Confocal- and electron-microscopic localization of FITC-albumin in H2O2-induced pulmonary edema

Fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) was used to detect sites of protein leakage in rat lungs perfused for 15-60 min with H2O2 (75, 150, and 300 microM). Leaky vessels were localized by confocal laser microscopy. Endothelial routes of protein leakage were identified by electron microscopy after photo-conversion of FITC-BSA to an osmiophilic diaminobenzidine product. Transport of FITC-BSA into the alveolar interstitium was assessed by immunogold labeling and anti-FITC antibodies. We detected leakage of FITC-BSA through transendothelial gaps in the pulmonary arterial endothelium after 30 min of perfusion with 300 microM H2O2 and after 60 min of perfusion with 150 microM H2O2. Junctional permeability and distribution of ZO-1 protein in the arterial endothelium were unchanged. Microvascular permeability to FITC-BSA was not increased in lungs perfused with H2O2. In lungs perfused with 300 microM H2O2, progressive extravasation of albumin was associated with significant increases in water content and perfusing pressure. We conclude that the pulmonary arterial endothelium is the primary target of circulating H2O2.