Role of EETs in Growth of Human Endothelial Cells

描述(申请人提供)：我们的初步数据显示，在两种血管生成的动物模型中，环氧二十碳三烯酸(EETs)是促血管生成的，Matrigel塞子植入大鼠皮下和鸡绒毛膜尿囊膜中。这项建议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，并确定执行这些功能的细胞信号机制。这将解决我们的长期目标，即在人类心脏和肺的病理性损伤期间诱导血管生成。我们发现EETs能刺激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(HLMECs)的生长和分化。信号调节因子丝裂原活化蛋白激酶磷酸酶-1(MKP-1)的水平也由环氧合酶的过度表达所诱导，环氧合酶催化HLMECs中EET的形成。使用威斯康星医学院开发的一种新的、灵敏的荧光分析方法，我们测量了人内皮细胞中的4个EET区域异构体，并证明了缓激肽刺激后HCAEC中EET的增加。此外，我们还克隆了人内皮环氧合酶2C9及其反义和大鼠2C11两种功能性环氧合酶的EDNA。环氧合酶已在腺病毒载体中重组，并可以90%的效率输送给原代HCAECs和HLMECs。利用这些数据和工具，本建议的目的是：(1)确定特定EET区域异构体和重组环氧合酶在存在和不存在特定抑制剂的情况下对HCAEC和HLMEC的生长；2)测量不同EET区域异构体、环氧合酶及其抑制剂处理后同一细胞在体外的管状形成；3)确定每个区域异构体在体内介导血管生成的效力；以及4)测试MKP-1在抑制p38丝裂原活化蛋白激酶途径以促进体内血管生成中的作用。这些目标结合了分析、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，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。它将影响EETs在心肌缺血和急性肺损伤期间和之后作为治疗剂的发展潜力。

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.