Autotaxin Lysophophatidic acid pathway in bronchiolitis obliterans post-lung tran

描述（由申请方提供）：继发于纤维化瘢痕形成和小气道闭塞的肺功能受损，称为闭塞性细支气管炎综合征（BOS），是肺移植后慢性移植物衰竭和死亡的主要原因。了解肺移植纤维化的机制途径是这一竞技场新的治疗方法的关键。来自人肺同种异体移植物的间充质细胞（MC）的研究使我们靶向MC动员和活化的新信号通路。在BOS患者的支气管肺泡灌洗（BAL）中观察到供体来源的MC数量增加。来自BOS肺的MC还表现出稳定的活化表型，其特征在于胶原蛋白和连环蛋白表达增加以及自分泌运动因子（ATX）分泌活性增加。ATX是一种分泌型溶血磷脂酶D，其从膜脂质产生溶血磷脂酸（LPA），一种在组织纤维化中起作用的生物活性脂质。我们已发表的工作揭示了一个独特的信号级联反应，其中通过LPA 1连接和随后的磷酸激酶-C介导的糖原合成酶激酶-3激活，LPA导致<$-连环蛋白稳定和转录激活。新的初步数据表明，自分泌ATX分泌和LPA 1连接有助于从人纤维化肺移植物分离的MC中观察到的稳定的β-连环蛋白和胶原上调。此外，注意到BOS患者的BAL样本中LPA水平较高。这些人类数据使我们提出了研究ATX-LPA-β-连环蛋白途径在BO发病机制中的体内作用的实验，并研究LPA 1拮抗作用是否可以成为这种疾病的潜在治疗选择。为了实现这些目标，我们建立了全肺小鼠原位肺移植模型，其中中度MHC错配导致第28天发生气道纤维化。在目的1中，我们建议使用该小鼠模型研究ATX和LPA在体内同种异体移植物的驻留MC和其他细胞组分中上调的时间过程。我们将利用BAC荧光标记Foxf 1的小鼠，Foxf 1是一种在胚胎肺间充质中观察到的转录因子，在肺MC中高度表达，以特异性地识别和研究移植驻留MC。这些研究也将告知我们移植物驻留MCs在纤维化中的作用。目的2将集中于了解<$-连环蛋白在MC分化中的作用，并剖析其影响细胞纤维化功能的机制。机制途径将在人类细胞中进行研究。然而，我们将利用报告小鼠（Axin 2LacZ）来跟踪同种异体移植物纤维化过程中的<$-连环蛋白转录活性，并测试LPA 1连接在MC <$-连环蛋白激活中的体内作用。在目标3中，我们将确定BAL样本中的LPA水平是否可以预测BOS的后续发展。重要的是，我们将测试LPA 1拮抗剂是否可以在小鼠原位单肺移植模型中预防同种异体移植物纤维化的发生或进展。拟议的工作将是BOS中ATX/LPA/LPA 1/<$-catenin信号传导的首次研究，并将为未来针对肺移植中该途径的临床试验提供重要的理论基础。

DESCRIPTION (provided by applicant): Impaired lung function secondary to fibrotic scarring and obliteration of small airways, termed bronchiolitis obliterans syndrome (BOS), is the major cause of chronic graft failure and mortality following lung transplantation. Understanding mechanistic pathways involved in lung allograft fibrogenesis is the key to novel therapeutic approaches in this arena. Studies of mesenchymal cells (MCs) from human lung allografts have led us to target a novel signaling pathway of MC mobilization and activation. An increase in number of donor-derived MCs is seen in the bronchoalveolar lavage (BAL) of patients with BOS. MCs from BOS lungs also demonstrated a stable activated phenotype marked by an increased collagen and ¿-catenin protein expression as well as an increased autotaxin (ATX) secretory activity. ATX is a secreted lysophopholipase D which generates lysophosphatidic acid (LPA), a bioactive lipid with a role in tissue fibrosis, from membrane lipids. Our published work has revealed a unique signaling cascade where through LPA1 ligation and subsequent phosphokinase-C mediated glycogen synthase kinase-3¿ activation, LPA leads to ¿-catenin stabilization and transcriptional activation. New preliminary data demonstrates that autocrine ATX secretion and LPA1 ligation contributes to stable ¿-catenin and collagen upregulation noted in MCs isolated from human fibrotic lung grafts. Furthermore, LPA levels were noted to be higher in BAL samples from patients with BOS. This human data has led us to propose experiments investigating the in vivo role of ATX-LPA-¿-catenin pathway in pathogenesis of BO and to investigate if LPA1 antagonism can be a potential therapeutic option in this disease. To achieve these aims we have established a whole lung mouse orthotopic lung transplant model where a moderate MHC mismatch leads to development of airway fibrosis by day 28. In Aim 1, we propose to investigate the time course of ATX and LPA upregulation in the resident MCs and other cellular components of the allograft in vivo using this murine model. We will utilize mice with BAC florescent labeling of Foxf1, a transcription factor seen in embryonic lung mesenchyme and shown to be highly expressed in lung MCs, to specifically identify and study graft-resident MCs. These studies will also inform us of the role of graft-resident MCs in fibrogenesis. Aim 2 will focus on understanding the role of ¿-catenin in MC differentiation and dissecting the mechanisms by which it affects the cells fibrotic functions. The mechanistic pathways will be studied in human cells. However, we will utilize a reporter mouse (Axin2LacZ) to follow ¿-catenin transcriptional activity during allograft fibrogenesis and test the in vivo rol of LPA1 ligation in MC ¿-catenin activation. In Aim 3 we will determine if LPA levels in BAL samples can predict subsequent development of BOS. Importantly, we will test if LPA1 antagonist can prevent onset or progression of allograft fibrosis in the murine orthotopic single lung transplant model. The proposed work will be the first investigation of ATX/LPA/LPA1/ ¿-catenin signaling in BOS and will provide important rationale for future clinical trials targeting this pathway in lung transplantation.