Mitochondrial-targeted DNA repair enzyme 8-oxoguanine DNA glycosylase 1 protects against ventilator-induced lung injury in intact mice.

This study tested the hypothesis that oxidative mitochondrial-targeted DNA (mtDNA) damage triggered ventilator-induced lung injury (VILI). Control mice and mice infused with a fusion protein targeting the DNA repair enzyme, 8-oxoguanine-DNA glycosylase 1 (OGG1) to mitochondria were mechanically ventilated with a range of peak inflation pressures (PIP) for specified durations. In minimal VILI (1 h at 40 cmH(2)O PIP), lung total extravascular albumin space increased 2.8-fold even though neither lung wet/dry (W/D) weight ratios nor bronchoalveolar lavage (BAL) macrophage inflammatory protein (MIP)-2 or IL-6 failed to differ from nonventilated or low PIP controls. This increase in albumin space was attenuated by OGG1. Moderately severe VILI (2 h at 40 cmH(2)O PIP) produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio and marked increases in BAL MIP-2 and IL-6, accompanied by oxidative mitochondrial DNA damage, as well as decreases in the total tissue glutathione (GSH) and GSH/GSSH ratio compared with nonventilated lungs. All of these injury indices were attenuated in OGG1-treated mice. At the highest level of VILI (2 h at 50 cmH(2)O PIP), OGG1 failed to protect against massive lung edema and BAL cytokines or against depletion of the tissue GSH pool. Interestingly, whereas untreated mice died before completing the 2-h protocol, OGG1-treated mice lived for the duration of observation. Thus mitochondrially targeted OGG1 prevented VILI over a range of ventilation times and pressures and enhanced survival in the most severely injured group. These findings support the concept that oxidative mtDNA damage caused by high PIP triggers induction of acute lung inflammation and injury.

Disrupted anabolic and catabolic processes may contribute to alcohol-accentuated SAIDS-associated wasting.

BACKGROUND: Alcohol abuse is a comorbid factor in many human immunodeficiency virus (HIV)-infected patients. Previously, we demonstrated that chronic binge alcohol accentuates loss of body mass at terminal stage of simian immunodeficiency virus (SIV) infection. The purpose of this study was to investigate changes in pathways that may contribute to muscle wasting in chronic binge alcohol-fed SIV-infected macaques. METHODS: The impact of chronic binge alcohol during SIV infection on insulin signaling and the ubiquitin (Ub)-proteasome system-regulators of protein synthesis and degradation-was examined in SIV-infected macaques. RESULTS: SIV infection induced an inflammatory and pro-oxidative milieu in skeletal muscle, which was associated with decreased insulin-stimulated phosphatidylinositol 3-kinase (PI-3k) activity and upregulated gene expression of mTOR and atrogin-1, and protein expression of Ub-proteasome system 19S base. Chronic binge alcohol accentuated the skeletal muscle pro-oxidative milieu and 19S base expression. Additionally, chronic binge alcohol increased skeletal muscle protein expression of protein-tyrosine phosphatase 1B (a negative regulator of insulin signaling) and 19S proteasome regulator non-ATPase (Rpn) 6 subunit and Rpn12, and suppressed PI-3K activity. Animals that were alcohol-fed and SIV-infected for >15 months had increased Ub-proteasome system activity. CONCLUSIONS: These data suggest negative modulation of insulin signaling coupled with enhanced Ub-proteasome system activity may be central mechanisms underlying chronic binge alcohol-induced accentuation of SIV-associated muscle wasting.

Interferon-gamma induced adipose tissue inflammation is linked to endothelial dysfunction in type 2 diabetic mice.

