Phytoestrogen α-Zearalanol Antagonizes Oxidized LDL-Induced Inhibition of Nitric Oxide Production and Stimulation of Endothelin-1 Release in Human Umbilical Vein Endothelial Cells

Abstract

Oxidative modification of low-density lipoprotein (LDL) leads to formation of the atherogenic molecule oxidized LDL (oxLDL), which is considered to be an important mediator for vascular endothelial dysfunction and atherosclerosis. It is speculated that reduced nitric oxide (NO) release/bioavailability and enhanced release of endothelin-1 (ET-1) may contribute to oxLDL-induced endothelial dysfunction. Estrogen may improve lipid profile and inhibit oxLDL-induced endothelial damage. However, estrogen replacement therapy has been suspended due to uncertainty in benefits versus risk (such as cancer progression) in postmenopausal women. This study was designed to evaluate the effect of a novel phytoestrogen, alpha-zearalanol (alpha-ZAL), on oxLDL-induced effect on NO and ET-1 production in human umbilical vein endothelial cells (HUVEC). HUVEC were incubated with oxLDL (50 microg/mL) for 24 h in the absence or presence of alpha-ZAL (0-1000 nM), 17beta-estradiol (E2, 10 nM), or the E2 receptor antagonist ICI182780 (1 microM). Levels of NO and ET-1 were measured by spectrophotometry and enzymatic immunoassay, respectively. NOS activity was evaluated by conversion of 3H-arginine to 3H-citrulline. Protein and mRNA expression of NOS and ET-1 were measured by Western blot and RT-PCR. Our results indicated that oxLDL significantly reduced NO release and NOS activity, and enhanced ET-1 pro-duction associated with reduced NOS3 (but not NOS2) expression and enhanced ET-1 mRNA expression. All these oxLDL-induced alterations were significantly attenuated or abolished by co-incubation with alpha-ZAL or E2, both through an E2 receptor-dependent mechanism. alpha-ZAL, E2, and ICI182780 had no effect on NO/ET-1 release, NOS activity, or expression of NOS and ET-1. These data suggested that the phytoestrogen alpha-ZAL, like E2, may effectively antagonize oxLDL-induced decrease in NO and increase in ET-1, which may be protective for endothelial function.

Reactive oxygen species mediate oxidized low-density lipoprotein-induced endothelin-1 gene expression via extracellular signal-regulated kinase in vascular endothelial cells

“…Confluent HUVEC cells were treated with oxLDL (35 mg/ml) for 24 h in the absence or presence of antioxidant NAC (10 mmol/l) before being collected for ROS, ET-1, protein and RNA analysis. Both dose (0-100 mg/ml) and time (0-48 h) response curves were constructed for oxLDL, which revealed a maximal effect on ET-1 production at 35 mg/ml with 24-h incubation in the absence of overt cytotoxicity (data not shown), consistent with our earlier report [6].…”

“…The pGL3-vector, luciferase and b-galactosidase enzyme assay kits were purchased from Promega (Madison, Wisconsin, USA). oxLDL was prepared as described previously [6].…”

“…Oxidized low-density lipoprotein (oxLDL), an important mediator for endothelial dysfunction and atherosclerosis, has been shown to act either independently or synergistically with ET-1 in inducing vascular smooth muscle cell proliferation by way of the redoxsensitive and mitogen-activated protein kinase (MAPK) pathways [5]. Although previous studies from our laboratories have indicated that oxLDL directly promotes gene expression and secretion of ET-1 [6], the precise regulatory mechanism of ET-1 gene induced by oxLDL in vascular endothelial cells still remains unclear.…”

Ellagic acid inhibits oxidized LDL-mediated LOX-1 expression, ROS generation, and inflammation in human endothelial cells

“…Decreased eNOS activity and enhanced release of endothelin-1 (ET-1) by oxLDL has been proposed to lead to an increase in oxidative stress, which would serve as a critical toxic mechanism for oxLDL in endothelial cells and is partially reversed by antioxidant treatment. 24 OxLDL antagonizes the vasodilatory effect of NO, and ROS and NO antagonize each other at the level of intracellular signaling; for example, NO released from eNOS inhibits cleavage of IB and NF-B activation, which is enhanced by ROS. 36 Consistent with a previous study that revealed that ellagic acid inhibits cytokine-induced adhesion molecule expression, 17 our data showed that the antiathrogenic effects of ellagic acid are due, at least in part, to the inhibition of adhesion molecules and subsequent monocytic adherence by moderating the reduction in eNOS expression caused by oxLDL.…”

“…24 To determine the effects of ellagic acid on oxLDL-induced ET-1 and IL-8 secretion, HUVECs were seeded in 24-well plates at 0.5 ϫ 10 5 cells. After 2 days, cells were pretreated with the indicated concentrations of ellagic acid for 2 hours followed by treatment with oxLDL (150 g/mL) for 24 hours.…”