MECHANICAL INJURY TO LUNG ENDOTHELIUM

肺内皮机械损伤

• 批准号: 6233721
• 负责人: JAMES Courtney PARKER
• 金额: $ 24.02万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2001-09-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6233721
• 关键词: CHO cells; calcium flux; cytokine; hemodynamics; inflammation; laboratory rat; lung injury; mechanical stress; membrane channels; transfection; vascular endothelium; vascular endothelium permeability; voltage /patch clamp

DESCRIPTION (Applicant's Abstract): Mechanical injury to the lung by high airway and vascular pressures cause increased vascular permeability and fluid extravasation in a number of disease states, but the segmental localization of vascular injury and cellular pathways are unknown. However, mechanical strain induced Ca2+ entry through stretch activated cation channels (SACC) appears to initiate endothelial cell (EC) retraction and adhesion release leading to vascular leak. We propose to establish SACC involvement in mechanical lung injury using three novel approaches with vertically integrated studies at the single channel, endothelial monolayer and intact lung levels. 1) Alveolar and extra-alveolar segmental permeabilities in isolated lungs will be assessed using segmental filtration coefficients (Kf) of arterial, venous an microvascular segments and electron microscopy under conditions which differentially alter segmental wall stresses. 2) Whole-cell voltage clamp and single-channel cell-attached patch clamp measurements of Ca2+ and monovalent cation entry currents evoked by cell deformation will be compared in cultured endothelial cells from rat pulmonary artery, vein and microvascular segments and Chinese Hamster Ovary (CHO) cells with and without transfection and expression of MID 1, a SACC gene product recently cloned from the yeast, Saccharomyces cerevisiae. 3) Pressure deformation induced increases in hydraulic conductance (Lp), decreases in protein reflection coefficient and changes in equivalent pore distribution in monolayers of the three EC phenotypes will be measured with and without expression of the MID1 gene product to determine the role of SACC in initiating the increased permeability as wet as certain cellular mechanism involved in the increase. Finally, the modulation of strain induced Ca2+ entry and permeability by cyclic nucleotides, cytoskeletal tone, cytokines and oxidants will be determined in the three experimental models to determine if polymodal gating and cellular feed back regulated SACC Ca2+ entry. At the end of this research we will unequivocally establish that SACC are critical for initiating strain induced vascular permeability increases and whether polymodal gating by cellular pathways and inflammatory mediators modulate this response to increase susceptibility to mechanical injury of patients with lung disease.

描述(申请人摘要)：高位致肺机械性损伤 呼吸道和血管压力导致血管通透性和液体增加 在许多疾病状态下的渗出，但节段性定位 血管损伤和细胞通路尚不清楚。然而，机械应变 通过拉伸激活的阳离子通道(SACC)诱导的钙内流似乎 启动内皮细胞(EC)回缩和黏附释放，导致 血管渗漏。我们建议建立SACC参与机械性肺 使用三种新的方法进行垂直整合的损伤研究 单通道、内皮细胞单层和完整的肺水平。1)牙槽骨和牙槽骨 将评估离体肺的肺泡外节段通透性。 利用动、静脉血管节段滤过系数(KF) 微血管节段和电子显微镜条件下的 以不同的方式改变分段墙应力。2)全电池电压钳位 单通道细胞附着式膜片钳测量钙离子和单价钙离子 由细胞变形引起的阳离子进入电流将在培养的 大鼠肺动、静脉和微血管段内皮细胞 和中国仓鼠卵巢(CHO)细胞经和未经转基因和 新近从酵母中克隆的SACC基因产物MID1的表达 酿酒酵母。3)压力变形引起的增加 水力传导性(Lp)，蛋白质反射系数和 三种EC单层中等效孔分布的变化 表型将在有和没有MID1基因表达的情况下进行测量 产品以确定SACC在启动通透性增加中的作用 由于某些细胞机制参与了增加，所以湿润。最后， 环核苷酸对应变诱导的钙离子内流和通透性的调节 细胞骨架张力、细胞因子和氧化剂将在这三个方面进行测定 确定多模式门控和细胞反馈的实验模型 调节SACC钙离子内流。在本研究结束时，我们将毫不含糊地 确定SACC在启动应变诱导血管中起关键作用 通透性增加以及通过细胞通路和 炎症介质调节这一反应以增加易感性 肺部疾病患者的机械损伤。