LOCATION OF PORE SYSTEMS IN CAPILLARY WALLS

毛细管壁中孔隙系统的位置

• 批准号: 2027971
• 负责人: GEORGE E PALADE
• 金额: $ 31.41万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2027971
• 关键词: 2,4 dinitrophenol; alpha 1 acid glycoprotein; antibody formation; capillary; caveolas; chimeric proteins; growth factor receptors; intercellular connection; ionophores; laboratory rat; membrane channels; membrane transport proteins; monoclonal antibody; platelet activating factor; radionuclides; radiotracer; transcytosis; transfection /expression vector; vascular endothelial growth factors; vascular endothelium permeability

DESCRIPTION (Adapted from Investigator's Abstract): The vascular endothelium performs critical functions in the physiology of mammals, men included. It mediates vast, continuous exchanges of water and hydrophilic and hydrophobic solutes (small as well as large molecules) between the blood-plasma and interstitial fluids. The survival of all cells of the tissues and organs of the body depends on these continuous exchanges. It participates in the control of coagulant and anticoagulant processes affecting the blood. It plays an essential role in the initial phases of inflammatory reactions. And it is involved in normal and pathological neovascularization (angiogenesis), including tumor vascularization on which depends tumor growth and metastasis. On these accounts, endothelial malfunction is the starting baseline for a large number of human pathological conditions. Work done under this grant has already provided useful information on cellular and molecular interactions involved in each of the critical functions listed above. It has also opened new vistas which the Principal Investigator proposes to explore during the continuation of the grant. He believes that increased understanding of these functions will help prevent, mitigate and if possible cure endothelial malfunctions at some time in the future. The studies are proposed under five Specific Aims. I. In the first specific aim, experiments utilizing alpha1-acidic glycoprotein (orosomucoid) as a tracer are proposed to reinforce the concept/process of transcytosis in the handling of macromolecules in continuous endothelia bounded by intercellular junctions. Morphological and cell fractionation methods will be employed to study the entrance and exit of the tracer via transendothelial channels formed by the fusion of Pvs or caveolae. Attempts will be made to identify two populations of caveolae, i.e., small and large ones, accommodating 40 angstrom and 250 angstrom dinitrophenylated probes, respectively. The studies will be extended also to identify the plasmalemmal protein(s) which anchor(s) the alpha1-acidic glycoproteins and constitute the fiber matrix. Further experiments to identify the factor(s) responsible for the fusion of the caveolae are also proposed. In these experiments, fusion proteins would be generated utilizing vectors containing cDNA for NEM sensitive factor (NSF) and SNAPs; their corresponding polyclonal antibodies will be generated, and used to study the distribution in the endothelial compartments. The distribution would be confirmed by using other approaches, such as, with the employment of specific monoclonal antibodies. II. In the second specific aim, the studies proposed will focus on fenestrated endothelia with or without diaphragms, and utilize the methodologies as described in specific aim # I. III. The third specific aim relates to the studies of structural modulations of caveolae induced by platelet activating factor (PAF) and vascular endothelial growth factor (VEGF). A particular attention will be paid to study the changes in the paracaveolar glycoprotein assembly, defined by the PI as "corset", upon perfusion of PAF and VEGF into the vasculature. Since their actions are mediated via their receptors, the experiments are outlined to study the receptor phosphorylation under the influence of these growth factors. The studies will be extended to investigate the influence of negative and possible positive modulators of transcytosis, and they include NEM, Na-nitroprusside, ANP and Ca++ ionophores. In these experiments, alterations, if any, in the interendothelial junctions will be investigated. IV. The specific aim # 4 deals with the biochemical characterization of glycoproteins enriched in the caveolar fractions isolated from pulmonary endothelia, utilizing cationized silica procedures. Their isolation will be facilitated by the use of various monoclonal antibodies. These studies would be extended to characterize the multiplicity of the functions of caveolae. V. In specific aim # 5, the characterization of fenestrated endothelia from diaphragmatic capillaries, e.g., peritubular capillaries will be carried out by utilizing the techniques described in above specific aims.

描述（改编自研究者摘要）：血管 内皮细胞在哺乳动物、男性的生理学中发挥着重要作用 包括。 它介导大量、持续的水和亲水性交换 和疏水性溶质（小分子和大分子）之间 血浆和间质液。 所有细胞的生存 身体的组织和器官依赖于这些不断的交换。 它 参与凝血和抗凝血过程的控制 影响血液。 它在初始阶段起着至关重要的作用 炎症反应。 它涉及正常和病理 新血管形成（血管生成），包括肿瘤血管形成 取决于肿瘤的生长和转移。 在这些帐户中，内皮细胞 故障是大量人类的起始基线 病理状况。 这笔赠款下完成的工作已经提供 有关每个细胞和分子相互作用的有用信息 上面列出的关键功能。 也开启了新的视野 首席研究员建议在继续进行探索 补助金。 他相信，加深对这些功能的了解将有助于 有助于预防、减轻甚至治愈某些部位的内皮功能障碍 未来的时间。 这些研究是根据五个具体目标提出的。 我。 第一个具体目标是利用 α1-酸性糖蛋白进行实验 提出（orosumucoid）作为示踪剂来强化以下概念/过程： 连续内皮细胞中大分子处理的转胞吞作用 以细胞间连接为界。 形态学和细胞分离 将采用方法来研究示踪剂的入口和出口 由 Pvs 或细胞膜窝融合形成的跨内皮通道。 尝试 将确定两个小窝群体，即小型和大型 可容纳 40 埃和 250 埃二硝基苯化探针， 分别。 研究范围还将扩大，以确定 锚定 α1 酸性糖蛋白的质膜蛋白和 构成纤维基质。 进一步的实验来确定因素 还提出了负责小窝融合的因素。 在这些 实验中，融合蛋白将利用含有 NEM 敏感因子 (NSF) 和 SNAP 的 cDNA；他们对应的 将生成多克隆抗体，并用于研究分布 在内皮室中。 分配将由以下方式确认 使用其他方法，例如使用特定的单克隆抗体 抗体。 二. 在第二个具体目标中，提出的研究将 重点关注有或没有隔膜的有窗内皮细胞，并利用 具体目标 # I.III 中描述的方法。 第三个具体目标 涉及由引起的小窝结构调制的研究 血小板活化因子（PAF）和血管内皮生长因子 （血管内皮生长因子）。 将特别关注研究变化 腔旁糖蛋白组装，被 PI 定义为“紧身胸衣”， 将 PAF 和 VEGF 灌注到脉管系统中。 既然他们的行动是 通过其受体介导，实验概述为研究 在这些生长因子的影响下受体磷酸化。 这 将扩大研究范围以调查负面和负面影响的影响 转胞吞作用可能的正调节剂，它们包括 NEM， 硝普钠、ANP 和 Ca++ 离子载体。 在这些实验中， 如果有的话，将调查内皮间连接处的变化。 四． 具体目标#4涉及生物化学特征 从肺中分离出的小穴组分中富含糖蛋白 内皮细胞，利用阳离子化二氧化硅程序。 他们将被孤立 通过使用各种单克隆抗体来促进。 这些研究 将被扩展以表征功能的多样性 小穴。 V. 在具体目标#5中，开窗的特征 来自膈毛细血管的内皮细胞，例如管周毛细血管 将利用上述具体描述的技术来进行 目标。