RESUMEN
BACKGROUND: Reprogramming of monocytes and macrophage manifests in hyperinflammatory responses and chronification of inflammation in atherosclerosis. Recent studies focused on epigenetic, transcriptional, and metabolic alterations that characterize trained immunity. However, the underlying effector mechanisms driving the hyperinflammatory response of reprogrammed macrophages remain unclear. We hypothesized that the plasma membrane of atherosclerotic lesion macrophages undergoes reprogramming to maintain inflammarafts, enlarged lipid rafts (LR) serving as a platform for assembly of inflammatory receptor complexes. METHODS: Single-cell suspensions from the aortae of Western diet-fed Ldlr-/- mice were gated for BODIPY-high foamy and BODIPY-low nonfoamy F4/80 macrophages by flow cytometry. Inflammarafts were characterized by increased levels of LR, TLR4 (toll-like receptor-4) localization to LR, TLR4 dimers, and the proximity between TLR2, TLR1, and CD36. In a cellular model of trained immunity, LR, TLR4 dimers, and the inflammatory response were measured in bone marrow-derived macrophages subjected to a 24-hour treatment with LPS (lipopolysaccharide) or OxLDL (oxidized low-density lipoprotein), followed by a 6-day wash-out period. RESULTS: Nonfoamy macrophages, which constituted ≈40% of macrophages in atherosclerotic lesions, expressed significantly higher levels of LR and TLR4 dimers, as well as proximity ligation signals for TLR4-LR, TLR2-CD36, and TLR2-TLR1 complexes, compared with foamy macrophages. These inflammaraft measures associated, to a different degree, with plasma cholesterol and inflammatory cytokines, as well as the size of the atherosclerotic lesions and necrotic cores. The bone marrow-derived macrophages trained with LPS simulated nonfoamy atherosclerotic lesion macrophages and continued to express inflammarafts and inflammatory genes for 6 days after LPS removal and displayed a hyperinflammatory response to Pam3CSK4, a TLR2/TLR1 agonist. OxLDL-exposed, lipid-laden macrophages did not express inflammarafts. CONCLUSIONS: Our data support the hypothesis that persistent inflammarafts in nonfoamy macrophages in atherosclerotic lesions serve as effectors of macrophage reprogramming into a hyperinflammatory phenotype.
Asunto(s)
Aterosclerosis , Células Espumosas , Ratones , Animales , Células Espumosas/patología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Lipopolisacáridos , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 1/metabolismo , Macrófagos/metabolismo , Aterosclerosis/patología , Lipoproteínas LDL/metabolismo , Antígenos CD36/genética , Antígenos CD36/metabolismoRESUMEN
In this Letter, affiliation number 1 was originally missing from the HTML; the affiliations were missing for author Ming-Yow Hung in the HTML; and the Fig. 4 legend erroneously referred to panels a-h, instead of a-g. These errors have been corrected online.
RESUMEN
Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes 1 . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL2-4. Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr-/- background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr-/- mice, Ldlr -/- E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis.
