Determination of lower radiation dose limit for automatic measurement of adipose tissue.

The purpose of this study was to determine the lower limit of radiation dose required to measure visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) volumes when a fat quantification and noise reduction techniques (NRTs) are combined. For this purpose, we utilized CT colonography (CTC) images taken at low doses and manually segmented VAT and SAT fat volumes as ground truth. In order to derive the acceptable precision of the measurements needed to estimate the lower limit of radiation dose, we estimated the effect of different positioning during CT scanning on fat measurements using manually segmented VAT and SAT against normal dose. As a result, the acceptable accuracy of SAT and VAT was found to be 94.5% and 85.2%, respectively. Using these thresholds, the lower radiation dose limit required to accurately measure SAT using 5.25-mm slice-thick images was 1.5 mGy of size-specific dose estimates (SSDE), while the lower radiation dose limit required to accurately measure VAT was 0.4 mGy of SSDE. The lower dose limit for SAT and VAT combined was 1.5 mGy, which was equivalent to an estimated effective dose of 0.38 mSv. Alternatively, without noise reduction, SAT could not achieve acceptable accuracy even for images with a slice thickness of 5.25 mm, while VAT required noise reduction for images with a slice thickness of 1.25 mm, but could achieve acceptable accuracy without noise reduction for images with a slice thickness of 5.25 mm.

Atherogenesis in the Carotid Artery with and without Interrupted Blood Flow of Two Hyperlipidemic Mouse Strains.

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 µm2/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.

Genetic analysis of a mouse cross implicates an anti-inflammatory gene in control of atherosclerosis susceptibility.

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.

Genetic linkage of hyperglycemia and dyslipidemia in an intercross between BALB/cJ and SM/J Apoe-deficient mouse strains.

BACKGROUND: Individuals with dyslipidemia often develop type 2 diabetes, and diabetic patients often have dyslipidemia. It remains to be determined whether there are genetic connections between the 2 disorders. METHODS: A female F2 cohort, generated from BALB/cJ (BALB) and SM/J (SM) Apoe-deficient (Apoe(-/-)) strains, was started on a Western diet at 6 weeks of age and maintained on the diet for 12 weeks. Fasting plasma glucose and lipid levels were measured before and after 12 weeks of Western diet. 144 genetic markers across the entire genome were used for quantitative trait locus (QTL) analysis. RESULTS: One significant QTL on chromosome 9, named Bglu17 [26.4 cM, logarithm of odds ratio (LOD): 5.4], and 3 suggestive QTLs were identified for fasting glucose levels. The suggestive QTL near the proximal end of chromosome 9 (2.4 cM, LOD: 3.12) was replicated at both time points and named Bglu16. Bglu17 coincided with a significant QTL for HDL (high-density lipoprotein) and a suggestive QTL for non-HDL cholesterol levels. Plasma glucose levels were inversely correlated with HDL but positively correlated with non-HDL cholesterol levels in F2 mice on either chow or Western diet. A significant correlation between fasting glucose and triglyceride levels was also observed on the Western diet. Haplotype analysis revealed that "lipid genes" Sik3, Apoa1, and Apoc3 were probable candidates for Bglu17. CONCLUSIONS: We have identified multiple QTLs for fasting glucose and lipid levels. The colocalization of QTLs for both phenotypes and the sharing of potential candidate genes demonstrate genetic connections between dyslipidemia and type 2 diabetes.

Inflammation and enhanced atherogenesis in the carotid artery with altered blood flow in an atherosclerosis-resistant mouse strain.

Ligation of the common carotid artery near its bifurcation in apolipoprotein E-deficient (Apoe-/- ) mice leads to rapid atherosclerosis development, which is affected by genetic backgrounds. BALB/cJ (BALB) mice are resistant to atherosclerosis, developing much smaller aortic lesions than C57BL/6 (B6) mice. In this study, we examined cellular events leading to lesion formation in carotid arteries with or without blood flow restriction of B6 and BALB Apoe-/- mice. Blood flow was obstructed by ligating the left common carotid artery near its bifurcation in one group of mice, and other group received no surgical intervention. Without blood flow interruption, BALB-Apoe-/- mice formed much smaller atherosclerotic lesions than B6-Apoe-/- mice after 12 weeks of Western diet (3,325 ± 1,086 vs. 81,549 ± 9,983 µm2 /section; p = 2.1E-7). Lesions occurred at arterial bifurcations in both strains. When blood flow was obstructed, ligated carotid artery of both strains showed notable lipid deposition, inflammatory cell infiltration, and rapid plaque formation. Neutrophils and macrophages were observed in the arterial wall of BALB mice 3 days after ligation and 1 week after ligation in B6 mice. CD4 T cells were observed in intimal lesions of BALB but not B6 mice. By 4 weeks, both strains developed similar sizes of advanced lesions containing foam cells, smooth muscle cells, and neovessels. Atherosclerosis also occurred in straight regions of the contralateral common carotid artery where MCP-1 was abundantly expressed in the intima of BALB mice. These findings indicate that the disturbed blood flow is more prominent than high fat diet in promoting inflammation and atherosclerosis in hyperlipidemic BALB mice.

