Mechanisms of endothelial dysfunction in obesity

Abstract

Obesity is a chronic disease, whose incidence is alarmingly growing, affecting not only adults but also children and adolescents. It is associated with severe metabolic abnormalities and increased cardiovascular morbidity and mortality. Adipose tissue secretes a great number of hormones and cytokines that not only regulate substrate metabolism but may deeply and negatively influence endothelial physiology, a condition which may lead to the formation of the atherosclerotic plaque. In this review, the physiology of the endothelium is summarised and the mechanisms by which obesity, through the secretory products of adipose tissue, influences endothelial function are explained. A short description of methodological approaches to diagnose endothelial dysfunction is presented. The possible pathogenetic links between obesity and cardiovascular disease, mediated by oxidative stress, inflammation and endothelial dysfunction are described as well.

Introduction

For centuries, obesity has been frequently regarded as a condition of healthiness and beauty. Unfortunately, the positive anthropological and sociological considerations toward obesity are the wrong sides of the coin because obesity is a serious health problem, especially in countries where economy is growing fast. As defined by Bray, obesity is a chronic disease in the same sense as hypertension and atherosclerosis [1]. Obesity predisposes to diabetes mellitus and to metabolic syndrome, conditions in which the distinct metabolic defect is insulin resistance; this abnormality contributes greatly to the pathophysiology of the metabolic abnormalities and their associated morbidity [2], [3]. There is now substantial evidence that the regional distribution of fat is important: excessive accumulation of fat in the upper body's region, or central obesity, is a better predictor of morbidity than excess fat in the lower body [4]. Thus, it is well established that obesity, in particular central obesity, appears to be the depot most associated with insulin resistance.

Obesity predisposes to diseases due to increased fat cell mass, such as diabetes, non-alcoholic fatty liver disease, cardiovascular disease and cancer, and to diseases due to increased fat mass, such as osteoarthritis and sleep apnea. Obesity is associated with premature death, i.e., obese people have an increased years of life lost: non-smoking women with a body mass index (BMI) > 25 kg/m² at age of 40 lose 3.3 years, while men 3.1 years; if the BMI is > 30 kg/m², women lose 7.1 years and men 5.8 [5]. As recently shown, the BMI is an important and independent predictor of mortality and, most importantly, a higher level of physical activity does not appear to negate the risk associated with adiposity [6].

The cause of increased morbidity and mortality, in obese people, is that all the major risk factors for coronary artery disease coexist, and this condition predisposes to premature cardiovascular disease (CVD). Obesity is indeed a component of the metabolic syndrome, a constellation of metabolic risk factors that consist of serum elevations of triglycerides, low levels of high-density lipoprotein, elevated blood pressure, elevated glucose associated with insulin resistance, a prothrombotic state and a proinflammatory state [7]. The metabolic hallmark of the metabolic syndrome is the presence of insulin resistance, i.e., a decreased sensitivity or responsiveness of peripheral tissues to the metabolic action of insulin. Insulin resistance per se and all the components of the metabolic syndrome are associated with altered functions of the endothelium, which ultimately lead to CVD. It appears that obesity is indeed associated with an excess of CVD.

The Framingham Study showed in 2005 men and 2521 women that the 28-year age-adjusted rate (per 100) of coronary heart disease (CHD) was 26.3 for a mean BMI of 21.6 kg/m² and 42.2 for a mean BMI of 31 in men, and 19.5 for a BMI of 20.4 and 28.8 for a BMI of 32.3 in women, respectively [8]. The Gothenburg Study, in a 12-year incidence period, showed, in a multivariate analysis, that the waist hip ratio (WHR) was the strongest predictor [9] of myocardial infarction in 1462 women [9]. In 1990, the Nurses Health Study, during an 8-year observation, clearly showed in a population of 121 700 females that obesity is a determinant of CHD; after control far cigarette smoking, which is essential to assess the true effect of obesity, even mild-to-moderate overweight increased the risk of CHD [10]. This study showed a relative risk of 3.3 for a BMI of > 29 kg/m² when compared to a BMI < 21; a negative effect of obesity remained appreciable after a multivariate correction for hypertension, diabetes and high cholesterol levels. Similarly, the Honolulu Heart Program demonstrated over a 20-year observation period that a mean subscapular skinfold thickness of 27.2 mm increased the risk of developing CHD in Japanese American [11]. The Rochester Coronary Heart Disease project suggested that both weight and BMI are mildly associated with angina [12]. The Paris Prospective Study has shown that increased BMI, along with resting heart rate, systolic or diastolic blood pressure, tobacco consumption, diabetes, cholesterol and parental history of sudden death, was an independent predictor of sudden death during follow-up [13].

