Effect of Tsc2 depletion on gene expression in isolated mouse lung cells.

To evaluate the impact of mTOR activation by Tsc2KD in MVPC on the microvascular endothelial cell population we utilized single cell transcriptomic analysis of fractionated lung tissue. Mouse lung samples were pooled and cell sorting was 2 days and 10 weeks post tamoxifen induction. Following annotation of the original 28 lung subclusters, capillary MVEC were localized to 7 subclusters. Temporal comparison of the MVEC populations demonstrated that in WT the population was transcriptionally stable from 2 days to 10 weeks postinduction, as would be expected during angiostasis. In contrast lung MVEC from the mice with mTOR+ Tsc2KD MVPC demonstrated significant increases in gene expression at 10 weeks relative to day 2 post-induction. Increased gene expression was related to mesenchymal stem cell differentiation, microvascular endothelial differentiation, mTOR activation, autophagy and apoptosis. These detailed complementary analyses identify mechanistic consequences of Tsc2KD and mTOR dysregulation in MVPC. To investigate how tsc2 knockdown in adult mouse lung ABCG2 positive microvascular progenitor cells we induced 2 (mixed background C57bl6/B129 strains) WILDTYPE or ABCG2 CREERT2 x floxed Tsc2 (Tsc2 knockdown) adult FEMALE mice 10-12 weeks of age with a single low dose of tamoxifen (0.5mg/18g) and isolated cells 2 days or 10 weeks later. Lungs were digested to form a single cell suspension and stained with CD45 antibody. Cells were sorted from WT and Tsc2KD strains for analysis as follows: 2 day cd45negative GFP negative, CD45 negative GFP positive and CD45 positive; 10 weeks cd45negative GFP negative, CD45 negative GFP positive .