Cuihua Zhang

Role of TNF-α in vascular dysfunction

Healthy vascular function is primarily regulated by several factors including EDRF (endothelium-dependent relaxing factor), EDCF (endothelium-dependent contracting factor) and EDHF (endothelium-dependent hyperpolarizing factor). Vascular dysfunction or injury induced by aging, smoking, inflammation, trauma, hyperlipidaemia and hyperglycaemia are among a myriad of risk factors that may contribute to the pathogenesis of many cardiovascular diseases, such as hypertension, diabetes and atherosclerosis. However, the exact mechanisms underlying the impaired vascular activity remain unresolved and there is no current scientific consensus. Accumulating evidence suggests that the inflammatory cytokine TNF (tumour necrosis factor)-α plays a pivotal role in the disruption of macrovascular and microvascular circulation both in vivo and in vitro. AGEs (advanced glycation end-products)/RAGE (receptor for AGEs), LOX-1 [lectin-like oxidized low-density lipoprotein receptor-1) and NF-κB (nuclear factor κB) signalling play key roles in TNF-α expression through an increase in circulating and/or local vascular TNF-α production. The increase in TNF-α expression induces the production of ROS (reactive oxygen species), resulting in endothelial dysfunction in many pathophysiological conditions. Lipid metabolism, dietary supplements and physical activity affect TNF-α expression. The interaction between TNF-α and stem cells is also important in terms of vascular repair or regeneration. Careful scrutiny of these factors may help elucidate the mechanisms that induce vascular dysfunction. The focus of the present review is to summarize recent evidence showing the role of TNF-α in vascular dysfunction in cardiovascular disease. We believe these findings may prompt new directions for targeting inflammation in future therapies.

Resveratrol attenuates mitochondrial oxidative stress in coronary arterial endothelial cells.

The production of hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) is a key event in the development of diabetic complications. Because resveratrol, a naturally occurring polyphenol, has been reported to confer vasoprotection, improving endothelial function and preventing complications of diabetes, we investigated the effect of resveratrol on mtROS production in cultured human coronary arterial endothelial cells (CAECs). The measurement of MitoSox fluorescence showed that resveratrol attenuates both steady-state and high glucose (30 mM)-induced mtROS production in CAECs, an effect that was prevented by the knockdown of the protein deacetylase silent information regulator 2/sirtuin 1 (SIRT1), an intracellular target of resveratrol. An overexpression of SIRT1 mimicked the effects of resveratrol, attenuating mtROS production. Similar results were obtained in CAECs transfected with mitochondria-targeted H(2)O(2)-sensitive HyPer-Mito fluorescent sensor. Amplex red assay showed that resveratrol and SIRT1 overexpression significantly reduced cellular H(2)O(2) levels as well. Resveratrol upregulated MnSOD expression and increased cellular GSH content in a concentration-dependent manner (measured by HPLC coulometric analysis). These effects were attenuated by SIRT1 knockdown and mimicked by SIRT1 overexpression. We propose that resveratrol, via a pathway that involves the activation of SIRT1 and the upregulation of antioxidant defense mechanisms, attenuates mtROS production, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

The role of inflammatory cytokines in endothelial dysfunction

Clinical and experimental data support a link between endothelial dysfunction and inflammation. Inflammatory cytokines are important protagonists in formation of atherosclerotic plaque, eliciting effects throughout the atherosclerotic vessel. Importantly, the development of atherosclerotic lesions, regardless of the risk factor, e.g., diabetes, hypertension, obesity, is characterized by disruption in normal function of the endothelial cells. Endothelial cells, which line the internal lumen of the vasculature, are part of a complex system that regulates vasodilation and vasoconstriction, growth of vascular smooth muscle cells, inflammation, and hemostasis, maintaining a proper blood supply to tissues and regulating inflammation and coagulation. Current concepts suggest that the earliest event in atherogenesis is endothelial dysfunction, manifested by deficiencies in the production of nitric oxide (NO) and prostacyclin. The focus of this review is to summarize recent evidence showing the effects of inflammation on vascular dysfunction in ischemic-heart disease, which may prompt new directions for targeting inflammation in future therapies.Clinical and experimental data support a link between endothelial dysfunction and inflammation. Inflammatory cytokines are important protagonists in formation of atherosclerotic plaque, eliciting effects throughout the atherosclerotic vessel. Importantly, the development of atherosclerotic lesions, regardless of the risk factor, e.g., diabetes, hypertension, obesity, is characterized by disruption in normal function of the endothelial cells. Endothelial cells, which line the internal lumen of the vasculature, are part of a complex system that regulates vasodilation and vasoconstriction, growth of vascular smooth muscle cells, inflammation, and hemostasis, maintaining a proper blood supply to tissues and regulating inflammation and coagulation. Current concepts suggest that the earliest event in atherogenesis is endothelial dysfunction, manifested by deficiencies in the production of nitric oxide (NO) and prostacyclin. The focus of this review is to summarize recent evidence showing the effects of inflammation on vascular dysfunction in ischemic-heart disease, which may prompt new directions for targeting inflammation in future therapies.

