POLARITY AND FATE OF VWF SECRETED FROM ENDOTHELIAL CELLS

这项建议将集中在血管紧张素Ⅱ分泌调节的研究， 来自内皮细胞的韦伯-帕拉德小体的维勒布兰德因子（vWf）， 重点是这种分泌途径的极性和 vWF发布后。 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 filters.药理学试剂将用于阻断细胞收缩， 迁移，可伴随促分泌素刺激发生，和 研究对vWf释放的表观极性的影响。 将采用显微镜技术直接观察这一过程 威宝-帕拉德公司的尸体 初步研究表明，一些vWf 从韦伯-帕拉德机构释放仍然与外部 细胞表面 这种相互作用的性质及其与 将使用双标记免疫荧光染色探索时间， 活细胞的荧光标记。 代谢标记研究将 进行以确定蛋白聚糖分子是否是韦伯的组分 Palade机构，如果它们在发布后仍然与细胞相关，并且如果 它们在细胞表面的vWf重组中起作用， release. 内皮细胞将在广泛的细胞外基质上培养。 牛角膜内皮细胞产生的基质和vWf的命运 以及从韦伯-帕拉德体释放后其裂解的多肽原， 关于其在细胞裂解物、细胞外 基质和培养基将使用该培养系统进行研究。 的 拟议的研究将提供更好的了解功能 vWf的韦伯-帕拉德体池和细胞过程的作用 引导极化分泌和引导分泌的蛋白质到它们的位点 的行动。

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.