RESUMO
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.
Assuntos
Aterosclerose , Placa Aterosclerótica , Humanos , Camundongos , Animais , Meios de Contraste , Dextranos/farmacologia , Células Espumosas/patologia , Aterosclerose/diagnóstico por imagem , Aterosclerose/tratamento farmacológico , Aterosclerose/patologia , Óxido Ferroso-Férrico/farmacologia , Imageamento por Ressonância Magnética/métodos , Macrófagos/patologia , Apoptose , Óxidos/farmacologiaRESUMO
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.
Assuntos
Acetilgalactosamina/química , Doenças Cardiovasculares/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Terapia Genética/métodos , Fígado/efeitos dos fármacos , Oligonucleotídeos/administração & dosagem , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Terapêutica com RNAiRESUMO
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.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/genética , Aterosclerose , Medula Óssea , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Aterosclerose/genética , Medula Óssea/metabolismo , Colesterol , Lipoproteínas LDL/metabolismo , Camundongos , Camundongos Knockout , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMO
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.
Assuntos
Apoptose , Células Gigantes/citologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Células 3T3 , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Diferenciação Celular , Deleção de Genes , Humanos , Imuno-Histoquímica , Lipídeos/química , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neutrófilos/metabolismo , Fenótipo , Placa Aterosclerótica/metabolismoRESUMO
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.
Assuntos
Aterosclerose/genética , Doenças Autoimunes/etiologia , Proteína 11 Semelhante a Bcl-2/deficiência , Inflamação/etiologia , Leucócitos/imunologia , Leucócitos/metabolismo , Receptores de LDL/deficiência , Animais , Apoptose/genética , Doenças Autoimunes/patologia , Proteína 11 Semelhante a Bcl-2/genética , Transplante de Medula Óssea , Modelos Animais de Doenças , Hiperlipidemias , Imunidade Humoral , Imunoglobulinas/imunologia , Inflamação/patologia , Contagem de Linfócitos , Camundongos , Camundongos Knockout , Receptores de LDL/genética , Esplenomegalia , Células Th1/imunologia , Células Th1/metabolismoRESUMO
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.
Assuntos
Arginase/metabolismo , Aterosclerose/enzimologia , Células da Medula Óssea/enzimologia , Células Espumosas/enzimologia , Leucócitos/enzimologia , Leucocitose/prevenção & controle , Macrófagos Peritoneais/enzimologia , Animais , Arginase/genética , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/patologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Transplante de Medula Óssea , Diferenciação Celular , Células Cultivadas , Colesterol/sangue , Feminino , Células Espumosas/efeitos dos fármacos , Células Espumosas/patologia , Predisposição Genética para Doença , Leucócitos/efeitos dos fármacos , Leucócitos/patologia , Leucocitose/sangue , Leucocitose/enzimologia , Leucocitose/genética , Lipoproteínas LDL/farmacologia , Ativação de Macrófagos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Placa Aterosclerótica , Receptores de LDL/deficiência , Receptores de LDL/genéticaAssuntos
Aterosclerose/prevenção & controle , Aterosclerose/fisiopatologia , Hipolipemiantes/uso terapêutico , Lipoproteínas HDL/fisiologia , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Aterosclerose/sangue , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/fisiopatologia , Colesterol/metabolismo , Expressão Gênica , Humanos , Lipoproteínas HDL/sangue , Macrófagos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/deficiência , Coração/fisiopatologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/prevenção & controle , Animais , Aterosclerose/metabolismo , Linfócitos B/citologia , Transplante de Medula Óssea , Feminino , Leucócitos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/metabolismoRESUMO
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.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Macrófagos Peritoneais/patologia , Monócitos/patologia , Placa Aterosclerótica/patologia , Proteínas Proto-Oncogênicas c-hck/deficiência , Proteínas Proto-Oncogênicas/deficiência , Quinases da Família src/deficiência , Animais , Apoptose , Adesão Celular , Extensões da Superfície Celular/ultraestrutura , Células Cultivadas , Células Endoteliais , Proteínas da Matriz Extracelular/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Migração e Rolagem de Leucócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Placa Aterosclerótica/enzimologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-hck/genética , Proteínas Proto-Oncogênicas c-hck/fisiologia , Quimera por Radiação , Receptores de LDL/deficiência , Receptores de LDL/genética , Receptores de LDL/fisiologia , Migração Transendotelial e Transepitelial , Quinases da Família src/genética , Quinases da Família src/fisiologiaRESUMO
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.
