Pseudomonas aeruginosa induces vascular endothelial growth factor synthesis in airway epithelium in vitro and in vivo

Abstract

Pseudomonas aeruginosa (PA) airway infection and bronchial blood vessel proliferation are features of bronchiectasis. Because vascular endothelial growth factor (VEGF)-A regulates angiogenesis, we hypothesised that PA infection induces VEGF synthesis in epithelium and peribronchial angiogenesis. Because epidermal growth factor receptor (EGFR) activation regulates VEGF synthesis in cancer, we also evaluated the roles of EGFR.Airway epithelial cells were incubated for 24 h with PA supernatants and VEGF concentrations were measured in culture medium by ELISA. C57BL/6N mice were instilled intratracheally with sterile agarose beads or with agarose beads coated with the PA strain PAO1 (mean¡SEM 6610 ?day -1 intraperitoneally). Epithelial immunostaining for VEGF and phosphorylated EGFR, and peribronchial vascularity, were quantified using morphometric analysis. VEGF expression was further assessed by western blot in mouse lung homogenates. PA supernatants induced dose-dependent VEGF synthesis in cultured airway epithelial cells, effects which were prevented by EGFR antagonists. In mice, persistent PAO1 infection increased immunostaining for VEGF and phosphorylated EGFR in airway epithelium, and resulted in increased peribronchial vascularity within 7 days. These effects were reduced by EGFR inhibition.Persistent PA infection induced VEGF synthesis in airway epithelium and peribronchial angiogenesis, at least in part via EGFR-dependent mechanisms.

CFTR dysfunction induces vascular endothelial growth factor synthesis in airway epithelium

“…Thus, the absence of increased peribronchial vascularity in Cftr-deficient mice should be interpreted cautiously. However, one might hypothesise that besides CFTR dysfunction, other factors inducing VEGF-A expression, such as bacterial infection [18], might be necessary to promote peribronchial angiogenesis. Finally, CFTR inhibition induced both increased VEGF-A mRNA and protein synthesis, suggesting a transcriptional effect.…”

“…Because EGFR phosphorylation is required for VEGF-A synthesis, which is induced by various stimuli in airway epithelial cells [18,29], we examined its role in VEGF-A synthesis induced by CFTR inhibition. Role of functional CFTR in VEGF-A synthesis in primary cultures of HAECs Primary cultures of HAEC isolated from freshly excised nasal polyps secreted VEGF-A during the first week of culture, which gradually decreased over time, confirming previously published data (see online supplementary fig.…”

“…Immunohistochemical staining was performed on 5-mm paraffin-embedded sections, as previously described [18]. The following primary antibodies were used: a polyclonal antibody to VEGF-A (A 20, 1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); a polyclonal antibody to the endothelial marker von Willebrand factor (vWF, 1:50; DakoCytomation, Carpinteria, CA, USA) to identify blood vessels.…”

“…Quantification of peribronchial vascularity and of VEGF-A epithelial immunostaining were performed by point counting, as previously described [4,18,19]. Volume occupied by VEGF-A positively stained cells in epithelium was expressed per volume of epithelium.…”

“…Culture of human airway epithelial cells NCI-H292 human airway epithelial cells (HAECs), which express functional CFTR [20] and produce VEGF-A [18], were plated at 10 5 cells?mL -1 in 24-well plates (BD Falcon; BD Biosciences, San Jose, CA, USA), and grown in RPMI 1640 medium containing 10% fetal calf serum (FCS), penicillin (100 U?mL -1 ), streptomycin (100 mg?mL -1 ) and HEPES (25 mM) at 37uC in a humidified 5% carbon dioxide waterjacketed incubator.…”