Published online before print January 2, 2007, doi: 10.1161/CIRCULATIONAHA.106.650671
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 2007;115:245-254.)
© 2007 American Heart Association, Inc.
Molecular Cardiology |
Tumor Necrosis Factor-
Induces Endothelial Dysfunction in Leprdb Mice
From the Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Tex (X.G., A.P., X.X., N.T.-S., S.C., C.Z.); and Department of Physiology, Louisiana State University Health Sciences Center, New Orleans (S.B., B.J.P., W.M.C.).
Correspondence to Cuihua Zhang, MD, PhD, Mechael E. DeBakey Institute, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4466. E-mail czhang{at}cvm.tamu.edu
Received July 9, 2006; accepted November 2, 2006.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background— We hypothesized that the inflammatory cytokine tumor necrosis factor-
(TNF) produces endothelial dysfunction in type 2 diabetes.
Methods and Results— In m Leprdb control mice, sodium nitroprusside and acetylcholine induced dose-dependent vasodilation, and dilation to acetylcholine was blocked by the NO synthase inhibitor NG-monomethyl-L-arginine. In type 2 diabetic (Leprdb) mice, acetylcholine- or flow-induced dilation was blunted compared with m Leprdb, but sodium nitroprusside produced comparable dilation. In Leprdb mice null for TNF (dbTNF–/dbTNF–), dilation to acetylcholine or flow was greater than in diabetic Leprdb mice and comparable to that in controls. Plasma concentration of TNF was significantly increased in Leprdb versus m Leprdb mice. Real-time polymerase chain reaction and Western blotting showed that mRNA and protein expression of TNF and nuclear factor-
B were higher in Leprdb mice than in controls. Administration of anti-TNF or soluble receptor of advanced glycation end products attenuated nuclear factor-B and TNF expression in the Leprdb mice. Immunostaining results show that TNF in mouse heart is localized predominantly in vascular smooth muscle cells rather than in endothelial cells and macrophages. Superoxide generation was elevated in vessels from Leprdb mice versus controls. Administration of the superoxide scavenger TEMPOL, NAD(P)H oxidase inhibitor (apocynin), or anti-TNF restored endothelium-dependent dilation in Leprdb mice. NAD(P)H oxidase activity, protein expression of nitrotyrosine, and hydrogen peroxide production were increased in Leprdb mice (compared with controls), but these variables were restored to control levels by anti-TNF.
Conclusion— Advanced glycation end products/receptor of advanced glycation end products and nuclear factor-B signaling play pivotal roles in TNF expression through an increase in circulating and/or local vascular TNF production in the Leprdb mouse with type 2 diabetes. Increases in TNF expression induce activation of NAD(P)H oxidase and production of reactive oxidative species, leading to endothelial dysfunction in type 2 diabetes.
Key Words: acetylcholine • coronary disease • inflammation • microcirculation • diabetes mellitus • endothelium • vasodilation
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Clinical and experimental studies have demonstrated that cardiac function is compromised and cardiovascular diseases are increased in type 2 diabetes, suggesting that alterations in cardiac tissue metabolism are responsible for this impairment.1 In diabetic humans, vasodilation of coronary arteries was also altered after pharmacological (acetylcholine) or mechanical (cold test) stimuli, but these abnormalities of large vessels were not associated with angiographic lesions and are independent of other cardiovascular risk factors,2 suggesting impaired endothelial function without any anatomic lesions. However, the exact mechanisms underlying type 2 diabetes–induced impaired vasodilation remain unresolved, and there is no scientific consensus.
