Cyclophilin A mediates the ox-LDL-induced activation and apoptosis of macrophages via autophagy

doi:10.1016/j.ijcard.2016.12.042

International Journal of Cardiology

Volume 230, 1 March 2017, Pages 142-148

https://doi.org/10.1016/j.ijcard.2016.12.042 Get rights and content

Abstract

Background

Oxidized low-density lipoprotein (ox-LDL) is the most common inflammatory factor that mediates the activation and apoptosis of macrophages. Cyclophilin A (CyPA) is expressed following oxidative stress, hypoxia, and infection. However, the role of CyPA in the activation and apoptosis of macrophages is unclear. The aims of the study were to determine whether CyPA mediates the ox-LDL-induced activation and apoptosis in RAW264.7 cells and to analyze potential mechanisms.

Methods and results

Through Western blot and ELISA test, the expression of CyPA induced by ox-LDL is time-dependent in RAW264.7 cells. Gene silencing of CyPA reduced the generation of lipid droplets in the cytoplasm and downregulated the expression of the surface markers of macrophage activation, namely, CD80, CD86, and major histocompatibility complex class 2 antigen. Cell apoptosis is significantly decreased and the level of anti-apoptosis protein bcl-2 is increased in CyPA silent cells compared with the control group. Finally, autophagy-related protein LC3-II/LC3-I ratio level significantly decreased in CyPA silent cells with less autophagosome formation while the blocked autophagy flux was recovered. The differences in the activation and apoptosis between CyPA silent cells and the control cells were inhibited by pre-treatment with class III PI 3-kinase inhibitor 3-MA.

Conclusions

These results indicate that CyPA mediates the ox-LDL-induced activation and apoptosis in RAW264.7 cells by regulating autophagy.

Introduction

Macrophages are key players in the initiation and development of atherosclerosis. Under inflammation, macrophages are attracted by proinflammatory signals that initially attach to the problematic arterial sites and infiltrate the intima [1]. Abnormal hemodynamic forces and the accumulation of oxidized lipids in the arterial wall contribute to the vascular injury. In the subendothelial layer, macrophages were activated by the local stimuli and were transformed to foam cells. In this process, surface marks such as CD80, CD86, and major histocompatibility complex (MHC) class 2 antigen were upregulated and proinflammatory cytokine such as TNF-α, IL6, and tissue factor (TF) were secreted [2], [3]. At the same time, the activated cells undergo apoptosis. Scavenger receptor CD36, mediates macrophage uptake and degradation of ox-LDL, may be one crucial to macrophage-derived foam cell apoptosis [4], [5].

Autophagy is a widely known physiological process that helps cells to maintain cellular homeostasis and function [6]. Autophagy may protect plaque cells against oxidative stress by degrading the damaged material, in particular, polarized mitochondria in the very early stages before cytochrome c release occurs [7]. In contrast, severe or continuous oxidative stress may lead to an autophagic type of death [8]. In addition to the level of autophagy, excessive or insufficient levels of autophagic flux can each contribute to heart disease pathogenesis [6]. However, the precise mechanism of the relationship between autophagy and apoptosis is rarely known.

Cyclophilin A (CyPA), a ubiquitously expressed protein belonging to the cyclophilin family, has been highlighted as a major secreted oxidative inducing stress factors in atherosclerosis [9]. Normally, an intracellular protein like CyPA can nonetheless be secreted from monocytes or macrophages in response to ROS. Significant CyPA expression has been identified in advanced atherosclerotic lesions, indicating its potential role in ROS-induced vascular inflammation [10].

The mechanism by which CyPA interacts with macrophages is unclear. Our previous studies have shown that CyPA is one of the key proinflammatory factors in monocytes, perhaps especially in response to ROS stimulation [11], [12]. Pathogenic formation of ROS during atherosclerosis progression is stimulated by oxidized lipoprotein. A recent study demonstrated that the efferocytosis of ox-LDL-induced apoptotic cells was significantly improved by macrophage autophagy [13]. Therefore, we hypothesized that cyclophilin A also mediates the ox-LDL-induced activation and apoptosis of macrophages via autophagy.

Section snippets

Cell culture

Murine macrophage RAW264.7 cell line was purchased from the Cell Bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cells were cultured in DMEM (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Wisent, Montreal , Canada) at 37 °C with 5% CO2 in a humidified atmosphere. The cells were passaged everyday in a relatively low-density as other half suspension cells to maintain its resting-state. When incubated with ox-LDL (100 g/L)

Transient transfection

SiRNA for CyPA

Ox-LDL-induced CyPA expression in macrophages

We first tested if ox-LDL treatment altered the production of CyPA. As shown in (Fig. 1a–b), the expression of CyPA greatly increased while intracellular CyPA reached the peak at 6 h and released to the supernatant time-dependent RAW264.7 cells.

CyPA mediated the markers of autophagy and autophagy flux induced by ox-LDL in macrophages

The siRNA-targeting CyPA and the negative control siRNA were transiently transfected into RAW264.7 cells and the result was evaluated by Western blot analysis. The result confirmed that CyPA expression was significantly decreased in the CyPA-silencing

Discussion

Studies have shown highly expressed CyPA in advanced atherosclerotic lesions. Within the plaques, macrophages can secrete large amounts of CyPA when stimulated by ROS [14]. The results of our experiment showed that ox-LDL, which had been indicated to induce autophagy and apoptosis in various cells [15], [16], [17], [18], upregulated the level of CyPA (peak at 6 h) and induced the secretion of CyPA in RAW264.7 cells. These findings strongly suggest CyPA is most likely to be broadly involved in

Conflict of interest

The authors report no relationships that could be construed as a conflict of interest.

Acknowledgments/grant support

This work was supported by grants from the National Natural Science Foundation of China (81200222, 81370409), the Foundation of Health Department of Jiangsu Province (H201435), the Foundation of 333 talent of Jiangsu Province (BRA2015208) and Social development of Zhenjiang (SH2015035).

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¹: Zhi-qiang Xue and Wei Yuan contributed equally to this article.

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