Gene therapy techniques are being developed as potential treatments for cardiovascular diseases. During the past decade, many gene transfer methods including viral transfer techniques have been developed, and some are being applied clinically in human gene therapy studies. Recently, we have developed a novel gene transfer method mediated by Hemagglutinating Virus of Japan (HVJ) -liposome, with which we have already reported several cases of successful gene transfer in vivo. Since the virus is inactivated by ultraviolet light, there is little potential for biological hazard with this method as compared to other viral gene transfer approaches. We also developed a novel strategy of gene therapy for cardiovascular diseases utilizing hepatocyte growth factor (HGF) which is an endothelial cell specific growth factor and an angiogenic growth factor. Based on these facts, we hypothesized that HGF may prevent restenosis after angioplasty through re-endothelialization and myocardial infarction through induction of angiogenesis. The present results provide evidence of the efficacy of supplemental therapy with HGF by gene transfer in cardiovascular diseases. These data suggest the efficacy of novel molecular therapeutic approaches as gene therapy for cardiovascular diseases such as restenosis and myocardial infarction.

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Elevated plasma levels of lipoprotein (a) [Lp (a)] constitutes an independent risk factor for coronary heart disease, stroke, and restenosis. Over the past years, our understanding of the genetics, metabolism and pathophysiology of Lp (a) have increased considerably. However, the precise mechanism (s) by which this atherogenic lipoprotein mediates the development of atherosclerosis remains unclear. This is partly due to the lack of appropriate animal models since apolipoprotein (a) [apo (a)], a distinct component of Lp (a) is found only in primates and humans. Development of transgenic mice expressing human apo (a) has provided an alternative means to investigate many aspects of Lp (a). However, human apo (a) in transgenic mice can not bind to murine apoB to form Lp (a) particles. In this aspect, we generated transgenic rabbits expressing human apo (a). In the plasma of transgenic rabbits, unlike the plasma of transgenic mice, about 80% of the apo (a) was associated with rabbit apo-B and was contained in the fractions with density 1.02-1.10 g/ml, indicating the formation of Lp (a). Our study suggests that transgenic rabbits expressing human apo (a) exhibit efficient assembly of Lp (a) and can be used as an animal model for the study of human Lp (a).

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We and other groups have recently demonstrated that oxidized low density lipoprotein (Ox-LDL) induces proliferation of macrophages in vitro. Since previous immunohistochemical studies demonstrated that macrophages and macrophage-derived foam cells proliferated in situ in atherosclerotic lesions, it seems reasonable to expect that the Ox-LDL-induced macrophage proliferation might be linked to the development of atherosclerotic lesions. Thus, clarification of the molecular cascades of Ox-LDL-induced macrophage proliferation is expected to enhance our knowledge of the pathogenesis of atherosclerosis. Recently, we demonstrated that the activation of PKC leads to release into the culture medium of granulocyte/macrophage colony-stimulating factor (GM-CSF) which plays an important role in Ox-LDL-induced macrophage proliferation. In this review article, we mainly show the role of GM-CSF in the Ox-LDL-induced macrophage proliferation. Moreover, based on our recent findings, we summarize the Ox-LDL-induced signaling pathway for macrophage proliferation.

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Receptors belonging to the low density lipoprotein receptor (LDLR) superfamily play important biological roles in addition to mediating the lipoprotein metabolism. The recent discovery of a novel mosaic LDLR family member by us (Yamazaki H, Bujo H, Kusunoki J, Seimiya K, Kanaki T, Morisaki N, Schneider WJ, and Saito Y J Biol Chem 271 : 24761-24768, 1996) and others, which we termed LR11, offers the opportunity to gain new insights into receptor multifunctionality. The expression of a 250-kDa mosaic LDLR family member, which we termed LR11 due to the presence of 11 ligand binding repeats, is markedly induced during the process of atherogenesis in two animal models. The highest induction of LR11 occurs in the intimal smooth muscle cells (SMCs) of atheromatous lesions. In agreement with the correlation of LR11 induction during increased cell proliferation in vivo, cultured SMCs showed a marked increase in LR11 expression in the proliferative phase. Furthermore, such proliferation-dependent expression of LR11 could be observed in a cultured neuroblastoma cell line, which was established to be a suitable in vitro model for proliferation and differentiation. Possible involvement of LR11 in the cellular proliferation sheds new light on the recently proposed novel functions of the LDL receptor gene family in atherosclerosis.

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The rabbit has been extensively utilized as an ideal model of atherosclerosis because of its size, easy manipulation, and extraordinary response to dietary cholesterol. The availability of spontaneously hypercholesterolemic model, Watanabe heritable hyperlipidemic rabbits (WHHL) and St. Thomas rabbits, has also provided insights into understanding human familiar hypercholesterolemia and atherosclerosis. With the advent of genetically engineered rabbits, transgenic rabbits have become a novel means to explore a number of proteins that are associated with cardiovascular diseases including atherosclerosis. To date, transgenes for human apo (a), apoA-I, apoB, apoE₂, apoE₃, hepatic lipase, lecithin : cholesterol acyltransferase (LCAT), lipoprotein lipase, 15-lipoxygenase, as well as for rabbit apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1), have been expressed in rabbits. In addition, human apoA-I, LCAT and apo (a) have been introduced into WHHL rabbits which have deficient LDL receptor function. All of these transgenes have been found to have significant effects on plasma lipoprotein metabolism or/and atherosclerosis. These studies have revealed new insights into the mechanisms responsible for the development of atherosclerosis. In this article, we provide a brief review on the rabbit model for the study of atherosclerosis with emphasis on transgenic rabbit models developed during the past few years.

