Discovery of a benzoxazine derivative promoting angiogenesis in vitro and in vivo.

Angiogenesis is a multi-step process that refers to the growth of new vessels from pre-existing ones. Endothelial proliferation, migration, and tube formation constitute a critical step in angiogenesis. Recently, we demonstrated that a novel benzoxazine derivative, 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (ABO) could improve the proliferation of human umbilical vein endothelial cells (HUVECs) without basic fibroblast growth factor (bFGF) and serum. In this study, we further tested its effect on endothelial angiogenesis with Matrigel assay, migration assay, and in vivo chick chorioallantoic membrane (CAM) assay. Our results showed that ABO effectively facilitated cell migration and promoted capillary-like tube formation in vitro and in vivo. To elucidate the underlying mechanisms, we examined intracellular reactive oxygen species (ROS) level/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and superoxide dismutase (SOD) activities, nitric oxide (NO) level/endothelial nitric oxide synthase (eNOS) activity, and mitochondrial membrane potential (MMP). Our data indicated that ABO depressed ROS with inhibition of NADPH oxidase instead of SOD activity, stimulated NO production and eNOS activation, and restored MMP in HUVECs. Our findings suggest that ABO is a promising tool for exploring the mechanisms of angiogenesis and may have a therapeutic potential in ischemic pathologies. J. Cell. Physiol. 223: 202-208, 2010. (c) 2009 Wiley-Liss, Inc.