Serum levels of high density lipoprotein cholesterol (HDL-C) are inversely correlated with coronary heart disease (CHD). Kinetic studies indicate that the mechanism for the variation in HDL levels associated with various pathophysiologic states includes changes in the fractional catabolic rate (FCR) and/or the synthesis rate of HDL and its major proteins apolipoprotein (apo) A-I and apo A-II. The antiatherogenic effects of HDL are thought to be mainly due to its role in reverse cholesterol transport. HDL is an assembly of heterogeneous particles. HDL enlarges when it takes up cellular cholesterol, and shrinks when HDL-cholesterol ester (CE) is transfered to low density lipoprotein (LDL) and very low density lipoprotein (VLDL) particles. The functional ability of HDL (to remove cellular cholesterol) has drawn considerable attention. The fractional esterification rate of cholesterol in HDL (FER_HDL) has been established as a functional assay of HDL, and reflects the size of HDL particles. We investigated the clinical significance of FER_HDL and its relationship to the quantity of HDL. FER_HDL values were inversely correlated with levels of HDL-C and large lipoprotein containing apo A-I (LpA-I). The association between FER_HDL and CHD changed with serum HDL-C levels : increased FER_HDL values significantly increased the risk of CHD when serum HDL-C levels were low, while there was no such relationship when HDL-C levels were high. We concluded that the combination of HDL-C levels and FER_HDL is a stronger indicator of CHD than either the HDL-C level (quantitative measure of HDL) or FER_HDL (functional measure of HDL) alone.

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Apolipoprotein (apo) A-I is composed of 243 amino acid residues that fold into amphipathic helixes, and plays a central role in the high density lipoprotein (HDL) metabolism. Familial apoA-I deficiency is a rare metabolic disorder of which three cases have been characterized at a molecular level in western Japan. However, in subjects with apoA-I deficiency, coronary artery disease was not always present. One apo A-I deficiency was compound heterozygous apoA-I mutant for a TATA box mutation and a structural nonsense mutation. To date, screening analysis in our laboratory has identified nine genetically-determined structural mutations of apo A-I. We have also characterized these apo A-I mutations, including apoA-I (Glu235del) Nichinan. Few structural mutations were associated with altered HDL cholesterol levels.

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Recent progress in molecular biology has led to the development of gene therapy as a new strategy to treat a variety of cardiovascular diseases. Targeted diseases range from single gene deficiency diseases to more complex diseases in adults such as restenosis after angioplasty. One obvious major target in the field of gene therapy is ischemic diseases such as myocardial infarction, angina and peripheral arterial diseases (i.e. ASO (arteriosclerosis obliterans)). In a large proportion of such patients, the anatomical extent and the distribution of arterial occlusive disease make the patients unsuitable for operative or percutaneous revascularization. Thus, the disease frequently follows an inexorable down-hill course. Of importance, there is no optimal medical therapy for severe ischemic hearts and critical ischemic limbs. Therefore, novel therapies are required to treat these patients. Recently, the efficacy of therapeutic angiogenesis using VEGF (vascular endothelial growth factor) gene transfer has been reported in human patients with critical limb ischemia and myocardial ischemia. Thus, the strategy for therapeutic angiogenesis using angiogenic growth factors should be considered for the treatment of patients with critical limb ischemia or myocardial infarction. The endothelial cell specificity of VEGF has been considered to be an important advantage for therapeutic angiogenesis, as endothelial cells represent the critical cellular element responsible for new vessel formation. Indeed, human gene therapy for ASO and angina has already begun in the USA, with surprising and beneficial effects. We have focused on hepatocyte growth factor (HGF), which is a mesenchyme-derived pleiotropic factor that regulates cell growth, cell motility, and morphogenesis in various types of cells. Recently, HGF is also considered to be a powertul growth tactor for endothelial cells. In this review, we described the potential gene therapy for ischemic diseases using HGF.

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Peroxizome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptor family of transcription factors that regulate adipocyte differentiation. Recent studies indicate that liganded PPAR not only promotes differentiation but also inhibits the activation of macrophages. Osteopontin, a component of extracellular matrix, is synthesized by macrophages in atherosclerotic plaques. In this study, we examined whether PPAR ligand regulates osteopontin gene expression in THP-1 cells, a cell line derived from human monocytic leukemia cells which can differentiate to macrophage upon stimulation with phorbol ester PMA. Northern blot analysis showed that osteopontin expression is markedly induced in response to PMA. Troglitazone, a PPAR ligand, dramatically attenuated the PMA-induced osteopontin expression. Transient transfection assays of the human osteopontin promoter/luciferase construct which contains a 5'-flanking region between -1500 and +87 relative to the transcription start site demonstrate that either treatment with troglitazone or cotransfection of PPARγ expression vector inhibits osteopontin promoter activity. These data indicate that troglitazone reduces osteopontin gene expression at transcriptional level through PPARγ activation, and suggest the role of troglitazone in inhibiting the ability of macrophages to produce extracellular matrix, which is particularly relevant to atherosclerotic plaque formation.