Asunto(s)
Aterosclerosis/tratamiento farmacológico , Aterosclerosis/metabolismo , Hipercolesterolemia/metabolismo , Inflamación/metabolismo , Fosfolípidos/antagonistas & inhibidores , Fosfolípidos/metabolismo , Animales , Estenosis de la Válvula Aórtica/tratamiento farmacológico , Estenosis de la Válvula Aórtica/metabolismo , Estenosis de la Válvula Aórtica/patología , Apoptosis , Aterosclerosis/inducido químicamente , Aterosclerosis/genética , Colesterol/administración & dosificación , Colesterol/farmacología , Progresión de la Enfermedad , Hígado Graso/tratamiento farmacológico , Hígado Graso/metabolismo , Hígado Graso/patología , Femenino , Hipercolesterolemia/patología , Inmunoglobulina M/genética , Inmunoglobulina M/inmunología , Inmunoglobulina M/uso terapéutico , Inflamación/tratamiento farmacológico , Inflamación/patología , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oxidación-Reducción , Fosfolípidos/química , Fosfolípidos/inmunología , Fosforilcolina/inmunología , Receptores de LDL/deficiencia , Receptores de LDL/genética , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/uso terapéuticoRESUMEN
Monocytes are associated with human cardiovascular disease progression. Monocytes are segregated into three major subsets: classical (cMo), intermediate (iMo), and nonclassical (nMo). Recent studies have identified heterogeneity within each of these main monocyte classes, yet the extent to which these subsets contribute to heart disease progression is not known. Peripheral blood mononuclear cells (PBMC) were obtained from 61 human subjects within the Coronary Assessment of Virginia (CAVA) Cohort. Coronary atherosclerosis severity was quantified using the Gensini Score (GS). We employed high-dimensional single-cell transcriptome and protein methods to define how human monocytes differ in subjects with low to severe coronary artery disease. We analyzed 487 immune-related genes and 49 surface proteins at the single-cell level using Antibody-Seq (Ab-Seq). We identified six subsets of myeloid cells (cMo, iMo, nMo, plasmacytoid DC, classical DC, and DC3) at the single-cell level based on surface proteins, and we associated these subsets with coronary artery disease (CAD) incidence based on Gensini score (GS) in each subject. Only frequencies of iMo were associated with high CAD (GS > 32), adj.p = 0.024. Spearman correlation analysis with GS from each subject revealed a positive correlation with iMo frequencies (r = 0.314, p = 0.014) and further showed a robust sex-dependent positive correlation in female subjects (r = 0.663, p = 0.004). cMo frequencies did not correlate with CAD severity. Key gene pathways differed in iMo among low and high CAD subjects and between males and females. Further single-cell analysis of iMo revealed three iMo subsets in human PBMC, distinguished by the expression of HLA-DR, CXCR3, and CD206. We found that the frequency of immunoregulatory iMo_HLA-DR+CXCR3+CD206+ was associated with CAD severity (adj.p = 0.006). The immunoregulatory iMo subset positively correlated with GS in both females (r = 0.660, p = 0.004) and males (r = 0.315, p = 0.037). Cell interaction analyses identified strong interactions of iMo with CD4+ effector/memory T cells and Tregs from the same subjects. This study shows the importance of iMo in CAD progression and suggests that iMo may have important functional roles in modulating CAD risk, particularly among females.
Asunto(s)
Enfermedad de la Arteria Coronaria , Humanos , Femenino , Masculino , Enfermedad de la Arteria Coronaria/metabolismo , Monocitos/metabolismo , Leucocitos Mononucleares , Caracteres Sexuales , Antígenos HLA-DR/metabolismoRESUMEN
PURPOSE OF REVIEW: Advances in single cell techniques revealed a remarkable diversity in macrophage gene expression profiles in atherosclerosis. However, the diversity of functional processes at the macrophage plasma membrane remains less studied. This review summarizes recent advances in characterization of lipid rafts, where inflammatory receptors assemble, in macrophages that undergo reprogramming in atherosclerotic lesions and in vitro under conditions relevant to the development of atherosclerosis. RECENT FINDINGS: The term inflammarafts refers to enlarged lipid rafts with increased cholesterol content, hosting components of inflammatory receptor complexes assembled in close proximity, including TLR4-TLR4, TLR2-TLR1 and TLR2-CD36 dimers. Macrophages decorated with inflammarafts maintain chronic inflammatory gene expression and are primed to an augmented response to additional inflammatory stimuli. In mouse atherosclerotic lesions, inflammarafts are expressed primarily in nonfoamy macrophages and less in lipid-laden foam cells. This agrees with the reported suppression of inflammatory programs in foam cells. In contrast, nonfoamy macrophages expressing inflammarafts are the major inflammatory population in atherosclerotic lesions. Discussed are emerging reports that help understand formation and persistence of inflammarafts and the potential of inflammarafts as a novel therapeutic target. SUMMARY: Chronic maintenance of inflammarafts in nonfoamy macrophages serves as an effector mechanism of inflammatory macrophage reprogramming in atherosclerosis.
