Adrian Rogers for help with imaging and flow cytometry. Authors’ Contributions D.V. necessary for the angiogenic responses elicited by dRP. The upregulation of VEGFR2 and NOX2-dependent stimulation of angiogenesis by dRP were confirmed in excisional wound and Matrigel plug vascularization assays using NOX2?/? mice. For the first time, we demonstrate that dRP acts intracellularly Isovitexin and stimulates superoxide anion generation by direct binding and activation of the NOX2 enzymatic complex. This study describes a novel molecular mechanism underlying the proangiogenic activity of dRP, which involves Isovitexin the Isovitexin sequential activation of NOX2 and NF-B and upregulation of VEGFR2. 28, 110C130. and (7, 24, 42, 49, 50, 58). The generation of dRP in eukaryotic cells is catalyzed by phosphorylases with specificity for different nucleosides. Three main enzymes have been characterized: thymidine phosphorylase (TP), uridine phosphorylase (UP), and purine nucleoside phosphorylase (PNP) (48). Nucleoside phosphorylases play a key role in nucleoside and pentose metabolism by degrading nucleosides into free nitrogen base and dRP, with dRP converted to deoxyribose-5-phosphate by phosphopentomutase (64). Several studies have suggested that nucleoside phosphorylases stimulate cancer angiogenesis in solid tumors and participate in the progression of the disease (27, 31, 62). Although regulation of nucleoside phosphorylases is largely unknown and their constitutive activity has been described (5), we previously presented data on the release of dRP by human platelets in response to cellular stimulation (67). In this study, we have evaluated the proangiogenic activity of dRP on human umbilical vein endothelial cells (HUVECs) using a variety of molecular techniques and have identified the NOX2-NF-B signaling axis that is engaged by dRP, resulting in the upregulation of VEGF receptor 2 (VEGFR2) expression and stimulation of angiogenic responses. This study is the most comprehensive and exhaustive characterization of dRP as a proangiogenic stimulus to date. Understanding the molecular mechanisms underlying the actions of dRP as a proangiogenic stimulus will have important applications in cancer, vascular, and regenerative medicine. Results Isovitexin dRP stimulates increased levels of ROS generation in an NOX-dependent manner We have previously described the release of dRP by human platelets (67). Using a quantitative liquid chromatographyCmass spectrometry (LC-MS) method, we quantified dRP released by human platelets and mouse macrophages. In platelet suspensions at physiological density ((Fig. 1A). The ability of dRP to induce the formation of capillary-like structures by endothelial cells (using low serum and growth factor-reduced Matrigel? (Fig. 1B and Supplementary Fig. S1A; Supplementary Data are available online at, while other pentoses were not effective (Supplementary Fig. S2). We also confirmed that dRP concentrations as low as 8?stimulate a significant increase in endothelial cell ROS formation (while 2?dRP produced a trend toward increased ROS formation without reaching statistical significance), as measured using dihydroethidium (DHE) after 1?h of treatment (Fig. 1C). Complete time courses of ROS generation at low micromolar dRP concentrations are shown in Supplementary Figure S1B. The dRP-dependent increase in ROS generation rates was abolished in the presence of 1?mN-acetyl-l-cysteine (NAC), 10?Mn(III)tetrakis(4-benzoic acid)porphyrin (MnTBAP), or 10?4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Tempol) (Fig. 1D). The link between oxidative stress and angiogenic activity of HUVECs and the role of ROS generation in the angiogenic response induced by dRP were then tested using the ROS scavenger NAC (71) and the superoxide dismutase (SOD) mimetics, MnTBAP (21) and Tempol (33). All three significantly impaired the tubulogenic activity of dRP (Fig. 1E). Other angiogenic responses induced by dRP (was quantified by LC-MS. Presented data are from six and three independent samples, respectively. Statistical significance was assessed by one-way ANOVA with Bonferroni test (*and 1?mand after 4?h of culture and quantification of tube number per optical field were performed using the Angiogenesis Analyzer plug-in of ImageJ. (C) ROS generation was analyzed with DHE staining for 1?h in response to Rabbit Polyclonal to SERPING1 concentrations of dRP ranging from 2?to 1 1?mand expressed as.