Visceral Fat as a Determinant of Fibrinolysis and Hemostasis

Table of Contents

ABSTRACT

An increased amount of deep abdominal visceral fat has generally been accepted as an important cardiovascular risk factor, and disturbances in hemostasis and fibrinolysis have been suggested to play a role. Fibrinogen and von Willebrand factor, representatives of the hemostatic system, and plasminogen activator inhibitor 1 (PAI-1), as the most important inhibitor of the fibrinolytic system, have been associated with visceral obesity, with the most convincing evidence found for the involvement of PAI-1. The association with fibrinogen and von Willebrand factor has been suggested to be merely a reflection of the association with inflammation and endothelial dysfunction. The fact that PAI-1 is secreted by adipose tissue has attracted much attention. The increase of PAI-1 in visceral obesity could be because visceral adipose tissue produces more PAI-1 compared with subcutaneous abdominal adipose tissue. The contribution of other cell types such as hepatocytes or endothelial cells is probably more important, with stimulation of PAI-1 production by different components of the metabolic syndrome. PAI-1 secretion by adipose tissue has been suggested to have a more local effect, playing a role in tissue remodeling during the development of obesity.

KEYWORDS

Visceral obesity - fibrinogen - vWF - PAI-1

Obesity, and abdominal obesity in particular, has been shown to be an important independent risk factor for cardiovascular morbidity and mortality.[1] In addition, abdominal obesity is characterized by a series of cardiovascular risk factors, such as insulin resistance/hyperinsulinemia, dyslipidemia (increased triglycerides; low high-density lipoprotein; and small, dense low-density lipoprotein cholesterol particles), glucose intolerance/type 2 diabetes, and high blood pressure-all components of the insulin resistance or metabolic syndrome. Recently, both the World Health Organization (WHO)[2] and the National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (NCEP-ATPIII)[3] published criteria for the definition of the metabolic syndrome. Central obesity and insulin resistance have also been linked to more recently defined risk factors for cardiovascular disease such as inflammation,[4] endothelial dysfunction,[5] and a prothrombotic tendency.[6]

Early studies on body fat distribution made a distinction between upper and lower body fat distribution, mostly assessed by the waist-to-hip ratio (WHR). However, in later years the importance of the relative amounts of subcutaneous (SAT) versus visceral abdominal adipose tissue (VAT) became more and more apparent, and the WHR or waist circumference cannot make a distinction between these two abdominal fat compartments. Imaging techniques such as computerized tomography (CT) or magnetic resonance imaging (MRI) can precisely quantify abdominal fat within or outside the peritoneum. However, these techniques are time consuming and not easy accessible, and in the case of CT scan, they expose the patient to radiation. The waist circumference has been accepted to be a useful alternative because a closer association with visceral fat accumulation has been shown compared with the WHR.[7]

In this review we focus on three components of the hemostatic/fibrinolytic system that have been associated with both obesity and an increased cardiovascular risk[6]: fibrinogen, von Willebrand factor (vWF), and plasminogen activator inhibitor (PAI-1).

FIBRINOGEN, OBESITY, AND VISCERAL FAT

Fibrinogen, synthesized in the liver, is considered to be an integral part of the hemostatic system, as well as an acute-phase protein associated with the inflammatory response.

Different studies have compared plasma fibrinogen levels in obese and nonobese subjects and found significantly higher levels in the obese.[8] [9] Our group[8] found a 40% increase in plasma fibrinogen levels in obese compared with age- and gender-matched nonobese individuals. Results from studies investigating the relationship between body fat distribution, using waist or WHR, and fibrinogen levels are not so clear, with some finding an association[10] [11] and others not.[12] [13] Studies investigating the relationship between the exact amount of visceral fat, as measured with CT scan or MRI, also found conflicting results. Cigolini et al.[14] found higher levels of fibrinogen in healthy men with high levels of visceral fat compared with men with low levels of visceral fat. Asakawa et al.[15] studied a group of Japanese male and female, mostly nonobese, type 2 diabetic subjects and found a correlation with visceral fat in female but not in male patients. Rattarasarn et al.[16] studied a small group of Thai lean and obese type 2 diabetic women and found no significant relationship between fibrinogen and visceral or subcutaneous fat after correction for percentage body fat. Finally, in a study of obese adolescents,[17] fibrinogen levels were significantly related to levels of visceral fat, but not to changes in visceral fat after physical training. Differences in size and ethnicity of the study populations and differences in correction for confounding factors could in part explain these discrepancies. Studies using the newly defined criteria for the metabolic syndrome found higher levels of fibrinogen in subjects with the metabolic syndrome.[18] [19] [20]

