Biphasic Regulation Of H₂O₂ On Angiogenesis Implicated NADPH Oxidase And Natural Antioxidant Pedicularioside G Inhibits Angiogenesis And Tumorigenesis In Vitro And In Vivo

Angiogenesis is a complex physiological and pathological process that includes endothelial cell activation, extracellular matrix degradation, endothelial cell migration, proliferation and final new vessels formation. Reactive oxygen species (ROS) are believed to play an important role in the regulation of angiogenesis. ROS is the product of aerobic metabolism. A huge amount of ROS result in several kinds of deseases including cancer, whereas ROS in a moderate level have been regarded as signal molecules. The important signaling role of ROS during angiogenesis and tumorigenesis is thought highly and current studies are focused on the sources of ROS involved in angiogenesis and tumorigenesis. Although there are several ways to produce ROS in cells, multicomponent non-phagocytic NADPH oxidase seems to be especially important and be the one takes charge of the ROS generation in endothelial cells.Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) mediated generation of reactive oxygen species (ROS) was originally identified as the powerful host defense machinery against microorganism in phagocytes. But after the mid-80s of last century, some reports indicated that some non-phagocytic cells, including endothelial cell, smooth muscle cell, and neurocyte also have the NADPH oxidase activity. It is reported that vascular NADPH oxidase is involved in atherosclerosis and diabetic vascular disorders.To clarify the role of ROS in angiogenesis and the potential mechanisms, the effects of extracellular H₂O₂, one kind of ROS, on endothelial cell proliferation, migration, apoptosis and angiogenesis in chicken embryo chorioallantoic membrane (CAM) as well as the role of NADPH oidase were studied. Meanwhile, A natural antioxidant, Pedicularioside G (Ped G), on angiogenesis, tumorigenesis and formation of reactive oxygen species were studied.Results: 1. Low concentration of H₂O₂ (10μM) stimulates endothelial cell proliferation and migration, while higher concentration inhibits them. For apoptosis, low concentrations of H₂O₂ (under 100μM for 24 h) have almost no influence on apoptosis, while 200μM stimulated apoptosis strongly. However, much higher concentrations of H₂O₂, 300, 400 and 500μM, decline apoptosis with concentration dependently because of the toxicity of high level H₂O₂.2. H₂O₂, 1 or 2 nmol/cm², increases the neovascularization in chicken chorioallantoic membrane slightly, and get the peak at 4 nmol/cm², then declines with the concentration of H₂O₂ increasing.3. Endothelial cell proliferation, migration, apoptosis and neovascularization in chicken chorioallantoic membrane were significantly suppressed by addition of DPI, the inhibitor of NADPH oxidase, which means NADPH oxidase is implicated and may be activated by H₂O₂ to produce O₂ to carry out the angiogenesis processes.4. Ped G inhibits two prerequisite angiogenic procedures, endothelial cell proliferation and migration, as well as neovascularization in vivo. Moreover, Ped G inhibits human hepatoma cells proliferation and migration in vitro along with tumor formation and growth in vivo. Therefore, Ped G has the properties of not only anti-angiogenesis but also anti-tumor growth and anti-tumorigenesis which might partially attribute to its antioxidative activity.In conclusion, ROS mediated by NADPH oxidase regulated the angiogenesis in vitro and in vivo biphasically. Once the ROS in cells is reduced by antioxidants, both angiogenesis and tumorigenesis in vitro and in vivo will be suppressed. The results of the current study laid a foundation for the therapy of angiogenesis-releated diseases.