Characterization of Bglu3, a mouse fasting glucose locus, and identification of Apcs as an underlying candidate gene

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.     

Complex inheritance of the 5-lipoxygenase locus influencing atherosclerosis in mice

We previously mapped a locus on chromosome 6 with a large effect (LOD > 6) on aortic lesion size in a (C57BL/6J x CAST/Ei) F(2) cross and identified arachidonate 5-lipoxygenase (5LO) as a candidate gene in this region. Subsequent studies with the 5LO knockout model showed effects on atherosclerosis and aortic aneurysms. We now report detailed genetic analysis of the chromosome 6 locus. We created a panel of overlapping and reciprocal subcongenic lines from the B6.CAST Ldlr(-/-) chromosome 6 congenic strain (CON6.Ldlr(-/-)) and analyzed aortic lesion size in different subcongenic lines. Our results revealed that there are at least two subregions, designated as Ath37 and Ath38 that affect the size of aortic lesions independently of 5LO. We also showed that homozygote 5LO null mice develop smaller atherosclerotic lesions. We conclude that the relation between the mouse chromosome 6 locus and atherosclerosis is complex and is due to at least two genes with large effects within this region. This complexity should be considered when interpreting results of knockout studies.     

Quantitative trait locus analysis of atherosclerosis in an intercross between C57BL/6 and C3H mice carrying the mutant apolipoprotein E gene

Inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) differ significantly in atherosclerosis susceptibility and plasma lipid levels on the apolipoprotein E-deficient (apoE-/-) background when fed a Western diet. To determine genetic factors contributing to the variations in these phenotypes, we performed quantitative trait locus (QTL) analysis using an intercross between the two strains carrying the apoE-/- gene. Atherosclerotic lesions at the aortic root and plasma lipid levels of 234 female F2 mice were analyzed after being fed a Western diet for 12 weeks. QTL analysis revealed one significant QTL, named Ath22 (42 cM, LOD 4.1), on chromosome 9 and a suggestive QTL near D11mit236 (20 cM, LOD 2.4) on chromosome 11 that influenced atherosclerotic lesion size. One significant QTL on distal chromosome 1, which accounted for major variations in plasma LDL/VLDL cholesterol and triglyceride levels, coincided with a QTL having strong effects on body weight. Plasma LDL/VLDL cholesterol or triglyceride levels of F2 mice were significantly correlated with body weight, but they were not correlated with atherosclerotic lesion sizes. These data indicate that atherosclerosis susceptibility and plasma cholesterol levels are controlled by separate genetic factors in the B6 and C3H mouse model and that genetic linkages exist between body weight and lipoprotein metabolism.     

Confirmation and high resolution mapping of an atherosclerosis susceptibility gene in mice on Chromosome 1

Previously, we demonstrated that Ath1 is a quantitative trait locus for aortic fatty streak formation, located on Chromosome (chr) 1, with susceptibility in C57BL/6J mice and resistance in C3H/HeJ and BALB/cJ mice fed an atherogenic diet. In this study, we find an atherosclerosis susceptibility locus in the same region of Chr 1 by constructing two congenic strains with the resistance phenotype transferred from different resistant strains, PERA/EiJ or SPRETUS/EiJ. By backcrossing one congenic strain to C57BL/6J and testing recombinant animals, we reduced the distance of the atherosclerosis susceptibility region to 2.3 cM between D1Mit14 and D1Mit10. Further testing of nine recombinant animals showed that eight of the nine were consistent with a further narrowing between D1Mit159 and D1Mit398 a distance of 0.66 cM. This region encompasses a number of potential candidate genes including the thiol-specific antioxidant gene Aop2, also known as peroxiredoxin 5 (Prdx5). AOP2 is capable of reducing hydroperoxides and lipid peroxides in the cell. To investigate Aop2 as a potential candidate, we mapped Aop2 in our backcross and localized it to the atherosclerosis susceptibility interval. We determined that Aop2 is highly expressed in atherosclerosis-related tissues including liver and heart. We also found an inverse correlation between Aop2 mRNA in liver and atherosclerosis phenotype for strains C57BL/6 and the resistant congenic derived from SPRETUS/EiJ. Since LDL oxidation has been implicated in the pathogenesis of this disease, and AOP2 possesses antioxidant activity, we suggest the role of Aop2 in atherosclerosis susceptibility needs to be further explored.     