Different mechanisms could be put forward explaining the increased levels of fibrinogen associated with visceral obesity or insulin resistance. First, adipose tissue produces interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α), which in turn could stimulate fibrinogen and C-reactive protein (CRP) production by the liver. Second, visceral obesity, insulin resistance, and the associated increase in free fatty acids (FFAs) could also lead to an increase in fibrinogen production. FFAs secreted from VAT are drained by the portal vein to the liver and could stimulate the production of both fibrinogen and CRP.[21] Different authors have stated that elevated levels of fibrinogen in (visceral) obesity should merely be seen as a marker of inflammation.[13] [22] Indeed, visceral obesity has also been found to be associated with CRP, another acute phase protein.[23]

VWF, OBESITY, AND VISCERAL FAT

vWF, a multimeric, high-molecular weight glycoprotein, is synthesized by endothelial cells and megakaryocytes and stored in the Weibel-Palade bodies of endothelial cells and in α-granules of platelets. In physiological conditions, vWF is mainly synthesized by the endothelial cell, making it a useful tool for the measurement of endothelial activation as a measure of atherogenic pathology. Different studies have shown disturbances in endothelial dependent vasodilatation in obese subjects,[24] [25] which seems to be closely related to an abdominal fat distribution as measured by the WHR[25] or the amount of visceral fat.[24] Studies investigating the influence of body fat distribution on vWF using waist or WHR found conflicting results. A study by De Pergola et al.,[26] using waist circumference, found an association with vWF, although most studies using WHR did not.[27] [28] [29]

In a recent study, we investigated the relationship between vWF:Ag and visceral fat in a group of 181 overweight and obese premenopausal women. Subjects were divided in quintiles according to their levels of visceral fat and we found that subjects with VAT in the highest quintile had significantly higher levels of vWF:Ag compared with subjects in the lowest quintile (171 ± 60% versus 129 ± 40%; p = 0.001), even after correction for percentage fat mass (p = 0.017) or insulin (p = 0.041). Stepwise multiple regression analysis showed VAT as the most important determinant of vWF:Ag levels, explaining 7% of plasma vWF:Ag levels. To the best of our knowledge, no other studies have been published on the relationship between vWF and VAT. Because vWF has not been shown to be produced by the adipocyte, the link between visceral obesity and vWF could be explained by the relationship of both factors with insulin resistance. A significant relationship has been found between vWF:Ag and HOMA as an indirect measure of insulin sensitivity.[12] Inflammatory mechanisms, believed to be associated with atherosclerosis, also increase vWF levels.[30] Because the relationship with most of the components of the metabolic syndrome is weak, different authors have stated that vWF cannot be seen as a true component of the metabolic syndrome.[20] [31] Indeed, two recent studies did not find higher levels of vWF in subjects with the metabolic syndrome compared with subjects without the metabolic syndrome.[19] [20]

PLASMINOGEN ACTIVATOR INHIBITOR, OBESITY, AND VISCERAL FAT

Plasminogen activator inhibitor (PAI-1) is the primary inhibitor of tissue-type plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA), limiting the conversion of plasminogen into plasmin. In plasma, PAI-1 inhibits the degradation of fibrin clots, contributing to an increased athero-thrombotic risk. In tissue, PAI-1 promotes accumulation of extracellular matrix (ECM) and regulates vascular remodeling. PAI-1 is secreted by a variety of cell types such as endothelial cells, smooth muscle cells, hepatocytes, platelets, kidney tubular cells, and adipocytes.[32]

PAI-1 has been shown to be increased in obese subjects,[26] correlating well with body mass index,[33] and with measures of body fat distribution such as waist[34] [35] and WHR.[34] Studies in different types of subjects have shown a significant relationship between visceral fat and PAI-1,[34] [36] [37] [38] which seems to be independent of insulin,[37] [38] insulin sensitivity,[38] triglycerides,[38] total fat mass,[36] [38] and age.[36]

We investigated the contribution of visceral fat to PAI-1 activity levels in two groups of 30 overweight and obese type 2 diabetic and nondiabetic subjects, matched for age, weight, body mass index (BMI), total fat mass, and total abdominal fat.[34] We found a significant correlation between PAI-1 activity and VAT in the whole group (r = 0.43; p = 0.001) and in the nondiabetic women (r = 0.[47] p = 0.009), and a borderline significant correlation was found in the diabetic women (r = 0.33; p = 0.08) (Fig. [1]). No correlation with SAT was found (p > 0.05). Stepwise regression analysis showed visceral fat as the most important determinant factor for PAI-1 in the whole group and in the nondiabetic group, explaining 19 and 22%, respectively, of the variability in plasma PAI-1 levels. In the diabetic group, fasting insulin was the most important determinant, contributing for 19%. These results confirm that visceral fat is more important than BMI, total body fat, or SAT in the determination of PAI-1 levels in overweight and obese diabetic and nondiabetic subjects.[34]

Figure 1 Relationship between visceral fat and plasminogen activator inhibitor-1 activity in two groups of 30 obese diabetic and obese nondiabetic women.

