Fine Mapping of the Mouse Ath28 Locus Yields Three Atherosclerosis Modifying Sub-Regions

A mouse strain intercross between Apoe−/− AKR/J and DBA/2J mice identified three replicated atherosclerosis quantitative trait loci (QTLs). Our objective was to fine map mouse atherosclerosis modifier genes within a genomic region known to affect lesion development in apoE-deficient (Apoe−/−) mice. We dissected the Ath28 QTL on the distal end of chromosome 2 by breeding a panel of congenic strains and measuring aortic root lesion area in 16-week-old male and female mice fed regular laboratory diets. The parental congenic strain contained ~9.65 Mb of AKR/J DNA from chromosome 2 on the DBA/2J genetic background, which had lesions 55% and 47% smaller than female and male DBA/2J mice, respectively (p < 0.001). Seven additional congenic lines identified three separate regions associated with the lesion area, named Ath28.1, Ath28.2, and Ath28.3, where the AKR/J alleles were atherosclerosis-protective for two regions and atherosclerosis-promoting for the other region. These results were replicated in both sexes, and in combined analysis after adjusting for sex. The congenic lines did not greatly impact total and HDL cholesterol levels or body weight. Bioinformatic analyses identified all coding and non-coding genes in the Ath28.1 sub-region, as well as strain sequence differences that may be impactful. Even within a <10 Mb region of the mouse genome, evidence supports the presence of at least three atherosclerosis modifier genes that differ between the AKR/J and DBA/2J mouse strains, supporting the polygenic nature of atherosclerosis susceptibility.

Buprenorphine Depresses Respiratory Variability in Obese Mice with Altered Leptin Signaling

Background Opiate-induced respiratory depression is sexually dimorphic and associated with increased risk among the obese. The mechanisms underlying these associations are unknown. The present study evaluated the two-tailed hypothesis that sex, leptin status, and obesity modulate buprenorphine-induced changes in breathing. Methods Mice (n = 40 male and 40 female) comprising four congenic lines that differ in leptin signaling and body weight were injected with saline and buprenorphine (0.3 mg/kg). Whole-body plethysmography was used to quantify the effects on minute ventilation. The data were evaluated using three-way analysis of variance, regression, and Poincaré analyses. Results Relative to B6 mice with normal leptin, buprenorphine decreased minute ventilation in mice with diet-induced obesity (37.2%; P &lt; 0.0001), ob/ob mice that lack leptin (62.6%; P &lt; 0.0001), and db/db mice with dysfunctional leptin receptors (65.9%; P &lt; 0.0001). Poincaré analyses showed that buprenorphine caused a significant (P &lt; 0.0001) collapse in minute ventilation variability that was greatest in mice with leptin dysfunction. There was no significant effect of sex or body weight on minute ventilation. Conclusions The results support the interpretation that leptin status but not body weight or sex contributed to the buprenorphine-induced decrease in minute ventilation. Poincaré plots illustrate that the buprenorphine-induced decrease in minute ventilation variability was greatest in mice with impaired leptin signaling. This is relevant because normal respiratory variability is essential for martialing a compensatory response to ventilatory challenges imposed by disease, obesity, and surgical stress.

Abstract 247: Gender-specific Angiogenesis Suppression By Btg2 May Be Mediated Through Suppressed Renin Expression

