The role of NF-kappaB in the angiogenic response of coronary microvessel endothelial cells

The activation of nuclear factor (NF)-kappaB by 12(R)-hydroxyeicosatrienoic acid [12(R)-HETrE], an arachidonic acid metabolite with potent stereospecific proinflammatory and angiogenic properties, was examined and its role in the angiogenic response was determined in capillary endothelial cells derived from coronary microvessels. Electrophoretic mobility-shift assay of nuclear protein extracts from cells treated with 12(R)-HETrE demonstrated a rapid and stereospecific time- and concentration-dependent increase in the binding activity of NF-kappaB, which was inhibitable by the antioxidants N-acetylcysteine, butylated hydroxyanisole, and pyrrolidine dithiocarbamate and was partially attenuated by the protein kinase C inhibitors, staurosporine and calphostin C. Neither 12(S)-HETrE nor other related eicosanoids--e.g., 12(R)-HETE, 12(S)-HETE, and leukotriene B4--stimulated the activation of NF-kappaB relative to 12(R)-HETrE, substantiating the claim for a specific receptor-mediated mechanism. 12(R)-HETrE stimulated the formation of capillary-like cords of microvessel endothelial cells distinguishable from a control; this effect was comparable to that observed with basic fibroblast growth factor (bFGF). Inhibition of NF-kappaB activation resulted in inhibition of capillary-like formation of endothelial cells treated with 12(R)-HETrE by 80% but did not affect growth observed with bFGF. It is suggested that 12(R)-HETrE's angiogenic activity involves the activation of NF-kappaB, possibly via protein kinase C stimulation and the generation of reactive oxygen intermediates for downstream signaling.