Interferon-gamma (IFNγ) has previously been associated with immuno-mediated inflammation in diet-induced obesity and type 1 diabetes. This study sought to define the role of IFNγ-induced adipose tissue inflammation in endothelial dysfunction in type 2 diabetes. We examined mesenteric adipose tissue (MAT) inflammation, and endothelial function of small mesenteric artery (SMA) in control mice (m Lepr(db)), diabetic mice (Lepr(db)), m Lepr(db) treated with IFNγ, and Lepr(db) treated with anti-IFNγ or anti-monocyte chemoattractant protein-1 (anti-MCP-1). mRNA and protein expression of IFNγ and MCP-1 were increased in MAT of Lepr(db), accompanied by increased T-lymphocyte and macrophage infiltration. Anti-IFNγ reduced MAT inflammatory cell infiltration and inflammatory cytokine expression in Lepr(db), while IFNγ treatment showed the opposite effects in m Lepr(db). Acetylcholine (ACh)-induced vasorelaxation of SMA was impaired in Lepr(db) versus m Lepr(db), but sodium nitroprusside (SNP)-induced vasorelaxation was comparable. Both anti-IFNγ and anti-MCP-1 improved endothelial function of Lepr(db), while IFNγ treatment impaired endothelial function of m Lepr(db). Superoxide production was higher in both MAT and SMA of Lepr(db) mice, and anti-IFNγ reduced MAT and SMA superoxide production. Macrophage accumulation in the adventitia of SMA, and mRNA expression of MCP-1 in SMA were increased in Lepr(db) and IFNγ-treated m Lepr(db), but reduced in anti-IFNγ treated Lepr(db). These findings suggest IFNγ has a key role in the regulation of visceral adipose tissue inflammatory response and endothelial dysfunction in type 2 diabetes.

Bariatric surgery reduces visceral adipose inflammation and improves endothelial function in type 2 diabetic mice.

OBJECTIVE: Bariatric surgery is emerging as an effective method to alleviate a multitude of medical conditions associated with morbid obesity and type 2 diabetes. However, little is known about the effects and mechanisms of bariatric surgery on visceral fat inflammation and endothelial dysfunction in type 2 diabetes. We hypothesize that bariatric surgery ameliorates interferon-γ-mediated adipose tissue inflammation/oxidative stress and improves endothelial function in type 2 diabetic mice. METHODS AND RESULTS: Control mice (m Lepr(db)) and diabetic mice (Lepr(db)) were treated with either sham surgery or improved gastric bypass surgery and then were evaluated at 5, 10, 20, and 30 days to assess postsurgical effects. Surgery reduced body weight, abdominal adiposity, blood glucose level, and food intake in Lepr(db). The surgery-induced decrease in visceral adiposity was accompanied by amelioration of T-lymphocytes and macrophage infiltration, as well as reduction in the expression of interferon-γ and other inflammatory cytokines in the mesenteric adipose tissue (MAT) of Lepr(db) mice. Furthermore, surgery improved endothelium-dependent, but not endothelium-independent, vasorelaxation in small mesenteric arteries (SMA) of Lepr(db) mice. The improvement in endothelial function was largely attenuated by nitric oxide synthase inhibitor (L-NAME) incubation. Interferon-γ treatment increased the mRNA expression of tumor necrosis factor-α in the MAT of control mice and incubation of SMA of control mice with tumor necrosis factor-α caused impairment of endothelial function. Superoxide production in MAT/SMA and nitrotyrosine protein level in SMA were elevated in diabetic mice. Surgery reduced MAT/SMA oxidative stress in Lepr(db) mice. CONCLUSIONS: The amelioration of adipose tissue inflammation and the improvement of endothelial function may represent important mechanisms that result in cardiovascular benefits after bariatric surgery.

Resveratrol improves left ventricular diastolic relaxation in type 2 diabetes by inhibiting oxidative/nitrative stress: in vivo demonstration with magnetic resonance imaging.