Clinical Perspective p 254
The ligand-activated transcription factor belonging to the nuclear receptor family, peroxisome proliferator-activated receptor-
,3 is a regulator of lipid and glucose metabolism and therefore is the target of insulin-sensitizing drugs, such as thiazolidinediones, which are frequently used to treat metabolic complications associated with type 2 diabetes mellitus.4 Despite these connections, there has been no link established between the vascular pathology of diabetes and the existent inflammation. Tumor necrosis factor (TNF) is a proinflammatory cytokine that has been implicated in the pathogenesis of septic, traumatic, and hypovolemic shock–associated cardiac dysfunction, as well as cardiovascular diseases such as acute myocardial infarction, chronic heart failure, atherosclerosis, viral myocarditis, and cardiac allograft rejection.5 A decrease in endothelium-dependent dilation occurs shortly after the generation of superoxide (O2–) radicals during reperfusion,6–9 suggesting that endothelial generation of O2– radicals acts as a triggering mechanism for endothelial dysfunction.8,9 Moreover, O2– can lead to formation of other reactive oxidative species (ROS) such as hydrogen peroxide (H2O2) and peroxynitrite (ONOO–). The interaction of advanced glycation end products (AGEs) with its receptor (RAGE) induces production of ROS, which can stimulate the cascade leading to nuclear factor-B (NF-B)–induced transcriptional events.10,11 NF-B induces expression of TNF.12 Despite these connections, there has been no link established between the vascular pathology of diabetes and the existent inflammation. Therefore, we propose that type 2 diabetes–induced coronary endothelial dysfunction is mediated by TNF and that further investigation will reveal the causal mechanisms. To test this, we evaluated the endothelium-dependent and -independent vasodilation, the circulating levels of TNF, and its expression at the wall of coronary arterioles in type 2 diabetes (Leprdb), type 2 diabetes null for TNF (dbTNF–/dbTNF–), and the lean control (m Leprdb) mice. We also tested the mechanisms by which TNF induces endothelial dysfunction, the role of NF-B and AGE/RAGE signaling in the expression of TNF, and the role of ROS (O2–, H2O2, ONOO–) in coronary arterioles in type 2 diabetes.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animal Models
The procedures followed were in accordance with approved guidelines set by the Laboratory Animal Care Committee at Texas A&M University. Heterozygote controls (m Leprdb), homozygote type 2 diabetes (Leprdb), and Leprdb null for TNF (dbTNF–/dbTNF–) mice were purchased from the Jackson Laboratory (Bar Harbor, Me) and maintained on a normal rodent chow diet. Our studies used 12- to 14-week-old, 15- to 25-g m Leprdb and 25- to 50-g Leprdb and dbTNF–/dbTNF– mice of either sex. We used the same strain (C57BL/6J) of m Leprdb and dbTNF–/dbTNF– mice to match the backgrounds of Leprdb mice. The cross (dbTNF–/dbTNF–) of Leprdb with TNF knockout mice is heterozygous for Leprdb and homozygous for TNF knockout mice (TNF–/–). These dbTNF–/dbTNF– mice show the phenotypes of hyperglycemia and obesity, the diabetic phenotype that is consistent with the penetrance of the leptin receptor mutation. The obese mice from the second round of breeding of Leprdb and TNF–/– were used in experimentation.
Measurement of Blood Parameters
Blood was obtained from vena cava after anesthesia with sodium pentobarbital (50 mg/kg IP) and exposure of the vein. Blood was collected, and the plasma was stored at –80 C° until analysis.
Plasma Concentration of TNF-
TNF was measured with the use of a commercial kit, BIO-Plex cytokine assay (BIO-Plex mouse 3-plex assay, Bio-Rad Laboratories, Hercules, Calif). TNF concentrations were automatically calculated by BIO-Plex Manager software with the use of a standard curve derived from a recombinant cytokine standard. Values were expressed as picograms per milliliter.
Blood Glucose
We used an Accu-check compact glucometer (Roche Diagnostic GmbH, Mannheim, Germany) for measuring blood glucose in m Leprdb, Leprdb, and dbTNF–/dbTNF– mice with food ad libitum at the same time (7 to 8 AM) every time. (Absence of a functional leptin receptor in Leprdb mouse makes food deprivation stressful; this precluded a fasting protocol.)
Lipid Level
Serum lipid level was measured with the Cholesterol/Cholesteryl Ester Quantitation Kit (Biovision) with the use of spectrophotometry.
Insulin Resistance
Blood (
1 mL) was obtained by cardiac puncture with a syringe containing 24 mmol/L EDTA. Insulin resistance was determined by using the homeostasis model assessment, which estimates steady state insulin resistance.
Blood Pressure
Blood pressure was monitored with the use of a MacLab/8 data acquisition system (AD Instruments, Milford, Mass) equipped with an ETH 400 transducer amplifier via the femoral artery catheterized with PE-10 polyethylene tubing.
mRNA Expression of TNF by Real-Time Polymerase Chain Reaction
Total RNA was extracted from isolated coronary arterioles with the use of Trizol reagent (Life Technologies Inc) and was processed directly to cDNA synthesis with SuperScript III Reverse Transcriptase (Life Technologies Inc). The primers of TNF were designed (primer 3 software) and synthesized (Qiagen).7,13 cDNA was amplified with a quantitative reverse transcription polymerase chain reaction kit with SYBR Green (Life Technologies Inc). Data were calculated by the 2–
CT method7 and presented as fold change of transcripts for TNF gene in Leprdb mice normalized to ß-actin, compared with m Leprdb mice (defined as 1.0-fold).
Treatment With TNF Neutralization or Soluble RAGE
The neutralizing antibody to TNF (anti-TNF)14 is 2E2 monoclonal antibody (2E2 monoclonal antibody 94021402; NCI Biological Resources Branch). At 12 to 14 weeks of age, all mice received the neutralizing anti-TNF (2E2 monoclonal antibody; 0.625 mg/mL per kilogram per day IP for 3 days). Dosage was based on our estimates of TNF expression (in the low nanogram or picogram range); this is able to neutralize this amount of TNF 10- to 100-fold.
Soluble RAGE (sRAGE), the extracellular two thirds of the receptor, binds AGEs and interferes with their ability to bind and activate cellular RAGE.15 We administered sRAGE (a gift from Dr Ann Marie Schmidt; 80 µg IP per mouse per day) to m Leprdb and Leprdb mice for 10 days to determine whether RAGE affects the expression of TNF-.
