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

Hypertension ◽  
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.

Abstract P083: Fetuin-a Induces Vascular Dysfunction Through Toll-like Receptor 4 And Nox1/4 Activation

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Karla B Neves ◽  
Rheure A Lopes ◽  
Anastasiya Strembitska ◽  
Ross Hepburn ◽  
Wendy Beattie ◽  
...  
Keyword(s):  
Vascular Function ◽  
Molecular Mechanisms ◽  
Vascular Dysfunction ◽  
Convincing Evidence ◽  
Ros Production ◽  
Toll Like Receptor ◽  
Vascular Effects ◽  
Toll Like Receptor 4 ◽  
Fetuin A ◽  
Wky Rats

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.

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

Hypertension ◽  
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.

Abstract 66: Altered Vascular Reactivity in Mice Overexpressing Adipocyte Mineralocorticoid Receptors - Role of Oxidative Stress and Rho Kinase

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Aurelie Nguyen Dinh Cat ◽  
Glaucia E Callera ◽  
Tayze T Antunes ◽  
Augusto C Montezano ◽  
Ying He ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Endothelial Dysfunction ◽  
Vascular Function ◽  
Vascular Reactivity ◽  
Vascular Dysfunction ◽  
Rho Kinase ◽  
Mesenteric Arteries ◽  
Resistance Arteries ◽  
Tissue Interactions

Aldosterone (aldo) plays a role in obesity and cardiovascular diseases, such as hypertension. We previously demonstrated that adipocyte-derived factors regulate vascular function and vascular smooth muscle cells signaling. Moreover, adipocytes express aldosterone synthase (CYP11B2) and produce aldo. The mineralocorticoid receptor (MR), which is responsible for aldo signaling, is also found in these cells, but its role in regulating adipose tissue interactions with the vasculature is unknown. In this study, we investigated mechanisms whether MR activation in adipocytes regulates vascular reactivity. Conditional transgenic mice that overexpress MR in an adipocyte-specific manner were studied. Vascular reactivity of resistance mesenteric arteries to acetylcholine (Ach), sodium nitroprusside and phenylephrine (Phe), in the absence or presence of fat conditioned medium (Fcm) from control and adipocyte overexpressing MR (MROE) mice, was performed by myography. In basal conditions, endothelial dysfunction was not observed in MROE or control (Ctr) mice. However, exposure of arteries from control mice to Fcm from MROE mice induces endothelial dysfunction (Ach 10 -6 M: 77.5±9.6% no Fcm vs. 49.8±7.5% Fcm, p<0.05), an effect blocked by N-acetyl-cysteine (an antioxidant) (Ach 10 -6 M: 82.2±6.6%). Resistance arteries from MROE mice had decreased Phe-induced contraction, compared to control mice (Phe 10 -5 M: 2.7±0.2 mN/mm Ctr vs. 1.7±0.2 mN/mm MROE, p<0.05). Rho Kinase activity, which regulates vascular contraction, is decreased in arteries and adipo tissue from MROE (mesenteric arteries, Ctr: 100±16.2% vs. MROE: 31.1±6.1%, arbitrary units, p<0.01; adipose tissue, Ctr: 100±12.6% vs. MROE: 51.3±9.3%, arbitrary units, p<0.01). In conclusion, MR in adipocytes may play an important role in the regulation of vascular function, through redox-sensitive pathways and activation of Rho kinase. Our study identifies novel mechanisms linking vascular/adipose tissue biology and aldo/MR activation, which may be particularly important in vascular dysfunction associated with hypertension and hyperaldosteronism.

