Toll-Like Receptor 4 Mutation Protects Obese Mice Against Endothelial Dysfunction by Decreasing NADPH Oxidase Isoforms 1 and 4Significance
Objective—To analyze the role of toll-like receptor 4 in modulating metabolism and endothelial function.
Approach and Results—Type 2 diabetic mice with mutated toll-like receptor 4 (DWM) were protected from hyperglycemia and hypertension, despite an increased body weight. Isometric tension was measured in arterial rings with endothelium. Relaxations to acetylcholine were blunted in aortae and mesenteric arteries of Leprdb/db mice, but not in DWM mice; the endothelial NO synthase dimer/monomer ratio and endothelial NO synthase phosphorylation levels were higher in DWM preparations. These differences were abolished by apocynin. Contractions to acetylcholine (in the presence of L-NAME) were larger in carotid arteries from Leprdb/db mice than from DWM mice and were inhibited by indomethacin and SC560, demonstrating involvement of cyclooxygenase-1. The release of 6-ketoprostaglandin F1α was lower in DWM mice arteries, implying lower cyclooxygenase-1 activity. Apocynin, manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin, catalase, and diethyldithiocarbamate inhibited endothelium-dependent contractions. The mRNA and protein levels of NADPH oxidase isoforms NOX1 and NOX4 were downregulated in DWM mice arteries. The in vivo and in vitro administration of lipopolysaccharide caused endothelial dysfunction in the arteries of wild-type, but not toll-like receptor 4–mutated mice.
Conclusions—Toll-like receptor 4 plays a key role in obesity and diabetes-associated endothelial dysfunction by increasing oxidative stress.
- endothelium-derived contracting factor
- nitric oxide
- proinflammatory cytokines
- systolic arterial blood pressure
Obesity and diabetes mellitus are associated with augmented circulating levels of free fatty acids, low-grade chronic inflammation,1 and endothelial dysfunction attributable to impaired release of endothelium-derived relaxing and augmented production of endothelium-derived contracting (EDCF) factors.2 Thus, the bioavailability of NO is reduced by obesity and diabetes mellitus,3 and relaxations attributable to endothelium-derived hyperpolarization (EDH) are impaired at the early stage of diabetes mellitus.4 Finally, increased release of endothelium-derived vasoconstrictor prostanoids and reactive oxygen species (ROS) also contributes to endothelial dysfunction in obesity and diabetes mellitus.5
Proinflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin 6 [IL-6], resistin, and lipocalin-2) link obesity to metabolic and vascular dysfunction.1 Bacterial endotoxin lipopolysaccharide (LPS) and saturated fatty acids share as pattern recognition target toll-like receptors 4 (TLR4), which activate inflammatory pathways, induce cytokine expression in the endothelium,6,7 and augment the expression of NADPH oxidase in macrophages.8 The absence of TLR4 protects mice against obesity-induced insulin resistance.7
It is unknown whether or not TLR4 signaling contributes to the impaired NO- and EDHF-mediated relaxations and the amplified EDCF-mediated contractions seen in obesity and diabetes mellitus. Therefore, the present experiments tested the hypothesis that TLR4 signaling mediates, at least in part, the deleterious effects of diet-induced and genetic obesity leading to endothelial dysfunction.
Materials and Methods
Materials and Methods are available in the online-only Supplement.
There were no significant differences in food intake between wild-type (WT) and TLR4−/− mice (Figure IA in the online-only Data Supplement). The high-fat diet evoked comparable increases in body weight in 12-week-old WT and TLR4−/− mice (Figure IB in the online-only Data Supplement). In DWM mice, the food intake and the body weight were higher starting at the age of 8 weeks (Figure IC in the online-only Data Supplement) compared with Leprdb/db mice. Both feeding (Figure IC in the online-only Data Supplement) and fasting (Figure ID in the online-only Data Supplement) glucose levels of DWM mice were significantly lower than those of Leprdb/db mice. After injection of glucose, the glycemia rose more slowly and returned back to normal more quickly in DWM mice than in Leprdb/db mice (Figure IE and IF in the online-only Data Supplement). After injection of insulin, blood glucose dropped more quickly in DWM mice than in Leprdb/db mice (Figure IG and IH in the online-only Data Supplement).
