Figure 1

Multi parameter characterization of in vitro angiogenesis in a co-culture model. (A) HUVECs formed capillary structure (cord) when plated on dermal fibroblast cells over the period of 12–14 days. The angiogenic cord structure was visualized by human CD31 staining (green), fibroblast cells were stained with an intermediate filament marker vimentin (red), while the nucleus of both cell types were visualized with Hoechst staining (blue). Cord formation was greatly enhanced by addition of 20 ng/ml VEGF (VEGF) to the culture, while 30 nM Sunitinib (CMPD) drastically inhibited cord formation as compared to the medium control (CTL). (B). Morphological features of the cord formed in the co-culture were captured and quantitated by high content image analysis using the ArrayScan platform adopting the tube formation bioapplication. 47 measurements were assessed, normalized using the Z-score across different treatment groups and analyzed by Principal Component Analysis (PCA). The first three principal components accounting for more than 80% of the variance were selected for sample scatter plotting. Blue are the control cultures (CTL); yellow, cultures treated with 20 ng/ml VEGF; and red, cultures treated with 30 nM Sunitinib (CMPD) treated samples. (C). The Z-scores of 47 morphological measurements were clustered in Spotfire Decision Site with the correlation coefficient being used as the distance metric. Complete linkage was used as the clustering algorithm. Red stands for higher and green for lower than mean (black) expression. (D). Six representative parameters were selected and plotted. Except for the cord width, the cord length, area, branching point, the number of nucleus within the cord, and a composite angiogenesis index (Angio Index) all manifested the anti and pro angiogenic effects from Sunitinib and VEGF, respectively. Data are expressed as Mean ± S.D.