Functional Assessment of Isolated Coronary Arterioles
The techniques for identification and isolation of coronary microvessels were described in detail previously.16 Briefly, coronary arterioles (40 to 100 µm in diameter) from m Leprdb, Leprdb, and dbTNF–/dbTNF– mice were carefully dissected for in vitro study.17 The contributions of the NO pathway in these vasodilations were examined by treating the vessels with the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) (10 µmol/L, 30-minute incubation).
To determine whether TNF was playing a role in endothelial injury in type 2 diabetes, endothelial-dependent dilation (acetylcholine, 0.1 nmol/L to 10 µmol/L), endothelial-independent dilation (sodium nitroprusside, 0.1 nmol/L to 10 µmol/L), and flow-induced dilation (NO-mediated, endothelial-dependent, but agonist-independent; 4 to 60 cm H2O) were assessed in coronary arterioles in m Leprdb, Leprdb, and Leprdb mice treated with anti-TNF and dbTNF–/dbTNF– mice. Flow is established by the production of a pressure drop across the vessel and is linearly related to the pressure drop. To determine the role of TNF and O2– anion in altered vasoactive responses in type 2 diabetes, the aforementioned vasodilatory functions were examined in the presence of the O2– scavenger TEMPOL (a membrane-permeable O2– dismutase mimetic; 1 mmol/L, 60-minute incubation). The contributions of NAD(P)H oxidase, xanthine oxidase, and mitochondrial respiratory chain in generating O2– were assessed by treating the vessels with an NAD(P)H oxidase inhibitor apocynin (10 µmol/L), a xanthine oxidase inhibitor allopurinol (10 µmol/L), or the mitochondrial respiratory chain inhibitor rotenone (1 µmol/L) for a 60-minute incubation, separately. Ebselen (10 µmol/L) and catalase (1000 U/mL) were also used (60-minute incubation) to determine whether ONOO– and H2O2 were involved in endothelial dysfunction. All drugs were administered extraluminally in these functional studies.
NAD(P)H Oxidase Activity
NAD(P)H oxidase activity was assayed in protein isolated from coronary arteriole extracts as initiated by the addition of 50 µmol/L N,N-dimethyl-9,9-biacridinium dinitrate (Lucigenin) (Sigma, St Louis, Mo) to the incubation mixture. Samples were counted immediately with the use of a tabletop luminometer, and fluorescence values were averaged from 2 minutes of stable readings for that sample. Samples were run in duplicate, and the NAD(P)H oxidase activity was normalized to the m Leprdb control group.
Protein Expression of TNF, Nitrotyrosine, and NF-B by Western Blot Analyses
Coronary arteries (4 to 6 vessels per sample) were separately homogenized and sonicated in lysis buffer (Cellytic MT Mammalian Tissue Lysis/Extraction Reagent, Sigma). Protein concentrations were assessed with the use of BCA Protein Assay Kit (Pierce Biotechnology, Inc, Rockford, Ill), and equal amounts of protein (40 µg) were separated by SDS-PAGE and transferred to nitrocellulose membranes (Hybond, Amersham Pharmacia Biotech,Uppsala, Sweden).18,19 TNF, nitrotyrosine (an indicator for peroxynitrite-mediated tissue injury), and NF-B protein expressions were detected with the use of TNF primary antibodies (Santa Cruz), nitrotyrosine primary antibodies (Abcam), and NF-B primary antibodies (Abcam) in m Leprdb, Leprdb, and Leprdb mice treated with anti-TNF (0.625 mg/mL per kilogram per day IP for 3 days), the NAD(P)H oxidase inhibitor apocynin (100 mg/kg per day IP for 3 days), or sRAGE (80 µg/d IP for 10 days; to antagonize RAGE signaling). Horseradish peroxi-dase–conjugated goat anti-mouse was used as the secondary antibody (Abcam). Signals were visualized by enhanced chemiluminescence (ECL, Amersham) and quantified by Quantity One (Bio-Rad Versadoc imaging system).
Immunohistochemical Analyses
To identify and localize proteins in sections of arteries or myocardial tissue, we used immunohistochemistry.19 Slides prepared from formalin-fixed hearts were incubated with blocking solution (BSA 3% in Tris buffer), then incubated with polyclonal goat antibody against TNF (R&D Systems, Minneapolis, Minn) and endothelial cell marker, von Willebrand factor (DakoCytomation), smooth muscle -actin (1A4; Calbiochem), or macrophages (mouse anti-rat CD68; Serotec), then incubated with a secondary fluorescent antibody (Alexa Fluor 488 and Alexa Fluor 568; Molecular Probes, Carlsbad, Calif). Sections were finally mounted in an antifading agent (Slowfade gold with DAPI, Molecular Probes), and then the slides were observed and analyzed with the use of a fluorescence microscope (Leica microscope with a x63 objective). For every section, a negative control (without primary antibody) was performed.
