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LOCATION OF PORE SYSTEMS IN CAPILLARY WALLS

毛细管壁中孔隙系统的位置

基本信息

批准号：
6164993
负责人：
GEORGE E PALADE
金额：
$ 34.36万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-03-01 至 2002-02-28
项目状态：
已结题

项目摘要

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、男性和女性的生理学中起着关键的作用，包括. 它介导了大量的，持续的水和亲水性物质的交换，和疏水溶质（小分子和大分子）之间的血浆和组织液。所有细胞的存活身体的组织和器官依赖于这些持续的交换。它参与凝血和抗凝过程的控制影响血液。它在早期阶段发挥着重要作用，炎症反应。它参与了正常和病理的新血管形成（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，内皮细胞故障是大量人类的起始基线，病理条件。在这项赠款下所做的工作已经提供了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。它还开辟了新的前景，主要研究者建议在继续研究期间，补助金他认为，增加对这些功能的了解，有助于预防、减轻和治愈某些情况下的内皮功能障碍时间在未来。这些研究是根据五个具体目标提出的。 I. 在第一个具体目标中，利用α 1-酸性糖蛋白的实验（orosomucoid）作为示踪剂，以加强概念/过程，胞吞作用在连续内皮细胞大分子处理中的作用以细胞间连接为界。形态学和细胞分级方法将被用来研究示踪剂的入口和出口，由Pvs或小窝融合形成的跨内皮通道。尝试将确定两个群体的小窝，即，小型和大型一个，容纳40埃和250埃的二硝基苯化探针，分别这些研究还将扩大到确定质膜蛋白，其锚定α 1-酸性糖蛋白，和构成纤维基质。确定因素的进一步实验负责融合的小窝也提出了。在这些在实验中，融合蛋白将利用含有 NEM敏感因子（NSF）和SNAP的cDNA;其相应的将产生多克隆抗体，并用于研究分布在内皮细胞中。分配将由以下人员确认：使用其他方法，例如，使用特异性单克隆抗体，抗体的二. 在第二个具体目标中，拟议的研究将重点关注有或无隔膜的有孔内皮，具体目标# I中所述的方法。三. 第三个具体目标涉及到研究的结构调制的小窝诱导血小板活化因子和血管内皮生长因子（VEGF）。将特别注意研究旁囊泡糖蛋白组装，PI定义为“紧身胸衣”，将PAF和VEGF灌注到脉管系统中。因为他们的行为介导的受体，实验概述了研究在这些生长因子的影响下受体磷酸化。的研究将扩大到调查负面的影响，转胞吞作用的可能的正调节剂，它们包括NEM，硝普钠、心钠素和钙离子载体。在这些实验中，将研究内皮间连接的改变（如果有的话）。四. 具体目标#4涉及以下生物化学表征：从肺组织分离的小窝组分中富集的糖蛋白内皮细胞，利用阳离子化二氧化硅程序。他们的隔离将是通过使用各种单克隆抗体来促进。这些研究将被扩展到表征的功能的多样性，小窝 V.在具体目标5中，来自血管毛细血管的内皮，例如，管周毛细血管将通过利用上述具体实施方式中描述的技术来执行目标。