170 Fetuin-a induces nox1-ros-dependent vascular dysfunction partially through toll like receptor 4 activation

Although studies demonstrate an important role for fetuin-A (FetA) in the inhibition of vascular calcification, convincing evidence suggests that fetuin-A is also involved in insulin resistance, inflammation and cardiovascular damage. The present study seeks to unravel FetA vascular effects and associated molecular mechanisms, focusing on oxidative stress and toll-like receptor 4 (TLR4). Vascular function studies were performed in mesenteric resistance arteries from WKY rats, wild-type, Nox1 KO, Nox4 KO and Ang II-dependent hypertensive mice (LinA3) and rat aortic endothelial cells (RAEC). ROS production (chemiluminescence, Amplex Red, ELISA) and pro-inflammatory markers expression (RT-PCR) were measured in VSMCs from WKY rats and RAEC. FetA impaired endothelium-dependent (LogEC50 7.320±0.08 M vs control 8.025±0.06) and endothelium-independent vasorelaxation (LogEC50 6.48±0.19 M vs control 7.38±0.12), p<0.05; effects blocked by tempol (superoxide dismutase mimetic), Nox1 inhibitor, ML171, and TLR4 inhibitor, CLI095. We did not observe any changes in contraction. FetA increased ROS production (62%) and peroxynitrite levels (158%) in VSMCs; while in RAEC, FetA increased ROS production (105%) followed by a decrease in H2O2 (62%) levels (p<0.05 vs control). FetA-induced effects on ROS were inhibited by ML171 and GKT137831 (Nox1/Nox4 inhibitor), as well as CLI095. Vascular dysfunction in arteries from Nox1 and Nox4 KO mice was unaffected by FetA. Activation of the FetA/TLR4/Nox axis led to an increase in IL-1β (190%), Il-6 (124%) and RANTES mRNA expression(116%) in RAEC, p<0.05 vs control. FetA enhanced vascular dysfunctionin LinA3 mice. Together, these results suggest that FetA through TLR4/Nox1 and 4-derived ROS leads to vascular dysfunction and inflammation, which may play an important role in the development of vascular injury during hypertension.

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Abstract 099: Fetuin-A and Toll-like Receptor 4 Regulate Vascular Function: Role of Nox1

Hypertension ◽

10.1161/hyp.66.suppl_1.099 ◽

2015 ◽

Vol 66 (suppl_1) ◽

Author(s):

Augusto C Montezano ◽

Karla B Neves ◽

Rheure A Lopes ◽

Susan Leckerman ◽

Anastasiya Strembitska ◽

...

Keyword(s):