Neointimal formation in two apolipoprotein E-deficient mouse strains with different atherosclerosis susceptibility

C57BL/6 (B6) and C3H/HeJ (C3H) are two commonly used mouse strains that differ markedly in atherosclerosis susceptibility. In this study, we determined plaque formation after removal of the endothelium in the two strains carrying the mutant apolipoprotein E gene (apoE(-/-)). At 10 weeks of age, male B6.apoE(-/-) and C3H.apoE(-/-) mice underwent endothelial denudation of the left common carotid artery. Two weeks after injury, B6.apoE(-/-) mice developed significantly larger neointimal lesions in the vessel than their C3H.apoE(-/-) counterparts, although they had comparable plasma cholesterol levels on a chow diet. Feeding of a Western diet aggravated lesion formation in both strains, but the increase was more dramatic in B6.apoE(-/-) mice than in C3H.apoE(-/-) mice. Immunohistochemical and histological analyses demonstrated the presence of macrophage foam cells in neointimal lesions. We then compared neointimal growth in F1 mice reconstituted with bone marrow from B6.apoE(-/-) and C3H.apoE(-/-) mice. No significant lesions were observed 2 weeks after endothelial denudation in the mice reconstituted with bone marrow from either donor. Thus, these data indicate that foam cell formation contributes to neointimal growth in the hyperlipidemic apoE(-/-) model and that neither endothelial cells nor blood cells alone explain the dramatic difference between B6 and C3H mice in plaque formation.     

Circulating adhesion molecules in apoE-deficient mouse strains with different atherosclerosis susceptibility

Recruitment of inflammatory cells in the arterial wall by vascular adhesion molecules plays a key role in development of atherosclerosis. Apolipoprotein E-deficient (apoE(-/-)) mice have spontaneous hyperlipidemia and develop all phases of atherosclerotic lesions. We sought to examine plasma levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and sP-selectin in two apoE(-/-) strains C57BL/6 (B6) and BALB/c with early or advanced lesions. Mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. On either diet, BALB/c.apoE(-/-) mice developed much smaller atherosclerotic lesions and displayed significantly lower levels of sVCAM-1 and sP-selectin than B6.apoE(-/-) mice. The Western diet significantly elevated sVCAM-1 levels in both strains and sP-selectin levels in B6.apoE(-/-) mice. BALB/c.apoE(-/-) mice exhibited 2-fold higher HDL cholesterol levels on the chow diet and 15-fold higher HDL levels on the Western diet than B6.apoE(-/-) mice, although the two strains had comparable levels of total cholesterol and triglyceride. Thus, increased atherosclerosis is accompanied by increases in circulating VCAM-1 and P-selectin levels in the two apoE(-/-) mouse strains, and the high HDL level may protect against atherosclerosis by inhibiting the expression of adhesion molecules in BALB/c.apoE(-/-) mice.     

Quantitative trait loci analysis for the differences in susceptibility to atherosclerosis and diabetes between inbred mouse strains C57BL/6J and C57BLKS/J.

Mice from the inbred strain C57BLKS/J (BKS) exhibit increased susceptibility to both diabetes and atherosclerosis compared to C57BL/6J (B6) mice. To determine whether the differences in diabetes and atherosclerosis are related, we carried out a cross between B6-db/db and BKS. We selected 99 female F2-db/db progeny, tested the progeny for plasma lipids, plasma glucose, and fatty-streak lesions, and used quantitative trait loci (QTL) analysis to identify the chromosomal regions associated with these phenotypes. No major QTL were found for total cholesterol, VLDL-cholesterol, or triglycerides. Two suggestive QTL were found for HDL-cholesterol (LOD scores of 2. 7 and 2.8), and two suggestive loci were found for plasma glucose (LOD scores of 2.3 and 2.0). Lesion size was not correlated with plasma lipid levels or glucose. Lesion size was determined by a locus at D12Mit49 with a LOD score of 2.5 and a significant likelihood ratio statistic. The gene for apolipoprotein apoB lies within the region, but apoB levels were similar in strains B6 and BKS. The QTL on Chr 12 was confirmed by constructing a congenic strain with BKS alleles in the QTL region on a B6 genetic background. We conclude that susceptibilities to diabetes and atherosclerosis are not conferred by the same genes in these strains and that a major gene on Chr 12, which we name Ath6, determines the difference in atherosclerosis susceptibility.     

