Egr-3 plays a role in VEGF-mediated neovascularization of ex vivo aortic explants and in vivo Matrigel plugs in mice. (A) Quantitative real-time PCR analysis of Egr-3 mRNA expression in VEGF-treated murine MS-1 cells transduced with adenovirus expressing miControl (Ad-miControl) or miRNA against Egr-3 (Ad-miEgr-3). Egr-3 expression is normalized to Cyclophilin A mRNA levels. The results show the mean ± SD of expression levels relative to miControl obtained from 3 independent experiments. *P < .001 compared with Ad-miControl. (B) C57/BL6 mice were injected intravenously with 10⁹ pfu Ad-miControl or Ad-miEgr-3. At 3 days later, a short segment of the aorta was removed, embedded in Matrigel, and incubated with MCDB131 medium containing 10⁹ pfu of Ad-miControl or Ad-miEgr-3. Outgrowth of neovessels from the aorta was observed under phase-contrast microscopy, and tube length was calculated by the use of a cell image analyzer. The mean ± SD were derived from 4 independent experiments. *P < .001 compared with Ad-miControl plus VEGF. (C) Matrigel containing 10⁹ pfu Ad-miControl or Ad-miEgr-3 was injected subcutaneously into the flank of C57/BL6 mice. Fourteen days later, Matrigel plugs were removed, and sections were immunostained with anti–PECAM-1 antibody. Broken line indicates the boundary between flank muscle and explanted Matrigel. Bar indicates 50 μm. The data are representative of 6 independent experiments. To quantify neoangiogenesis, 1% Evans blue was injected intravenously into mice. At 10 minutes later, Matrigel plugs were removed and incubated in formamide. The amount of Evans blue dye was quantified by OD₆₂₀ and normalized to weight of Matrigel. The mean ± SD were derived from 6 Matrigel plugs in each condition. *P < .01 compared with Ad-miControl.