Characterization of Ath29, a major mouse atherosclerosis susceptibility locus, and identification of Rcn2 as a novel regulator of cytokine expression.
ABSTRACT: Ath29 is an atherosclerosis susceptibility locus on chromosome 9 identified in an intercross between C57BL/6 (B6) and C3H/HeJ (C3H) apolipoprotein E-deficient (apoE(-/-)) mice. This locus was subsequently replicated in two separate intercrosses that developed early or advanced atherosclerotic lesions. The objective of this study was to characterize Ath29 through construction and analysis of a congenic strain and identify underlying candidate genes. A congenic line was constructed by introgressing the chromosomal segment harboring Ath29 from C3H.apoE(-/-) into B6.apoE(-/-) mice. Congenic mice developed significantly smaller early and advance atherosclerotic lesions than B6.apoE(-/-) mice. Microarray analysis revealed 317 genes to be differentially expressed in the aorta of congenic mice compared with B6.apoE(-/-) mice. Pathway analysis of these genes suggested the Ca(2+) signaling pathway to be implicated in regulating atherosclerosis susceptibility. Rcn2 is located underneath the linkage peak of Ath29 and involved in Ca(2+) signaling. Multiple single-nucleotide polymorphisms between B6 and C3H mice were detected within and surrounding Rcn2 with one single-nucleotide polymorphism falling within an upstream cAMP response element. Immunostaining demonstrated its expression in atherosclerotic lesions. Knockdown of Rcn2 with small interfering RNAs resulted in significant reductions in both baseline and oxidized phospholipid-induced VCAM-1 and monocyte chemoattractant protein-1 expression by endothelial cells. Ath29 is confirmed to be a major atherosclerosis susceptibility locus affecting both early and advanced lesion formation in mice, and Rcn2 is identified as a novel regulator of cytokine expression.
Project description:C3H/HeJ (C3H) mice develop much smaller atherosclerotic lesions than C57BL/6 (B6) mice when deficient in apolipoprotein E (apoE?(/)?) or fed an atherogenic diet. The 2 strains differ in H2 haplotypes, with B6 having H2(b) and C3H having H2(k). C3.SW-H2(b)/SnJ (C3.SW) is a congenic strain of C3H/HeJ in which H2(k) is replaced with H2(b).We performed bone marrow transplantation and found that atherosclerosis-resistant C3.SW.apoE?(/)? mice reconstituted with bone marrow from either C3.SW.apoE?(/)? or B6.apoE?(/)? mice after lethal irradiation had significantly larger atherosclerotic lesions than B6.apoE?(/)? mice receiving identical treatments and much larger lesions than C3H.apoE?(/)? mice reconstituted with syngeneic bone marrow. For syngeneic transplantation, C3.SW.apoE?(/)? mice exhibited a 21-fold increase in lesion size over C3H.apoE?(/)? mice (152 800±21 937 versus 7060±2290 ?m²/section) and a near 4-fold increase over B6.apoE?(/)? mice (40 529±4675 ?m²/section). C3.SW.apoE?(/)? mice reconstituted with syngeneic marrow exhibited enhanced lesion formation relative to those reconstituted with B6 marrow (152 800±21 937 versus 107 000±9374 ?m²/section; P=0.067). Sublethal irradiation led to a 6-fold increase of lesion size in C3.SW.apoE?(/)? mice (9795±2804 versus 1550±607 ?m²/section; P=0.008). Wild-type C3.SW mice reconstituted with apoE(+/+) or apoE?(/)? bone marrow had significantly larger atherosclerotic lesions than C3H mice receiving identical treatments on an atherogenic diet.These results indicate that gene(s) within the H2 region have a dramatic impact on radiation-enhanced atherosclerosis, and their effect is conveyed partially through bone marrow-derived cells.
