TRAF6 mediates high glucose-induced endothelial dysfunction

doi:10.1016/j.yexcr.2018.07.014

Experimental Cell Research

Volume 370, Issue 2, 15 September 2018, Pages 490-497

https://doi.org/10.1016/j.yexcr.2018.07.014 Get rights and content

Highlights

•
TRAF6 mediated high glucose-induced endothelial dysfunction.
•
NF-κB- and AP-1-dependent signaling served as mechanism.
•
Targeting TRAF6 may delay progression of vascular diseases during diabetes mellitus and atherosclerosis.

Abstract

To investigate the role of tumor necrosis factor-associated factor 6 (TRAF6) in high glucose-induced endothelial cell dysfunction. Human aortic endothelial cells (HAECs) were cultured in high glucose medium, and TRAF6 expression was assayed by quantitative real-time Polymerase Chain Reaction (PCR) and western blotting. The effect of TRAF6 on in vitro endothelial cell viability, apoptosis, migration, and endothelial–monocyte adhesion was investigated by gene knockdown. The expression of TRAF6 and related adhesion molecules was assayed in a mouse streptozotocin-induced type I diabetes model. The signaling pathways associated with TRAF6 effects on endothelial cells were investigated in high glucose HAEC cultures. Culture of HAECs in high glucose medium significantly increased TRAF6 mRNA and protein expression in a time dependent manner. High glucose markedly reduced HAEC viability, apoptosis, and migration, and these effects was significantly reversed by TRAF6 knockdown. High glucose significantly increased intercellular adhesion of THP-1 monocytic cells and HAECs via upregulation of ICAM-1 and VCAM-1 expression, and TRAF6 knockdown attenuated the effect on THP-1 cell adhesion. TRAF6, ICAM-1, and VCAM-1 expression were increased in aorta tissue of mice with streptozotocin-induced diabetes. The free radical scavenger N-acetyl-L-cysteine attenuated TRAF6 expression in HAECs cultured in high glucose medium, and TRAF6 knockdown inhibited high glucose-induced IκB-α degradation and JNK phosphorylation. TRAF6 mediated high glucose-induced endothelial dysfunction via NF-κB- and AP-1-dependent signaling. Targeting TRAF6 may delay progression of vascular diseases during diabetes mellitus and atherosclerosis.

Introduction

Diabetes mellitus (DM) is characterized by chronic hyperglycemia, and impaired insulin secretion or action, or a combination of both, and is often associated with the development of vascular complications [1]. The prevalence of DM is about 2–5% in most western countries, and is rapidly increasing in Asian countries because of ongoing life style changes [2]. Effective strategies for DM treatment are urgently needed.

The simple endothelium that lines the internal surface of blood vessels is important for maintaining vascular tone and structure [3]. Substantial clinical and experimental evidence suggests that both DM and insulin resistance are responsible for endothelial changes that reduce the anti-atherogenic function of vascular endothelium [4], [5], [6], but the underlying mechanisms are not clear.

Previous studies have described TNF receptor associated factor (TRAF) mediated inflammatory responses [7], [8], [9]. TRAF6 acts downstream of cytokine and toll-like receptors (TLRs) to regulate the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [10], which plays a key role in the progression of inflammation-related diseases [11], [12], [13]. However, the activity of TRAF6 in DM at the molecular has not been determined.

We investigated the role of TRAF6 in vitro in a high glucose-induced model of endothelial cell dysfunction and in vivo in a type I diabetes mouse model. We found that TRAF6 mediated high glucose-induced endothelial dysfunction via NF-κB- and activator protein (AP)-1-dependent signaling. Targeting TRAF6 in patients with DM and atherosclerosis might delay progression of vascular diseases.

Section snippets

Ethical approval of the study protocol

All research involving human participants was approved by the Institutional Review Board of Shanghai Jiaotong University School of Medicine, Shanghai, China. All participants gave written informed consent. The study protocol was approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine. The study was conducted following international guidelines for animal experimentation.

Cell culture

Human aortic endothelial cells (HAECs) were purchased from Lonza Co.

High glucose induces TRAF6 expression in HAECs

qPCR and western blotting revealed that high glucose significantly increased TRAF6 mRNA (Fig. 1A) and protein (Fig. 1B) expression in HAEC after 30 min of incubation, and that the levels remained elevated even after 8 h.

TRAF6 knockdown significantly reduces high glucose-induced endothelial cell dysfunction

Because high glucose elevated TRAF6 expression, we investigated the relationship of TRAF6 expression and endothelial cell dysfunction by gene knockdown in cell culture models (Fig. 2A). High glucose markedly reduced HAEC viability (Fig. 2B) and apoptosis (Fig. 2C), but those

Discussion

We demonstrated that culture in high glucose medium significantly increased TRAF6 mRNA and protein expression in HAEC in a time dependent manner. In the functional assay, we found that high glucose markedly reduced HAEC viability, apoptosis, and migration, and that these effects were significantly reversed by TRAF6 knockdown. Moreover, high glucose significantly increased adhesion of THP-1 monocytes by upregulating ICAM-1 and VCAM-1 expression, whereas TRAF6 knockdown attenuated that effect.

Conclusion

In conclusion, TRAF6 mediated high glucose-induced endothelial dysfunction via NF-κB and AP-1-dependent signaling. Treatments targeting TRAF6 might delay progression of vascular diseases in patients with DM and atherosclerosis.

Acknowledgement

None.

Disclosure of conflict

None.

Funding

This work was supported by the National Natural Science Foundation of China (Grant number 81300177, 81770282), Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (Grant number 20152232), National Natural Science Foundation of China (Grant number 81300177) and Shanghai Jiao Tong University (YG2013MS52), Project supported by Shanghai Jiao Tong University Affiliated Sixth People's Hospital (1715).

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¹: These authors contributed equally to this work.

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TRAF6 mediates high glucose-induced endothelial dysfunction

Highlights

Abstract

Introduction

Section snippets

Ethical approval of the study protocol

Cell culture

High glucose induces TRAF6 expression in HAECs

TRAF6 knockdown significantly reduces high glucose-induced endothelial cell dysfunction

Discussion

Conclusion

Acknowledgement

Disclosure of conflict

Funding

Biochim. Biophys. Acta

Nutr. Res.

Biochim. Biophys. Acta

Pharmacol. Rep.: PR

Free Radic. Biol. Med.

Mol. Cell. Endocrinol.

Atherosclerosis

Neuroscience

Cell

The American Diabetes Association (ADA) has been actively involved in the development and dissemination of diabetes care standards, guidelines, and related documents for many years