RESUMEN
Sequence polymorphisms in a 58-kilobase (kb) interval on chromosome 9p21 confer a markedly increased risk of coronary artery disease (CAD), the leading cause of death worldwide. The variants have a substantial effect on the epidemiology of CAD and other life-threatening vascular conditions because nearly one-quarter of Caucasians are homozygous for risk alleles. However, the risk interval is devoid of protein-coding genes and the mechanism linking the region to CAD risk has remained enigmatic. Here we show that deletion of the orthologous 70-kb non-coding interval on mouse chromosome 4 affects cardiac expression of neighbouring genes, as well as proliferation properties of vascular cells. Chr4(Delta70kb/Delta70kb) mice are viable, but show increased mortality both during development and as adults. Cardiac expression of two genes near the non-coding interval, Cdkn2a and Cdkn2b, is severely reduced in chr4(Delta70kb/Delta70kb) mice, indicating that distant-acting gene regulatory functions are located in the non-coding CAD risk interval. Allele-specific expression of Cdkn2b transcripts in heterozygous mice showed that the deletion affects expression through a cis-acting mechanism. Primary cultures of chr4(Delta70kb/Delta70kb) aortic smooth muscle cells exhibited excessive proliferation and diminished senescence, a cellular phenotype consistent with accelerated CAD pathogenesis. Taken together, our results provide direct evidence that the CAD risk interval has a pivotal role in regulation of cardiac Cdkn2a/b expression, and suggest that this region affects CAD progression by altering the dynamics of vascular cell proliferation.
Asunto(s)
Deleción Cromosómica , Cromosomas de los Mamíferos/genética , Enfermedad de la Arteria Coronaria/genética , Animales , Aorta/patología , Proliferación Celular , Células Cultivadas , Senescencia Celular/genética , Cromosomas Humanos Par 9/genética , Enfermedad de la Arteria Coronaria/patología , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/deficiencia , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Embrión de Mamíferos/embriología , Regulación de la Expresión Génica/genética , Predisposición Genética a la Enfermedad/genética , Humanos , Ratones , Miocitos del Músculo Liso/patología , Análisis de SupervivenciaRESUMEN
No evidence of premature vascular disease is found in apolipoprotein A-I(Milano) (apoA-I(M)) human carriers, despite very low high density lipoprotein (HDL) cholesterol levels. Whether apoA-I(M) may impart a "gain of function" in atherosclerosis protection compared to wild-type apoA-I is hotly debated. To address this question, knock-in mice expressing human apoA-I or apoA-I(M) were crossed with atherosclerosis-susceptible mice expressing the human apoB/A-II transgene (h-B/A-II/A-I(Hu/Hu) and h-B/A-II/A-I(M)(Hu/Hu)). On a chow diet, h-B/A-II/A-I(M)(Hu/Hu) mice were characterized by low HDL cholesterol levels compared to h-B/A-II/A-I(Hu/Hu) mice (35.65+/-8.00 mg/dl versus 58.09+/-13.50mg/dl, respectively; p<0.005). Gender differences in response to high fat diet were observed in both h-B/A-II/A-I(M)(Hu/Hu) and h-B/A-II/A-I(Hu/Hu) lines. h-B/A-II/A-I(M)(Hu/Hu) females had higher total cholesterol levels compared to h-B/A-II/A-I(Hu/Hu) females (895.08+/-183.07 mg/dl versus 544.43+/-116.42 mg/dl; p<0.05) and developed larger atherosclerotic lesions (148,260+/-78,924 microm(2) versus 54,132+/-43,204 microm(2), respectively; p<0.05). On the contrary, no difference in mean lesion area was found between h-B/A-II/A-I(M)(Hu/Hu) and h-B/A-II/A-I(Hu/Hu) males (19,779+/-6,098 microm(2) versus 15,706+/-13,095 microm(2); p=0.685). Our data suggest that, in the atherosclerosis-susceptible human apoB/A-II mouse model, expression of the human apoA-I(M) gene does not have protective advantage over that of the apoA-I gene.
Asunto(s)
Apolipoproteína A-I/genética , Aterosclerosis/genética , Expresión Génica , Animales , Apolipoproteína A-I/sangre , Aterosclerosis/sangre , Aterosclerosis/patología , Western Blotting , ADN/genética , Dieta Aterogénica , Modelos Animales de Enfermedad , Femenino , Genotipo , Humanos , Masculino , Ratones , Ratones Noqueados , Nefelometría y Turbidimetría , Reacción en Cadena de la Polimerasa , Factores de Riesgo , Factores SexualesRESUMEN
OBJECTIVE: The apolipoprotein(a) [apo(a)] gene locus is the major determinant of the circulating concentration of the atherothrombogenic lipoprotein Lp(a). In vitro analysis of the intergenic region between the apo(a) and plasminogen genes revealed the presence of a putative apo(a) transcription control region (ACR) approximately 20 kb upstream of the apo(a) gene that significantly increases the minimal promoter activity of the human apo(a) gene. METHODS AND RESULTS: To examine the function of the ACR in its natural genomic context, we used the Cre-loxP recombination system to generate 2 nearly identical apo(a)-yeast artificial chromosome transgenic mouse lines that possess a single integration site for the human apo(a) transgene in the mouse genome but differ by the presence or absence of the ACR enhancer. Analysis of the 2 groups of animals revealed that the deletion of the ACR was associated with 30% reduction in plasma and mRNA apo(a) levels. Apo(a)-yeast artificial chromosome transgenic mice with and without the ACR sequence were similar in all other aspects of apo(a) regulation, including liver-specific apo(a) expression and alteration in expression levels in response to sexual maturation and a high-fat diet. CONCLUSIONS: This study provides the first experimental in vivo evidence for a functional role of the ACR enhancer in determining levels of apo(a) expression.