Abstract 651: It Takes Two to Grow: Angiogenesis Requires Sufficient VEGF Responses Plus Correct Spatio-temporal Organization of Redox Signaling by PON2

Abstract

Angiogenesis describes the growing of new blood vessels from existing ones and is critical for e.g. developmental processes, vascular regeneration, atherosclerosis and tumors. Therapeutic compounds interfere with angiogenesis mostly through extracellular activities, typically by targeting either the most relevant pro-angiogenic ligand, vascular endothelial growth factor (VEGF), or its dominant receptor, VEGFR2. Intracellular signal transmission is less clear, but redox signaling i.e. formation of reactive oxygen species (ROS) has been established as regulator of endothelial functions. Thus we tested a role for paraoxonase-2 (PON2), a vascular anti-oxidative enzyme. This protein has two, seemingly independent effects on angiogenesis. We observed that endothelial cells (ECs) from PON2-/- mice expressed significantly less VEGFR2, likely through an interaction with the VEGFR2-controlling transcription factor GTF2i. As a result, VEGF-stimulated PON2-/- ECs showed weaker downstream effects, such as AKT activation, Calcium release and tube formations in in vitro angiogenesis assays. Sprouting of endothelial cells from aortic rings of PON2-/- mice was also reduced. In addition to this process, a ROS-dependent mechanism was found. Strikingly, 3D analyses of ECs sproutings from aortic rings revealed that in controls, ROS-producing hotspots located to both tip regions and branch points of endothelial sproutings, coinciding with high mitochondrial activity and migratory functions. The intermittent regions of proliferating areas had low ROS levels. This finding implies the presence of a tightly regulated, spatio-temporal ROS production that adjusts 3D sprouting by controlling migration and proliferation. Importantly, PON2 deficiency led to more ROS, but it also disintegrated the underlying 3D ROS arrangement. In PON2-/- sproutings, ROS hotspots were comparatively unordered, causing faster migrating (but not proliferating) ECs that produced less sproutings. Together, these findings reveal a mechanism that controls EC sprouting in a spatio-temporally organized and ROS-mediated manner. Interestingly, PON2 is an enzyme that combines the control of this process and responses to VEGF.