The B apolipoproteins, apo-B48 and apo-B100, are key proteins in mammalian lipoprotein metabolism and are components of all classes of lipoproteins considered to be atherogenic. Our laboratory has generated an array of genetically modified mice for studying apo-B biology. Using gene targeting in mouse embryonic stem cells, we have generated apo-B-deficient mice. Heterozygotes had low plasma levels of apo-B and cholesterol; homozygotes died early in embryonic development, most likely because the absence of lipoprotein secretion by the yolk sac interfered with the delivery of lipid nutrients to the developing embryo. We have also generated human apo-B transgenic mice with an 80-kb genomic DNA fragment spanning the entire human apo-B gene ; those mice had markedly increased plasma levels of low density lipoprotein cholesterol and exhibited increased susceptibility to atherosclerosis. The human apo-B transgenic mice have also yielded insights regarding the regulation of apo-B expression in different tissues. Although the 80-kb transgene contained nearly 20 kb of 5' and 3' flanking sequences and was expressed at high levels in the liver, no transgene expression was detectable in the intestine. Subsequent transgenic mouse studies have demonstrated that the expression of the apo-B gene in the intestine is controlled by DNA sequences that are very distant from the structural gene. Transgenic mice have also proved useful for studying apo-B structure/function relationships. By expressing mutant forms of human apo-B in transgenic mice, we have examined the structural features of the apo-B molecule that are required for lipoprotein (a) formation. We have demonstrated that the carboxyl-terminal cystine residue of apo-B100, cysteine-4326, is required for apo-B100's disulfide linkage with apo (a) to form lipoprotein (a). Finally, we have used gene-targeting techniques to generate mice that synthesize exclusively apo-B48 (apo-B48-only mice) and mice that synthesize exclusively apo-B100 (apo-B100-only mice) : These mice have helped to clarify the unique metabolic roles of the two apo-B proteins. J Atheroscler Thromb, 1996 ; 3 : 62-74.
Apoptosis is a mode of cell death in which intrinsic cellular mechanisms participate in the demise of the cell. The modulation of endothelial apoptosis may play a role in atherosclerosis, angiogenesis, vascular remodeling and other pathophysiological states. Control of cell death is mediated by the state of activation of a death pathway as well as by the levels of anti-apoptotic proteins. The final common pathway of many, if not all, triggers of apoptosis involves activation of cysteine proteases. The Bcl-2 family, in contrast, appears to play a major role in protection against apoptosis. The role of these mechanisms in modulating endothelial cell apoptosis under various conditions is discussed. J Atheroscler Thromb, 1996; 3 : 75-80.
The molecular mechanism by which hypolipidemic fibrates and antidiabetic thiazolidinediones exert their hypotriglyceridemic action are discussed. Increased activity of lipoprotein lipase (LPL), a key lipolytic enzyme, and decreased levels of apolipoprotein C-III (apo C-III) seem to explain the hypotriglyceridemic effects of compounds. Both fibrates and thiazolidinediones exert their action by activating transcription factors of the peroxisome proliferator activated receptor (PPAR) family, thereby modulating the expression of the LPL and apo C-II genes. First, treatment of rats with PPARα activators, such as fibrates induced LPL mRNA and activity selectively in the liver. In contrast, the thiazolidinediones, which are high affinity ligands for PPARγ, have no effect on liver, but induce LPL mRNA and activity levels in adipose tissue. In hepatocytes, fibrates, unlike the thiazolidinediones, induce LPL mRNA levels, whereas in preadipocyte cell lines the PPARγ ligand induces LPL mRNA levels much quicker and to a higher extent than fibrates. Second, apo C-III mRNA and protein production strongly decrease in livers of fibrate-but not thiazolidinedione-treated animals. Fibrates also reduced apo C-III production in primary cultures of rat and human hepatocytes. The modulation of the expression of the LPL and apo C-III genes by either PPARα or γ activators, correlates with the tissue-specific distribution of the respective PPARs : PPARγ expression is restricted to adipose tissues, whereas PPARα is expressed predominantly in liver. In both the LPL and apo C-III genes, sequence elements responsible for the modulation of their expression by activated PPARs have been identified which supports that the transcriptional regulation of these genes by fibrates and thiazolidinediones contributes significantly to their hypotriglyceridemic effects in vivo. Whereas thiazolidinediones predominantly affect adipocyte LPL production through activation of PPARγ, fibrates exert their effects mainly in the liver via a PPARα-mediated reduction in apo C-III production. This tissue-specific transcriptional regulation of genes involved in lipid metabolism by PPAR activators and/or ligands might have important therapeutic implications. J Atheroscler Thromb, 1996; 3 : 81-89.