The interaction between CHD and obesity has been confirmed by the PROCAM Study [14].

The association between obesity and CHD becomes more robust when the distribution of fat is considered. Several studies have confirmed that the abdominal adiposity is an independent risk for CHD [15], [16], [17]. The relationship between obesity and CHD is operative not only in the elderly population but also in children and adolescents [18], [19].

Obesity is also a risk factor for cerebrovascular disease, although its negative role appears more clearly in women than in men [20]. The ARIC (Atherosclerosis Risk in Communities) Study found that, in diabetic patients, the relative risk for ischemic stroke was 1.74 for a 0.11 increment of WHR [21].

Finally, obesity appears to be an independent predictor of peripheral vascular disease (PVD) [22]. Therefore, from the available data, obesity is a significant predictor of CVD: this condition begins when the risk factors, which coexist in obese people, induce the endothelial dysfunction, which appears when the endothelium loses its physiological properties, i.e., the tendency to promote vasodilation, fibrinolysis and antiaggregation.

Vascular endothelial cells play a major role in maintaining cardiovascular homeostasis in health. In addition to providing a physical barrier between the vessel wall and lumen, the endothelium secretes a number of mediators that regulate platelet aggregation, coagulation, fibrinolysis and vessel tone. Endothelial cells secrete an array of mediators, which can alternatively mediate either vasoconstriction, such as endothelin-1 and thromboxane A2, or vasodilation such as nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarizing factor (EDHF) (Fig. 1) [23]. NO is the major contributor to endothelium-dependent relaxation in conduit arteries, whereas the contribution of EDHF predominates in smaller resistance vessels [24]. l-Arginine, the physiologic precursor of NO, is carried within the endothelial cells by facilitated transport mediated by the y+ system carrier [25]. Intracellular l-arginine concentrations in endothelial cells range between 0.1 and 0.8 mM; within the cells, l-arginine can be converted to l-citrulline and NO, or to l-ornithine and urea. Evidence suggests that there is a complex compartmentalization of l-arginine within endothelial cells: one compartment is accessible to NOS; in another compartment, the recycling of l-citrulline to l-arginine takes place and, in an additional compartment, l-arginine derives from protein breakdown [26]. The conversion of l-arginine to NO is catalysed by a family of enzymes, the NO synthases (NOS). Three NOS isoforms have been identified: endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible or inflammatory NOS (iNOS) [27]. These enzymes have a ~ 50% sequence homology, and catalyse the NADPH and O₂-dependent oxidation of l-arginine to NO and citrulline. NOS are flavohaem enzymes that are active only as dimers. The dimerization activates the enzyme by sequestering iron, generating high-affinity binding sites for arginine and the essential cofactor tetrahydrobiopterin (BH4), and allowing electron transfer from the reductase-domain flavins to the oxygenase-domain haem [28]. Activity is also dependent on bound calmodulin. In addition, eNOS activity can also be regulated by post-translational modifications: these modifications occur through the phosphorylation of Ser1179, which increases the activity of the enzyme [27]. Several kinases can phosphorylate this site, including protein kinase A, protein kinase C and serine/threonine kinase Akt. Myristoylation and palmitoylation maintain the localization of eNOS to caveolae, discrete microdomains of the plasma membrane, where eNOS is bound to caveolin which keeps the enzyme inactive [29]. Activation of endothelial acetylcholine receptors activates phospholipase C (PLC) that catalyzes the production of inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG) from phosphatidylinositol 4,5-biphosphate (PIP₂). The IP₃-induced increase in intracellular Ca²⁺ activates calmodulin that binds to eNOS, which dissociates from caveolin and translocates to the cytoplasm. Phosphorylation of eNOS by protein kinase A (PKA) inactivates the enzyme, which then relocates to the membrane caveolin. It has been shown that insulin has vasodilatory properties: this effect takes place because this hormone can stimulate NO synthesis, in vivo through the activation of post-receptor pathways, that involve phosphatidylinositol-3 kinase (PI₃K) and Akt [30], [31]. In insulin-resistant states, such as in obesity, the alterations of insulin post-receptor pathways impair not only metabolic, but also vascular effects of the hormone.

