit both being equally potent inhibitors of PSGL-1/P-selectin interaction (IC50= 7-12 µM). CONCLUSIONS: Our data suggest that the sulfatide/P-selectin interaction implicates multiple binding pockets, which only partly overlap with that of PSGL-1. These observations open ways to selectively interfere with sulfatide/P-selectin-dependent platelet aggregation without affecting PSGL-1-dependent cell adhesion.
RESUMEN
The hepatocyte-specific asialoglycoprotein receptor (ASGPR) is an ideal candidate for targeted drug delivery to the liver due to its high capacity for substrate clearance from circulation together with its well-conserved expression and function across species. The development of GalNAc-siRNA conjugates, in which a synthetic triantennary N-acetylgalactosamine-based ligand is conjugated to chemically modified siRNA, has enabled efficient, ASGPR-mediated delivery to hepatocytes. To investigate the potential impact of variations in receptor expression on the efficiency of GalNAc-siRNA conjugate delivery, we evaluated the pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates in multiple pre-clinical models with reduced receptor expression. Despite greater than 50% reduction in ASGPR levels, GalNAc conjugate activity was retained, suggesting that the remaining receptor capacity was sufficient to mediate efficient uptake of potent GalNAc-siRNAs at pharmacologically relevant dose levels. Collectively, our data support a broad application of the GalNAc-siRNA technology for hepatic targeting, including disease states where ASGPR expression may be reduced.
Asunto(s)
Acetilgalactosamina , Receptor de Asialoglicoproteína/genética , Regulación de la Expresión Génica , Interferencia de ARN , ARN Interferente Pequeño/genética , Acetilgalactosamina/química , Animales , Receptor de Asialoglicoproteína/química , Receptor de Asialoglicoproteína/metabolismo , Modelos Animales de Enfermedad , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Femenino , Silenciador del Gen , Hepatocitos/metabolismo , Humanos , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Ratones , Ratones Noqueados , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Interferente Pequeño/químicaRESUMEN
Proapoptotic Bcl-2 family member Bim is particularly relevant for deletion of autoreactive and activated T and B cells, implicating Bim in autoimmunity. As atherosclerosis is a chronic inflammatory process with features of autoimmune disease, we investigated the impact of hematopoietic Bim deficiency on plaque formation and parameters of plaque stability. Bim -/- or wild type bone marrow transplanted ldlr -/- mice were fed a Western type diet (WTD) for 5 or 10 weeks, after which they were immunophenotyped and atherosclerotic lesions were analyzed. Bim -/- transplanted mice displayed splenomegaly and overt lymphocytosis. CD4+ and CD8+ T cells were more activated (increased CD69 and CD71 expression, increased interferon gamma production). B cells were elevated by 147%, with a shift towards the pro-atherogenic IgG-producing B2 cell phenotype, resulting in a doubling of anti-oxLDL IgG1 antibody titers in serum of bim -/- mice. Bim -/- mice displayed massive intraplaque accumulation of Ig complexes and of lesional T cells, although this did not translate in changes in plaque size or stability features (apoptotic cell and macrophage content). The surprising lack in plaque phenotype despite the profound pro-atherogenic immune effects may be attributable to the sharp reduction of serum cholesterol levels in WTD fed bim -/- mice.
