Fig. 1.

CREB mediates VEGFC-induced angiogenic events. A. C57BL/6 mice pups after exposure to 75% oxygen from P7 to P12 were returned to room air to develop the relative hypoxia. Eyes from pups left in normoxia or at various time periods of hypoxia were enucleated, retinas isolated and extracts were prepared. An equal amount of protein from normoxic and various time periods of hypoxic retinal extracts was analyzed by Western blotting for VEGFA, VEGFB, VEGFC and VEGFD levels using their specific antibodies and normalized to β-tubulin. B–D. Quiescent HRMVECs were treated with and without VEGFC (100 ng/ml) and DNA synthesis (B), migration (C), or tube formation (D) were measured. E. An equal amount of protein from control and the indicated time periods of VEGFC (100 ng/ml)-treated HRMVECs was analyzed by Western blotting for pCREB levels using its phospho-specific antibodies and normalized to CREB. F. Cells were transduced with Ad-GFP or Ad-KCREB (40 moi) and 48 h later cell extracts were prepared and analyzed for over expression of GFP or KCREB by Western blotting using their specific antibodies and normalized to β-tubulin. G–I. All the conditions were the same as in panel F except that after transduction with the adenovirus, cells were quiesced and subjected to VEGFC (100 ng/ml)-induced DNA synthesis (G), migration (H), or tube formation (I). J. All the conditions were the same as in panel F except that after transduction, cells were labeled with Cell Tracker Green, coated onto Cytodex beads, embedded in a 3D-fibrin gel in EGM2 medium for 3 days and sprouts were observed under fluorescent microscope. The images were captured using MRm camera. The bar graphs represent quantitative analysis of three independent experiments. The values are presented as Mean ± SD. * p < 0.01 vs control, normoxia or Ad-GFP; ** p < 0.01 vs Ad-GFP + VEGFC. Scale bar represents 50 μm.