Assuntos
Aterosclerose/genética , Aterosclerose/patologia , Células da Medula Óssea/metabolismo , Receptores de LDL/deficiência , Receptores de Esteroides/deficiência , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Aterosclerose/terapia , Biomarcadores , Colesterol/sangue , Colesterol/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Células Espumosas/patologia , Expressão Gênica , Mediadores da Inflamação/metabolismo , Contagem de Leucócitos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Gás de Mostarda , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Receptores de LDL/genética , Receptores de Esteroides/genética , Fatores de Tempo , Triglicerídeos/sangueRESUMO
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.
Assuntos
Aterosclerose/etiologia , Medula Óssea/metabolismo , Dieta , Epigênese Genética , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Contagem de Células Sanguíneas , Medula Óssea/patologia , Transplante de Medula Óssea , Metilação de DNA , Dieta Ocidental/efeitos adversos , Modelos Animais de Doenças , Hematopoese/genética , Leucócitos/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Placa Aterosclerótica/genética , Receptores de LDL/deficiência , Receptores de LDL/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Aterosclerose/genética , Hemorragia/genética , Neutrófilos/metabolismo , Placa Aterosclerótica/genética , Receptores CXCR4/genética , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Adesão Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Progressão da Doença , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Regulação da Expressão Gênica , Vetores Genéticos , Hemorragia/metabolismo , Hemorragia/patologia , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Camundongos , Camundongos Knockout , Neutrófilos/patologia , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Transdução de SinaisRESUMO
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.
Assuntos
Hidrocarbonetos Fluorados/uso terapêutico , Macrófagos/efeitos dos fármacos , Receptores Nucleares Órfãos/agonistas , Placa Aterosclerótica/tratamento farmacológico , Sulfonamidas/uso terapêutico , Animais , Apolipoproteínas E/genética , Transplante de Medula Óssea , Contagem de Células , VLDL-Colesterol/sangue , Dieta , Modelos Animais de Doenças , Feminino , Hidrocarbonetos Fluorados/administração & dosagem , Hidrocarbonetos Fluorados/farmacologia , Receptores X do Fígado , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Placa Aterosclerótica/sangue , Placa Aterosclerótica/patologia , Receptores de LDL/genética , Índice de Gravidade de Doença , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacologia , Triglicerídeos/sangueRESUMO
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.
Assuntos
Aterosclerose/metabolismo , Colesterol/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de LDL/deficiência , Receptores OX40/metabolismo , Fatores de Necrose Tumoral/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Células Apresentadoras de Antígenos/metabolismo , Proliferação de Células , Células Cultivadas , Fator de Transcrição GATA3/metabolismo , Imunoglobulina E/sangue , Imunoglobulina M/imunologia , Interleucina-33 , Interleucina-4/metabolismo , Interleucina-5/biossíntese , Interleucinas/biossíntese , Ativação Linfocitária/imunologia , Masculino , Mastócitos/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligante OX40 , Receptores de LDL/genética , Células Th2/imunologia , Fatores de Necrose Tumoral/imunologiaRESUMO
The CD40/CD40L dyad is deemed to play a central role in several inflammatory processes, including atherosclerosis. As CD40 is overexpressed in atherosclerotic lesions, it constitutes a promising candidate for targeted imaging approaches. Here we describe the design of a novel, selective peptide ligand for CD40 by phage display. A synthetic peptide corresponding with the phage insert NP31 displayed nanomolar affinity for CD40. Affinity was further enhanced by mutimeric presentation of NP31. An essential 11-mer peptide motif was identified by truncation and alanine scan studies. Enriched phage selectively bound human CD40 and homed to inflammatory joints in a murine model of rheumatoid arthritis. NP31 ablated VEGF and IL-6 transcriptional activation and partially inhibited IL-6 production by CD40L-activated endothelial cells. Notably, NP31 did not only alter the biodistribution profile of a streptavidin scaffold but also markedly increased accumulation of the carrier in atherosclerotic aortic lesions of aged ApoE(-/-) mice in a CD40-dependent manner. This potent and selective peptide ligand has potential for targeted imaging and drug delivery approaches in CD40-dependent inflammatory disorders such as atherosclerosis.
