ENDOTHELIAL MODULATION OF LUNG VASCULAR PERMEABILITY

肺血管通透性的内皮调节

• 批准号: 6399960
• 负责人: RICHARD C SCHAEFFER
• 金额: $ 3.11万
• 依托单位: SIDNEY KIMMEL CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-15 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6399960
• 关键词: SDS polyacrylamide gel electrophoresis; calcium; charge coupled device camera; cyclic AMP; cytoskeletal proteins; cytoskeleton; digital imaging; electron microscopy; fluorescent dye /probe; intracellular; microcirculation; protein kinase A; protein kinase C; pulmonary artery; pulmonary circulation; radioimmunoassay; second messengers; solute; thrombin; vascular endothelium permeability; SDS polyacrylamide gel electrophoresis; calcium; charge coupled device camera; cyclic AMP; cytoskeletal proteins; cytoskeleton; digital imaging; electron microscopy; fluorescent dye /probe; intracellular; microcirculation; protein kinase A; protein kinase C; pulmonary artery; pulmonary circulation; radioimmunoassay; second messengers; solute; thrombin; vascular endothelium permeability

A persistent increase in lung endothelial permeability to protein is a hallmark of the Adult Respiratory Distress Syndrome. Moreover, inflammatory mediator-initiated increase in the Area/Unit pathlength (Arho/Ax) of the Endothelial Junctional Space is the foundation of elevated solute permeability of the microvascular wall. The long term objectives of this research are to better understand the endothelial mechanisms that modulate pulmonary microvascular permeability to macromolecules. Intracellular level of cyclic nucleotide, adenosine 3',5' cyclic monophosphate (cAMP), or the activities of protein kinase C and A, and intracellular free Ca2+ concentration ([Ca2+]) will be specifically manipulated in vitro. The hypotheses to be tested are: 1) Agonist-induced changes of intracellular second messengers modulate endothelial morphology leading to the discrete Solute Permeability Mechanism: Restricted Diffusion or Convection and 2) Different mediator induced distinct cellular responses by discrete spatiotemporal alterations in [Ca2+] of cytoskeleton protein will be assessed in specific aims 1-3. Specific aim 4 will test the Crone Hypothesis. These aims will be tested using bovine lung endothelial cells from two distinct locations: large vessel (pulmonary artery endothelial cells, AEC) and macrovessel (MEC). Monolayers of AEC and MEC will be used as cellular models of the lung microvascular barrier to solutes. The following assays will be used: 1) The solute permeability mechanisms that define the barrier properties of endothelial monolayer will be measured for each experimental condition, 2) Endothelial surface area will be measured from differential interference contrast (DIC) digital images using Image-1 software (universal Imaging Corp), 3) Agonist-induced spatiotemporal alterations of intracellular [Ca2+] will be measured by fura-2 fluorescence digital images. 4) Agent-induced rearrangement of endothelial F-actin/myosin II will be measure by fluorescence digital images in living cells, 5) Electron microscopic analysis by rotary shadowing of the endothelial cytoskeleton will be performed, 6) Intracellular [cAMP] and [cGMP] will be measured by radioimmunoassay, 7) Protein kinase activities will be measured by P-labelled immunoaffinity protein SDS polyacrylamide gel electrophoresis.

肺内皮渗透性持续增加是一种 成人呼吸窘迫综合征的标志。 而且， 炎症中介体引起的区域/单位路径长度增加 （Arho/ax）内皮连接空间的基础 微血管壁的溶质渗透率升高。 长期 这项研究的目标是更好地了解内皮 调节肺微血管通透性的机制 大分子。 循环核苷酸，腺苷的细胞内水平 3'，5'环状单磷酸（CAMP）或蛋白激酶的活性 C和A，以及细胞内游离Ca2+浓度（[Ca2+]）将为 在体外特异性操纵。 要测试的假设是：1） 激动剂引起的细胞内第二使者的变化调节 内皮形态导致离散溶质渗透性 机制：限制扩散或对流和2）不同的中介者 通过离散时空引起的独特的细胞反应 将评估[Ca2+]的[Ca2+]的变化。 具体目标1-3。 特定的目标4将检验Crone假设。 这些 目标将使用来自两个不同的牛肺内皮细胞进行测试 位置：大血管（肺动脉内皮细胞，AEC）和 Macrovessel（MEC）。 AEC和MEC的单层将用作细胞 肺微血管屏障的模型。 下列 将使用测定：1）定义的溶质渗透率机制 将测量每个内皮单层的屏障特性 实验条件，2）将从 使用Image-1的差分干扰对比度（DIC）数字图像 软件（通用成像公司），3）激动剂引起的时空 细胞内[Ca2+]的改变将通过Fura-2测量 荧光数字图像。 4）代理引起的重排 内皮F-肌动蛋白/肌球蛋白II将通过荧光数字测量 活细胞中的图像，5）旋转的电子显微镜分析 将执行内皮细胞骨架的阴影，6） 细胞内[CAMP]和[CGMP]将通过放射免疫测定法测量7） 蛋白激酶活性将通过p标记的免疫亲和力测量 蛋白SDS聚丙烯酰胺凝胶电泳。