Ox-LDL Causes Endothelial Cell Injury Through ASK1/NLRP3-Mediated Inflammasome Activation via Endoplasmic Reticulum Stress

Abstract

Objective

This study was to investigate the mechanism of inflammatory pathology modification induced by ox-LDL in endothelial cells.

Methodology

In this study, we firstly investigated the efflux of cholesterol of endothelial cells under the treatment of ox-LDL, and cell proliferation, ROS production, cell apoptosis was measured. Further, proteins of ASK1, NLRP3 inflammasomes and endoplasmic reticulum stress response were detected. Afterwards, ASK1 inhibitor (GS-4997) or endoplasmic reticulum stress (ERS) inhibitor (4-PBA) was used to measure the performance of endothelial cells.

Results

In this study, endothelial cells were treated with ox-LDLs alone or in combination with a GS-4997 or 4-PBA. Results showed that ox-LDLs attenuated the efflux of cholesterol from endothelial cells in a dose-dependent manner. Ox-LDLs inhibited the proliferation of endothelial cells, and induced their apoptosis and production of reactive oxygen species (ROS). Additionally, ox-LDLs upregulated the levels of phosphorylated ASK1, ERS-related proteins (chop, p-PERK, GRP78, and p-IRE-1), and inflammation-associated proteins (NLRP3, IL-1β, and caspase 1) in endothelial cells. Moreover, we proved that GS-4997 could partly reverse ox-LDL-mediated cell proliferation, apoptosis, ROS production, and inflammation in endothelial cells, and increase cholesterol efflux. We also found that 4-PBA could attenuate the effects of ox-LDLs on endothelial cell cholesterol efflux, proliferation, apoptosis, ROS production, and inflammation.

Conclusion

Our results suggest that cholesterol efflux from endothelial cells is reduced by ox-LDLs, and these reductions in cholesterol efflux are accompanied by increased NLRP3 inflammasome signaling, ASK1 and higher levels of endoplasmic reticulum stress. Our results suggest this axis as potential targets for treating atherosclerosis.

Keywords:

• ox-LDL
• endoplasmic reticulum stress
• cholesterol efflux
• inflammatory corpuscles
• endothelial cell
• apoptosis signal-regulating kinase 1

Abbreviations

ASK1, apoptotic signal-regulating kinase 1; ABCA1, ATP binding cassette subfamily A member 1; ABCG1, ATP binding cassette subfamily G member 1; CHOP, C/EBP-homologous protein; CVD, cardiovascular disease; ER, endoplasmic reticulum; ERS, endoplasmic reticulum stress; NLRP3, NLR family pyrin domain containing 3; FITC, fluorescein isothiocyanate; GRP78, glucose-regulated protein 78 kD; IL-18, interleukin 18; IL-1β, interleukin 1β; IRE-1, Inositol-requiring enzyme-1; LD, low dose; MD, middle dose; HD, high dose; PERK, protein kinase R (PKR)-like endoplasmic reticulum kinase; PI, propidium iodide; ROS, reactive oxygen species; TNF-α, tumor necrosis factor α; UPR, unfolded protein response.

Data Sharing Statement

All data generated or analyzed during this study are included in this published article.

Author Contributions

LWH designed the experiments; LWH and YP performed the experiments and collected the data; LWH and RX analyzed and interpreted the data; XDL validated the data analysis; LWH and ZLL drafted the manuscript and ZLL and XDL revised and approved the manuscript. All authors approved the manuscript before submission. All authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.