Fig 2: RT-PCR quantification of Del-1 mRNA expression in various mouse tissues during sepsis. After 24 h of CLP, relative quantification of Del-1 expression shows that CLP significantly decreased Del-1 mRNA expression in adrenal gland, heart, brain, and lung tissue, but not kidney. The data are shown as a ratio of the control, which is set as 1.0. The data are shown as the mean ± SEM (n = 5 mice per experimental group and n = 3 mice per control group). *P < 0.05, Mann-Whitney U test (Student’s t-test in D). CLP: cecal ligation and puncture; Del-1: developmental endothelial locus-1; RT-PCR: real-time polymerase chain reaction.

Fig 3: Sections of lung from CLP and control mice stained for heparan sulfate, Del-1, and glycocalyx. Scale bars, 50 µm. (A) The endothelial glycocalyx was mostly intact, and heparan sulfate and Del-1 were spatially distributed in the endothelial cell and extracellular matrix. (B) After 24 h of CLP, the endothelial glycocalyx was almost completely degraded with less formation of heparan sulfate and Del-1. CLP: cecal ligation and puncture; Del-1: developmental endothelial locus-1; HS: heparan sulfate.

Fig 4: Del-1 interferes with binding of av integrin to LAP. (A) Binding of avß6 integrin (100 nM) to immobilized BSA, Del-1 (100 nM), and LAP (100 nM), and binding of avß6 integrin to immobilized LAP in the presence of Del-1, as assessed in a solid-phase binding assay. Data are representative of three independent experiments, each with similar results, and are expressed as the mean ± SD (n = 4 per group). ***p < 0.001; Student's t-test. (B) Adhesion of avß6-overexpressing HEK293T cells or human small airway alveolar epithelial primary cells (HSAEpCs) onto increasing concentrations of immobilized LAP protein was assessed. Alternatively, adhesion of cells to immobilized inactive TGF-ß derived from conditioned medium was assessed. Data are expressed as the mean ± SD (n = 3). *p < 0.05; Mann-Whitney U-test. (C) Adhesion of avß6 integrin-overexpressing HEK 293T cells or HSAEpC to immobilized LAP is impaired by Del-1 (1, 5, and 10 µg/mL), EGF1-3 (5 µg/mL), or an avß6-blocking antibody (5 µg/mL). Data are expressed as the mean ± SD (n = 3). *p < 0.05; Mann-Whitney U-test. (D) Representative co-immunoprecipitation assay to validate the interaction between av integrin and Del-1 and the interaction between av integrin and LAP in the absence or presence of Del-1. HEK293T cells overexpressing avß6 integrin after lentiviral transduction were incubated for 6 h with Del-1 (20 nM) or LAP (20 nM) and then processed for immunoprecipitation and western blotting. Data are representative of three independent experiments, each with similar results.

Fig 5: Supplementation with Del-1 attenuates the pathological characteristics of PF in mice. (A) Representative ex vivo micro-computed tomography (µ-CT) scans of lung sections from WT and Del-1-/- mice with BLM (1 U/kg)-induced PF, which were supplemented intravenously with bovine serum albumin (BSA; 50 µg/dose) or soluble Del-1 (sDel-1; 50 µg/dose) at 5, 9, and 13 days post-BLM administration (dpa). Lung sections were taken at 21 dpa. Dashed lines indicate fibrotic regions. Quantification of fibrosis in the µ-CT images is shown in the right panel. Data are represented as the mean ± SEM (n = 3 mice per group). *p < 0.05; n.s., not significant; Mann-Whitney U-test. (B) Survival analysis of WT and Del-1-/- mice with BLM-induced PF (1.5 U/kg BLM) that were supplemented with BSA or sDel-1 (intravenous injection, 50 µg/dose sDel-1 at 5, 9, and 13 dpa [n = 12 mice per group]). *p < 0.0001; log-rank test. (C) The levels of active TGF-ß and LAP in the lungs of mice with BLM-induced PF that were supplemented with Fc or Del-1-Fc (intravenous injection; 50 µg/dose at 5, 9, and 13 dpa). The lung lysates were collected at 14 dpa and subject to ELISA. Data are expressed as mean ± SEM (n = 3 mice per group). *p < 0.05, ***p < 0.001; Mann-Whitney U-test. (D) Representative western blots to detect the expression of a-SMA and the level of phosphorylated Smad3 in WT mice with BLM-induced PF (2 U/kg BLM) supplemented with Fc or Del-1-Fc (intravenous injection; 50 µg/dose Fc or Del-1-Fc at 14, and 18 dpa). Lung lysates were collected at 21 dpa. Densitometric data are shown in the right panels. Data are expressed as mean ± SEM (n = 3–4 mice per group). **p < 0.01, ***p < 0.001; Student's t-test. (E) Hydroxyproline analysis of lung tissues from WT mice with BLM-induced PF (2 U/kg BLM) supplemented with Fc or Del-1-Fc, as in (D). Data are expressed as the mean ± SEM (n = 3–4 mice per group). *p < 0.05, ***p < 0.001; Student's t-test. (F) Hydroxyproline analysis of lung and skin tissues from WT mice with systemic fibrosis (8 U/kg/dose BLM, daily subcutaneous injection for 15 days) supplemented with Fc or Del-1-Fc (intravenous injection; 50 µg/dose Del-1-Fc at 7, 11, 15, and 19 dpa after the first BLM instillation). Tissues were harvested at 23 days after the first BLM instillation. Data are expressed as the mean ± SEM (n = 3 mice per group). *p < 0.05; Mann-Whitney U-test.