RESUMO
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.
Assuntos
Doenças Cardiovasculares , Dislipidemias , Humanos , Doenças Cardiovasculares/terapia , Doenças Cardiovasculares/tratamento farmacológico , Edição de Genes , Dislipidemias/tratamento farmacológico , Dislipidemias/genética , Hipolipemiantes/uso terapêutico , HepatócitosRESUMO
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
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.
Assuntos
Aterosclerose/prevenção & controle , HDL-Colesterol/sangue , Metabolismo Energético , Fígado/metabolismo , Síndrome Metabólica/sangue , Proteínas de Transferência de Fosfolipídeos/deficiência , Receptores Depuradores Classe B/deficiência , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/patologia , Ésteres do Colesterol/administração & dosagem , Ésteres do Colesterol/sangue , Modelos Animais de Doenças , Feminino , Intolerância à Glucose/sangue , Intolerância à Glucose/genética , Hipertrigliceridemia/sangue , Hipertrigliceridemia/genética , Masculino , Síndrome Metabólica/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/sangue , Obesidade/genética , Proteínas de Transferência de Fosfolipídeos/genética , Placa Aterosclerótica , Receptores Depuradores Classe B/genéticaRESUMO
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.
Assuntos
Acetilgalactosamina , Receptor de Asialoglicoproteína/genética , Regulação da Expressão Gênica , Interferência de RNA , RNA Interferente Pequeno/genética , Acetilgalactosamina/química , Animais , Receptor de Asialoglicoproteína/química , Receptor de Asialoglicoproteína/metabolismo , Modelos Animais de Doenças , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Feminino , Inativação Gênica , Hepatócitos/metabolismo , Humanos , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Camundongos Knockout , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/químicaRESUMO
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
RATIONALE: RNA-binding proteins are critical post-transcriptional regulators of RNA and can influence pre-mRNA splicing, RNA localization, and stability. The RNA-binding protein Quaking (QKI) is essential for embryonic blood vessel development. However, the role of QKI in the adult vasculature, and in particular in vascular smooth muscle cells (VSMCs), is currently unknown. OBJECTIVE: We sought to determine the role of QKI in regulating adult VSMC function and plasticity. METHODS AND RESULTS: We identified that QKI is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Quaking viable mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering noncontractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome, where it interacts with the myocardin pre-mRNA and regulates the splicing of alternative exon 2a. This post-transcriptional event impacts the Myocd_v3/Myocd_v1 mRNA balance and can be modulated by mutating the quaking response element in exon 2a of myocardin. Furthermore, we identified that arterial damage triggers myocardin alternative splicing and is tightly coupled with changes in the expression levels of distinct QKI isoforms. CONCLUSIONS: We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.
Assuntos
Proliferação de Células , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Animais , Lesões das Artérias Carótidas/metabolismo , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Movimento Celular , Reestenose Coronária/metabolismo , Reestenose Coronária/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Hiperplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Quaking , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Neointima , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Interferência de RNA , Proteínas de Ligação a RNA/genética , Transativadores/genética , Transativadores/metabolismo , TransfecçãoRESUMO
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
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
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.
Assuntos
Acetilgalactosamina/química , Inativação Gênica , Hepatócitos/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Animais , Camundongos , Camundongos Endogâmicos C57BL , Estrutura MolecularRESUMO
BACKGROUND: In mice, the scavenger receptor class B type I (SR-BI) is essential for the delivery of high-density lipoprotein (HDL) cholesterol to the liver and steroidogenic organs. Paradoxically, elevated HDL cholesterol levels are associated with increased atherosclerosis in SR-BI-knockout mice. It is unclear what role SR-BI plays in human metabolism. METHODS: We sequenced the gene encoding SR-BI in persons with elevated HDL cholesterol levels and identified a family with a new missense mutation (P297S). The functional effects of the P297S mutation on HDL binding, cellular cholesterol uptake and efflux, atherosclerosis, platelet function, and adrenal function were studied. RESULTS: Cholesterol uptake from HDL by primary murine hepatocytes that expressed mutant SR-BI was reduced to half of that of hepatocytes expressing wild-type SR-BI. Carriers of the P297S mutation had increased HDL cholesterol levels (70.4 mg per deciliter [1.8 mmol per liter], vs. 53.4 mg per deciliter [1.4 mmol per liter] in noncarriers; P<0.001) and a reduced capacity for efflux of cholesterol from macrophages, but the carotid artery intima-media thickness was similar in carriers and in family noncarriers. Platelets from carriers had increased unesterified cholesterol content and impaired function. In carriers, adrenal steroidogenesis was attenuated, as evidenced by decreased urinary excretion of sterol metabolites, a decreased response to corticotropin stimulation, and symptoms of diminished adrenal function. CONCLUSIONS: We identified a family with a functional mutation in SR-BI. The mutation carriers had increased HDL cholesterol levels and a reduction in cholesterol efflux from macrophages but no significant increase in atherosclerosis. Reduced SR-BI function was associated with altered platelet function and decreased adrenal steroidogenesis. (Funded by the European Community and others.).