Measurement of O2–
Dihydroethidium
The production of O2– was evaluated in isolated coronary arterioles (40 to 100 µm) with the oxidative fluorescent dye dihydroethidium.17 Dihydroethidium fluorescence for O2– in both endothelial and smooth muscle layers of vessels was measured in m Leprdb, Leprdb, or Leprdb mice treated with anti-TNF. Images were obtained with the use of a Nikon fluorescence microscope (605-nm long-pass filter). Control and experimental tissues were placed on the same slide and processed under the same conditions.
Electron Paramagnetic Resonance Spectroscopy
Superoxide quantification from the electron paramagnetic resonance spectra was determined in the homogenate (4 to 6 isolated coronary arterioles) as described previously.17
Measurement of H2O2
Production of H2O2 was determined by using the Assay Kit (R&D Systems). Serum was obtained from m Leprdb, Leprdb, and Leprdb mice treated with catalase (1000 U/mL IP for 3 days) and Leprdb mice treated with anti-TNF (0.625 mg/mL IP per kilogram per day for 3 days). The H2O2 concentration was then determined by measuring the optical density of the solution in each well (microplate reader set to 550 nm).
Data Analysis
At the end of each experiment, the vessel was relaxed with 100 µmol/L sodium nitroprusside to obtain its maximal diameter at 60 cm H2O intraluminal pressure.17 All diameter changes in response to agonists were normalized to the vasodilation in response to 100 µmol/L sodium nitroprusside and expressed as a percentage of maximal dilation. All data are presented as mean±SEM. Statistical comparisons of vasomotor responses under various treatments were performed with 2-way ANOVA, and intergroup differences were tested with Bonferroni inequality. Significance was accepted at P<0.05.
The authors had full access to and take responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Plasma Concentrations of Glucose, Blood Pressure, Body Weights, Abdominal Girth, Lipid Level, and Insulin Resistance
Plasma parameters were measured at 12 to 14 weeks in different strains of mice (Table).
|
Plasma Concentration, mRNA, and Protein Expression of TNF
Figure 1 shows the plasma concentration and mRNA expression of TNF in isolated coronary arterioles of m Leprdb and Leprdb mice.
|
Cellular Source of TNF Expression in Type 2 Diabetes
Markers for endothelial cells (von Willebrand factor; Figure 2), vascular smooth muscle cells (-actin; Figure 2), or macrophages (mouse anti-rat CD68; data not shown) along with TNF to establish the cell type expressing the TNF show that TNF in Leprdb mice hearts was localized in vascular smooth muscle cells (Figure 2). Experiments were performed without the primary antibodies to test whether or not staining specificity was related to the nonspecific binding of the secondary antibodies, which showed no staining in heart sections, indicating that the signals were due to specific binding of the primary antibody.
|
Role of TNF in Type 2 Diabetes–Induced Vascular Dysfunction
To show NO dependency of acetylcholine-dependent dilation in m Leprdb mice, we studied responses to the agonist before and after treatment with L-NMMA (Figure 3). Dilation to acetylcholine was significantly attenuated after administration of L-NMMA in m Leprdb mice (Figure 3), which indicates that vasodilation to acetylcholine was NO mediated. In dbTNF–/dbTNF– or anti-TNF–treated Leprdb mice, acetylcholine-induced vasodilation was greater than that in diabetic Leprdb mice and was comparable to that in m Leprdb controls (Figures 3 and 4
), which provides further support for our hypothesis that TNF plays a key role in endothelial dysfunction in diabetes.
|
|
Roles of ROS in Type 2 Diabetes–Induced Vascular Dysfunction
To establish the pathway for O2– production, we administered O2– scavenger with TEMPOL (Figure 5A), the NAD(P)H oxidase inhibitor apocynin (Figure 5B), the xanthine oxidase inhibitor allopurinol (10 µmol/L; data not shown), or the mitochondrial respiratory chain inhibitor rotenone (1 µmol/L; data not shown) to determine whether vasodilation to acetylcholine would be restored in Leprdb mice. Administration of TEMPOL, apocynin, or anti–TNF- restored impaired vasodilation to acetylcholine in Leprdb mice, but allopurinol or rotenone did not (Figure 5A and 5B).
|
We also examined the roles of H2O2 with H2O2 inhibitor catalase (1000 U/mL) and ONOO– with the ONOO– scavenger ebselen (10 µmol/L; glutathione peroxidase mimetic) in TNF-induced endothelial dysfunction during diabetes (Figure 5).
