Yoonjung Park

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.

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.

Feed-forward signaling of TNF-α and NF-κB via IKK-β pathway contributes to insulin resistance and coronary arteriolar dysfunction in type 2 diabetic mice

We hypothesized that the interaction between tumor necrosis factor-alpha (TNF-alpha)/nuclear factor-kappaB (NF-kappaB) via the activation of IKK-beta may amplify one another, resulting in the evolution of vascular disease and insulin resistance associated with diabetes. To test this hypothesis, endothelium-dependent (ACh) and -independent (sodium nitroprusside) vasodilation of isolated, pressurized coronary arterioles from mLepr(db) (heterozygote, normal), Lepr(db) (homozygote, diabetic), and Lepr(db) mice null for TNF-alpha (db(TNF-)/db(TNF-)) were examined. Although the dilation of vessels to sodium nitroprusside was not different between Lepr(db) and mLepr(db) mice, the dilation to ACh was reduced in Lepr(db) mice. The NF-kappaB antagonist MG-132 or the IKK-beta inhibitor sodium salicylate (NaSal) partially restored nitric oxide-mediated endothelium-dependent coronary arteriolar dilation in Lepr(db) mice, but the responses in mLepr(db) mice were unaffected. The protein expression of IKK-alpha and IKK-beta were higher in Lepr(db) than in mLepr(db) mice; the expression of IKK-beta, but not the expression of IKK-alpha, was attenuated by MG-132, the antioxidant apocynin, or the genetic deletion of TNF-alpha in diabetic mice. Lepr(db) mice showed an increased insulin resistance, but NaSal improved insulin sensitivity. The protein expression of TNF-alpha and NF-kappaB and the protein modification of phosphorylated (p)-IKK-beta and p-JNK were greater in Lepr(db) mice, but NaSal attenuated TNF-alpha, NF-kappaB, p-IKK-beta, and p-JNK in Lepr(db) mice. The ratio of p-insulin receptor substrate (IRS)-1 at Ser307 to IRS-1 was elevated in Lepr(db) compared with mLepr(db) mice; both NaSal and the JNK inhibitor SP-600125 reduced the p-IRS-1-to-IRS-1 ratio in Lepr(db) mice. MG-132 or the neutralization of TNF-alpha reduced superoxide production in Lepr(db) mice. In conclusion, our results indicate that the interaction between NF-kappaB and TNF-alpha signaling induces the activation of IKK-beta and amplifies oxidative stress, leading to endothelial dysfunction in type 2 diabetes.

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.

Effects of interventions on oxidative stress and inflammation of cardiovascular diseases

Excessive oxidative stress and low-grade chronic inflammation are major pathophysiological factors contributing to the development of cardiovascular diseases (CVD) such as hypertension, diabetes and atherosclerosis. Accumulating evidence suggests that a compromised anti-oxidant system can lead to excessive oxidative stress in cardiovascular related organs, resulting in cell damage and death. In addition, increased circulating levels of pro-inflammatory cytokines, such as tumor necrosis factor α, interleukin-6 and C-reactive protein, are closely related to morbidity and mortality of cardiovascular complications. Emerging evidence suggests that interventions including nutrition, pharmacology and exercise may activate expression of cellular anti-oxidant systems via the nuclear factor erythroid 2-related factor 2-Kelch-like ECH-associated protein 1 signaling pathway and play a role in preventing inflammatory processes in CVD. The focus of the present review is to summarize recent evidence showing the role of these anti-oxidant and anti-inflammatory interventions in cardiovascular disease. We believe that these findings may prompt new effective pathogenesis-oriented interventions, based on the exercise-induced protection from disease in the cardiovascular system, aimed at targeting oxidant stress and inflammation.

Exercise training improves endothelial function via adiponectin-dependent and independent pathways in type 2 diabetic mice

Type 2 diabetes (T2D) is a leading risk factor for a variety of cardiovascular diseases including coronary heart disease and atherosclerosis. Exercise training (ET) has a beneficial effect on these disorders, but the basis for this effect is not fully understood. This study was designed to investigate whether the ET abates endothelial dysfunction in the aorta in T2D. Heterozygous controls (m Lepr(db)) and type 2 diabetic mice (db/db; Lepr(db)) were either exercise entrained by forced treadmill exercise or remained sedentary for 10 wk. Ex vivo functional assessment of aortic rings showed that ET restored acetylcholine-induced endothelial-dependent vasodilation of diabetic mice. Although the protein expression of endothelial nitric oxide synthase did not increase, ET reduced both IFN-γ and superoxide production by inhibiting gp91(phox) protein levels. In addition, ET increased the expression of adiponectin (APN) and the antioxidant enzyme, SOD-1. To investigate whether these beneficial effects of ET are APN dependent, we used adiponectin knockout (APNKO) mice. Indeed, impaired endothelial-dependent vasodilation occurred in APNKO mice, suggesting that APN plays a central role in prevention of endothelial dysfunction. APNKO mice also showed increased protein expression of IFN-γ, gp91(phox), and nitrotyrosine but protein expression of SOD-1 and -3 were comparable between wild-type and APNKO. These findings in the aorta imply that APN suppresses inflammation and oxidative stress in the aorta, but not SOD-1 and -3. Thus ET improves endothelial function in the aorta in T2D via both APN-dependent and independent pathways. This improvement is due to the effects of ET in inhibiting inflammation and oxidative stress (APN-dependent) as well as in improving antioxidant enzyme (APN-independent) performance in T2D.