Asunto(s)
Aterosclerosis/genética , Enfermedades Autoinmunes/etiología , Proteína 11 Similar a Bcl2/deficiencia , Inflamación/etiología , Leucocitos/inmunología , Leucocitos/metabolismo , Receptores de LDL/deficiencia , Animales , Apoptosis/genética , Enfermedades Autoinmunes/patología , Proteína 11 Similar a Bcl2/genética , Trasplante de Médula Ósea , Modelos Animales de Enfermedad , Hiperlipidemias , Inmunidad Humoral , Inmunoglobulinas/inmunología , Inflamación/patología , Recuento de Linfocitos , Ratones , Ratones Noqueados , Receptores de LDL/genética , Esplenomegalia , Células TH1/inmunología , Células TH1/metabolismoRESUMEN
BACKGROUND AND AIMS: Arginase1 (Arg1), an M2 macrophage marker, plays a critical role in a number of immunological functions in macrophages, which are the main cell type facilitating atherosclerotic lesion development. Arg1 uses the substrate l-arginine to create l-ornithine, a precursor molecule required for collagen formation and vascular smooth muscle cell differentiation. By reducing l-arginine availability, Arg1 limits the production of nitric oxide (NO), a pro-atherogenic factor in macrophages. In endothelial cells, conversely, NO is strongly anti-atherogenic. However, until now, the role of Arg1 in atherosclerosis is largely unknown. The aim of this study is to specifically investigate the effect of Arg1 deletion in hematopoietic cells on atherosclerosis susceptibility. METHODS: Ldlr KO mice were transplanted with Arg1flox/flox;Tie2-Cre (Arg1 KO) bone marrow (BM) or wildtype (WT) BM. After 8 weeks of recovery on chow diet, recipients mice were fed a Western-Type Diet (WTD) for 10 weeks to induce atherosclerosis. RESULTS: After 10-week WTD challenge, blood leukocyte counts were decreased by 25% (p < 0.001), and spleen leukocytes were decreased by 35% (p = 0.05) in Ldlr KO mice transplanted with Arg1 KO BM compared to mice transplanted with WT BM. The decrease in leukocytes was due to lower B lymphocyte counts. However, oxLDL-specific antibodies were increased in plasma of Ldlr KO mice transplanted with Arg1 KO BM compared to WT BM transplanted controls, whereas oxLDL-specific IgM was not affected. On the other hand, peritoneal foam cells in Arg1 KO BM recipients were increased 3-fold (p < 0.001) compared to WT BM recipients. No change in blood cholesterol was found. Despite changes in leukocyte counts and macrophage foam cell formation, we did not observe differences in atherosclerotic plaque size or plaque macrophage content in the aortic root. Surprisingly, there was also no difference in plaque collagen content, indicating that absence of macrophage Arg1 function does not reduce plaque stability. CONCLUSIONS: Deletion of Arg1 in hematopoietic cells adversely affects blood leukocyte counts and increases foam cell formation. However, no effects on atherosclerosis could be demonstrated, indicating that hematopoietic Arg1 function is not a decisive factor in atherosclerotic plaque formation.
Asunto(s)
Arginasa/metabolismo , Aterosclerosis/enzimología , Células de la Médula Ósea/enzimología , Células Espumosas/enzimología , Leucocitos/enzimología , Leucocitosis/prevención & control , Macrófagos Peritoneales/enzimología , Animales , Arginasa/genética , Aterosclerosis/sangre , Aterosclerosis/genética , Aterosclerosis/patología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/patología , Trasplante de Médula Ósea , Diferenciación Celular , Células Cultivadas , Colesterol/sangre , Femenino , Células Espumosas/efectos de los fármacos , Células Espumosas/patología , Predisposición Genética a la Enfermedad , Leucocitos/efectos de los fármacos , Leucocitos/patología , Leucocitosis/sangre , Leucocitosis/enzimología , Leucocitosis/genética , Lipoproteínas LDL/farmacología , Activación de Macrófagos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Placa Aterosclerótica , Receptores de LDL/deficiencia , Receptores de LDL/genéticaAsunto(s)
Aterosclerosis/prevención & control , Aterosclerosis/fisiopatología , Hipolipemiantes/uso terapéutico , Lipoproteínas HDL/fisiología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Aterosclerosis/sangre , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/fisiopatología , Colesterol/metabolismo , Expresión Génica , Humanos , Lipoproteínas HDL/sangre , Macrófagos/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND AND AIMS: We explored the role of ATP-binding cassette transporter A1 (Abca1), in post-myocardial infarction (MI) cardiac injury. METHODS: In Abca1(-/-) mice, wild type (WT) mice, and WT mice transplanted with Abca1(-/-) or WT bone marrow, an MI was induced in vivo. Furthermore, an ex vivo MI was induced in isolated Abca1(-/-) and WT hearts. RESULTS: Twenty-four hours and two weeks after in vivo MI induction, MI size was reduced in Abca1(-/-) (-58%, p = 0.007; -59%, p = 0.03) compared to WT. Ex vivo MI induction showed no effect of Abca1(-/-) on infarct size. Interestingly, two weeks after MI, Abca1(-/-) mice showed higher circulating levels of B-cells (+3.0 fold, p = 0.02) and T-cells (+4.2 fold, p = 0.002) compared to WT. Bone marrow-specific Abca1(-/-) tended to reduce infarct size (-43%, p = 0.12), suggesting a detrimental role for hematopoietic Abca1 after MI. CONCLUSIONS: Although Abca1 has a protective role in atherosclerosis, it exerts detrimental effects on cardiac function after MI.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/deficiencia , Corazón/fisiopatología , Infarto del Miocardio/genética , Infarto del Miocardio/prevención & control , Animales , Aterosclerosis/metabolismo , Linfocitos B/citología , Trasplante de Médula Ósea , Femenino , Leucocitos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/metabolismoRESUMEN
