Because various inhibitor doses and assays were used, a large number of cells was required. Therefore, most of the studies were performed using cultured bovine RECs. They were very similar to human RECs in response to growth factors. For each angiogenic assay, cells were treated with growth factor combinations, with or without signaling inhibitors, as described. The inhibitors were as follows: H7 (blocks various protein kinases; Seikagaku America, East Falmouth, MA), H89 (blocks protein kinase [PK]A and PKG, also affects CK2
29 ; Seikagaku America), calphostin C (blocks PKC; BioMol, Plymouth Meeting, PA), LY379196 (blocks PKC-β; from author MBG), KN-93 (blocks Ca
2+/calmodulin kinase II; Seikagaku America), CKI-7 (blocks casein kinase (CK)1, also affects CK2 at high doses; Seikagaku America), wortmannin (blocks phosphoinositide 3 [P13]) kinase; Sigma-Aldrich), LY294002 (CK2+PI3 kinase; Sigma-Aldrich), emodin (1,3,8-trihydroxy-6-methylanthraquinone; Sigma-Aldrich), DRB (5,6-dichloro-1-β-o-ribofuranosyl benzimidazole; BioMol), and TBB (4,5,6,7-tetrabromobenzotriazole; synthesized according to protocol described by Büchel
30 ; all block CK2 with TBB being the most specific to date
31 32 33 34 35 36 ), PD98059 (blocks mitogen-activated protein [MAP] kinase kinase [MEK])/extracellular signal-regulated kinase [ERK], SB202190 (blocks p38 MAP kinase), SB203580 (blocks p38 MAP kinase and MAP kinase-activated protein [MAPKAP] kinase), and SB202474 (an inactive compound used as a negative control for MAP kinase inhibitors). The last four inhibitors were part of MAP kinase inhibitor set I (Calbiochem, San Diego, CA). In preliminary experiments, inhibitors of CK2 and some other kinases, apigenin (4′,5,7-trihydroxyflavone), and quercetin (3,5,7,3′,4′-pentahydroxyflavone, both from Sigma-Aldrich), were also used. Quercetin was further used in pilot animal experiments because of its lack of toxicity to cultured cells, animals, and humans.
37 38 In some experiments, the matrix metalloproteinase (MMP) inhibitor galardin (GM6001; BioMol) and a transcription inhibitor, actinomycin D (Sigma-Aldrich), were used. Working concentrations of all inhibitors and assays in which they were tested are summarized in
Table 1 .