Project description:PURPOSE:Atherosclerosis in the carotid arteries is a common cause of ischemic stroke. We examined atherogenesis in the left carotid artery with and without interrupted blood flow of C57BL/6 (B6) and C3H-Apoe-deficient (Apoe-/-) mouse strains. METHODS:Blood flow was interrupted by ligating the common carotid artery near its bifurcation in one group of mice and another group was not interrupted. RESULTS:Without interference with blood flow, C3H-Apoe-/- mice developed no atherosclerosis in the carotid artery, while B6-Apoe-/- mice formed advanced atherosclerotic lesions (98,019 ± 10,594 ?m2/section) after 12 weeks of a Western diet. When blood flow was interrupted by ligating the common carotid artery near its bifurcation, C3H-Apoe-/- mice showed fatty streak lesions 2 weeks after ligation, and by 4 weeks fibrous lesions had formed, although they were smaller than in B6-Apoe-/- mice. Neutrophil adhesion to endothelium and infiltration in lesions was observed in ligated arteries of both strains. Treatment of B6-Apoe-/- mice with antibody against neutrophils had little effect on lesion size. CONCLUSIONS:These findings demonstrate the dramatic influences of genetic backgrounds and blood flow on atherogenesis in the carotid artery of hyperlipidemic mice.
Project description:Bglu3 is a quantitative trait locus for fasting glucose on distal chromosome 1 identified in an intercross between C57BL/6 (B6) and C3H/HeJ (C3H) apolipoprotein E-deficient (apoE(-/-)) mice. This locus was subsequently replicated in two separate mouse intercrosses. The objective of this study was to characterize Bglu3 through construction and analysis of a congenic strain and identify underlying candidate genes. Congenic mice were constructed by introgressing a genomic region harboring Bglu3 from C3H.apoE(-/-) into B6.apoE(-/-) mice. Mice were started with a Western diet at 6 wk of age and maintained on the diet for 12 wk. Gene expression in the liver was analyzed by microarrays. Congenic mice had significantly higher fasting glucose levels and developed more significant glucose intolerance compared with B6.apoE(-/-) mice on the Western diet. Microarray analysis revealed 336 genes to be differentially expressed in the liver of congenic mice. Further pathway analysis suggested a role for acute phase response signaling in regulating glucose intolerance. Apcs, encoding an acute phase response protein serum amyloid P (SAP), is located underneath the linkage peak of Bglu3. Multiple single nucleotide polymorphisms between B6 and C3H mice were detected within and surrounding Apcs. Apcs expression in the liver was significantly higher in congenic and C3H mice compared with B6 mice. The Western diet consumption led to a gradual rise in plasma SAP levels, which was accompanied by rising fasting glucose in both B6 and C3H apoE(-/-) mice. Expression of C3H Apcs in B6.apoE(-/-) mice aggravated glucose intolerance. Bglu3 is confirmed to be a locus affecting diabetes susceptibility, and Apcs is a probable candidate gene.
Project description:Fc? receptors (Fc?Rs) are classified as activating (Fc?RI, III, and IV) and inhibitory (Fc?RII) receptors. We have reported that deletion of activating Fc?Rs in apolipoprotein E (apoE) single knockout mice attenuated atherosclerosis. In this report, we investigated the hypothesis that deficiency of inhibitory Fc?RIIb exacerbates atherosclerosis.ApoE-Fc?RIIb double knockout mice, congenic to the C57BL/6 (apoE-Fc?RIIbB6 (-/-)), were generated and atherosclerotic lesions were assessed. In contrary to our hypothesis, when compared with apoE single knockout mice, arterial lesions were significantly decreased in apoE-Fc?RIIbB6 (-/-) male and female mice fed chow or high-fat diets. Chimeric mice generated by transplanting apoE-Fc?RIIbB6 (-/-) marrow into apoE single knockout mice also developed reduced lesions. CD4(+) T cells from apoE-Fc?RIIbB6 (-/-) mice produced higher levels of interleukin-10 and transforming growth factor-? than their apoE single knockout counterparts. As our findings conflict with a previous report using apoE-Fc?RIIb129/B6 (-/-) mice on a mixed genetic background, we investigated whether strain differences contributed to the anti-inflammatory response. Macrophages from Fc?RIIb129/B6 (-/-) mice on a mixed genetic background produced more interleukin-1? and MCP-1 (monocyte chemoattractant protein-1) in response to immune complexes, whereas congenic Fc?RIIbB6 (-/-) mice generated more interleukin-10 and significantly less interleukin-1?. Interestingly, the expression of lupus-associated slam genes, located in proximity to fcgr2b in mouse chromosome 1, is upregulated only in mixed Fc?RIIb129/B6 (-/-) mice.Our findings demonstrate a detrimental role for Fc?RIIb signaling in atherosclerosis and the contribution of anti-inflammatory cytokine responses in the attenuated lesions observed in apoE-Fc?RIIbB6 (-/-) mice. As 129/sv genome-derived lupus-associated genes have been implicated in lupus phenotype in Fc?RIIb129/B6 (-/-) mice, our findings suggest possible epistatic mechanism contributing to the decreased lesions.
