Abstract
Introduction: Angiogenesis is a feature of chronic asthma with the levels of multiple pro-angiogenic factors, including connective tissue growth factor (CTGF) and vascular growth factor (VEGF), increased in the lungs of patients with asthma. Preliminary data from our lab suggests that the pro-angiogenic effect of asthmatic airway smooth muscle cells is lost if CTGF is immuno-precipitated from the media but not restored by supplementation with recombinant CTGF. These data indicate that CTGF may be exerting these pro-angiogenic effects by binding to another protein. Aim: To identify other factor/s that bind to CTGF.
Methods: Plates were coated with CTGF C-terminal fragment (amino acids 253 to 350) (90 nM) then blocked with 1% BSA. The potential binding proteins were biotinylated and added to the CTGF-coated wells. The amount of protein bound to CTGF was detected using streptavidin conjugated HRP and TMB.
Results: On dose response curves, angiopoietin (Ang)-1 (34±10 units (U)/nmol protein) and Slit3 (20±5 U/nmol protein) had the strongest association constants, with Von Willebrand factor, basic fibroblast growth factor and TNF-α all having moderate affinity (range of 8-15 U/nmol protein) and VEGF-A165 (5±4 U/nmol protein) and VEGF-D (3±0.7 U/nmol protein) showing low association/high disassociation kinetics. VEGF-A121, VEGF-C, Ang-2, PDGF-BB, TGFβ1, placenta growth factor, EGF, IL1β, IL17 and human chorionic gonadotropin had little or no binding to CTGF.
Conclusion: Agents aimed at preventing blood vessel formation improve airway hyperresponsiveness and asthma symptoms. Identifying the binding partner for CTGF could help develop a new therapeutic approach for targeting asthma.
- Copyright ©ERS 2015