Identification of Mep1a as a susceptibility gene for atherosclerosis in mice.

Atherosclerosis is the underlying cause of heart attack, ischemic stroke and peripheral arterial disease, and genetic factors involved remain mostly unidentified. We previously identified a significant locus on mouse chromosome 17 for atherosclerosis, Ath49, in an intercross between BALB/c and SM strains. Ath49 partially overlaps in the confidence interval with Ath22 mapped in an AKR × DBA/2 intercross. Bioinformatics analysis prioritized Mep1a, encoding meprin 1α metalloendopeptidase, as a likely candidate gene for Ath49. To prove causality, Mep1a-/-Apoe-/- mice were generated and compared with Mep1a+/+Apoe-/- mice for atherosclerosis development. Mep1a was found abundantly expressed in atherosclerotic lesions but not in healthy aorta and liver of mice. Mep1a-/- Apoe-/- mice exhibited significant reductions in both early and advanced lesion sizes. Loss of Mep1a led to decreased necrosis but increased macrophage and neutrophil contents in advanced lesions, reduced plasma levels of CXCL5 and an oxidative stress biomarker. In addition, Mep1a-/- mice had significantly reduced triglyceride levels on a chow diet. Thus, Mep1a is a susceptibility gene for atherosclerosis and aggravates atherosclerosis partially through action on oxidative stress and inflammation.

Deep Learning-based Quantification of Abdominal Subcutaneous and Visceral Fat Volume on CT Images.

RATIONALE AND OBJECTIVES: Develop a deep learning-based algorithm using the U-Net architecture to measure abdominal fat on computed tomography (CT) images. MATERIALS AND METHODS: Sequential CT images spanning the abdominal region of seven subjects were manually segmented to calculate subcutaneous fat (SAT) and visceral fat (VAT). The resulting segmentation maps of SAT and VAT were augmented using a template-based data augmentation approach to create a large dataset for neural network training. Neural network performance was evaluated on both sequential CT slices from three subjects and randomly selected CT images from the upper, central, and lower abdominal regions of 100 subjects. RESULTS: Both subcutaneous and abdominal cavity segmentation images created by the two methods were highly comparable with an overall Dice similarity coefficient of 0.94. Pearson's correlation coefficients between the subcutaneous and visceral fat volumes quantified using the two methods were 0.99 and 0.99 and the overall percent residual squared error were 5.5% and 8.5%. Manual segmentation of SAT and VAT on the 555 CT slices used for testing took approximately 46 hours while automated segmentation took approximately 1 minute. CONCLUSION: Our data demonstrates that deep learning methods utilizing a template-based data augmentation strategy can be employed to accurately and rapidly quantify total abdominal SAT and VAT with a small number of training images.

Data on genetic linkage of oxidative stress with cardiometabolic traits in an intercross derived from hyperlipidemic mouse strains.

The data presented here are related to the research article, entitled Genetic linkage of oxidative stress with cardiometabolic traits in an intercross derived from hyperlipidemic mouse strains, published in Atherosclerosis 2019 Dec 3;293:1-10 (D. Fuller, A.T. Grainger, A. Manichaikul, W. Shi). The supporting materials include original genotypic and phenotypic data obtained from 266 female F2 mice derived from an intercross between C57BL/6 (B6) and BALB/cJ (BALB) Apoe-/- mice. F2 mice were fed 12 weeks of Western diet, starting at 6 weeks of age. Plasma levels of HDL, LDL cholesterol, triglycerides, glucose and malondialdehyde (MDA) and atherosclerosis in the aortic root and the left carotid artery were measured. 127 microsatellite markers across the entire genome were genotyped. The data is provided in the format ready for QTL analysis with J/qtl and MapManager QTX.

Genetic linkage of oxidative stress with cardiometabolic traits in an intercross derived from hyperlipidemic mouse strains.

