H-2g–induced rat aortic ring angiogenesis in vitro is inhibited by inhibitors of JAK2 and PI3 kinase. Rat aortic rings were placed on a Matrigel drop in 48-well plates and covered with an additional drop of Matrigel. Serum-free EGM medium (400 μL) with various concentrations of H-2g was added to each well. bFGF (10 nM) was used as a positive control. (A) H-2g (100 nM) induced rat aortic ring sprouting.AG490, a JAK2 inhibitor, and LY294002, a PI3K inhibitor, inhibited this sprouting. (B) Quantitatively, JAK2 blocking resulted in 94% inhibition of EC sprouting, whereas PI3K inhibition resulted in 82% inhibition. (C) We incorporated antisense and sense ODNs (10 μg/mL) in the Matrigel, and in the media throughout the experiments. Consistent with the chemical inhibitor effect, JAK2 and PI3K antisense ODNs inhibited microvessel sprouting, as did NFκB decoy ODNs. (D) Quantitatively, NFκB decoy ODN transfection resulted in 87% inhibition, compared with scrambled NFκB ODN; JAK2 antisense ODN transfection resulted in 78% inhibition compared with JAK2 sense ODN; and PI3K antisense ODN transfection resulted in 76% inhibition compared with PI3K sense ODN (decoy or antisense ODN transfection, □; scrambled or sense ODN transfection, ▪). Results represent 4 independent experiments. Results are expressed as the mean number of sprouting plus or minus SEM from 4 independent experiments. ^*Represents a significant difference (P < .05) between the respective groups. The pictures were taken with a Nikon Coolpix 4500 camera (Nikon, Tokyo, Japan) and an Olympus CK40 microscope (Olympus, Melville, NY), objective × 40/0.55 numerical aperture. Photoshop 5.5 (Adobe, San Jose, CA) was then used to process the images.