Project description:We have previously identified a quantitative trait locus (QTL) for atherosclerosis susceptibility on proximal chromosome 10 (Chr10) (Ath11) in independent crosses of FVB and C57BL/6 (B6) mice on the apolipoprotein E (ApoE-/-) and LDL receptor (LDLR-/-) deficient backgrounds. The aims of the current study were to (1) test a novel strategy for validating QTLs using interval-specific congenic strains that were heterozygous (F1) across the genome, (2) validate the Chr10 QTL, and (3) to assess whether the phenotype is transferable by bone marrow transplantation.We generated Chr10 (0 to 21 cM) interval-specific mice on the F1.ApoE-/- background by crossing congenic FVB.ApoE-/-Chr10(B6/FVB) with B6.ApoE-/-, and B6.ApoE-/-Chr10(B6/FVB) with FVB.ApoE-/- mice. Lesion size was significantly larger in the resultant F1.ApoE-/-Chr10(FVB/FVB) mice compared to F1.ApoE-/-Chr10(B6/FVB) and F1.ApoE-/-Chr10(B6/B6) mice, validating the Chr10 QTL. The effect of the congenic interval was more robust on the F1.ApoE-/- than on the FVB.ApoE-/- and B6.ApoE-/- backgrounds. Bone marrow transplantation in congenic mice showed that the effect of the proximal Chr10 interval was not transferable by bone marrow-derived cells.A novel strategy of congenic strains on an F1 background proved useful to validate an atherosclerosis susceptibility QTL on mouse proximal Chr10.
Project description:BACKGROUND AND AIMS:We previously demonstrated that Apoe-/- mice on DBA/2 vs. AKR genetic background have >10-fold larger atherosclerotic lesions. Prior quantitative trait locus mapping via strain intercrossing identified a region on chromosome 17, Ath26, as the strongest atherosclerosis-modifying locus. We aimed to confirm Ath26, identify candidate genes, and validate the candidate gene effects on atherosclerosis. METHODS:We bred chromosome 17 interval congenic mice to confirm that Ath26 locus contains atherosclerosis modifying gene(s). Bone marrow derived macrophage transcriptomics was performed to identify candidate genes at this locus whose expression was correlated with lesions in a strain intercross. The Cyp4f13 candidate gene was tested via a gene knockout approach and in vivo and ex vivo phenotype analyses. RESULTS:A congenic mouse strain containing the DBA/2 interval on chromosome 17 on the AKR Apoe-/- background demonstrated that this interval conferred increased lesion area. Transcriptomic analysis of bone marrow macrophages identified that expression of the Cyp4f13 gene, mapping to this locus, was highly associated with lesion area in an F2 cohort. AKR vs. DBA/2 macrophages had less Cyp4f13 mRNA expression, and their livers had lower leukotriene B4 (LTB4) 20-hydroxylase enzymatic activity. A Cyp4f13 knockout allele was bred onto the DBA/2 Apoe-/- background and this conferred less enzymatic activity, decreased macrophage migration in response to LTB4, and smaller aortic root atherosclerotic lesions. CONCLUSIONS:Allelic differences in the Cyp4f13 gene may in part be responsible for the Ath26 QTL conferring larger lesions in DBA/2 vs. AKR Apoe-/- mice.
