Sequence polymorphisms in a 58-kilobase (kb) interval on chromosome 9p21 confer a markedly increased risk of coronary artery disease (CAD), the leading cause of death worldwide. The variants have a ...substantial effect on the epidemiology of CAD and other life-threatening vascular conditions because nearly one-quarter of Caucasians are homozygous for risk alleles. However, the risk interval is devoid of protein-coding genes and the mechanism linking the region to CAD risk has remained enigmatic. Here we show that deletion of the orthologous 70-kb non-coding interval on mouse chromosome 4 affects cardiac expression of neighbouring genes, as well as proliferation properties of vascular cells. Chr4 70kb/ 70kb mice are viable, but show increased mortality both during development and as adults. Cardiac expression of two genes near the non-coding interval, Cdkn2a and Cdkn2b, is severely reduced in chr4 70kb/ 70kb mice, indicating that distant-acting gene regulatory functions are located in the non-coding CAD risk interval. Allele-specific expression of Cdkn2b transcripts in heterozygous mice showed that the deletion affects expression through a cis-acting mechanism. Primary cultures of chr4 70kb/ 70kb aortic smooth muscle cells exhibited excessive proliferation and diminished senescence, a cellular phenotype consistent with accelerated CAD pathogenesis. Taken together, our results provide direct evidence that the CAD risk interval has a pivotal role in regulation of cardiac Cdkn2a/b expression, and suggest that this region affects CAD progression by altering the dynamics of vascular cell proliferation.
Recent studies have indicated that the scavenger receptor class B type I (SR-BI) may play an important role in the uptake of high density lipoprotein (HDL) cholesteryl ester in liver and ...steroidogenic tissues. To investigate the in vivo effects of liver-specific SR-BI overexpression on lipid metabolism, we created several lines of SR-BI transgenic mice with an SR-BI genomic construct where the SR-BI promoter region had been replaced by the apolipoprotein (apo)A-I promoter. The effect of constitutively increased SR-BI expression on plasma HDL and non-HDL lipoproteins and apolipoproteins was characterized. There was an inverse correlation between SR-BI expression and apoA-I and HDL cholesterol levels in transgenic mice fed either mouse chow or a diet high in fat and cholesterol. An unexpected finding in the SR-BI transgenic mice was the dramatic impact of the SR-BI transgene on non-HDL cholesterol and apoB whose levels were also inversely correlated with SR-BI expression. Consistent with the decrease in plasma HDL and non-HDL cholesterol was an accelerated clearance of HDL, non-HDL, and their major associated apolipoproteins in the transgenics compared with control animals. These in vivo studies of the effect of SR-BI overexpression on plasma lipoproteins support the previously proposed hypothesis that SR-BI accelerates the metabolism of HDL and also highlight the capacity of this receptor to participate in the metabolism of non-HDL lipoproteins.
Both in vitro and in vivostudies of scavenger receptor class B type I (SR-BI) have implicated it as a likely participant in the metabolism of HDL cholesterol. To investigate the effect of SR-BI on ...atherogenesis, we examined two lines of SR-BI transgenic mice with high (10-fold increases) and low (2-fold increases) SR-BI expression in an inbred mouse background hemizygous for a human apolipoprotein (apo) B transgene. Unlike non-HDL cholesterol levels that minimally differed in the various groups of animals, HDL cholesterol levels were inversely related to SR-BI expression. Mice with the low expression SR-BI transgene had a 50% reduction in HDL cholesterol, whereas the high expression SR-BI transgene was associated with 2-fold decreases in HDL cholesterol as well as dramatic alterations in HDL composition and size including the near absence of α-migrating particles as determined by two-dimensional electrophoresis. The low expression SR-BI/apo B transgenics had more than a 2-fold decrease in the development of diet-induced fatty streak lesions compared with the apo B transgenics (4448 ± 1908 μm2/aorta to 10133 ± 4035 μm2/aorta; p < 0.001), whereas the high expression SR-BI/apo B transgenics had an atherogenic response similar to that of the apo B transgenics (14692 ± 7238 μm2/aorta) but 3-fold greater than the low SR-BI/apo B mice (p < 0.001). The prominent anti-atherogenic effect of moderate SR-BI expression provides in vivosupport for the hypothesis that HDL functions to inhibit atherogenesis through its interactions with SR-BI in facilitating reverse cholesterol transport. The failure of the high SR-BI/apo B transgenics to have similar or even greater reductions in atherogenesis suggests that the changes resulting from extremely high SR-BI expression including dramatic changes in lipoproteins may have both pro- and anti-atherogenic consequences, illustrating the complexity of the relationship between SR-BI and atherogenesis.