Angiogenesis is the formation of new microvessels from existing vascular beds. AngII, a downstream product of renin, has been shown to be essential mediator of skeletal muscle angiogenesis in Dahl Salt Sensitive (SS) and Sprague Dawley rats, C57BL/6 mice, and human endothelial cells in vitro. Using partial chromosome introgression from the Brown Norway (BN) rat into the SS rat we have created two congenic lines with small (<300 Kbp) BN substitutions that differ by 23 Kbp. The congenic regions define an angiogenesis locus that contains one gene, Btg2 . Sanger sequencing revealed no sequence variants in exons of Btg2 between the BN and SS strains. Angiogenesis was measured using an in vivo electrical stimulation model of one hindlimb, with the contralateral leg acting as the control, in males and females of both new congenic strains. Males from Btg2 BN have angiogenesis (TA=20.0±4.5% increase in vessel density in stimulated leg relative to unstimulated leg, EDL=15.8±5.8%,) while Btg2 SS males did not have angiogenesis (TA=5.1±2.2%, EDL=4.4±2.7%). Females of both strains had angiogenesis: Btg2 BN (TA=15.8±4.3%, EDL=3.4%), Btg2 SS (TA=14.5±2.0%, EDL=14.6±2.5%). Stimulation significantly increased Btg2 expression in both males and females. Btg2 SS males had a significantly greater increase in Btg2 mRNA in the stimulated muscle relative to unstimulated than Btg2 BN males (5.0±0.9 versus 2.1± 0.3), but there was no difference in stimulated females (Btg2 BN 2.5±0.2, Btg2 SS 3.2±1.5). To test the hypothesis that Btg2 impacted renin expression, we cloned the renin proximal promoter into a vector to drive luciferase, and co-transfected HEK-293 cells with this and vector(s) expressing Btg2 or Hoxb9 and Btg2 . Normalized luciferase activity was 4.52±0.30 (arbitrary units) with an empty vector control and suppressed with the Btg2 vector to 1.45±0.07. Co-expression of Btg2 and Hoxb9 lead to a further reduction in luciferase activity to 0.40±0.04. These data suggest the elevated Btg2 expression observed in Btg2 SS strain may be acting to suppress renin expression through renin proximal promoter transcription factor Hoxb9 , leading to an inhibited angiogenic response.

Abstract 568: Congenic Mapping of Hypertension in the Nephrectomy Model of Chronic Kidney Disease.

Introduction: We previously reported a locus on mouse chromosome 11 (chrom 11) linked to development of hypertension (HTN) after sub-total nephrectomy (Nx). To begin fine mapping, we used the 129S6 (129) susceptible strain and the C57BL/6 (B6) resistant strain to generate 4 reciprocal congenic lines carrying ~ 2-LOD intervals of the linked locus for recombinant progeny testing. The intervals are defined by microsatellite markers 1-5 and markers 1-6, corresponding to regions defined by markers D11Mit2 and D11Mit177, and D11Mit2 and D11Mit285, respectively. Through brother-sister matings, the 4 lines are as follows: 1) 129 background, homozygous for B6 segment defined by markers 1-5 (129 Chrom11:1-5ofB6 ), 2) 129 background, homozygous for 129 segment defined by markers 1-5 (129 Chrom11:1-5of129 ), 3) B6 background, homozygous for 129 segment defined by markers 1-6 (B6 Chrom11:1-6of129 ) and 4) B6 background, homozygous for B6 segment defined by markers 1-6 (B6 Chrom11:1-6ofB6 ). Methods: Sub-total Nx was performed on male and female congenic mice at ~ 8 weeks of age. Beginning 4 weeks after surgery, using tail cuff manometer, mice were trained for 2 weeks, after which systolic blood pressure (SBP) was recorded daily for 2 weeks. Results: Table 1 summarizes the average SBP of the congenic lines. Conclusion: These findings confirm the effect of this chrom 11 region is dependent on the 129 genetic background, and suggest that interactions between this 129 locus and other loci in the 129 genome are necessary for the development of HTN after nephron mass reduction. Moreover, the influence of this region on chrom 11 appears to be dependent on sex.

Strain-specific modifier genes of Cecr2-associated exencephaly in mice: genetic analysis and identification of differentially expressed candidate genes

Although neural tube defects (NTDs) are common in humans, little is known about their multifactorial genetic causes. While most mouse models involve NTDs caused by a single mutated gene, we have previously described a multigenic system involving susceptibility to NTDs. In mice with a mutation in Cecr2, the cranial NTD exencephaly shows strain-specific differences in penetrance, with 74% penetrance in BALB/cCrl and 0% penetrance in FVB/N. Whole genome linkage analysis showed that a region of chromosome 19 was partially responsible for this difference in penetrance. We now reveal by genetic analysis of three subinterval congenic lines that the chromosome 19 region contains more than one modifier gene. Analysis of embryos showed that although a Cecr2 mutation causes wider neural tubes in both strains, FVB/N embryos overcome this abnormality and close. A microarray analysis comparing neurulating female embryos from both strains identified differentially expressed genes within the chromosome 19 region, including Arhgap19, which is expressed at a lower level in BALB/cCrl due to a stop codon specific to that substrain. Modifier genes in this region are of particular interest because a large portion of this region is syntenic to human chromosome 10q25, the site of a human susceptibility locus.