Dissecting mechanisms of alveolar repair failure and lung destruction in lymphangioleiomyomatosis

淋巴管肌瘤病(LAM)是一种罕见的、进行性的、不可治愈的疾病，可导致呼吸衰竭、淋巴管异常和肾脏肿瘤，几乎只影响女性。LAM的产生是由于控制mTOR的基因功能丧失，mTOR是一种特定细胞类型的关键细胞调节因子，称为LAM细胞。持续的mTOR激活允许LAM细胞增殖，在不利条件下存活，并入侵肺部和淋巴组织，造成组织损伤。在LAM中，肺不断受损，导致进行性呼吸困难和肺衰竭，但导致这种肺损伤的机制尚不清楚。肺部不断暴露在环境中的有害化学物质和颗粒物中，通常能够在发生时修复低水平的损伤。然而，由于衰老或疾病，这些修复机制变得效率较低，导致肺功能受损。在这项工作中，我们将测试衰老的想法，衰老是一种生物过程，通常会减少衰老过程中发育的异常细胞的能力，但由于mTOR的激活，LAM会过早地发生衰老。我们认为衰老的LAM细胞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，这种mTOR诱导的衰老减少了导致LAM肺损伤的肺泡修复。我们已经证明，LAM肺结节和周围肺泡内的细胞都表达与衰老相关的多个基因和蛋白质标记物。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，我们将检测肺泡上皮细胞基因在LAM中的表达，并使用单细胞RNA测序来控制肺，这可以测量肺移植时采集的LAM肺的单个细胞群体的基因表达。为了研究早期LAM，我们还将通过激光捕获显微切割诊断性活检组织来检测肺泡细胞和LAM结节中的基因表达，以分离特定的细胞群。通过使用基因表达分析工具，我们将研究在LAM的上皮细胞中哪些信号通路是活跃的，以及这些信号通路如何与细胞死亡和修复有关。接下来，使用上游调节因子分析，这是一种预测哪些介体导致特定基因变化的工具，我们将检查LAM结节中的哪些可溶性蛋白影响上皮细胞修复，以及这与mTOR激活如何相关。使用40份具有相关临床数据的个体肺活检，我们将确认这些基因在人类组织中的变化，以及重要的是，这些蛋白质如何与疾病持续时间和疾病进展(通过肺功能的预期丧失来衡量)有关。一旦我们了解了哪些LAM细胞来源的因子会导致肺泡修复减少，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调节的，以及它们如何通过使用药物或基因编辑来阻断个别蛋白质来影响体外的肺泡细胞修复。然后，我们将在我们实验室开发的LAM小鼠模型中检查这些过程。该模型将使我们能够在整个生物体中检查这些变化，并评估阻止关键蛋白质和过程，包括mTOR激活和衰老如何影响肺损伤。这些实验将为LAM和其他肺部疾病提供新的线索，在这些疾病中，mTOR激活有助于肺损伤。利用这些数据来了解肺损伤是如何发生的，我们希望改进LAM和其他潜在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.