BACKGROUND: Leukocyte migration is critical for the infiltration of monocytes and accumulation of monocyte-derived macrophages in inflammation. Considering that Hck and Fgr are instrumental in this process, their impact on atherosclerosis and on lesion inflammation and stability was evaluated. METHODS AND RESULTS: Hematopoietic Hck/Fgr-deficient, LDLr(-/-) chimeras, obtained by bone marrow transplantation, had smaller but, paradoxically, less stable lesions with reduced macrophage content, overt cap thinning, and necrotic core expansion as the most prominent features. Despite a Ly6C(high)-skewed proinflammatory monocyte phenotype, Hck/Fgr deficiency led to disrupted adhesion of myeloid cells to and transmigration across endothelial monolayers in vitro and atherosclerotic plaques in vivo, as assessed by intravital microscopy, flow cytometry, and histological examination of atherosclerotic arteries. Moreover, Hck/Fgr-deficient macrophages showed blunted podosome formation and mesenchymal migration capacity. In consequence, transmigrated double-knockout macrophages were seen to accumulate in the fibrous cap, potentially promoting its focal erosion, as observed for double-knockout chimeras. CONCLUSIONS: The hematopoietic deficiency of Hck and Fgr led to attenuated atherosclerotic plaque formation by abrogating endothelial adhesion and transmigration; paradoxically, it also promoted plaque instability by causing monocyte subset imbalance and subendothelial accumulation, raising a note of caution regarding src kinase-targeted intervention in plaque inflammation.
Asunto(s)
Quimiotaxis de Leucocito/fisiología , Macrófagos Peritoneales/patología , Monocitos/patología , Placa Aterosclerótica/patología , Proteínas Proto-Oncogénicas c-hck/deficiencia , Proteínas Proto-Oncogénicas/deficiencia , Familia-src Quinasas/deficiencia , Animales , Apoptosis , Adhesión Celular , Extensiones de la Superficie Celular/ultraestructura , Células Cultivadas , Células Endoteliales , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Rodamiento de Leucocito , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis , Placa Aterosclerótica/enzimología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-hck/genética , Proteínas Proto-Oncogénicas c-hck/fisiología , Quimera por Radiación , Receptores de LDL/deficiencia , Receptores de LDL/genética , Receptores de LDL/fisiología , Migración Transendotelial y Transepitelial , Familia-src Quinasas/genética , Familia-src Quinasas/fisiologíaRESUMEN
Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.
Asunto(s)
Acetilgalactosamina/química , Silenciador del Gen , Hepatocitos/química , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Animales , Ratones , Ratones Endogámicos C57BL , Estructura MolecularRESUMEN
INTRODUCTION: Oxysterol binding protein Related Proteins (ORPs) mediate intracellular lipid transport and homeostatic regulation. ORP8 downregulates ABCA1 expression in macrophages and cellular cholesterol efflux to apolipoprotein A-I. In line, ORP8 knockout mice display increased amounts of HDL cholesterol in blood. However, the role of macrophage ORP8 in atherosclerotic lesion development is unknown. METHODS AND RESULTS: LDL receptor knockout (KO) mice were transplanted with bone marrow (BM) from ORP8 KO mice and C57Bl/6 wild type mice. Subsequently, the animals were challenged with a high fat/high cholesterol Western-type diet to induce atherosclerosis. After 9 weeks of Western-Type diet feeding, serum levels of VLDL cholesterol were increased by 50% in ORP8 KO BM recipients compared to the wild-type recipients. However, no differences were observed in HDL cholesterol. Despite the increase in VLDL cholesterol, lesions in mice transplanted with ORP8 KO bone marrow were 20% smaller compared to WT transplanted controls. In addition, ORP8 KO transplanted mice displayed a modest increase in the percentage of macrophages in the lesion as compared to the wild-type transplanted group. ORP8 deficient macrophages displayed decreased production of pro-inflammatory factors IL-6 and TNFα, decreased expression of differentiation markers and showed a reduced capacity to form foam cells in the peritoneal cavity. CONCLUSIONS: Deletion of ORP8 in bone marrow-derived cells, including macrophages, reduces lesion progression after 9 weeks of WTD challenge, despite increased amounts of circulating pro-atherogenic VLDL. Reduced macrophage foam cell formation and lower macrophage inflammatory potential are plausible mechanisms contributing to the observed reduction in atherosclerosis.
