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【摘要】
Objective— C3H/HeJ (C3H) mice are extremely resistant to atherosclerosis. To identify the genetic factors involved in lesion initiation, we studied a cross between C3H and the susceptible strain C57BL/6J (B6) on a hyperlipidemic (apolipoprotein E–null) background.
Methods and Results— Whereas a previous cross in mice fed a Western diet for 16 weeks revealed a very complex inheritance pattern with many significant lesion QTLs, the present cross, on a chow diet, revealed a single major locus on chromosome 9 (lod=5.0, Ath29 *), and a suggestive locus on chromosome 4 (lod=2.6, Ath8 ). QTLs for plasma HDL, total cholesterol, and triglyceride levels were found on chromosome 1 over the ApoA2 gene. Neither of the lesion QTLs were associated with differences in plasma lipid levels or other systemic risk factors, consistent with the concept that genetic factors affecting cellular functions of the vessel wall are important determinants of atherosclerosis susceptibility. We generated a congenic strain for Ath29 and confirmed its contribution to lesion development. Toll-like receptor 4 ( Tlr4 ), the lipopolysaccharide (LPS) receptor, is located in the Ath8 region and is known to be defective in C3H/HeJ mice. We constructed a congenic strain carrying a normal Tlr4 gene on the C3H Apoe -null background and found that the defective Tlr4 does not contribute significantly to lesion resistance during early lesion development.
Conclusions— We identified one major QTL on chromosome 9, Ath29, for early lesion development in the BXH ApoE –/– cross fed on a chow diet and confirmed its contribution in congenic mice. We have also determined that Tlr4 on the C3H ApoE –/– background does not contribute to early lesion development. * Ath29 is referred to as Ath22 in Su et al 2006.
An F2 intercross between C57BL/6J and C3H/HeJ on the ApoE –/– background, fed on a chow diet and euthanized at 12 weeks exhibited one significant QTL on chromosome 9, Ath29 and a suggestive QTL on chromosome 4, Ath8. Congenics for Ath29 confirmed the contribution of the locus to lesion development.
【关键词】 atherosclerosis quantitative trait locus CH/HeJ
Introduction
The genes underlying a number of Mendelian forms of atherosclerosis, such as familial hypercholesterolemia, have been identified, but the genetic factors involved in the common forms, explaining about 50% of all deaths in Western populations, remain largely unknown. 1 One approach to the problem has been to study inbred strains of mice differing in atherosclerosis susceptibility. During the past 20 years, a number of strains of mice with varying susceptibility to the disease have been studied. 2 C3H/HeJ (C3H) mice have proven to be particularly resistant to the disease. 3 Whereas C57BL/6J (B6) and most other strains develop large, human-like atherosclerotic lesions on the background of the hyperlipidemia-inducing apolipoprotein E–null ( Apoe –/– ) mutation, 4 C3H mice develop almost no lesions unless placed on a high-fat, Western diet. 5 The resistance of C3H mice appears to be mediated in part by the failure of endothelial cells to respond to oxidized lipids. Thus, whereas oxidized LDL induces the expression of a variety of inflammatory genes such as monocyte chemotactic protein 1 ( Mcp-1 ) and vascular cell adhesion molecule (VCAM) ( Vcam-1 ) in aortic endothelial cells (ECs) from B6, ECs from C3H mice are almost totally resistant to the induction. 5–7 Thus, studies of C3H mice provide an opportunity to identify novel mechanisms underlying susceptibility to atherosclerosis.
We now report the mapping of chromosomal loci contributing to atherosclerosis resistance in C3H mice on an Apoe –/– background. We studied 12-week-old mice maintained on a chow diet to assess genetic contributions to early lesion development. As in a previous study of mice maintained on a high-fat, high-cholesterol diet, the major locus identified in our study was on chromosome 9. 8 C3H/HeJ mice carry a defective allele of Toll-like receptor 4 ( Tlr4 ), a gene that has been implicated in resistance to atherosclerosis in human studies 9 as well as mice. 10 Using a congenic mouse strain, we show that the Tlr4 gene does not have a significant impact on early atherosclerosis on the C3H background. We have also isolated the major locus contributing to atherosclerosis susceptibility on chromosome 9 as a congenic strain and characterized its effect on atherosclerosis. This strain should make fine mapping of the gene feasible.
