Elsevier

Atherosclerosis

Volume 143, Issue 1, March 1999, Pages 81-90
Atherosclerosis

Expression of plasminogen activator inhibitor-1 in human adipose tissue: a role for TNF-α?

https://doi.org/10.1016/S0021-9150(98)00281-0Get rights and content

Abstract

Elevated plasminogen activator inhibitor-1 (PAI-1) plasma levels, responsible for reduced fibrinolysis, are associated with animal and human obesity and with increased cardiovascular disease. The expression of PAI-1 has been found recently in animal and human adipose tissue. Factors and mechanisms regulating such an expression remain to be elucidated. In omental and/or subcutaneous biopsies from obese non-diabetic patients, incubated in Medium 199, we have confirmed that human adipose tissue expresses PAI-1 protein and mRNA; furthermore we have demonstrated that such an expression is clearly evident also in collagenase isolated human adipocytes and that it is stimulated by incubation itself and enhanced by exogenous human tumor necrosis factor-alpha (h-TNF-α). Since human adipose tissue produces TNF-α, to further characterize the relationship of PAI-1 to TNF-α, human fat biopsies were also incubated with Pentoxifylline (PTX) or Genistein, both known to inhibit endogenous TNF-α through different mechanisms. PTX caused a dose-dependent decrease of basal PAI-1 protein release, reaching 80% maximal inhibitory effect at 10−3 M, the same inhibitory effect caused by Genistein at 100 μg/ml. This was associated to a marked inhibition of PAI-1 mRNA and of endogenous TNF-α production. Furthermore, when human fat biopsies were incubated in the presence of polyclonal rabbit neutralizing anti-human TNF-α antibody (at a concentration able to inhibit 100 UI/ml human TNF-α activity), a modest but significant decrease of the incubation induced expression of PAI-1 mRNA was observed (19.8±19.0% decrease, P=0.04, n=7). In conclusion, the results of this study demonstrate that PAI-1 expression is present in human isolated adipocytes and that it is enhanced in human adipose tissue in vitro by exogenous TNF-α. Furthermore our data support the possibility of a main role of endogenous TNF-α on human adipose tissue PAI-1 expression. This cytokine, produced by human adipose tissue and causing insulin resistance, may be a link in the clinical relationship between insulin-resistance syndrome and increased PAI-1 plasma levels.

Introduction

Type-1 plasminogen activator inhibitor (PAI-1) is the main circulating inhibitor of fibrinolysis [1]. Elevated levels of PAI-1 have been previously associated with increased risk for both venous [2], [3], [4] and arterial [5], [6], [7], [8], [9] thrombosis. However, there is now agreement that elevated PAI-1 levels are not a proven pathogenic cause of venous thrombosis, whilst arguments in favour of a role of PAI-1 in arteriosclerosis and arterial thrombosis (smooth muscle cell migration, neointima formation, tissue remodeling, vascular wound healing, reaction to arterial injury) have recently been brought up by Carmeliet et al. [10], [11], [12]

Obesity, and particularly abdominal obesity, is an independent risk factor for cardiovascular disease [13]. Increased visceral fat, with or without obesity, is part of a metabolic syndrome including insulin resistance, impaired glucose tolerance or type-2-diabetes mellitus, hypertriglyceridemia and low HDL-cholesterol, hypertension [13], [14], [15].

Recently, increased PAI-1 activity has been associated with the metabolic syndrome [16], [17], [18] and independent, highly significant correlations between direct measurements of visceral fat and plasma PAI-1 levels have been demonstrated in humans [19], [20], suggesting a direct role of adipose tissue in the determination of plasma PAI-1 levels. The hypothesis that increased plasma PAI-1 levels in obese human subjects may be the result of PAI-1 release from the increased mass of adipose tissue is very attractive and is supported by studies in animals and in cell lines in vitro. In fact, previous studies on the tissue distribution of PAI-1 in mice showed high concentrations of PAI-1 mRNA in the epididymal fat pad [21], [22]; furthermore, both PAI-1 mRNA and protein were found to be actively produced by 3T3-L1 cells after differentiation into adipocytes [20], [23]. PAI-1 expression in human adipose tissue has been recently documented as well [24].

In murine fat tissue, PAI-1 expression is markedly influenced by the cytokine tumor necrosis factor-alpha (TNF-α), as shown by in vivo and in vitro studies [22], while no effect of TNF-α could be found on human adipose tissue PAI-1 production [24].

