A three-dimensional in vitro model of angiogenesis in the airway mucosa.

Bronchial asthma is an inflammatory disease characterized by chronic intermittent bronchoconstriction. A key feature of the disease is structural changes in the airway wall (airway remodeling) consistent with tissue growth and chronic wound healing including angiogenesis. The epithelium directs mesenchymal processes during both embryogenesis and wound healing, and thus we hypothesized that the bronchial epithelium plays a critical role in directing angiogenesis. To study angiogenesis in the airways, we have developed a three-dimensional (3-D) in vitro model of the airway mucosa that consists of normal differentiated human bronchial epithelial cells (NHBE), normal human lung fibroblasts (NHLF), and human umbilical vein endothelial cells (HUVEC). The HUVEC are coated on dextran beads and suspended in a fibrin gel approximately 2mm beneath a confluent monolayer of NHLF which are just beneath the confluent monolayer of differentiated NHBE. In the presence of fibroblasts, visible capillaries reaching lengths of up to 1mm sprout from the HUVEC-coated beads. Over 11 days in culture, the bronchial epithelium produces transforming growth factor-beta2 (TGFbeta2, 60pg/ml), significantly increases vascular endothelial growth factor (VEGF) more than 6-fold to a concentration of 1.85ng/ml, but does not significantly impact total network formation. Exogenous TGFbeta2 stimulates VEGF production in a dose-dependent fashion (0-400pg/ml) through a MAPK-dependent pathway, but also inhibits capillary network formation. We conclude that the bronchial epithelium produces biologically relevant concentrations of VEGF and TGFbeta2 in a 3-D model of the airway mucosa that may be useful in probing mechanisms of angiogenesis in asthma.