Analyses of ischemia-induced signals in cardiac myocytes

本课题的目的是阐明碱性螺旋-环-螺旋-PER/ARNT/SIM(BHLHPAS)蛋白家族成员缺氧诱导因子1α(HIF-1α)在缺血再灌注心肌中的功能意义。重点分析了缺氧诱导因子-1α靶基因血管内皮生长因子和诱导型一氧化氮合酶在心肌细胞中的表达。1)血管内皮生长因子在心肌细胞中的表达分析：利用培养的新生大鼠心肌细胞，我们观察到脂多糖(Sugishita Y等)促进了血管内皮生长因子的表达。Biochem BiPhys res Commun，2000)或腺苷(通过其A2受体)。另一方面，我们分析了鸡血管内皮生长因子在培养10日龄鸡胚胎心室肌细胞中的表达(Sugishita Y，et al.2)鸡胚胎成纤维细胞诱导型一氧化氮合酶基因表达的分析：我们已经获得的数据表明，核因子-κB和Ets-1都参与了脂多糖诱导的鸡胚胎成纤维细胞诱导型一氧化氮合酶基因的反式激活(Takahashi T，et al.3)鸡缺氧诱导因子-1α基因的克隆及表达分析：本研究采用RT-α和RACE方法从鸡胚成纤维细胞中克隆了鸡缺氧诱导因子-1基因的编码区。然后，我们检测了鸡HIF-1α在不同胚胎组织中的表达。鸡HIF-1α与人HIF-1αα的氨基酸序列同源性为79%，与另一成员内皮PAS蛋白1(EPAS1)的同源性仅为49%。HIF-1α在胎盘发育过程中大量表达，而在肝脏中几乎不表达，与EPAS1α的表达模式有很大不同，提示HIF-1 mRNA可能参与胚胎心血管的发育(Takahashi T等)。《生物化学与生物物理通讯》，2001，正在出版中)。

The objective of this project was to elucidate functional significance of hypoxia-inducible factor 1α (HIF-1α), a member of the basic-helix-loop-helix-Per/ARNT/Sim (bHLH-PAS) protein family, in the ischemic-reperfused myocardium. Particularly, we analyzed expression of vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS), which are among target genes of HIF-1α, in cardiac myocytes.1) Analyses of VEGF expression in cardiac myocytes : Using cultured neonatal rat myocytes, we have observed that expression of VEGF was augmented by lipopolysaccaharide (LPS) (Sugishita Y, et al. Biochem Biophys Res Commun, 2000) or adenosine (via its A_2 receptor). On the other hand, we have analyzed expression of the chick VEGF in cultured 10-day-old chick embryonic ventricular myocytes (CEVM) (Sugishita Y, et al. J Mol Cell Caardiol, 2000).2) Analyses of the chick iNOS gene expression in CEVM : We have obtained the data showing that both NF-κB and Ets-1 are involved in the LPS-induced transactivation of the chick iNOS geen in CEVM (Takahashi T, et al. Mol Cell Biochem, submitted).3) Molecular cloning and expressional analyses of the chick HIF-1α cDNA in CEVM : We have cloned cDNAs encoding the chick HIF-1α from CEVM with RT-PCR and Race methods. We then examined expression of the chick HIF-1α mRNA species in various embryonic tissues. The deduced amino acid sequence of the chick HIF-1α cDNA showed 79% identity with that of the human HIF-1α cDNA.In contrast, sequence homology between the chick HIF-1α and endothelial PAS protein 1 (EPAS1), another member of the bHLH-PAS proteins, was only low (49%). HIF-1α mRNA was expressed abundantly in CEVM, but scarcely in the liver, which was quite different from expression pattem of EPAS1 mRNA.These data suggest that HIF-1α may be involved in embryonic cardiovascular development (Takahashi T, et al. Biochem Biophys Res Commun, 2001, in press).