RESUMO
OBJECTIVE: Susceptibility to atherosclerosis is genetically complex, and modifier genes that do not operate via traditional risk factors are largely unknown. A mouse genetics approach can simplify the genetic analysis and provide tools for mechanistic studies. METHODS AND RESULTS: We previously identified atherosclerosis susceptibility QTL (Athsq1) on chromosome 4 acting independently of systemic risk factors. We now report confirmation of this locus in congenic strains carrying the MOLF-derived susceptibility allele in the C57BL/6J-Ldlr(-/-) genetic background. Homozygous congenic mice exhibited up to 4.5-fold greater lesion area compared to noncongenic littermates (P<0.0001). Analysis of extracellular matrix composition revealed prominent accumulation of versican, a presumed proatherogenic matrix component abundant in human lesions but almost absent in the widely-used C57BL/6 murine atherosclerosis model. The results of a bone marrow transplantation experiment suggested that both accelerated lesion development and versican accumulation are mediated, at least in part, by macrophages. Interestingly, comparative mapping revealed that the Athsq1 congenic interval contains the mouse region homologous to a widely-replicated CHD locus on human chromosome 9p21. CONCLUSIONS: These studies confirm the proatherogenic activity of a novel gene(s) in the MOLF-derived Athsq1 locus and provide in vivo evidence for a causative role of versican in lesion development.
Assuntos
Aterosclerose/genética , Aterosclerose/metabolismo , Locos de Características Quantitativas , Versicanas/metabolismo , Animais , Aterosclerose/etiologia , Transplante de Medula Óssea , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Especificidade da Espécie , Versicanas/genéticaRESUMO
BACKGROUND: The formation of an occluding thrombus on a ruptured or eroded atherosclerotic plaque is the hallmark event leading to acute coronary syndromes, myocardial infarction, and sudden death in humans. However, other species are highly resistant to plaque complications, and the specific processes predisposing to plaque destabilization and thrombosis are poorly understood. METHODS AND RESULTS: Mice carrying a null mutation of a gene regulating intracellular cholesterol transport (the Niemann-Pick C1 [Npc1] gene) were crossed with apolipoprotein E (Apoe) knockout mice to examine the effect of Npc1 on atherosclerotic lesion formation. Double-mutant mice showed greater lesion area compared with Apoe-/- littermates. Remarkably, the double mutants also developed large, protruding thrombi associated with the plaques and prominent medial degradation with inflammatory cell infiltration into the adventitia. Genetic studies suggested that the BALB background was permissive for plaque complications compared with C57BL/6J, and a BALB susceptibility locus was mapped by linkage analysis to chromosome 6. Examination of clotting parameters in double-knockout mice revealed that native clotting times were shortened and thrombin-antithrombin complex and soluble CD40 ligand levels were elevated compared with wild-type controls. In addition, cathepsin K was induced in Npc1-/- macrophages, and cathepsin K immunostaining and elastase activity were increased in proximal aortas of double-mutant mice compared with controls. CONCLUSIONS: A defect in intracellular cholesterol trafficking caused by the Npc1 null mutation predisposes to increased lesion formation, atherothrombosis, and medial degradation. Plaque complications may require a procoagulant state and an increased protease activity, leading to plaque destabilization.
Assuntos
Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Proteínas/genética , Trombose/genética , Trombose/patologia , Túnica Média/patologia , Animais , Apolipoproteínas E/fisiologia , Aterosclerose/metabolismo , Colesterol/genética , Colesterol/metabolismo , Predisposição Genética para Doença , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Mutação , Proteína C1 de Niemann-Pick , Proteínas/fisiologia , Trombose/enzimologia , Túnica Média/metabolismoRESUMO
Cystathionine-ß-synthase (CBS) deficiency is a human genetic disease causing homocystinuria, thrombosis, mental retardation, and a suite of other devastating manifestations. Early detection coupled with dietary modification greatly reduces pathology, but the response to treatment differs with the allele of CBS. A better understanding of the relationship between allelic variants and protein function will improve both diagnosis and treatment. To this end, we tested the function of 84 CBS alleles previously sequenced from patients with homocystinuria by ortholog replacement in Saccharomyces cerevisiae. Within this clinically associated set, 15% of variant alleles were indistinguishable from the predominant CBS allele in function, suggesting enzymatic activity was retained. An additional 37% of the alleles were partially functional or could be rescued by cofactor supplementation in the growth medium. This large class included alleles rescued by elevated levels of the cofactor vitamin B6, but also alleles rescued by elevated heme, a second CBS cofactor. Measurement of the metabolite levels in CBS-substituted yeast grown with different B6 levels using LC-MS revealed changes in metabolism that propagated beyond the substrate and product of CBS. Production of the critical antioxidant glutathione through the CBS pathway was greatly decreased when CBS function was restricted through genetic, cofactor, or substrate restriction, a metabolic consequence with implications for treatment.