BACKGROUND AND AIMS: Oxidative stress is associated with cardiometabolic traits in observational studies, yet the underlying causal relationship remains unclear. Apolipoprotein E-deficient (Apoe-/-) mice develop significant hyperlipidemia and hyperglycemia on a Western diet. Here we conducted linkage analysis to investigate genetic connections between cardiometabolic traits and oxidative stress. METHODS: 266 female F2 mice were generated from an intercross between C57BL/6 (B6) and BALB/c (BALB) Apoe-/- mice and fed 12 weeks of Western diet. Plasma levels of HDL, LDL cholesterol, triglycerides, glucose and malondialdehyde (MDA) and atherosclerosis in aortic root and left carotid artery were measured. 127 microsatellite markers across the genome were genotyped. RESULTS: One significant locus at 78.3 cM on chromosome (Chr) 1 (LOD score: 3.85), named Mda1, and two suggestive loci near 60.3 cM on Chr1 (LOD score: 2.32, named Mda2 due to replication in a separate cross) and 19.6 cM on Chr4 (LOD score: 2.34) were identified for MDA levels. Mda1 coincided precisely with loci for LDL, triglyceride, glucose, and body weight and overlapped with a locus for atherosclerosis in the aortic root. Plasma LDL, triglyceride, and glucose explained 25.5, 19.2, and 24.2% of the variation in MDA levels of F2 mice, respectively. After correction for triglyceride or LDL, QTLs for MDA on Chr1 and Chr4 disappeared. QTLs on Chr1 disappeared, remained on Chr4, and additional QTLs on Chr12 and Chr13 were detected after correction for glucose. The QTL on Chr12, named Mda3, had a significant LOD score of 8.034 and peaked 62.22 at cM. CONCLUSIONS: We demonstrated a causative role for cardiometabolic traits in oxidative stress and identified hyperlipidemia and hyperglycemia as a major driver of oxidative stress.

Deep learning-based quantification of abdominal fat on magnetic resonance images.

Obesity is increasingly prevalent and associated with increased risk of developing type 2 diabetes, cardiovascular diseases, and cancer. Magnetic resonance imaging (MRI) is an accurate method for determination of body fat volume and distribution. However, quantifying body fat from numerous MRI slices is tedious and time-consuming. Here we developed a deep learning-based method for measuring visceral and subcutaneous fat in the abdominal region of mice. Congenic mice only differ from C57BL/6 (B6) Apoe knockout (Apoe-/-) mice in chromosome 9 that is replaced by C3H/HeJ genome. Male congenic mice had lighter body weight than B6-Apoe-/- mice after being fed 14 weeks of Western diet. Axial and coronal T1-weighted sequencing at 1-mm-thickness and 1-mm-gap was acquired with a 7T Bruker ClinScan scanner. A deep learning approach was developed for segmenting visceral and subcutaneous fat based on the U-net architecture made publicly available through the open-source ANTsRNet library-a growing repository of well-known neural networks. The volumes of subcutaneous and visceral fat measured through our approach were highly comparable with those from manual measurements. The Dice score, root-mean-square error (RMSE), and correlation analysis demonstrated the similarity between two methods in quantifying visceral and subcutaneous fat. Analysis with the automated method showed significant reductions in volumes of visceral and subcutaneous fat but not non-fat tissues in congenic mice compared to B6 mice. These results demonstrate the accuracy of deep learning in quantification of abdominal fat and its significance in determining body weight.

Polygenic Control of Carotid Atherosclerosis in a BALB/cJ × SM/J Intercross and a Combined Cross Involving Multiple Mouse Strains.

Atherosclerosis in the carotid arteries is a major cause of ischemic stroke, which accounts for 85% of all stroke cases. Genetic factors contributing to carotid atherosclerosis remain poorly understood. The aim of this study was to identify chromosomal regions harboring genes contributing to carotid atherosclerosis in mice. From an intercross between BALB/cJ (BALB) and SM/J (SM) apolipoprotein E-deficient (Apoe-/-) mice, 228 female F2 mice were generated and fed a "Western" diet for 12 wk. Atherosclerotic lesion sizes in the left carotid artery were quantified. Across the entire genome, 149 genetic markers were genotyped. Quantitative trait locus (QTL) analysis revealed eight loci for carotid lesion sizes, located on chromosomes 1, 5, 12, 13, 15, 16, and 18. Combined cross-linkage analysis using data from this cross, and two previous F2 crosses derived from BALB, C57BL/6J and C3H/HeJ strains, identified five significant QTL on chromosomes 5, 9, 12, and 13, and nine suggestive QTL for carotid atherosclerosis. Of them, the QTL on chromosome 12 had a high LOD score of 9.95. Bioinformatic analysis prioritized Arhgap5, Akap6, Mipol1, Clec14a, Fancm, Nin, Dact1, Rtn1, and Slc38a6 as probable candidate genes for this QTL. Atherosclerotic lesion sizes were significantly correlated with non-HDL cholesterol levels (r = 0.254; p = 0.00016) but inversely correlated with HDL cholesterol levels (r = -0.134; p = 0.049) in the current cross. Thus, we demonstrated the polygenic control of carotid atherosclerosis in mice. The correlations of carotid lesion sizes with non-HDL and HDL suggest that genetic factors exert effects on carotid atherosclerosis partially through modulation of lipoprotein homeostasis.