The mRNA expressions of IL1-β, IL-6, and TNF-α were significantly lower in the epididymal fat of DWM mice compared with Leprdb/db mice (Figure II in the online-only Data Supplement).
Systolic arterial blood pressures were comparable between 16-week-old lean WT and TLR4−/− mice. WT mice were fed high-fat diet, and Leprdb/db mice fed standard chow exhibited an elevated systolic arterial blood pressure (Figure IIIA in the online-only Data Supplement). DWM mice showed significantly lower systolic arterial blood pressures than Leprdb/db mice (Figure IIIA in the online-only Data Supplement).
Mutation of TLR4
Relaxations to increasing concentrations of acetylcholine (10−10 to 10−4 mol/L) were reduced significantly in aortae of 16-week-old WT mice compared with TLR4−/− preparations (Figure 1).
Increasing concentrations of acetylcholine (in the presence of L-NAME [10−4 mol/L; nonselective NOS inhibitor] and indomethacin [10−5 mol/L; nonselective cyclooxygenase inhibitor] to assess responses attributable to EDH)26 caused relaxations in mesenteric arteries, which were significantly larger in preparations of TLR4−/− than in those of control mice (Figure 1).
In the presence of L-NAME (10−4 mol/L, to optimize endothelium-dependent contractions),18 acetylcholine induced increases in tension in the quiescent carotid arteries of WT and TLR4−/− mice (Figure 1). These contractions were significantly attenuated in TLR4−/− mice arteries.
After 12 weeks of high-fat diet, relaxations to acetylcholine (10−10 to 10−4 mol/L) were significantly impaired in aortae or mesenteric arteries (in the presence of L-NAME and indomethacin) of WT mice (Figure 1). The high-fat diet did not significantly affect relaxations to acetylcholine in either aortae or mesenteric arteries of TLR4−/− mice (Figure 1).
Contractions induced by acetylcholine in the presence of L-NAME were enhanced significantly in WT carotid arteries after high-fat feeding (Figure 1).
Double Mutation of Leptin Receptor and TLR4
Vascular Smooth Muscle
Contractions to 60 mmol/L KCl were not significantly different in the aortae of 12-week-old Leprdb/db and DWM mice (1.83±0.49 and 1.79±0.36 g, respectively). Likewise, contractions to increasing concentration of U46619 were not significantly different in Leprdb/db and DWM mice aortae without endothelium (Figure IIIC in the online-only Data Supplement).
Relaxations to increasing concentrations of sodium nitroprusside were not significantly different between Leprdb/db and DWM mice aortae (Figure IIIB in the online-only Data Supplement).
NO-Dependent Relaxations and eNOS Coupling
The relaxations to acetylcholine (10−10 to 10−4 mol/L) were significantly larger in DWM compared with Leprdb/db mice aortae (Figure 2A). After incubation with apocynin (10−4 mol/L; antioxidant), the relaxations to acetylcholine were potentiated only in Leprdb/db mice aortae, and the difference in relaxations between the 2 strains was no longer significant (Figure 2A). The relaxations to acetylcholine were abolished by L-NAME (10−4 mol/L) in the aortae of both strains (Figure IIIE in the online-only Data Supplement).
The basal and the acetylcholine-evoked phosphorylation of endothelial NO synthase (eNOS) at Serine 1177 was significantly higher in DWM than in Leprdb/db mice aortae (Figure 2B). Apocynin enhanced the acetylcholine-evoked phosphorylation of eNOS only in Leprdb/db preparations. The expression of total eNOS was comparable in Leprdb/db and DWM preparations and not affected by apocynin (Figure 2B).
The eNOS dimer/monomer ratio was significantly higher in DWM than in Leprdb/db mice aortae (Figure 2C). Apocynin increased the eNOS dimer levels and decreased those of eNOS monomer in Leprdb/db aortae to comparable levels as those observed in DWM preparations (Figure 2D). The eNOS dimer and monomer levels were not altered significantly by apocynin in DWM preparations (Figure 2D).