Materials and Methods
Mice and Diets
C57BL/6J Apoe –/– (B6 Apoe –/– ) mice were purchased from the Jackson Laboratory, Bar Harbor, Maine and C3H/HeJ Apoe –/– (C3H Apoe –/– ) mice were bred by backcrossing B6 Apoe –/– to C3H/HeJ for 10 generations as previously described. 5 All mice were fed ad libidum and maintained on a 12-hour light/dark cycle. F2 mice were generated by crossing B6 Apoe –/– with C3H Apoe –/– and subsequently intercrossing the F1s. F2 mice were fed Purina Chow (Ralston-Purina Co) containing 4% fat and euthanized at 12 weeks of age. Before being euthanized, they were fasted overnight, anesthetized with Isoflurane, and bled through the retro-orbital sinus. Plasma was stored at –80C.
Tlr4 Congenic Studies
To produce C3H. Apoe –/– Tlr4 lps–n (normal response to lipopolysaccharade ) congenic mice, C3H Apoe –/–, which are Tlr4 lps–d (defective LPS response) because of a spontaneous mutation, were intercrossed with C3H/DiSnA Apoe +/+ Tlr4 lps–n mice 11 and C3H Apoe –/– Tlr4 lps–n F2 offspring were selected. Genotyping for Apoe and Tlr4 was done by polymerase chain reaction (PCR). PCR for Apoe was performed as previously described. 12 The Tlr4 primer sequences are as follows: F 5'tcagaatgaggactgggtga3' R 5'tcaaagatacaccaacggctc3'. The Tlr4 PCR product was digested with NlaIII (CATG); the Tlr4 lps–d allele contains a unique restriction enzyme site yielding a 98-bp product. The progeny of the F1 heterozygotes gave rise to approximately a 1:2:1 ratio of Tlr4 lps–n, Tlr4 lps–n/d, and Tlr4 lps–d respectively. C3H/DiSnA mice were obtained from Dr Peter Demant, Department of Molecular Biology, Roswell Park Memorial Institute, Buffalo, NY, who had bred the substrain in his laboratory. The Tlr4 lps–d allele arose on the C3H/HeJ strain after the separation of the two colonies.
Histological Analyses
The aorta was sectioned and lesions were quantified as previously described. 13 After the mice were euthanized, the heart and proximal aorta were excised and washed in PBS. The apex and lower half of the ventricles were cut off. The remaining specimen was embedded in Tissue-Tek (Miles), frozen on dry ice, and stored at –80C until sectioning. Serial cryosections were prepared through the ventricle until the aortic valves appeared. From then on, every fifth 10-µm section was collected on poly- D -lysine-coated slides until the aortic sinus was completely sectioned. Sections were stained with hematoxylin and oil red O, which specifically stains lipids. Slides were examined by light microscopy. The average fatty streak lesion area was quantified throughout the aortic sinus using an ocular with a grid. Forty sections per mouse were quantified and averaged to yield a score that was used for QTL analysis.
