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Relation between TNF-α in Adipose Tissue and Plasminogen Activator Inhibitor-1 in Plasma

Author: CaoYanLi
Tutor: ZhangJin
School: China Medical University
Course: Internal Medicine
Keywords: Obesity Omental adipose tissue Cardiovascular diseases Adipocytokines Tumor necrosis factor-alpha Plasminogen activator inhibitor-1 Early growth response gene-1 Extracellular signal-regulated kinases 1/2
CLC: R589.2
Type: PhD thesis
Year: 2008
Downloads: 262
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IntroductionObesity now present the biggest health challenges worldwide because of its increasing prevalence rate each year. Many epidemiological studies have shown that obesity is associated with a high risk of obesity-related comorbidites, such as type 2 diabetes, cardiovascular disease. However, the underlying mechanism for this is largely unknown. Adipose tissue secretes bioactive peptides, termed ’adipocytokines’, which act locally and distally through autocrine, paracrine and endocrine effects. These include tumor necrosis factors (TNF)-α, IL-6, CRP, resistin or adiponectin, which are involved in the pathogenesis of vascular diseases and may represent a link between obesity and atherosclerosis.Evidence is mounting to suggest that adipocytokines may directly influence endothelial function through their proinflammatory properties. While the majority of the data are based on in vitro studies, they shed light on the molecular links between obesity, insulin resistance, and endothelial dysfunction. The secretary products of adipose tissue contribute to the elevated risks of cardiovascular diseases, and these effects appear to be independent of their effects on insulin resistance and diabetes. The effects of adipocytokines on vascular homeostasis are different. Recently more attention to TNF-αin the development of obesity related cardiovascular diseases are being paid.The animal studies on TNF-αand development of atherosclerosis have produced mixed results. Although reducing TNF-αlevel in apoE-deficient mice resulted in significant decrease of atherosclerosis lesions, in a wild-type background, it produced no improvements. However, mice deficient for the p55 TNF-αreceptor exhibited accelerated atherosclerosis. Finally, although TNF-αis thought to play a role in the progression of ischemia-related congestive heart failure, anti-TNF-αtherapy has shown no benefits for congestive heart failure progression in patients. Despite these conflicting results, there remains a great interest in testing anti-TNF-αtherapies for cardioprotective effects.Plasminogen activator inhibitor-1 (PAI-1) is a kind of inhibitor of serine protein enzyme, and a key factor in fibrinolysis. PAI-1 was an important inhibitor of fibrinolytic activity, and it could disturb normal fibrin clearance mechanisms and promote thrombosis. PAI-1 was dramatically increased in obesity, and it was an independent risk factor of the development of cardiovascular diseases. After intravenous injection of TNF-αto human beings, the level of PAI-1 in plasma was increased. In vitro, TNF-αcould increase the expression of PAI-1 in both endothelial cells and adipocytes. Thus, TNF-αmay be contribute to cardiovascular diseases through increasing the expression of PAI-1.Early growth response gene-1 (egr-1) was originally identified as one of the immediate early genes associated with many kinds of stimulations, include growth factor, cell factor, ischemia, physical energy and trauma, etc. All of these stimulations were associated with vascular diseases. McCaffrey demonstrated Egr-1 expression levels in atherosclerotic lesions after vascular injury were elevated. Anti-Egr-1 may decrease the expression of Egr-1 in the lesion of atherosclerosis through inhibiting smooth muscle cells migration and proliferation. Hence, our study would observed the expression of Egr-1 in endothelial cells affected by TNF-α, to discuss the mechanisms about TNF-αinduced cardiovascular diseases.Our study is aimed to discuss the effect of adipocytokine TNF-αin obesity related cardiovascular complications and the mechanism. Therefore, we set out to examine the association between the expression of TNF-αprotein in omental and subcutaneous adipose tissue and homeostasis model assessment insulin resistance (HOMA-IR), lipids, and plasma PAI-1 levels in female and male respectively, to discuss the relationship between TNF-αand insulin resistance or cardiovascular diseases. Then in cell culture, we observed the