Fine mapping of Ath6, a quantitative trait locus for atherosclerosis in mice

Ath6 is a novel quantitative trait locus associated with differences in susceptibility to atherosclerosis between C57BL/6J (B6) and C57BLKS/J (BKS) inbred mouse strains. Combining data from an intercross and a backcross (1593 meioses) between mice from B6 and BKS strains and from The Jackson Laboratory interspecific backcross panels, (C57BL/6J ×Mus spretus) F₁× C57BL/6J and (C57BL/6J × SPRET/Ei) F₁× SPRET/Ei, we constructed a consensus genetic map and narrowed Ath6 to a 1.07 ± 0.26 cM interval between the anonymous DNA marker D12Pgn4 and the gene Nmyc1. This region is near the proximal end of murine Chromosome (Chr) 12, which is homologous to the human chromosomal region 2p24-p25. Marker order in the Ath6 region was concordant among the two crosses and The Jackson Laboratory interspecific backcross panels. This high resolution map rules out candidate genes encoding apolipoprotein B, syndecan 1, and Adam17. The two Ath6 crosses have a combined potential resolution of 0.06 cM. Received: 12 September 2000 / Accepted: 22 February 2001     

Elimination of macrophage-specific apolipoprotein E reduces diet-induced atherosclerosis in C57BL/6J male mice

Apolipoprotein (apo)E is synthesized in atherosclerotic lesions by macrophages, however, its role in lesions is not known. Whereas apoE could exacerbate atherosclerosis by promoting macrophage uptake of cholesterol-rich lipoproteins or modulating protective inflammatory responses, it could also restrict lesion formation by facilitating cholesterol efflux out of lesions. The role of apoE was examined in lethally irradiated male C57BL/6J wild-type (WT) mice that were repopulated with bone marrow cells (BMT) from either identical C57BL/6J mice (WT+WT BMT) or C57BL/6J apoE-deficient mice (WT+E-/- BMT). This enabled us to compare normal mice with mice possessing macrophages that did not express apoE. The participation of macrophage-derived apoE in atherosclerosis was assessed by placing the mice on an atherogenic diet. Male WT+E-/- BMT mice had significantly reduced lesion area in the aortic valves (P < 0.01) compared with male WT+WT BMT mice ( approximately 22,000 vs. approximately 49,000 microm2/section, respectively). Further evaluation revealed that plasma cholesterol, lipoprotein cholesterol distribution, and plasma apoE were similar between the two groups, indicating that these known risk factors did not account for the differences in lesion area. However, the two groups were distinguished by the amount of apoE found in the lesions. ApoE antigen was expressed abundantly in WT+WT BMT lesions, whereas WT+E-/- BMT lesions contained little apoE. These findings indicate that the majority of apoE in lesions is synthesized locally by resident macrophages, and suggest that locally produced apoE can promote diet-induced atherosclerosis in male wild-type mice.     

Variation in Galr1 expression determines susceptibility to exocitotoxin-induced cell death in mice

Inbred strains of mice differ in their susceptibility to excitotoxin-induced cell death, but the genetic basis of individual variation in differential susceptibility is unknown. Previously, we identified a highly significant quantitative trait locus (QTL) on chromosome 18 that influenced susceptibility to kainic acid-induced cell death ( Sicd1 ). Comparison of susceptibility to seizure-induced cell death between reciprocal congenic lines for Sicd1 and parental background mice indicates that genes influencing this trait were captured in both strains. Two positional gene candidates, Galr1 and Mbp , map to 55 cM, where the Sicd1 QTL had been previously mapped. Thus, this study was undertaken to determine if Galr1 and/or Mbp could be considered as candidate genes. Genomic sequence comparison of these two functional candidate genes from the C57BL/6J (resistant at Sicd1 ) and the FVB/NJ (susceptible at Sicd1 ) strains showed no single-nucleotide polymorphisms. However, expression studies confirmed that Galr1 shows significant differential expression in the congenic and parental inbred strains. Galr1 expression was downregulated in the hippocampus of C57BL/6J mice and FVB.B6- Sicd1 congenic mice when compared with FVB/NJ or B6.FVB- Sicd1 congenic mice. A survey of Galr1 expression among other inbred strains showed a significant effect such that 'susceptible' strains showed a reduction in Galr1 expression as compared with 'resistant' strains. In contrast, no differences in Mbp expression were observed. In summary, these results suggest that differential expression of Galr1 may contribute to the differences in susceptibility to seizure-induced cell death between cell death-resistant and cell death-susceptible strains.     

