RESUMO
OBJECTIVE: The cAMP second messenger system, a major stress-response pathway, plays essential roles in normal cardiovascular functions and in pathogenesis of heart diseases. Here, we test the hypothesis that the Epac1 (exchange protein directly activated by cAMP 1) acts as a major downstream effector of cAMP signaling to promote atherogenesis and represents a novel therapeutic target. Approach and Results: To ascertain Epac1's function in atherosclerosis development, a triple knockout mouse model (LTe) was generated by crossing Epac1-/- mice with atherosclerosis-prone LDb mice lacking both Ldlr and Apobec1. Deletion of Epac1 led to a significant reduction of atherosclerotic lesion formation as measured by postmortem staining, accompanied by attenuated macrophage/foam cell infiltrations within atherosclerotic plaques as determined by immunofluorescence staining in LTe animals compared with LDb littermates. Primary bone marrow-derived macrophages were isolated from Epac1-null and wild-type mice to investigate the role of Epac1 in lipid uptake and foam cell formation. ox-LDLs (oxidized low-density lipoproteins) stimulation of bone marrow-derived macrophages led to elevated intracellular cAMP and Epac1 levels, whereas an Epac-specific agonist, increased lipid accumulation in wild-type, but not Epac1-null, bone marrow-derived macrophages. Mechanistically, Epac1 acts through PKC (protein kinase C) to upregulate LOX-1 (ox-LDL receptor 1), a major scavenger receptor for ox-LDL uptake, exerting a feedforward mechanism with ox-LDL to increase lipid uptake and propel foam cell formation and atherogenesis. CONCLUSIONS: Our study demonstrates a fundamental role of cAMP/Epac1 signaling in vascular remodeling by promoting ox-LDL uptake and foam cell formation during atherosclerosis lesion development. Therefore, Epac1 represents a promising, unexplored therapeutic target for atherosclerosis.
Assuntos
Aorta Torácica/metabolismo , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Células Espumosas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Placa Aterosclerótica , Receptores Depuradores Classe E/metabolismo , Desaminase APOBEC-1/deficiência , Desaminase APOBEC-1/genética , Animais , Aorta Torácica/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/patologia , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Células Espumosas/patologia , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/deficiência , Receptores de LDL/genética , Sistemas do Segundo Mensageiro , Células THP-1 , Remodelação VascularRESUMO
BACKGROUND/AIMS: Oxidative modifications of low-density lipoprotein (ox-LDL) play a key role in initial steps of atheroprogression possibly via specific scavenger receptors on inflammatory and endothelial cells. Amongst others, CD68 might play a crucial role in this leading to fatty streak formation. METHODS: Different CD68-Fc fusion proteins were cloned, expressed and tested in vitro for their oxLDL binding properties as a decoy for endogenous oxLDL. Physiological functions were tested in foam cell assays with human monocytes in culture and by binding oxLDL from human blood. The best suited candidate FcIgG2-FL-CD68 was injected twice weekly in LDL receptor and ApoBec deficient mice (LDLR-/-/Apobec-/-), and the oxLDL content was measured in peripheral blood, in different cell types of the spleen and aortic wall by specific oxLDL antibodies using flow cytometry. RESULTS: Different variants of the CD68-Fc bound to copper-oxided LDL (oxLDL), LDL and to a lesser extent HDL with different efficacy in an ELISA based binding assay in vitro. Native oxLDL content in human blood derived from patients with extended atherosclerosis was reduced after passage through a specific protein G column conjugated with the different CD68-Fc fusion proteins. Foam cell formation from human peripheral blood monocyte-platelet co-culture was reduced by the most effective CD68-Fc fusion proteins. oxLDL was not increased in the blood but markedly increased in the vessel wall from LDLR-/-/Apobec-/- mice at an early stage of atherosclerosis. Platelet-like cells in the vessel well contributed most to the increase in tissue oxLDL. FcIgG2-FL-CD68, reduced oxLDL content of aortic vessel wall cells from LDLR-/-/Apobec-/- mice. However a tissue specific reduction on the oxLDL content in peripheral blood, the spleen or cells from the aortic vessel by FcIgG2-FL-CD68 could not be shown. CONCLUSION: Platelets contribute to increased tissue oxLDL in the aortic wall but not in peripheral blood. CD68 seems to play a role in the oxLDL metabolism in the vessel wall at early stages of atherosclerosis. FcIgG2-FL-CD68 could serve as a novel therapeutic option to modify the oxLDL content in the vessel wall.