Abstract P217: Nox Compartmentalization, Protein Oxidation and ER Stress in Vascular Smooth Muscle Cells in Hypertension

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Livia L Camargo ◽  
Augusto C Montezano ◽  
Adam Harvey ◽  
Sofia Tsiropoulou ◽  
Katie Hood ◽  
...  
Keyword(s):  
Er Stress ◽  
Protein Oxidation ◽  
Vascular Function ◽  
Vascular Reactivity ◽  
Stress Proteins ◽  
Vascular Dysfunction ◽  
Mesenteric Arteries ◽  
Resistance Arteries ◽  
Post Translational Modification ◽  
Wistar Kyoto

In hypertension, activation of NADPH oxidases (Noxs) is associated with oxidative stress and vascular dysfunction. The exact role of each isoform in hypertension-associated vascular injury is still unclear. We investigated the compartmentalization of Noxs in VSMC from resistance arteries of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Expression of Nox1 and Nox4 was increased in SHR cells (96.6±28.7% and 48.2±21.2% vs WKY, p<0.05), as well as basal ROS levels measured by chemiluminescence (110.2±26.4% vs WKY, p<0.05) and amplex red (105.2±33.2% vs WKY, p<0.05). Phosphorylation of unfolded protein response activators, PERK and IRE1α, and expression of ER chaperone BiP were elevated in SHR cells (p<0.05 vs WKY), indicating activation of ER stress response. Immunoblotting after organelle fractionation demonstrated that Noxs are expressed in an organelle-specific manner, with Nox1, 2 and 4 present in plasma membrane, ER and nucleus, but not in mitochondria. In SHR cells, NoxA1ds (Nox1 inhibitor, 10μM) and GKT136901 (Nox1/4 inhibitor, 10μM) decreased AngII-induced ROS levels (p<0.001 vs Ctl). Additionally, mito-tempol (mitochondrial-targeted antioxidant, 50nM) and 4-PBA (ER stress inhibitor, 1mM) decreased basal ROS levels in SHR cells (p<0.05 vs Ctl). Furthermore, oxidation of the antioxidant enzymes Peroxiredoxins (Prx) was increased in SHRSP compared to WKY (2.51±0.14 vs 0.56±0.07, p<0.001). One-dimensional isoelectric focusing revealed that cytosolic Prx2 and mitochondrial Prx3 were more oxidized in SHRSP than WKY cells. Using a biotin-tagged dimedone-based probe (DCP-Bio) we identified oxidation of ER stress proteins BiP and IRE1. To investigate the effect of protein oxidation in vascular function, vascular reactivity was evaluated in isolated mesenteric arteries. Inhibition of general oxidation (DTT 1mM; Emax: 111.7±33.1) and peroxiredoxin (Conoidin A 10nM; Emax: 116.0±7.3) reduces vascular contraction in response to noradrenalin in WKY rats (Emax: 166.6±30.2; p<0.05). These findings suggest an important role for Nox1/4 in redox-dependent organelle dysfunction and post-translational modification of proteins, processes that may play an important role in vascular dysfunction in hypertension.

Endothelial dysfunction in Type 2 diabetes correlates with deregulated expression of the tail-anchored membrane protein SLMAP

2005 ◽  
Vol 289 (1) ◽  
pp. H206-H211 ◽  
Author(s):  
Hong Ding ◽  
Andrew G. Howarth ◽  
Malarvannan Pannirselvam ◽  
Todd J. Anderson ◽  
David L. Severson ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Endothelial Dysfunction ◽  
Vascular Function ◽  
Vascular Dysfunction ◽  
Microvascular Disease ◽  
Mesenteric Arteries ◽  
Mrna Levels ◽  
Important Indicator ◽  
Membrane Function

The Type 2 diabetic db/ db mouse experiences vascular dysfunction typified by changes in the contraction and relaxation profiles of small mesenteric arteries (SMAs). Contractions of SMAs from the db/ db mouse to the α1-adrenoceptor agonist phenylephrine (PE) were significantly enhanced, and acetylcholine (ACh)-induced relaxations were significantly depressed. Drug treatment of db/ db mice with a nonthiazolidinedione peroxisome prolifetor-activated receptor-γ agonist and insulin sensitizing agent 2-[2-(4-phenoxy-2-propylphenoxy)ethyl]indole-5-acetic acid (COOH) completely prevented the changes in endothelium-dependent relaxation, but, with the discontinuation of therapy, endothelial dysfunction returned. Dysfunctional SMAs were found to specifically upregulate the expression of a 35-kDa isoform of sarcolemmal membrane-associated protein (SLMAP), which is a component of the excitation-contraction coupling apparatus and implicated in the regulation of membrane function in muscle cells. Real-time PCR revealed high SLMAP mRNA levels in the db/ db microvasculature, which were markedly downregulated during COOH treatment but elevated again when drug therapy was discontinued. These data reveal that the microvasculature in db/ db mice undergoes significant changes in vascular function with the endothelial component of vascular dysfunction specifically correlating with the overexpression of SLMAP. Thus changes in SLMAP expression may be an important indicator for microvascular disease associated with Type 2 diabetes.