Assuntos
Insuficiência Adrenal/genética , Aterosclerose/genética , HDL-Colesterol/sangue , Colesterol/metabolismo , Mutação de Sentido Incorreto , Receptores Depuradores Classe B/genética , Adolescente , Glândulas Suprarrenais/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Artérias Carótidas/anatomia & histologia , Colesterol/sangue , Análise Mutacional de DNA , Feminino , Heterozigoto , Homeostase/genética , Humanos , Hidrocortisona/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Linhagem , Ativação Plaquetária/genética , Triglicerídeos/sangue , Adulto JovemRESUMO
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
Leukocyte chemotaxis is deemed instrumental in initiation and progression of atherosclerosis. It is mediated by G-protein-coupled receptors (e.g., CCR2 and CCR5), the activity of which is controlled by G-protein-coupled receptor kinases (GRKs). In this study, we analyzed the effect of hematopoietic deficiency of a potent regulator kinase of chemotaxis (GRK2) on atherogenesis. LDL receptor-deficient (LDLr(-/-)) mice with heterozygous hematopoietic GRK2 deficiency, generated by bone marrow transplantation (n=15), displayed a dramatic attenuation of plaque development, with 79% reduction in necrotic core and increased macrophage content. Circulating monocytes decreased and granulocytes increased in GRK2(+/-) chimeras, which could be attributed to diminished granulocyte colony-forming units in bone marrow. Collectively, these data pointed to myeloid cells as major mediators of the impaired atherogenic response in GRK2(+/-) chimeras. LDLr(-/-) mice with macrophage/granulocyte-specific GRK2 deficiency (LysM-Cre GRK2(flox/flox); n=8) failed to mimic the aforementioned phenotype, acquitting these cells as major responsible subsets for GRK2 deficiency-associated atheroprotection. To conclude, even partial hematopoietic GRK2 deficiency prevents atherosclerotic lesion progression beyond the fatty streak stage, identifying hematopoietic GRK2 as a potential target for intervention in atherosclerosis.
Assuntos
Aterosclerose/patologia , Quinase 2 de Receptor Acoplado a Proteína G/genética , Receptores de LDL/genética , Animais , Apoptose , Aterosclerose/genética , Feminino , Citometria de Fluxo , Camundongos , Camundongos Knockout , FagocitoseRESUMO
RATIONALE: Nuclear factor of activated T-cells (NFAT) is importantly implicated in pathological cardiac remodeling and vascular lesion formation. NFAT functionality is mainly regulated by calcineurin, a Ca(2+)-dependent multi-effector phosphatase. Calcineurin inhibitors such as cyclosporine A (CsA) were shown to be effective in the treatment of restenosis and vascular inflammation but with adverse side effects. OBJECTIVE: This prompted the design of more selective inhibitors such as VIVIT and inhibitors of NFAT-calcineurin association, which unfortunately have a poor potency precluding clinical use. METHODS AND RESULTS: Here, we describe the rational design of a potent bipartite inhibitor of NFAT-calcineurin interaction, MCV1, which targets two separate calcineurin docking motifs. Modeling, site-directed mutagenesis, and functional studies demonstrated that MCV1 acts by allosteric modulation of calcineurin. Comparable to CsA, MCV1 prevents NFAT activation at nanomolar potency without impairing calcineurin phosphatase activity, nuclear factor-κB nuclear import, and general cell signaling. In contrast, CsA but not MCV1-activated basal level extracellular signal-regulated kinases activity and prevented nuclear import of calcineurin, independent of NFAT activation. In vivo MCV1 abrogated NFAT-mediated T-cell activation in a model of PMA-elicited peritonitis, whereas topical application of MCV1 markedly reduced neointima formation in a mouse model of restenosis. CONCLUSIONS: We designed a bipartite NFAT inhibitor that is more potent than VIVIT and more selective than CsA. MCV1 constitutes not only a powerful tool to unravel NFAT function but also a potential drug candidate for the treatment of diseases implicating NFAT activation.
