Fig. 3.

EC specific deletion of Sox17 (Sox17^EC−/−) in mice prevents endothelial regeneration. a Schematic diagram of Sox17^fl/fl mice crossed with Scl-CreERT2 to delete Sox17 in ECs. After tamoxifen feeding for five days and rest for another four weeks, mice are challenged with LPS (sub-lethal 8 mg/kg, i.p.) for analysis. b Western blot analysis of Sox17 protein expression in isolated ECs obtained from flushed lungs of Sox17^EC−/− and control mice. n = 4. c Quantification of b shows 80% deletion of Sox17 in ECs of Sox17^EC−/− mice compared to control mice. n = 4. d Time course of lung transvascular permeability following LPS challenge in Sox17^EC−/− and Sox17^fl/fl mice. n = 4. While control mouse lungs showed increased endothelial permeability at day 1 post-LPS and then recovered to baseline by day 5, Sox17^EC−/− mice showed prolonged endothelial barrier leakiness post-LPS. e Time course of changes in lung transvascular permeability following LPS challenge was also carried out in Sox17^fl/fl mice crossed with Cdh5-CreERT2 mice. n = 4. Similar as in d, these Sox17^EC−/− mice also showed persistent leakiness post-LPS while control mice fully recovered. f Survival curve of LPS challenge in Sox17^EC−/− and control mice. n = 8 per group. At this sub-lethal dose, all control mice survived whereas half of Sox17^EC−/− mice died on day 2 post-LPS with increased mortality on day 3. By day 5, the death rate for control mice is 0 while for Sox17^EC−/− mice is 60%. g Flow cytometry analysis of CD31⁺CD45⁻ ECs among whole lung population in mice following injury. n = 4. In contrast to control mice in which CD31⁺CD45⁻ EC population gradually recovered with day 3 post-LPS after initial loss of ECs, Sox17^EC−/− mice showed significantly delayed restoration of ECs post-LPS period. h Quantification of BrdU+ nuclei in each field of 425 μm² area in flushed lung cryo-sections from mice following injury. n = 4 mice per group and 6 replicates per sample. Slides are co-stained with CD31-AF488, BrdU-APC, and DAPI. At day 3 post-LPS, the control group showed a significantly higher number of BrdU+ECs compared to baseline. However, Sox17^EC−/− mice showed markedly reduced level of BrdU+ECs, indicating reduced EC proliferation. i To assess whether expression of Sox17 in ECs can restore lung endothelial integrity, studies were performed in Sox17^EC−/− mice to overexpress Sox17 protein. We used a mixture of 50 μg plasmid (mouse Cdh5 promoter—Flag —Sox17) encapsulated in liposomes, which were injected i.v. 3 h after LPS challenge. j At day 3 post-LPS, liposome vector-treated Sox17^EC−/− mice showed marked EC barrier leakiness as assessed by lung transvascular permeability of albumin whereas the Sox17-rescued mice showed markedly reduced endothelial permeability. n = 4. OE, overexpression. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using two-tailed Student’s t-test for (c, j), two-way ANOVA with Bonferroni post-tests for (d, e, g, h) and Log-rank (Mantel-Cox) test for (f)