Lipid Measurements
Plasma triglycerides, total cholesterol, HDL, and free fatty acids were assayed as previously described. 13
Genome Scan
A 10 cM genome scan was performed using microsatellite markers. DNA (concentration 15 ng/µL) was isolated using a standard phenol-chloroform extraction protocol and amplified by touchdown PCR using fluorescently labeled primers from Research Genetics (Huntsville, Ala). PCR products then underwent capillary electrophoresis (Applied Biosystems), and genotypes were determined using Genotyper (Applied Biosystems). The list of microsatellites are as follows: D1mit64, D1mit278, D1mit322, D1mit216, D1mit386, D1mit217, D1mit101, D1mit33, D1mit268, D1mit540, D1mit150, D1mit362, D1mit512, D2mit1, D2mit92, D2mit41, D2mit487, D2mit396, D2mit412, D2mit286, D2mit200, D3mit328, D3mit203, D3mit169, D3mit184, D3mit292, D3mit128, D3mit19, D4mit101, D4mit111, D4mit327, D4mit31, D4mit71, D4mit42, D4mit344, D5mit193, D5mit13, D5mit55, D5mit394, D5mit396, D5mit25, D5mit95, D5mit98, D6mit50, D6mit74, D6mit384, D6mit284, D6mit37, D6mit340, D7mit316, D7mit321, D7mit66, D7mit291, D8mit29, D8mit244, D8mit56, D9mit297, D9mit25, D9mit301, D9mit141, D9mit259, D9mit156, D9mit239, D9mit33, D9mit136, D9mit16, D9mit151, D10mit168, D10mit31, D10mit42, D10mit10, D10mit145, D11mit306, D11mit206, D11mit155, D11mit31, D11mit39, D12mit12, D12mit34, D12mit17, D13mit198, D13mit63, D13mit113, D13mit314, D13mit287, D13mit260, D13mit35, D14mit174, D14mit157, D14mit68, D14mit199, D14mit136, D15mit199, D15mit204, D15mit167, D15mit3, D15mit31, D15mit43, D16mit55, D16mit4, D16mit76, D16mit86, D17mit171, D17mit101, D17mit178, D17mit183, D17mit155, D18mit19, D18mit120, D18mit58, D18mit123, D18mit161, D18mit33, D18mit49, D18mit4, D18mit144, D19mit59, D19mit42, D19mit96, D19mit40, D19mit13, D19mit90, Dxmit89, Dxmit166, Dxmit1, Dxmit10, Dxmit222.
Linkage and Data Analysis
Genotype and phenotype data were imported into Windows QTL Cartographer version 2.0 14 and a linkage map was created according to the marker location specified in the Mouse Genome Informatics website (www.informatics.jax.org) following the procedures outlined in the accompanying manual. The marker map was consistent with an empirically generated map and was scanned for double recombinants. Interval mapping was performed using model H3:H0, which accounts for both additive and dominant effects. Phenotypic traits were transformed as needed to normalize the residuals. This involved 3 SD) were omitted. Significance was assessed based on permuting the data 1000 times using the Zmapqtl function (model 6) for a genome-wide P <0.05. The corresponding lod score was 3.5 for all traits.
Analysis of variance (ANOVA) between markers and traits was calculated using Statview v5.0 (SAS Institute Inc). Correlation between traits was calculated using the Spearman rank correlation. Data were then graphed in Sigma Plot (SPSS Inc).
Results
Genetic Determinants of Atherosclerosis for Early Lesions
To identify QTLs for early atherosclerotic lesions, which reflect differences in lesion initiation, B6 Apoe –/– and C3H Apoe –/– mice were intercrossed to generate a population of 135 F2 females. The F2 mice were fed a chow diet, euthanized at 12 weeks of age, and lesions at the aortic sinus were quantified. The distribution of lesion sizes across the population ranged from 0 to 80,000 µm 2 /section (supplemental Figure Ia, available online at http://atvb.ahajournals.org). A genome scan with an average density of 10 cM was performed using microsatellite markers. One significant (lod=5.0) QTL for atherosclerosis was found on chromosome 9 at about 27 cM ( Figure 1 a). Two suggestive QTLs were found on chromosome 4 (lod=2.6) at 28 cM ( Figure 1 b) and on chromosome 10 at 58 cM (lod=2.5; supplemental Figure IIa). The B6 allele conferred susceptibility in all cases.
Figure 1. Atherosclerosis QTLs on chromosome 9 (a) and chromosome 4 (b). Insets: lesions were regressed against peak markers genotypes; D9mit25(a) and D4mit111(b). For D9mit25(a), n=25, 24, and 18 for genotypes b, h, and c, respectively. Individuals with the b genotype had significantly larger lesions than those with the h genotype ( P =0.014) and the c genotype ( P =0.0015). Those with c and h genotypes were not significantly different ( P =0.34). For D4mit111(b), n=28, 72, and 34 for genotypes b, h, and c, respectively. Individuals with the b genotype had significantly larger lesions than those with the h genotype ( P =0.034) and the c genotype ( P =0.0087). Those with c and h genotypes were not significantly different ( P =0.33).
