ENDOTHELIAL CELL THROMBOXANE A2 RECEPTOR

内皮细胞血栓A2受体

基本信息

批准号：
2223337
负责人：
J Anthony Ware
金额：
$ 17.01万
依托单位：
BETH ISRAEL DEACONESS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-08-01 至 1999-07-31
项目状态：
已结题

项目摘要

Thromboxane A2(TxA2) and related endoperoxides cause vasoconstriction and platelet aggregation following their binding to specific sites (putative TxA2 receptors) on vascular smooth muscle cells and platelets. Such sites have not been described in human endothelial cells. The overall hypothesis to be examined in these studies is that binding of endoperoxides to TxA2 receptors on human endothelial cells has potentially important functional effects. Two secondary hypotheses are that the expression of the mRNA encoding the thromboxane A2 receptor is altered by factors likely to be relevant in vascular diseases and that the variation in the molecular structure of the cytoplasmic domain might confer differences in the intracellular mediators and functional events associated with thromboxane A2 binding. The first aim of the proposal is to determine the physiologic and functional effects of TxA2 binding human endothelial cells. The release of prostacyclin and the expression of P-selectin on the cell surface, and the production of endothelial- derived relaxing factor by cultured endothelial cells will be measured following stimulation with endoperoxide analogs. The ability of TxA2 binding to induce expression of the protooncogenes c-fos, c-myc, and c- jun/AP1 and proliferation of endothelial cells will be assessed, as will the TxA2 stimulated expression of mRNA encoding plasminogen activator inhibitors. The second aim of this proposal is to determine the signal transduction mechanism responsible for these functional effects. Calcium rise following endoperoxide analogs will be measured, and the possibility that this rise results from internal mobilization as well as influx via a Ca2+ channel will be assessed. The effect of activation of individual protein kinase C isozymes will be assessed as will the contributions of Ca2+ and PKC activation to desensitization of the TxA2 receptor following occupancy. The third aim s to characterize the expression of the gene encoding the human endothelial thromboxane A2 receptor in endothelial cells stimulated with cytokines and growth factors. A full length cDNA encoding this receptor has been isolated form a human endothelial cell library, and will be labelled so the expression of Txa@ receptor mRNA in human endothelial cells following stimulation with cytokines growth factors and exposure to shear stress can be studies. Finally, the full length cDNA encoding the endothelial TxA2 receptor will be overexpressed in both Chines Hamster Ovary cells and in endothelial like cell lines devoid of thromboxane A2 receptors, so the functional events and signal transduction can be assessed in autologous cells. Such information may permit development of more selective and potent therapy for thrombosis and other vascular disorders.

血栓烷A2（TXA2）和相关内氧化物引起血管收缩和与特定位点结合后的血小板聚集（推定 TXA2受体）在血管平滑肌细胞和血小板上。这样的在人内皮细胞中尚未描述部位。总体在这些研究中要研究的假设是结合在人内皮细胞上对TXA2受体的内氧化物具有潜在的重要功能效应。两个次要假设是编码血栓烷A2受体的mRNA的表达是因可能与血管疾病相关的因素而改变，并且细胞质结构域分子结构的变化可能赋予细胞内介体和功能事件的差异与血栓烷A2结合有关。提案的第一个目的是确定TXA2结合的生理和功能效应人内皮细胞。前列环蛋白的释放和表达细胞表面上的P-选择素，并产生内皮的生产将测量通过培养的内皮细胞得出的放松因子用内氧化物类似物刺激后。 TXA2的能力结合诱导原子基因C-FOS，C-MYC和C-的表达将评估JUN/AP1和内皮细胞的增殖 TXA2刺激了编码纤溶酶原激活剂的mRNA表达抑制剂。该提案的第二个目的是确定信号导致这些功能效应的转导机制。钙将测量后过氧化物类似物的增加，并可能性这是由于内部动员以及通过将评估CA2+通道。个体激活的影响蛋白激酶C同工酶将被评估 CA2+和PKC激活TXA2受体脱敏占用。表征基因表达的第三个目标在内皮中编码人内皮血栓烷A2受体用细胞因子和生长因子刺激细胞。全长cDNA 编码该受体已被隔离，形成了人类内皮细胞库，并将被标记为TXA@受体mRNA的表达刺激细胞因子生长后的人体内皮细胞因素和暴露于剪切应力的可能是研究。最后，全部编码内皮TXA2受体的长度cDNA将过表达在Chines仓鼠卵巢细胞中，在内皮等细胞系中没有血栓烷A2受体，因此功能事件和信号可以在自体细胞中评估转导。这样的信息可能允许开发更有力和有效的血栓形成疗法和其他血管疾病。