effect of TNF-αon HUVEC, as well as the expression of Egr-1 in HUVEC, to discuss the mechanism of TNF-αin cardiovascular disease.Materials and methods1. Lean and central obesity subjects involved in surgery were recruited at surgery department. Participants in a fasting state underwent anthropometric evaluation. Anthropometric measurements included weight, height, waist circumferences.2. Fasting plasma glucose concentration (glucose oxidase method), serum cholesterol (CHOD-PAP method), serum triglycerides (GPO-PAP method), and high-density lipoprotein cholesterol (IRC method) of all the patients were estimated. Fasting serum insulin concentrations were determined with chemiluminescence method using automated immunoassay system. The homeostasis model assessment insulin resistance (HOMA-IR) index derives an estimate of whole-body insulin sensitivity from fasting glucose and insulin concentrations: HOMA-IR=fasting insulin (mU/l)×fasting plasma glucose (mM)/22.5.3. The expression of TNF-αprotein in omental and subcutaneous fat was quantified by using immunohistochemistry and western blot method.4. PAI-1 in human plasma was determined by specific enzyme-linked immuno-sorbent assay (ELISA) method.5. We used HUVEC culture method, and examined the effect of TNF-αand glucose on cell preincubated. And also examined the effect of PD98059 on cell.6. The expression of Egr-1 and ERK1/2 protein in HUVEC was quantified by using western blot method.7. The level of PAI-1 in cell culture solution was quantified by using ELISA method.Results1. The basic anthropometric and metabolic characteristics of the subjects enrolled in this study are presented that BMI, waist circumference, fasting plasma insulin, HOMA-IR, triglycerides, total cholesterol, and plasma PAI-1 were higher in obese than in lean subjects in both male and female. HDL-cholesterol were lower in obese than in lean subjects.2. TNF-αprotein levels were significantly increased in obese compared with lean subjects in both omental and subcutaneous adipose tissue.3. Within each fat depot, there was a positive correlation between adipose cells size and TNF-αexpression (omental: r=0.779, P<0.01; subcutaneous: r=0.452, P<0.05) in female obese subjects. While in male obese subjects, positive correlation only was found in omental adipose tissue (r=0.828, P<0.01).4. In female obese subjects, omental TNF-αprotein levels showed a close association with most of the parameters studied: fasting glucose (r=0.541, P<0.05); fasting insulin (r=0.599, P<0.01); HOMA-IR (r=0.546, P<0.05); triglycerides (r=0.469, P<0.05); HDL-cholesterol (r=-0.759, P<0.01). There was not a similar statistically significant relationship between subcutaneous TNF-αprotein levels and these parameters in obese women. In obese men, no significant correlations between TNF-αprotein and HOMA-IR were apparent in either adipose depot. Although we found correlations between omental TNF-αprotein levels and glucose (r=0.762, P<0.01); insulin (r=0.622, P<0.05); triglycerides (r=0.650, P<0.05); HDL-cholesterol (r=-0.880, P<0.01) in the male obese population. There was no correlation between subcutaneous TNF-αprotein levels and glucose, insulin, triglycerides, HDL-cholesterol in men.5. In both female and male obesity subjects, omental TNF-αprotein levels showed a close association with plasma PAI-1 levels (female, n=20, r=0.763, P<0.01; male, n=12, r=0.760, P<0.01).6. 25mmol/l glucose and TNF-αincreased Egr-1 protein levels: Both glucose and TNF-αincreased Egr-1 protein levels at 60 minutes, but with different time course. TNF-αinduced Egr-1 expression was transient, peaking at 60 minutes, whereas Egr-1, induced by glucose, remained elevated until 240 minutes.7. When cells were incubated with both glucose and TNF-α, Egr-1 expression level was increased 2.64 fold and 2.34 fold (P<0.05) respectively, higher than that in cells incubated with either glucose or TNF-αalone. It suggested that glucose and TNF-αhad an additive effect on Egr-1 expression.8. Cells pretreatment with MEK inhibitors PD98059 (20μmol/l) downregulated control group, glucose group and TNF-αgroup induced Egr-1 expression 16.92%, 13.99% and 63.08% respectively (P<0.05). 10ng/ml TNF-αincreased phosphorylated ERK1/2 levels 1.76 fold (P<0.05) after 25mmol/l glucose pretreatment, but glucose did not enhance ERKl/2 activation after TNF-αtreatment.9. 