Fig. 1.

Genetic participants of the angiogenic switch. (A) We hypothesized that global analysis of transcriptional perturbation induced by positive and negative regulators of angiogenic balance may provide a rational algorithm to define the genetic participants of the angiogenic switch. Human microvascular endothelial cells were treated for 4 h with the endogenous angiogenesis inhibitor endostatin (200 ng/ml) to mimic the shift of the angiogenic balance toward an antiangiogenic (Off) state. Conversely, the proangiogenic (On) state in endothelium was emulated by using proangiogenic stimulators VEGF (10 ng/ml), bFGF (20 ng/ml), or combined VEGF (10 ng/ml) and bFGF (20 ng/ml). The inverse-regulation pattern of genes after pro- and antiangiogenic treatment was used as the selection criteria to predict the genes involvement in the angiogenic process. (B) Using significance analysis of microarrays, 2,370 transcripts with significant inverse-expression patterns were selected (P < 0.01). From these 2,370 transcripts, 1,140 were down-regulated after endostatin treatment and up-regulated after VEGF/bFGF treatment (categorized to participate in proangiogenic signaling, example genes in green box), whereas the remaining 1,230 transcripts were oppositely regulated (categorized to participate in antiangiogenic signaling, red box; see also SI Text). Each row represents log² expression ratios of an individual gene (see color code) and the columns indicate each respective treatment (in quadruplicates, 1–4). (C) Real-time quantitative RT-PCR confirmation of inverse regulation of six selected example genes in endothelial cells after endostatin vs. VEGF/bFGF treatment. Bars are means ± SD from three independent measurements and show relative expression levels compared with untreated control (P < 0.01).