Endothelial Dysfunction ◽

Protein Oxidation ◽

Vascular Function ◽

Vascular Dysfunction ◽

Rho Kinase ◽

Mesenteric Arteries ◽

Ros Production ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Fetuin A

Fetuin-A (FetA) regulates calcium and phosphate homeostasis. It is also an agonist to toll-like receptor 4 (TLR4) and is related to insulin resistance and inflammation. FetA has also been associated with endothelial dysfunction, which is regulated by oxidative stress. Mechanisms whereby FetA influences vascular function are unknown. We hypothesized that FetA through TLR4 and ROS production induces vascular dysfunction. Mesenteric arteries and vascular cells from WKY rats were studied. Vascular function was analysed by wire myography in the presence or absence of FetA (50 ng/mL) and/or CLI095 (CLI - 10-6M - TLR4 inhibitor). Levels of reactive oxygen species (ROS) were measured by chemiluminescence, Amplex Red (H2O2) and ELISA (nitrotyrosine) Protein oxidation and levels were measured by immunoblotting. WKY vessels exposed to FetA were less sensitive to acetylcholine (Ach)-induced and sodium nitroprusside (SNP)-induced relaxation, while sensitivity to phenylephrine was increased by FetA; an effect blocked by N-acetylcysteine (antioxidant) and ML171 (Nox1 inhibitor). Inhibition of TLR4 blocked FetA effects on endothelial-dependent relaxation and contraction, but not on endothelial-independent relaxation. FetA increased ROS production (131±49.2%), but decreased H2O2 intracellular levels (63±14%) in endothelial cells (EC) (vs. veh, p<0.05); an effect blocked by CLI095. ROS production (66±12.2%), as well as, H2O2 (45±8%) and ONOO- (105±31.6%) levels, were increased by FetA in VSMCs (vs. veh, p<0.05). Protein oxidation was increased by FetA in VSMCs (103±26% vs. veh, p<0.05). In EC, eNOS inactivation (136±38%) and JNK activation (84±5%) were increased by FetA (vs. veh, p<0.05). In VSMCs, Rho kinase activity was increased (200±25% vs. veh, p<0.05) at 30 min; while myosin light chain (MLC) activation was only increased (25±3.56% vs. veh, p<0.05) at 15 min. In summary, FetA influences vascular function through Nox1-ROS dependent mechanisms. FetA-induced endothelial dysfunction and contractile responses involve TLR4. Our findings identify a novel system whereby FetA differentially influences vascular function through Nox1-ROS and TLR4. Vascular responses to FetA may depend on the specific pathway activated.

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Toll-Like Receptor 4 Upregulation by Angiotensin II Contributes to Hypertension and Vascular Dysfunction through Reactive Oxygen Species Production

PLoS ONE ◽

10.1371/journal.pone.0104020 ◽

2014 ◽

Vol 9 (8) ◽

pp. e104020 ◽

Cited By ~ 60

Author(s):

Priscila R. De Batista ◽

Roberto Palacios ◽

Angela Martín ◽

Raquel Hernanz ◽

Cindy T. Médici ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Angiotensin Ii ◽

Reactive Oxygen Species Production ◽

Vascular Dysfunction ◽

Reactive Oxygen ◽

Oxygen Species ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Species Production

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Abstract P340: Molecular Mechanisms of Fetuin-a-induced Vascular Dysfunction and Inflammation: Role of Oxidative Stress and Toll-like Receptor 4

Hypertension ◽

10.1161/hyp.68.suppl_1.p340 ◽

2016 ◽

Vol 68 (suppl_1) ◽

Author(s):

Augusto C Montezano ◽

Delyth Graham ◽

Rhian M Touyz

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Endothelial Cells ◽

Vascular Function ◽

Cell Metabolism ◽

Ros Production ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Fetuin A ◽

Tlr4 Activation

Fetuin-A (FetA) is an endogenous agonist to toll-like receptor 4 (TLR4) and regulates insulin resistance and inflammation. FetA has been associated with endothelial dysfunction during metabolic diseases. Exact mechanisms whereby FetA influences vascular function in pathological conditions remain unknown, but we demonstrated that FetA regulates vascular function by Nox1 and TLR4 activation. Here we hypothesized that FetA, through changes in cell metabolism and activation of TLR4-Nox1 axis induces ROS formation and inflammation in hypertension. Normotensive (WKY) and hypertensive (SHRSP) vascular cells, as well as human microvascular endothelial cells, were stimulated with FetA (50 ng/mL). ROS production was measured by lucigenin and Amplex red, while gene expression was assessed by qPCR. FetA increased ROS production (131±49.2%), decreased H 2 O 2 intracellular levels (63±14%) and increased gene levels of IL6 (2 fold), IL1β (1 fold), RANTES (1 fold) and MMP2/9 (2 fold) in rat endothelial cells ( vs. veh, p<0.05); all effects were blocked by TLR4 inhibitor (CLI095) and Nox1 inhibitor (ML171). FetA increased JNK (184±19%), but not p38 MAPK, activation in endothelial cells. In VSMCs, FetA-induced TLR4-dependent ROS generation was similar in WKY (136±9%) and SHRSP (144±14%) (p<0.05 vs veh). However, while IL6 gene expression was increased by FetA in WKY (4 fold) and SHRSP (0.5 fold), IL-1β gene levels were only increased by FetA in SHRSP (1 fold) derived VSMCs (p<0.05). CLI095 inhibited FetA effects on IL6 expression; however, TLR4 inhibition did not block FetA effects on IL-1β gene levels. In human endothelial cells, FetA increased ROS levels, was inhibited by CLI095 and a glucose-6-phosphate dehydrogenase (G6PD) inhibitor (6-aminonicotinamide), suggesting that FetA effects may be related to control of cell metabolism. In conclusion, FetA seems to regulate ROS and pro-inflammatory responses by TLR4, Nox1 and G6PD in endothelial cells. In VSMCs, FetA effects on oxidative stress and markers of cell injury are partially dependent on TLR4 activation and may involve other molecular partners.

