TNF-α Activates High-Mobility Group Box 1 - Toll-Like Receptor 4 Signaling Pathway in Human Aortic Endothelial Cells

Background/Aims: Toll-like receptor 4 (TLR4) interacts with endogenous substances as well as lipopolysaccharide. We explored whether TLR4 is implicated in tumor necrosis factor-α (TNF-α) signal transduction in human aortic endothelial cells. Methods: The pathway was evaluated by transfection of siRNAs, immunoprecipitation and Western blot analysis. Results: TNF-α activated spleen tyrosine kinase (Syk) within 10 min, which led to endothelin-1 (ET-1) production. TLR4 was also rapidly activated by TNF-α stimulation, as shown by recruitment of interleukin-1 receptor-associated kinase 1 to TLR4 and its adaptor molecule, myeloid differentiation factor 88 (MyD88). siRNA depletion of TLR4 markedly attenuated TNF-α-induced Syk activation and ET-1 production. TLR4 inhibitor (CLI-095), TLR4-neutralizing antibody and siRNA depletion of MyD88 also attenuated TNF-α-induced Syk activation. Syk was co-immunoprecipitated with TLR4, and TNF-α activated Syk bound to TLR4. High-mobility group box 1 (HMGB1) was rapidly released and associated with TLR4 after TNF-α stimulation with a peak at 5 min, which was prevented by N-acetylcysteine, an antioxidant. Glycyrrhizin (HMGB1 inhibitor), HMGB1-neutralizing antibody and siRNA depletion of HMGB1 all suppressed TNF-α-induced Syk activation and ET-1 production. Conclusion: Upon TNF-α stimulation, TLR4 is activated by HMGB1 that is immediately released after the generation of reactive oxygen species, and plays a crucial role in the signal transduction.

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Abstract 97: High mobility group box-1 Promotes Restenosis Via Toll like receptor-4 Signal Pathway

Circulation Research ◽

10.1161/res.115.suppl_1.97 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Jingjing Cai ◽

Hong Yuan ◽

Qingde Wang ◽

Timothy R. Billiar ◽

Alex F. Chen

Keyword(s):

High Mobility ◽

Arterial Injury ◽

High Mobility Group ◽

Sterile Inflammation ◽

Mouse Strains ◽

Dependent Manner ◽

Toll Like Receptor ◽

Inhibitory Peptide ◽

Toll Like Receptor 4 ◽

Tnf Α

Background: High-mobility group box 1 (HMGB1) is an endogenous molecule released during cell stress and death termed damage-associated molecular patterns (DAMPs). HMGB1 activates the pattern recognition receptor, toll-like receptor 4 (TLR4), and induces sterile inflammation. However, how HMGB1 and TLR4 affect restenosis, the major complication following balloon and stent intervention clinically, remains unknown. We tested the hypothesis that HMGB1 released following acute arterial injury promotes intimal hyperplasia (IH), a hallmark of restenosis, via TLR4 signaling pathway. Methods and Results: Wire injury of the carotid artery in C57BL/6 wild-type (WT) mice significantly increased intima-to-media ratio in 4 weeks. Global deletion of HMGB1 using an inducible knockout mouse strain prevented IH and vessel remodeling. IH decreased by over 50% in WT mice treated with a HMGB1 neutralizing antibody. Of the mouse strains deficient in putative receptors and co-regulator for HMGB1 (TLR4-/-, TLR2-/-, RAGE-/- and CD14-/-), TLR4-/- mice showed the greatest inhibition of IH after injury. Both TLR4 adaptors MYD88 and TRIF synergistically participated in the inflammatory response to vascular injury. HMGB1 antibody-treated mice and TLR4-/- mice showed a marked decrease in monocytic recruitment following injury. Mice with selective depletion of TLR4 from macrophages (TLR4-/--Mø) exhibited similar level of IH inhibition and macrophage infiltration, compared to the global TLR4-/- mice. In vitro, disulfide HMGB1 concentration-dependently promoted smooth muscle cell (SMC) migration and MCP-1/CCR2 expression, which were abolished by treatment with TLR4 inhibitory peptide. Moreover, conditioned media from HMGB1-treated macrophage induced SMC proliferation, which was blunted by blocking TLR4 on macrophage, but not SMCs. Finally, HMGB1 increased cytokine (TNF-α and IL-6), chemokine (MCP-1) and mitogen (PDGF-A) levels in macrophage in a TLR4-dependent manner. Conclusion: These findings demonstrate, for the first time, that HMGB1 released following acute arterial injury promotes restenosis via SMC migration and MCP-1/CCR2 production as well as macrophage-released TNF-α, IL-6, MCP-1 and PDGF-A in SMC through the TLR4-MyD88/TRIF signal cascade.