High-density lipoprotein (HDL) contains two major proteins, apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II), comprising about 70% and 20% of the total HDL protein mass, respectively. ...HDL exists in human plasma in two main forms, one containing apoA-I with apoA-II (AI/AII-HDL) and another containing apoA-I without apoA-II (AI-HDL). A strong inverse relationship exists between total plasma HDL concentration and atherosclerosis, but the results of studies examining the relationship between AI-HDL and AI/AII-HDL and atherosclerosis have been conflicting. To determine whether these two HDL populations have different effects on atherogenesis, human apoA-I (AI) and human apoA-I and apoA-II (AI/AII) transgenic mice were produced in an atherosclerosis-susceptible strain. Following an atherogenic diet, despite similar total cholesterol and HDL cholesterol concentrations, the area of atherogenic lesions in the AI/AII mice was 15-fold greater than in the AI animals. These studies show that the protein composition of HDL significantly affects its role in atherogenesis and that AI-HDL is more antiatherogenic than AI/AII-HDL.
Ionizing radiation promotes formation of reactive oxygen species, including the superoxide anion (O2). To evaluate whether O2 or O2 perturbations may contribute to the known atherogenic effects of ...radiation, we examined aortic lesion formation in irradiated C57BL/6 mice and evaluated the effects of CuZn-superoxide dismutase (CuZn-SOD) overexpression. Ten-week-old mice were exposed to a 2-, 4-, or 8-Gy dose of 250-keV x-rays to the upper thorax and then placed on a high-fat diet for 18 weeks. Based on quantitative lipid staining of serial sections of the proximal aorta, mean lesion area was increased with increasing radiation dose and was 3-fold greater in 8-Gy-irradiated than sham-irradiated mice (7800 +/- 2140 versus 2635 +/- 709 micro signm, P<0.05). These effects were absolutely dependent on a high-fat diet, which had to be introduced within 1 to 2 weeks of the radiation exposure, suggesting the early involvement of atherogenic lipoproteins that were elevated in response to the diet. The importance of radiation-induced oxidative stress was supported by the observation of a 2-fold lower mean lesion area in irradiated CuZn-SOD transgenic mice than in their irradiated, nontransgenic littermates (3026 +/- 1590 versus 6102 +/- 1834 micro signm, P<0.05). Lucigenin-enhanced chemiluminescence, used as an index of aortic O2 concentrations, was significantly elevated in the postradiation period, and this response was reduced in CuZn-SOD transgenics. On the basis of these results, we propose that radiation may be a useful tool for initiating oxidative or redox-regulated events that promote atherogenesis and for testing the antiatherogenic properties of antioxidants. (Arterioscler Thromb Vasc Biol. 1999;19:1387-1392.)
Studies in vitro have shown that copper-zinc superoxide dismutase (CuZn-SOD) inhibits a number of events putatively involved in atherogenesis, including cell-mediated oxidation of LDL. To investigate ...whether increased activity of CuZn-SOD reduces atherogenesis in vivo, we examined diet-induced fatty streak formation in CuZn-SOD transgenic mice (n = 24) as compared with their nontransgenic littermates (n = 28). Transgenic animals were originally created by introduction of an EcoRI-BamHI human genomic DNA fragment containing the CuZn-SOD gene and its regulatory elements into B6SJL zygotes. For the current studies, the transgene was bred for 12 generations into the atherosclerosis-susceptible C57BL/6 background. Animals were fed atherogenic diets (15% fat, 1.25% cholesterol, 0.5% Na cholate) starting at 10 weeks of age and extending for 18 weeks. At the end of the diet period, aortic SOD activity was two-fold higher in transgenics than nontransgenics (mean + /- SE46.7 +/- 5.8 versus 20.1 +/- 2.4 units/mg of protein, P < .001). Levels of protein-bound amino acid oxidation products (meta-, ortho-, and dityrosine) were either similar or lower in aorta and heart from transgenics as compared with nontransgenics, suggesting that amplification of CuZn-SOD activity above the normal complement had modest inhibitory effects on basal oxidative stress in these tissues. CuZn-SOD overexpression did not reduce the extent of lesion development as analyzed by quantitative lipid staining of serial sections of the proximal aorta; mean lesion areas (+/- SE) were 997 +/- 478 and 943 +/- 221 micro in transgenics and nontransgenics, respectively. Notably, the range of values for lesion area was 2.2-fold greater in transgenics (0-8403 versus 0-3868 micro in nontransgenics). Moreover, within this group, lesion area showed a significant positive correlation with SOD activity (r = .611, P < .03). These results do not support an antiatherogenic effect of CuZn-SOD over expression, and the possibility that high tissue SOD activity may potentiate atherogenesis in fat-fed atherosclerosis-susceptible mice. (Arterioscler Thromb Vasc Biol. 1997;17:1734-1740.)
OBJECTIVE—The apolipoprotein(a) apo(a) gene locus is the major determinant of the circulating concentration of the atherothrombogenic lipoprotein Lp(a). In vitro analysis of the intergenic region ...between the apo(a) and plasminogen genes revealed the presence of a putative apo(a) transcription control region (ACR) approximately 20 kb upstream of the apo(a) gene that significantly increases the minimal promoter activity of the human apo(a) gene.
