The autophagy-lysosome pathway: A novel mechanism involved in the processing of oxidized LDL in human vascular endothelial cells

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Abstract

Oxidized low-density lipoprotein (ox-LDL) is involved in the pathogenesis of atherosclerosis and atherosclerotic plaque rupture by promoting lipid accumulation, proinflammatory responses, and cell death. LDL is mainly oxidized in the subendothelial layer of the vascular wall and then can be taken up by vascular endothelial cells. However, little is known about the intracellular processing of the damaged LDL. Previous studies found that autophagy is involved in degrading oxidized proteins under oxidative stress conditions in Arabidopsis thaliana, while ox-LDL can activate autophagy in EA.hy926 endothelial cells, suggesting a possible role of autophagy in the degradation of ox-LDL by endothelial cells. The present study showed that ox-LDL aggregated in human umbilical vein endothelial cells (HUVECs) and brought about an increase in the formation of autophagosomes and autolysosomes. Ox-LDL-induced increase in the autophagic level was blocked by treatment with the autophagy inhibitor 3-methyladenine and increased by the autophagy inducer rapamycin, while the aggregation of Dil-labled ox-LDL was increased by 3-methyladenine and decreased by rapamycin. In addition, Dil-labeled ox-LDL colocalized with the autophagy marker MDC, microtubule-associated protein light chain 3 (MAP1-LC3), and lysosome-associated membrane protein 2a (lamp2a). HUVECs treated with Dil-labeled-ox-LDL showed a much greater degree of overlap of MAP1-LC3 and Lamp2a than control. The results suggest that ox-LDL activates the autophagic lysosome pathway in HUVECs through the LC3/beclin1 pathway, leading to the degradation of ox-LDL.

Introduction

Lipoprotein endothelial cell interactions have been implicated in the initiation of atherosclerosis [1], [2]. Recently, much attention has focused on the role of modified lipoproteins, especially low-density lipoprotein (LDL), in the initiation and propagation of atherosclerosis. Certain modified forms of LDL, e.g., oxidized LDL, are taken up by lectin-like oxidized low-density lipoprotein-1 (LOX-1) and scavenger receptors that are present on endothelial cells [3], [4], [5]. However, little is known about how these cells process the damaged LDL.

There are two major proteolytic systems for bulk degradation in eukaryotes: the proteasome, and vacuolar autophagy. In mammalian cells, the ubiquitin–proteasome pathway carry out the degradation of oxidized proteins in conditions of mild oxidative stress. Moderately oxidized proteins are substrates for the proteasome, but heavily oxidized proteins are no longer degradable, accumulate, and may become toxic [6], [7]. Ox-LDL alters the activity of the ubiquitin proteasome pathway [6]. When the proteasome is impaired, autophagy provides a possible alternate pathway for clearing aggregated ubiquitinated proteins [8]. In Arabidopsis thaliana, autophagy has been shown recently to act in the degradation of oxidized proteins following severe oxidative stress [9].

Autophagy is a reparative, life-sustaining process by which cytoplasmic components are sequestered in double-membrane vesicles and degraded on fusion with lysosomes. Several studies have demonstrated the presence of increased numbers of autophagosomes in EA.hy926 endothelial cells [10], [11]. However, the effect of ox-LDL on the level of autophagy in human vascular endothelial cells (HUVECs) is still unknown. Previous reports have shown that ox-LDL causes the unfolded protein response, which induces autophagy leading to the degradation of misfolded proteins [12]. However, it is not clear if ox-LDL is degraded by this process. Although it is involved in the metabolism of unoxidized lipids [13], [14], [15], there is still no direct evidence to support a role for autophagy in the removal of ox-LDL.

Here, we hypothesized that autophagy in HUVECs is activated by ox-LDL, and such activation may contribute to degradation of ox-LDL through lysosomes. To investigate the autophagic mechanisms in the metabolism of ox-LDL, the present study observed activation of autophagy and lysosomes after ox-LDL exposure, the effects of autophagy inducer rapamycin and inhibitor 3-methyladenine (3-MA) on ox-LDL-induced alterations in the expression of autophagy-related proteins, and aggregation of ox-LDL in HUVECs. The results suggest, primarily, that an autophagic mechanism contributes to the metabolism of ox-LDL.

Section snippets

Cell culture

HUVEC-2c cultures were maintained in 5% CO2 at 37 °C in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 44 mM NaHCO3, 4 mM Hepes and 2 mM glutamine. Subconfluent cultures were stimulated under complete culture medium for 0, 6, 12, and 24 h before fixation or harvesting. Treatment conditions and compounds used were ox-LDL (100 μg/ml), rapamycin (10 nM, Sigma–Aldrich), 3-methyladenine (3-MA; 10 mM, Sigma–Aldrich), leupeptin (20 μM, Sigma–Aldrich) and Pepstatin A (10 μM, Sigma–Aldrich).

Autophagy induction by ox-LDL in HUVECs

Ox-LDL treatment intensifies autophagy in EA.hy926 cells under serum starvation [10]. In the present work, we investigated whether the exposure of ox-LDL also induces autophagy in human umbilical vein endothelial cells (HUVEC) leading to the degradation of ox-LDL.

LC3-II and beclin1 are two well-known markers of autophagy. We first verified whether these two proteins are upregulated in response to stimulation of ox-LDL on endothelial cells. HUVECs cultivated as monolayers were stimulated with

Acknowledgments

This work is supported by the Suzhou Social Progress Foundation (SZD0875), and Plans for Graduate Research and Innovation in Colleges and Universities of Jiangsu Province (CX09B_036Z). We also thank Professor IC Bruce for critical reading of the manuscript.

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