Hyperactive mTORC1 in Lung Mesenchyme Induces Endothelial Cell Dysfunction and Pulmonary Vascular Remodeling

Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease caused by tuberous sclerosis complex 1/2 (TSC1/2) gene mutations resulting in activation of the mechanistic target of rapamycin complex 1 (mTORC1). A subset of LAM patients develops pulmonary vascular remodeling and pulmonary hypertension. To model LAM disease, we utilized an mTORC1 gain-of-function mouse model with a Tsc2 knock-out (Tsc2KO) specific to lung mesenchyme (Tbx4LME-CreTsc2fl/fl), similar to the mesenchyme specific genetic alterations seen in human disease. As early as 8 weeks of age, ECs from Tbx4LME-CreTsc2fl/fl mice exhibited marked transcriptomic changes despite absence of morphological changes to the distal lung microvasculature. In contrast, 1 year old Tbx4LME-CreTsc2fl/fl mice spontaneously developed pulmonary vascular remodeling with increased medial thickness. We subsequently performed single cell RNA-sequencing of 1 year old mouse lung and identified paracrine ligands originating from Tsc2KO mesenchyme impacting arterial endothelial cells. These cells contained transcriptionally altered genes including those in pathways associated with blood vessel remodeling, highlighting the pathogenic importance of the mesenchymal-endothelial cell axis. Whole murine lung single cells suspension in one year Tbx4Tsc2 WT and KO; samples were CD45 depleted with 10% spiked back into CD45- fractions and then loaded on 10x genomic platform for single cell RNA sequencing淋巴管肌瘤病（Lymphangioleiomyomatosis, LAM）是一种进展性囊性肺疾病，由结节性硬化症复合体1/2（TSC1/2）基因突变引发雷帕霉素机制靶标复合体1（mTORC1）激活所导致。部分LAM患者会出现肺血管重构与肺动脉高压。为构建LAM疾病模型，我们采用了肺间质特异性Tsc2基因敲除（Tsc2KO）的mTORC1功能获得性小鼠模型（Tbx4LME-CreTsc2fl/fl），该模型可模拟人类疾病中所见的间质特异性遗传改变。早在8周龄时，尽管远端肺微血管未出现形态学改变，Tbx4LME-CreTsc2fl/fl小鼠的内皮细胞已呈现显著的转录组学变化。与之相反，12月龄的Tbx4LME-CreTsc2fl/fl小鼠会自发出现肺血管重构，伴随中膜厚度增加。后续我们对12月龄小鼠的肺组织开展了单细胞RNA测序（single cell RNA-sequencing），鉴定出源自Tsc2KO间质细胞的旁分泌配体，此类配体可作用于动脉内皮细胞。这些细胞存在转录水平改变的基因，其中涵盖与血管重构相关的通路，这凸显了间质-内皮细胞轴（mesenchymal-endothelial cell axis）在疾病致病过程中的关键作用。本研究的样本为12月龄Tbx4Tsc2野生型（Wild Type, WT）与敲除型（Knock Out, KO）小鼠的全肺单细胞悬液；样本先经CD45细胞耗竭（CD45 depleted）处理，随后将10%的细胞回掺至CD45阴性组分中，最后通过10x Genomics平台完成单细胞RNA测序。