Asunto(s)
Aterosclerosis , Placa Aterosclerótica , Animales , Ratones , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 2/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Aterosclerosis/metabolismo , Células Espumosas/metabolismo , Placa Aterosclerótica/patologíaRESUMEN
OBJECTIVE: Atherosclerotic lesions are often characterized by accumulation of OxLDL (oxidized low-density lipoprotein), which is associated with vascular inflammation and lesion vulnerability to rupture. Extracellular AIBP (apolipoprotein A-I binding protein; encoded by APOA1BP gene), when secreted, promotes cholesterol efflux and regulates lipid rafts dynamics, but its role as an intracellular protein in mammalian cells remains unknown. The aim of this work was to determine the function of intracellular AIBP in macrophages exposed to OxLDL and in atherosclerotic lesions. Approach and Results: Using a novel monoclonal antibody against human and mouse AIBP, which are highly homologous, we demonstrated robust AIBP expression in human and mouse atherosclerotic lesions. We observed significantly reduced autophagy in bone marrow-derived macrophages, isolated from Apoa1bp-/- compared with wild-type mice, which were exposed to OxLDL. In atherosclerotic lesions from Apoa1bp-/- mice subjected to Ldlr knockdown and fed a Western diet, autophagy was reduced, whereas apoptosis was increased, when compared with that in wild-type mice. AIBP expression was necessary for efficient control of reactive oxygen species and cell death and for mitochondria quality control in macrophages exposed to OxLDL. Mitochondria-localized AIBP, via its N-terminal domain, associated with E3 ubiquitin-protein ligase PARK2 (Parkin), MFN (mitofusin)1, and MFN2, but not BNIP3 (Bcl2/adenovirus E1B 19-kDa-interacting protein-3), and regulated ubiquitination of MFN1 and MFN2, key components of mitophagy. CONCLUSIONS: These data suggest that intracellular AIBP is a new regulator of autophagy in macrophages. Mitochondria-localized AIBP augments mitophagy and participates in mitochondria quality control, protecting macrophages against cell death in the context of atherosclerosis.
Asunto(s)
Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Lipoproteínas LDL/toxicidad , Macrófagos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitofagia/efectos de los fármacos , Fosfoproteínas/metabolismo , Racemasas y Epimerasas/metabolismo , Animales , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Apoptosis/efectos de los fármacos , Aterosclerosis/genética , Aterosclerosis/patología , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagosomas/patología , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Modelos Animales de Enfermedad , Células HEK293 , Células Hep G2 , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fosfoproteínas/genética , Racemasas y Epimerasas/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
HIV infection has a profound effect on "bystander" cells causing metabolic co-morbidities. This may be mediated by exosomes secreted by HIV-infected cells and containing viral factors. Here we show that exosomes containing HIV-1 protein Nef (exNef) are rapidly taken up by macrophages releasing Nef into the cell interior. This caused down-regulation of ABCA1, reduction of cholesterol efflux and sharp elevation of the abundance of lipid rafts through reduced activation of small GTPase Cdc42 and decreased actin polymerization. Changes in rafts led to re-localization of TLR4 and TREM-1 to rafts, phosphorylation of ERK1/2, activation of NLRP3 inflammasome, and increased secretion of pro-inflammatory cytokines. The effects of exNef on lipid rafts and on inflammation were reversed by overexpression of a constitutively active mutant of Cdc42. Similar effects were observed in macrophages treated with exosomes produced by HIV-infected cells or isolated from plasma of HIV-infected subjects, but not with exosomes from cells and subjects infected with ΔNef-HIV or uninfected subjects. Mice injected with exNef exhibited monocytosis, reduced ABCA1 in macrophages, increased raft abundance in monocytes and augmented inflammation. Thus, Nef-containing exosomes potentiated pro-inflammatory response by inducing changes in cholesterol metabolism and reorganizing lipid rafts. These mechanisms may contribute to HIV-associated metabolic co-morbidities.