Asunto(s)
Aterosclerosis/genética , Aterosclerosis/patología , Células de la Médula Ósea/metabolismo , Receptores de LDL/deficiencia , Receptores de Esteroides/deficiencia , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Aterosclerosis/terapia , Biomarcadores , Colesterol/sangre , Colesterol/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Células Espumosas/patología , Expresión Génica , Mediadores de Inflamación/metabolismo , Recuento de Leucocitos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Gas Mostaza , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Receptores de LDL/genética , Receptores de Esteroides/genética , Factores de Tiempo , Triglicéridos/sangreRESUMEN
Alterations in DNA methylation patterns in peripheral blood leukocytes precede atherosclerotic lesion development in mouse models of atherosclerosis and have been linked to cardiovascular death in patients. The aim of this study is to investigate the long-term changes induced by WTD feeding on BM cells and the consequences for atherosclerosis susceptibility. Hereto, WTD BM or Chow BM was transplanted into LDLR KO mice on chow. BM from WTD BM recipient mice exhibited hypomethylation of CpG regions in the genes encoding Pu.1 and IRF8, key regulators of monocyte proliferation and macrophage differentiation. In agreement, in blood, the numbers of leukocytes were 40% (P<0.05) higher as a result of an increase in F4/80(+) monocytes (3.4-fold; P<0.01). An increase of CD11c(++) cells was also found (2.4-fold; P<0.05). Furthermore, spleens were enlarged, and the percentage of F4/80(+) cells expressing CD86 was induced (1.8-fold; P<0.01), indicating increased activation of splenic macrophages. Importantly, mice reconstituted with WTD BM showed a significant, 1.4-fold (P<0.05) increase in aortic root plaque size in the absence of changes in serum cholesterol. We conclude that WTD challenge induces transplantable epigenetic changes in BM, alterations in the hematopoietic system, and increased susceptibility to atherosclerosis. Manipulation of the epigenome, when used in conjunction with blood lipid reduction, could thus prove beneficial to treat cardiovascular disorders.
Asunto(s)
Aterosclerosis/etiología , Médula Ósea/metabolismo , Dieta , Epigénesis Genética , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Recuento de Células Sanguíneas , Médula Ósea/patología , Trasplante de Médula Ósea , Metilación de ADN , Dieta Occidental/efectos adversos , Modelos Animales de Enfermedad , Hematopoyesis/genética , Leucocitos/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Placa Aterosclerótica/genética , Receptores de LDL/deficiencia , Receptores de LDL/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
AIMS: The SDF-1α/CXCR4 dyad was previously shown by us and others to be instrumental in intimal hyperplasia as well as early stage atherosclerosis. We here sought to investigate its impact on clinically relevant stages of atherosclerosis in mouse and man. METHODS AND RESULTS: Immunohistochemical analysis of CXCR4 expression in human atherosclerotic lesions revealed a progressive accumulation of CXCR4(+) cells during plaque progression. To address causal involvement of CXCR4 in advanced stages of atherosclerosis we reconstituted LDLr(-/-) mice with autologous bone marrow infected with lentivirus encoding SDF-1α antagonist or CXCR4 degrakine, which effects proteasomal degradation of CXCR4. Functional CXCR4 blockade led to progressive plaque expansion with disease progression, while also promoting intraplaque haemorrhage. Moreover, CXCR4 knockdown was seen to augment endothelial adhesion of neutrophils. Concordant with this finding, inhibition of CXCR4 function increased adhesive capacity and reduced apoptosis of neutrophils and resulted in hyperactivation of circulating neutrophils. Compatible with a role of the neutrophil CXCR4 in end-stage atherosclerosis, CXCR4 expression by circulating neutrophils was lowered in patients with acute cardiovascular syndromes. CONCLUSION: In conclusion, CXCR4 contributes to later stages of plaque progression by perturbing neutrophil function.
