mTert induction in p21-positive cells counteracts capillary rarefaction and pulmonary emphysema

Lung diseases develop when telomeres are shortened beyond a critical point. We have constructed a mouse model in which the catalytic subunit of telomerase (mTert), or its catalytically inactive form (mTertCI), is expressed from the p21Cdkn1a promoter. We found that this particular expression of mTert reduces senescence of endothelial cells (EC) in lungs of aged mice, as well as emphysema and pulmonary perivascular fibrosis. We also show that mTert counteracts the decline in capillary density in aged mice and promotes the maintenance of high numbers of Cd34+ cells, identified as a subclass of endothelial cells with proliferative capacity. In line with these results, young p21+/Tert mice treated with a VEGF receptor inhibitor combined with hypoxia are also protected against senescence and emphysema induced by this treatment. The catalytic activity of mTert is required for all the effects observed. However, and unexpectedly, we found that both mTert and mTertCI expression significantly reduced p21 levels in the lungs of aged mice. mTert thus protects against age-related and induced loss of capillary vessels and subsequent lung emphysema. Overall design: Droplet-based single-cell RNA-sequencing on 3 samples of total cells from mouse lung tissue. Three genotypes were simultaneously analyzed. Single-cell 3'-RNA-Seq samples were prepared using single cell V2 reagent kit and loaded in the Chromium controller according to standard manufacturer protocol (10x Genomics, PN-120237) to capture 6.000 cells. Briefly, dissociated lung cells are encapsulated using microfluidic device. RNAs are captured on beads coated of oligos containing an oligo-dTTT, UMIs and a specific barcode. After reverse transcription, cDNAs are washed, PCR-amplified and washed again before analysis on a Bioanalyzer (Agilent) for quality control. Finally, libraries are prepared following standard Illumina protocol and sequenced on a NovaSeq sequencer (Illumina). Raw sequences are demultiplexed and reads are mapped onto the mm10 reference genome using the v2.3 Cell Ranger pipeline (10X Genomics) to generate a count matrix for each sample. The digital matrices were filtered by cell type (on clusters composed of the same cell type), to remove low-quality cells with low UMI counts and cells with relatively high mitochondrial DNA content. Outlier analysis was performed with perCellQCMetrics from the scatter package. An upper cutoff was manually determined for each sample based on a plot of gene count versus UMI count or % of mito-chondrial genes, to have at least 1000 UMIs, number of transcripts ranging between 1000 and 30000 and at most 14% mitochondrial transcripts. The quality was consistent across samples, and differences in RNA and gene content could be ascribed to cell-type-specific effects. DGE matrices from all sam-ples, sequenced at different time, were then merged and subsequently normalized using the deconvo-lution normalization method in the scran R package in order to correct for differences in read depth and library size inside and between samples.

当端粒（telomere）缩短至临界阈值以下时，便会引发肺部疾病。本研究构建了一款小鼠模型，该模型可通过p21Cdkn1a启动子介导端粒酶催化亚基（telomerase catalytic subunit, mTert）或其催化失活变体（catalytically inactive form, mTertCI）的表达。研究发现，该特异性表达的mTert可延缓老年小鼠肺组织内皮细胞（endothelial cell, EC）的衰老，同时减轻肺气肿（emphysema）与肺血管周围纤维化（pulmonary perivascular fibrosis）病变。本研究同时证实，mTert能够抵消老年小鼠肺毛细血管密度（capillary density）的下降，并促进维持高数量的CD34阳性细胞（Cd34+ cells）——这类细胞被鉴定为具有增殖能力的内皮细胞亚群。与上述结果一致，经血管内皮生长因子（vascular endothelial growth factor, VEGF）受体抑制剂联合低氧（hypoxia）处理的年轻p21+/Tert小鼠，同样可免受该处理诱导的细胞衰老与肺气肿损伤。本研究观测到的所有保护效应，均依赖于mTert的催化活性。但令人意外的是，本研究同时发现，在老年小鼠肺组织中，mTert与mTertCI的表达均可显著降低p21的表达水平。由此可见，mTert可抵御衰老相关及诱导性的毛细血管丢失，并阻止后续肺气肿的发生。

实验整体设计：针对小鼠肺组织总细胞的3份样本进行液滴法单细胞RNA测序（droplet-based single-cell RNA-sequencing），同时分析3种不同基因型（genotype）的样本。单细胞3'-RNA测序（single-cell 3'-RNA-Seq）文库的制备采用单细胞V2试剂盒，并依照厂商标准操作流程（10x Genomics，货号PN-120237）在Chromium控制器上进行上机操作，以捕获约6000个细胞。简言之，解离后的肺细胞通过微流控装置（microfluidic device）进行包裹；RNA会被包被有寡核苷酸链的磁珠（beads）捕获，该寡核苷酸链包含oligo-dT序列、唯一分子标识符（unique molecular identifier, UMI）以及特异性条形码（specific barcode）。逆转录（reverse transcription）完成后，对cDNA（complementary DNA, cDNA）进行洗涤、聚合酶链式反应（polymerase chain reaction, PCR）扩增，随后再次洗涤，之后使用生物分析仪（Agilent Bioanalyzer）进行质量检测。最终，依照Illumina标准流程制备测序文库，并在NovaSeq测序仪（NovaSeq sequencer, Illumina）上完成测序。

原始测序序列经解多重（demultiplexing）处理后，使用v2.3版本的Cell Ranger分析流程（10x Genomics）将reads比对至mm10参考基因组（mm10 reference genome），为每份样本生成基因计数矩阵（count matrix）。随后基于细胞类型对数字表达矩阵进行过滤：以同类型细胞组成的聚类为单位，剔除UMI计数偏低、线粒体DNA（mitochondrial DNA）占比过高的低质量细胞。使用scatter软件包中的perCellQCMetrics工具进行异常值分析（outlier analysis）。针对每份样本，基于基因计数与UMI计数的散点图、线粒体基因占比图手动确定上限阈值，最终保留至少1000个UMI、转录本数量介于1000至30000之间、线粒体转录本占比不超过14%的细胞。所有样本的质量均保持一致，RNA与基因含量的差异可归因于细胞类型特异性的表达特征。将不同测序时间点获取的所有样本的差异基因表达（differential gene expression, DGE）矩阵进行合并，随后使用scran R包中的反卷积归一化方法进行归一化处理，以校正样本内部及样本间的测序深度与文库大小差异。