Asunto(s)
Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismo , Animales , Efecto Espectador , Colesterol/metabolismo , Exosomas/metabolismo , Exosomas/virología , Células HEK293 , VIH-1 , Humanos , Inflamación/metabolismo , Inflamación/virología , Microdominios de Membrana/metabolismo , Microdominios de Membrana/virología , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Células RAW 264.7 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptor Toll-Like 4/metabolismo , Receptor Activador Expresado en Células Mieloides 1/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
OBJECTIVE: AIBP (apolipoprotein A-I binding protein) is an effective and selective regulator of lipid rafts modulating many metabolic pathways originating from the rafts, including inflammation. The mechanism of action was suggested to involve stimulation by AIBP of cholesterol efflux, depleting rafts of cholesterol, which is essential for lipid raft integrity. Here we describe a different mechanism contributing to the regulation of lipid rafts by AIBP. Approach and Results: We demonstrate that modulation of rafts by AIBP may not exclusively depend on the rate of cholesterol efflux or presence of the key regulator of the efflux, ABCA1 (ATP-binding cassette transporter A-I). AIBP interacted with phosphatidylinositol 3-phosphate, which was associated with increased abundance and activation of Cdc42 and rearrangement of the actin cytoskeleton. Cytoskeleton rearrangement was accompanied with reduction of the abundance of lipid rafts, without significant changes in the lipid composition of the rafts. The interaction of AIBP with phosphatidylinositol 3-phosphate was blocked by AIBP substrate, NADPH (nicotinamide adenine dinucleotide phosphate), and both NADPH and silencing of Cdc42 interfered with the ability of AIBP to regulate lipid rafts and cholesterol efflux. CONCLUSIONS: Our findings indicate that an underlying mechanism of regulation of lipid rafts by AIBP involves PIP-dependent rearrangement of the cytoskeleton.
Asunto(s)
Citoesqueleto de Actina/enzimología , Colesterol/metabolismo , Microdominios de Membrana/enzimología , Racemasas y Epimerasas/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismo , Citoesqueleto de Actina/genética , Animales , Células HeLa , Humanos , Microdominios de Membrana/genética , Ratones , Fosfatidilinositol 3-Quinasa/metabolismo , Transducción de Señal , Células THP-1 , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Lipid rafts are organized plasma membrane microdomains, which provide a distinct level of regulation of cellular metabolism and response to extracellular stimuli, affecting a diverse range of physiologic and pathologic processes. This Thematic Review Series focuses on Biology of Lipid Rafts rather than on their composition or structure. The aim is to provide an overview of ideas on how lipid rafts are involved in regulation of different pathways and how they interact with other layers of metabolic regulation. Articles in the series will review the involvement of lipid rafts in regulation of hematopoiesis, production of extracellular vesicles, host interaction with infection, and the development and progression of cancer, neuroinflammation, and neurodegeneration, as well as the current outlook on therapeutic targeting of lipid rafts.