Assuntos
Ligante de CD40/metabolismo , Biblioteca de Peptídeos , Sequência de Aminoácidos , Animais , Aorta/patologia , Artrite Reumatoide/diagnóstico , Aterosclerose/metabolismo , Aterosclerose/patologia , Ligante de CD40/genética , Linhagem Celular , Humanos , Inflamação , Camundongos , Ligação ProteicaRESUMO
OBJECTIVE: In search of molecular imaging modalities for specific detection of inflammatory atherosclerotic plaques, we explored the potential of targeting scavenger receptor-AI (SR-AI), which is highly expressed by lesional macrophages and linked to effective internalization machinery. APPROACH AND RESULTS: Ultrasmall superparamagnetic iron oxide particles were conjugated to a peptidic SR-AI ligand (0.371 mol Fe/L and 0.018 mol PP1/L). In vitro incubation of human or murine macrophages with SR-AI-targeted USPIO led to significantly higher iron uptake in vitro than with nontargeted USPIO, as judged by quantitative atomic absorption spectroscopy and Perl's staining. Incremental uptake was strictly mediated by SRs. SR-AI-targeted USPIO displayed accelerated plasma decay and a 3.5-fold increase (P=0.01) in atherosclerotic plaque accumulation on intravenous injection into apolipoprotein E-deficient mice compared with nontargeted USPIO. In addition, atherosclerotic humanized LDLr(-/-) chimeras with leukocyte expression of human SR-AI showed a significant improvement in contrast-to-noise ratio (2.7-fold; P=0.003) in the atherosclerotic aortic arch plaques 24 hours after injection of SR-AI-targeted USPIO compared with chimeras with leukocyte SR-AI deficiency. CONCLUSIONS: Collectively, our data provide several lines of evidence that SR-AI-targeted molecular imaging of USPIO-based contrast agents holds great promise for in situ detection of inflammatory plaques in manifest atherosclerosis.
Assuntos
Dextranos , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Receptores Depuradores Classe A/metabolismo , Animais , Apolipoproteínas E/genética , Células Cultivadas , Dextranos/farmacocinética , Modelos Animais de Doenças , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Nanopartículas , Transdução de Sinais/fisiologia , Espectrofotometria Atômica/métodosRESUMO
AIMS: Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1(-/-)) deficiency on leukocyte subsets relevant to atherosclerosis. METHODS AND RESULTS: LDL receptor deficient mice that were transplanted with Sgpl1(-/-) bone marrow showed disrupted S1P gradients translating into lymphopenia and abrogated lymphocyte mitogenic and cytokine response as compared to controls. Remarkably however, Sgpl1(-/-) chimeras displayed mild monocytosis, due to impeded stromal retention and myelopoiesis, and plasma cytokine and macrophage expression patterns, that were largely compatible with classical macrophage activation. Collectively these two phenotypic features of Sgpl1 deficiency culminated in diminished atherogenic response. CONCLUSIONS: Here we not only firmly establish the critical role of hematopoietic S1P lyase in controlling S1P levels and T cell trafficking in blood and lymphoid tissue, but also identify leukocyte Sgpl1 as critical factor in monocyte macrophage differentiation and function. Its, partly counterbalancing, pro- and anti-inflammatory activity spectrum imply that intervention in S1P lyase function in inflammatory disorders such as atherosclerosis should be considered with caution.
Assuntos
Aldeído Liases/deficiência , Aterosclerose/enzimologia , Placa Aterosclerótica/enzimologia , Receptores de LDL/deficiência , Aldeído Liases/genética , Animais , Aterosclerose/imunologia , Aterosclerose/patologia , Células da Medula Óssea/enzimologia , Diferenciação Celular , Feminino , Hematopoese , Contagem de Linfócitos , Linfopenia/enzimologia , Linfopenia/imunologia , Lisofosfolipídeos/metabolismo , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/fisiologia , Camundongos , Camundongos Knockout , Neutrófilos/enzimologia , Fenótipo , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/patologia , Receptores de LDL/genética , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Baço/metabolismoRESUMO
BACKGROUND: The zinc finger protein Znf202 is a transcriptional suppressor of lipid related genes and has been linked to hypoalphalipoproteinemia. A functional role of Znf202 in lipid metabolism in vivo still remains to be established. METHODOLOGY AND PRINCIPAL FINDINGS: We generated mouse Znf202 expression vectors, the functionality of which was established in several in vitro systems. Next, effects of adenoviral znf202 overexpression in vivo were determined in normo- as well as hyperlipidemic mouse models. Znf202 overexpression in mouse hepatoma cells mhAT3F2 resulted in downregulation of members of the Apoe/c1/c2 and Apoa1/c3/a4 gene cluster. The repressive activity of Znf202 was firmly confirmed in an apoE reporter assay and Znf202 responsive elements within the ApoE promoter were identified. Adenoviral Znf202 transfer to Ldlr-/- mice resulted in downregulation of apoe, apoc1, apoa1, and apoc3 within 24 h after gene transfer. Interestingly, key genes in bile flux (abcg5/8 and bsep) and in bile acid synthesis (cyp7a1) were also downregulated. At 5 days post-infection, the expression of the aforementioned genes was normalized, but mice had developed severe hepatosteatosis accompanied by hypercholesterolemia and hypoalphalipoproteinemia. A much milder phenotype was observed in wildtype mice after 5 days of hepatic Znf202 overexpression. Interestingly and similar to Ldl-/- mice, HDL-cholesterol levels in wildtype mice were lowered after hepatic Znf202 overexpression. CONCLUSION/SIGNIFICANCE: Znf202 overexpression in vivo reveals an important role of this transcriptional regulator in liver lipid homeostasis, while firmly establishing the proposed key role in the control of HDL levels.
