REGULATION OF ENDOTHELIAL CELL ECTO-ADPASE ACTIVITY

我们实验室最近的研究提出了内皮细胞的概念， 细胞血栓调节 血小板活化是血管性疾病的结果 损伤导致内皮细胞（EC）以定向的方式应答， 限制或逆转血小板聚集的闭塞性后果 积累 这种限制或逆转是由EC通过3 单独的血栓调节系统：类二十烷酸，内皮源性 舒张因子（EDRF/NO），以及最重要的是，内皮细胞外 核苷酸酶 后者迅速代谢血小板释放的ADP 从而使血小板恢复到静止状态。 建议的研究 该项目将侧重于控制系统和机制， 血管血栓调节的调节。 重点将放在 由于我们之前的研究 强调了这个酶系统。 为此，我们将确定机制， 细胞因子，如IL-1b、TNF-α、IFN-γ和IL-13， 腺苷三磷酸活性和基因表达。 此外，我们将评估 EC外膜-ADDRESS的转录调控通过5 '- 调节区，包括细胞因子应答元件。 我们 与项目II的合作对于成功完成 这些目标，因为博士。Hajjar和D. Hajjar在这方面有专长 的研究。 由于在EC中的初步研究表明IL-13诱导 磷酸化的Janus激酶Jak-2，我们将研究是否IL- 13. EC ADDRESS活性的上调与Jak-13的激活有关。 信号转导的STAT途径。 与Project IV的合作 对于实现这些目标至关重要，因为Hempstead博士 在这一发展中的学科中有相当多的专业知识。 的研究 双脂氧合酶产物，脂氧素A4， 将被扩展到评估其上调EC ADDRESS的能力， 活动 此外，重要的是确定LXA 4是否可以 阻止或逆转EC ADDRESS活性的血栓前下调 是由细胞因子暴露引起的 为了进一步阐明 LXA 4作为一种抗血栓的类花生酸，我们将分离EC特异性LXA 4， 受体cDNA，使用我们最近的完全开放阅读框架（ORF）， 获得髓样LXA 4受体作为探针。 EC特异性LXA 4受体 然后将在哺乳动物中进行生物化学和功能表征， 表达系统。 Pomerantz和D.哈扎尔在 类花生酸分子生物学将是实现这些目标所必需的。 了解控制EC ADP酶活性的机制可能会导致 开发合理有效的抗血栓治疗形式， 可以在血栓形成的最早阶段实施。

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.