After incubation with L-NAME (10−4 mol/L) and indomethacin (5×10−6 mol/L), acetylcholine caused concentration-dependent relaxations of mesenteric arteries, which were larger in DWM than in Leprdb/db preparations (Figure IV in the online-only Data Supplement). In both cases, the relaxations were attenuated significantly by either 1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole (10−7 mol/L; intermediate-conductance calcium-activated potassium channels channel blocker) or 6,12,19,20,25,26-hexahydro- 5,27:13,18:21,24-Trietheno-11,7-metheno-7H-dibenzo[b,m][1,5,12,16]tetraazacyclotricosine-5,13-diium ditrifluoroacetate hydrate (10−7 mol/L; small-conductance calcium-activated potassium channels channel blocker) and nearly abolished by the combination of these 2 inhibitors (Figure IV in the online-only Data Supplement). Incubation with apocynin potentiated the relaxations in Leprdb/db, but not in DWM mice arteries, and in the presence of the antioxidant, the relaxations were no longer significantly different between the 2 types of preparations (Figure IV in the online-only Data Supplement).
In the presence of L-NAME, acetylcholine evoked concentration-dependent contractions in quiescent carotid arteries of 12-week-old Leprdb/db mice (Figure 3A). The increases in tension caused by acetylcholine were significantly smaller in DWM preparations (Figure 3A). The contractions were abolished by apocynin (Figure 3A and 3C). Removal of the endothelium, as well as incubation with indomethacin, SC560 (3×10−7 mol/L; selective cyclooxygenase [COX]-1 inhibitor) and S18886 (10−7 mol/L; thromboxane-prostanoids receptor antagonist), but not NS398 (10−6 mol/L; preferential COX-2 inhibitor), significantly inhibited the acetylcholine-induced contractions (Figure V in the online-only Data Supplement). Diphenyliodium (10−5 mol/L; flavoenzyme oxidoreductases inhibitor), diethyldithiocarbamate (10−4 mol/L; superoxide dismutase inhibitor), catalase-polyethylene glycol (1200 U mL−1), and manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin (10−4 mol/L; cell permeable superoxide dismutase mimetic) significantly decreased the endothelium-dependent contractions to acetylcholine in both type of preparations, whereas superoxide dismutase (120UmL−1) and deferoxamine (10−4 mol/L; inhibitor of formation of hydroxyl radicals) had no significant effect (Figure 3C).
The mRNA expression and protein levels of COX-1 were comparable in Leprdb/db and DWM mice carotid arteries (Figure 3B).
The production of 6-ketoPGF1α under basal conditions and during stimulation with acetylcholine (10−6 mol/L) was significantly lower in DWM than in Leprdb/db mice carotid arteries (Figure 4A). Incubation with apocynin abolished the difference in the production of 6-ketoPGF1α (Figure 4A).
NADPH Oxidase and Superoxide Anion Production
The extracellular free radical presence, estimated by lucigenin-enhanced chemiluminescence, was lower under basal conditions in membrane fractions of DWM than in Leprdb/db preparations (Figure 4E). Acetylcholine increased the ROS production in preparations of Leprdb/db, but not of DWM mice, but this response was not affected significantly by indomethacin (Figure 4E).
The glucose and insulin tolerances were comparable in WT mice 4 hours after the in vivo intraperitoneal injection of either vehicle or LPS (25 mg/kg; Figure VI in the online-only Data Supplement).
The acetylcholine-induced concentration-dependent relaxations were not significantly different between the aortae of 8-week-old WT and TLR4−/− mice (Figure 5). The in vivo injection of LPS significantly attenuated the relaxations to acetylcholine in WT, but not in TLR4−/− preparations (Figure 5). The acetylcholine-induced relaxations in WT aortae were normalized by apocynin (10−4 mol/L), which did not significantly affect the response in TLR4−/− preparations (Figure 5). The injection of LPS did not significantly affect the relaxations to sodium nitroprusside (Figure VII in the online-only Data Supplement).
In vitro incubation with LPS (10−6 mol/L for 4 hours) significantly attenuated the relaxations to acetylcholine in WT, but not in TLR4−/− aortae (Figure 6).
