RESUMEN
RATIONALE: The high morbidity/mortality of atherosclerosis is typically precipitated by plaque rupture and consequent thrombosis. However, research on underlying mechanisms and therapeutic approaches is limited by the lack of animal models that reproduce plaque instability observed in humans. OBJECTIVE: Development and use of a mouse model of plaque rupture that reflects the end stage of human atherosclerosis. METHODS AND RESULTS: On the basis of flow measurements and computational fluid dynamics, we applied a tandem stenosis to the carotid artery of apolipoprotein E-deficient mice on high-fat diet. At 7 weeks postoperatively, we observed intraplaque hemorrhage in ≈50% of mice, as well as disruption of fibrous caps, intraluminal thrombosis, neovascularization, and further characteristics typically seen in human unstable plaques. Administration of atorvastatin was associated with plaque stabilization and downregulation of monocyte chemoattractant protein-1 and ubiquitin. Microarray profiling of mRNA and microRNA (miR) and, in particular, its combined analysis demonstrated major differences in the hierarchical clustering of genes and miRs among nonatherosclerotic arteries, stable, and unstable plaques and allows the identification of distinct genes/miRs, potentially representing novel therapeutic targets for plaque stabilization. The feasibility of the described animal model as a discovery tool was established in a pilot approach, identifying a disintegrin and metalloprotease with thrombospondin motifs 4 (ADAMTS4) and miR-322 as potential pathogenic factors of plaque instability in mice and validated in human plaques. CONCLUSIONS: The newly described mouse model reflects human atherosclerotic plaque instability and represents a discovery tool toward the development and testing of therapeutic strategies aimed at preventing plaque rupture. Distinctly expressed genes and miRs can be linked to plaque instability.
Asunto(s)
Estenosis Carotídea/genética , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica/métodos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , MicroARNs/genética , Placa Aterosclerótica/genética , Animales , Estenosis Carotídea/tratamiento farmacológico , Estenosis Carotídea/patología , Dieta Alta en Grasa/efectos adversos , Evaluación Preclínica de Medicamentos/métodos , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/biosíntesis , Placa Aterosclerótica/tratamiento farmacológico , Placa Aterosclerótica/patologíaRESUMEN
OBJECTIVE: Atherosclerosis is driven by inflammatory reactions that are shared with the innate immune system. Toll-like receptor-9 (TLR9) is an intracellular pattern recognition receptor of the innate immune system that is currently under clinical investigation as a therapeutic target in inflammatory diseases. Here, we investigated whether TLR9 has a role in the development of atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. APPROACH AND RESULTS: Newly generated double-knockout ApoE(-/-):TLR9(-/-) mice and control ApoE(-/-) mice were fed a high-fat diet from 8 weeks and effects on lesion size, cellular composition, inflammatory status, and plasma lipids were assessed after 8, 12, 15, and 20 weeks. All 4 time points demonstrated exacerbated atherosclerotic lesion severity in ApoE(-/-):TLR9(-/-) mice, with a corresponding increase in lipid deposition and accumulation of macrophages, dendritic cells, and CD4(+) T cells. Although ApoE(-/-):TLR9(-/-) mice exhibited an increase in plasma very low-density lipoprotein/low-density-lipoprotein cholesterol, the very low-density lipoprotein/low-density lipoprotein:high-density lipoprotein ratio was unaltered because of a parallel increase in plasma high-density lipoprotein cholesterol. As a potential mechanism accounting for plaque progression in ApoE(-/-):TLR9(-/-) mice, CD4(+) T-cell accumulation was further investigated and depletion of these cells in ApoE(-/-):TLR9(-/-) mice significantly reduced lesion severity. As a final translational approach, administration of a TLR9 agonist (type B CpG oligodeoxynucleotide 1668) to ApoE(-/-) mice resulted in a reduction of lesion severity. CONCLUSIONS: Genetic deletion of the innate immune receptor TLR9 exacerbated atherosclerosis in ApoE(-/-) mice fed a high-fat diet. CD4(+) T cells were identified as potential mediators of this effect. A type B CpG oligodeoxynucleotide TLR9 agonist reduced lesion severity, thus identifying a novel therapeutic approach in atherosclerosis.
Asunto(s)
Aterosclerosis/prevención & control , Receptor Toll-Like 9/fisiología , Animales , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Apolipoproteínas E/deficiencia , Aterosclerosis/sangre , Aterosclerosis/etiología , Aterosclerosis/inmunología , Aterosclerosis/patología , Linfocitos T CD4-Positivos/patología , Células Cultivadas , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Grasas de la Dieta/toxicidad , Progresión de la Enfermedad , Lipoproteínas HDL/sangre , Lipoproteínas VLDL/sangre , Macrófagos Peritoneales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oligodesoxirribonucleótidos/farmacología , Oligodesoxirribonucleótidos/uso terapéutico , Distribución Aleatoria , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/deficiencia , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/inmunologíaRESUMEN
PreImplantation factor (PIF) is a 15-amino acid peptide endogenously secreted by viable embryos, regulating/enabling maternal (host) acceptance/tolerance to the "invading" embryo (allograft) all-while preserving maternal immunity to fight infections. Such attributes make PIF a potential therapeutic agent for chronic inflammatory diseases. We investigated whether PIF's immunomodulatory properties prevent progression of atherosclerosis in the hyper-cholesterolaemic ApoE-deficient murine model. Male, high-fat diet fed, ApoE-deficient (ApoE-/-) mice were administered either PBS, scrambled PIF (0.3-3 mg/kg) or PIF (0.3-3 mg/kg) for seven weeks. After treatment, PIF (3 mg/kg)-treated ApoE-/- mice displayed significantly reduced atherosclerosis lesion burden in the aortic sinus and aortic arch, without any effect on lipid profile. PIF also caused a significant reduction in infiltration of macrophages, decreased expression of pro-inflammatory adhesion molecules, cytokines and chemokines in the plaque, and reduced circulating IFN-γ levels. PIF preferentially binds to monocytes/neutrophils. In vitro, PIF attenuated monocyte migration (MCP-1-induced chemotaxis assay) and in vivo in LPS peritonitis model. Also PIF prevented leukocyte extravasation (peritonitis thioglycollate-induced model), demonstrating that PIF exerts its effect in part by modulation of monocyte function. Inhibition of the potassium channel KCNAB3 (Kv1.3) and of the insulin degrading enzyme (IDE) was demonstrated as potential mechanism of PIF's immunomodulatory effects. In conclusion, PIF regulates/lowers inflammation and prevents atherosclerosis development without affecting circulating lipids. Overall our findings establish PIF as a strong immunomodulatory drug candidate for atherosclerosis therapy.