Abstract WMP109: Activation of the Brain Renin-Angiotensin System (RAS) Causes Selective Cerebrovascular Dysfunction

Stroke ◽  
2016 ◽  
Vol 47 (suppl_1) ◽  
Author(s):  
Travice M De Silva ◽  
Justin Grobe ◽  
Frank M Faraci
Keyword(s):  
Vascular Function ◽  
Vascular Dysfunction ◽  
Tap Water ◽  
Cerebral Arteries ◽  
Rho Kinase ◽  
Renin Angiotensin System ◽  
Mesenteric Arteries ◽  
The Impact ◽  
The Brain

Hypertension is a leading risk factor for cerebrovascular disease. Although many changes during hypertension may be mediated by the RAS, most hypertensive patients have normal or reduced levels of circulating renin, suggesting that local tissue RAS play a key role. Surprisingly, little is known regarding the impact of the brain RAS on the cerebral vasculature. We tested the hypothesis that activation of the central RAS using deoxycorticosterone (DOCA)-salt (which simultaneously suppresses the peripheral RAS) alters vascular function. Male C57Bl/6 mice treated with DOCA were given both tap water and water with 0.9% salt for 3 weeks, followed by measurements of vessel reactivity. Mean arterial pressure was elevated modestly after DOCA-salt (79±2 vs 95±3 mmHg; P<0.01). In DOCA-salt treated mice, endothelium-dependent dilation of isolated middle cerebral arteries was almost abolished compared to shams, whereas dilation in mesenteric arteries was normal. This striking effect in brain extended to small parenchymal arterioles. Endothelium-independent vasodilation was similar in all groups. Analysis of mRNA revealed that expression of renal renin was markedly reduced by DOCA-salt while expression of RAS components (eg, AGT, ACE) were increased in both brain and cerebral arteries. In NZ44 reporter mice that express GFP driven by the angiotensin II type 1A receptor (AT1AR) promoter, DOCA-salt significantly increased AT1R-GFP protein expression in cerebral arteries. In cerebral arterioles in vivo, local inhibition of AT1R, mineralocorticoid receptors (MR), or Rho kinase (including ROCK2) reversed endothelial dysfunction in DOCA-salt treated mice. These findings suggest for the first time that activation of the brain and the cerebrovascular RAS profoundly, but selectively, impairs vascular function in brain. Our findings identify AT1R, MR and Rho kinase as key contributors to vascular dysfunction in brain in a clinically relevant model of hypertension.

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

Heart ◽  
2017 ◽  
Vol 103 (Suppl 5) ◽  
pp. A119.3-A120
Author(s):  
Augusto Montezano ◽  
Rheure Lopes ◽  
Karla Neves ◽  
Delyth Graham ◽  
Rhian M Touyz
Keyword(s):  
Vascular Dysfunction ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Fetuin A

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

Heart ◽  
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

scholarly journals Lycopene Inhibits Toll-Like Receptor 4-Mediated Expression of Inflammatory Cytokines in House Dust Mite-Stimulated Respiratory Epithelial Cells

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3127
Author(s):  
Jiyeon Choi ◽  
Joo Weon Lim ◽  
Hyeyoung Kim
Keyword(s):  
Epithelial Cells ◽  
A549 Cells ◽  
Cytokine Expression ◽  
Ros Production ◽  
Toll Like Receptor ◽  
Respiratory Epithelial Cells ◽  
Toll Like Receptor 4 ◽  
Mitochondrial Ros ◽  
Tlr4 Activation ◽  
Respiratory Epithelial

