POLARITY AND FATE OF VWF SECRETED FROM ENDOTHELIAL CELLS

这项提案将重点研究von的调节分泌。 血管内皮细胞韦贝尔-帕拉德小体产生的Willebrand因子(VWF)， 重点是这条分泌途径的两极和 发布后的VWF。使用多种促分泌剂的极性研究 将使用在多孔膜上培养的内皮细胞进行 过滤器。药物将被用来阻止细胞回缩和 迁移，这可能伴随着促分泌剂刺激而发生，以及 研究其对VWF表观释放极性的影响。 将使用微观技术来直接可视化这一过程 韦贝尔-帕莱德的身体释放。初步研究表明，一些VWF 从韦贝尔-帕拉德遗体中释放出来的遗体与外部 细胞表面。这种相互作用的性质及其与 将探索时间，使用双标记免疫荧光染色和 活细胞的荧光标记。新陈代谢标签研究将是 以确定蛋白多糖分子是否为韦伯的成分 Palade小体，如果它们在释放后仍然与细胞相关，并且如果 它们在细胞表面VWF的重组中起作用 放手。内皮细胞将在广泛的细胞外培养 牛角膜内皮细胞产生的基质与VWF的命运 及其裂解的原多肽从韦贝尔-帕拉德小体释放后， 关于它在细胞裂解物、细胞外 将利用该培养系统对基质和培养基进行研究。这个 拟议的研究将提供对功能的更多了解 VWF的Webel-Palade体库和细胞突起的作用 引导极化分泌和将分泌的蛋白质定向到它们的位置 行动的一部分。

This proposal will focus on studies of the regulated secretion of von Willebrand factor (vWf) from the Weibel-Palade bodies of endothelial cells, with emphasis on the polarity of this secretory pathway and on the fate of vWf following release. Polarity studies using a variety of secretagogues will be conducted using endothelial cells cultured on porous membrane filters. Pharmacologic agents will be used to block cell retraction and migration, which can occur concomitant with secretagogue stimulation, and to study the resulting effect on the apparent polarity of release of vWf. Microscopic techniques will be employed to directly visualize the process of Weibel-Palade body release. Preliminary studies reveal that some vWf released from Weibel-Palade bodies remains associated with the external cell surface. The nature of this interaction and its reorganization with time will be explored, using double-label immunofluorescence staining and fluorescence labeling of live cells. Metabolic-labeling studies will be conducted to determine if proteoglycan molecules are constituents of Weibel Palade bodies, if they remain cell-associated following release, and if they function in the reorganization of vWf at the cell surface following release. Endothelial cells will be cultured on an extensive extracellular matrix produced by bovine corneal endothelial cells, and the fate of vWf and its cleaved propolypeptide following release from Weibel-Palade bodies, with regard to its compartmentation between cell lysate, extracellular matrix and culture medium will be studied using this culture system. The proposed studies will provide a greater understanding of the functional role of the Weibel-Palade body pool of vWf and of the cellular processes directing polarized secretion and directing secreted proteins to their site of action.