ApoE-/-Fas-/- C57BL/6 mice: a novel murine model simultaneously exhibits lupus nephritis, atherosclerosis, and osteopenia

To establish a mouse model of accelerated atherosclerosis in lupus, we generated apolipoprotein E-deficient (apoE(-/-)) and Fas(lpr/lpr) (Fas(-/-)) C57BL/6 mice. On a normal chow diet, 5 month old apoE(-/-)Fas(-/-) mice had enlarged glomerular tuft areas, severe proteinuria, increased circulating autoantibody levels, and increased apoptotic cells in renal and vascular lesions compared with either single knockout mice. Also, double knockout mice developed increased atherosclerotic lesions but decreased serum levels of total and non-HDL cholesterol compared with apoE(-/-)Fas(+/+) littermates. Moreover, female apoE(-/-)Fas(-/-) mice had lower vertebral bone mineral density (BMD) and bone volume density (BV/TV) than age-matched female apoE(-/-)Fas(+/+) mice. Compared with apoE(-/-)Fas(+/+) and apoE(+/+)Fas(-/-) mice, apoE(-/-)Fas(-/-) mice had decreased circulating oxidized phospholipid (OxPL) content on apoB-100 containing lipoprotein particles and increased serum IgG antibodies to OxPL, which were significantly correlated with aortic lesion areas (r = 0.58), glomerular tuft areas (r = 0.87), BMD (r = -0.57), and BV/TV (r = -0.72). These results suggest that the apoE(-/-)Fas(-/-) mouse model might be used to study atherosclerosis and osteopenia in lupus. Correlations of IgG anti-OxPL with lupus-like disease, atherosclerosis, and bone loss suggested a shared pathway of these disease processes.     

Ath-2, a Second Gene Determining Atherosclerosis Susceptibility and High Density Lipoprotein Levels in Mice

Strain C57BL/6J and A/J differ at two genes determining atherosclerosis susceptibility. The first gene, Ath-1, was described earlier and this report characterizes Ath-2. The alleles at Ath-2 are r for resistance and s for susceptibility to atherosclerosis. The resistant phenotype in female mice is characterized by high plasma high density lipoprotein-cholesterol levels (74 mg/dl +/- SEM 2) and very few lesions/mouse after 14 weeks of consumption of an atherogenic diet (0.1 +/- SEM 0.1 in a predetermined region of the aorta). The susceptible phenotype in female mice is characterized by low levels of high density lipoprotein-cholesterol (35 mg/dl +/- SEM 1) and 1.2 lesions/mouse +/- SEM 0.2 in the same region of the aorta. In Ath-2 heterozygotes, resistance is dominant to susceptibility. Recombinant inbred strains derived from C57BL/6 and A were characterized for Apoa 1, Apoa 2 and susceptibility to atherosclerosis. Ath-1 and Ath-2 interact with each other so that resistant alleles at either locus confer a resistant phenotype to the animal. The map position of Ath-2 is not known, but Ath-2 does not map near genes determining the apolipoproteins for A-I, A-II, or E.     

A torrid zone on mouse chromosome 1 containing a cluster of recombinational hotspots

Within the 2.38-Mb Ath1 region of mouse chromosome 1, 42 of 45 genetic crossovers from crosses between C57BL/6J (B6) and either C3H/HeJ (H) or Mus spretus (SPRET) occurred in four zones (A-D); zone A, 100 kb long, contained a cluster of at least four recombination hotspots. F1 sperm assays indicate that within this "torrid zone" the most active hotspot (A3) can initiate recombination on H and SPRET but not B6 chromosomes. The A3 DNA sequence contains a (G/C)TTT repeat, long stretches of A or T, and a cyclic variation in AT content. Recombination was drastically reduced in a cross between B6 and a B6.SPRET Ath1 congenic strain, but was unaffected in a B6 x B6.H Ath1 congenic cross. Similar nonrandom clustering of hotspots has been observed in yeast and the major histocompatibility complexes of human and mouse. To the extent that torrid zones are a general feature of mammalian genomes, they have considerable implications for genetic mapping strategies in both human populations and mouse crosses.     