25mmol/l glucose or 10ng/ml TNF-αmay increase the levels of PAI-1 in endothelial cells culture solution. When pretreatment with PD98059, the levels of PAI-1 decreased 30.43% and 66.67% respectively (P<0.05) in glucose group and TNF-αgroup.DiscussionObesity is a heterogeneous condition with respect to regional distribution of fat tissue; visceral obesity refers to fat accumulation within omental and mesenteric fat depots, whereas peripheral obesity generally refers to subcutaneous fat accumulation. Many epidemiological studies have shown that visceral obesity is associated with a high risk of obesity-related comorbidites, such as insulin resistance, type 2 diabetes, cardiovascular disease, and dyslipidemia, than is peripheral obesity. However, the underlying mechanism for this pathophysiological difference is largely unknown. Visceral adipocytes express higher levels of interleukin-6, interleukin-8, plasminogen activator inhibitor 1 (PAI-1), and angiotensinogen than subcutaneous adipocytes. Presumably, distinctive biological properties of visceral fat contribute to the increased pathogenecity of central obesity.The expression of tumor necrosis factor-alpha (TNF-α) in adipose tissue is increased in human obesity, and TNF-αis proposed as molecular link between obesity and insulin resistance. Alessi MC et al found that TNF-αmRNA in the omental is higher than in the sc adipose tissue. Our results confirm this data and additionally show that difference by the groups of male and female.Since a strong correlation has previously been demonstrated between TNF-αexpression and adipocytes size, we were interested to determine whether our data on subcutaneous and omental adipocytes confirmed this. We found that subcutaneous fat cells were larger than omental fat cells in obese female. In obese male, there was no significance difference between the two depots fat cell size. While within each depot, there is a positive relationship was found between TNF-αprotein and fat cell size in the two adipose depots in obesity except subcutaneous fat in obese male, a finding consistent with previous reports. Obesity, characterized by adipocytes hypertrophy, is a major cause of insulin resistance, type 2 diabetes, hyperlipidaemia and hypertension, clustering of risk factors for atherosclerosis. Thus, we indicate that hypertrophic adipocytes with increased expressions of adipokines causing insulin resistance, such as TNF-α, are associated with insulin resistance which may be a central mechanism to cause life style-related disease. However, the use of isolated adipose tissue rather than whole adipose tissue controlled for differences in the ratio of adipocytes to adventitial tissue within the individual biopsies.The key role of TNF-αin the genesis of insulin resistance has been reported in humans and in animal models especially in relation to obesity associated with insulin resistance. In the fatty tissue of obese animals with insulin resistance and type 2 diabetes, the TNF-αconcentration is elevated. Neutralization of TNF-αin vivo dramatically increased the insulin sensitivity of these obese animals, resulting in increased glucose uptake in peripheral tissues. These observations confirmed the role of TNF-αas a key mediator of insulin resistance in obesity. In our study, omental TNF-αprotein concentration was associated with the serum insulin concentrations in obese group. Moreover, it was significantly associated with insulin resistance in obese women, which manifested as correlation of omental TNF-αlevel with HOMA-IR. From our observations it can be concluded that omental TNF-αprotein plays an important role in the development of insulin resistance in obesity. Four large prospective studies have shown that hyperinsulinemia is a predictor of coronary artery disease. The greatest association of hyperinsulinemia with coronary artery disease has been found in Finland in a population with a very high frequency of coronary artery disease. Results of a prospective investigation of 2103 men clearly showed that high fasting insulin concentrations are an independent predictor of cardiovascular diseases. Additionally, increased TNF-αlevel have been associated with high triglycerides concentrations in metabolic syndrome. In the present study we found that in obesity omental TNF-αprotein was not only positively associated with triglycerides, also negatively with HDL-cholesterol levels, which reflected an increased risk of heart disease. More importantly, our data showed that omental TNF-αprotein was significantly associated with plasma PAI-1 levels. To date, TNF-αis known to be effective for stimulation of PAI-1 release in many cell types, especially adipocytes. PAI-1 could