In mesenteric arteries (in the presence of L-NAME and indomethacin) of 8-week-old WT and TLR4−/− mice, acetylcholine induced comparable concentration-dependent and endothelium-dependent relaxations (Figure 5). The in vivo injection of LPS reduced the relaxations to acetylcholine in WT, but not in TLR4−/− arteries (Figure 5). The in vitro administration of apocynin prevented this inhibitory effect of LPS (Figure 5).
In vitro incubation with LPS significantly reduced the relaxations to acetylcholine in WT, but not in TLR4−/− arteries (Figure 6).
In the presence of L-NAME (10−4 mol/L), acetylcholine evoked comparable and minimal contractions in rings with endothelium of carotid arteries of lean WT mice and TLR4−/− mice (Figure 5). After the in vivo injection of LPS, it induced significantly larger increases in tension in WT, but not in TLR4−/− preparations, which were inhibited by apocynin (Figure 5). However, in vivo injection of LPS did not significantly affect the contractions to the thromboxane-prostanoids receptor agonist U46619 (Figure VIII in the Supplement).
After in vitro incubation with LPS, acetylcholine induced significantly larger increases in tension in WT preparations only (Figure 6).
The current experiments were undertaken to determine whether or not TLR4, a major target for the initiation of inflammatory responses,13 contributes to the endothelial dysfunction resulting from diet-induced obesity and diabetes mellitus. Responses of arteries from WT mice were compared with those obtained in preparations from mice with mutated, nonfunctional TLR4, subjected to a high-fat diet. Furthermore, a unique animal model was created, with combined nonfunctional mutation of the leptin and TLR4 receptors. Although these DWM mice displayed a significantly heavier body weight, they were protected from hypertension and hyperglycemia demonstrating that overweight, per se, does not cause these pathological changes. They can be attributed to the decreased production of cytokines illustrated by the lower expression of TNF-α, IL-1β, and IL-6 in the DWM mice adipose tissue. The lower plasma level of free fatty acids and cholesterol, also attributable to the TLR4 mutation,14 explained why these animals did not develop the equivalent of a metabolic syndrome. The enhanced insulin sensitivity in DWM mice directly illustrates the link between TLR4 and insulin resistance.
The present study confirms that high-fat diet14,15 and diabetes mellitus16 increase arterial blood pressure in mice and demonstrates that loss of function of TLR4 prevents obesity- and diabetes-induced hypertension but does not affect this parameter in animals receiving standard diet.
Three types of endothelium-dependent responses were investigated in the present study.
First, NO-mediated acetylcholine-induced relaxations were determined in aortae in which, endothelium-dependent relaxations are attributable exclusively to the production of NO by eNOS.17 The involvement of NO was confirmed by the abolition of the responses by L-NAME, a nonselective inhibitor of NO synthases. In the aortae of 16-week-old WT mice given high-fat diet, or those of Leprdb/db mice, relaxations to acetylcholine, but not those to sodium nitroprusside, were blunted, demonstrating endothelial dysfunction. However, such relaxations were not altered in preparations of TLR4−/− or DWM mice, suggesting that TLR4 loss-of-function protects against the reduction in NO bioavailability attributable to diet-induced obesity and diabetes mellitus. The latter reduction is attributable to TLR4-induced impairment of eNOS activation. This conclusion is supported by the Western blotting data in Leprdb/db mice demonstrating a reduced phosphorylation both under basal conditions and on stimulation with acetylcholine, and an increased eNOS monomer level, 2 measures of the activation of the enzyme.17 In mice, uncoupling of eNOS can be caused by hypercholesterolemia.18 The present study demonstrates that loss of function of TLR4 rescues diabetic obese mice from the impaired relaxations. The likely mechanism underlying this effect is a reduced superoxide anion production leading to prevention of eNOS uncoupling.19–21 This interpretation is supported by the observation that in Leprdb/db aortae the antioxidant apocynin, which scavenges superoxide anions,10 recoupled eNOS allowing phosphorylation of the enzyme on exposure to acetylcholine, thus potentiating relaxations to the muscarinic agonist.