Assuntos
Desaminase APOBEC-1/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Plaquetas/metabolismo , Lipoproteínas LDL/genética , Desaminase APOBEC-1/deficiência , Animais , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Plaquetas/citologia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Modelos Animais de Doenças , Células Espumosas/citologia , Células Espumosas/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/análise , Lipoproteínas LDL/deficiência , Lipoproteínas LDL/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
BACKGROUND: Plasma low-density lipoprotein (LDL) cholesterol is implicated in abdominal aorta (AA) and aortic dissection (AD); however, its role in the pathogenesis of AA and AD, a disease with a high mortality rate, is unknown. The existing animal models such as apolipoprotein E-deficient (Apoe-/-) mice cannot reproduce all the conditions of AA/AD, including elevated LDL-cholesterol levels and spontaneous atheroma formation; therefore, a more reliable in vivo model is required. Here, we analyzed angiotensin II (Ang II)-induced mice with combined deficiency of the LDL receptor and the catalytic component of the apolipoprotein B-edisome complex (Ldlr-/-/Apobec1-/- [WKO]) to understand AA formation and AD occurrence in relation to plasma lipid composition. METHODS: AAs and ADs were created in 18- to 22- week-old male Apoe-/- and Ldlr-/-/Apobec1-/- mice by Ang II infusion. Immunostaining allowed assessment of smooth muscle cells and mural monocytes/macrophages. RESULTS: Ldlr-/-/Apobec1-/- mice had elevated LDL-cholesterol levels characteristic for human type IIa hyperlipidemia, resulting in atherogenesis, which promoted mortality, AA formation, and AD development. Interestingly, variations in the distribution of atheromas and inflammatory sites between Apoe-/- and Ldlr-/-/Apobec1-/- mice depending on lipid profiles resulted in differences in AA formation and AD occurrence in the thoracic aorta. CONCLUSIONS: Our results indicate the presence of a pathogenic pathway involving serum lipid composition that plays a key role in AA formation and AD occurrence in Ang II-induced mice.
Assuntos
Angiotensina II , Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/induzido quimicamente , LDL-Colesterol/sangue , Hipercolesterolemia/sangue , Desaminase APOBEC-1/deficiência , Desaminase APOBEC-1/genética , Animais , Aorta Torácica/patologia , Aneurisma da Aorta Torácica/sangue , Aneurisma da Aorta Torácica/patologia , Biomarcadores/sangue , Modelos Animais de Doenças , Predisposição Genética para Doença , Hipercolesterolemia/complicações , Hipercolesterolemia/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Fenótipo , Placa Aterosclerótica , Receptores de LDL/deficiência , Receptores de LDL/genética , Fatores de Tempo , Regulação para CimaRESUMO
The homozygous form of familial hypercholesterolemia (HoFH) is an excellent model for developing in vivo gene therapy in humans. The success of orthotropic liver transplantation in correcting the metabolic abnormalities in HoFH suggests that the correction of low-density lipoprotein receptor (LDLR) expression in hepatocytes via gene therapy should be sufficient for therapeutic efficacy. Vectors based on adeno-associated virus serotype 8 (AAV8) have been previously developed for liver-targeted gene therapy of a number of genetic diseases, including HoFH. In preparation for initiating a Phase 1 clinical trial of AAV8-mediated LDLR gene therapy for HoFH, a combined pharmacology/toxicology study was conducted in a mouse model of HoFH. No dose-limiting toxicities were found at or below 6.0 × 1013 GC/kg. Therefore, the maximally tolerated dose is greater than the highest dose that was tested. Mild and transient liver pathology was noted at the highest dose. Therefore, the no effect dose was greater than or equal to the middle dose of 7.5 × 1012 GC/kg. The minimally effective dose was determined to be ≤7.5 × 1011 GC/kg, based on stable reductions in cholesterol that were considered to be clinically significant. This translates to a therapeutic window of ≥80-fold for the treatment of HoFH.
Assuntos
Desaminase APOBEC-1/genética , Dependovirus/genética , Terapia Genética , Vetores Genéticos/administração & dosagem , Hiperlipoproteinemia Tipo II/terapia , Fígado/patologia , Receptores de LDL/genética , Desaminase APOBEC-1/deficiência , Animais , Colesterol/sangue , Modelos Animais de Doenças , Feminino , Vetores Genéticos/toxicidade , Homozigoto , Humanos , Hiperlipoproteinemia Tipo II/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/deficiênciaRESUMO
Vectors based on adeno-associated virus serotype 8 (AAV8) have been evaluated in several clinical trials of gene therapy for hemophilia B with encouraging results. In preparation for a Phase 1 clinical trial of AAV8 gene therapy for the treatment of homozygous familial hypercholesterolemia (HoFH), the safety of the clinical candidate vector, AAV8.TBG.hLDLR, was evaluated in wild-type rhesus macaques and macaques heterozygous for a nonsense mutation in the low-density lipoprotein receptor (LDLR) gene (LDLR+/-). Intravenous infusion of 1.25 × 1013 GC/kg of AAV8.TBG.hLDLR expressing the human version of LDLR was well tolerated and associated with only mild histopathology that was restricted to the liver and sporadic, low-level, and transient elevations in transaminases. Some animals developed T cells to both capsid and the hLDLR transgene, although these adaptive immune responses were most evident at the early time points from peripheral blood and in mononuclear cells derived from the liver. This toxicology study supports the safety of AAV8.TBG.hLDLR for evaluation in HoFH patients, and provides some context for evaluating previously conducted clinical trials of AAV8 in patients with hemophilia.