Figure 3.

Vaspin reverses pulmonary endothelial cell barrier dysfunction in vivo and in vitro, with no effect on pulmonary endothelial cell adherens junctions and the actin cytoskeleton following LPS insult. Mice were systemically instilled with Ad-vaspin or Ad-β-gal as a control (3×10⁷ PFU/mouse, 3 days) and were subjected to an intratracheal injection with LPS (5 mg/kg) to establish a mouse model of ARDS. Mice were intratracheally injected with PBS as a control. (A) Total BALF protein concentrations, (B) Evans blue dyed-albumin extravasation and (C) wet/dry ratios in a murine model of ARDS were measured 4 h post-LPS injection (n=5 mice from each group assessed in triplicate). (D) Expression levels of VE-cadherin and β-catenin were detected in lung tissues by western blot analysis (n=5 mice from each group assessed in triplicate). Relative protein expression levels were semi-quantified by measuring corresponding band intensities, and values were expressed relative to GADPH. HPMECs were pretreated with rh-vaspin (10 ng/ml) or PBS as a control for 24 h, and were then exposed to PBS or LPS (100 ng/ml) for 4 h. (E) Influx of FITC-dextran was measured by permeability assay (n=5 cultures from each group assessed in triplicate). mRNA expression levels of (F) VE-cadherin and (G) β-catenin in the HPMECs lysate were assessed by quantitative polymerase chain reaction (n=5 cultures from each group assessed in triplicate). (H) Actin cytoskeleton distribution was assessed by phalloidin staining (n=3 cultures from each group assessed in triplicate). Data are presented as the mean ± standard deviation. ^*P<0.05. Ad-β-gal, adenoviral vector expressing β-galactosidase; Ad-vaspin, adenoviral vector expressing vaspin; ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; FITC, fluorescein isothiocyanate; HPMECs, human pulmonary microvascular endothelial cells; LPS, lipopolysaccharide; rh-vaspin, recombinant human vaspin; VE-cadherin, vascular endothelial-cadherin.