Mechanical stress regulation of lung growth & remodeling

肺高压和肺泡膜切除术是导致机械应激刺激肺细胞增殖和细胞外基质产生的肺部疾病的例子。在目前的研究中，钙周期蛋白基因的表达被认为是传递新细胞分裂信号的机械转导途径中的一个重要事件。更具体地说，钙周期蛋白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。为了验证这一假说，我们将使用机械应变增加的隔离灌流肺模型来研究钙周期蛋白和相关的肺S100基因表达的水平和位置与肺细胞增殖的关系。此外，还将检测因机械骨骼组织和细胞形状而刺激增殖的成纤维细胞中钙周期蛋白和S100基因的表达。通过测量细胞骨架亚细胞部分中的钙周期蛋白水平以及与原肌球蛋白的相关性，将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自分泌活性将被测试为该途径中可能的调控步骤。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)将测量在高或低肺充气状态下换气的隔离灌流大鼠肺组织中总的前胶原和原弹性蛋白的合成2)将测量在ECM蛋白(纤维连接蛋白、弹性蛋白、层粘连蛋白)上培养的机械应变的成纤维细胞A1前胶原和原弹性蛋白的基因表达(3)将通过破坏微丝、微管或中间丝来分析细胞形态和细胞骨架组织的作用，估计ECM与细胞接触的位置的数量，以及鉴定细胞表面存在的ECM受体(整合素)的丰度和特异性。特定的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多肽来确定。4)最后，我们将在我们的机械应变肺模型中测试ECM多肽配体下调应变诱导的前胶原和原弹性蛋白表达的能力。这项研究将进一步深入了解在我们综合呼吸生理学部门目前正在研究的几个生理模型中观察到的可能的应变诱导的增殖和细胞外基质重塑。

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.