Resveratrol is a natural phytophenol that exhibits cardioprotective effects. This study was designed to elucidate the mechanisms by which resveratrol protects against diabetes-induced cardiac dysfunction. Normal control (m-Lepr(db)) mice and type 2 diabetic (Lepr(db)) mice were treated with resveratrol orally for 4 wk. In vivo MRI showed that resveratrol improved cardiac function by increasing the left ventricular diastolic peak filling rate in Lepr(db) mice. This protective role is partially explained by resveratrol's effects in improving nitric oxide (NO) production and inhibiting oxidative/nitrative stress in cardiac tissue. Resveratrol increased NO production by enhancing endothelial NO synthase (eNOS) expression and reduced O(2)(·-) production by inhibiting NAD(P)H oxidase activity and gp91(phox) mRNA and protein expression. The increased nitrotyrosine (N-Tyr) protein expression in Lepr(db) mice was prevented by the inducible NO synthase (iNOS) inhibitor 1400W. Resveratrol reduced both N-Tyr and iNOS expression in Lepr(db) mice. Furthermore, TNF-α mRNA and protein expression, as well as NF-κB activation, were reduced in resveratrol-treated Lepr(db) mice. Both Lepr(db) mice null for TNF-α (db(TNF-)/db(TNF-) mice) and Lepr(db) mice treated with the NF-κB inhibitor MG-132 showed decreased NAD(P)H oxidase activity and iNOS expression as well as elevated eNOS expression, whereas m-Lepr(db) mice treated with TNF-α showed the opposite effects. Thus, resveratrol protects against cardiac dysfunction by inhibiting oxidative/nitrative stress and improving NO availability. This improvement is due to the role of resveratrol in inhibiting TNF-α-induced NF-κB activation, therefore subsequently inhibiting the expression and activation of NAD(P)H oxidase and iNOS as well as increasing eNOS expression in type 2 diabetes.

Direct relationship between levels of TNF-alpha expression and endothelial dysfunction in reperfusion injury.

We previously found that myocardial ischemia/reperfusion (I/R) initiates expression of tumor necrosis factor-alpha (TNF) leading to coronary endothelial dysfunction. However, it is not clear whether there is a direct relationship between levels of TNF expression and endothelial dysfunction in reperfusion injury. We studied levels of TNF expression by using different transgenic animals expressing varying amounts of TNF in I/R. We crossed TNF overexpression (TNF(++/++)) with TNF knockout (TNF(-/-)) mice; thus we have a heterozygote population of mice with the expression of TNF "in between" the TNF(-/-) and TNF(++/++) mice. Mouse hearts were subjected to 30 min of global ischemia followed by 90 min of reperfusion and their vasoactivity before and after I/R was examined in wild type (WT), TNF(-/-), TNF(++/++) and TNF heterozygote (TNF(-/++), cross between TNF(-/-) and TNF(++/++)) mice. In heterozygote TNF(-/++) mice with intermediate cardiac-specific expression of TNF, acetylcholine-induced or flow-induced endothelial-dependent vasodilation following I/R was between TNF(++/++) and TNF(-/-) following I/R. Neutralizing antibodies to TNF administered immediately before the onset of reperfusion-preserved endothelial-dependent dilation following I/R in WT, TNF(-/++) and TNF(++/++) mice. In WT, TNF(-/++) and TNF(++/++) mice, I/R-induced endothelial dysfunction was progressively lessened by administration of free-radical scavenger TEMPOL immediately before initiating reperfusion. During I/R, production of superoxide (O(2) (.-)) was greatest in TNF(++/++) mice as compared to WT, TNF(-/++) and TNF(-/-) mice. Following I/R, arginase mRNA expression was elevated in the WT, substantially elevated in the TNF(-/++) and TNF(++/++) mice and not affected in the TNF(-/-) mice. These results suggest that the level of TNF expression determines arginase expression in endothelial cells during myocardial I/R, which is one of the mechanisms by which TNF compromises coronary endothelial function in reperfusion injury.

Role of TNF-alpha-induced reactive oxygen species in endothelial dysfunction during reperfusion injury.

