Figure 1

Validation of bioactivity of VEGF expressed by HEK cells. In (A), the pro-angiogenic action of HEK-VEGF cells was checked using a chamber model. After 5 d of subcutaneous implantation of a chamber containing HEK or HEK-VEGF cells on the back of mice, a vascular cast was made by i.v. injection of a solution of carmine red and gelatin, and angiogenesis was quantified by carmine content of the dorsal skin. Representative photographs taken from the dorsal skin before tissue digestion evidence an increased number of blood vessels in HEK-VEGF treated mice, and these newly formed vessels were tortuous and morphologically distinct from preexisting vessels. After digestion, the dye content of the samples was assayed spectrophotometrically and the results were then expressed quantitatively as mg dye content as shown in the graphic. Further, HEK nodules formed upon the CAM after grafting of HEK or HEK-VEGF cells were reacted with specific antibodies against VEGF and EPO as positive control. EPO expression was detected in both HEK (B) and HEK-VEGF (C) nodules. VEGF was detected only in HEK-VEGF (E) nodules, not in HEK nodules (D). N = nodule, and arrows indicate the CAM tissue subjacent to the cell nodule. Bars = 50 µm.