Experiment
An important clinical measurement of vascular remodeling is carotid intima thickening (IMT). In response to variations in hemodynamics parameters the vessel wall remodels to compensate for changes in blood flow. The goal of this study was to identify a genomic locus that housed candidate genes that affect the inflammatory response during carotid IMT response that is associated with increased risks such as stroke, myocardial infarction and peripheral vascular disease.
In response to low blood flow SJL mice develop significantly greater IMT accompanied by an increased leukocyte infiltration in comparison to C3HeB/FeJ (C3H/F) mice. There is also an increase in CD45+ cells in the intima of SJL relative to C3H/F mice. The current study utilized a previously characterized (C3HeB/FeJ x SJL/J) cross for QTL analysis of the CD45+ staining in the N2 progeny. A nearly significant locus (significant LOD=2.4) was detected on Chromosome 11 and a second suggestive locus was detected on Chromosome 9, LOD=1.5, p<0.067.
QTL interval mapping revealed QTL Leuf1, leukocyte filtration 1, mapping with peak marker D11Mit231 on Chromosome 11, LOD=2.3, p<0.05 for the CD45+ locus which coincided with a previously reported peak for QTL Intim2 (intima modifier 2), [ J:159788]. Since there was a clear overlap, congenic mice were created to study the regulation of IMT by the CD45+ locus. Two congenic mouse lines were created by introgressing the Intim2 Chr 11 locus from the SJL onto the C3H/F background using marker-assisted genotyping. Both congenic lines, C3FeJ.SJL-Intim2/Bcb (C3H/F.SJL.11.1) and C3FeJ.SJL-Intim2/2Bcb (C3H/F.SJL.11.2) contained the entire Intim2 QTL but 11.2 contained additional SJL genomic material proximal to the peak locus marker D11Mit231. [Fig 3A.]
Partial carotid ligation was performed to create a low flow environment on the congenic mice, and quantified vessel wall compartment volumes to determine the contribution of the Intim2 and Leuf1 loci on IMT. An increase in intima volume was not observed along the length of the carotids in the congenic mice. Intima formation in the congenic mice was comparable to C3H/F mice suggesting that the C3H/F genome functions to protect the vascular from intima formation. CD45+ staining was observed only in areas of the congenic mice where the vessel wall had remodeled leading to the conclusion that leukocyte infiltration only partially explained the intima trait. The Intim2 and Leuf1 QTL play a role in recruiting or retaining inflammatory cells during vascular remodeling. By itself the Leuf1 locus within the Intim2 QTL only partially explains the entire IMT phenotype, appearing to contribute to about 8% of the trait.
The overlapping Intim2 and Leuf1 loci suggest that the genomic region houses causal genes that promote IMT. Using the JaxLab phenome and SNP database a list of approximately 140 genes with SNPs differing between strains SJL and C3H/F were identified in the region of the Leuf1 locus (17.0 63.0 Mb). Two genes on the list, Efemp1 and Pnpt1, were also identified in previously published microarray data [J:159788]. In an expression QTL (eQTL) analysis of macrophages from C3H/F mice, Pnpt1 expression was significantly increased relative to other strains.
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