Reduction of isoprostanes and regression of advanced atherosclerosis by apolipoprotein E

Apolipoprotein E is a multifunctional protein synthesized by hepatocytes and macrophages. Plasma apoE is largely liver-derived and known to regulate lipoprotein metabolism. Macrophage-derived apoE has been shown to reduce the progression of atherosclerosis in mice. We tested the hypothesis that liver-derived apoE could directly induce regression of pre-existing advanced atherosclerotic lesions without reducing plasma cholesterol levels. Aged low density lipoprotein (LDL) receptor-deficient (LDLR(-/-)) mice were fed a western-type diet for 14 weeks to induce advanced atherosclerotic lesions. One group of mice was sacrificed for evaluation of atherosclerosis at base line, and two other groups were injected with a second generation adenoviruses encoding human apoE3 or a control empty virus. Hepatic apoE gene transfer increased plasma apoE levels by 4-fold at 1 week, and apoE levels remained at least 2-fold higher than controls at 6 weeks. There were no significant changes in plasma total cholesterol levels or lipoprotein composition induced by expression of apoE. The liver-derived human apoE gained access to and was retained in arterial wall. Compared with base-line mice, the control group demonstrated progression of atherosclerosis; in contrast, hepatic apoE expression induced highly significant regression of advanced atherosclerotic lesions. Regression of lesions was accompanied by the loss of macrophage-derived foam cells and a trend toward increase in extracellular matrix of lesions. As an index of in vivo oxidant stress, we quantitated the isoprostane iPF(2 alpha)-VI and found that expression of apoE markedly reduced urinary, LDL-associated, and arterial wall iPF(2 alpha)-VI levels. In summary, these results demonstrate that liver-derived apoE directly induced regression of advanced atherosclerosis and has anti-oxidant properties in vivo that may contribute to its anti-atherogenic effects.     

Effects of MHC and gender on lupus-like autoimmunity in Nba2 congenic mice

The lupus-like disease that develops in hybrids of NZB and NZW mice is genetically complex, involving both MHC- and non-MHC-encoded genes. Studies in this model have indicated that the H2d/z MHC type, compared with H2d/d or H2z/z, is critical for disease development. C57BL/6 (B6) mice (H2b/b) congenic for NZB autoimmunity 2 (Nba2), a NZB-derived susceptibility locus on distal chromosome 1, produce autoantibodies to nuclear Ags, but do not develop kidney disease. Crossing B6.Nba2 to NZW results in H2b/z F1 offspring that develop severe lupus nephritis. Despite the importance of H2z in past studies, we found no enhancement of autoantibody production or nephritis in H2b/z vs H2b/b B6.Nba2 mice, and inheritance of H2z/z markedly suppressed autoantibody production. (B6.Nba2 x NZW)F1 mice, compared with MHC-matched B6.Nba2 mice, produced higher levels of IgG autoantibodies to chromatin, but not to dsDNA. Although progressive renal damage with proteinuria only occurred in F1 mice, kidneys of some B6.Nba2 mice showed similar extensive IgG and C3 deposition. We also studied male and female B6.Nba2 and F1 mice with different MHC combinations to determine whether increased susceptibility to lupus among females was also expressed within the context of the Nba2 locus. Regardless of MHC or the presence of NZW genes, females produced higher levels of antinuclear autoantibodies, and female F1 mice developed severe proteinuria with higher frequencies. Together, these studies help to clarify particular genetic and sex-specific influences on the pathogenesis of lupus nephritis.     

Overexpression of Prdx6 reduces H2O2 but does not prevent diet-induced atherosclerosis in the aortic root

The mammalian 1-Cys peroxiredoxin (Prdx6) is a unique member of the peroxiredoxin family of proteins capable of protecting cells from metal-catalyzed oxidative damage. We recently identified Prdx6 as a candidate for the quantitative trait locus Ath1, a gene responsible for a difference in diet-induced atherosclerosis susceptibility in mice. To investigate the role of Prdx6 in atherosclerosis, we generated transgenic mice that overexpress the Prdx6 allele from the Ath1-resistant 129/SvJ strain on an Ath1-susceptible C57BL/6J background. These mice expressed significantly elevated levels of Prdx6 mRNA and protein in multiple tissues including liver, aorta, and peritoneal macrophages, which accumulated significantly lower levels of hydrogen peroxide, revealing an enhanced antioxidant activity in these mice. However, overexpression of Prdx6 had no protective effect on LDL oxidation in vitro, and transgenic mice fed an atherogenic diet for 10 weeks did not possess an increased resistance to atherosclerosis nor did they maintain the high prediet plasma HDL levels consistent with the Ath1-resistant phenotype. In addition, the Prdx6 allele from the susceptible strain was shown to have a higher antioxidant activity than that of the resistant strains. These data suggest that the increased peroxidase activity attributable to Prdx6 overexpression in transgenic mice is not sufficient to protect mice from atherosclerosis, and that Prdx6 is not likely to be the gene underlying Ath1.     