Type 2 Diabetes–Induced Superoxide Production in Coronary Arterioles in Type 2 Diabetes
Figure 6A shows dihydroethidium fluorescence imaging of O2– in coronary arterioles. Setting the scanning threshold to obtain a clear background image of the blood vessel allowed identification of the smooth muscle and endothelial layers. In control conditions (nondiabetic, ie, m Leprdb mice), dihydroethidium oxidative fluorescence revealed sparse levels of O2– throughout the vessel wall. Figure 6B shows the results from electron paramagnetic resonance spectroscopy to quantify the production of O2– and reflects agreement with the results obtained by dihydroethidium and discussed above.
|
Type 2 Diabetes Increased NAD(P)H Oxidase Activity
We examined NAD(P)H oxidase activity from isolated coronary arterioles in m Leprdb and Leprdb mice (Figure 7A). The treatment of anti-TNF (0.625 mg/mL per kilogram per day IP for 3 days) or apocynin (100 mg/kg per day IP for 3 days) did not affect NAD(P)H oxidase activity in m Leprdb mice (data not shown).
|
NF-B Expression Increased and sRAGE Decreased TNF Expression in Type 2 Diabetes
Western blotting shows the protein expression NF-B p65 (Figure 7B) in isolated coronary arterioles of m Leprdb, Leprdb, and Leprdb mice treated with apocynin or anti-TNF. Apocynin or anti-TNF decreased the expression of NF-B in Leprdb mice to levels similar to those observed in m Leprdb. Figure 7C shows TNF protein expression in m Leprdb, Leprdb, sRAGE-treated m Leprdb, and sRAGE-treated Leprdb mice. sRAGE significantly reduced TNF expression in the Leprdb mice from the untreated Leprdb mice.
Type 2 Diabetes–Induced ONOO– and H2O2 Production in Coronary Arterioles Isolated From Leprdb Mice
Western blot analysis (Figure 8A) for nitrotyrosine in homogenates from m Leprdb, Leprdb, and anti-TNF–treated Leprdb mice revealed significantly higher levels of nitrotyrosine in Leprdb mice, which was reduced to control (m Leprdb) levels by anti-TNF. Figure 8B shows the elevations in H2O2 production in Leprdb mice compared with the control m Leprdb strain. Treatment with anti-TNF or catalase reduced H2O2 production in the Leprdb mice.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Our results suggest that the inflammatory cytokine TNF and AGE/RAGE signaling lead to oxidative stress via NAD(P)H activation and perhaps via activating NF-
B, which in turn may lead to TNF expression inducing endothelial dysfunction in the Leprdb mouse, which is a model for obesity and type 2 diabetes. Importantly, our findings support the concept that TNF plays a pivotal role in endothelial dysfunction in type 2 diabetes based on the following observations: antibody neutralization of TNF prevented coronary endothelial dysfunction and reduced ONOO– and O2– generation and formation of nitrotyrosine in Leprdb mice. Blockade of NAD(P)H oxidase mimicked the actions of anti-TNF on endothelial function in Leprdb mice. Molecular evidence indicated that the expressions of TNF, NF-B, and NAD(P)H oxidase activity were significantly increased in Leprdb mice; however, sRAGE decreased TNF protein expression, anti-TNF attenuated NF-B expression, and administration of anti-TNF to Leprdb mice resulted in NAD(P)H oxidase levels comparable to those in the control. Our findings are consistent with TNF involvement by activating NAD(P)H oxidase in endothelial dysfunction in type 2 diabetes.
Impaired Coronary Control in Type 2 Diabetes
Recent evidence supports the idea that the effect of TNF is NO dependent by producing a rapid inhibitory action on NO synthase in the endothelium via activation of a sphingomyelinase/ceramide signaling pathway; this mechanism purportedly mediates the action of TNF, thereby contributing to vascular endothelial dysfunction in coronary circulation under different pathological conditions with increased cytokines.9,17–19 TNF expression was significantly increased in Leprdb mice; anti-TNF restored NO-mediated coronary arterial dilation in Leprdb mice but did not affect the endothelium-dependent vasodilation in lean controls. Our previous study20 shows that administration of anti-TNF protected endothelial dysfunction induced by TNF in isolated vessels from lean control rats, confirming that the monoclonal anti-TNF14 was specific. Impaired endothelium-dependent vasodilation was restored in Leprdb mice after the treatment with anti-TNF, demonstrating the pivotal role of this inflammatory cytokine in the vascular pathology of type 2 diabetes. This result is in agreement with those of previous studies showing that TNF can decrease the release of endothelial NO and induce impairment of endothelium-dependent relaxation in a variety of vascular beds.14,19
In the present study, endothelium-dependent vasodilation was attenuated in coronary arterioles from Leprdb versus m Leprdb control mice. The present results provide direct evidence that type 2 diabetes is associated with impaired agonist-induced NO production and NO-mediated dilation in the coronary microcirculation. We found increased TNF mRNA expression (6-fold), plasma concentration of TNF (6-fold), and protein expression of TNF (>2.5-fold in small coronary arteries), but sRAGE decreased TNF expression in Leprdb mice. Immunostaining results showed that TNF in Leprdb mice heart is localized in vascular smooth muscle cells. We believe that ROS production in vascular endothelial cells and smooth muscle cells by TNF would limit NO bioavailability and reduce NO-dependent dilation. It is provocative to note that despite the similarities in glucose, body weights, lipid level, insulin resistance, and blood pressure in diabetic animals, endothelial function was better in dbTNF–/dbTNF– and in Leprdb mice treated with anti-TNF. This suggests that AGE/RAGE signaling plays a pivotal role in attenuating TNF protein expression, and TNF is the key cytokine that induced endothelial dysfunction in type 2 diabetes.