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.

Physical activity opposes coronary vascular dysfunction induced during high fat feeding in mice

Sedentary and high‐fat diet lifestyles are associated with greater prevalence of obesity and type 2 diabetes in humans, both of which independently increase atherosclerosis. High‐fat diet in sedentary individuals produces endothelial dysfunction in blood vessels as a first step toward coronary arteriosclerosis. We observed preservation of coronary arteriolar vascular function when mice began voluntary running in wheels at the start of a high‐fat diet. We further showed that mechanisms by which running opposed the detrimental effects of high‐fat diet on vascular function included maintenance of eNOS phosphorylation, leptin sensitivity, and redox balance in mouse coronary arterioles. The results provide evidence for how physical activity is an effective therapy to oppose the development of atherosclerosis in the first place.

Coronary and Aortic Endothelial Function Affected by Feedback Between Adiponectin and Tumor Necrosis Factor α in Type 2 Diabetic Mice

Objective—To verify that adiponectin and tumor necrosis factor (TNF)-&agr; reciprocally regulate their expression, thereby synergistically affecting both coronary and aortic endothelial dysfunction in type 2 diabetic mice. Methods and Results—We examined endothelium-dependent and endothelium-independent vasodilation/vasorelaxation of coronary arterioles and aortas in control mice, diabetic mice (Leprdb), and Leprdb treated with adiponectin or neutralizing antibody to TNF-&agr; (anti–TNF-&agr;). Endothelium-dependent vasodilation to acetylcholine in both coronary arterioles and aortas was blunted in Leprdb compared with control mice. Endothelium-independent vasodilation to sodium nitroprusside was comparable. Adiponectin and anti–TNF-&agr; improved acetylcholine-induced vasodilation of coronary arterioles and aortas in Leprdb without affecting dilator response to sodium nitroprusside. Adiponectin protein expression was significantly reduced, and TNF-&agr; protein expression was significantly greater, in coronary arterioles and aortas of Leprdb compared with control mice. Immunofluorescence staining results indicate that adiponectin was colocalized with endothelial cells. Anti–TNF-&agr; treatment upregulated adiponectin protein expression in Leprdb coronary arterioles and aortas. Adiponectin administration reduced TNF-&agr; protein expression in Leprdb. Although adiponectin receptor 1 protein expression in coronary arterioles and aortas was similar between control and diabetic mice, adiponectin receptor 2 protein expression was significantly reduced in Leprdb. Both adiponectin and anti–TNF-&agr; inhibited I&kgr;B&agr; phosphorylation and nuclear factor &kgr;B protein expression in Leprdb, suggesting that adiponectin and TNF-&agr; signaling may converge on nuclear factor &kgr;B to reciprocally regulate their expression. Conclusion—A reciprocal suppression occurs between adiponectin and TNF-&agr; that fundamentally affects the regulation of coronary and aortic endothelial function in type 2 diabetic mice.

Vascular dysfunction in type 2 diabetes: emerging targets for therapy

Vascular dysfunction associated with Type 2 diabetes is initially manifested in the pre-diabetic condition and continuously expressed as this complex disease progresses to include other cardiovascular complications that collectively increase patient risk to morbidity and mortality. Many factors are known to affect vascular function and this review focuses on the role of adipokines and obesity in this process. Growing evidence suggests that adipose-derived adipokines, such as cytokines, chemokines and hormones, plays a significant role in the regulation of vascular function. Inhibition of vascular reactive oxygen species (ROS) formation and lowering plasma free fatty acid level are all potential therapeutic targets for type 2 diabetes-induced vascular dysfunction. Bariatric surgery is a relatively new and more aggressive treatment for the morbidly obese patient that also results in an instant and obvious improvement of vascular function through as yet unexplained mechanisms. These therapies show great promise for the prevention and cure of diabetes-induced vascular dysfunction.