Lysophosphatidylcholine-induced modulation of Ca²⁺-activated K⁺channels contributes to ROS-dependent proliferation of cultured human endothelial cells

Abstract

Proliferation of endothelial cells plays a crucial role in the process of atherosclerotic plaque destabilization. The major component of oxidized low-density lipoprotein lysophosphatidylcholine (LPC) has been shown to promote endothelial proliferation by increasing the production of reactive oxygen species (ROS). Since K⁺ channels are known to control the cell cycle, we investigated the role of Ca²⁺-activated K⁺ channels (BK_Ca) in the regulation of LPC-induced endothelial proliferation and ROS generation. A significant increase of cell growth induced by LPC (20 μmol/l; cell counts (CCs): +87%, thymidin incorporation: +89%; n = 12, P < 0.01) was observed, which was inhibited by the BK_Ca inhibitor iberiotoxin (IBX; 100 nmol/l), by the NAD(P)H-oxidase inhibitor diphenyleneiodonium (5 μmol/l) and by transfection with antisense (AS) oligonucleotides against NAD(P)H oxidase, whereas N^G-monomethyl-l-arginine (l-NMMA) further increased LPC-induced cell growth. Using the patch-clamp technique a significant increase of BK_Ca open-state probability (control: 0.004 ± 0.002; LPC: 0.104 ± 0.035; n = 21, P < 0.05) by LPC was observed. Using dichlorofluorescein fluorescence microscopy a significant increase of ROS induced by LPC was reported, that was blocked by IBX and Ca²⁺ antagonists. Intracellular Ca²⁺ measurements revealed a capacitative Ca²⁺ influx caused by LPC. Bioactivity of nitric oxide (NO) was measured using a [³H]-cGMP radioimmunoassay. LPC significantly decreased acetylcholine-induced NO synthesis. LPC significantly increased cGMP levels in endothelial cells transfected with AS, which was blocked by IBX. In conclusion, our results demonstrate that LPC activates BK_Ca thereby increasing ROS production which induces endothelial proliferation. In addition LPC-induced BK_Ca-activation contributes to increased cGMP levels, if ROS production is prevented by AS.

Introduction

Atherosclerosis can be seen as a chronic inflammatory disease, in which increased cellular turnover on the one hand and apoptotic/necrotic cell death on the other hand takes place within the vascular wall [1], [2], [3]. Besides hypertension, diabetes mellitus, and nicotine abuse, low-density lipoproteins (LDLs) play a major role in the pathogenesis of atherosclerosis. The oxidative modification of LDL, during which lysophosphatidylcholine (LPC) accumulates within the LDL particle, seems to be a key event in this process [4].

It has been shown in an animal-atherosclerosis model, that endothelium-dependent vasorelaxation is impaired, if endothelium-derived nitric oxide (NO) or its biological activity is decreased [5], [6]. The vascular production of reactive oxygen species (ROS) is dramatically increased in atherosclerotic arteries, which is of great importance since superoxide is known to inactivate NO in a chemical reaction during which peroxynitrite is formed [7]. Therefore, oxidative inactivation of NO is regarded as an important cause of its decreased biological activity [8].

Another well recognized feature in atherosclerotic lesions is intimal angiogenesis. Angiogenesis is implicated in the development of atherosclerosis and associated clinical syndromes in the coronary circulation [9], [10]. It has been suggested that the vascular wall of these new blood vessels is weak and therefore subject to rupture. Intramural hemorrhage from these microvessels may precipitate the clinical complications of atherosclerosis with sudden expansion and rupture of the plaque in association with arterial dissection, ulceration, or thrombosis [11].

It has previously been shown that oxidized LDL (oxLDL) and LPC induce endothelial proliferation by increasing the superoxide production of the NAD(P)H oxidase [12]. Recently, we have reported that modulation of Ca²⁺-activated K⁺ channels (BK_Ca) is involved in oxLDL-induced endothelial proliferation and NO production [13]. BK_Ca have been shown to regulate the membrane potential and thereby influence intracellular Ca²⁺ levels. Endothelial cell functions are strongly influenced by changes of the intracellular Ca²⁺ homeostasis [14]. In endothelial cells, which do not express voltage-gated Ca²⁺ channels [15] changes of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) are mainly due to Ca²⁺ release from internal stores or through transmembrane Ca²⁺ influx, which depends on membrane hyperpolarization [14].

In this study, we investigated whether LPC modulates endothelial BK_Ca thereby influencing the intracellular Ca²⁺ homeostasis, and whether this ion channel is involved in the LPC-induced changes of ROS generation, cell proliferation, and NO bioactivity.