Role of EETs in Growth of Human Endothelial Cells

EET 在人内皮细胞生长中的作用

• 批准号: 6895193
• 负责人: MEETHA M MEDHORA
• 金额: $ 33.75万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6895193
• 关键词: angiogenesis; biological signal transduction; cell cycle proteins; cell differentiation; cell growth regulation; cell proliferation; enzyme inhibitors; enzyme mechanism; gene therapy; high performance liquid chromatography; immunocytochemistry; isomer; laboratory rat; mass spectrometry; matrigel; microtubules; mitogen activated protein kinase; morphometry; oxygenases; phosphorylation; recombinant proteins; tissue /cell culture; vascular endothelium; vasodilators; western blottings

DESCRIPTION (provided by applicant): Our preliminary data show that epoxyeicosatrienoic acids (EETs) are pro-angiogenic in two animal models for angiogenesis, matrigel plugs embedded subcutaneously in rats and chick chorioallantoic membranes. The aims of this proposal are to measure regioisomer specific EET-mediated growth and capillary morphogenesis of human endothelial cells derived from the lung and heart and to define cellular signaling mechanisms that carry out these functions. This will address our long term goal to induce angiogenesis during pathological injury to the human heart and lung. We present evidence that growth and tubular differentiation of primary cultures of human coronary artery endothelial cells (HCAECs) and human lung microvascular endothelial cells (HLMECs) are stimulated by EETs. Levels of the signal regulator, mitogen-activated protein kinase phosphatase-1 (MKP-1) are also induced by over-expresssion of epoxygenase enzymes that catalyze formation of EETs in HLMECs. Using a novel, sensitive fluorescent assay developed at the Medical College of Wisconsin we have measured 4 EET regiosiomers in human endothelial cells and demonstrated increase in EETs in HCAECs after stimulation with bradykinin. In addition, we have cloned eDNA for 2 functional epoxygenase enzymes, the human endothelial epoxygenase 2C9 and its antisense, as well as rat 2C11. The epoxygenases have been recombined in adenoviral vectors and can be delivered to primary HCAECs and HLMECs with >90% efficiency. Using these data and tools the aims of this proposal are: (1) to determine growth of HCAEC and HLMECs by specific EET-regiosiomers and by over-expression of recombinant epoxygenases in the presence and absence of specific inhibitors 2) measure tube formation in the same cells in vitro after treatment with different EET regioisomers, epoxygenases and their inhibitors 3) determine potency of each regioisomer to mediate angiogenesis in vivo and 4) test the role of MKP-1 in inactivating the p38 mitogen-activated protein kinase pathway to promote tubular differentiation of human endothelial cells and angiogenesis in vivo. These aims combine analytical, molecular, pharmacological and whole animal protocols to coordinate a timely, detailed, and focused research effort that will evaluate the role of EETs in growth and differentiation of human endothelial cells as well as angiogenesis in animals. It will impact on the potential for development of EETs as therapeutic agents during and after cardiac ischemia and acute lung injury.

描述（由申请人提供）：我们的初步数据表明，环氧二十碳三烯酸（EAE）在两种血管生成动物模型中具有促血管生成作用，即大鼠皮下包埋的基质胶塞和鸡绒毛尿囊膜。该提案的目的是测量区域异构体特异性EET介导的人肺和心脏内皮细胞的生长和毛细血管形态发生，并定义执行这些功能的细胞信号传导机制。这将解决我们在人类心脏和肺的病理损伤期间诱导血管生成的长期目标。我们目前的证据表明，生长和肾小管分化的原代培养的人冠状动脉内皮细胞（HCAECs）和人肺微血管内皮细胞（HLMEC）的刺激，由依普利。信号调节因子丝裂原活化蛋白激酶磷酸酶-1（MKP-1）的水平也由催化HLMEC中EkB形成的环氧合酶的过表达诱导。使用威斯康星州医学院开发的一种新的、灵敏的荧光测定法，我们测量了人内皮细胞中的4种EET区域异构体，并证明了用缓激肽刺激后HCAEC中EET的增加。此外，我们已经克隆了2个功能性环氧合酶的eDNA，人内皮环氧合酶2C 9及其反义，以及大鼠2C 11。环氧合酶已经重组在腺病毒载体中，并且可以以>90%的效率递送至原代HCAEC和HLMEC。利用这些数据和工具，本提案的目标是：（1）通过特异性EET区域异构体和通过在存在和不存在特异性抑制剂的情况下重组环氧合酶的过表达来确定HCAEC和HLMEC的生长; 2）在体外测量用不同EET区域异构体处理后相同细胞中的管形成，表氧化酶及其抑制剂，3）测定每种区域异构体在体内介导血管生成的效力，和4）测试MKP-1在灭活p38促分裂原中的作用，激活蛋白激酶途径，促进人内皮细胞肾小管分化和体内血管生成。这些目标结合了联合收割机分析、分子、药理学和整个动物方案，以协调及时、详细和集中的研究工作，从而评估内皮素在人内皮细胞生长和分化以及动物血管生成中的作用。这将影响到开发的潜力，作为治疗药物的心脏缺血和急性肺损伤期间和之后。