Plasma lipid levels were measured to determine whether any correlation existed between lesion size and lipid levels. QTL analysis was also performed on lipid levels to identify loci that regulate lipids and to determine whether any lipid QTLs colocalized with the lesion QTLs. Using the Spearman rank correlation, the correlation coefficient ( ) was calculated between lesion size and LDL/very low-density lipoprotein, high-density lipoprotein, total cholesterol, triglycerides, and free fatty acids. No significant correlations were found between any of the lipid traits and lesions (supplemental Table I). Significant QTLs on chromosome 1 at 92 cM were found for HDL (lod=7.1) and triglycerides (lod=4.4; Figure 2 ). A suggestive QTL for total cholesterol (lod=3.0) was also identified at the same locus. A suggestive QTL for HDL (lod=3.8) and triglycerides (lod=2.5) was found on chromosome 10 at 14 cM. Hence, no QTL overlaps were found between lesions and lipids (supplemental Figure IIa and IIb).
Figure 2. QTLs for total cholesterol (TC), HDL-cholesterol (HDL), and triglycerides (TG) on chromosome 1 over the ApoA2 locus.
Confirmation and Characterization of the Chromosome 9 Locus
We constructed mice congenic for the chromosome 9 locus by introgressing that region of C3H onto a B6 Apoe –/– background by backcrossing for 8 generations. As determined by genotyping, the congenic region extended from 15 to 61 cM between markers D9mit297 and D9mit16. Female congenics had a mean lesion size of 9094±1871 µm 2 per section and B6 Apoe –/– littermate controls had a mean size of 36319±4518 µm 2 per section. Thus, the lesions in the female congenics were about 25% the size of the controls ( P <0.0001). Male congenics had a mean lesion size of 11375±1649 µm 2 per section and control B6 Apoe –/– had a size of 20896±3481 µm 2 per section. Thus, the congenic lesions of the males were about 54% the size of the controls ( P =0.013). These results confirm the contribution of this locus to atherosclerosis ( Figure 3 ). The majority of studies of atherosclerosis in mice are consistent with our sex-biased results; female mice almost always develop larger lesions than male mice, and the reasons underlying this sex bias are not known. The congenic mice resemble C3H Apoe –/– mice in their lesion development. Male and female C3H Apoe –/– mice exhibit extremely small lesions that are statistically not different in size, likewise for our congenics. We speculate that the chromosome 9 region harbors an allele that may act more strongly in females compared with males.
Figure 3. Comparison of B6 Apoe –/– mice with B6 Apoe –/– congenic for the chromosome 9 QTL. Female B6 Apoe –/– mice versus B6 Apoe –/– congenic P <0.0001; male B6 Apoe –/– mice versus B6 Apoe –/– congenic P =0.013. The bars indicate the mean values.
Tlr4 on the C3H Background Does Not Significantly Influence Lesion Development
Tlr4, located on chromosome 4 at 33 cM, was just distal to the suggestive chromosome 4 peak. Because Tlr4 has been associated with atherosclerosis in humans 9 and mice 10 and is known to be defective in C3H/HeJ, 15 we investigated the role of Tlr4 in atherosclerosis in C3H/HeJ mice. We generated C3H Apoe –/– mice that carry the functional Tlr4 lps–n gene from C3H/DisNA by intercrossing the strains. The mean lesion size for C3H Apoe –/– Tlr4 lps–n was 384±127 µm 2 /section, that of C3H Apoe –/– Tlr4 lps–d was 221±105 µm 2 per section, and that of B6 Apoe –/– Tlr4 lps–n was 15580±1220 µm 2 /section. Thus, the congenic C3H Apoe –/– Tlr4 lps–n showed no significant difference in lesion size compared with C3H Apoe –/– Tlr4 lps–d, though both of these groups were significantly different from B6 Apoe –/– Tlr4 lps–n ( P <0.0001) ( Figure 4 ). These data suggest that Tlr4 does not contribute significantly to the extreme resistance of C3H mice to early lesion development, and that Tlr4 is not a cause of the difference in lesion initiation between C3H and B6.