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ADAM17-Mediated Ectodomain Shedding of Toll-Like Receptor 4 as a Negative Feedback Regulation in Lipopolysaccharide-Activated Aortic Endothelial Cells

Cellular Physiology and Biochemistry ◽

10.1159/000487876 ◽

2018 ◽

Vol 45 (5) ◽

pp. 1851-1862 ◽

Cited By ~ 13

Author(s):

Won Seok Yang ◽

Jin Ju Kim ◽

Mee Jeong Lee ◽

Eun Kyoung Lee ◽

Su-Kil Park

Keyword(s):

Endothelial Cells ◽

P38 Mapk ◽

Ectodomain Shedding ◽

Toll Like Receptor ◽

Tlr4 Expression ◽

Toll Like Receptor 4 ◽

Negative Feedback Regulation ◽

Mapk Activation ◽

Aortic Endothelial Cells ◽

Aortic Endothelial

Background/Aims: Lipopolysaccharide (LPS)-activated monocytes/macrophages develop endotoxin tolerance in part by reducing cell surface toll-like receptor 4 (TLR4) through cluster of differentiation 14 (CD14)-dependent endocytosis. In case of endothelial cells, CD14 is expressed in low copy numbers as compared with monocytes/macrophages. Thus, we explored how endothelial cells regulate TLR4 expression after LPS stimulation. Methods: Cultured human aortic endothelial cells (HAECs) were treated with LPS. TLR4 expression was analyzed by Western blot analysis and immunofluorescence staining. A disintegrin and metalloprotease 17 (ADAM17) activity was measured using a fluorescent substrate. Results: TLR4 in cell lysate began to decrease within 30 min of LPS treatment with a maximal reduction at 2 h, and it was accompanied by an increase of N-terminal fragment of TLR4 in culture supernatant, indicating ectodomain shedding of the receptor. LPS activated p38 mitogen-activated protein kinase (p38 MAPK) and ADAM17, while LPS-induced ADAM17 activation was inhibited by SB203580, a p38 MAPK inhibitor. LPS-induced ectodomain shedding of TLR4 was attenuated by siRNA depletion of ADAM17 as well as TAPI-2 (an inhibitor of ADAM family) and SB203580. LPS pretreatment resulted in a blunted response of p38 MAPK activation to further LPS stimulation. In the cells depleted of ADAM17, LPS-induced p38 MAPK activation was prolonged and LPS-induced intercellular adhesion molecule-1 expression was potentiated. Conclusion: HAECs respond to LPS by rapid shedding of the ectodomain of TLR4 and thereby reduce the responsiveness to subsequent LPS exposure. ADAM17, downstream of p38 MAPK, is implicated in the ectodomain cleavage of TLR4.

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Circulating high mobility group box-1 and toll-like receptor 4 expressions increase the risk and severity of epilepsy

Brazilian Journal of Medical and Biological Research ◽

10.1590/1414-431x20197374 ◽

2019 ◽

Vol 52 (7) ◽

Cited By ~ 3

Author(s):

Minchen Kan ◽

Lihong Song ◽

Xueqiang Zhang ◽

Jing Zhang ◽

Pingping Fang

Keyword(s):

High Mobility ◽

High Mobility Group ◽

Toll Like Receptor ◽

Toll Like Receptor 4

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Retracted : Baicalin relieves TNF‐α‐evoked injury in human aortic endothelial cells by up‐regulation of miR‐145

Phytotherapy Research ◽

10.1002/ptr.6566 ◽

2019 ◽

Vol 34 (4) ◽

pp. 836-845 ◽

Cited By ~ 2

Author(s):

Ling Li ◽

Min Liu ◽

Lianman He ◽

Shanshan Wang ◽

Shuxian Cui

Keyword(s):

Endothelial Cells ◽

Aortic Endothelial Cells ◽

Tnf Α ◽

Human Aortic Endothelial Cells ◽

Aortic Endothelial

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ASK1 Mediates Apoptosis and Autophagy during oxLDL-CD36 Signaling in Senescent Endothelial Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/2840437 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

KyoungJoo Cho ◽

Seung Ho Choi

Keyword(s):

Endothelial Cells ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Neutralizing Antibody ◽

Aortic Endothelial Cells ◽

Cd36 Expression ◽

Signaling Axis ◽

Induced Apoptosis ◽

Cluster Of Differentiation ◽

Human Aortic Endothelial Cells

Vessel damage by oxidized low-density lipoprotein (oxLDL) increases reactive oxygen species (ROS) and the membrane receptor cluster of differentiation 36 (CD36), involving various vascular pathological processes. In this study, the role of apoptosis signal-regulating kinase 1 (ASK1) as a cellular effector via the oxLDL-CD36 signaling axis, and its related mechanism as a downstream responder of CD36, was investigated in senescent human aortic endothelial cells (HAECs). To inhibit oxLDL-triggered vascular damage, HAECs and monocytes were treated with the CD36-neutralizing antibody or the ASK1 inhibitor NQDI-1. The oxLDL-triggered increases in ROS and CD36 elevated active ASK1 in the senescent HAECs. The ROS increase induced apoptosis, whereas CD36 neutralization or ASK1 inhibition protected against cell death. The blocking of CD36 increased senescent HAEC autophagy. In monocytes, oxLDL also induced CD36 expression and autophagy, the latter of which still occurred following ASK1 inhibition but not after CD36 neutralization. These findings suggest that oxLDL exposure activates ASK1, as a CD36 downstream responder, to accelerate apoptosis, particularly in senescent HAECs. ASK1’s involvement in monocytic autophagy was due to endoplasmic reticulum stress resulting from the oxLDL load, suggesting that oxLDL loading on aged vessels causes atherosclerotic endothelial dysfunction mediated by active ASK1.

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