Vasoprotective effects of resveratrol and SIRT1: attenuation of cigarette smoke-induced oxidative stress and proinflammatory phenotypic alterations

The dietary polyphenolic compound resveratrol, by activating the protein deacetylase enzyme silent information regulator 2/sirtuin 1 (SIRT1), prolongs life span in evolutionarily distant organisms and may mimic the cytoprotective effects of dietary restriction. The present study was designed to elucidate the effects of resveratrol on cigarette smoke-induced vascular oxidative stress and inflammation, which is a clinically highly relevant model of accelerated vascular aging. Cigarette smoke exposure of rats impaired the acetylcholine-induced relaxation of carotid arteries, which could be prevented by resveratrol treatment. Smoking and in vitro treatment with cigarette smoke extract (CSE) increased reactive oxygen species production in rat arteries and cultured coronary arterial endothelial cells (CAECs), respectively, which was attenuated by resveratrol treatment. The smoking-induced upregulation of inflammatory markers (ICAM-1, inducible nitric oxide synthase, IL-6, and TNF-alpha) in rat arteries was also abrogated by resveratrol treatment. Resveratrol also inhibited CSE-induced NF-kappaB activation and inflammatory gene expression in CAECs. In CAECs, the aforementioned protective effects of resveratrol were abolished by knockdown of SIRT1, whereas the overexpression of SIRT1 mimicked the effects of resveratrol. Resveratrol treatment of rats protected aortic endothelial cells against cigarette smoking-induced apoptotic cell death. Resveratrol also exerted antiapoptotic effects in CSE-treated CAECs, which could be abrogated by knockdown of SIRT1. Resveratrol treatment also attenuated CSE-induced DNA damage in CAECs (comet assay). Thus resveratrol and SIRT1 exert antioxidant, anti-inflammatory, and antiapoptotic effects, which protect the endothelial cells against the adverse effects of cigarette smoking-induced oxidative stress. The vasoprotective effects of resveratrol will likely contribute to its antiaging action in mammals and may be especially beneficial in pathophysiological conditions associated with accelerated vascular aging.

Resveratrol Improves Endothelial Function. Role of TNFα and Vascular Oxidative Stress

Objective—Oxidative stress plays an important role in type 2 diabetes–related endothelial dysfunction. We hypothesized that resveratrol protects against oxidative stress–induced endothelial dysfunction in aortas of diabetic mice by inhibiting tumor necrosis factor α (TNFα)-induced activation of NAD(P)H oxidase and preserving phosphorylation of endothelial nitric oxide synthase (eNOS). Methods and Results—We examined endothelial-dependent vasorelaxation to acetylcholine (ACh) in diabetic mice (Leprdb) and normal controls (m Leprdb). Relaxation to ACh was blunted in Leprdb compared with m Leprdb, whereas endothelial-independent vasorelaxation to sodium nitroprusside (SNP) was comparable. Resveratrol improved ACh-induced vasorelaxation in Leprdb without affecting dilator response to SNP. Impaired relaxation to ACh in Leprdb was partially reversed by incubating the vessels with NAD(P)H oxidase inhibitor apocynin and a membrane-permeable superoxide dismutase mimetic TEMPOL. Dihydroethidium (DHE) staining showed an elevated superoxide (O2·−) production in Leprdb, whereas both resveratrol and apocynin significantly reduced O2·− signal. Resveratrol increased nitrite/nitrate levels and eNOS (Ser1177) phosphorylation, and attenuated H2O2 production and nitrotyrosine (N-Tyr) content in Leprdb aortas. Furthermore, resveratrol attenuated the mRNA and protein expression of TNFα. Genetic deletion of TNFα in diabetic mice (dbTNF−/dbTNF−) was associated with a reduced NAD(P)H oxidase activity and vascular O2·− production and an increased eNOS (Ser1177) phosphorylation, suggesting that TNFα plays a pivotal role in aortic dysfunction in diabetes by inducing oxidative stress and reducing NO bioavailability. Conclusions—Resveratrol restored endothelial function in type 2 diabetes by inhibiting TNFα-induced activation of NAD(P)H oxidase and preserving eNOS phosphorylation, suggesting the potential for new treatment approaches to promote vascular health in metabolic diseases.

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 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.

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.