Selective macrophage ascorbate deficiency suppresses early atherosclerosis

To test whether severe ascorbic acid deficiency in macrophages affects progression of early atherosclerosis, we used fetal liver cell transplantation to generate atherosclerosis-prone apolipoprotein E-deficient (apoE(-/-)) mice that selectively lacked the ascorbate transporter (SVCT2) in hematopoietic cells, including macrophages. After 13 weeks of chow diet, apoE(-/-) mice lacking the SVCT2 in macrophages had surprisingly less aortic atherosclerosis, decreased lesion macrophage numbers, and increased macrophage apoptosis compared to control-transplanted mice. Serum lipid levels were similar in both groups. Peritoneal macrophages lacking the SVCT2 had undetectable ascorbate; increased susceptibility to H(2)O(2)-induced mitochondrial dysfunction and apoptosis; decreased expression of genes for COX-2, IL1β, and IL6; and decreased lipopolysaccharide-stimulated NF-κB and antiapoptotic gene expression. These changes were associated with decreased expression of both the receptor for advanced glycation end products and HIF-1α, either or both of which could have been the proximal cause of decreased macrophage activation and apoptosis in ascorbate-deficient macrophages.     

Lipid retention in the arterial wall of two mouse strains with different atherosclerosis susceptibility

LDL deposition in the subendothelium of arterial walls is the initial event in the development of atherosclerosis. The deposited LDL undergoes oxidative modification by arterial wall cells to become oxidized LDL and consequently contributes to atherosclerotic formation. Using mouse strains C57BL/6J (B6) and C3H/HeJ (C3H), which differ markedly in susceptibility to atherosclerosis, we determined whether variation in subendothelial retention of apolipoprotein B (apoB)-containing lipoproteins constitutes a genetic component in atherosclerosis. Lipoprotein retention was quantitated by Western blot analysis to detect the presence of apoB in aortic walls before foam cells developed. In both dietary and apoE-deficient models, B6 mice exhibited up to a 2-fold increase of apoB in the aortic wall compared with C3H mice. This increase could not be attributed to differences in plasma lipid levels of the two strains. In vitro, endothelial cells from C3H mice took up more acetylated and oxidized LDL but not native LDL and converted more native LDL to oxidized LDL than did endothelial cells from B6 mice. C3H mice expressed more scavenger receptor A in their aortic wall than B6 mice. Thus, variation in the subendothelial retention of apoB-containing lipoproteins cannot explain the dramatic difference in atherosclerosis susceptibility between B6 and C3H mice, and endothelial cells may play a role in alleviating lipid accumulation in arterial walls.     

Ex vivo evaluation of a novel polyiodinated compound for early detection of atherosclerosis

Atherosclerosis is a primary cause of heart disease and stroke; it is the underlying cause of about 50% of all deaths in Western countries. It is known that early detection of atherosclerotic lesions would significantly reduce the risk of mortality. The objective of this study was to develop a radioimaging method for early detection of atherosclerotic plaques. A novel polyiodinated cholesterol analog, cholesteryl 1,3-diiopanoate glyceryl ether (C2I, patent pending), was synthesized and radiolabeled with 125I. 125I-C2I was incorporated into acetylated low-density lipoprotein (AcLDL), which is considered to be an atherosclerotic plaque-seeking carrier. 125I-C2I was also prepared as a chylomicron-like emulsion. Transgenic mice deficient in apoE and low-density lipoprotein receptors (LDLR), known as apoE/LDLR double knockout, were used as an animal model of early atherosclerosis. 125I-C2I/AcLDL or 125I-C2I emulsion was injected into the apoE/LDLR knockout mice via the tail vein, and the mice were killed humanely 24 h after injection. Various tissues including aorta were removed and radioactivity was determined. The aorta samples were also imaged to determine the accumulation of radioactivity from C2I. The images were compared to the atherosclerotic lesions revealed by histological studies. It was found that both 125I-C2I/AcLDL and 125I-C2I emulsion resulted in accumulation of radioactivity at the site of early atherosclerotic lesions, and they therefore may be useful for early detection of atherosclerosis.