Asunto(s)
Microdominios de Membrana/metabolismo , HumanosRESUMEN
Lipid rafts regulate the initiation of cellular metabolic and signaling pathways by organizing the pathway components in ordered microdomains on the cell surface. Cellular responses regulated by lipid rafts range from physiological to pathological, and the success of a therapeutic approach targeting "pathological" lipid rafts depends on the ability of a remedial agent to recognize them and disrupt pathological lipid rafts without affecting normal raft-dependent cellular functions. In this article, concluding the Thematic Review Series on Biology of Lipid Rafts, we review current experimental therapies targeting pathological lipid rafts, including examples of inflammarafts and clusters of apoptotic signaling molecule-enriched rafts. The corrective approaches include regulation of cholesterol and sphingolipid metabolism and membrane trafficking by using HDL and its mimetics, LXR agonists, ABCA1 overexpression, and cyclodextrins, as well as a more targeted intervention with apoA-I binding protein. Among others, we highlight the design of antagonists that target inflammatory receptors only in their activated form of homo- or heterodimers, when receptor dimerization occurs in pathological lipid rafts. Other therapies aim to promote raft-dependent physiological functions, such as augmenting caveolae-dependent tissue repair. The overview of this highly dynamic field will provide readers with a view on the emerging concept of targeting lipid rafts as a therapeutic strategy.jlr;61/5/687/F1F1f1.
Asunto(s)
Microdominios de Membrana/efectos de los fármacos , Terapia Molecular Dirigida/métodos , Animales , Humanos , Microdominios de Membrana/metabolismoRESUMEN
Activation of microglia and astrocytes secondary to inflammatory processes contributes to the development and perpetuation of pain with a neuropathic phenotype. This pain state presents as a chronic debilitating condition and affects a large population of patients with conditions like rheumatoid arthritis and diabetes, or after surgery, trauma, or chemotherapy. Here, we review the regulation of lipid rafts in glial cells and the role they play as a key component of neuroinflammatory sensitization of central pain signaling pathways. In this context, we introduce the concept of an inflammaraft (i-raft), enlarged lipid rafts harboring activated receptors and adaptor molecules and serving as an organizing platform to initiate inflammatory signaling and the cellular response. Characteristics of the inflammaraft include increased relative abundance of lipid rafts in inflammatory cells, increased content of cholesterol per raft, and increased levels of inflammatory receptors, such as toll-like receptor (TLR)4, adaptor molecules, ion channels, and enzymes in lipid rafts. This inflammaraft motif serves an important role in the membrane assembly of protein complexes, for example, TLR4 dimerization. Operating within this framework, we demonstrate the involvement of inflammatory receptors, redox molecules, and ion channels in the inflammaraft formation and the regulation of cholesterol and sphingolipid metabolism in the inflammaraft maintenance and disruption. Strategies for targeting inflammarafts, without affecting the integrity of lipid rafts in noninflammatory cells, may lead to developing novel therapies for neuropathic pain states and other neuroinflammatory conditions.
Asunto(s)
Microdominios de Membrana/metabolismo , Neuroglía/patología , Dolor/patología , Animales , Humanos , Inflamación/patología , Proteínas de la Membrana/metabolismoRESUMEN
PURPOSE OF REVIEW: Recent studies demonstrate an important role of the secreted apolipoprotein A-I binding protein (AIBP) in regulation of cholesterol efflux and lipid rafts. The article discusses these findings in the context of angiogenesis and inflammation. RECENT FINDINGS: Lipid rafts are cholesterol-rich and sphingomyelin-rich membrane domains in which many receptor complexes assemble upon activation. AIBP mediates selective cholesterol efflux, in part via binding to toll-like receptor-4 (TLR4) in activated macrophages and microglia, and thus reverses lipid raft increases in activated cells. Recent articles report AIBP regulation of vascular endothelial growth factor receptor-2, Notch1 and TLR4 function. In zebrafish and mouse animal models, AIBP deficiency results in accelerated angiogenesis, increased inflammation and exacerbated atherosclerosis. Spinal delivery of recombinant AIBP reduces neuraxial inflammation and reverses persistent pain state in a mouse model of chemotherapy-induced polyneuropathy. Inhalation of recombinant AIBP reduces lipopolysaccharide-induced acute lung injury in mice. These findings are discussed in the perspective of AIBP's proposed other function, as an NAD(P)H hydrate epimerase, evolving into a regulator of cholesterol trafficking and lipid rafts. SUMMARY: Novel findings of AIBP regulatory circuitry affecting lipid rafts and related cellular processes may provide new therapeutic avenues for angiogenic and inflammatory diseases.