The presence of TNF-α in adipose tissue has recently received a great deal of attention. This cytokine is expressed in human adipocytes of both normal and, at higher level, obese subjects [25], [26], [27]. TNF-α influences the lipid and glucose metabolism in fat cells and causes insulin resistance [25], [28] by inhibiting very early steps of insulin action at post-receptor-binding level [29]. As a result, TNF-α is now considered a key component of the obesity–diabetes link [30]. To confirm this, protection from obesity-induced insulin resistance has been recently demonstrated in TNF-α-deficient obese mice [31].

On the basis of findings reported in literature, we hypothesized that TNF-α may influence PAI-1 expression in human adipose tissue, the cytokine being a possible patho–physiological link in the clinical relationship between insulin-resistance syndrome and increased PAI-1 plasma levels. Therefore, the present study aimed at verifying whether TNF-α can influence PAI-1 production in human adipose tissue.

Section snippets

Subjects and tissue sample acquisition

Forty non-neoplastic, overweight (BMI 28±5 Kg/mq) subjects were recruited to obtain fat biopsies. Twelve were surgical patients (explorative laparotomy, gallbladder disease, abdominal hernia) and from seven of them both subcutaneous and omental adipose tissue could be obtained. The remaining 28 subjects were ambulatory patients from our metabolic unit and, in that case, only subcutaneous fat tissue was taken by needle biopsy, according to Kolaczynski et al. [32]. All were non-diabetic as

Production of PAI-1 protein in culture

When human subcutaneous and omental adipose tissue fragments were incubated in basal medium, PAI-1 protein level in conditioned medium increased with incubation time, as shown in Fig. 1, left panel (after 1 h incubation: 0.28±0.07 ng/ml; after 8 h incubation: 6.22±2.10 ng/ml; P<0.05, n=7). Therefore, the rate of PAI-1 release increased during 8 h incubation from 0.28±0.07 ng/ml/h (mean±S.D.) per gram adipose tissue in the first hour, to an average of 0.78±0.28 ng/ml/h in 8 h. The effect of

Discussion

This study confirms that human adipose tissue from obese individuals expresses PAI-1 mRNA and releases PAI-1 protein in culture. Similar results were previously obtained in vitro and in vivo in animals [21], [22], in 3T3-L1 preadipocytes cell line, capable of developing into mature adipocytes under proper stimulation [20], [23] and, recently, in human adipose tissue [24]. The demonstration that human adipose tissue produces PAI-1 can contribute to explain the pathophysiology of the elevation of

Acknowledgements

The authors are indebted to Dr Marina Botto (Department of Rheumatology, Hammersmith Hospital, London), for help during all of the steps of this study. The authors also express their gratitude to Professor Elena Tremoli and Dr Luciana Mussoni of the Institute of Pharmacological Sciences, University of Milan, for their constructive advice and the PAI-1 cDNA probe; to Dr Giovanna Agostino for preliminary mRNA analysis and, together with Dr Ketti Boschini, for assistance in our metabolic ward; to

References (41)

  • V.W.M. van Hinsbergh et al.

    Genistein reduces tumor necrosis factor-α induced plasminogen activator Inhibitor-1 transcription but not Urokinase expression in human endothelial cells

    Blood

    (1994)
  • M. Colucci et al.

    Generation in plasma of a fast-acting inhibitor of plasminogen activator in response to endotoxin stimulation

    J. Clin. Invest.

    (1985)
  • B. Wiman et al.

    The fibrinolytic enzyme system and its role in the etiology of thrombotic disease

    Semin. Thromb. Haemost.

    (1990)
  • A. Hamsten et al.

    Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction

    N. Engl. J. Med.

    (1985)
  • I.B. Sundell et al.

    Interrelationship between plasma levels of plasminogen activator inhibitor, tissue plasminogen activator, lipoprotein (a) and established cardiovascular risk factors in a North Swedish population

    Atherosclerosis

    (1989)
  • P. Carmeliet et al.

    Insights in vessel development and vascular disorders using targeted inactivation and transfer of vascular endothelial growth factor, the tissue factor receptor, and the plasminogen system

    Ann. N.Y. Acad. Sci.

    (1997)
  • P. Carmeliet et al.

    Inhibitory role of plasminogen activator-1 in arterial wound healing and neointima formation: a gene targeting and gene transfer study in mice

    Circulation

    (1997)
  • P.M. Farrehi et al.

    Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice

    Circulation

    (1998)
  • P. Björntorp

    Abdominal fat distribution and disease: an overview of epidemiological data

    Ann. Med.

    (1992)
  • R.A. De Fronzo et al.

    Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease

    Diabetes Care

    (1991)
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