Takefumi Nojiri

Endothelial PAS Domain Protein 1 Gene Promotes Angiogenesis Through the Transactivation of Both Vascular Endothelial Growth Factor and Its Receptor, Flt-1

Endothelial PAS domain protein 1 (EPAS1) is a basic-helix–loop–helix/PAS domain transcription factor that is expressed preferentially in vascular endothelial cells. EPAS1 shares high homology with hypoxia-inducible factor-1&agr; (HIF-1&agr;) and is reported to transactivate vascular endothelial growth factor (VEGF), fetal liver kinase-1 (Flk-1), and Tie2 promoters. In this study, we analyzed the role of EPAS1 in the process of angiogenesis. Using microarray technology, we looked for target genes regulated by EPAS1 in vascular endothelial cells. A total of 130 genes were upregulated by EPAS1, including fms-like tyrosine kinase-1 (Flt-1). Reporter analysis using human Flt-1 promoter and gel mobility shift assays showed that the heterodimer of EPAS1 and aryl hydrocarbon receptor nuclear translocator binds directly to HIF-1–binding site upstream of Flt-1 promoter and transactivates it. Small interfering RNA targeted to EPAS1 but not HIF-1&agr; attenuated desferrioxamine-induced Flt-1 mRNA expression, thus EPAS1 is thought to play an essential role in hypoxic induction of Flt-1 gene. Furthermore, using mouse wound healing models, we demonstrated that adenovirus-mediated delivery of EPAS1 gene significantly induced the expression of VEGF, Flt-1, Flk-1, and Tie2 mRNA at the wound site and promoted mature angiogenesis. The proportion of the number of mural cells in newly formed vessels was significantly higher in EPAS1-treated wound area than VEGF-treated area. In conclusion, EPAS1 promotes Flt-1 gene expression and induces mRNA expression of VEGF, Flk-1, and Tie2, leading to enhancement of mature angiogenesis in vivo. Thus, EPAS1 may contribute to the construction of mature vessels by modulating the coordinated expressions of VEGF, Flt-1, Flk-1, and Tie2.

Genetic variations of matrix metalloproteinase-1 and -3 promoter regions and their associations with susceptibility to myocardial infarction in Japanese

Matrix metalloproteinases (MMPs) are involved in plaque rupture, which is the main pathological cause of myocardial infarction (MI). Recently, several genetic studies have demonstrated that MMP-1 1G/2G polymorphism and MMP-3 5A/6A polymorphism modify each transcriptional activity in allele specific manners. Within this context, we conducted case-control studies to examine whether these genetic polymorphisms are associated with susceptibility to MI. Two groups comprising patients with MI (group-1 164 patients, group-2 302 patients) were compared with control group comprising 335 patients without cardiovascular diseases. The MMP-3 5A allele was more frequent in patients with MI than in the control subjects (P=0.018 MI group-1, P=0.0059 MI group-2), whereas there was no disease association for MMP-1 genotypes. Logistic regression analyses revealed that MMP-3 5A/6A polymorphism was associated with susceptibility to MI [odds ratio(OR) (95% confidential interval) 1.67 (1.02-2.74); P=0.042, MI group-1; 1.61 (1.12-2.23); P=0.0095, MI group-2]. Other important findings were that there was strong linkage disequilibrium between these polymorphisms, which are located closely on chromosome 11q.22, and that the 5A-1G haplotype was a genetic risk factor for MI (OR 1.97 P=0.0082, MI group-1 OR 1.51 P=0.017, MI group-2). Taken together, the present findings suggest that genetic variations in these MMP genes and especially their haplotype may be useful genetic markers for determining susceptibility to MI in Japanese.

A common Ile 823 Met variant of ATP-binding cassette transporter A1 gene (ABCA1) alters high density lipoprotein cholesterol level in Japanese population

Recently, variants in ATP-binding cassette transporter A1 (ABCA1) were demonstrated to be associated with increased level of high density lipoprotein cholesterol (HDL-C) and decreased risk of coronary artery disease (CAD) in Caucasians. However, this is not universally applicable due to the ethnic or environmental differences. In this context, to clarify the effect of ABCA1 in Japanese, we evaluated the phenotypic effects of I/M 823 and R/K 219 variants on the plasma level of HDL-C in 410 patients recruited in our hospital. Subjects with M 823 allele had significantly higher level of HDL-C than those without M823 allele (49.0+/-15.1 vs. 44.9+/-11.5 mg/dl, respectively, P<0.05). This statistical significance did not change even after multiple regression analysis. In contrast, there was no difference in HDL-C level among the genotypes in R/K 219 polymorphism. Further, in our study population an inverse relationship was shown to exist between HDL-C level and incidence of CAD. However, no positive association was observed between those variants and susceptibility to CAD. In this study, we provide evidence that I/M 823 variant, not R/K 219 variant, in ABCA1 is one of the determinants of HDL-C level, suggesting the importance of this gene on lipid metabolism in Japanese.

Circulating malondialdehyde modified LDL is a biochemical risk marker for coronary artery disease

Oxidatively modified low density lipoprotein (OxLDL) plays an important role in the development of atherosclerosis as its uptake by macrophages and smooth muscle cells leads to formation of foam cells which is a critical step in the evolution of the pathological state.1,2 Circulating OxLDL concentrations may therefore reflect the state of pathological atherosclerosis, and be a possible biochemical risk marker for coronary artery disease (CAD). Numerous efforts have been directed at detecting OxLDL concentrations in the circulation for this reason, but technical difficulties have hampered detection of minute amounts of OxLDL. To overcome these limitations, we focused on circulating malondialdehyde modified LDL (MDA-LDL), a chemical modification thought to reflect naturally occurring oxidation of LDL,3,4 and developed a sensitive immunoassay of circulating MDA-LDL concentrations. The diagnostic performance of MDA-LDL in CAD was compared against known lipid markers. This comparison revealed, for the first time, that MDA-LDL is superior, thus suggesting that MDA-LDL may be a promising tool for the biochemical detection of CAD. Consenting patients with CAD defined as having greater than 75% stenosis in one or more arteries on coronary angiography were enrolled, as were normal control subjects which included patients with normal coronary angiograms, and subjects who were admitted for regular health examinations and had: (1) no history of CAD; (2) normal renal function; (3) normal ECG and chest x ray. Blood was drawn under fasting conditions and centrifuged within four hours. Stabilising reagent containing sucrose and EDTA was added and samples were stored at −20°C until the time of …