Airway and Lung Vascular Remodeling in COPD

描述(由申请人提供)： 退伍军人的烟草使用和慢性阻塞性肺疾病(COPD)患病率显著高于非退伍军人。气道和肺血管重构是COPD发病机制中最关键的过程。COPD患者活化的炎症细胞和其他肺细胞或烟草烟雾本身释放出大量的活性氧簇(ROS)。ROS参与了呼吸道和肺血管重塑的发生。然而，关于ROS和TS是如何导致这些病理改变的，人们知之甚少。我们发现ROS和TS激活了支气管和肺动脉平滑肌细胞(BSMCs和PASMCs)中的Calain，并介导了PASMCs中细胞的增殖和I型胶原的合成。钙蛋白酶是一类钙依赖的非溶酶体中性半胱氨酸内切酶家族，在哺乳动物细胞中通过对底物蛋白的有限降解发挥作用，包括BSMCs和PASMCs。我们最近首次报道，全局抑制钙蛋白酶可以防止慢性缺氧和野百合碱引起的肺血管重构。在初步实验中，我们发现特异性的Calain抑制剂MDL28170可以阻止ROS诱导的BSMCs和PASMCs细胞增殖和I型胶原蛋白水平的增加。转化生长因子(TGF)/Smad通路在气道和肺血管重构中发挥重要作用。我们已经报道，calain通过裂解潜伏的TGF1来激活TGF1。本研究旨在研究一种新的假说，即在TS诱导的COPD模型中，Calain通过激活细胞内TGF1来介导ROS和TS诱导的气道和肺血管平滑肌细胞的增殖和胶原合成。在加入或不加入H_2O_2和TS的情况下，分别测定BSMCs和PASMCs中钙蛋白酶的蛋白和活性、细胞增殖和胶原合成。钙蛋白酶的活性将通过特定的抑制物、siRNA和过度表达的calastatin基因来操纵。在过氧化氢和TS处理的细胞中，将研究一种新的内分泌TGF1途径。此外，在COPD动物模型中，将使用创新的可诱导的全局和平滑肌特异性Calain基因敲除小鼠系来评估calain在呼吸道和肺血管重建中的作用。最后，在TS诱导的COPD大鼠模型中，将使用特定的Calain抑制剂MDL28170进行药理学靶向治疗。这一建议是新颖的，因为它将确定Calain是呼吸道和肺血管重构的媒介，而Calain通过激活BSMCs和PASMCs的细胞内TGF1来发挥这一作用。更好地理解这一机制，将为在COPD、肺动脉高压和肺心病等肺部疾病的治疗中操纵钙蛋白酶活性提供强有力的理论基础。该项目的完成将有助于找到治疗慢性阻塞性肺疾病的新方法，进而改善退伍军人的生活和护理。

DESCRIPTION (provided by applicant): Tobacco use and prevalence of chronic obstructive pulmonary disease (COPD) are significantly higher among veterans than non-veteran population. Airway and pulmonary vascular remodeling is the most critical process in the pathogenesis of COPD. Large amounts of reactive oxygen species (ROS) are released from activated inflammatory cells and other lung cells in COPD or from tobacco smoke (TS) per se. ROS are implicated in the development of airway and pulmonary vascular remodeling. However, little has been known about how ROS and TS lead to these pathological alterations. We have found that ROS and TS activates calpain in bronchial and pulmonary artery smooth muscle cells (BSMCs and PASMCs) and that calpain mediates cell proliferation and collagen-I synthesis in PASMCs. Calpain is a family of calcium-dependent non- lysosomal neutral cysteine endopeptidases that act via limited proteolysis of substrate proteins in mammalian cells, including BSMCs and PASMCs. We have recently reported for the first time that global inhibition of calpain prevents pulmonary vascular remodeling induced by chronic hypoxia and monocrotaline. In preliminary experiments, we found that the specific calpain inhibitor, MDL28170, prevented ROS-induced increases in cell proliferation and protein levels of collagen-I in BSMCs and PASMCs. Transforming growth factor (TGF)/Smad pathway have been shown to play important roles in airway and pulmonary vascular remodeling. We have reported that calpain activates TGF1 by cleaving latent TGF1. This proposal is to study a novel hypothesis that calpain mediates ROS- and TS-induced cell proliferation and collagen synthesis in airway and pulmonary vascular smooth muscle cells via activation of intracellular TGF1 in TS-induced COPD. The protein and activity of calpain, cell proliferation and collagen synthesis will be determined in BSMCs and PASMCs with or without treatment of H2O2 and TS. Calpain activity will be manipulated using specific inhibitor, siRNA and over-expression of calpastatin genes. A novel intracrine TGF1 pathway will be studied in H2O2- and TS-treated cells. Moreover, the role of calpain in airway and pulmonary vascular remodeling will be evaluated in a COPD animal model using innovative inducible global and smooth muscle-specific calpain knockout mouse line. Finally, calpain will be pharmacologically targeted using a specific calpain inhibitor MDL28170 in a TS-induced COPD rat model. This proposal is novel because it will identify calpain as a mediator in airway and pulmonary vascular remodeling and calpain serves this role by activating intracellular TGF1 in BSMCs and PASMCs. A better understanding of the mechanistic insight will provide a strong rationale for manipulating calpain activities in the treatment of lung diseases such as COPD, pulmonary hypertension and cor pulmonale. Completion of this project will help find novel treatments for COPD and in turn improve the life and care of veterans.