We hypothesized that neutralization of TNF-alpha at the time of reperfusion exerts a salubrious role on endothelial function and reduces the production of reactive oxygen species. We employed a mouse model of myocardial ischemia-reperfusion (I/R, 30 min/90 min) and administered TNF-alpha neutralizing antibodies at the time of reperfusion. I/R elevated TNF-alpha expression (mRNA and protein), whereas administration of anti-TNF-alpha before reperfusion attenuated TNF-alpha expression. We detected TNF-alpha expression in vascular smooth muscle cells, mast cells, and macrophages, but not in the endothelial cells. I/R induced endothelial dysfunction and superoxide production. Administration of anti-TNF-alpha at the onset of reperfusion partially restored nitric oxide-mediated coronary arteriolar dilation and reduced superoxide production. I/R increased the activity of NAD(P)H oxidase and of xanthine oxidase and enhanced the formation of nitrotyrosine residues in untreated mice compared with shams. Administration of anti-TNF-alpha before reperfusion blocked the increase in activity of these enzymes. Inhibition of xanthine oxidase (allopurinol) or NAD(P)H oxidase (apocynin) improved endothelium-dependent dilation and reduced superoxide production in isolated coronary arterioles following I/R. Interestingly, I/R enhanced superoxide generation and reduced endothelial function in neutropenic animals and in mice treated with a neutrophil NAD(P)H oxidase inhibitor, indicating that the effects of TNF-alpha are not through neutrophil activation. We conclude that myocardial ischemia initiates TNF-alpha expression, which induces vascular oxidative stress, independent of neutrophil activation, and leads to coronary endothelial dysfunction.

Maturation-induces endothelial dysfunction via vascular inflammation in diabetic mice.

We hypothesized that maturation-induced vascular inflammation produces endothelial dysfunction in type II diabetes and TNFalpha plays a key role in triggering inflammation in the development of diabetes. In control (Db/db) mice aged 6, 12, 18 and 24 weeks, sodium nitroprusside (SNP) and acetylcholine (ACh) induced dose-dependent vasodilation, and dilation to ACh was blocked by the NO synthase inhibitor N (G)-monomethyl-L: -arginine. In type II diabetic (db/db) mice at age of 12, 18 and 24 weeks, ACh or flow-induced dilation was blunted compared to Db/db; endothelial function is normal at 6 weeks of age in db/db Vs. control mice, but SNP produced comparable dilation at age of 6, 12, 18 and 24 weeks. Decrements in endothelial function in db/db mice progressively increased from 6-12 to 18-24 weeks. Administration of neutralizing antibodies to TNFalpha ameliorated endothelial dysfunction in db/db mice aged 12, 18 and 24 weeks. The effect was most prominent in the younger animals. Plasma concentration, expression of TNFalpha and TNFalpha receptor 1 (TNFR1) were elevated in coronary arterioles, even at the age of 6 weeks before the development of diabetes in db/db mice compared to control mice. Superoxide production was lower in Db/db mice compared to db/db mice and increments in superoxide production in db/db mice progressively increased from 6-12 to 18-24 weeks. NAD(P)H oxidase inhibitor apocynin attenuated superoxide production in db/db mice at 12 weeks of age, mitochondria respiratory chain inhibitor rotenone attenuated superoxide production at 24 weeks in db/db and Db/db mice, but the combination of apocynin and rotenone reduced superoxide production at 18 weeks for db/db and Db/db mice. The expression of TNFalpha and its receptors increase progressively with maturation in concert with the development of diabetes. Incremental increases in TNFalpha/TNFR1 expression induces activation and production of superoxide via NAD(P)H oxidase and/or mitochondria respiratory chain, leading to endothelial dysfunction progressing to the development of type II diabetes.

Anti-LOX-1 rescues endothelial function in coronary arterioles in atherosclerotic ApoE knockout mice.

BACKGROUND: We hypothesized that atherosclerosis inhibits NO-mediated endothelium-dependent dilation of coronary arterioles through interaction of ox-LDL with its receptor, LOX-1, through the production of O2ÿ- in endothelial cells. METHODS AND RESULTS: We assessed the role of ox-LDL in endothelial dysfunction in a murine model of atherosclerosis (ApoE KO mice). Coronary arterioles from WT control and ApoE KO mice were isolated and pressurized without flow. Although dilation of vessels to endothelium-independent vasodilator SNP was not altered between ApoE KO and WT mice, dilation to the endothelium-dependent agonist, ACh was reduced in ApoE KO versus WT mice. Impaired vasodilation to ACh in ApoE KO mice is partially restored by NAD(P)H oxidase inhibitor, apocynin or DPI. Messenger RNA expression for NAD(P)H oxidases was higher in ApoE KO mice than that in WT and anti-LOX-1 treated ApoE KO mice. Anti-LOX-1, given in vivo, restored NO-mediated coronary arteriolar dilation in ApoE KO mice, but did not affect the endothelium-dependent vasodilation in controls. CONCLUSIONS: These results suggest that ox-LDL impairs endothelium-dependent NO-mediated dilation of coronary arterioles by activation of a signaling cascade involving LOX-1 and NAD(P)H oxidase expression.

Tumor necrosis factor-alpha induces endothelial dysfunction in Lepr(db) mice.

BACKGROUND: We hypothesized that the inflammatory cytokine tumor necrosis factor-alpha (TNF) produces endothelial dysfunction in type 2 diabetes. METHODS AND RESULTS: In m Lepr(db) control mice, sodium nitroprusside and acetylcholine induced dose-dependent vasodilation, and dilation to acetylcholine was blocked by the NO synthase inhibitor N(G)-monomethyl-L-arginine. In type 2 diabetic (Lepr(db)) mice, acetylcholine- or flow-induced dilation was blunted compared with m Lepr(db), but sodium nitroprusside produced comparable dilation. In Lepr(db) mice null for TNF (db(TNF-)/db(TNF-)), dilation to acetylcholine or flow was greater than in diabetic Lepr(db) mice and comparable to that in controls. Plasma concentration of TNF was significantly increased in Lepr(db) versus m Lepr(db) mice. Real-time polymerase chain reaction and Western blotting showed that mRNA and protein expression of TNF and nuclear factor-kappaB were higher in Lepr(db) mice than in controls. Administration of anti-TNF or soluble receptor of advanced glycation end products attenuated nuclear factor-kappaB and TNF expression in the Lepr(db) 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 Lepr(db) mice versus controls. Administration of the superoxide scavenger TEMPOL, NAD(P)H oxidase inhibitor (apocynin), or anti-TNF restored endothelium-dependent dilation in Lepr(db) mice. NAD(P)H oxidase activity, protein expression of nitrotyrosine, and hydrogen peroxide production were increased in Lepr(db) mice (compared with controls), but these variables were restored to control levels by anti-TNF. CONCLUSIONS: Advanced glycation end products/receptor of advanced glycation end products and nuclear factor-kappaB signaling play pivotal roles in TNF expression through an increase in circulating and/or local vascular TNF production in the Lepr(db) 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.

Tumor necrosis factor-alpha induces endothelial dysfunction in the prediabetic metabolic syndrome.

Inflammation is a condition that underscores many cardiovascular pathologies including endothelial dysfunction, but no link is yet established between the vascular pathology of the metabolic syndrome with a particular inflammatory cytokine. We hypothesized that impairments in coronary endothelial function in the obese condition the prediabetic metabolic syndrome is caused by TNF-alpha overexpression. To test this, we measured endothelium-dependent (acetylcholine) and -independent vasodilation (sodium nitroprusside) of isolated, pressurized coronary small arteries from lean control and Zucker obese fatty (ZOF, a model of prediabetic metabolic syndrome) rats. In ZOF rats, dilation to ACh was blunted compared with lean rats, but sodium nitroprusside-induced dilation was comparable. Superoxide (O2*-) generation was elevated in vessels from ZOF rats compared with lean rats, and administration of the O2*- scavenger TEMPOL, NAD(P)H oxidase inhibitor (apocynin), or anti-TNF-alpha restored endothelium-dependent dilation in the ZOF rats. Real-time PCR and Western blotting revealed that mRNA and protein of TNF-alpha were higher in ZOF rats than that in lean rats, whereas eNOS protein levels were reduced in the ZOF versus lean rats. Immunostaining showed that TNF-alpha in ZOF rat heart is localized in endothelial cells and vascular smooth muscle cells. Expression of NAD(P)H subunits p22 and p40-phox were elevated in ZOF compared with lean animals. Administration of TNF-alpha more than 3 days also induced expression of these NAD(P)H subunits and abrogated endothelium-dependent dilation. In conclusion, the results demonstrate the endothelial dysfunction occurring in the metabolic syndrome is the result of effects of the inflammatory cytokine TNF-alpha and subsequent production of O2*-.

