Mechanical stress regulation of lung growth & remodeling

肺生长的机械应力调节

• 批准号: 6589834
• 负责人: Ellen C Breen
• 金额: $ 24.62万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6589834
• 关键词: calcium binding protein; cell cycle; cell growth regulation; cell proliferation; collagen; elastin; epidermal growth factor; extracellular matrix; extracellular matrix proteins; fibroblasts; gene expression; genetic regulatory element; intermediate filaments; isolation perfusion; laboratory rat; lung; lung ischemia /hypoxia; mechanical stress; microfilaments; microtubules; tropomyosin

Pulmonary hypertension and pseumonectomy are examples of pulmonary conditions that implicate mechanical stress as a stimulus for lung cell proliferation and extracellular matrix production. In the present study, calcyclin gene expression is proposed to be an essential event in the mechanotransduction pathway signaling new cell division. More specifically, calcyclin is thought to function as a regulatory protein controlling changes in cell shape and cytoskeletal organization required for the cell cycle to progress. To test this hypothesis an isolated-perfused lung model of increased mechanical strain will be used to correlate the level and location of calcyclin and related lung S100 gene expression with pulmonary cell proliferation. Calcyclin and S100 gene expression will also be measured in fibroblasts stimulated to proliferate in response to mechanical skeletal organization and cell shape. Cytoskeletal involvement will be confirmed by measuring calcyclin levels in the cytoskeletal subcellular fraction and association with tropomyosin. Furthermore, transcriptional activation via cis-acting promoter elements and PDGF autocrine activity will be tested as possible regulatory control steps in this pathway. Pulmonary fibroblasts are also proposed to play an important role in strain-induced ECM remodeling. Therefore, 1) Total pro-collagen and tropoelastin synthesis will be measured in isolated perfused rat lungs ventilated at high or low states of lung inflation 2) The gene expression of a1 (I) pro-collagen and tropoelastin will be measured in mechanically strained fibroblasts cultured on ECM proteins (fibronectin elastin, laminin( 3) The role of cell shape and cytoskeletal organization will be analyzed by disruption of microfilaments, microtubules or intermediate filaments; estimating the number of ECM- cell contact sites; and identifying the abundance and specificity of ECM receptors (integrins) present on the cell surface. The functional role of specific ECM-fibroblast interactions will be determined by inhibition with fibronectin, laminin, or elastin peptides. 4) Finally, the ability of ECM peptide ligands to down regulate strain induced pro-collagen and tropoelastin expression will be tested in our mechanical strain lung model. This research will give further insight into possible strain induced proliferation and ECM remodeling observed in several physiological models currently under investigation in our integrative respiratory physiology division.

肺动脉高压和假单细胞切除术是肺部疾病的例子，涉及机械应力作为肺细胞增殖和细胞外基质生产的刺激。在本研究中，钙调蛋白基因表达被认为是机械转导途径中信号传导新细胞分裂的重要事件。更具体地说，钙调素被认为是一种调节蛋白，控制细胞周期进展所需的细胞形状和细胞骨架组织的变化。为了验证这一假设，将使用机械应变增加的分离灌注肺模型来将钙调素的水平和位置以及相关的肺S100基因表达与肺细胞增殖联系起来。钙调素和S100基因表达也将在受刺激的成纤维细胞中被测量，以响应机械骨骼组织和细胞形状的增殖。通过测量细胞骨架亚细胞部分中的钙调素水平以及与原肌球蛋白的关联，可以证实细胞骨架的参与。此外，通过顺式作用启动子元件的转录激活和PDGF自分泌活性将作为这一途径中可能的调节控制步骤进行测试。肺成纤维细胞也被认为在应变诱导的ECM重塑中发挥重要作用。因此，1)将在高或低肺膨胀状态下通气的离体灌注大鼠肺中测量总前胶原和tropoelastin合成2)将在ECM蛋白（纤维连接蛋白、弹性蛋白、层粘连蛋白）上培养的机械张力成纤维细胞中测量a1 (I)前胶原和tropoelastin的基因表达3)将通过微丝、微管或中间丝的破坏来分析细胞形状和细胞骨架组织的作用；估计ECM细胞接触点的数量；并鉴定存在于细胞表面的ECM受体（整合素）的丰度和特异性。特异性ecm -成纤维细胞相互作用的功能作用将通过抑制纤维连接蛋白、层粘连蛋白或弹性蛋白肽来确定。4)最后，我们将在机械应变肺模型中测试ECM肽配体下调应变诱导的前胶原蛋白和tropoelastin表达的能力。这项研究将进一步深入了解在我们的综合呼吸生理部门正在研究的几种生理模型中观察到的可能的应变诱导增殖和ECM重塑。