Abstract 1460: Nox2 Signalling via p21 Cip1 and p53 in Endothelial Cell Cycle Regulation

Abstract

The abilities of endothelial cells to proliferate, to be quiescent in monolayer and to undergo apoptosis during remodeling are important determinants relating to angiogenesis, wound healing and many diseases including atherosclerosis. During ischemia, angioplasty or organ transplantation, endothelial cells experience sustained nutrient deprivation (starvation), which causes cell oxidative stress and apoptosis. Endothelial cells express constitutively an NADPH oxidase, which is a major source of superoxide production. The catalytic subunit of NADPH oxidase has several isoforms (Nox1–5). However, their individual roles in endothelial function remain unknown. In this study, we investigated the role of Nox2 in nutrient deprivation-induced cell cycle arrest and apoptosis. In proliferating human dermal microvascular EC (HMEC1), Nox2 mRNA expression was low relative to Nox4 (Nox2:Nox4~1:13), but was upregulated 24h after starvation and increased to 8±3.5-fold at 36h of starvation as detected by quantitative real-time PCR. Accompanying the upregulation of Nox2, there was a 2.28±0.18-fold increase in O 2 .− production detected by tiron (O 2 −. scavenger)-inhibitable lucigenin (5 μM)-chemiluminescence; a dramatic induction of p21 cip1 and p53 protein expression detected by immunoblotting; cell cycle arrest and the onset of cell apoptosis detected by propidium-iodide FACS analysis (all P<0.05). All these changes were inhibited significantly by adding apocynin (an NADPH oxidase inhibitor), or by in vitro deletion of Nox2 expression using full-length antisense Nox2 cDNA, or in coronary microvascular endothelial cells isolated from Nox2 knockout mice. In Nox2 knockout cells, although there was a 3.8±0.5-fold increase in Nox4 mRNA expression after 36h of starvation (P<0.01), neither O 2 .− production nor the p21 cip1 and p53 expressions were increased significantly and only 0.46% of cells were apoptotic compared to ~12% apoptotic cells found in wild-type control cells cultured under the same condition. In conclusion, Nox2-derived O 2 .− , through the modulation of p21 cip1 and p53 expression, participates in the regulation of endothelial cell cycle control and apoptosis.