Figure 4. Comparison of aortic root lesion area between C3H Apoe –/– Tlr4 lps–d mice (n=7) with congenic C3H Apoe –/– Tlr4 lps–n (n=44) and B6 Apoe –/– Tlr4 lps–n (n=29) 9- to 10-week-old mice were studied. C3H Apoe –/– Tlr4 lps–d vs C3H Apoe –/– Tlr4 lps–n P =0.92. C3H Apoe –/– Tlr4 lps–d or C3H Apoe –/– Tlr4 lps–n vs B6 Apoe –/– Tlr4 lps–n P <0.0001.
Discussion
C3H mice are unusually resistant to atherosclerosis, and we now report a genetic analysis of a cross between strain C3H and the susceptible strain B6 on the background of hyperlipidemia because of an Apoe –/– mutation. Several significant conclusions have emerged. First, the cross does not replicate the Ath1 locus identified using C57BL/6J (B6) x C3H/HeJ (C3H) recombinant inbred (RI) strains fed a cholic acid diet. Second, a major locus on chromosome 9 was identified and confirmed using a congenic strategy. Third, the Tlr4 gene was shown to not be a significant factor in the resistance of C3H mice to atherosclerosis. Finally, a chromosome 1 locus for plasma lipids identified in a BXH cross on a chow diet was replicated. Below, we discuss these points in turn.
Ath1 was the first locus identified for atherosclerosis susceptibility in mice in BXH and B6XBALB/c recombinant inbred strains. 16 It was determined to be the major locus located on chromosome 1 at 90 cM contributing to differences in diet-induced atherosclerosis in female mice. Recent studies suggest that variations in Tnfsf4 underlie Ath1. 17 The reason for our failure to replicate the Ath1 locus is unclear. It is possible that QTLs found on the high fat cholic acid diet are different from those found in Apoe –/– mice fed chow diet, or we may not have been sufficiently powered to detect it. Studies of BXH recombinant inbred strains maintained on a cholic acid diet performed in our laboratory were not consistent with a chromosome 1 location for a major atherosclerosis susceptibility gene. 6 It is noteworthy that the early study treated atherosclerosis as a nominal rather than quantitative trait. 16 Another BXH Apoe –/– F2 intercross fed on a 12-week Western diet also failed to replicate the locus. 8 The major locus contributing to atherosclerosis in this hyperlipidemic cross was on chromosome 9, which is designated Ath29. More recently, we performed a second cross with those strains maintained on a Western diet for 16 weeks and observed a number of additional loci contributing to lesion development. 18 The chromosome 9 locus in the present study likely replicates the Ath29 QTL previously found in both BXH Apoe –/– F2 crosses fed the Western diet.
There are many notable genes within the 95% confidence interval for Ath29 (17 to 33 cM). Of the 529 genes in the interval, more than 100 of them are olfactory receptors. A short list of candidate genes can be found in the Table. Although lipid metabolism genes are not likely to be causal for atherosclerosis in this cross, we note that the Apoa1, Apoa4, Apoc3, Apoa5 cluster, which has been associated with familial combined hypercholesterolemia, 19 falls in the region. Another gene, Sortilin-related Receptor, Sorl1, has structural homology with the Ldlr family, 20 and has been shown to bind both apolipoprotein E and lipoprotein lipase ( Lpl ) and is expressed in the lesions of Apoe null mice. 21 Nicotinamide n-methyltransferase ( Nnmt ) is involved in the metabolism of homocysteine, whose levels in the plasma are an independent risk factor for CAD. Plasma homocysteine levels were linked to the locus containing Nnmt in Spanish families. 22 Lysyl oxidase like 1 ( Loxl1 ) is involved in the deposition and maintenance of elastin fibers. The Loxl1 null mouse has vascular abnormalities, among other defects. 23 Loxl1 serves as a crosslinking enzyme and an element of the scaffold to ensure spatially defined deposition of elastin.
