Trimethylamine N-oxide induces inflammation and endothelial dysfunction in human umbilical vein endothelial cells via activating ROS-TXNIP-NLRP3 inflammasome

https://doi.org/10.1016/j.bbrc.2016.11.017Get rights and content

Highlights

  • TMAO triggered ROS and activate TXNIP-NLRP3 inflammasome.

  • eNOS/NO were obviously inhibited by TMAO.

  • TMAO-mediated abnormities were abrogated by siRNA-TXNIP or NLRP3, especially followed by NAC.

Abstract

Recent research demonstrates that the choline-derived metabolite trimethylamine-N-oxide (TMAO) levels are strongly associated with atherosclerosis and cardiovascular risks. The NLRP3 inflammasome responds to exogenous and endogenous danger signals involved in the development of atherosclerosis. Moreover, thioredoxin-interactive protein (TXNIP) activation is a key event linked to NLRP3 inflammasome via reactive oxygen species (ROS). Whether TMAO prime NLRP3 inflammasome via ROS-TXNIP pathway remains unclear. This study observed the expression of TXNIP-NLRP3 inflammasome stimulated by TMAO in human umbilical vein endothelial cells (HUVECs), aiming to elucidate the mechanism by which the TMAO may contribute to inflammation and endothelial dysfunction. Our data showed that TMAO significantly triggered oxidative stress and activated TXNIP-NLRP3 inflammasome whereat inflammatory cytokines interleukin (IL)-1β and IL-18 were released in a dose- and time-dependent manner, but endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) were inhibited. Moreover, TMAO-mediated effects were observably reversed by ROS inhibitor N-acetylcysteine (NAC) treatment or siRNA-mediated knockdown TXPIN and NLRP3. Taken together, our results firstly reveal that TMAO induces inflammation and endothelial dysfunction via activating ROS-TXNIP-NLRP3 inflammasome, suggest a likely mechanism for TMAO-dependent enhancement in atherosclerosis and cardiovascular risks.

Introduction

The dietary choline and l-carnitine when reaching the gut are metabolized by microbiota and produce an intermediate compound known as trimethylamine (TMA) [1]. TMA is absorbed in turn and travels via the portal circulation to the liver, where it is oxidized by enzyme flavin-containing monooxygenases (FMO), primarily FMO3, to trimethlyamine-N-oxide (TMAO) [2]. Previous studies observed significant positive associations of TMAO plasma levels with cardiovascular risk and heart failure [3], impaired glucose tolerance in mice [4], and recently also colorectal cancer [5]. TMAO was recently identified as a promoter of atherosclerosis partly by promoting the formation of foam cells from macrophage, but an important question that remains is how this small molecule alter systemic homoeostasis and subsequent events [6].

Growing evidences suggest that atherosclerosis are mediated by the inflammasome, a large multiprotein complex in the cytosol and a component of the innate immune system, which acts as both a sensor and a regulator of the inflammatory response. The nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, the best characterized inflammasome to date, contains the NLRP3 associated with the adapter protein, apoptosis associated speck-like protein (ASC), and cysteinyl aspartate specific proteinase-1 (caspase-1) [7]. NLRP3 interacts with ASC to cleave caspase-1, leading to maturation and secretion of the pro-inflammatory cytokines IL-1β and IL-18, which cause inflammatory responses and play a vital role in promoting the development of lipid plaques and destabilizing the plaques of atherosclerosis [8]. However, whether NLRP3 inflammasome is involved in the TMAO-mediated pathophysiological changes remains unknown.

Oxidative stress and inflammation are inseparably linked as each causes and intensifies the other, which play a critical role in pathogenesis of atherosclerosis. Inflammasome activators, such as cholesterol crystals, saturated fatty acids and reactive oxygen species (ROS), induced the dissociation of thioredoxin-interactive protein (TXNIP) from thioredoxin (TRX) and the direct interaction between TXNIP and NLRP3, leading to the activation of the NLRP3 inflammasome [9]. It has been widely recognized that the production of ROS leads to the activation of the NLRP3 inflammasome via the ROS-sensitive TXNIP protein and TXNIP-NLRP3 activation is involved in the pathogenesis of cardiovascular diseases [10]. Some compounds may confer protective effects by targeting TXNIP-NLRP3 pathway, such as N-acetylcysteine (NAC), an antioxidant that acts as a free radical scavenger and eliminates ROS in oxidative stress [11]. Based on these findings, the present study was designed to investigate the effects of TMAO on ROS-TXINP-NLRP3 inflammasome pathway, the expression of endothelial nitric oxide synthase (eNOS) and the production of nitric oxide (NO) in human umbilical vein endothelial cells (HUVECs), and then observed the changes of the players using NAC or Small interfering RNAs (siRNA)-mediated knockdown TXPIN and NLRP3, aiming to elucidate the role of TMAO in pathogenesis of atherosclerosis and provide some new insights into treatment of TMAO-associated complications.

Section snippets

Cell culture and treatment

HUVECs were isolated from the vein of a normal human umbilical cord and cultured in DMEM-F12 medium (Hyclone, UT, USA) supplemented with 10% fetal bovine serum (Gibco, CA, USA), 2 mmol/L l-glutamine (Beyotime), 5U/mL heparin (Sigma-Aldrich, MO, USA), 100U/mL penicillin and 100 μg/mL streptomycin (Beyotime, China) and 50 μg/mL Endothelial Cell growth supplement (Sigma-Aldrich, MO, USA). The cells were seeded and grown in 6-well plates at concentrations of 1 × 105 cells/ml under humidified 5% CO2

TMAO induced oxidative stress, inflammatory conditions, and endothelial dysfunction in HUVECs

The quantitative assay showed that intracellular ROS (Fig. 1A) and MDA (Fig. 1B) in the different concentrations of TMAO stimulus group were obviously higher than NC group, the highest relative production of these factors was observed in the 300 μmol/L TMAO group after stimulation of 6 h; while the levels of SOD (Fig. 1C) were significantly decreased by TMAO treatment (p < 0.05). Meanwhile, the inflammatory cytokine IL-1β (Fig. 1D) and IL-18 (Fig. 1E) release from HUVECs was obviously increased

Discussion

TMAO is a metabolite of phosphatidyl choline and l-carnitine, both abundant in red meat [12]. Previous report showed that administration of TMAO was strongly correlated with plaque size; TMAO enhanced uptake of cholesterol in peritoneal macrophages, a critical step in atherosclerosis [13]. However, TMAO also possesses several unique and intriguing biophysical properties, it is well established that TMAO plays a protective role in cell homeostasis in numerous animal species. For example, cells

Acknowledgments

This work was supported by the Youth Innovation and Medical Research of Sichuan Province (YIMRSP) grant from the Sichuan Medical Association Fund of China (grant numbers: Q15043). We would like to thank the Center of Peripheral Vascular Surgery (Affiliated Hospital of Southwest Medical University). The authors also thank BioMed Proofreading LLC for English expression polished.

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