disturb normal fibrin clearance mechanisms and promote thrombosis. The elevated omental TNF-αprotein observed during central obesity and the positive correlation observed between omental TNF-αprotein and PAI-1 levels have led us to characterize omental TNF-αprotein as an important factor in the pathophysiology of obesity-related cardiovascular diseases.In conclusion, these results indicated that TNF-αmay be the link between the cluster of visceral obesity, insulin resistance, and cardiovascular comorbidities. Further studies should identify the molecular mechanisms underlying such events, which may lead to effective therapeutic strategies designed to protect against atherosclerosis in obese patients.The effect of different concentrations of glucose and TNF-αon Egr-1 expression suggest that glucose and TNF-αall increased the expression of Egr-1, and the two factors additively increased Egr-1 expression. TNF-αactivate the expression of ERK1/2 and Egr-1 in the HUVEC. TNF-αinduced Egr-1 expression through ERK1/2 activation in HUVEC.Egr-1 was originally identified as an immediate-early gene that is rapidly induced in response to a variety of stimuli, including growth factors, cytokines, hypoxia, physical forces, and injury, all of which are implicated in the progress of vascular diseases. Recent studies demonstrated that Egr-1 expression is elevated in both atherosclerotic lesions after vascular injury. Interestingly, reduction of Egr-1 expression by Egr-1 antisense technology decreased intimal through inhibiting smooth muscle cell migration and proliferation. Taken together, these findings suggest that Egr-1 is the key mediator in orchestrating the functional characteristics of the vessel wall after injury. And TNF-αmay be increase Egr-1 expression in endothelial cells to contribute the development of cardiovascular diseases in obesity.The importance of TNF-αand the ERK1/2 pathway in the pathogenesis of vascular alterations, however, is well accepted and underscored by recent studies showing reduced vascular lesion formation in TNF-αdeficient mice receiving ERK1/2 antisense treatment. ERK1/2 serves as a key signaling molecule for migration, proliferation and other cellular responses that contribute to atherosclerotic of the arterial wall. Inhibition of the ERK1/2 pathway, therefore, constitutes a logical pharmacological approach for protecting the vasculature from atherosclerotic damage. To date, no selective ERK1/2 inhibitor is clinically available.Although MEK inhibitors inhibited glucose-induced Egr-1 expression,we ruled out the involvement of ERK1/2, because glucose did not increase TNF-αrelated ERK1/2 activity. Rukhsana reported that glucose induced Egr-1 expression was mediated by PKC activation. MEK inhibitors may inhibiting other signaling pathways, such as PKC pathway, then inhibited glucose-induced Egr-1 expression. The datas suggest that TNF-αand glucose may regulate Egr-1 expression through different ways, with glucose mediating its effects through one of the classical PKC isoforms and TNF-αacting through the ERK1/2 pathway.Thus, TNF-αmay play a key role in the development of accumulation of visceral adipose tissue, insulin resistance and dyslipidemia. Further studies should identify the molecular mechanisms underlying such events, which may lead to effective therapeutic strategies designed to protect against atherosclerosis in obese patients. Our studies conclude that Egr-1 play an important role in the development of cardiovascular complications, especially in the process of atherosclerosis. TNF-αand glucose-induced the higher expression of Egr-1 maybe the initial event in the diabetes related cardiovascular complications, and the inhibition of the pathway may provide basic datas for the therapy of cardiovascular complications.Conclusion1. The higher expression of TNF-αin omental adipose tissue is associated with the development of obesity related cardiovascular diseases in obesity subjects.2. Omental adipose tissue may through secret TNF-αto stimulate lipolysis, increase free fatty acids, hence accelerates the development of insulin resistance, hyperlipidemia and cardiovascular diseases. TNF-αalso stimulates the expression of PAI-1 in adipocytes and endothelial cells, increases the level of plasma PAI-1, which increases the risk of cardiovascular diseases.3. TNF-αmay increase the expression of ERK1/2 and Egr-1 in endothelial cells, and TNF-αmay regulate Egr-1 expression through the ERK1/2 pathway.

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