Assuntos
HDL-Colesterol/sangue , Fígado Gorduroso/complicações , Hiperlipidemias/complicações , Proteínas Repressoras/fisiologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Fígado Gorduroso/sangue , Hiperlipidemias/sangue , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Proteínas Repressoras/metabolismoRESUMO
Lysophosphatidic acid (LPA), a bioactive lysophospholipid, accumulates in the atherosclerotic plaque. It has the capacity to activate mast cells, which potentially exacerbates plaque progression. In this study, we thus aimed to investigate whether LPA contributes to plaque destabilization by modulating mast cell function. We here show by an imaging mass spectrometry approach that several LPA species are present in atherosclerotic plaques. Subsequently, we demonstrate that LPA is a potent mast cell activator which, unlike other triggers, favors release of tryptase. Local perivascular administration of LPA to an atherosclerotic carotid artery segment increases the activation status of perivascular mast cells and promotes intraplaque hemorrhage and macrophage recruitment without impacting plaque cell apoptosis. The mast cell stabilizer cromolyn could prevent intraplaque hemorrhage elicited by LPA-mediated mast cell activation. Finally, the involvement of mast cells in these events was further emphasized by the lack of effect of perivascular LPA administration in mast cell deficient animals. We demonstrate that increased accumulation of LPA in plaques induces perivascular mast cell activation and in this way contributes to plaque destabilization in vivo. This study points to local LPA availability as an important factor in atherosclerotic plaque stability.
Assuntos
Inflamação/metabolismo , Lisofosfolipídeos/metabolismo , Mastócitos/metabolismo , Placa Aterosclerótica/patologia , Animais , Apoptose/efeitos dos fármacos , Artérias Carótidas/diagnóstico por imagem , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Células Cultivadas , Cromolina Sódica/farmacologia , Inflamação/patologia , Macrófagos/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/patologia , Camundongos , Placa Aterosclerótica/diagnóstico por imagem , Placa Aterosclerótica/metabolismo , Radiografia , Triptases/metabolismoRESUMO
OBJECTIVE: Despite common disbelief that neutrophils are involved in atherosclerosis, evidence is accumulating for a causal role of neutrophils in atherosclerosis. CC chemokine ligand (CCL)3 is an inflammatory chemokine and its expression is significantly increased during atherosclerotic lesion formation in mice. It has recently been shown that under conditions of inflammation neutrophils can migrate along a CCL3 gradient. In this study, we aimed to elucidate the role of leukocyte-derived CCL3 in atherogenesis. METHODS AND RESULTS: Irradiated low density lipoprotein receptor(-/-) mice, reconstituted with CCL3(-/-) or littermate bone marrow showed markedly reduced CCL3 response to lipopolysaccharide treatment, establishing the critical relevance of leukocytes as source of CCL3. Hematopoietic deficiency of CCL3 significantly reduced aortic sinus lesion formation by 31% after 12 weeks of western-type diet. Interestingly, whereas plaque macrophage, collagen, and vascular smooth muscle cell content were unchanged, neutrophil adhesion to and presence in plaques was significantly attenuated in CCL3(-/-) chimeras. These mice had reduced circulating neutrophil numbers, which could be ascribed to an increased neutrophil turnover and CCL3(-/-) neutrophils were shown to be less responsive toward the neutrophil chemoattractant CXC chemokine ligand 1. CONCLUSIONS: Our data indicate that under conditions of acute inflammation leukocyte-derived CCL3 can induce neutrophil chemotaxis toward the atherosclerotic plaque, thereby accelerating lesion formation.