Analyses of ischemia-induced signals in cardiac myocytes

本项目旨在阐明缺氧诱导因子1α (HIF-1α)在缺血再灌注心肌中的功能意义，HIF-1α是碱性-螺旋-环-螺旋- per /ARNT/Sim （bHLH-PAS）蛋白家族的成员。我们特别分析了HIF-1α靶基因血管内皮生长因子（VEGF）和诱导型一氧化氮合酶（iNOS）在心肌细胞中的表达。1)心肌细胞中VEGF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（LPS）增强了VEGF的表达（Sugishita Y, et al.）。Biochem Biophys Res common, 2000)或腺苷（通过A_2受体）。另一方面，我们分析了鸡VEGF在培养的10日龄鸡胚胎心室肌细胞（CEVM）中的表达（Sugishita Y, et al.）。journal of Mol Cell cardiol, 2000)。2)鸡CEVM中iNOS基因表达分析：我们获得的数据显示NF-κB和Ets-1都参与了lps诱导的鸡CEVM中iNOS基因的转激活（Takahashi T, et al.）。Mol Cell Biochem，已提交)。3)鸡HIF-1α cDNA在CEVM中的分子克隆及表达分析：利用RT-PCR和Race方法从CEVM中克隆了鸡HIF-1α的编码cDNA。然后，我们检测了鸡HIF-1α mRNA在不同胚胎组织中的表达。鸡HIF-1α cDNA的氨基酸序列与人HIF-1α cDNA的同源性达到79%。相比之下，鸡HIF-1α与内皮PAS蛋白1 (EPAS1) （bHLH-PAS蛋白的另一个成员）之间的序列同源性仅为49%。HIF-1α mRNA在CEVM中表达丰富，而在肝脏中表达较少，这与EPAS1 mRNA的表达模式有很大不同。这些数据表明HIF-1α可能参与胚胎心血管发育（Takahashi等）。生物化学，生物物理研究，2001，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).