Xue Gao

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.

TNF-α Contributes to Endothelial Dysfunction by Upregulating Arginase in Ischemia/Reperfusion Injury

Background—We tested whether tumor necrosis factor (TNF)-&agr; increases arginase expression in endothelial cells as one of the primary mechanisms by which this inflammatory cytokine compromises endothelial function during ischemia-reperfusion (I/R) injury. Methods and Results—Mouse hearts were subjected to 30 minutes of global ischemia followed by 90 minutes of reperfusion and their vasoactivity before and after I/R was examined in wild-type (WT), tumor necrosis factor knockout (TNF−/−), and TNF 1.6 (TNF++/++) mice. In WT mice, dilation to the endothelium-dependent vasodilator ACh was blunted in I/R compared with sham control. L-arginine or arginase inhibitor NOHA restored NO-mediated coronary arteriolar dilation in WT I/R mice. O2− production was reduced by eNOS inhibitor, L-NAME, or NOHA in WT I/R mice. In TNF−/− mice, I/R did not alter Ach-induced vasodilation and O2− production compared with sham mice. The increase in arginase expression that occurs during I/R in WT mice was absent in TNF−/− mice. Arginase expression was confined largely to the endothelium and independent of inflammatory cell invasion. Arginase activity was markedly lower in TNF−/−, but higher in WT I/R than that in WT sham mice. Conclusions—Our data demonstrate TNF-&agr; upregulates expression of arginase in endothelial cells, which leads to O2− production then induces endothelial dysfunction in I/R injury.

AGE/RAGE produces endothelial dysfunction in coronary arterioles in Type 2 diabetic mice

We hypothesized that impaired nitric oxide (NO)-dependent dilation (endothelial dysfunction) in type 2 diabetes results, in part, from elevated production of superoxide (O(2)(*-)) induced by the interaction of advanced glycation end products (AGE)/receptor for AGE (RAGE) and TNF-alpha signaling. We assessed the role of AGE/RAGE and TNF-alpha signaling in endothelial dysfunction in type 2 diabetic (Lepr(db)) mice by evaluation of endothelial function in isolated coronary resistance vessels of normal control (nondiabetic, m Lepr(db)) and diabetic mice. Although dilation of vessels to the endothelium-independent vasodilator sodium nitroprusside (SNP) was not different between diabetic and control mice, dilation to the endothelium-dependent agonist acetylcholine (ACh) was reduced in diabetic vs. control mice. The activation of RAGE with RAGE agonist S100b eliminated SNP-potentiated dilation to ACh in Lepr(db) mice. Administration of a soluble form of RAGE (sRAGE) partially restored dilation in diabetic mice but did not affect dilation in control mice. The expression of RAGE in coronary arterioles was markedly increased in diabetic vs. control mice. We also observed in diabetic mice that augmented RAGE signaling augmented expression of TNF-alpha, because this increase was attenuated by sRAGE or NF-kappaB inhibitor MG132. Protein and mRNA expression of NAD(P)H oxidase subunits including NOX-2, p22(phox), and p40(phox) increased in diabetic compared with control mice. sRAGE significantly inhibited the expression of NAD(P)H oxidase in diabetic mice. These results indicate that AGE/RAGE signaling plays a pivotal role in regulating the production/expression of TNF-alpha, oxidative stress, and endothelial dysfunction in type 2 diabetes.

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.

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.