Asunto(s)
Aterosclerosis/genética , Hemorragia/genética , Neutrófilos/metabolismo , Placa Aterosclerótica/genética , Receptores CXCR4/genética , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Adhesión Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Progresión de la Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Regulación de la Expresión Génica , Vectores Genéticos , Hemorragia/metabolismo , Hemorragia/patología , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Ratones , Ratones Noqueados , Neutrófilos/patología , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Transducción de SeñalRESUMEN
While numerous studies have aimed to develop strategies to inhibit the development and progression of atherosclerosis, recent attention has focussed on the regression of pre-existing atherosclerotic plaques. As important regulator of total body cholesterol homeostasis, the liver X receptor (LXR) could possibly be an important target to induce regression. Here, we describe the effect of LXR activation by the synthetic agonist T0901317 on lesion regression in different mouse models with early fatty streak lesions or advanced collagen-rich lesions. Although T0901317 caused a dramatic increase in plasma (V)LDL levels in low-density lipoprotein (LDL) receptor knockout mice, no further increase in lesion size was observed, which points to beneficial LXR activity in the vascular wall. In normolipidemic C57BL/6 mice with cholate diet-induced atherosclerotic lesions, T0901317 treatment improved plasma lipoprotein levels and induced lesion regression (-43%, p<0.05). Apolipoprotein E (APOE) reconstitution in APOE knockout mice by means of bone marrow transplantation dramatically improved plasma lipoprotein profiles and resulted in a marked regression of initial (-45%, p<0.001) and advanced lesions (-23%, p<0.01). Atherosclerosis regression was associated with a decrease in the absolute macrophage content (-84%, p<0.001). T0901317 supplementation further decreased the size of early (-71%, p<0.001 vs baseline; -48%, p<0.01 vs chow diet alone) and more advanced atherosclerotic lesions (-36%, p<0.001 and -17%, p=0.06 respectively). In conclusion, our study highlights the potential of LXR agonist T0901317 to stimulate removal of macrophages from atherosclerotic lesions ultimately leading to a highly significant plaque regression of both early and advanced atherosclerotic lesions.
Asunto(s)
Hidrocarburos Fluorados/uso terapéutico , Macrófagos/efectos de los fármacos , Receptores Nucleares Huérfanos/agonistas , Placa Aterosclerótica/tratamiento farmacológico , Sulfonamidas/uso terapéutico , Animales , Apolipoproteínas E/genética , Trasplante de Médula Ósea , Recuento de Células , VLDL-Colesterol/sangre , Dieta , Modelos Animales de Enfermedad , Femenino , Hidrocarburos Fluorados/administración & dosificación , Hidrocarburos Fluorados/farmacología , Receptores X del Hígado , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Placa Aterosclerótica/sangre , Placa Aterosclerótica/patología , Receptores de LDL/genética , Índice de Severidad de la Enfermedad , Sulfonamidas/administración & dosificación , Sulfonamidas/farmacología , Triglicéridos/sangreRESUMEN
Patients suffering from cardiovascular disease have well-established atherosclerotic lesions, rendering lesion regression of therapeutic interest. The OX40 (TNFRSF4)-OX40 ligand (OX40L; TNFSF4) pathway is important for the proliferation and survival of T cells, stimulates B cells, and is associated with cardiovascular disease. We hypothesized that interference with the OX40-OX40L pathway, in combination with decreases in cholesterol, may induce regression of atherosclerosis. LDLr(-/-) mice were fed a Western-type diet for 10 wk, after which they received chow diet and were treated with anti-OX40L or PBS for 10 wk. A significant regression of lesions was observed in the aorta and aortic arch of anti-OX40L-treated mice compared with control mice. Interference of the OX40-OX40L pathway reduced Th2 responses, as shown by decreases in GATA-3 and IL-4 levels. Also, IgE levels were decreased, as demonstrated by reduced mast cell presence and activation. Notably, IL-5 production by T and B1 cells was increased, thus enhancing atheroprotective oxidized low-density lipoprotein-specific IgM production. The increase in IL-5 production and IgM was mediated by IL-33 production by APCs upon OX40L blockade. We conclude that interruption of the OX40-OX40L signaling pathway, combined with decreases in dietary cholesterol, induces the regression of atherosclerosis through induction of IL-5-producing T cells and oxidized low-density lipoprotein-specific IgM and reductions in Th2 and mast cells.