Superoxide Generated by Lysophosphatidylcholine Induces Endothelial Nitric Oxide Synthase Downregulation in Human Endothelial Cells

Abstract

We examined the mechanism through which lysophosphatidylcholine (LPC) induces endothelial nitric oxide (eNOS) downregulation. Human umbilical vein endothelial cells (HUVECs) were treated with LPC (50-150 µM) for 0.5-2 h or the reactive oxygen species (ROS) donors, xanthine/xanthine oxidase (X/XO), 1,4-hydroquinone (HQ) or tert-butylhydroperoxide (TBHP) for 2 h. Protein levels of eNOS, superoxide dismutase1 (SOD1), catalase, and phospho-extracellular signal regulated kinase 1/2 (pERK 1/2) were assessed using immunoblotting. LPC treatment reduced SOD1 levels but increased catalase levels. The superoxide donors X/XO and HQ showed similar effects. The hydroperoxide donor TBHP increased SOD1 levels but did not change catalase levels. LPC concentration- and time-dependently decreased eNOS levels, but this effect was blocked by antioxidants and SOD and potentiated by the SOD1 inhibitor, ammonium tetrathiomolybdate. LPC and X/XO inhibited ERK1/2 phosphorylation, whereas TBHP stimulated phosphorylation. Taken together, these data indicate that LPC induces superoxide overload in HUVECs via SOD1 inhibition and downregulates phospho-ERK1/2 and eNOS levels.

Oleoyl-Lysophosphatidylcholine Limits Endothelial Nitric Oxide Bioavailability by Induction of Reactive Oxygen Species

“…Based on the fact that the critical micellar concentration for LPC 16:0 was estimated to be between 7 and 50 µM (depending on the conditions like temperature, salt concentration, pH or presence of proteins or lipids [45]–[47]) one can only speculate whether free LPC under given experimental conditions exist as single molecules or micelles. Previous studies reported both decrease and increase in eNOS levels and NO bioavailability in cells exposed to LPC 16:0 for periods between 2 and 24 h [21]–[26]. In contrast to these studies, the effects of LPC 18:1 on NO bioavailability in our experimental model comprise exclusively rapid molecular events, independent of induction of mRNA or protein synthesis.…”

“…However minute free LPC might appear in phases of excessive lipolysis and concomitant saturation of albumin and carrier proteins with fatty acids (FA) and LPC, leading to interaction of this free LPC with cells [20]. The vascular function of the mostly studied, saturated LPC 16:0 is discussed controversially: Both has been described: a decrease as well as increase in eNOS synthesis and NO production [21]–[26] and consistently, a promoted or impaired endothelium-dependent relaxation [27]–[29].…”

Contradictory Effects of Superoxide and Hydrogen Peroxide on KCa3.1 in Human Endothelial Cells

“…1B). In addition, we examined the effects of LPC, a molecule involved in superoxide generation in endothelial cells [3], on K Ca 3.1 expression (Fig. 1C).…”

“…Moreover, endothelial cells themselves generate ROS by stimulation with various substances in plasma [1,2]. ROS play a key role in the physiological and pathological processes in endothelial cells; hydrogen peroxide upregulates endothelial NO synthase (eNOS) [3], and serves as an endothelium-derived hyperpolarizing factor (EDHF) that mediates vascular relaxation [4,5]. Conversely, superoxide impacts endothelial function by downregulating the expression of eNOS [3], thus mediating vascular contraction [6,7].…”

“…ROS play a key role in the physiological and pathological processes in endothelial cells; hydrogen peroxide upregulates endothelial NO synthase (eNOS) [3], and serves as an endothelium-derived hyperpolarizing factor (EDHF) that mediates vascular relaxation [4,5]. Conversely, superoxide impacts endothelial function by downregulating the expression of eNOS [3], thus mediating vascular contraction [6,7]. In addition, excessive ROS production damages endothelial cells, leading to endothelial dysfunction [1].…”