Antiangiogenic and antimitotic effects of aspirin in hypoxia–reoxygenation modulation of the LOX-1-NADPH oxidase axis as a potential mechanism.

Hypoxia–reoxygenation (HR) is a primary driver of angiogenesis in both atherogenesis and tumorigenesis. The main target of hypoxia-driven proangiogenic signaling is adherens junctions responsible for contact inhibition of endothelial cells. We analyzed the effects of hypoxia (8–12 hours) followed by a brief period of reoxygenation (2 hours) (HR) on angiogenesis and integrity of adherens junction in cultured human umbilical vein endothelial cells as well as the effects of aspirin on modulation of human umbilical vein endothelial cells' response to HR. Cells exposed to HR displayed considerable enhancement of tube formation (angiogenesis) on matrigel. Immunocytostaining of near-confluent cells revealed that HR caused disruption of adherens junctions and internalization of their components VE-cadherin, p120 catenin, and b-catenin. Additionally, HR resulted in the appearance of binucleated cells, and VE-cadherin in colocalization with b-catenin was found to be positioned between the separating nuclei. Presence of aspirin (acetylsalicylic acid, 1 mM) resulted in preservation of adherens junctions on the cellular membrane and prevented angiogenesis as well as mitosis. HR caused upregulation LOX-1, the p47(phox) subunit of NADPH, while reducing transcription of endothelial nitric oxide synthase. Aspirin had no effect on endothelial nitric oxide synthase and canceled the transcriptional activation of the LOX-1 and p47(phox) subunit of NADPH oxidase. Based on these data, we hypothesize that aspirin preserves the integrity of adherens junctions and thus blunts angiogenic response to HR through downregulation of LOX-1 and the LOX-1-mediated p47(phox) component of NADPH oxidase transcription, thus preventing NADPH oxidase assembly and function.