Role of EDHF in type 2 diabetes-induced endothelial dysfunction

Endothelium-derived hyperpolarizing factor (EDHF) plays a crucial role in modulating vasomotor tone, especially in microvessels when nitric oxide-dependent control is compromised such as in diabetes. Epoxyeicosatrienoic acids (EETs), potassium ions (K+), and hydrogen peroxide (H2O2) are proposed as EDHFs. However, the identity (or identities) of EDHF-dependent endothelial dilators has not been clearly elucidated in diabetes. We assessed the mechanisms of EDHF-induced vasodilation in wild-type (WT, normal), db/db (advanced type 2 diabetic) mice, and db/db mice null for TNF (dbTNF-/dbTNF-). In db/db mice, EDHF-induced vasodilation [ACh-induced vasodilation in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME, 10 micromol/l) and prostaglandin synthase inhibitor indomethacin (Indo, 10 mumol/l)] was diminished after the administration of catalase (an enzyme that selectively dismutates H2O2 to water and oxygen, 1,000 U/ml); administration of the combination of charybdotoxin (a nonselective blocker of intermediate-conductance Ca2+-activated K+ channels, 10 micromol/l) and apamin (a selective blocker of small-conductance Ca2+-activated K+ channels, 50 micromol/l) also attenuated EDHF-induced vasodilation, but the inhibition of EETs synthesis [14,15-epoxyeicosa-5(Z)-enoic acid; 10 mumol/l] did not alter EDHF-induced vasodilation. In WT controls, EDHF-dependent vasodilation was significantly diminished after an inhibition of K+ channel, EETs synthesis, or H2O2 production. Our molecular results indicate that mRNA and protein expression of interleukin-6 (IL-6) were greater in db/db versus WT and dbTNF-/dbTNF- mice, but neutralizing antibody to IL-6 (anti-IL-6; 0.28 mg.ml(-1).kg(-1) ip for 3 days) attenuated IL-6 expression in db/db mice. The incubation of the microvessels with IL-6 (5 ng/ml) induced endothelial dysfunction in the presence of l-NAME and Indo in WT mice, but anti-IL-6 restored ACh-induced vasodilation in the presence of L-NAME and Indo in db/db mice. In db(TNF-)/db(TNF-) mice, EDHF-induced vasodilation was greater and comparable with controls, but IL-6 decreased EDHF-mediated vasodilation. Our results indicate that EDHF compensates for diminished NO-dependent dilation in IL-6-induced endothelial dysfunction by the activation of H2O2 or a K+ channel in type 2 diabetes.

Role of TNF-α-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.

Role of MCP-1 in tumor necrosis factor-α-induced endothelial dysfunction in type 2 diabetic mice

Tumor necrosis factor-alpha (TNF-alpha) upregulates the expression of monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules in type 2 diabetes. We hypothesized that TNF-alpha and MCP-1 may interact to contribute to the evolution of vascular inflammation and endothelial dysfunction in coronary arterioles in type 2 diabetes. To test this hypothesis, we administered anti-MCP-1 to block MCP-1 signaling in genetically modified mice with type 2 diabetes (Lepr(db)) and in heterozygote (m Lepr(db)) lean control. Anti-MCP-1 partially restored vasodilation to the endothelium-dependent vasodilator acetylcholine in isolated, cannulated, and pressurized coronary arterioles in Lepr(db) mice but did not affect vasodilation in m Lepr(db) mice. Anti-MCP-1 attenuated superoxide production and the protein expression of nitrotyrosine, which is an indicator of peroxynitrite production, in isolated coronary arterioles of Lepr(db) mice. Immunostaining results showed that the expression of MCP-1 and vascular cellular adhesion molecule-1 is colocalized with endothelial cells and macrophages. Anti-TNF-alpha or anti-MCP-1 markedly reduced macrophage infiltration and the number of MCP-1-positive endothelium in Lepr(db) mice. The neutralization of TNF-alpha or anti-MCP-1 reduced the expression of adhesion molecules, suggesting that proinflammatory cytokines interact to amplify the signaling process that leads to vascular dysfunction. These findings demonstrate that the endothelial dysfunction occurring in type 2 diabetes is the result of the effects of the inflammatory cytokine TNF-alpha and TNF-alpha-related signaling, including the expression of MCP-1 and adhesion molecules, which further exacerbates vessel inflammation and oxidative stress.

Tumor Necrosis Factor- 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.