Sox17 is required for endothelial regeneration following inflammation-induced vascular injury

Abstract

Repair of the endothelial cell barrier after inflammatory injury is essential for tissue fluid homeostasis and normalizing leukocyte transmigration. However, the mechanisms of endothelial regeneration remain poorly understood. Here we show that the endothelial and hematopoietic developmental transcription factor Sox17 promotes endothelial regeneration in the endotoxemia model of endothelial injury. Genetic lineage tracing studies demonstrate that the native endothelium itself serves as the primary source of endothelial cells repopulating the vessel wall following injury. We identify Sox17 as a key regulator of endothelial cell regeneration using endothelial-specific deletion and overexpression of Sox17. Endotoxemia upregulates Hypoxia inducible factor 1α, which in turn transcriptionally activates Sox17 expression. We observe that Sox17 increases endothelial cell proliferation via upregulation of Cyclin E1. Furthermore, endothelial-specific upregulation of Sox17 in vivo enhances lung endothelial regeneration. We conclude that endotoxemia adaptively activates Sox17 expression to mediate Cyclin E1-dependent endothelial cell regeneration and restore vascular homeostasis.

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

Fig. 2

Activation of Sox17 at onset of EC regeneration and Sox17-mediated Cyclin E1 expression. a qPCR analysis of gene expression in sorted CD31⁺ cells from mTmG-Scl mice before and after injury (12 mg/kg i.p.). Sox17, Vegfr2, and Ccne1 increased significantly at day 2 post-LPS compared to baseline. n = 3. Color scale: the fold change increases from red to white to green color. b Western blot analysis in fresh isolated ECs from wild-type mice and quantification c showed a 5-fold increase in Sox17 protein expression within 1 day following injury compared to baseline and followed by recovery within 3 days post-LPS. n = 3. d, e Western blot analysis of cultured HLMVECs in which Sox17 was overexpressed showed 2.5x fold increase in Cyclin E1 protein expression relative to control cells. n = 3. OE, overexpression. f Representation of the CCNE1 promoter region with Sox17 binding sites (circled numbers) and their sequences. g HLMVECs were retrovirally transduced with Sox17 or control plasmid for 3 days, and Ch-IP assay followed by qPCR was performed to amplify Sox17 binding sites in the CCNE1 promoter. n = 3. h 293T cells were transfected with a Sox17 overexpression plasmid containing CCNE1 luciferase reporter constructs. Luciferase values were normalized to Renilla luciferase control reporter values. A schematic representation of corresponding deletion constructs is presented in the right panel. n = 3 and duplicates per sample. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using one-way ANOVA for (c) and two-way ANOVA with Bonferroni post-tests for (e, g, h)

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)

Fig. 4

soHIF-1α signaling induces Sox17 expression. a MPO activity of flushed lung sample from mice challenged with LPS (12 mg/kg i.p.) for 6 and 24 h. n = 3. b Western blot analysis of wild-type mice lung before and after LPS-induced injury (12 mg/kg i.p.) and its quantification c showed that HIF-1α protein expression increased within 6 h post-LPS and remained increased until day 2. n = 3. d Western blot analysis in freshly isolated ECs from Hif1a^fl/fl mice and quantification e showed significantly increased Sox17 protein expression after injury when compared to baseline levels. n = 3. f Western blot analysis in freshly isolated ECs from Hif1a^EC−/− mice and quantification g showed no significant difference in Sox17 protein expression before and after injury. n = 3. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using one-way ANOVA for (b, c, e, g)

Fig. 5

HIF-1α activates transcription of Sox17. a Western blot analysis in control HLMVECs and HLMVECs for which CRISPR/Cas9 was used to delete HIFs. ECs were treated with the HIF prolyl hydroxylase inhibitor DMOG to induce HIF expression. DMOG (1 mM) increased HIF-1α and HIF-2α protein expression in control ECs but not in ECs lacking HIF-1α or HIF-2α. Induction of HIF expression was coupled to Sox17 upregulation. n = 3. b Quantification of a showed that protein expression of HIF-1α and HIF-2α in ECs was significantly increased by DMOG treatment. Sox17 showed a 2.5-fold increase in Sox17 expression control DMSO treated ECs. The increase in Sox17 was significantly reduced in HIF-1α-deleted ECs but preserved in HIF-2α-deleted ECs, indicating the importance of HIF-1α in mediating Sox17 expression. n = 3. c Representation of the SOX17 promoter region with 3 HREs indicated by circled numbers, and their respective sequences are displayed. d HLMVECs were exposed to normoxia or 1% O2 (hypoxia) for 8 h. Ch-IP assay followed by qPCR was performed to amplify the HRE regions in the SOX17 promoter. Studies were performed in ECs exposed to either normoxia or hypoxia. n = 3. e 293T cells were transfected with a HIF-1α overexpression plasmid containing SOX17 luciferase reporter constructs. Luciferase values were normalized to Renilla luciferase control reporter values. A schematic representation of corresponding deletion constructs is presented in the right panel. n = 3 and duplicates per sample. Results show that hypoxia activation of SOX17 HRE3 was required for Sox17 expression. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using two-way ANOVA with Bonferroni post-tests for (b, d, e)

Fig. 6

Overexpression of Sox17 in ECs induces EC proliferation and regeneration. Mixture of 50 μg plasmid with 100 μl liposomes was injected i.v. 3 h after LPS challenge (12 mg/dose i.p.) in wild-type mice. This plasmid has a Flag-tag added to the N-terminus of Sox17 protein coding region and expression is under the regulation of a mouse Cdh5 promoter. a Confocal microscopy of flag staining with CD31 and DAPI co-staining for nuclei in lung cryo-sections from mice receiving a control vector or a Sox17-construct to over-express Sox17. Scale bar = 50 μm (original panel) and 20 μm (enlarged panel). n = 6. OE, overexpression. b Co-localization coefficient for the fraction of Flag in CD31⁺ cells assesses the transgene expression in the endothelium. The Pearson correlation coefficient is significantly increased in Sox17-overexpressing mice compared to control mice. n = 6. c Western blot analysis and its quantification d showed a significant increase in the flag and Cyclin E1 expression in the pulmonary endothelial cells of mice with 3 days of Sox17 overexpression compared to vector mice. n = 3. e Quantification of BrdU⁺ nuclei in each field of 425 μm² area in lung cryo-sections from vector-overexpressing and Sox17-overexpressing mice. n = 5 per group and 6 technical replicates per sample. Slides are co-stained with CD31-AF594, BrdU-AF488, and DAPI. Both groups show increased BrdU⁺ ECs at day 3 post-LPS as compared to baseline and the response was significantly greater in mice in which ECs overexpressed Sox 17. f Lung transvascular albumin permeability pre-LPS and post-LPS challenge in mice overexpressing endothelial Sox17 and control mice. n = 5. Mice overexpressing Sox17 in ECs showed significantly reduced vascular leakiness post-LPS when compared to control mice. g Survival curve of LPS challenge in control mice and mice over-expressing Sox17 in the endothelium. n = 11 per group. At this lethal dose of LPS (20 mg/kg), the death rate for control mice is 60% while for Sox17-overexpressed mice is 10%. h Model. LPS induces tissue hypoxia due to local oxygen depletion by infiltrating activated neutrophils, thereby stabilizing HIF-1α resulting in upregulation Sox17 expression and Sox17 mediated expression of Cyclin E1. This activates cell cycle re-entry and EC proliferation, and restoration of endothelial integrity. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using two-way ANOVA with Bonferroni post-tests for (d–f) and Log-rank (Mantel-Cox) test for (g)