Analyses of ischemia-induced signals in cardiac myocytes

心肌细胞缺血诱导信号的分析

• 批准号: 11470159
• 负责人: TAKAHASHI Toshiyuki
• 金额: $ 4.1万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470159/
• 关键词: myocardial ischemia-reperfusion; hypoxia-inducible factor 1α; vascular endothelial growth factor; inducible nitric oxide synthase; lipopolysaccharide; adenosine; chick embryo; cardiac myocytes; RT-PCR法; RACE法; endothelial PAS protein 1; cDNA クロ

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).

本研究旨在探讨缺氧诱导因子1α（hypoxia-inducible factor 1α，HIF-1α）在心肌缺血再灌注损伤中的作用。特别地，我们分析了心肌细胞中血管内皮生长因子（VEGF）和诱导型一氧化氮合酶（iNOS）的表达，它们是HIF-1α的靶基因之一。利用培养的新生大鼠心肌细胞，我们观察到血管内皮生长因子的表达增加脂多糖（LPS）（Sugishita Y，et al. Biochem Biophys Res Commun，2000）或腺苷（通过其A_2受体）。另一方面，我们分析了培养的10日龄鸡胚心室肌细胞（CEVM）中鸡VEGF的表达。（Sugishita Y等，J Mol Cell Caardiol，2000）。2）CEVM中鸡iNOS基因表达的分析：结果表明，NF-κB和Ets-1均参与LPS诱导的鸡胚CEVM中iNOS基因的转录激活3）鸡HIF-1α cDNA的克隆及在CEVM中的表达分析：采用RT-PCR和Race方法从CEVM中克隆了鸡HIF-1 α 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的同源性为79%，而与bHLH-PAS蛋白的另一成员内皮PAS蛋白1（EPAS 1）的同源性较低（49%）。HIF-1α mRNA在CEVM中大量表达，而在肝脏中几乎不表达，这与EPAS 1 mRNA的表达模式有很大不同，提示HIF-1α可能参与了胚胎心血管的发育（Takahashi T，et al. Biochem Biophys Res Commun，2001，in press）。

项目成果

Sugishita Y, Shimizu T, Takahashi T, et al.: "Lipopolysaccharide augments expression and secretion of vascular endothelial growth factor in rat ventricular myocytes."Biochem Biophys Res Commun. 268. 657-662 (2000)

Sugishita Y、Shimizu T、Takahashi T 等人：“脂多糖增强大鼠心室肌细胞中血管内皮生长因子的表达和分泌。”Biochem Biophys Res Commun。

Takahashi T,Sugishita Y,Nojiri T, et al.: "Cloning of hypoxia-inducible factor 1α cDNA from chick embryonic ventricular myocytes."Biochem Biophys Res Commun. (印刷中). (2001)

Takahashi T、Sugishita Y、Nojiri T 等人：“从鸡胚胎心室肌细胞中克隆缺氧诱导因子 1α cDNA。”Biochem Biophys Res Commun（印刷中）。

Takahashi T, Sugishita Y, Nojiri T, et al.: "Cloning of hypoxia-inducible factor 1α cDNA from chick embryonic ventricular myocytes."Biochem Biophys Res Commun. (in press). (2001)

Takahashi T、Sugishita Y、Nojiri T 等人：“从鸡胚胎心室肌细胞中克隆缺氧诱导因子 1α cDNA。”Biochem Biophys Res Commun（印刷中）。

Sugishita Y, Takahashi T, Shimizu T, et al.: "Expression of genes encoding vascular endothelial growth factor and its FIK-1 receptor in the chick embryonic heart."J Mol Cell Cardiol. 32. 1039-1051 (2000)

Sugishita Y、Takahashi T、Shimizu T 等人：“在鸡胚胎心脏中编码血管内皮生长因子及其 FIK-1 受体的基因的表达。”J Mol Cell Cardiol。

Sugishita Y,Takahashi T,Shimizu T, et al.: "Expression of genes encoding vascular endothelial growth factor and its Flk-1 receptor in the chick embryonic heart."J Mol Cell Cardiol. 32. 1039-1051 (2000)

Sugishita Y、Takahashi T、Shimizu T 等人：“鸡胚心脏中血管内皮生长因子及其 Flk-1 受体编码基因的表达。”J Mol Cell Cardiol。