House dust mites (HDM) are critical factors in airway inflammation. They activate respiratory epithelial cells to produce reactive oxygen species (ROS) and activate Toll-like receptor 4 (TLR4). ROS induce the expression of inflammatory cytokines in respiratory epithelial cells. Lycopene is a potent antioxidant nutrient with anti-inflammatory activity. The present study aimed to investigate whether HDM induce intracellular and mitochondrial ROS production, TLR4 activation, and pro-inflammatory cytokine expression (IL-6 and IL-8) in respiratory epithelial A549 cells. Additionally, we examined whether lycopene inhibits HDM-induced alterations in A549 cells. The treatment of A549 cells with HDM activated TLR4, induced the expression of IL-6 and IL-8, and increased intracellular and mitochondrial ROS levels. TAK242, a TLR4 inhibitor, suppressed both HDM-induced ROS production and cytokine expression. Furthermore, lycopene inhibited the HDM-induced TLR4 activation and cytokine expression, along with reducing the intracellular and mitochondrial ROS levels in HDM-treated cells. These results collectively indicated that the HDM induced TLR4 activation and increased intracellular and mitochondrial ROS levels, thus resulting in the induction of cytokine expression in respiratory epithelial cells. The antioxidant lycopene could inhibit HDM-induced cytokine expression, possibly by suppressing TLR4 activation and reducing the intracellular and mitochondrial ROS levels in respiratory epithelial cells.

Abstract 665: Activation of Histone Deacetylase 2: A Novel Strategy for Reversing Vascular Dysfunction in Atherogenesis

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Deepesh Pandey ◽  
Gautam Sikka ◽  
Yehudis Bergman ◽  
Jae H Kim ◽  
Sungwoo Ryoo ◽  
...  
Keyword(s):  
Endothelial Dysfunction ◽  
Endothelial Function ◽  
Cell Activation ◽  
Vascular Dysfunction ◽  
Hdac Inhibitors ◽  
Ros Production ◽  
Promoter Regions ◽  
Endothelial Cell Activation ◽  
Novel Therapy ◽  
Histone Deacetylase 2

Arginase 2 is a critical target in atherosclerosis as it regulates both endothelial NO, fibrosis and inflammation. The increase in Arg2 activity with endothelial cell activation is dependent on both early post-translational dependent mechanisms as well as a later increase in Arg 2 expression. The regulators of Arg2 transcription in the endothelium have not been characterized. The goal of current study is to determine the role of specific HDACs in the regulation of endothelial Arg2 transcription and thereby endothelial function. The global HDAC inhibitor, trichostatin (TSA) both time and concentration-dependently increased Arg2 mRNA, protein levels and activity in both HAECs and mouse aortic rings, a process that leads to Arg2-dependent endothelial dysfunction. TSA and atherogenic stimulus enhances activity of common promoter regions of Arg 2. All non-selective Class I HDAC inhibitors (TSA, Scriptaid, varinostat) enhanced Arg2 expression, while only the, the HDCA 1 and 2 selective inhibitor, mocetinostat (MGCD) enhances Arg2 expression. Overexpression of HDAC 2, 3 or 8 in HAECs have no effect on Arg 2 expression while HDAC2 cDNA overexpression concentration-dependently suppresses Arg2 expression. Conversely, siRNA knockdown of HDAC2 enhances Arg2 expression. Additionally like TSA, mouse aortic rings pre-incubated with MGCD resulted in endothelial dysfunction. Finally HDAC inhibition with TSA decreased endothelial NO and increased ROS production in an arginase-inhibitable manner. In conclusion, HDAC2 is critical regulator of Arg2 expression thereby regulating endothelial NO and ROS production, and consequently endothelial function. Overexpression or activation of HDAC2 thus represents a novel therapy for the prevention and treatment of endothelial dysfunction and atherosclerosis.

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