Fig. 1

miR-27b augments VEGF-induced angiogenesis and recruitment of the bone marrow -derived cells. Extracellular matrix (Matrigel) supplemented with Heparin (60 U/ml) to retain growth factors, was injected in mice to generate subcutaneous gel plugs. VEGF, miR-27b mimics (27b) or negative control RNAi (NC), were added to Matrigel as indicated. The plugs were harvested and processed for analysis after 10 days. (a) Sections of cryopreserved Matrigel plugs were stained for the endothelial marker, CD31 (red) and cell nuclei visualized with DAPI (blue). (b) Fluorescence images (×20, a minimum of 10 per condition) were taken and CD31-positive vascular area measured using Elements software (Nikon). The data is presented as box plot with whiskers; median values are shown. P value was calculated using Kruskal-Wallis test. Note increased vascularization in the presence of miR-27b. (c) Mice were lethally irradiated and reconstituted with bone marrow (BM) harvested from mice with ubiquitous expression of actin-GFP transgene. After 6-week recovery, mice were used in Matrigel plug assay for angiogenesis, as in (a). GFP-expressing BM-derived cells (BMDCs) are shown in green. (d) The recruitment of the GFP-positive BMDCs to the Matrigel (green fluorescence area) was measured using Elements software. The measurements from at least eight ×20 fields per condition are graphed as box plot with whiskers; median values are shown. P value is calculated using Wilcoxon Signed Rank test. Note elevated BMDC recruitment in response to miR-27b. (e) Dual immunofluorescence for the endothelial marker, CD31 (red) and GFP-positive BMDCs (green). Note minimal co-localization between CD31 and GFP (yellow)