Dissecting mechanisms of alveolar repair failure and lung destruction in lymphangioleiomyomatosis

淋巴管平滑肌瘤病（LAM）是一种罕见的，进行性和无法治愈的疾病，导致呼吸衰竭，淋巴异常和肾脏肿瘤，几乎只影响妇女。LAM的产生是由于控制mTOR的基因功能丧失，mTOR是一种称为LAM细胞的特定细胞类型的关键细胞调节因子。mTOR的持续激活使LAM细胞能够在不利条件下繁殖、存活并侵入肺部和淋巴组织，造成组织损伤。在LAM中，肺部持续受损，导致进行性呼吸困难和肺萎陷，但导致这种肺损伤的机制尚不清楚。肺部持续暴露在环境中的有害化学物质和颗粒中，通常能够在发生低水平损伤时进行修复。然而，由于衰老或疾病，这些修复机制变得不那么有效，导致肺功能受损。在这项工作中，我们将测试衰老的观点，衰老是一个生物学过程，通常会降低在衰老过程中发育的异常细胞的能力，在LAM中由于mTOR激活而过早发生。我们认为衰老的LAM细胞会产生诱导周围细胞衰老的因子，包括通常负责替换受损的肺泡上皮细胞的肺泡II型细胞。因此，mTOR诱导的衰老减少了肺泡修复，导致LAM中的肺损伤。我们已经证明，LAM肺结节和周围肺泡内的细胞表达与衰老相关的多种基因和蛋白质标记。在我们的LAM动物模型中，衰老标志物随着时间的推移而增加，并与进行性肺损伤有关。在这项工作中，我们将系统地研究mTOR激活如何导致LAM衰老。为此，我们将检测LAM中的肺泡上皮细胞基因表达，并使用单细胞RNA测序控制肺，该测序允许测量肺移植时取自LAM肺的单个细胞群的基因表达。为了研究早期LAM，我们还将通过激光捕获显微解剖在诊断活检组织中分离特定细胞群来测量肺泡细胞和LAM结节中的基因表达。通过使用基因表达分析工具，我们将研究LAM中上皮细胞中哪些信号通路是活跃的，以及这些信号通路与细胞死亡和修复的关系。接下来，使用上游调节因子分析（一种预测哪些介质导致特定基因变化的工具），我们将检查来自LAM结节的哪些可溶性蛋白影响上皮细胞修复以及这与mTOR激活的关系。使用40个具有相关临床数据的个体肺活检，我们将确认人体组织中的这些基因变化，重要的是，这些蛋白质如何与疾病持续时间和通过预期肺功能丧失测量的疾病进展相关。一旦我们了解了哪些LAM细胞衍生因子导致肺泡修复减少，我们将使用我们开发的新型3D组织培养系统来研究这些过程是如何被调节的，以及它们如何通过使用药物或基因编辑来阻断单个蛋白质来影响肺泡细胞的体外修复。然后，我们将在实验室开发的LAM小鼠模型中检查这些过程。该模型将使我们能够检查整个生物体中的这些变化，并评估阻断关键蛋白质和过程（包括mTOR激活和衰老）如何影响肺损伤。这些实验将为LAM和其他mTOR激活导致肺损伤的肺部疾病提供新的线索。利用这些数据来了解肺损伤是如何发生的，我们希望改善LAM和潜在的其他破坏性肺部疾病的治疗方法。

Lymphangioleiomyomatosis (LAM) is a rare, progressive and incurable disease causing respiratory failure, lymphatic abnormalities and renal tumours which almost exclusively affects women. LAM arises due to loss of function of genes which control mTOR, a key cellular regulator in a specific cell type called the LAM cell. Constant mTOR activation allows LAM cells to multiply, survive in adverse conditions and invade the lungs and lymphatic tissue to cause tissue damage. In LAM, the lungs are continually damaged leading to progressive breathlessness and lung collapse, but the mechanisms causing this lung damage are not understood. The lungs are continually exposed to harmful chemicals and particles in the environment and are normally able to repair low level damage as it occurs. However due to aging or disease, these repair mechanisms become less efficient resulting in impaired lung function. In this work we will test the idea that senescence, a biological process that normally reduces the capacity of abnormal cells which develop during aging, occurs prematurely in LAM as a result of mTOR activation. We think that senescent LAM cells produce factors that induce senescence in surrounding cells, including alveolar type II cells which are normally responsible for replacing damaged alveolar epithelial cells. This mTOR induced senescence therefore reduces alveolar repair contributing to lung damage in LAM. We have already shown that cells within both LAM lung nodules and the surrounding alveoli express multiple genes and protein markers associated with senescence. In our animal model of LAM, senescence markers increase over time and are associated with progressive lung damage. In this work we will systematically examine how mTOR activation leads to senescence in LAM. To do this we will examine alveolar epithelial cell gene expression in LAM and control lungs using single cell RNA sequencing which allows measurement of gene expression in individual cell populations from LAM lungs taken at lung transplants. To study early LAM we will also measure gene expression in alveolar cells and LAM nodules by laser capture microdissection in diagnostic biopsy tissue to isolate specific cell populations. By using gene expression analysis tools we will examine which signaling pathways are active in epithelial cells in LAM and how these relate to cell death and repair. Next, using upstream regulator analysis, a tool which predicts which mediators result in specific gene changes we will examine which soluble proteins from LAM nodules affect epithelial cell repair and how this is related to mTOR activation. Using 40 individual lung biopsies with linked clinical data we will confirm these gene changes in human tissue and importantly, how these proteins are related to duration of disease and disease progression measured by prospective loss of lung function. Once we have understood which LAM cell derived factors result in reduced alveolar repair we will use novel 3D tissue culture systems we have developed to study how these processes are regulated and how they affect alveolar cell repair in vitro by using drugs or gene editing to block individual proteins. We will then examine these processes in a mouse model of LAM we have developed in our lab. The model will allow us to examine these changes in a whole organism and assess how blocking key proteins and processes, including mTOR activation and senescence affects lung damage. These experiments will shed new light on LAM and other lung diseases where mTOR activation contributes to lung damage. Using these data to understand how lung damage occurs we hope to improve treatments for LAM and potentially other destructive lung diseases.