Asunto(s)
Microdominios de Membrana/metabolismo , Neovascularización Fisiológica , Fosfoproteínas/metabolismo , Animales , Aterosclerosis/metabolismo , Colesterol/metabolismo , Humanos , Inflamación/metabolismoRESUMEN
Atherosclerosis is associated with increased lipid peroxidation, leading to generation of multiple oxidation-specific epitopes (OSEs), contributing to the pathogenesis of atherosclerosis and its clinical manifestation. Oxidized cholesteryl esters (OxCEs) are a major class of OSEs found in human plasma and atherosclerotic tissue. To evaluate OxCEs as a candidate biomarker, we generated a novel mouse monoclonal Ab (mAb) specific to an OxCE modification of proteins. The mAb AG23 (IgG1) was raised in C57BL6 mice immunized with OxCE-modified keyhole limpet hemocyanin, and hybridomas were screened against OxCE-modified BSA. This method ensures mAb specificity to the OxCE modification, independent of a carrier protein. AG23 specifically stained human carotid artery atherosclerotic lesions. An ELISA method, with AG23 as a capture and either anti-apoAI or anti-apoB-100 as the detection Abs, was developed to assay apoAI and apoB-100 lipoproteins that have one or more OxCE epitopes. OxCE-apoA or OxCE-apoB did not correlate with the well-established oxidized phospholipid-apoB biomarker. In a cohort of subjects treated with atorvastatin, OxCE-apoA was significantly lower than in the placebo group, independent of the apoAI levels. These results suggest the potential diagnostic utility of a new biomarker assay to measure OxCE-modified lipoproteins in patients with CVD.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Apolipoproteína A-I/metabolismo , Apolipoproteína B-100/metabolismo , Ésteres del Colesterol/sangre , Ésteres del Colesterol/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Animales , Ésteres del Colesterol/inmunología , Humanos , Ratones , Oxidación-ReducciónRESUMEN
RATIONALE: Angiogenesis improves perfusion to the ischemic tissue after acute vascular obstruction. Angiogenesis in pathophysiological settings reactivates signaling pathways involved in developmental angiogenesis. We showed previously that AIBP (apolipoprotein A-I [apoA-I]-binding protein)-regulated cholesterol efflux in endothelial cells controls zebra fish embryonic angiogenesis. OBJECTIVE: This study is to determine whether loss of AIBP affects angiogenesis in mice during development and under pathological conditions and to explore the underlying molecular mechanism. METHODS AND RESULTS: In this article, we report the generation of AIBP knockout (Apoa1bp-/-) mice, which are characterized of accelerated postnatal retinal angiogenesis. Mechanistically, AIBP triggered relocalization of γ-secretase from lipid rafts to nonlipid rafts where it cleaved Notch. Consistently, AIBP treatment enhanced DLL4 (delta-like ligand 4)-stimulated Notch activation in human retinal endothelial cells. Increasing high-density lipoprotein levels in Apoa1bp-/- mice by crossing them with apoA-I transgenic mice rescued Notch activation and corrected dysregulated retinal angiogenesis. Notably, the retinal vessels in Apoa1bp-/- mice manifested normal pericyte coverage and vascular integrity. Similarly, in the subcutaneous Matrigel plug assay, which mimics ischemic/inflammatory neovascularization, angiogenesis was dramatically upregulated in Apoa1bp-/- mice and associated with a profound inhibition of Notch activation and reduced expression of downstream targets. Furthermore, loss of AIBP increased vascular density and facilitated the recovery of blood vessel perfusion function in a murine hindlimb ischemia model. In addition, AIBP expression was significantly increased in human patients with ischemic cardiomyopathy. CONCLUSIONS: Our data reveal a novel mechanistic connection between AIBP-mediated cholesterol metabolism and Notch signaling, implicating AIBP as a possible druggable target to modulate angiogenesis under pathological conditions.