Candidate Genes Underlying Ath29
Cultured B6 aortic endothelial cells are highly responsive to oxidized LDL, exhibiting induction of chemokines and adhesion molecules, whereas C3H endothelial cells are relatively unresponsive. 7 In addition, reciprocal bone marrow transplants between B6 Apoe –/– and C3H Apoe –/– mice did not alter lesion size in the recipient mice, indicating that variations in monocyte function were not involved. Evidence supporting the arterial wall involvement was also found when B6 Apoe –/– and C3H Apoe –/– aortic segments were transplanted into F1 mice and the donor aortic segments of B6 mice developed significantly larger lesions than those of C3H mice. 24 A number of inflammatory genes are located at the Ath29 locus, including: IL 10 receptor alpha ( Il10ra ), IL 18 ( Il18 ), TIR domain-containing adaptor protein ( Tirap ), and platelet-activating factor acetylhydrolase 1b2 Pafah1b2. Among these, only Il18 has previously been associated with atherosclerosis. Il18 is expressed in human lesions, particularly in unstable plaques. 25 Serum concentrations of IL18 were significantly higher in patients who had a fatal cardiac event compared with patients with CAD who did not have a fatal event during the 3.9 year follow-up period. 26 Haplotypes of Il18 have also been associated with CAD mortality. 27
A suggestive QTL for lesions was found on chromosome 4, located at 27.8 cM, replicating Ath8. 28 Tlr4, at 33 cM, was at the distal end of the peak and is known to be the LPS receptor, an inflammatory response gene. 29 A polymorphism of Tlr4 has been associated with atherosclerosis in human studies, 9 and knock out mouse models have implicated it in lesion formation. 10 Tlr4 has also been implicated as the receptor for oxidized products of 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphorylcholine, which play a role in the induction of Il-8. 30 The C3H/HeJ strain is resistant to atherosclerosis and has a nonfunctional Tlr4 allele, and thus Tlr4 is a candidate for the resistance of the C3H/HeJ strain to atherosclerosis. To test this possibility, we constructed a congenic C3H Apoe –/– strain with the functional Tlr4 lps–n allele donated by the C3H/DisNA mouse and found that these mice were also highly resistant to atherosclerosis. Lesion size was not significantly different from that of Tlr4 lps–d mice. From this we can conclude that Tlr4 does not appear to play a significant role in early lesion development in C3H/HeJ mice. Another gene under the chromosome 4 peak is Angptl3, which is a candidate gene for atherosclerosis in the SM/J x NZB/B1NJ cross. 31 An Angptl3 null mouse exhibited decreased lesions by 3-fold in KK san mice 32 and SNPs in the promoter region of ANGPTL3 have been associated with atherosclerosis in humans. 31
Apoa2 is a candidate gene underlying the total cholesterol, triglyceride, and HDL QTLs on chromosome 1. Apoa2 is polymorphic between B6 and C3H and variation in sequence confers higher translational efficiency to the C3H allele, 33 resulting in approximately 30% more plasma APOA2 in C3H. Transgenic Apoa2 mice have increased plasma HDL and total cholesterol levels as well as increased atherosclerosis. 34 No association with atherosclerosis was observed between Apoa2 alleles and atherosclerosis in this cross, perhaps because of the hyperlipidemic Apoe –/– background. Sterol O-acyltransferase ( Soat1 ) is another candidate for the lipid QTLs on chromosome 1. Also known as acyl coenzyme A (CoA): cholesterol acyltransferase, it is an endoplasmic reticulum protein that forms cholesterol esters from cholesterol. It is also involved in adrenocortical lipid depletion in AKR/J mice. 35
In conclusion, the mapping and genetic isolation of the chromosome 9 locus conferring atherosclerosis resistance of C3H mice should allow fine mapping of the region and the identification of the underlying gene. This task should be aided by the recent development of an expression quantitative trait locus database for C3H X B6 mice. 36
Acknowledgments
We thank Pingzi Wen for assisting with the genotyping, Sarada Charugundla and Lawrence Castellani for performing the lipid assays, and Minori Imura for assisting in the construction of the C3H. ApoE –/– Tlr4 lps–d congenic.
Sources of Funding
This work was supported by NIH grants HL30568 and HL28481.
Disclosures
None.
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作者单位：Departments of Human Genetics (S.S.W., L.V., S.G., M.T.W., A.J.L.), Medicine (W.S., X.W., A.J.L.), and Pathology and Laboratory Medicine (X.W., T.A.D.), University of California at Los Angeles. Present address for W.S.: Department of Radiology, University of Virginia, Charlottesville.