Assuntos
Lesões das Artérias Carótidas/tratamento farmacológico , Artéria Carótida Primitiva/efeitos dos fármacos , Estenose das Carótidas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Fatores de Transcrição NFATC/antagonistas & inibidores , Peptídeos/farmacologia , Peritonite/tratamento farmacológico , Linfócitos T/efeitos dos fármacos , Motivos de Aminoácidos , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Células COS , Calcineurina/metabolismo , Lesões das Artérias Carótidas/imunologia , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Artéria Carótida Primitiva/imunologia , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Estenose das Carótidas/imunologia , Estenose das Carótidas/metabolismo , Estenose das Carótidas/patologia , Chlorocebus aethiops , Ciclosporina/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Desenho de Fármacos , Células HEK293 , Humanos , Hiperplasia , Imunossupressores/química , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Peptídeos/química , Peritonite/imunologia , Peritonite/metabolismo , Recidiva , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Linfócitos T/imunologia , Linfócitos T/metabolismo , TransfecçãoRESUMO
OBJECTIVE: We determined the physiological consequences of adrenocortical-specific deletion of scavenger receptor BI (SR-BI) function in C57BL/6 wild-type mice. METHODS AND RESULTS: One adrenal from 10-day-old SR-BI knockout (KO) mice or wild-type controls was transplanted under the renal capsule of adrenalectomized C57BL/6 recipient mice. The fasting plasma corticosterone level increased over time in transplanted mice. Corticosterone values in SR-BI KO transplanted mice remained ≈50% lower (P<0.001) as compared with wild-type transplanted mice, which coincided with adrenocortical lipid depletion. A 6.5-fold higher (P<0.01) plasma adrenocorticotropic hormone level was present in SR-BI KO transplanted mice reminiscent of primary glucocorticoid insufficiency. On feeding with cholic acid-containing high cholesterol/high fat diet, SR-BI KO transplanted mice exhibited a 26% (P<0.05) reduction in their liver triglyceride level. Hepatic myosin regulatory light chain interacting protein/inducible degrader of the low-density lipoprotein receptor mRNA expression was 48% (P<0.01) decreased in adrenal-specific SR-BI KO mice, which was paralleled by a marked decrease (-46%; P<0.01) in proatherogenic very-low-density and low-density lipoprotein levels. CONCLUSIONS: Adrenal-specific disruption of SR-BI function induces glucocorticoid insufficiency and lowers plasma very-low-density and low-density lipoprotein levels in atherogenic diet-fed C57BL/6 mice. These findings further highlight the interaction between adrenal high-density lipoprotein-cholesterol uptake by SR-BI, adrenal steroidogenesis, and the regulation of hepatic lipid metabolism.
Assuntos
Glândulas Suprarrenais/metabolismo , Insuficiência Adrenal/metabolismo , Corticosterona/deficiência , Lipoproteínas LDL/sangue , Lipoproteínas VLDL/sangue , Receptores Depuradores Classe B/deficiência , Glândulas Suprarrenais/transplante , Insuficiência Adrenal/sangue , Insuficiência Adrenal/genética , Adrenalectomia , Hormônio Adrenocorticotrópico/sangue , Animais , Colesterol na Dieta/sangue , Corticosterona/sangue , Dieta Aterogênica , Regulação para Baixo , Jejum/sangue , Feminino , Regulação da Expressão Gênica , Genótipo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , RNA Mensageiro/metabolismo , Receptores Depuradores Classe B/genética , Transdução de Sinais , Fatores de Tempo , Triglicerídeos/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.
