Identification of a Genetic Locus on Chromosome 11 That Regulates Leukocyte Infiltration in Mouse Carotid Artery
ABSTRACT OBJECTIVE: We demonstrated that inflammatory cells and intima-media thickening are increased in carotids exposed to low-blood flow in the SJL/J (SJL) strain compared with other mouse strains. We hypothesized that the extent of inflammation associated with intima-media thickening is a genetically regulated trait.Approach and Results-We performed a whole genome approach to measure leukocyte infiltration in the carotid intima as a quantitative trait in a genetic cross between C3HeB/FeJ (C3H/F) and SJL mice. Immunostaining for CD45+ (a pan-specific leukocyte marker) was performed on carotids from C3H/F, SJL, F1, and N2 progeny to measure leukocyte infiltration. We identified a nearly significant quantitative trait locus for CD45+ on chromosome (chr) 11 (17 cM, LOD=2.3; significance was considered at threshold P=0.05). Interval mapping showed that the CD45+ locus on chr 11 accounted for 8% of the variation in the C3H/FxSJL backcross. Importantly, the CD45+ locus colocalized with the intima-modifier 2 (Im2) locus, which controls 17% of intima variation. We created 2 Im2 congenic lines of mice (C3H/F.SJL.11.1 and C3H/F.SJL.11.2) to better understand the regulation of intima-media thickening by the chr 11 locus. The C3H/F.SJL.11.1 congenic mouse showed ≈30% of the SJL trait, confirming that CD45+ cell infiltration contributed to the intima trait. CONCLUSIONS: We discovered a novel locus on chr 11 that controls leukocyte infiltration in the carotid. Importantly, this locus overlaps with our previously published Im2 locus on chr 11. Our study reveals a potential mechanistic relationship between leukocyte infiltration and intima-media thickening in response to decreased blood flow.
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ABSTRACT: Carotid intima formation is a significant risk factor for cardiovascular disease. C3H/FeJ (C3H/F) and SJL/J (SJL) inbred mouse strains differ in susceptibility to immune and vascular traits. Using a congenic approach we demonstrated that the Intima modifier 2 (Im2) locus on chromosome 11 regulates leukocyte infiltration. We sought to determine whether inflammation was due to changes in circulating immune cells or activation of vascular wall cells in genetically pure Im2 (C3H/F.SJL.11.1) mice. Complete blood counts showed no differences in circulating monocytes between C3H/F and C3H/F.SJL.11.1 compared to SJL mice. Aortic vascular cell adhesion molecule-1 (VCAM-1) total protein levels were dramatically increased in SJL and C3H/F.SJL.11.1 compared to C3H/F mice. Immunostaining of aortic endothelial cells (EC) showed a significant increase in VCAM-1 expression in SJL and C3H/F.SJL.11.1 compared to C3H/F under steady flow conditions. Immunostaining of EC membranes revealed a significant decrease in EC size in SJL and C3H/F.SJL.11.1 versus C3H/F in regions of disturbed flow. Vascular permeability was significantly higher in C3H/F.SJL.11.1 compared to C3H/F. Our results indicate that Im2 regulation of leukocyte infiltration is mediated by EC inflammation and permeability. RNA sequencing and pathway analyses comparing genes in the Im2 locus to C3H/F provides insight into candidate genes that regulate vascular wall inflammation and permeability highlighting important genetic mechanisms that control vascular intima in response to injury.Physiological Genomics 07/2014; 46(17). DOI:10.1152/physiolgenomics.00048.2014 · 2.81 Impact Factor