Effect of impairment of CREB function on lung microvessel permeability. (A-E) Expression of dn-CREB mutant in lungs exaggerate pulmonary edema formation. Mice were injected retro-orbitally with liposome encapsulating either vector or dn-CREB mutant. After 48 hours after transfection, either PAR1 agonist (1 mg/kg) or control peptide was injected intravenously into mice (A-C), or mice were exposed to nebulized LPS (D-E). Lung vascular permeability was determined by quantifying EBAE or wet-dry weight ratio at the indicated times. (A) Representative images of PAR1-induced Evans blue accumulation in the lungs-transducing vector or dn-CREB mutant. (B-E) Plot shows mean ± SEM of changes in EBAE and wet-dry weight ratio after PAR1 peptide administration or LPS exposure in control and dn-CREB-transdcucing lungs (n = 4). Asterisk (*) indicates values different from control vector group value at time 0, and double asterisk (**) indicates values different from corresponding PAR1 peptide treated control vector group (P < .05). (F) Control vector or dn-CREB–transducing lungs were harvested after treatment with either control peptide or PAR1 peptide at indicated times. Lung lysates were immunoblotted with the indicated antibodies to determine PAR1-induced phosphorylation of CREB and MYPT1. Immunoblot with anti-CREB antibody was used to confirm the overexpression of CREB in dn-CREB mutant lungs. (G-H) Plot shows mean ± SEM of fold-increase in CREB phosphorylation and MYPT1 activity in lungs-transducing dn-CREB mutant. Asterisk (*) indicates significance from its control vector group at time 0 (P < .05), and double asterisk (**) indicates significance from the corresponding PAR1 peptide–treated control vector mice group (P < .05).