Assuntos
Doenças das Artérias Carótidas/prevenção & controle , Artéria Carótida Primitiva/imunologia , Quimiocina CCL3/deficiência , Quimiotaxia de Leucócito , Leucócitos/imunologia , Infiltração de Neutrófilos , Neutrófilos/imunologia , Animais , Apoptose , Transplante de Medula Óssea , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/imunologia , Doenças das Artérias Carótidas/patologia , Artéria Carótida Primitiva/patologia , Adesão Celular , Células Cultivadas , Quimiocina CCL3/genética , Quimiocina CXCL1/metabolismo , Ciclofosfamida , Gorduras na Dieta , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutropenia/induzido quimicamente , Neutropenia/imunologia , Placa Aterosclerótica , RNA Mensageiro/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Fatores de Tempo , Irradiação Corporal TotalRESUMO
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
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.
Assuntos
Aterosclerose , Quimiocinas CC , Receptores CCR6 , Animais , Humanos , Aterosclerose/imunologia , Aterosclerose/metabolismo , Camundongos , Receptores CCR6/metabolismo , Receptores CCR6/genética , Quimiocinas CC/metabolismo , Quimiocinas CC/genética , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Células Jurkat , Placa Aterosclerótica/imunologia , Camundongos Knockout , Masculino , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Feminino , Camundongos Knockout para ApoERESUMO
In vitro studies have suggested that HDL and apoB-containing lipoproteins can provide cholesterol for synthesis of glucocorticoids. Here we assessed adrenal glucocorticoid function in LCAT knockout (KO) mice to determine the specific contribution of HDL-cholesteryl esters to adrenal glucocorticoid output in vivo. LCAT KO mice exhibit an 8-fold higher plasma free cholesterol-to-cholesteryl ester ratio (P < 0.001) and complete HDL-cholesteryl ester deficiency. ApoB-containing lipoprotein and associated triglyceride levels are increased in LCAT KO mice as compared with C57BL/6 control mice (44%; P < 0.05). Glucocorticoid-producing adrenocortical cells within the zona fasciculata in LCAT KO mice are devoid of neutral lipids. However, adrenal weights and basal corticosterone levels are not significantly changed in LCAT KO mice. In contrast, adrenals of LCAT KO mice show compensatory up-regulation of genes involved in cholesterol synthesis (HMG-CoA reductase; 516%; P < 0.001) and acquisition (LDL receptor; 385%; P < 0.001) and a marked 40-50% lower glucocorticoid response to adrenocorticotropic hormone exposure, endotoxemia, or fasting (P < 0.001 for all). In conclusion, our studies show that HDL-cholesteryl ester deficiency in LCAT KO mice is associated with a 40-50% lower adrenal glucocorticoid output. These findings further highlight the important novel role for HDL as cholesterol donor for the synthesis of glucocorticoids by the adrenals.
Assuntos
Glândulas Suprarrenais/metabolismo , Técnicas de Inativação de Genes , Glucocorticoides/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Animais , Ésteres do Colesterol/metabolismo , Corticosterona/metabolismo , Hepatócitos/metabolismo , Lipoproteínas HDL/metabolismo , Camundongos , Regulação para CimaRESUMO
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
Cholesteryl ester transfer protein (CETP) activity results in a proatherogenic lipoprotein profile. In cholestatic conditions, farnesoid X receptor (FXR) signaling by bile acids (BA) is activated and plasma HDL cholesterol (HDL-C) levels are low. This study tested the hypothesis that FXR-mediated induction of CETP contributes to this phenotype. Patients with cholestasis and high plasma BA had lower HDL-C levels and higher plasma CETP activity and mass compared with matched controls with low plasma BA (each P < 0.01). BA feeding in APOE3*Leiden transgenic mice expressing the human CETP transgene controlled by its endogenous promoter increased cholesterol within apoB-containing lipoproteins and decreased HDL-C (each P < 0.01), while hepatic CETP mRNA expression and plasma CETP activity and mass increased (each P < 0.01). In vitro studies confirmed that FXR agonists substantially augmented CETP mRNA expression in hepatocytes and macrophages dependent on functional FXR expression (each P < 0.001). These transcriptional effects are likely mediated by an ER8 FXR response element (FXRE) in the first intron. In conclusion, using a translational approach, this study identifies CETP as novel FXR target gene. By increasing CETP expression, FXR activation leads to a proatherogenic lipoprotein profile. These results have clinical relevance, especially when considering FXR agonists as emerging treatment strategy for metabolic disease and atherosclerosis.