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。 血管活性导致血小板活化 损伤导致内皮细胞 (EC) 以定向方式做出反应 限制或逆转血小板的闭塞后果 积累。 这种限制或逆转是由 EC 通过 3 实现的 独立的血栓调节系统：类二十烷酸，内皮衍生的 松弛因子 (EDRF/NO)，最重要的是，内皮细胞外 核苷酸酶。 后者快速代谢血小板释放的 ADP 从而使血小板恢复到静息状态。 提出的研究 该项目将重点关注负责的控制系统和机制 血管血栓调节的调节。 将重点关注 自从我们之前的研究以来，关于内皮细胞外 ADP 酶活性 强调了这个酶系统。 为此，我们将确定机制 IL-1b、TNF-a、IFN-g 和 IL-13 等细胞因子通过其调节 ADP酶活性和基因表达。 此外，我们将评估 EC ecto-ADPase 5'-元件的转录调节 调节区，包括细胞因子反应元件。 我们的 与项目 II 的合作对于成功完成 这些目标，因为博士。 K. Hajjar 和 D. Hajjar 拥有该领域的专业知识 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 IL- 13 EC ADPase 活性的上调与 Jak- 的激活有关 信号转导的STAT途径。 我们与 Project IV 的合作 对于实现这些目标至关重要，因为亨普斯特德博士已经 在这个发展中的学科中拥有丰富的专业知识。 研究的 双脂氧合酶产物脂氧素 A4 的抗血栓功能， 将扩展以评估其上调 EC ADPase 的能力 活动。 此外，确定 LXA4 是否能够 预防或逆转 EC ADPase 活性的血栓前下调 由细胞因子暴露引起。 为进一步阐明其功能 LXA4作为一种抗血栓类二十烷酸，我们将分离出EC特异性的LXA4 受体 cDNA，使用我们最近的完整开放阅读框（ORF） 获得骨髓LXA4受体作为探针。 EC特异性LXA4受体 然后将在哺乳动物中进行生化和功能表征 表达系统。 博士的专业知识。 Pomerantz 和 D. Hajjar 在 类二十烷酸分子生物学对于实现这些目标至关重要。 了解控制 EC ADPase 活性的机制可能会导致 开发合理有效的抗血栓治疗方法 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.