Fig. 1.

Lineage tracing analysis of lung EC injury induced by endotoxemia and kinetics of regeneration. a We carried out studies to establish a model of endotoxemia (LPS) induced EC injury in lungs followed by progressive recovery of endothelium. Studies were made in mTmG double fluorescent lineage tracing mice using endothelial-enhanced Scl-CreERT2. After tamoxifen induction, Cre recombinase in ECs translocates to the nucleus and induces EGFP expression. b 2 photon imaging and 3D surface reconstruction of lungs from mTmG-Scl mice at baseline and days post-LPS-induced vascular injury (LPS is given i.p. at a dose of sub-lethal 12 mg/kg i.p.). Red indicates non-ECs and green indicates ECs. Scale bar = 20 μm. n = 3. Same lung sample in 3D structure movie were shown in Videos S1 and S2 for baseline and post-injury day 1. c Quantification of the surface area of EGFP⁺ (EGFP positive) cells relative to total surface areas (EGFP⁺ TdTomato). EGFP⁺ cell surface area, reflecting EC population, is 50% of total lung cell population at baseline. The EC population significantly decreases at post-LPS day 1 and progressively recovers by day 4. n = 3. d Flow cytometry analysis of ECs shown as percent of CD31⁺CD45⁻ (CD31 positive, CD45 negative) cells. n = 4. In the non-leukocyte fraction (exclusion of CD45⁺ cells), CD31⁺ ECs, were significantly reduced within first the 24 h post-LPS-induced injury and then gradually recovered within 7 days. e Flow cytometry analysis of EGFP⁺ cells percentage among whole lung population. n = 5. Similar to d, EGFP⁺ cells also showed a marked loss of ECs induced by LPS within the first 24 h and then a full restored population by day 7 post-LPS. *P < 0.05 and ***P < 0.001 versus day 0. Data are shown as mean ± SEM. Analysis was performed using one-way ANOVA