Second, the involvement of endothelium-dependent hyperpolarization was assessed by determining the inhibitory effect of incubation with 1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole and 6,12,19,20,25,26-hexahydro- 5,27:13,18:21,24-Trietheno-11,7-metheno-7H-dibenzo[b,m][1,5,12,16]tetraazacyclotricosine-5,13-diium ditrifluoroacetate hydrate on relaxations to acetylcholine, obtained in the presence of L-NAME and indomethacin.22 The present findings confirm that EDH-mediated relaxations are impaired in arteries of obese and diabetic mice.2 Activation of TLR4 triggers the production of inflammatory cytokines.7 In arteries of diabetic mice, TNF-α deficiency reverses the blunting of EDH-mediated relaxations.4 Taken in conjunction with the reduced level of TNF-α, IL-1β, and IL-6 observed in the adipose tissue of DWM mice, the prevention of the impairment of EDH-mediated relaxations observed in their arteries illustrates that TLR4 signaling contributes to this aspect of endothelial dysfunction resulting from obesity and diabetes mellitus.
An elevated glucose level inhibits acetylcholine-induced, EDH-mediated responses in the rat mesenteric artery because of the increased production of ROS.23 The administration of the antioxidant apocynin prevented the reduction in EDH-mediated relaxation, suggesting that the impaired response in arteries with functional TLR4 signaling also results from an increased ROS production.
Third, the aberrant production of vasoactive factors causing endothelial dysfunction includes the increased release of EDCFs from endothelial cells.24 In the mouse, EDCF-mediated responses are most pronounced in the carotid artery,25 and therefore this preparation was selected to examine endothelium-dependent contractions. Various arachidonic acid metabolites, including thromboxane A2, prostaglandin F2α, and prostacyclin, contribute to endothelium-dependent contractions that are ultimately attributable to activation of thromboxane-prostanoids receptors of the vascular smooth muscle.26 In the present study, the levels of 6-ketoPGF1α, the stable metabolite of prostacyclin, were measured to estimate the production of EDCF.24,26,27
The findings that the endothelium-dependent contractions to acetylcholine were abolished by indomethacin and a thromboxane-prostanoids receptor antagonist (S18886) demonstrate an EDCF-mediated response.24,26,27 Augmented endothelium-dependent contractions were observed in carotid arteries of older or high-fat fed WT and Leprdb/db mice, but not in TLR4−/− and DWM preparations, implying an amplification of the phenomenon by functional TLR4. The acetylcholine-induced endothelium-dependent contractions were blocked by a selective COX-1, but not by a COX-2 inhibitor, confirming the key role of COX-1 in EDCF-mediated responses in mice.28 TLR4 mutation did not significantly affect the COX-1 expression at either the mRNA or protein levels, implying that it does not modulate the presence of the enzyme. However, because the production of 6-ketoPGF1α was increased in Leprdb/db arteries, TLR4 must upregulate COX-1 activity. The antioxidant apocynin reduced the production of 6-ketoPGF1α, and hence the endothelium-dependent contractions,24,26,27 suggesting that ROS play a key role in activating cyclooxygenase to generate prostanoids when TLR4 is functional. Thus, the reduced endothelium-dependent contractions in the carotid arteries of the DWM mice are consistent with the lower production of ROS suggested by the lucigenin-chemiluminescence measurements in isolated membrane preparations of these blood vessels.28,29 In small mesenteric arteries of diabetic mice, the enhanced contractile activity is also associated with increased oxidative stress and cyclooxygenase production.16 The lower ROS production in DWM mice arteries is accompanied by a lesser mRNA expression and protein presence of NOX1 and NOX4, the major isoforms of NADPH oxidase30 that contribute to oxidative stress in the vascular wall. LPS, the prototypical agonist of TLR4, stimulates the production of superoxide anions in cultured endothelial cells, and this response is reduced by silencing RNAs for either NOX1 or NOX4 (Figure IX in the online-only Data Supplement). These observations indirectly support the interpretation that the decreased ROS production in DWM mice arteries may well result from a lesser activity of NADPH oxidase.
