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：（改编自申请人的摘要。）拟议的研究 旨在检查内皮细胞的机制 撤回。 指导这项工作的假设是内皮细胞 (EC) 回缩需要两个事件才能形成回缩间隙： 皮质细胞质中的溶解或凝胶-溶胶转变 EC 收缩装置的激活。 乙氯维诺 (ECV) 导致成人呼吸窘迫综合征（ARDS）的化学介质， 组胺和凝血酶，两种重要的炎症介质 增加血管通透性将被用来检验他们的假设。 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。 这些研究的目的是确定是否撤回 和间隙形成与 EC 肌球蛋白轻链相关 磷酸化。 MLC磷酸化与胞质的关系 Ca2+阳性，细胞变形能力和单层通透性将 决定。 此外，酶（MLCK 或 PKC 或两者）负责 将鉴定激动剂刺激的单层中的磷酸化MLC。 环单磷酸腺苷的升高及其合成物的用途 肌球蛋白轻链激酶的肽抑制剂将用于抑制 EC 回缩、MLC 磷酸化和细胞变形性。 的作用 凝溶胶蛋白，一种 Ca2+ 阳性激活的肌动蛋白切断蛋白，在变化中 将研究肌动蛋白网络的结构。 该小组将 将 EGTA 抗性 1:1 肌动蛋白/凝溶胶蛋白复合物与肌动蛋白相关联 切断激动剂刺激的单层细胞的活性。 课程时间为 肌动蛋白/凝溶胶蛋白复合物的形成和肌动蛋白切断活性将 与Ca2+正动员、细胞变形能力和EC相比 撤回。 透化的 EC 制剂将用于测试 凝溶胶蛋白对 EC 回缩的影响。 不同浓度的凝溶胶蛋白 将添加到这些制剂中以及对肌动蛋白网络的影响和 确定撤回。 共焦荧光显微镜将用于 定义肌动蛋白网络的三维特征 特别注意肌动蛋白的外围边缘。 的结构 静息和刺激 EC 中的细胞骨架将根据 肌动蛋白、肌球蛋白、MLCK 和凝溶胶蛋白的分布。