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Pseudohomozygous familial hypercholesterolemia is a rare condition of unknown etiology. Sitosterolemia is a rare autosomal recessively inherited disorder that is characterized by premature coronary artery disease, cutaneous xanthomas, and increased plasma plant sterols and 5a-stanols. Only a few cases of both sitosterolemia and pseudohomozygous familial hypercholesterolemia have been reported. In this study, we report two sisters with both conditions. With a low-cholesterol diet (<250 mg/day), serum cholesterol concentration decreased rapidly to an almost normal level and cutaneous xanthomas gradually regressed and finally disappeared ; however, plant sterol levels did not change during the period. Plant sterols should be measured in patients considered to have pseudohomozygous familial hypercholesterolemia. The two conditions in this family may have been the results of a single gene mutation. The findings also indicate that low cholesterol diet therapy is effective for the treatment of hypercholesterolemia but not of sitosterolemia in this family.

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Stimulation of vascular endothelial cells by tumor necrosis factor α (TNFα) plays a critical role in the pathogenesis of inflammation and vascular diseases. Changes in the gene expression profile in cultured human umbilical vein endothelial cells (HUVEC) treated with TNFα was analyzed with high-density oligonucleotide arrays comprised of 35, 000 genes. TNFα stimulation profoundly induced genes involved in signal transduction, leukocyte adhesion and chemoattraction. ICAM-1 mRNA (fold change 111.9) was most profoundly induced followed by TNFα receptor-associated factor 1 (TRAF1) (95.5), Bcl3 (71.8), IL8 (65. 4), fractalkaine (62.4), E-selectin (48.0), lymphotoxin, β (41.3) and VCAM-1 (31.7). In addition to these previously known genes, 18 poorly characterized or novel genes known as ESTs profoundly induced by TNFα. Initial sequencing analysis identified three of these the genes for squalene epoxydase, chromodomain helicase DNA binding protein 4, and CLP respectively. Further analysis of these genes will provide important information about TNF α signaling and function in vascular endothelial cells.

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We studied the molecular basis of familial lipoprotein lipase (LPL) deficiency in a new Japanese kindred. The proband was a four-month-old infant with severe hyperchylomicronemia. In postheparin plasma, LPL activity was virtually absent, although LPL mass was detectable. Single strand conformational polymorphism (SSCP) analysis showed an abnormal band with exon 5 of the LPL gene that was amplified by PCR from the proband's genomic DNA. DNA sequence analysis of the amplified fragment demonstrated that the proband was homozygous for a G-to-A change at nucleotide position 818 resulting in the substitution of glutamic acid for glycine at codon 188. Although this is among the first Gly188Glu mutations identified in Japanese, the missense mutation has previously been reported as a prevalent cause of familial LPL deficiency worldwide and has been proposed to have a common origin. However, DNA haplotype analysis with either restriction fragment length polymorphism (RFLP) or microsatellite markers revealed that the DNA haplotype of the proband was not identical to the haplotype previously reported as common to the other patients with the Gly188Glu mutation. These results add the Gly188Glu mutation to the growing list of LPL gene mutations underlying familial LPL deficiency in Japanese and indicate that the origin of the Gly188Glu mutation is not necessarily common but would be multicentric at least in part.

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This work was undertaken to examine the relationship between thyroid hormone and serum leptin concentration. This study included 368 Japanese female subjects (27 were affected with pretreatment hyperthyroidism, 68 with hyperthyroidism during treatment, 19 with pretreatment hypothyroidism, 57 with hypothyroidism during treatment and 197 euthyroid control subjects) and 60 control male subjects. In the control group, serum leptin levels in males were lower than those recorded in females (mean±SD ; 4.6±4.1 vs 9.5±6.4 ng/ml, p<0.001). The leptin values correlated well with body mass index (BMI) and body fat mass (BFM) in both control male and female subjects (p <0.001 for each). The serum leptin levels in pretreatment female patients with hyperthyroidism were significantly lower than those in the pretreatment patients with primary hypothyroidism and control female subjects (6.4±3.0 vs 9.7±6.3, 9.5±6.4 ng/ml ; p<0.05, 0.02, respectively), but after adjusting for BMI and BFM, the difference was mainly due to the significantly different BMI and BFM. Furthermore, serum leptin did not change significantly during the treatment in hyper-and hypothyroidism. There was no correlation between serum leptin and thyroid hormones or lipids levels in female patients with thyroid disorders. Adiposity and gender were the major determinants of leptin concentration, but thyroid hormones did not appear to play any relevant role in leptin synthesis and secretion in human.

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Although the prognosis of fatty liver depends on its causes, we feel from our clinical experience that fatty liver with hypertriglyceridemia has a good prognosis and responds well to treatment. In this study, 600 mg/day of pantethine was administered to 16 outpatients with fatty liver and hypertriglyceridemia for six months or longer to examine whether the drug improved fatty liver using abdominal plain computed tomography (CT). Nine of the 16-pantethine patients were no longer diagnosed as having fatty liver after the study period. An x² test indicated the significant disappearance of fatty liver. At the same time, the visceral fat calculated from the CT image passing the umbilical region was also significantly reduced. On the contrary, the subcutaneous fat area tended to increase, so the ratio of the visceral-to-subcutaneous fat area was reduced significantly. This indicates triglycerides may be pooled in the body as hepato-visceral fat and subcutaneous fat, and that pantethine may transfer fat from the liver and viscera to the subcutaneous tissue. This suggests that visceral fat deposition and fatty liver occurring with hypertriglyceridemia may have a common basis, probably excessive matrixes, and that pantethine may simultaneously improve the two conditions.

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