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The rapid endocytic uptake of fetuin‐A by adherent tumor cells is mediated by Toll‐like receptor 4 (TLR4)

FEBS Open Bio ◽

10.1002/2211-5463.13008 ◽

2020 ◽

Vol 10 (12) ◽

pp. 2722-2732

Author(s):

Portia L. Thomas ◽

Gladys Nangami ◽

Tanu Rana ◽

Adam Evans ◽

Stephen D. Williams ◽

...

Keyword(s):

Tumor Cells ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Fetuin A

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35 Fetuin-A and toll-like receptor 4 regulate vascular function: role of Nox1

Heart ◽

10.1136/heartjnl-2015-308734.35 ◽

2015 ◽

Vol 101 (Suppl 6) ◽

pp. A12.1-A12

Author(s):

KB Neves ◽

RAM Lopes ◽

S Leckerman ◽

A Strembitska ◽

C Jenkins ◽

...

Keyword(s):

Vascular Function ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Fetuin A

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Mesenteric artery responsiveness to acetylcholine and phenylephrine in cirrhotic rats challenged with endotoxin: the role of TLR4

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2014-0515 ◽

2015 ◽

Vol 93 (6) ◽

pp. 475-483 ◽

Cited By ~ 3

Author(s):

Sattar Ostadhadi ◽

Seyed-Mahdi Rezayat ◽

Shahram Ejtemaei-Mehr ◽

Seyed-Mohammad Tavangar ◽

Vahid Nikoui ◽

...

Keyword(s):

Mesenteric Artery ◽

Low Dose ◽

Vascular Dysfunction ◽

Mrna Levels ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Vascular Bed ◽

Duct Ligation ◽

Immunohistochemical Studies

Cirrhosis is associated with vascular dysfunction and endotoxemia. These experiments were designed to investigate the hypothesis that the administration of a low-dose of lipopolysaccharide (LPS) worsens vascular dysfunction in rats subjected to bile-duct ligation (BDL), and to determine whether LPS initiates changes in vascular Toll-like receptor 4 (TLR4) expression. Four weeks after BDL, the animals were given an intraperitoneal injection of either saline or LPS (1.0 mg/kg body mass). Three hours later, the superior mesenteric artery was isolated, perfused, and then subjected to the vasoconstriction and vasodilatation effects of phenylephrine and acetylcholine, respectively. Our results show that phenylephrine-induced vasoconstriction decreased in the cirrhotic vascular bed (BDL rats) compared with the vascular bed of the sham-operated animals, and that the LPS injections in the cirrhotic (BDL) rats worsened this response. LPS injection administered to the sham-operated animals had no such effect. On the other hand, both the BDL procedure and the LPS injection increased acetylcholine-induced vasorelaxation, but LPS administration to the BDL rats had no effect on this response. The mRNA levels of TLR4 did not change, but immunohistochemical studies showed that TLR4 localization switched from the endothelium to vascular smooth muscle cells following chronic BDL. In conclusion, acute endotoxemia in cirrhotic rats is associated with hyporesponsiveness to phenylephrine and tolerance to the effects of acetylcholine. Altered localization of TLR4 may be responsible for these effects.

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