METHODS AND RESULTS—To examine the function of the ACR in its natural genomic context, we used the Cre-lox P recombination system to generate 2 nearly identical apo(a)–yeast artificial chromosome transgenic mouse lines that possess a single integration site for the human apo(a) transgene in the mouse genome but differ by the presence or absence of the ACR enhancer. Analysis of the 2 groups of animals revealed that the deletion of the ACR was associated with 30% reduction in plasma and mRNA apo(a) levels. Apo(a)–yeast artificial chromosome transgenic mice with and without the ACR sequence were similar in all other aspects of apo(a) regulation, including liver-specific apo(a) expression and alteration in expression levels in response to sexual maturation and a high-fat diet.
CONCLUSIONS—This study provides the first experimental in vivo evidence for a functional role of the ACR enhancer in determining levels of apo(a) expression.
To accelerate the biological annotation of novel genes discovered in sequenced regions of mammalian genomes, we are creating large deletions in the mouse genome targeted to include clusters of such ...genes. Here we describe the targeted deletion of a 450-kb region on mouse chromosome 11, which, based on computational analysis of the deleted murine sequences and human 5q orthologous sequences, codes for nine putative genes. Mice homozygous for the deletion had a variety of abnormalities, including severe hypertriglyceridemia, hepatic and cardiac enlargement, growth retardation, and premature mortality. Analysis of triglyceride metabolism in these animals demonstrated a several-fold increase in hepatic very-low density lipoprotein triglyceride secretion, the most prevalent mechanism responsible for hypertriglyceridemia in humans. A series of mouse BAC and human YAC transgenes covering different intervals of the 450-kb deleted region were assessed for their ability to complement the deletion induced abnormalities. These studies revealed that OCTN2, a gene recently shown to play a role in carnitine transport, was able to correct the triglyceride abnormalities. The discovery of this previously unappreciated relationship between OCTN2, carnitine, and hepatic triglyceride production is of particular importance because of the clinical consequence of hypertriglyceridemia and the paucity of genes known to modulate triglyceride secretion.
Based on the assumption that severe alterations in the expression of genes known to be involved in high-density lipoprotein (HDL) metabolism may affect the expression of other genes, we screened an ...array of >5000 mouse expressed sequence tags for altered gene expression in the livers of two lines of mice with dramatic decreases in HDL plasma concentrations. Labeled cDNA from livers of apolipoprotein AI (apoAI)-knockout mice, scavenger receptor BI (SR-BI) transgenic mice, and control mice were cohybridized to microarrays. Two-sample t statistics were used to identify genes with altered expression levels in the knockout or transgenic mice compared with control mice. In the SR-BI group we found nine array elements representing at least five genes that were significantly altered on the basis of an adjusted P value < 0.05. In the apoAI-knockout group, eight array elements representing four genes were altered compared with the control group (adjusted P < 0.05). Several of the genes identified in the SR-BI transgenic suggest altered sterol metabolism and oxidative processes. These studies illustrate the use of multiple-testing methods for the identification of genes with altered expression in replicated microarray experiments.
No evidence of premature vascular disease is found in apolipoprotein A-I
Milano (apoA-I
M) human carriers, despite very low high density lipoprotein (HDL) cholesterol levels. Whether apoA-I
M may ...impart a “gain of function” in atherosclerosis protection compared to wild-type apoA-I is hotly debated. To address this question, knock-in mice expressing human apoA-I or apoA-I
M were crossed with atherosclerosis-susceptible mice expressing the human apoB/A-II transgene (h-B/A-II/A-I
Hu/Hu and h-B/A-II/A-I
M
Hu/Hu). On a chow diet, h-B/A-II/A-I
M
Hu/Hu mice were characterized by low HDL cholesterol levels compared to h-B/A-II/A-I
Hu/Hu mice (35.65
±
8.00
mg/dl versus 58.09
±
13.50
mg/dl, respectively;
p
<
0.005). Gender differences in response to high fat diet were observed in both h-B/A-II/A-I
M
Hu/Hu and h-B/A-II/A-I
Hu/Hu lines. h-B/A-II/A-I
M
Hu/Hu females had higher total cholesterol levels compared to h-B/A-II/A-I
Hu/Hu females (895.08
±
183.07
mg/dl versus 544.43
±
116.42
mg/dl;
p
<
0.05) and developed larger atherosclerotic lesions (148,260
±
78,924
μm
2 versus 54,132
±
43,204
μm
2, respectively;
p
<
0.05). On the contrary, no difference in mean lesion area was found between h-B/A-II/A-I
M
Hu/Hu and h-B/A-II/A-I
Hu/Hu males (19,779
±
6098
μm
2 versus 15,706
±
13,095
μm
2;
p
=
0.685). Our data suggest that, in the atherosclerosis-susceptible human apoB/A-II mouse model, expression of the human
apoA-I
M
gene does not have protective advantage over that of the
apoA-I gene.
PDF
