Fig. 2.
p18 promotes tube formation in vitro and in vivo. Equivalent numbers of LMVEC were transiently transfected with GFP, p18wt, or p18N39 cDNA. A: transfected cells were plated directly onto Matrigel-coated wells for 48 h. Cells were then treated with VEGF (50 ng/ml) for a further 6 h, and images of branching were captured (Ai) and in vitro vessel formation was quantified by using Angiogenesis Analyzer in ImageJ, expressed as number of junctions formed (arrow) per field of view (Aii). Images were captured at ×10 magnification, scale bar 20 μm. B: de novo vessel formation, by transfected endothelial cells, was examined by using in vivo Matrigel plugs in rats. Vessel formation was determined by examining presence of blood vessels containing red blood cells within the Matrigel plug. Plugs were removed 4 days after injection, formalin-fixed, sliced in paraffin, and stained with hematoxylin and eosin. Images were captured by using Aperio ScanScope CS (Bi, left) and number of vessels per field of view were quantified (Bii). Staining with von Willebrand factor (vWF) antibody demonstrated that the appearance of red blood cells coincided with formation of blood vessels within the plug (Bi, right). Images were captured at ×20 magnification, scale bar 200 μm. Arrows demonstrate examples of neovasculogenesis within plugs. Data are presented as means ± SD; n = 4 (A), n = 5 (B); *P < 0.05 vs. vehicle, #P < 0.05 vs. GFP.