Figure 2 Schematic representation of possible links between visceral fat and the different components of the hemostatic and fibrinolytic system.

The relationship between changes in visceral fat levels and changes in plasma PAI-1 levels associated with weight loss has also been studied. In the study by Janand-Delenne et al.,[37] changes in PAI-1 were more related to changes in VAT than to changes in SAT, insulin, or triglycerides. Kockx et al.[36] only found a relationship with changes in visceral fat in women and not in men. In addition, in the latter study, the relationship found in women disappeared after adjustment for total fat mass. Studies investigating changes in PAI-1 mRNA expression found that changes in plasma PAI-1 levels after weight loss are not related to changes in PAI-1 mRNA expression in abdominal subcutaneous fat cells.[39] [40] Although Bastard et al.[39] found an increase in PAI-1 mRNA and protein concentrations in subcutaneous adipose tissue during a very low calorie diet, Mavri et al.,[40] found a significant decrease in plasma and abdominal subcutaneous fat PAI-1 levels after a low-calorie diet. The results from these studies seem to indicate that changes in visceral fat are more important than changes in subcutaneous fat in the lowering of plasma PAI-1 levels associated with weight loss. However, other effects of weight loss such as improvement of insulin sensitivity or decrease of triglycerides could be equally or even more important.

Strong associations have been found between PAI-1 and the different components of the metabolic syndrome such as visceral obesity, blood pressure, insulin, proinsulin, triglycerides, high-density lipoprotein cholesterol, FFAs, and small, dense low-density lipoprotein particles,[6] with the strongest correlations found with insulin and triglycerides. Juhan Vague et al.[33] were the first to suggest, more than 10 years ago, that PAI-1 should be considered a true component of the insulin resistance or metabolic syndrome. However, in the recently defined criteria of the metabolic syndrome,[2] [3] increased levels of PAI-1 were not included. Studies using this newly defined WHO[19] or NCEP-ATPIII criteria[18] found higher levels of PAI-1 in subjects with the metabolic syndrome compared with subjects without the metabolic syndrome, confirming the statement of Juhan Vague et al.[33]

Although the association between PAI-1 and visceral obesity and the metabolic syndrome is very clear, the precise mechanisms are not completely elucidated yet. The increased levels of PAI-1 could be a result of the obese state itself, with overexpression of PAI-1 in (visceral) adipose tissue, or of the relationship with insulin resistance or other components of the insulin resistance syndrome. An important question in the search for the possible mechanisms is to identify the most important inducers of PAI-1 expression by the adipocyte and other cell types such as endothelial cells and hepatocytes in subjects with visceral obesity or the metabolic syndrome (Fig. 2). In vitro studies have shown that the different components of the insulin-resistance syndrome such as insulin,[41] glucose,[42] and lipids[43] are able to induce PAI-1 expression in endothelial cells, hepatocytes, or vascular smooth muscle cells. PAI-1 expression in these cells is also up-regulated by the inflammatory cytokines TNF-α and TGF-β[44] and by CRP.[45] Angiotensin II, the active component of the renin-angiotensin-aldosteron-system, and an important growth factor, has also been shown to stimulate PAI-1 expression in endothelial cells and smooth muscle cells.[46]

In the last few years, the fact that adipose tissue itself produces PAI-1 has attracted much attention.[47] [48] [50] The same inducers found to stimulate PAI-1 expression in nonadipocytes have been found to induce PAI-1 expression in adipose tissue such as triglycerides,[51] glucose,[52] TGF-β,[48] [51] [53] [54] glucocorticoids,[55] and angiotensin II.[56] For insulin, results are rather contradictory, with some studies finding a positive effect on PAI-1 production,[57] [58] but others not finding such an effect.[55] [59] Differences in handling, the samples could partly explain these variations. Among the PAI-1 inducers, the proinflammatory cytokines TGF-β and TNF-α and angiotensin II may play an important role, especially because they have been found to be produced by the adipocyte itself.[56] [60] Studies on the effect of TNF-α on PAI-1 expression in adipose tissue found conflicting results, with some finding a stimulatory effect[47] [50] and others finding no effect,[48] [54] [61] or even a reduction in PAI-1 release from preadipocytes.[62]

