ABSTRACT
Nitric Oxide (NO) and Reactive oxygen species (ROS) are endogenous regulators of angiogenesis-related events as endothelial cell proliferation and survival, but NO/ROS defect or unbalance contribute to cancers. We recently designed a novel photoactive inhibitor of NO-Synthases (NOS) called NS1, which binds their NADPH site in vitro. Here, we show that NS1 inhibited NO formed in aortic rings. NS1-induced NO decrease led to an inhibition of angiogenesis in a model of VEGF-induced endothelial tubes formation. Beside this effect, NS1 reduced ROS levels in endothelial and melanoma A375 cells and in aorta. In metastatic melanoma cells, NS1 first induced a strong decrease of VEGF and blocked melanoma cell cycle at G2/M. NS1 decreased NOX(4) and ROS levels that could lead to a specific proliferation arrest and cell death. In contrast, NS1 did not perturb melanocytes growth. Altogether, NS1 revealed a possible cross-talk between eNOS- and NOX(4) -associated pathways in melanoma cells via VEGF, Erk and Akt modulation by NS1 that could be targeted to stop proliferation. NS1 thus constitutes a promising tool that modulates NO and redox stresses by targeting and directly inhibiting eNOS and, at least indirectly, NADPH oxidase(s), with great potential to control angiogenesis.
Subject(s)
Enzyme Inhibitors/pharmacology , Human Umbilical Vein Endothelial Cells/metabolism , Light , Melanoma/metabolism , NADP/pharmacology , Nitric Oxide/metabolism , Reactive Oxygen Species/metabolism , Animals , Aorta/cytology , Aorta/drug effects , Aorta/metabolism , Apoptosis , Blotting, Western , Cell Cycle , Cell Proliferation , Electron Spin Resonance Spectroscopy , Flow Cytometry , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/drug effects , Humans , Male , Melanoma/drug therapy , Melanoma/pathology , Mice , Mice, Inbred C57BL , NADP/analogs & derivatives , NADPH Oxidase 4 , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/metabolism , Neovascularization, Pathologic , Nitric Oxide Synthase Type I/antagonists & inhibitors , Nitric Oxide Synthase Type I/metabolism , Nitric Oxide Synthase Type III/antagonists & inhibitors , Nitric Oxide Synthase Type III/metabolism , Signal Transduction/drug effects , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A/metabolismABSTRACT
OBJECTIVE: We investigated the impact of hypoxia-reoxygenation on endothelial relaxation and aimed to clarify the role of transient receptor potential cation channels V4 (TRPV4) and gap junctions in the protective effect associated with hypoxic preconditioning on the vascular function. METHODS AND RESULTS: By mimicking ischemia-reperfusion in C57BL/6 male mice in vivo, we documented a reduced NO-mediated relaxation and an increased endothelium-derived hyperpolarization (EDH[F])-mediated relaxation. Hypoxic preconditioning, however, restored NO relaxation and further improved the EDH(F) response. We also examined specifically 2 major effectors of the EDH(F) pathway, transient receptor potential cation channels V4 and connexins. We found that in endothelial cells, expression and activity of transient receptor potential cation channels V4 were increased by hypoxic stimuli independently of preconditioning which was interestingly associated with an increase of structural caveolar component caveolin-1 at membrane locations. Gap junctions, however, seemed to directly support EDH(F)-driven preconditioning as connexin 40 and connexin 43 expression increased and as in vivo carbenoxolone treatment completely inhibited the EDH(F) pathway and significantly reduced the protection afforded by preconditioning for the concomitant NO-mediated relaxation. CONCLUSIONS: Our work provides evidence on how transient receptor potential cation channels V4 and connexins might participate in preserving vasorelaxation under hypoxia and restoring the NO-mediated pathway in hypoxic preconditioning conditions pointing out caveolae as a common signaling location.
