Lipid accumulation and lysosomal pathways contribute to dysfunction and apoptosis of human endothelial cells caused by 7-oxysterols

https://doi.org/10.1016/j.bbrc.2011.05.071Get rights and content

Abstract

Endothelial dysfunction and cell death play an important role in pathogenesis of atherosclerosis. 7-Oxysterols, the major cytotoxic component found in oxidized low-density lipoprotein, are toxic to endothelial cells. However, the pathways and molecular mechanism involved in the process remain incompletely understood. In this study, we first investigate whether 7β-hydroxycholesterol (7βOH) or 7-ketocholesterol (7keto) induces apoptosis of human endothelial cell line (HUVEC-CS). We then examine possible involved pathways by focusing on cellular lipid, lysosomal pathways, cellular oxidative stress and mitochondrial pathways. Our results for the first time showed that 7-oxysterols induced apoptotic cell death of HUVEC-CS after 24 h, which was preceded by early lipid accumulation (6 h) and lysosomal membrane permeabilization (6−12 h). Afterward, levels of reactive oxygen species, mitochondrial membrane permeabilization, and lysosomal cathepsin were increased assayed by immuno-cytochemistry and blotting. Notably, the exposure to 7βOH or 7keto induced expressions and secretion of isoforms of von Willebrand factor (VWF). We conclude that apoptosis of HUVEC-CS induced by 7βOH or 7keto mediates by early lysosomal lipid accumulation and oxidative lysosomal pathways, which results in induction and release of VWF. The results suggest the cell death induced by 7-oxysterols may contribute to endothelial dysfunction and atherothrombosis.

Highlights

► 7βOH or 7keto induces early lipid accumulation and lysosomal membrane permeabilization. ► 7βOH or 7keto induces secretion of lysosomal cathepsin D, and up-regulation and release of VWF. ► Oxidative stress and cytosolic release of cytochrome c are involved in apoptosis of endothelial cells caused by 7βOH or 7keto.

Introduction

Inflammation and thrombosis play major roles in the pathogenesis and acute cardiac events of atherosclerosis. Cell injury and cell death appear to promote plaque rupture and thrombus formation [1]. Endothelial cell (EC) death in atherosclerotic lesions may also contribute to atherothrombosis [2]. In vivo, endothelial cell apoptosis has been implicated in advanced atherosclerosis. Increased levels of EC apoptosis have been reported in atherosclerotic internal carotid artery plaque [3] and even symptomatic high-grade carotid artery sclerotic patients [4].

Apoptosis of endothelial cells has been associated with inflammatory cytokines, oxidized low-density lipoprotein (oxLDL), and oxidative stress [2]. It has been shown that circulating levels of autoantibodies to oxLDL inversely correlate with endothelial function in resistance arteries in men with coronary heart disease [5]. Oxysterols constitute the major toxic component in oxLDL and are present in human atheroma lesions [6], [7]. 7β-Hydroxycholesterol (7βOH) in circulation has been related to the progression of atherosclerotic disease. Increased level of 7βOH has been associated with a high risk of cardiovascular disease [8] and coronary atherosclerotic patients [9]. 7βOH and 7-ketocholesterol (7keto) are cytotoxic to different types of arterial cells, such as smooth muscle cells, macrophages and endothelial cells [10], [11], [12]. Moreover, equivalent concentration of oxysterols as found in plasma of patients with vascular disease were toxic to cultured endothelial cells [13]. Activation of mitochondrial pathway in oxysterol-mediated apoptosis has been well documented, including endothelial cells [14]. Recently the lysosomal pathway and related cathepsins in the apoptosis machinery have been further recognized in a number of cell models [15]. However, whether lysosomal pathway and related cathepsins are involved in the 7-oxysterol induced apoptosis of endothelial cells and how 7-oxysterol influence cellular lipid metabolisms in the process remain unexplored.

von Willebrand factor (VWF) is generally regarded as a marker of dysfunction or injury of endothelial cells [16]. In plasma of patients with inflammatory vascular disease expression of VWF increases [17], which has been suggested as a predictor of asymptomatic atherosclerosis [18]. Although exposure to oxLDL induces release of VWF from endothelial cells [19], whether 7-oxysterols, as major toxic lipids found in oxLDL, alter expression and secretion of endothelial VWF remain unknown.

In the present study, we investigated the effects of 7βOH and 7keto on human umbilical vein endothelial cell line (HUVEC-CS), mainly focusing on cellular lipid, cell death pathways and VWF expression.

Section snippets

Cell culture and experimental conditions

The human endothelial cell line (HUVEC-CS) was from American Type Culture Collection (Manassas, USA). The cells were cultured in 25 cm2 gelatin-coated tissue-culture flasks in DMEM culture medium with 20% fetal bovine serum, 10 units penicillin G and 10 μg streptomycin (GIBCO, Paisley, UK).

The cells were sub-cultivated when they were confluent and seeded into 35 mm2 gelatin-coated cultured dishes with or without coverslips and were cultured for 1–2 days before experiments. For the experiments, the

The 7βOH is more cytotoxic to endothelial cells than 7keto

The potential toxicity of 7βOH or 7keto to HUVEC-CS was analyzed by assessment of morphological changes following Giemsa or AV/PI staining. As compared to control cells (Fig. 1A), 7βOH (Fig. 1B) and 7keto (Fig. 1C) induced cell shrinkage and nuclear condensation (arrows), and apoptotic body formation (arrow heads). The summarized results showed that there were no notable apoptosis at 12 h after treatment with 7βOH or 7keto (Fig. 1D). A significant increase in apoptosis was observed after 24 h,

Discussion

Increased plasma levels of 7-oxysterols and apoptosis of endothelial cells contribute to development and progression of atherosclerotic lesions and myocardial injury. In the present study, we found that both 7βOH and 7keto caused apoptosis of endothelial cells associated with early accumulation of cellular lipids and activation of lysosomal apoptosis pathway, which was followed by cellular oxidative stress, mitochondrial damage, up-regulation and release of VWF or lysosomal cathepsin D.

Acknowledgments

This work was supported by the Swedish Heart Lung Foundation, the County Council of Östergötland (ALF), the ÖLL Research Foundation, and research founds of Torsten och Ragnar Söderbergs, Gamla Tjänarinnor and Olle Engkvist.

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