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/fisiología , Proteínas Portadoras/biosíntesis , Neovascularización Fisiológica/fisiología , Fosfoproteínas/biosíntesis , Receptores Notch/biosíntesis , Transducción de Señal/fisiología , Regulación hacia Arriba/fisiología , Animales , Miembro Posterior/irrigación sanguínea , Miembro Posterior/metabolismo , Miembro Posterior/patología , Humanos , Isquemia/metabolismo , Isquemia/patología , Ratones , Ratones Noqueados , Racemasas y Epimerasas , Retina/metabolismo , Retina/patología , Pez CebraRESUMEN
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
Elevated plasma LDL cholesterol is the dominant risk factor for the development of atherosclerosis and cardiovascular disease. Deficiency in the LDL receptor (LDLR) is a major cause of familial hypercholesterolemia in humans, and the LDLR knockout mouse is a major animal model of atherosclerosis. Here we report the generation and characterization of an ldlr mutant zebrafish as a new animal model to study hypercholesterolemia and vascular lipid accumulation, an early event in the development of human atherosclerosis. The ldlr mutant zebrafish were characterized by activated SREBP-2 pathway and developed moderate hypercholesterolemia when fed a normal diet. However, a short-term, 5-day feeding of ldlr mutant larvae with a high-cholesterol diet (HCD) resulted in exacerbated hypercholesterolemia and accumulation of vascular lipid deposits. Lomitapide, an inhibitor of apoB lipoprotein secretion, but not the antioxidant probucol, significantly reduced accumulation of vascular lipid deposits in HCD-fed ldlr mutant larvae. Furthermore, ldlr mutants were defective in hepatic clearance of lipopolysaccharides, resulting in reduced survival. Taken together, our data suggest that the ldlr knockout zebra-fish is a versatile model for studying the function of the LDL receptor, hypercholesterolemia, and related vascular pathology in the context of early atherosclerosis.
Asunto(s)
Modelos Animales de Enfermedad , Hipercolesterolemia/metabolismo , Metabolismo de los Lípidos , Lípidos , Mutación , Receptores de LDL/genética , Pez Cebra/genética , Pez Cebra/metabolismo , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/metabolismoRESUMEN
Apolipoprotein A-I binding protein (AIBP) has been shown to augment cholesterol efflux from endothelial cells and macrophages. In zebrafish and mice, AIBP-mediated regulation of cholesterol levels in the plasma membrane of endothelial cells controls angiogenesis. The goal of this work was to evaluate metabolic changes and atherosclerosis in AIBP loss-of-function and gain-of-function animal studies. Here, we show that Apoa1bp-/-Ldlr-/- mice fed a high-cholesterol, high-fat diet had exacerbated weight gain, liver steatosis, glucose intolerance, hypercholesterolemia, hypertriglyceridemia, and larger atherosclerotic lesions compared with Ldlr-/- mice. Feeding Apoa1bp-/-Ldlr-/- mice a high-cholesterol, normal-fat diet did not result in significant differences in lipid levels or size of atherosclerotic lesions from Ldlr-/- mice. Conversely, adeno-associated virus-mediated overexpression of AIBP reduced hyperlipidemia and atherosclerosis in high-cholesterol, high-fat diet-fed Ldlr-/- mice. Injections of recombinant AIBP reduced aortic inflammation in Ldlr-/- mice fed a short high-cholesterol, high-fat diet. Conditional overexpression of AIBP in zebrafish also reduced diet-induced vascular lipid accumulation. In experiments with isolated macrophages, AIBP facilitated cholesterol efflux to HDL, reduced lipid rafts content, and inhibited inflammatory responses to lipopolysaccharide.jlr Our data demonstrate that AIBP confers protection against diet-induced metabolic abnormalities and atherosclerosis.