The Major Source of O2– Production in Type 2 Diabetes
Although there are multiple intracellular sources for formation of oxygen free radicals (eg, mitochondria, xanthine oxidase, NAD(P)H oxidase), our results support the idea that the major enzyme activated by TNF in type 2 diabetes is NAD(P)H oxidase. We can state with conviction that the major source of O2– was NAD(P)H oxidase in type 2 diabetes because the NAD(P)H oxidase inhibitor apocynin significantly reduced O2– production as measured by dihydroethidium fluorescence and electron paramagnetic resonance. Moreover, the antagonism of NAD(P)H oxidase virtually normalized endothelium-dependent vasodilatation. Our results strongly suggest that the pathway for TNF-induced endothelial dysfunction is mediated by activation of NF-B, NAD(P)H oxidase, and the subsequent production of O2–. On the contrary, we did not find any improvement in endothelial function after incubation with rotenone and allopurinol, suggesting that mitochondria and the xanthine oxidase system are not likely to be the source of O2– production. Guzik et al21 measured O2– production in diabetic and nondiabetic vessels in response to a range of potential oxidase inhibitors.13 Their results show that oxypurinol and rotenone had minimal or modest effect on O2– production, whereas diphenylene iodonium, an inhibitor of flavin-containing oxidases such as NAD(P)H oxidases, abolished O2– production. Our results demonstrate that the production of TNF is basal to the process of eliciting this oxidative stress. Guzik et al21 reported that the protein expression of the NAD(P)H oxidase subunits p22, p47, and p67-phox are significantly increased in diabetic human tissue versus normal control. We measured NAD(P)H oxidase activity by determining reductions in superoxide production after inhibition of NAD(P)H oxidase. This supports our functional results, in which the experiments with apocynin suggest that NAD(P)H oxidase might be a major enzymatic vascular source of ROS in Leprdb mice.
O2– production is postulated to be linked to TNF. Our results also provide insight into the basis for the endothelial dysfunction induced by type 2 diabetes, namely, oxidative stress. The present study indicates that the model of type 2 diabetes increases TNF, which stimulates endothelial generation of O2– through activation of NAD(P)H oxidase in the endothelium and contributes to the endothelial dysfunction. To our knowledge, this is the first functional study to link the mechanism(s) of the model of type 2 diabetes—in terms of endothelial dysfunction—to TNF, the subsequent activation of NAD(P)H oxidase, and thus the production of O2– in coronary artery endothelium. The link between TNF overexpression and O2– production has been investigated by Zhang et al,13 who found that tiron, a cell-permeable O2– scavenger, and polyethylene glycol–superoxide dismutase prevented TNF-induced impairment of endothelium-dependent vasorelaxation in coronary arterioles. Others18,19 have shown that TNF activates sphingosine kinase and, in vascular smooth muscle, sphingosine kinase activation leads to NADPH activation. Our results are consistent with these studies and support our hypotheses that TNF and O2– are connected in the production of oxidative stress and endothelial dysfunction in type 2 diabetes. Our results also indicate that there is a link among NF-B, NAD(P)H oxidase, and TNF in type 2 diabetes. Specifically, the results suggest that AGE/RAGE and TNF signaling in the diabetic mouse lead to oxidative stress via NAD(P)H activation, activating NF-B, which in turn may activate more TNF expression. The implication of this scheme is that oxidative stress begets further oxidative stress in the obese diabetic animal, and this may explain the development and evolution of vascular pathology in this condition.
Role of ONOO– Anion and H2O2 in Type 2 Diabetes
Oxygen-derived free radicals impair endothelium-dependent relaxation, and NO formed in the endothelium is inactivated by superoxide anion radical O2– to form a stable peroxynitrite anion, ONOO–. The ONOO– anion can produce further oxidative damage to cells because of its inherent potency and stability. Previous studies22 have shown that O2– may be dismutated (spontaneously or enzymatically) to H2O2, which is another oxidant and therefore another possible mediator of oxidant injury. Our data show that H2O2 is also involved in this mechanism because the H2O2 inhibitor catalase partially protected the impaired vasodilation induced by acetylcholine in type 2 diabetes. In addition, the reaction of NO and O2– produces the formation of ONOO–, which is a very potent oxidant. We cannot measure this species because of its very short half-life but can measure an index of its formation by performing immunohistochemical analyses and Western blotting for nitrotyrosine. Our results show that the production of ONOO– (nitrotyrosine) and H2O2 is higher in Leprdb mice than in controls and that anti-TNF reduces the production of ONOO– and H2O2 in diabetic mice, which indicates that TNF may also contribute to increasing ONOO– and H2O2 in addition to O2–, and like O2–, ONOO–, and H2O2 may also participate in endothelial dysfunction in type 2 diabetes. Additionally, apocynin and catalase greatly attenuated the signals for NAD(P)H oxidase activity and H2O2, thus showing specificity for the particular ROS in these experiments. Our study shows the existence of a causal link between TNF expression and O2– production in the endothelial dysfunction occurring in type 2 diabetes; increased O2– also leads to the formation of ONOO–, so that O2–, ONOO–, and H2O2 all limit the bioavailability of NO in type 2 diabetes.
