Role of EETs in Growth of Human Endothelial Cells

描述（由申请人提供）：我们的初步数据显示，环氧二碳三烯酸（EETs）在两种血管生成动物模型中具有促进血管生成的作用，即皮下植入大鼠和鸡绒毛膜尿囊膜的基质塞。本提案的目的是测量来自肺和心脏的人内皮细胞的区域异构体特异性eet介导的生长和毛细血管形态发生，并确定执行这些功能的细胞信号传导机制。这将解决我们的长期目标，即在人类心肺病理性损伤期间诱导血管生成。我们提供的证据表明，EETs可以刺激人冠状动脉内皮细胞（HCAECs）和人肺微血管内皮细胞（HLMECs）的生长和小管分化。信号调节因子、丝裂原活化蛋白激酶磷酸酶-1 （MKP-1）的水平也可通过在HLMECs中催化eet形成的环氧化酶的过度表达而诱导。利用威斯康星医学院开发的一种新型、灵敏的荧光测定方法，我们测量了人内皮细胞中的4个EET区域异构体，并证明在缓激肽刺激后hcaec中的EET增加。此外，我们还克隆了2种功能性环氧合酶的eDNA，即人内皮环氧合酶2C9及其反义酶，以及大鼠环氧合酶2C11。环氧合酶已在腺病毒载体上重组，并能以90%的效率递送至原发性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区域异构体和在存在和不存在特异性抑制剂的情况下过表达重组环氧合酶来测定HCAEC和HLMECs的生长情况；(2)在体外用不同的EET区域异构体处理同一细胞后，测量管的形成情况；3)确定每个区域异构体在体内介导血管生成的效力；4)检测MKP-1在体内灭活p38丝裂原激活的蛋白激酶途径，促进人内皮细胞小管分化和血管生成的作用。这些目标结合了分析、分子、药理学和全动物方案，以协调及时、详细和集中的研究工作，以评估EETs在人类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eet在心脏缺血和急性肺损伤期间和之后作为治疗药物的发展潜力。

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.