Project description:Nearly all genetic crosses generated from Apoe-/- or Lldlr-/- mice for genetic analysis of atherosclerosis have used C57BL/6 J (B6) mice as one parental strain, thus limiting their mapping power and coverage of allelic diversity. SM/J-Apoe -/- and BALB/cJ-Apoe -/- mice differ significantly in atherosclerosis susceptibility. 224 male F2 mice were generated from the two Apoe -/- strains to perform quantitative trait locus (QTL) analysis of atherosclerosis. F2 mice were fed 5 weeks of Western diet and analyzed for atherosclerotic lesions in the aortic root. Genome-wide scans with 144 informative SNP markers identified a significant locus near 20.2 Mb on chromosome 10 (LOD score: 6.03), named Ath48, and a suggestive locus near 49.5 Mb on chromosome 9 (LOD: 2.29; Ath29) affecting atherosclerotic lesion sizes. Using bioinformatics tools, we prioritized 12 candidate genes for Ath48. Of them, Tnfaip3, an anti-inflammatory gene, is located precisely underneath the linkage peak and contains two non-synonymous SNPs leading to conservative amino acid substitutions. Thus, this study demonstrates the power of forward genetics involving the use of a different susceptible strain and bioinformatics tools in finding atherosclerosis susceptibility genes.
Project description:Inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) exhibit a marked difference in atherosclerotic lesion formation when deficient in apolipoprotein E (apoE(-/-)), and the arterial wall has been identified as a source of the difference in atherosclerosis susceptibility. In the present study, differences in gene expression in aortic walls of the two strains were analyzed by microarrays. Total RNA was extracted from the aorta of 6-wk-old female B6 and C3H apoE(-/-) mice fed a chow or Western diet. There were 1,514 genes in chow fed mice and 590 genes in Western fed mice that were found to be differentially expressed between the two strains. Pathway analysis of differentially expressed genes suggested a role for the calcium signaling pathway in regulating atherosclerosis susceptibility. Oxidized LDL (oxLDL) induced a dose-dependent rise in cytosolic calcium levels in B6 endothelial cells. oxLDL-induced monocyte chemoattractant protein-1 production was inhibited by pretreatment with calcium chelator EGTA or intracellular calcium trapping compound BAPTA, indicating that calcium ions mediate the effect of oxLDL on monocyte chemoattractant protein-1 induction. The present findings demonstrate involvement of the calcium signaling pathway in the inflammatory process of atherogenesis.
Project description: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-alpha-induced protein 3 or Tnfaip3) gene, which is responsible for NF-kappaB 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 x 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-kappaB target genes, such as vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and macrophage-colony-stimulating factor, and elevated plasma levels of NF-kappaB-driven cytokines. These findings suggest that A20 diminishes atherosclerosis by decreasing NF-kappaB activity, thereby modulating the proinflammatory state associated with lesion development.
Project description:The accelerated development of atherosclerosis with increased risk of cardiovascular disease in systemic lupus erythematosus (SLE) patients is not well understood. An appropriate mouse model would greatly help to understand the mechanisms of this association. We have therefore combined the ApoE(-/-) model of atherosclerosis with three different murine models of SLE. We found that induction of cGVH in B6.ApoE(-/-) mice, breeding a Fas null gene onto the B6.ApoE(-/-) mice, and breeding the ApoE(-/-) defect onto MRL/lpr mice all caused a modest increase of atherosclerosis at 24 weeks of age compared to B6.ApoE(-/-) controls. B cells in B6.ApoE(-/-) mice had certain phenotypic differences compared to congenic C57BL/6 mice, as indicated by high expression of MHC II, Fas, CD86, and by increased number of cells bearing marginal zone phenotype. Furthermore, B6ApoE(-/-) mice had significant titers of anti-oxLDL and anti-cardiolipin autoantibodies compared to their B6 counterparts. Our studies also indicate that, following induction of cGVH, marginal zone B cells in B6.ApoE(-/-) are depleted, and there is considerable increase in anti-oxLDL and anti-cardiolipin abs along with secretion of lupus-specific autoantibodies, such as anti-dsDNA and anti-chromatin abs. Histological sections showed that cGVH and/or Fas deficiency could exacerbate atherosclerosis. The production of anti-oxLDL and anti-cardiolipin in ApoE(-/-) mice was also increased. These observations define a connection between induction of lupus-like symptoms and development of severe atherosclerosis in ApoE deficient lupus mouse models.