REGULATION OF ENDOTHELIAL CELL ECTO-ADPASE ACTIVITY

内皮细胞胞外 ADP 酶活性的调节

基本信息

批准号：
6336649
负责人：
Aaron Jacob Marcus
金额：
$ 28.8万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-08-01 至 2001-07-31
项目状态：
已结题

项目摘要

Recent research in our laboratory has led to the concept of endothelial cell Thromboregulation. Platelet activation as a consequence of vascular injury causes endothelial cells (EC) to respond in a manner directed toward limitation or reversal of the occlusive consequences of platelet accumulation. Such limitation or reversal is achieved by EC via 3 separate Thromboregulatory systems: Eicosanoids, endothelium-derived relaxing factor (EDRF/NO), and, most importantly, an endothelial ecto- nucleotidase. The latter rapidly metabolizes platelet-released ADP thereby restoring platelets to the resting state. Research proposed in this project will focus on control systems and mechanisms responsible for modulation of vascular Thromboregulation. Major emphasis will be placed on endothelial ecto-ADPase activity since our previous research has highlighted this enzyme system. To this end we will determine mechanisms by which cytokines, such as IL-1b, TNF-a, IFN-g, and IL-13, regulate ADPase activity and gene expression. In addition, we will assess transcriptional regulation of EC ecto-ADPase by elements of the 5'- regulatory region, including cytokine response elements. Our collaborations with Project II will be crucial to successful completion of these aims, since Drs. K. Hajjar and D. Hajjar have expertise in this area of research. Since preliminary studies in EC indicated that IL-13 induces phosphorylation of the Janus kinase Jak-2, we will investigate whether IL- 13 upregulation of EC ADPase activity is related to activation of the Jak- STAT pathway of signal transduction. Our collaboration with Project IV will be essential for achieving these objectives, since Dr. Hempstead has considerable expertise in this developing discipline. Studies of the antithrombotic function of the double lipoxygenase product, lipoxin A4, will be extended to evaluate its capacity for upregulation of EC ADPase activity. Furthermore, it will be important to determine whether LXA4 can prevent or reverse the prothrombotic downregulation of EC ADPase activity resulting from cytokine exposure. To further elucidate the function of LXA4 as an antithrombotic eicosanoid, we will isolate the EC-specific LXA4 receptor cDNA, using the full open reading frame (ORF) of our recently obtained myeloid LXA4 receptor as a probe. The EC-specific LXA4 receptor will then be biochemically and functionally characterized in mammalian expression system(s). The expertise of Drs. Pomerantz and D. Hajjar in eicosanoid molecular biology will be essential for achieving these aims. An understanding of mechanisms controlling EC ADPase activity may lead to development of rational effective forms of antithrombotic therapy which can be implemented at the very earliest stages of thrombus formation.

我们实验室的最新研究导致了内皮的概念细胞血栓调节。血小板激活是由于血管的损伤导致内皮细胞（EC）以某种方式做出反应限制或逆转血小板的闭塞后果积累。 EC通过3实现了这种限制或逆转单独的血小板调节系统：类花生酸，内皮衍生放松因子（EDRF/no），最重要的是，内皮ecto- 核苷酸酶。后者迅速代谢血小板发行的ADP 从而将血小板恢复到静止状态。研究提出了该项目将着重于控制系统和负责的机制血管血管调节的调节。重点将放置自从我们以前的研究已经强调了该酶系统。为此，我们将确定机制通过哪些细胞因子（例如IL-1b，TNF-A，IFN-G和IL-13）调节 ADPase活性和基因表达。此外，我们将评估通过5'-元素对EC ecto-ADPase的转录调节调节区域，包括细胞因子反应元件。我们的与II项目的合作对于成功完成至关重要这些目标，因为Drs。 K. Hajjar和D. Hajjar在这方面有专业知识研究。由于EC的初步研究表明IL-13诱导 Janus激酶JAK-2的磷酸化，我们将研究是否IL- 13 EC ADPase活性的上调与Jak-的激活有关信号转导的统计途径。我们与项目IV的合作由于Hempstead博士拥有在这项发展的学科中，具有丰富的专业知识。研究双脂氧合酶产物Lipoxin A4的抗血栓形成功能，将扩展以评估其上调EC ADPase的能力活动。此外，确定LXA4是否可以防止或逆转EC ADPase活性的脑血栓形成下调由细胞因子暴露导致。为了进一步阐明 lxa4作为抗血栓形成黄酸酯，我们将隔离EC特异性LXA4 受体cDNA，使用我们最近的完整开放阅读框（ORF）获得髓样LXA4受体作为探针。 EC特异性LXA4受体然后，将在哺乳动物中以生化和功能为特征表达系统。博士的专业知识。 Pomerantz和D. Hajjar 类eicosanoid分子生物学对于实现这些目标至关重要。对控制EC ADPase活性的机制的理解可能导致开发合理有效形式的抗血栓疗法的形式可以在血栓形成的最早阶段实现。