The finding that the endothelium-dependent contractions were prevented by the antioxidant apocynin5 and the inhibitor of flavoenzyme oxidoreductases (including NADPH oxidase)30 diphenyliodium31 is consistent with the key initial role of ROS in the EDCF-mediated contraction. The inhibitory effects of the cell permeable agents, diethyldithiocarbamate, catalase-polyethylene glycol, and manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin, observed in the present experiments imply that the dismutation of superoxide anions to H2O2 is also required. The observation that the addition of exogenous superoxide dismutase, which does not permeate cell membranes, did not prevent the endothelium-dependent contractions to acetylcholine, in confirmation of earlier studies in the rat aorta,12 indicates that the dismutation occurs intracellularly. The comparable contractions to exogenous H2O2 observed in the carotid arteries of Leprdb/db and DWM mice suggest that augmented levels of free radicals in the endothelial cells rather than their direct effect on vascular smooth muscle play a crucial role in the dysfunction observed in Leprdb/db preparations.
The present study thus demonstrates that loss-of-function in TLR4 protects mice from endothelial dysfunction resulting from obesity, whether genetic or diet induced, by both potentiating endothelium-dependent relaxations and decreasing endothelium-dependent contractions. These protective effects of the TLR4 mutation can be attributed to a reduced production of ROS because the different aspects of endothelial dysfunction observed in obese/diabetic mice with functional TLR4 are prevented by apocynin. Elevated ROS formation in the vascular wall is a key feature of cardiovascular disease and a likely contributor to endothelial dysfunction and vascular inflammation.32 ROS generated by NOX are the initial source of endothelial dysfunction in diabetes mellitus.5,9,19 NOX1 contributes significantly to gastrointestinal inflammation, hypertension, and restenosis after angioplasty.33
LPS is the prototypical ligand for TLR414 and triggers the signaling cascade leading to the activation of nuclear factor-κB and the production of proinflammatory cytokines, including TNF-α and IL-1β 8. Indeed, mutation of the TLR4 gene prevents LPS signal transduction in vitro and in vivo.34 Chronic elevation of the circulating levels of LPS exists in obese subjects and contributes to insulin resistance and its related cardiometabolic complications.35,36 The present experiments demonstrate that LPS administration in vivo to lean WT mice does not change glucose tolerance and insulin sensitivity but impairs endothelium-dependent relaxations and enhances endothelium-dependent contractions, mimicking the vascular phenotype observed in obese/diabetic mice. However, LPS fails to elicit these vascular responses in mice with mutation of TLR4, implying that the derogative effects of LPS on vascular tone are TLR4 dependent. These findings suggest that TLR4 activation-induced endothelial dysfunction is a direct effect and not secondary to metabolic changes. This conclusion is strengthened by the demonstration that in vitro administration of LPS had identical effects on endothelial function as its in vivo injection, and this again only in WT arteries. The ex vivo reversal by apocynin of these responses induced by LPS indicates again that ROS are the key link between TLR4 and endothelial dysfunction.
In conclusion, the present findings suggest that activation of TLR4 promotes the transcription of NADPH oxidase 1 and 4, resulting in elevated reactive oxidative stress. In endothelial cells, the increased level of ROS reduces eNOS coupling leading to a reduced NO-production and bioavailability, impairs EDH-mediated responses, and increases the activity of COX-1 with augmented EDCF-mediated contractions. These effects combine to initiate endothelial dysfunction in arteries of obese and diabetic mice. The present study identifies TLR4 as being responsible for the endothelial dysfunction resulting from obesity and diabetes mellitus.
We thank the late Dr Francosis Li for his contribution to the experiments.
Sources of Funding
This project is supported by the University of Hong Kong, the Research Grant Council of the Hong Kong Special Administrative Region (HKU777208M, NHKU 735/08, and HKU4/CRF/10), and the World Class University program (R31-20029) funded by the Ministry of Education, Science and Technology, South Korea.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.112.301087/-/DC1.
- Received February 27, 2012.
- Accepted January 22, 2013.
- © 2013 American Heart Association, Inc.
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This study reveals that loss-of-function of toll-like receptor 4 in mice can alleviate dietary obesity- and diabetes-associated endothelial dysfunction by decreasing the production of reactive oxygen species (generated by NADPH oxidase isoforms 1 and 4) and vasoconstrictor prostaglandins (generated by cyclooxygenase-1) in endothelial cells. Because endothelial dysfunction precedes several cardiovascular complications, toll-like receptor 4 inactivation may be a therapeutic target for the prevention and treatment of vascular disease associated with obesity and diabetes mellitus.