In conclusion, we found that TNF overexpression impairs endothelium-dependent vasodilation in coronary arterioles of type 2 diabetic mice, and the impaired endothelial function can be restored toward normal by administration of TNF antibodies. The mechanism by which TNF affects endothelial function is through increased superoxide production by NAD(P)H oxidases, which in turn leads to a reduced NO bioactivity by direct scavenging. These results confirmed that TNF plays a pivotal role in the vascular pathology of type 2 diabetes and provides new findings in the understanding of interactions between TNF and inflammation, diabetes, and atherosclerosis. These findings may provide further insight into a novel therapeutic target for cardiovascular diseases associated with elevated levels of TNF.
|
Acknowledgments |
|---|
We thank Dr Ann Marie Schmidt for her generous proprietary gift of sRAGE.
Sources of Funding
This study was supported by grants from Pfizer Atorvastatin Research Award (2004-37), an American Heart Association Scientist Development grant (110350047A), and a National Institutes of Health grant (RO1-HL077566) to Dr Cuihua Zhang.
Disclosures
None.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Mahgoub MA, Abd-Elfattah AS. Diabetes mellitus and cardiac function. Mol Cell Biochem. 1998; 180: 59–64.[CrossRef][Medline]
[Order article via Infotrieve]
- Nitenberg A, Paycha F, Ledoux S, Sachs R, Attali JR, Valensi P. Coronary artery responses to physiological stimuli are improved by deferoxamine but not by L-arginine in non-insulin-dependent diabetic patients with angiographically normal coronary arteries and no other risk factors. Circulation. 1998; 97: 736–743.
[Abstract/Free Full Text] - Verges B. Clinical interest of PPARs ligands. Diabetes Metab. 2004; 30: 7–12.[Medline]
[Order article via Infotrieve]
- Moller DE. New drug targets for type 2 diabetes and the metabolic syndrome. Nature. 2001; 414: 821–827.[CrossRef][Medline]
[Order article via Infotrieve]
- Meldrum DM, Cleveland JC, Cain BS. Increased myocardial tumor necrosis factor-alpha in a crystalloid-perfused model of cardiac ischemia-reperfusion injury. Ann Thorac Surg. 1998; 65: 439–443.
[Abstract/Free Full Text] - Downey JM, Omar B, Ooiwa H. Superoxide dismutase therapy for myocardial ischemia. Free Radic Res Commun. 1991; 12–13: 703–720.
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2–CT method. Methods. 2001; 25: 402–408.[CrossRef][Medline]
[Order article via Infotrieve]
- Salvemini D, Cuzzocrea S. Superoxide, superoxide dismutase and ischemic injury. Curr Opin Investig Drugs. 2002; 3: 886–895.[Medline]
[Order article via Infotrieve]
- Lefer AM, Ma XL. Cytokines and growth factors in endothelial dysfunction. Crit Care Med. 1993; 21 (suppl): S9–S14.[Medline]
[Order article via Infotrieve]
- Zou W, Amcheslavsky A, Takeshita S, Drissi H, Bar-Shavit Z. TNF-alpha expression is transcriptionally regulated by RANK ligand. J Cell Physiol. 2005; 202: 371–378.[CrossRef][Medline]
[Order article via Infotrieve]
- Tanaka N, Yonekura H, Yamagishi S, Fujimori H, Yamamoto Y, Yamamoto H. The receptor for advanced glycation end products is induced by the glycation products themselves and tumor necrosis factor-alpha through nuclear factor-kappa B, and by 17beta-estradiol through Sp-1 in human vascular endothelial cells. J Biol Chem. 2000; 275: 25781–25790.
[Abstract/Free Full Text] - De Martin R, Hoeth M, Hofer-Warbinek R. The transcription factor NF-kappa B and the regulation of vascular cell function. Arterioscler Thromb Vasc Biol. 2000; 20: E83–E88.
- Zhang DX, Yi FX, Zou AP, Li PL. Role of ceramide in TNF-alpha induced impairment of endothelium-dependent vasorelaxation in coronary arteries. Am J Physiol. 2002; 283: H1785–H1794.
- Lattime EC, Stutman O. Thymic lymphomas mediate non-MHC-restricted, TNF-dependent lysis of the murine sarcoma WEHI-164. Cell Immunol. 1991; 136: 69–79.[CrossRef][Medline]
[Order article via Infotrieve]
- Feng L, Matsumoto C, Schwartz A, Schmidt AM, Stern DM, Pile-Spellman J. Chronic vascular inflammation in patients with type 2 diabetes: endothelial biopsy and RT-PCR analysis. Diabetes Care. 2005; 28: 379–384.