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We attempted to find atherosclerotic plaques including a large lipid core and thin fibrous cap in twice-injured arterial specimens obtained from high cholesterol diet (HCD) -fed rabbits. Rabbits fed a HCD were subjected to carotid artery injury using a balloon catheter. After 2 or 4 weeks of cholesterol feeding, a second mild injury was induced in the same region as the first injury. The rabbits were given a standard diet for 2 weeks after the second injury. Typical atherosclerotic plaques with a fibrous cap formed by smooth muscle cells and extracellular matrix overlying a core formed by macrophage foam cells were observed in the lesion. Gelatin proteolytic activities were found in homogenates containing either media or intima from the injured artery, and activated matrix metalloproteinase-2 (MMP2) was detected. With prolongation of the HCD feeding period (interval between injuries) from 2 weeks to 4 weeks, typical plaque was observed more frequently. Furthermore, the neointimal area and the macrophage foam cells area increased, as did gelatin-proteolytic activity. Since the typical atherosclerotic plaques observed in the present study have some histopathological and pathogenic characteristics in common with unstable atherosclerotic plaque, we expect that the typical atherosclerotic plaque found in the present study will be useful for basic studies of plaque stabilization and prevention of acute coronary syndromes.

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Lipid abnormalities in diabetic patients, particularly in those with nephropathy, may be partially due to deteriorating atherosclerosis. Therefore, strict control of the lipid metabolism in addition to glycemic control is desirable. Whether or not lipid lowering drugs prevent albuminuria in diabetic patients in the long term remains unclear. This study involved 71 NIDDM patients with hypercholesterolemia (group A : n = 37, group B : N = 34). The effect of bezafibrate (group A) or pravastatin (group B) on the cholesterol (CH) content of apolipoprotein Al, B100 containing particles or remnant-like particles (RLP) or urinary albumin excretion was studied over 4 years. The CH content in apolipoprotein B100 particles after treatment with either bezafibrate or pravastatin decreased significantly (group A : 24.7%, group B : 26.6%). The CH content in RLP after treatment with bezafibrate showed a significant decrease (67.9%). Apolipoprotein Al after treatment with bezafibrate showed a significant increase (10.9%). Apolipoprotein B100 after treatment with either drug de-creased significantly (group A : 19.8%, group B : 23.4%). The urinary albumin excretion rate after treatment with either drug showed no significant change over 4 years. Bezafibrate and pravastatin appear to be useful in the preventive treatment of albuminuria as well as in lowering lipid levels in NIDDM patients

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Zinc finger proteins are known to mediate various transcriptional control mechanisms and other cellular functions in human cells. Tumor necrosis factor alpha (TNFα) induces a variety of genes in human endothelial cells including A20, an antiapoptotic zinc finger protein. In order to identify other zinc finger protein genes induced by TNFα, we studied the gene expression profile of human umbilical vein endothelial cells (HUVECs) stimulated by TNFα by means of oligonucleotide microarrays. Among the 155 genes encoding zinc finger motif, the level of EST M88357 mRNA encoding a novel designated EZFIT (endothelial zinc finger protein induced by TNFα) was induced most profoundly (>19 fold). The EZFIT gene is located on the chromosome 19q13.4. Isolation of the full length cDNA coding sequence by PCR using primers architected from the genomic sequence revealed that EZFIT has 490 amino acids which contain 13 C2H2 zinc finger motifs. Among 24 human organs and cell types studied, EZFIT mRNA was found to be most highly expressed in the placenta followed by the brain, testis, pancreas, heart, small intestine, muscle, uterus, prostate and peripheral blood leukocytes. EZFIT mRNA was not detected in the liver, lung, colon, stomach, the salivary gland or the thyroid gland.

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We studied the effects of a widely-used sulfonylurea, gliclazide, on the oxidizability of low-density lipoprotein (LDL) and the development of experimental atherosclerosis in cholesterol-fed rabbits. Daily oral administration of gliclazide (20 mg/kg/day) tended to inhibit the aortic atherosclerosis induced by feeding a 1% cholesterol-diet for 10 weeks, although it did not affect diet-induced hyperlipidemia. The administration of gliclazide tended to inhibit the increase of serum thiobarbituric acid-reacting substances (TBARS) by cholesterol feeding and to increase the lag time of the conjugated-diene formation of LDL subjected to in vitro oxidation by copper ion, although without significance. The present study suggests that gliclazide may have antioxidative properties in vivo, and have further beneficial effects for the treatment of diabetes mellitus by inhibiting the oxidation of LDL. J Atheroscier Thromb, 2000 ; 7 : 104-109.

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The Kyushu Lipid Intervention Study (KLIS) aimed to investigate the effects of pravastatin in the primary prevention of coronary events and cerebral infarction in Japanese men aged 45-74 years with serum total cholesterol of ≥ 220 mg/dl (5.69 mmol/l). The coronary events included myocardial infarction, coronary artery surgery and angioplasty, cardiac death, and sudden death. A total of 5, 640 patients were recruited, and 3, 061 and 2, 579 were allocated to the pravastatin (10-20 mg/day) and conventional treatment, respectively. They were followed up for 5 years on average. Due to unsuccessful randomization, a protocol-based analysis was carried out with adjustment for coronary risk factors at baseline using the Cox proportional hazards model. For several reasons (serum total cholesterol of≥300 mg/dl, use of drugs excluded in protocol, etc.), 2, 219 men in the pravastain group and 1, 634 in the conventional treatment group were used in the analysis. The baseline total cholesterol levels were 254 mg/dl in the pravastain group and 243 mg/dl in the conventional treatment group. Serum total cholesterol levels fell by 15% in the pravastain group and by 8% with the conventional treatment group. Adjusted relative risks for pravastatin versus conventional treatment were : coronary events 0.86 (one-side p= 0.23), cerebral infarction 0.78 (p= 0.13), and the two outcomes combined 0.81 (p= 0.08). These findings add to evidence that pravastatin use is beneficial in the prevention of coronary events and cerebral infarction in hypercholesterolemic Japanese patients, and suggest that pravastatin may be more protective against cerebral infarction.

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