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Up-regulation of inflammatory responses is considered a driving force of atherosclerotic lesion development. One key regulator of inflammation is the A20 (also called TNF-␣-induced protein 3 or Tnfaip3) gene, which is responsible for NF-B termination and maps to an atherosclerosis susceptibility locus revealed by quantitative trait locus-mapping studies at mouse proximal chromosome 10. In the current study, we examined the role of A20 in atherosclerotic lesion development. At the aortic root lesion size was found to be increased in C57BL/6 (BG) apolipoprotein E-deficient (ApoE ؊/؊ ) mice haploinsufficient for A20, compared with B6 ApoE ؊/؊ controls that expressed A20 normally (60% in males and 23% in females; P < 0.001 and P < 0.05, respectively). In contrast, lesion size was found to be decreased in F 1 (B6؋FVB/N) mice overexpressing A20 by virtue of containing an A20 BAC transgene compared with nontransgenic controls (30% in males, P < 0.001, and 17% in females, P ‫؍‬ 0.02). The increase in lesions in the A20 haploinsufficient mice correlated with increased expression of proatherosclerotic NF-B target genes, such as vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and macrophage-colony-stimulating factor, and elevated plasma levels of NF-B-driven cytokines. These findings suggest that A20 diminishes atherosclerosis by decreasing NF-B activity, thereby modulating the proinflammatory state associated with lesion development.A therosclerosis is a chronic inflammatory disease influenced by many genes, and mouse models are useful for identifying these genes and determining their mechanisms of action. We previously carried out an intercrosses between atherosclerosissensitive apolipoprotein E-deficient (ApoE Ϫ/Ϫ ) C57BL/6 (B6) and atherosclerosis-resistant ApoE Ϫ/Ϫ FVB mice, and by quantitative trait locus mapping identified an atherosclerosissusceptibility locus on proximal chromosome 10 (1, 2). A candidate gene in this region, A20, encodes a broadly expressed cytoplasmic protein that inhibits both TNF-␣-and IL-1␤/Tolllike receptor (TLR)-induced NF-B activation through mediating the destruction of receptor-interacting serine/threonine protein kinase 1 (RIP) and TNF receptor-associated factor 6 in their respective signaling complexes (3, 4). Up-regulation of NF-B activity leads to increased expression of many genes with established roles in atherosclerosis, including cytokines, chemokines, adhesion molecules, acute phase proteins, and regulators of apoptosis and cell proliferation. NF-B terminates its own activation by inducing the expression of I B␣ and A20. A20-deficient (A20 Ϫ/Ϫ ) mice die prematurely because of cachexia and spontaneous inflammation in several organs. This phenotype correlates with the failure to properly terminate TNF-induced NF-B activity in fibroblasts isolated from A20 Ϫ/Ϫ mice (5). We previously determined that A20 derived from B6 and FVB mice differs in a single amino acid residue (E627A; B6 vs. FVB). We showed this finding to be functionally significant by demonstrating...

Rationale Quantitative trait locus mapping of an intercross between C57.Apoe−/− and FVB.Apoe−/− mice revealed an atherosclerosis locus controlling aortic root lesion area on proximal chromosome 10, Ath11. In a previous work, subcongenic analysis showed Ath11 to be complex with proximal (10a) and distal (10b) regions. Objective To identify the causative genetic variation underlying the atherosclerosis modifier locus Ath11 10b. Methods and Results We now report subcongenic J, which narrows the 10b region to 5 genes, Myb, Hbs1L, Aldh8a1, Sgk1, and Raet1e. Sequence analysis of these genes revealed no amino acid coding differences between the parental strains. However, comparing aortic expression of these genes between F1.Apoe−/− Chr10SubJ(B/F) and F1.Apoe−/− Chr10SubJ(F/F) uncovered a consistent difference only for Raet1e, with decreased, virtually background, expression associated with increased atherosclerosis in the latter. The key role of Raet1e was confirmed by showing that transgene-induced aortic overexpression of Raet1e in F1.Apoe−/− Chr10SubJ(F/F) mice decreased atherosclerosis. Promoter reporter constructs comparing C57 and FVB sequences identified an FVB mutation in the core of the major aortic transcription start site abrogating activity. Conclusions This nonbiased approach has revealed Raet1e, a major histocompatibility complex class 1–like molecule expressed in lesional aortic endothelial cells and macrophage-rich regions, as a novel atherosclerosis gene and represents one of the few successes of the quantitative trait locus strategy in complex diseases.

Objective Ath11, an atherosclerosis susceptibility locus on proximal chromosome 10 (0–21cM) revealed in a cross between apolipoprotein E deficient C57BL/6 (B6) and FVB mice, was recently confirmed in congenic mice. The objectives of this study were to assess how Ath11 affects lesion development and morphology, to determine aortic gene expression in congenics, and to narrow the congenic interval. Methods and Results Assessing lesion area over time in congenic mice showed that homozygosity for the FVB allele increased lesion area at 6 weeks persisting through to 24 weeks of age. Staining of aortic root sections at 16 weeks did not reveal obvious differences between congenics. Aortic expression-array analysis at 6 weeks revealed 97 >2 fold regulated genes, including one gene in the QTL interval, Aldh8a1, and two gene clusters regulated by Hnf4α and Esr1. Analysis of lesion area in 11 subcongenic strains revealed two narrowed regions, 10a (21 genes) acting in females and 10b (7 genes) acting in both genders. Conclusions Ath11 appears to act early in lesion formation with significant effects on aortic gene expression. This QTL is genetically complex containing a female specific region 10a from 0 to 7.3 Mb, and a gender independent region 10b from 20.1 to 21.9 Mb.