[Abstract/Free Full Text] - Kuo L, Davis MJ, Chilian WM. Myogenic activity in isolated subepicardial and subendocardial coronary arterioles. Am J Physiol. 1988; 255: H1558–H1562.[Medline]
[Order article via Infotrieve]
- Zhang C, Xu X, Potter BJ, Wang W, Kuo L, Michael L, Bagby GJ, Chilian MW. TNF-alpha contributes to endothelial dysfunction in ischemia/reperfusion injury. Arterioscler Thromb Vasc Biol. 2006; 26: 475–480.
[Abstract/Free Full Text] - Keller M, Lidington D, Vogel L. Sphingosine kinase functionally links elevated transmural pressure and increased reactive oxygen species formation in resistance arteries. FASEB J. 2006; 20: 702–704.
[Abstract/Free Full Text] - Zhang C, Hein TW, Wang W, Ren Y, Shipley RD, Kuo L. Activation of JNK and xanthine oxidase by TNF- impairs nitric oxide-mediated dilation of coronary arterioles. J Mol Cell Cardiol. 2006; 40: 247–257.[CrossRef][Medline]
[Order article via Infotrieve]
- Picchi A, Gao X, Potter BJ. TNF alpha induces endothelial dysfunction in the prediabetic metabolic syndrome. Circ Res. 2006; 99: 69–77.
[Abstract/Free Full Text] - Guzik TJ, Mussa S, Gastaldi D, Sadowsky J, Ratnatunga C, Pillai R, Channon KM. Mechanisms of increased vascular superoxide production in humans diabetes mellitus: role of NADPH oxidase and endothelial nitric oxide synthase. Circulation. 2002; 105: 1656–1662.
[Abstract/Free Full Text] - Cai H. Hydrogen peroxide regulation of endothelial function: origins, mechanisms, and consequences. Cardiovasc Res. 2005; 68: 26–36.
[Abstract/Free Full Text]
CLINICAL PERSPECTIVE
Diabetes is one of the leading risk factors for the development of coronary artery and peripheral vascular diseases. Before vascular disease develops in diabetics, endothelial dysfunction occurs. In fact, endothelial dysfunction appears to be a hallmark underlying vascular disease of many etiologies. We believe that understanding endothelial dysfunction is critical because the progression of vascular disease may be halted if endothelial dysfunction is rectified. Our goal was to delineate a potential cause of endothelial dysfunction by testing the hypothesis that tumor necrosis factor- (TNF-) induces inflammation responsible for endothelial dysfunction in type 2 diabetes. Our data revealed that endothelial function was normal in diabetic mice that were lacking TNF (TNF knockout in the Leprdb diabetic mouse). Moreover, we observed that diabetic mice have elevated expression of TNF, suggesting that this inflammatory cytokine produces, or at least contributes to, endothelial dysfunction in diabetes. We also found that the endothelial dysfunction by TNF in diabetes was related to excess production of the free radical superoxide. Finally, we observed that advanced glycosylation end products and the receptor for these products seem to amplify TNF expression in diabetes; thus, TNF and advanced glycation end products/receptor of advanced glycation end products signaling play pivotal roles in endothelial dysfunction in type 2 diabetes. Furthermore, our study may provide new approaches for the treatment of vasculopathy in type 2 diabetes, such as the possible use of antibodies against TNF or its receptors or decoy receptors for this inflammatory agent.
|
Footnotes |
|---|
*The first 3 authors contributed equally to this work.
This article has been cited by other articles:
 |
|
 |
|
  M. Tesauro, F. Schinzari, V. Rovella, D. Melina, N. Mores, A. Barini, M. Mettimano, D. Lauro, M. Iantorno, M. J. Quon, et al. Tumor Necrosis Factor-{alpha} Antagonism Improves Vasodilation During Hyperinsulinemia in Metabolic Syndrome Diabetes Care, July 1, 2008; 31(7): 1439 - 1441. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Belmadani, D. I. Palen, R. A. Gonzalez-Villalobos, H. A. Boulares, and K. Matrougui Elevated Epidermal Growth Factor Receptor Phosphorylation Induces Resistance Artery Dysfunction in Diabetic db/db Mice Diabetes, June 1, 2008; 57(6): 1629 - 1637. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Jebelovszki, C. Kiraly, N. Erdei, A. Feher, E. T. Pasztor, I. Rutkai, T. Forster, I. Edes, A. Koller, and Z. Bagi High-fat diet-induced obesity leads to increased NO sensitivity of rat coronary arterioles: role of soluble guanylate cyclase activation Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2558 - H2564. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. G. Abraham and A. Kappas Pharmacological and Clinical Aspects of Heme Oxygenase Pharmacol. Rev., March 1, 2008; 60(1): 79 - 127. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||