Figure 2.

TanFe (transcellular activator of nuclear FOXF1 expression) increases FOXF1 (Forkhead Box F1) protein amounts in vivo. (A) Schematic shows treatment of C57Bl/6 mice with LPS and TanFe. LPS was administered intratracheally on Day 0. TanFe or vehicle was injected intraperitoneally on Days 0, 1, and 2 (5 mg/kg body weight). (B) TanFe increases FOXF1 protein amounts in LPS-injured mouse lungs. Total lung protein was prepared 6 days after LPS injury and analyzed by Western blot for FOXF1, FLK1, FLT1, CDH5 (VE cadherin), and β-Actin. Samples were obtained from individual mice (n = 3 mice per group). (C and D) Immunostaining for endomucin shows that TanFe increases the capillary density in alveolar regions of LPS-treated mice. Mice were harvested on Day 6 after LPS injury and lung paraffin sections were stained for endomucin (green). Slides were counterstained with DAPI (blue). Ten random slides were used to quantitate the staining (n = 3 mice per group). Magnification is ×400. (E) Endothelial permeability assay shows that TanFe protects endothelial barrier function after LPS lung injury. Evans blue dye was injected intraperitoneally, and lung tissue was collected 4 hours after injection (n = 5 mice in each group). Before the lung harvest, vasculature was perfused with saline to remove intravascular Evans blue dye. (F–H) TanFe inhibits accumulation of inflammatory cells in BAL fluid (BALF) of LPS-injured mice. BALF was collected on Day 3 after LPS lung injury and analyzed for total number of cells and for neutrophil counts (n = 5 mice in each group). (I) Hematoxylin and eosin staining shows that TanFe decreases lung inflammation in the alveolar region of LPS-injured mice. Mice were harvested on Day 6 after LPS injury (n = 3 mice per group). TanFe or vehicle was given on Days 0, 1, and 2. Magnification is ×400. *P < 0.05, **P < 0.01, and ***P < 0.001. n.s. = not significant.