It has been suggested that adipocytes from the visceral depot produce more PAI-1 mRNA compared with adipocytes from subcutaneous tissue,[48] [50] [51] [53] possibly explaining the relationship between visceral obesity and increased plasma PAI-1 levels. However, other studies did not find a difference[54] [63] or found even higher mRNA levels for subcutaneous than for visceral adipose tissue.[64] Methodological factors such as handling of the samples (freshly isolated tissue versus tissue after incubation), subject type (obese versus nonobese), duration time of incubation, expression of results, and method of measurement of mRNA should be taken in consideration while analyzing these different study results.[65] Indeed, a recent study by He et al.[66] showed that PAI-1 mRNA expression in fresh adipose tissue obtained immediately after surgery was similar in omental and subcutaneous adipose tissue. However, when adipose tissue fragments were cultured, PAI-1 mRNA and PAI-1 production were significantly higher in omental than in subcutaneous adipose tissue. Bastelica et al.[67] found that PAI-1 is mainly expressed in the stromal area and in small cells in close contact with adipocytes. Stromal cells could be monocytes, smooth muscle cells, or preadipocytes. Visceral fat seems to express up to five times more PAI-1-producing stromal cells compared with subcutaneous adipose tissue. Fain et al.[68] showed that the formation of PAI-1 by adipocytes was 38% of that by the nonfat cells of adipose tissue. Another important source of PAI-1 in adipose tissue could be vascular endothelial cells: visceral fat tissue is much more vascularized compared with subcutaneous adipose tissue and this could lead to higher levels of PAI-1 in subjects with visceral obesity.[65] It is also possible that the relation between visceral fat and PAI-1 is an indirect one through the association with FFA, as both insulin resistance and visceral obesity are related to increased levels of FFA. Moreover, direct effects of VLDL and FFA on PAI-1 gene expression have been documented.[69]

Although it has been shown that adipose tissue is able to secrete PAI-1, it is still not clear what the exact physiological relevance is of the synthesis of a fibrinolytic inhibitor by adipose tissue. Because plasmin is involved in tissue remodeling, and given the fact that during the development of obesity, adipose tissue undergoes extensive remodeling involving adipogenesis, angiogenesis, and ECM remodeling, it has been suggested that the fibrinolytic system could play a role in the regulation of adipose tissue growth.[70] [71] Animal studies showed that a high-fat diet results in a higher weight gain in PAI-1 knockout mice,[70] whereas PAI-1 transgenic mice with an overexpression of PAI-1[71] and plasminogen knockout mice fed a high-fat diet[72] gained less weight. In contrast, Schäfer et al.[73] found a reduction of obesity in the ob/ob mice by disruption of the PAI-1 gene. However, the studies used different types of animal models (nutritionally induced obesity[70] [71] versus ob/ob mouse[73]), indicating a role for leptin, which is absent in the ob/ob mice.

It has been argued that PAI-1 secreted by adipose tissue contributes significantly to plasma PAI-1 levels. Alessi et al.[74] recently showed that plasma PAI-1 levels are more closely related to fat accumulation and PAI-1 expression in the liver than in adipose tissue. The authors suggest that in insulin resistance, fatty liver is an important site of PAI-1 production.

CONCLUSIONS

Visceral obesity, an important component of the insulin resistance or metabolic syndrome, is clearly related to increased levels of fibrinogen, vWF:Ag, and PAI-1. However, it is most likely that different mechanisms are responsible for these disturbances. In a recent statistical clustering analysis,[13] PAI-1 levels were found to cluster mainly with insulin resistance markers such as body weight, body fat distribution, and fasting insulin, whereas fibrinogen loaded mainly with markers of inflammation such as CRP. PAI-1 may be considered a true component of the metabolic syndrome, whereas elevated levels of fibrinogen and vWF could merely reflect another disease state associated with the metabolic syndrome, such as inflammation or endothelial dysfunction.

Although it is clear that adipose tissue is able to produce PAI-1, much still has to be learned about the exact origin of the increased plasma PAI-1 levels found in visceral obesity and the exact inducers of PAI-1 expression by the various possible cell types. PAI-1 secreted in adipose tissue could have a local effect on adipose tissue growth and development, whereas PAI-1 secreted by other cells types could play a more important role in the determination of circulating plasma PAI-1 levels and the associated athero-thrombotic risk.

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