Calcium deposits account for most of the dry weight of atherosclerotic lesions. Previously considered uncommon, vascular calcification is now known to be present in 80% of significant lesions and in at least 90% of patients with coronary artery disease. Previously considered a passive process, it is increasingly recognized as an active, regulated process. Previously considered benign, it is now becoming recognized as a major risk factor for cardiovascular events, and a major contributor to systolic hypetension, heart failure, plaque rupture and stenosis. To confirm the similarity of vascular calcifiation with embryonic osteogenesis, we demonstrated the expression of bone morphogenetic protein in calcified human lesions, and we developed an in vitro model of vascular calcification that provides a useful experimental system for elucidating the molecular regulation of this process, which we have shown to include alkaline phosphatase induction and expression of bone matrix proteins and differentiation factors. Understanding the regulatory mechanisms of vacular calcification will allow future therapeutic approaches to prevent and possibly reverse this disease and its clinical consequences. J Atheroscier Thromb, 1996; 3 90-94.
This report describes the design and baseline results of the Kyushu Lipid Intervention Study (KLIS). The study aims to test the hypothesis that the long-term reduction of serum total cholesterol by pravastatin will lead to a decrease in coronary heart disease (CHD) events. The trial was designed to include a random 6, 000 male patients aged 45-74 years with serum total cholesterol of 220 mg/dl (5.69 mmol/l) or greater and without a history of myocardial infarction, coronary surgery or angioplasty, to undertake either pravastatin or conventional treatment (including hypolipidemic drugs other than HMG-CoA reductase inhibitors, probucol and bezafibrate), and to follow up each patient for 5 years. Primary endpoints are fatal and nonfatal myocardial infarction, coronary bypass surgery and angioplasty, cardiac death, and sudden and unexpected death. During the period from May 1990 to September 1993, a total of 5, 640 male patients aged 45-74 were recruited by 902 participating physicians throughout Kyushu. Randomization was, however, neglected by study physicians ; the numbers of patients enrolled were 3, 061 in the pravastatin group and 2, 579 in the conventional treatment group. Patients allocated to the pravastatin treatment were generally unfavorable regarding coronary risk factors. Baseline mean levels of serum total cholesterol were 259 mg/dl (6.70 mmol/l) in the pravastatin group and 246 mg/dl (6.36 mmol/l) in the conventional treatment group (p<0.001). Although the trial was regarded as a prospective observational study, the KLIS provides valuable quantitative data regarding cholesterol lowering and reduction in CHD events as well as safety data of the long-term use of a statin in Japanese men with hypercholesterolemia. J Atheroscler Thromb, 1996; 3 : 95-104.
The purpose of this study was to compare average cholesterol levels between Seattle based Japanese Americans and three other populations : U.S. population, native Japanese population and native Japanese urban workers. A total of 1, 466 Japanese Americans (724 men and 742 women) participated in cardiovascular disease screening in the Seattle area during 1989-94. Data sources for comparisons are from the Third National Health and Nutrition Examination Survey for 1988-91, the results of the National Cardiovascular Disease Examination Survey in Japan for 1990, and cardiovascular disease screening conducted by the Epidemiological Arteriosclerosis Research Institute in Japan for 1989. Total cholesterol and triglyceride levels of Seattle Japanese American men and women were highest among the four populations. Among men, high density lipoprotein cholesterol (HDL-C) levels for Seattle Japanese Americans and native Japanese were similar and fell between those of urban Japanese workers and the U.S. population. In women, the average HDL-C levels were highest in the Japanese urban workers, second highest in Seattle Japanese Americans, and lowest in both the U.S. population and native Japanese population. These differences in lipid levels may be caused by both genetic and environmental factors, which are now under investigation. J Atheroscler Thromb, 1996; 3 : 105-113.
This study was to examine the effects of estrogen replacement on atherosclerosis formation in ovariectomized cholesterol-fed rabbits. We also examined serum levels of nitrite/nitrate, stable metabolites of nitric oxide, to investigate the involvement of nitric oxide. Female New Zealand White rabbits were ovariectomized and divided into 3 groups; 1) fed a normal diet (ND group, n=5), 2) fed a 1% cholesterol diet (CD group, n=6), or 3) fed a 1% cholesterol diet and received estrogen replacement (CD + E group, n=7). After 3 months, the rabbits were sacrificed to examine atherosclerosis formation. Atherosclerosis was not observed in ND. The oil red O-positive area in the aorta was significantly greater in CD than in CD + E (CD, 17.3±2.2; CD+E, 9.3±0.8%, p<0.05). Stenosis of the coronary artery was also significantly greater in CD than in CD+E (CD, 30.6±9.7; CD+E, 6.7±2.9%, p<0.05). There was no significant difference in serum lipids between CD and CD+E. Serum nitrite/ nitrate levels were significantly lower in CD than in ND (ND, 37.6±3.6 ; CD, 25.3±3.1μM, p<0.05). There was a non-significant trend towards higher nitrite/nitrate levels after estrogen replacement (CD+E, 34.4±3.8μM, p=0.08vs.CD). These results suggest that direct actions on vascular wall including nitric oxide production contribute to the antiatherogenic effects of estrogen. J Atheroscler Thromb, 1996 ; 3 : 114-119.