NO-sensitive guanylyl cyclase (NO sensitive GC) is the most important receptor for the signalling molecule NO [32]. The latter stimulates cyclic guanosin monophosphate (cGMP) production by activating soluble GC, perhaps by binding to the heme moiety of the enzyme. cGMP mediates most of its intracellular effects through the activation of specific cGMP-dependent protein kinases (PKG). Several families of phosphodiesterases (PDE-I-VI) act as regulatory switches by catalyzing the degradation of cGMP to guanosine-5′-monophosphate (5′-GMP). The NO/cGMP signalling cascade is crucial in the cardiovascular system, where it controls smooth muscle relaxation, and inhibition of platelet aggregation; cyclic nucleotide PDEs hydrolyze cGMP and thus terminate their action [33]. Of note, it has been recently reported that, in obese leptin deficient mice, NO-cGMP signaling pathway is significantly altered in ventricular myocytes [34].

NO has several important effects on the vasculature. First, it maintains basal tone by relaxing vascular smooth muscle cells; it also inhibits platelet adhesion, activation, secretion and aggregation and promotes platelet disaggregation [35]. In addition to these effects, endothelial-derived NO inhibits leukocyte adhesion to the endothelium and inhibits smooth muscle cell migration and proliferation: therefore, NO is a powerful inhibitor of all these mechanisms that ultimately lead to neointimal proliferation and atherosclerosis.

Endothelium contributes to the regulation of blood pressure and blood flow by releasing not only NO but also several other compounds, which contribute both to vasodilation and vasoconstriction. Endothelium produces a less well-characterized compound known as EDHF that promotes vascular smooth muscle relaxation and vasodilation by activating ATP-sensitive potassium channels, smooth muscle sodium–potassium ATPase or both. The exact nature of EDHF however remains elusive. Among the more recent candidates to explain endothelium-dependent hyperpolarization, gap junction, epoxyeicosatrienoic acids (EETs), potassium ions and hydrogen peroxide are the major contenders [36].

Adipose tissue is a secretory factory: it can produce a significant amount of compounds able to affect endothelial function, the most important being leptin, resistin, tumor necrosis factor (TNF) α, interleukin (IL) 6, monocyte chemoattractant protein (MCP)-1, plasminogen activator inhibitor (PAI)-1, adiponectin and the proteins of the renin–angiotensin system [37]. The proteins of the renin–angiotensin system and PAI-1 will not be discussed in this review. Recent evidence indicates that there is a strong interaction between the secretory proteins of adipocytes, called adipokines, and endothelium; thus, it appears that the ability of adipokines to directly affect vascular homeostasis may represent an important mechanistic basis of cardiovascular disease in patients with obesity [38], [39]. In this review, we will consider recent findings on the effect of leptin, resistin, adiponectin, IL-6 and TNFα on endothelium.

Leptin is a 167-amino acid protein expressed mainly by adipocytes and released in the blood in proportion to the size of adipose tissue; leptin action in the CNS promotes weight loss by decreasing food intake and increasing energy expenditure. Recent studies have shown that leptin has a broad range of effects on vascular homeostasis [40]. Leptin can exert a pressor effect through the activation of sympathetic nervous system: this effect is probably due to a central neural action of this hormone because intracerebroventricular administration of leptin mimics the effects of systemic administration [41]. Leptin also affects endothelial function. In vitro studies have shown that leptin causes oxidative stress in cultured endothelial cells by increasing the generation of reactive oxygen species (ROS) [42]. Leptin also has been shown to stimulate the secretion of proinflammatory cytokines (e.g., tumor necrosis factor α, interleukin-6) that are known not only to promote hypertension but also to affect the endothelial function [43], [44].