Accelerated atherogenesis in completely ligated common carotid artery of apolipoprotein E-deficient mice.

Complete ligation of the common carotid artery near its bifurcation induces neointimal formation due to smooth muscle cell proliferation in normolipidemic wild-type mice, but it was unknown what would happen to hyperlipidemic apolipoprotein E-deficient (Apoe-/-) mice. Examination of these mice revealed rapid development of atherosclerotic lesions in completely ligated carotid arteries within 4 weeks. Mice were fed a Western diet, starting 1 week before ligation, or a chow diet. Foam cell lesions formed as early as 1 week after ligation in mice fed the Western diet and 2 weeks in mice fed the chow diet. Fibrous lesions comprised of foam cells and smooth muscle cells and more advance lesions containing neovessels occurred at 2 and 4 weeks after ligation, respectively, in the Western diet group. Lesions were larger and more advanced in the Western diet group than the chow group. Neutrophil infiltration was observed in growing intimal lesions in both diet groups, while CD8+ T cells were found in lesions of chow-fed mice. This study demonstrates that Apoe-/- mice develop the entire spectrum of atherosclerosis in ligated carotid arteries in an accelerated manner and this model could be a valuable tool for investigating the development and therapy of atherosclerosis.

Data on genetic analysis of atherosclerosis identifies a major susceptibility locus in the major histocompatibility complex of mice.

The data presented here are related to the research article, entitled Genetic analysis of atherosclerosis identifies a major susceptibility locus in the major histocompatibility complex of mice, published in Atherosclerosis 2016;254:124 (A.T. Grainger, M.B. Jones, J. Li, M.H. Chen, A. Manichaikul, W. Shi, 2016) [1]. The supporting materials include original genotypic and phenotypic data obtained from 206 female F2 mice derived from an intercross between BALB and SMJ inbred mice. The F2 mice were fed 12 weeks of Western diet, starting at 6 weeks of age. Atherosclerotic lesion size in the aortic root of each mouse is the sum of the top 8 lesion areas. The data is provided in the format required for determining QTLs using two independent programs, J/QTL and PLINK.

Genetic analysis of atherosclerosis identifies a major susceptibility locus in the major histocompatibility complex of mice.

BACKGROUND AND AIMS: Recent genome-wide association studies (GWAS) have identified over 50 significant loci containing common variants associated with coronary artery disease. However, these variants explain only 26% of the genetic heritability of the disease, suggesting that many more variants remain to be discovered. Here, we examined the genetic basis underlying the marked difference between SM/J-Apoe-/- and BALB/cJ-Apoe-/- mice in atherosclerotic lesion formation. METHODS: 206 female F2 mice generated from an intercross between the two Apoe-/- strains were fed 12 weeks of western diet. Atherosclerotic lesion sizes in the aortic root were measured and 149 genetic markers genotyped across the entire genome. RESULTS: A significant locus, named Ath49 (LOD score: 4.18), for atherosclerosis was mapped to the H2 complex [mouse major histocompatibility complex (MHC)] on chromosome 17. Bioinformatic analysis identified 12 probable candidate genes, including Tnfrsf21, Adgrf1, Adgrf5, Mep1a, and Pla2g7. Corresponding human genomic regions of Ath49 showed significant association with coronary heart disease. Five suggestive loci on chromosomes 1, 4, 5, and 8 for atherosclerosis were also identified. Atherosclerotic lesion sizes were significantly correlated with HDL but not with non-HDL cholesterol, triglyceride or glucose levels in the F2 cohort. CONCLUSIONS: We have identified the MHC as a major genetic determinant of atherosclerosis, highlighting the importance of inflammation in atherogenesis.