Airway and Lung Vascular Remodeling in COPD

描述（由申请人提供）： 烟草使用和慢性阻塞性肺疾病（COPD）的患病率显着高于退伍军人比非退伍军人人口。气道和肺血管重构是COPD发病机制中最关键的过程。大量活性氧（ROS）从COPD中活化的炎性细胞和其他肺细胞或从烟草烟雾（TS）本身释放。ROS参与气道和肺血管重塑的发展。然而，很少有人知道ROS和TS如何导致这些病理变化。我们发现ROS和TS激活支气管和肺动脉平滑肌细胞（BSMCs和PASMCs）中的钙蛋白酶，并且钙蛋白酶介导PASMCs中的细胞增殖和胶原-I合成。钙蛋白酶是钙依赖性非溶酶体中性半胱氨酸内肽酶家族，其通过哺乳动物细胞（包括BSMC和PASMC）中底物蛋白的有限蛋白水解起作用。我们最近首次报道了钙蛋白酶的全面抑制可防止慢性缺氧和野百合碱诱导的肺血管重构。在初步的实验中，我们发现，特异性钙蛋白酶抑制剂，MDL 28170，防止ROS诱导的细胞增殖和胶原蛋白-I在BSMCs和PASMCs的蛋白水平的增加。转化生长因子（Transforming growth factor，TGF）/Smad信号通路在气道和肺血管重构中起重要作用。我们已经报道了钙蛋白酶通过切割潜伏的TGF-1激活TGF-1。本研究旨在研究一种新的假说，即在TS诱导的COPD中，钙蛋白酶通过激活细胞内TGF-1介导ROS和TS诱导的气道和肺血管平滑肌细胞增殖和胶原合成。将在有或没有H2 O2和TS处理的BSMCs和PASMCs中测定钙蛋白酶的蛋白和活性、细胞增殖和胶原合成。将使用特异性抑制剂、siRNA和钙蛋白酶抑制蛋白基因的过表达来操纵钙蛋白酶活性。一种新的内分泌TGF 1途径将在过氧化氢和TS处理的细胞进行研究。此外，钙蛋白酶在气道和肺血管重塑中的作用将在COPD动物模型中使用创新的可诱导的整体和平滑肌特异性钙蛋白酶敲除小鼠系进行评估。最后，在TS诱导的COPD大鼠模型中，将使用特异性钙蛋白酶抑制剂MDL 28170靶向钙蛋白酶。该提议是新颖的，因为它将鉴定钙蛋白酶作为气道和肺血管重塑中的介体，并且钙蛋白酶通过激活BSMC和PASMC中的细胞内TGF 1来发挥该作用。更好地理解的机制的见解将提供一个强有力的理由操纵钙蛋白酶活性在肺部疾病，如COPD，肺动脉高压和肺心病的治疗。该项目的完成将有助于找到COPD的新疗法，从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.