iRhom2 promotes atherosclerosis through macrophage inflammation and induction of oxidative stress

https://doi.org/10.1016/j.bbrc.2018.07.133Get rights and content

Highlights

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    Increase of iRhom2 contributes to ox-LDL-incubated macrophages activation.

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    Down-regulation of iRhom2 expression attenuates inflammation in ox-LDL-incubated macrophages inflammatory infiltration.

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    iRhom2-knockdown alleviates oxidative stress in ox-LDL-incubated macrophages activation.

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    iRhom2 is expressed highly in HFD-fed ApoE−/- mouse artery.

Abstract

Atherosclerosis is a complex chronic inflammatory disease that is characterized by the formation of lipid-rich plaques on the inner walls of the arteries. Inactive rhomboid protein 2 (iRhom2) was recently determined as a necessary regulator for the shedding of tumor necrosis factor-alpha (TNF-α) in immune cells. In the present study, we aimed to explore the effects of iRhom2 on the inflammatory response and oxidative stress induced by low-density lipoprotein (ox-LDL) in RAW264.7 and THP-1-derived macrophages. The expression levels of iRhom2 were also investigated in apolipoprotein E knockout (ApoE−/-) mice fed a high-fat diet (HFD). iRhom2 was significantly induced by ox-LDL in macrophages, as confirmed by Western blotting and RT-qPCR analysis. Furthermore, iRhom2 knockdown showed significant suppressive effects on the activation of ox-LDL-induced RAW264.7 and THP-1-derived macrophages through reducing TACE and TNFR2 expressions, and the inactivation of the IκBα/NF-κB signaling pathway. A reduction in reactive oxygen species (ROS) generation, malondialdehyde (MDA) levels, and nitric oxide (NOX) activity and an increase in glutathione peroxidase (GSH-Px) activity were determined in the absence of iRhom2 expression. In addition, the NF-E2 related factor-2 (Nrf-2)/heme oxygenase-1 (HO-1) pathway was also upregulated in ox-LDL-treated macrophages subjected to iRhom2 inhibition. Moreover, suppression of iRhom2 expression inactivated PI3K/AKT pathway activation, contributing to ROS reduction in ox-LDL-stimulated macrophages. iRhom2 was also significantly expressed in ApoE−/- mice fed HFD. Finally, we observed increased serum levels of TNF-α, TNFR1, and TNFR2 in patients with coronary artery atherosclerosis as compared to healthy volunteers. In conclusion, our findings suggested that iRhom2 played a key role in the pathogenesis of atherosclerosis, and that iRhom2 might be a potential therapeutic target against atherosclerosis.

Introduction

Atherosclerosis has been reported as the leading cause of death worldwide [1]. Atherosclerosis is a chronic inflammatory disease that is characterized by the accumulation of lipids and fibrous elements in large arteries [2,3]. Oxidized low density lipoproteins (ox-LDLs) and macrophages inflammation play essential roles in the progression of atherosclerosis by enhancing intracellular lipid accumulation and foam cell formation [4]. ox-LDL results in ROS production and the secretion of inflammatory factors, which further contribute to atherosclerosis development [5]. Moreover, advanced atherosclerosis has been confirmed in atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−) mice fed a high fat diet [6]. However, the molecular mechanisms by which ox-LDL-induced inflammation and elevated oxidative stress, as well as atherosclerotic lesion pathogenesis are not fully understood.

iRhom2, also known as Rhbdf2, is an inactive member of the rhomboid intramembrane proteinase family that was recently determined to be an important modulator for the release of TNF-α through crosstalk with TNF-α convertase (TACE) [7]. Studies have demonstrated that the elevated TNF-α, caused by iRhom2 in inflammatory or rheumatoid arthritis models, indicated a positive feedback process in iRhom2/TACE/TNF-α signaling [8,9]. In addition, a deficiency of iRhom2 contributed to the suppression of TNF-α-mediated hepatic dyslipidemia [10]. Hence, these observations suggest that iRhom2 might be useful in inactivating TACE, reducing TNF-α secretion, and decreasing inflammatory responses. The role of macrophages in initiating the inflammatory response has been confirmed. While a high expression of iRhom2 occurs in activated macrophages, the process of TACE trafficking in response to ox-LDL treatment has not been investigated.

The present study aimed to explore the effects of iRhom2 on atherosclerosis progression in vitro and in vivo. The findings indicated that iRhom2 was significantly enhanced in ox-LDL-treated macrophages. The suppression of iRhom2 alleviated the inflammatory response and oxidative stress in ox-LDL-treated macrophages. In vivo, iRhom2 was significantly expressed in the aortic tissues of ApoE−/− mice fed HFD. We proposed that iRhom2 might be a potential target for preventing atherosclerosis development.

Section snippets

Human participants

20 patients with coronary artery atherosclerosis and 20 healthy participants were recruited into the study. The healthy participants were free from the history of cardiovascular diseases. With the history of chest pain or angina as the diagnosis hints, the coronary artery atherosclerosis was diagnosed by angiography with our established routine procedures. The demographic data of human participants were listed in Supplementary Table 1. Those with severe autoimmune diseases, organ diseases,

Increase in iRhom2 contributes to ox-LDL-incubated macrophage activation

As shown in Fig. 1A and B, cell viability was respectively reduced in RAW264.7 and THP-1 cells to 49.13 ± 1.35% and 50.28 ± 2.66% in response to 100 μg/ml ox-LDL treatment compared to the Control group. Consequently, 100 μg/ml ox-LDL was used in follow-up experiments. As shown in Fig. 1C and D, a significant increase of iRhom2, TACE, and TNFR2 expression was evident in RAW264.7 and THP-1 cells stimulated with ox-LDL. Consistently, immunofluorescent analysis indicated that ox-LDL incubation

Discussion

Rhomboids, a conserved subfamily of proteins linked to rhomboid intramembrane serine proteases, are reported to have various functions related to protein-factor release, mitochondrial reconstitution, apoptosis, as well as growth factor-mediated signaling transduction [8,9,15]. Accordingly, rhomboids participate in disease pathologies, such as rheumatoid and inflammatory arthritis, tumors, hepatic injury, and bowel disease [15]. Recently, inactive rhomboid protein 2 (iRhom2, also known as

Acknowledgment

We would like to thank LetPub (www.letpub.com) for providing linguistic assistance during the preparation of this manuscript.

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    The first two authors contributed equally to this work.

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