Asunto(s)
Aterosclerosis/metabolismo , Proteínas Portadoras/metabolismo , Síndrome Metabólico/metabolismo , Fosfoproteínas/metabolismo , Animales , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfoproteínas/deficiencia , Racemasas y Epimerasas , Receptores de LDL/deficiencia , Receptores de LDL/metabolismoRESUMEN
OBJECTIVE: Nonclassical monocytes (NCM) function to maintain vascular homeostasis by crawling or patrolling along the vessel wall. This subset of monocytes responds to viruses, tumor cells, and other pathogens to aid in protection of the host. In this study, we wished to determine how early atherogenesis impacts NCM patrolling in the vasculature. APPROACH AND RESULTS: To study the role of NCM in early atherogenesis, we quantified the patrolling behaviors of NCM in ApoE-/- (apolipoprotein E) and C57BL/6J mice fed a Western diet. Using intravital imaging, we found that NCM from Western diet-fed mice display a 4-fold increase in patrolling activity within large peripheral blood vessels. Both human and mouse NCM preferentially engulfed OxLDL (oxidized low-density lipoprotein) in the vasculature, and we observed that OxLDL selectively induced NCM patrolling in vivo. Induction of patrolling during early atherogenesis required scavenger receptor CD36, as CD36-/- mice revealed a significant reduction in patrolling activity along the femoral vasculature. Mechanistically, we found that CD36-regulated patrolling was mediated by a SFK (src family kinase) through DAP12 (DNAX activating protein of 12KDa) adaptor protein. CONCLUSIONS: Our studies show a novel pathway for induction of NCM patrolling along the vascular wall during early atherogenesis. Mice fed a Western diet showed increased NCM patrolling activity with a concurrent increase in SFK phosphorylation. This patrolling activity was lost in the absence of either CD36 or DAP12. These data suggest that NCM function in an atheroprotective manner through sensing and responding to oxidized lipoprotein moieties via scavenger receptor engagement during early atherogenesis.
Asunto(s)
Aterosclerosis/metabolismo , Antígenos CD36/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Arteria Femoral/metabolismo , Rodamiento de Leucocito , Monocitos/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/patología , Antígenos CD36/deficiencia , Antígenos CD36/genética , Dieta Occidental , Modelos Animales de Enfermedad , Células Endoteliales/patología , Endotelio Vascular/patología , Arteria Femoral/patología , Predisposición Genética a la Enfermedad , Humanos , Microscopía Intravital , Lipoproteínas LDL/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/patología , Fenotipo , Transducción de Señal , Factores de Tiempo , Familia-src Quinasas/metabolismoRESUMEN
The oxidation hypothesis of atherosclerosis proposes that oxidized LDL is a major causative factor in the development of atherosclerosis. Although this hypothesis has received strong mechanistic support and many animal studies demonstrated profound atheroprotective effects of antioxidants, which reduce LDL oxidation, the results of human clinical trials with antioxidants were mainly negative, except in selected groups of patients with clearly increased systemic oxidative stress. We propose that even if reducing lipoprotein oxidation in humans might be difficult to achieve, deeper understanding of mechanisms by which oxidized LDL promotes atherosclerosis and targeting these specific mechanisms will offer novel approaches to treatment of cardiovascular disease. In this review article, we focus on oxidized cholesteryl esters (OxCE), which are a major component of minimally and extensively oxidized LDL and of human atherosclerotic lesions. OxCE and OxCE-protein covalent adducts induce profound biological effects. Among these effects, OxCE activate macrophages via toll-like receptor-4 (TLR4) and spleen tyrosine kinase and induce macropinocytosis resulting in lipid accumulation, generation of reactive oxygen species and secretion of inflammatory cytokines. Specific inhibition of OxCE-induced TLR4 activation, as well as blocking other inflammatory effects of OxCE, may offer novel treatments of atherosclerosis and cardiovascular disease. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder.