Barry J. Potter

Tumor Necrosis Factor-α 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-&agr; 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 (&OV0151;) generation was elevated in vessels from ZOF rats compared with lean rats, and administration of the &OV0151; scavenger TEMPOL, NAD(P)H oxidase inhibitor (apocynin), or anti–TNF-&agr; restored endothelium-dependent dilation in the ZOF rats. Real-time PCR and Western blotting revealed that mRNA and protein of TNF-&agr; 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-&agr; 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-&agr; 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-&agr; and subsequent production of &OV0151;.

Oleate uptake by cardiac myocytes is carrier mediated and involves a 40-kD plasma membrane fatty acid binding protein similar to that in liver, adipose tissue, and gut.

Uptake of [3H]oleate by canine or rat cardiac myocytes is saturable, displays the countertransport phenomenon, and is inhibited by phloretin and trypsin. Cardiac myocytes contain a basic (pI approximately 9.1) 40-kD plasma membrane fatty acid binding protein (FABPPM) analogous to those recently isolated from liver, adipose tissue, and gut, unrelated to the 12-14-kD cytosolic FABP in these same tissues. An antibody to rat liver FABPPM selectively inhibits specific uptake of [3H]oleate by rat heart myocytes at 37 degrees C, but has no influence on nonspecific [3H]oleate uptake at 4 degrees C or on specific uptake of [3H]glucose. Uptake of long-chain free fatty acids by cardiac muscle cells, liver, and adipose tissue and absorption by gut epithelial cells is a facilitated process mediated by identical or closely related plasma membrane FABPs.

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

Background— We hypothesized that the inflammatory cytokine tumor necrosis factor-α (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-&kgr;B were higher in Leprdb mice than in controls. Administration of anti-TNF or soluble receptor of advanced glycation end products attenuated nuclear factor-&kgr;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-&kgr;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.

Hydrogen Peroxide A Feed-Forward Dilator That Couples Myocardial Metabolism to Coronary Blood Flow

Objective—We tested the hypothesis that hydrogen peroxide (H2O2), the dismutated product of superoxide (O2·−), couples myocardial oxygen consumption to coronary blood flow. Accordingly, we measured O2·− and H2O2 production by isolated cardiac myocytes, determined the role of mitochondrial electron transport in the production of these species, and determined the vasoactive properties of the produced H2O2. Methods and Results—The production of O2·− is coupled to oxidative metabolism because inhibition of complex I (rotenone) or III (antimycin) enhanced the production of O2·− during pacing by about 50% and 400%, respectively; whereas uncoupling oxidative phosphorylation by decreasing the protonmotive force with carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) decreased pacing-induced O2·− production. The inhibitor of cytosolic NAD(P)H oxidase assembly, apocynin, did not affect O2·− production by pacing. Aliquots of buffer from paced myocytes produced vasodilation of isolated arterioles (peak response 67±8% percent of maximal dilation) that was significantly reduced by catalase (5±0.5%, P<0.05) or the antagonist of Kv channels, 4-aminopyridine (18±4%, P<0.05). In intact animals, tissue concentrations of H2O2 are proportionate to myocardial oxygen consumption and directly correlated to coronary blood flow. Intracoronary infusion of catalase reduced tissue levels of H2O2 by 30%, and reduced coronary flow by 26%. Intracoronary administration of 4-aminopyridine also shifted the relationship between myocardial oxygen consumption and coronary blood flow or coronary sinus po2. Conclusions—Taken together, our results demonstrate that O2·− is produced in proportion to cardiac metabolism, which leads to the production of the vasoactive reactive oxygen species, H2O2. Our results further suggest that the production of H2O2 in proportion to metabolism couples coronary blood flow to myocardial oxygen consumption.

TNF-α 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-&agr; (TNF-&agr;) 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-&agr;. TNF-&agr; expression was increased >4-fold after I/R. To determine whether TNF-&agr; 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 &mgr;mol/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 (&OV0151;) scavenger (TEMPOL), or with the inhibitors of xanthine oxidase (allopurinol, oxypurinol), or previous administration of anti-TNF-&agr; restored endothelium-dependent dilation in the I/R group and reduced I/R-stimulated &OV0151; production in arteriolar endothelial cells. Activation of xanthine oxidase with I/R was prevented by allopurinol or anti–TNF-&agr;. Conclusions—These results suggest that myocardial I/R initiates expression of TNF-&agr;, which induces activation of xanthine oxidase and production of &OV0151;, leading to coronary endothelial dysfunction.

Tumor Necrosis Factor-α Induces Endothelial Dysfunction in Leprdb Mice

Background— We hypothesized that the inflammatory cytokine tumor necrosis factor-α (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-&kgr;B were higher in Leprdb mice than in controls. Administration of anti-TNF or soluble receptor of advanced glycation end products attenuated nuclear factor-&kgr;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-&kgr;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.

IS PRIMARY BILIARY CIRRHOSIS AN IMMUNE COMPLEX DISEASE

Large immune complexes are present in the circulation of patients with primary biliary cirrhosis and result in the activation of complement by the classical pathway. Such large complexes are capable of producing tissue damage. The granulomatous lesions surrounding the small bile-ducts within the liver of patients with primary biliary cirrhosis and the vasculitis, rheumatoid arthritis, and associated lesions are all compatible with immune complex injury. It is postulated that such large complexes could be formed in the vicinity of the bile-ducts by an antigen absorbed from the bile or biliary epithelium. Complexes reaching the systemic circulation might be responsible for the associated extra-hepatic diseases.

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 resveratrols 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.

Reduction of Immune Complexes and Immunoglobulins Induced by D-Penicillamine in Primary Biliary Cirrhosis

Penicillamine has an effect on immune complexes and immunoglobulins both in vivo and in vitro. We therefore studied the effect of penicillamine on immune complexes and immunoglobulins in primary biliary cirrhosis. Twenty-eight patients were randomly allocated into a treatment group receiving 600 to 900 mg of penicillamine, or a control group, and followed for a maximum of 24 months. After 12 and 24 months, serum immune complexes had fallen significantly in treated patients as compared to controls (P less than 0.05, P less than 0.01). Treatment reduced IgA, IgG and IgM concentrations, with IgM being significantly different from controls at six, 12 and 24 months (P less than 0.01). Over 24 months, serum aspartate transaminase levels fell in treated patients but rose in controls (P less than 0.01). Bilirubin concentrations increased at a slower rate in treated patients. Penicillamine may favorably influence the course of primary biliary cirrhosis by its immunologic action in addition to its copper-chelating action.

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.