TNF-alpha contributes to endothelial dysfunction in ischemia/reperfusion injury.

BACKGROUND: Despite the importance of endothelial function for coronary regulation, there is little information and virtually no consensus about the causal mechanisms of endothelial dysfunction in myocardial ischemia/reperfusion (I/R) injury. Because tumor necrosis factor-alpha (TNF-alpha) is reportedly expressed during ischemia and can induce vascular inflammation leading to endothelial dysfunction, we hypothesized that this inflammatory cytokine may play a pivotal role in I/R injury-induced coronary endothelial dysfunction. METHODS AND RESULTS: To test this hypothesis, we used a murine model of I/R (30 minutes/90 minutes) in conjunction with neutralizing antibodies to block the actions of TNF-alpha. TNF-alpha expression was increased >4-fold after I/R. To determine whether TNF-alpha abrogates endothelial function after I/R, we assessed endothelial-dependent (ACh) and endothelial-independent (SNP) vasodilation. In sham controls, ACh induced dose-dependent vasodilation that was blocked by the nitric oxide synthase (NOS) inhibitor L-NMMA (10 micromol/L), suggesting a key role for NO. In the I/R group, dilation to ACh was blunted, but SNP-induced dilation was preserved. Subsequent incubation of vessels with the superoxide (O2*-) scavenger (TEMPOL), or with the inhibitors of xanthine oxidase (allopurinol, oxypurinol), or previous administration of anti-TNF-alpha restored endothelium-dependent dilation in the I/R group and reduced I/R-stimulated O2*- production in arteriolar endothelial cells. Activation of xanthine oxidase with I/R was prevented by allopurinol or anti-TNF-alpha. CONCLUSIONS: These results suggest that myocardial I/R initiates expression of TNF-alpha, which induces activation of xanthine oxidase and production of O2*-, leading to coronary endothelial dysfunction.

Molecular regulation of iron homeostasis and resistance to infection in alcoholics.

Chronic alcohol abuse is associated with both an altered response to infection and deranged iron homeostasis. While both clinical manifestations are well known, the inter-relationships between alcohol and iron and the response to infection are not. The recent identification of a plethora of iron regulatory and transport proteins has now begun to explain these relationships. This article outlines the current state of knowledge on cellular iron homeostasis, with particular reference to the iron regulatory proteins (IRP1, IRP2 and HFE) and the iron membrane transport proteins, two of which have been shown to be members of the natural resistance- associated macrophage protein family (Nramp1 and 2). Following this introduction, the response of the body to infection, in terms of iron withholding is discussed at the cellular level, especially in terms of the macrophage and its cytokine-mediated responses. Prior alterations to body iron status are also considered in this section. The effect of alcohol alone on the body's response to infection is then outlined, principally in terms of the macrophage function and cytokine regulation. These are then combined to correlate the clinical and experimental observations with known derangements produced by the individual insults of alcohol and altered iron homeostasis. on the response to infection. Particular attention is paid not only to cytokine/chemokine actions, but also to the consequences of the altered production of reactive oxygen and nitrogen species. Finally, the possible mechanisms by which alcohol and altered iron homeostasis lead to tissue damage during infection.