Myocardin in the pathogenesis of pleural remodeling

胸膜损伤通常会导致胸膜表面的广泛重塑，在严重的情况下会导致 胸膜纤维化(PF)。肺炎可由细菌性肺炎、创伤、辐射暴露和 与石棉有关的胸膜损伤。严重时，肺泡炎可导致限制性肺部疾病或纤维胸腔。带PF的 肺限制在医疗实践中很常见，但目前的治疗方法并不令人满意和有效 目前还没有药物治疗。PF的特征是α-平滑肌肌动蛋白(α-SMA)的增殖 表达肌成纤维细胞。这些肌成纤维细胞通过增加胸膜厚度促进胸膜增厚。 细胞外基质沉积。肌成纤维细胞的扩张在很大程度上是由于间充质转化(MT)。 常驻胸膜间皮细胞，称为MesoMT。我们的初步数据显示转化生长因子-β诱导 肌钙蛋白的表达和活性。此外，肌钙蛋白及其效应蛋白，平滑肌肌球蛋白， 在我们的小鼠胸膜损伤模型中，Calponin和KIF5A表达上调。相反，心肌梗死- 调节减弱转化生长因子-β介导的系膜MT标志物α-SMA的诱导，胶原和 纤维连接蛋白与胸膜纤维化。我们还报道了KIF5A在转化生长因子-β诱导的PF和危重症中表达上调。 用来分泌胶原蛋白。基于这些观察和强有力的初步数据，我们强烈推断 Myocardin的表达和激活在PF的进展中起重要作用。在这个项目中，我们将 测试中心假设，肌钙蛋白及其效应蛋白的激活和上调是关键 活化的PMC表型获得和PF进展的决定因素。我们的目标是 使用最先进的细胞、生化、分子、生理学和成像技术来测试这一假设。 人类(H)和小鼠(M)原代PMCs将被用来定义MesoMT的调节机制。 将使用三种小鼠模型来评估Myocardin和KIF5信号在PF：Carbon中的作用 布莱克/博莱霉素诱导PF，肺炎链球菌诱导的脓胸/PF，转化生长因子-β诱导的PF。 我们的具体目标是：1)明确Myocardin诱导HPMCs系膜MT的机制；2)明确Myocardin诱导的HPMCs的MesoMT KIF5在MesoMT和HPMCs沉积ECM中的激活机制和3)确定KIF5的贡献 肌钙蛋白及相关效应蛋白对PF体内新基质沉积的影响。我们将使用 新的小鼠纤维化胸膜损伤模型，包括通过间皮标记实现命运映射的小鼠 分析，分子，生化和免疫组织化学技术，我们拥有专业知识和 最先进的CT成像和肺功能分析，以实现这些目标。机制(S)由 目前尚不清楚哪种转化生长因子-β和肌钙蛋白调节MesoMT和Pf，具有潜在的重要意义 我们对胸膜组织的发病机制和寻找新的治疗方法的能力认识上的差距 目标。这项提案解决了每一个差距，并可预见地确定了可能 开发用于改善PF/纤维胸腔积液患者的预后。

Pleural injury often leads to extensive remodeling of the pleural surfaces, which in severe cases leads to pleural fibrosis (PF). PF can occur as a result of bacterial pneumonia, trauma, radiation exposure and asbestos-related pleural injury. When severe, PF can cause restrictive lung disease or fibrothorax. PF with lung restriction is often seen in medical practice but current treatment is unsatisfactory and effective pharmacotherapy is not available. PF is characterized by proliferation of alpha-smooth muscle actin (α-SMA) expressing myofibroblasts. These myofibroblasts contribute to the thickening of the pleura via increased extracellular matrix deposition. The expansion of myofibroblasts is largely due to mesenchymal transition (MT) of resident pleural mesothelial cells, termed MesoMT. Our preliminary data show that TGF-β induces myocardin expression and activity. Further, myocardin and its effector proteins, smooth muscle myosin, calponin, and KIF5A are upregulated in our mouse models of pleural injury. Conversely, myocardin down- regulation attenuates TGF-β mediated induction of MesoMT marker, α-SMA, secretion of collagen and fibronectin and pleural fibrosis. We also reported that KIF5A is upregulated in TGF-β induced PF and critical for collagen secretion. Based on these observations and strong preliminary data, we strongly infer that myocardin expression and activation substantively contribute to the progression of PF. In this project, we will test the central hypothesis that the activation and upregulation of myocardin and its effector proteins are critical determinants in the acquisition of the activated PMC phenotypes and the progression of PF. Our objective is to test this postulate using state of the art cellular, biochemical, molecular, physiologic and imaging techniques. Primary human (H) and mouse (M) PMCs will be used to define mechanisms by which MesoMT is regulated. Three murine models will be used to assess the role of myocardin and KIF5 signaling in PF: carbon black/bleomycin (CBB) induced PF, Streptococcus pneumoniae-induced empyema/PF and TGF-β induced PF. Our specific aims are: 1) To define the mechanism of myocardin-induced MesoMT of HPMCs, 2) to define the mechanism of KIF5 activation in MesoMT and ECM deposition by HPMCs and 3) to define the contribution of myocardin and related effector proteins to neo-matrix deposition in the progression of PF in vivo. We will use new murine models of fibrosing pleural injury, including mice with mesothelial labelling to enable fate-mapping analyses, molecular, biochemical and immunohistochemical techniques with which we have expertise and state of the art CT imaging and pulmonary function analyses to accomplish these aims. The mechanism(s) by which TGF-β and myocardin regulate MesoMT and PF are currently unclear, representing potentially important gaps in our understanding of the pathogenesis of pleural organization and our ability to identify new therapeutic targets. This proposal addresses each of these gaps and predictably will identify novel targets that could be developed to improve outcomes of patients with PF/fibrothorax.