Mice were euthanized and 0.8 ml of dispase (1 mg/ml; Invitrogen, Paisley, UK) was instilled intratracheally, after which lungs were excised, finely minced, and digested with dispase at 37°C for 45 min. Samples were then triturated by pipetting and filtered through a 40-μm sieve (BD Biosciences, Oxford, UK), to produce a lung single-cell suspension. Lung endothelial cells (LEC) were obtained by a two-step magnetic affinity cell-sorting procedure (Miltenyi Biotech, Surrey, UK), comprising of a first step of leukocyte depletion using biotinylated anti-CD45 MAb (eBioscience, Hatfield, UK) followed by LEC enrichment with biotinylated anti-CD31 (PECAM-1) MAb (eBioscience). Purified LEC (CD31+, CD45−) were cultured in gelatin-coated flasks in DMEM with 10% FCS, 12 U/ml heparin, 25 mM HEPES, 75 μg/ml endothelial cell growth supplement (Sigma, Poole, UK), 1% sodium pyruvate, 1% nonessential amino acids, and penicillin-streptomycin, passaged with trypsin-EDTA, and used for experiments between passages 3 and 10. The phenotype of cultured cells was determined by flow cytometry on the basis of staining for CD31 and other surface markers constitutively expressed on endothelial cells including: VE-cadherin, ICAM-1, ICAM-2, endoglin, and CD34, as well as expression of inducible markers VCAM-1 and E-selectin.