ENDOTHELIAL CELL RETRACTION AND EDEMA OF ARDS

ARDS 的内皮细胞收缩和水肿

• 批准号: 2222485
• 负责人: Robert B. Wysolmerski
• 金额: $ 11.48万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2222485
• 关键词: actins; adult respiratory distress syndrome; alcohols; cow; cytoskeletal proteins; fluorescence microscopy; gelsolin; histamine; human tissue; inflammation; myosin light chain kinase; phosphorylation; pulmonary artery; pulmonary edema; thrombin; tissue /cell culture; vascular endothelium; vascular endothelium permeability

DESCRIPTION: (Adapted from the applicant's abstract.) The proposed studies are directed to an examination of the mechanism of endothelial cell retraction. The hypothesis directing this work is that endothelial cell (EC) retraction requires two events for the formation of retractive gaps: a dissolution or gel-sol transition in the cortical cytoplasm and the activation of the EC contractile apparatus. Ethchlorvynol (ECV), a chemical mediator which causes Adult Respiratory Distress Syndrome (ARDS), and histamine and thrombin, two important mediators of inflammation that increase vascular permeability will be used to test their hypothesis. A tissue culture model utilizing human vein and bovine pulmonary artery endothelial cells will be used to simulate the retraction that occurs in vivo in ARDS. The studies have been designed to determine if retraction and gap formation are associated with EC myosin light chain phosphorylation. The relationships between MLC phosphorylation, cytosolic Ca2 positive, cellular deformability and monolayer permeability will be determined. In addition, the enzymes (MLCK or PKC or both) responsible for phosphorylating MLC in agonist stimulated monolayers will be identified. Elevation of cyclic adenosine monophosphate and the use of synthetic peptide inhibitors to myosin light chain kinase will be employed to inhibit EC retraction, MLC phosphorylation and cellular deformability. The role of gelsolin, a Ca2 positive activated actin severing protein, in the changes in the structure of the actin network will be investigated. The group will correlate the EGTA resistant 1:1 actin/gelsolin complexes and actin severing activity from agonist stimulated monolayers. The time course for formation of actin/gelsolin complexes and actin severing activity will be compared with Ca2 positive mobilization, cellular deformability and EC retraction. The permeabilized EC preparation will be used to test the effects of gelsolin on EC retraction. Varying concentrations of gelsolin will be added to these preparations and the effect on the actin network and retraction determined. Confocal fluorescence microscopy will be used to define the three-dimensional characteristics of the actin network with particular attention to the peripheral rim of actin. The structure of the cytoskeleton in resting and stimulated EC will be defined with respect to the disposition of actin, myosin, MLCK and gelsolin.

描述：（根据申请人的摘要进行了改编。）拟议的研究 针对内皮细胞机理的检查 撤回。 指导这项工作的假设是内皮细胞 （EC）撤回需要两个事件以形成缩回间隙： 皮质细胞质中的溶解或凝胶 - 酚过渡和 EC收缩设备的激活。 乙醇（ECV），a 引起成人呼吸窘迫综合征（ARDS）的化学介质， 和组胺和凝血酶，两个重要的炎症介质 增加血管通透性将用于检验其假设。 一个 利用人静脉和牛肺动脉的组织培养模型 内皮细胞将用于模拟发生在 体内ards。 研究旨在确定是否撤回 和间隙形成与EC肌球蛋白轻链有关 磷酸化。 MLC磷酸化，胞质的关系 CA2阳性，细胞可变形性和单层渗透性将是 决定。 另外，负责的酶（MLCK或PKC或两者） 将鉴定出激动剂刺激的单层中的磷酸化MLC。 循环腺苷单磷酸盐的升高和合成的使用 肌球蛋白轻链激酶的肽抑制剂将用于抑制 EC回缩，MLC磷酸化和细胞可变形性。 的作用 Gelsolin，一种Ca2阳性激活肌动蛋白切断蛋白，在变化中 将研究肌动蛋白网络的结构。 小组会 将抗EGTA抗EGTA 1：1肌动蛋白/凝胶蛋白复合物和肌动蛋白相关联 激动剂刺激了一单层的活动。 时间课程 肌动蛋白/凝胶蛋白复合物和肌动蛋白切断活性的形成将是 与CA2阳性动员，细胞可变形性和EC相比 撤回。 透化的EC制剂将用于测试 凝胶素对EC缩回的影响。 不同浓度的凝胶素 将添加到这些准备工作中，以及对肌动蛋白网络的影响 缩回确定。 共聚焦荧光显微镜将用于 使用 特别注意肌动蛋白的外围边缘。 结构 在静止和刺激的EC中的细胞骨架将根据 肌动蛋白，肌球蛋白，MLCK和Gelsolin的处置。