However, it has been recently shown that leptin may have direct vascular effects that tend to decrease arterial pressure. Lembo et al. and Vecchione et al. have shown that vasorelaxation evoked by leptin is heterogeneous and related to a predominant role of the EDHF mechanisms [45], [46]; this vasodilatory effect, independent from NO, has also been confirmed by Matsuda et al. in human coronary arteries [47]. However, other groups have demonstrated that leptin can induce vasodilation through the stimulation of NO [48], [49]. Interestingly, Mastronardi et al. have hypothesized that the leptin-induced release of NO is not only determined by a direct effect on vascular endothelium, but also by an indirect effect on adipocytes: these cells, under leptin control, may indeed have a major role in NO release by activating NO synthase [50].

Resistin is a recently discovered adipokine that has been suggested to play a role in the development of insulin resistance and obesity [51]. Resistin appears to be produced during adipogenesis and inhibits glucose uptake in skeletal muscle cells in animal models. Verma et al. have shown that endothelial cells, incubated with human recombinant resistin, resulted in an increase in endothelin-1 release, with no change in NO production [52]. Additionally, they found that resistin-treated cells showed increased expression of vascular cellular adhesion molecules (VCAM)-1 and MCP-1. These data suggest that resistin directly activates endothelial cells by promoting endothelin-1 release and upregulating adhesion molecules. However, further studies are needed to determine the biological significance of resistin vascular effects in vivo in humans.

More consistent appear the data so far gathered on the effect of adiponectin on vascular endothelium. Adiponectin, a 30-kDa polypeptide highly and specifically expressed in differentiated adipocytes, circulates at high levels in the bloodstream [53]. A strong and consistent inverse association between adiponectin and both insulin resistance and inflammatory states has been established [54]. Within the vascular wall, adiponectin has several effects that are mediated via increased phosphorylation of the insulin receptor, activation of AMP activated protein kinase (AMPK) and modulation of the nuclear factor κB pathway [55]. In vitro studies have shown that it inhibits monocyte adhesion by decreasing expression of adhesion molecules, inhibits macrophage transformation to foam cells and decreases proliferation of migrating smooth muscle cells in response to growth factors. Adiponectin has also the ability to stimulate the production of NO; Chen et al. found that directly stimulates production of NO in endothelial cells, using phosphatidylinositol 3-kinase-dependent pathways involving phosphorylation of eNOS at Ser1179 by AMPK [56]. Moreover, adiponectin can also induce angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signalling within endothelial cells [57], [58].

Thus, adiponectin has strong antiatherogenic properties, which have been confirmed also in vivo, in humans. Ouchi et al. have analyzed the endothelial function in 202 hypertensive patients, and found that plasma adiponectin level was highly correlated with the vasodilator response to reactive hyperemia, a NO-mediated phenomenon [59]. They also found that, in adiponectin-KO mice, the endothelial function in response to acetylcholine was significantly reduced in adiponectin-KO mice compared with WT mice; conversely, hypoadiponectinemia is associated with a blunted endothelial function and coronary artery disease [60]. Interestingly, it has been recently shown that the action of adiponectin on vascular endothelium in humans appears to be independent of its link with insulin sensitivity [61].

MCP-1 is a chemokine that recruits monocytes to sites of inflammation, is expressed and secreted by adipose tissue [62]. MCP-1 expression in obese mice expressed 10- to 100-fold more MCP-1 mRNA than the liver, suggesting that the adipose tissue may be a major source of the increased plasma levels of MCP-1 observed in these animals [63]. The pathological role of MCP-1 expression in adipose tissue is not understood. MCP-1 has a direct angiogenic effect on endothelial cells [64]: it was recently observed that MCP-1 accelerates wound healing, a process that depends on blood vessel growth [65]. Despite a growing number of information, yet MCP-1 effect on endothelial function in human obesity has to be completely undisclosed.

A growing body of evidence implicates adipose tissue in general, and visceral adiposity in particular, as key regulators of inflammation. Adipose tissue secretes proinflammatory cytokines such as TNFα and IL-6, which seem to play a major role in affecting both endothelial function and glucose metabolism [66]. Growing evidence has pointed to a causative relationship between inflammation and insulin resistance. TNFα mediates insulin resistance as a result of obesity in many rodent obesity models [67]. Recently, MCP-1 was also shown to impair adipocyte insulin sensitivity [63]. Most importantly, recent articles demonstrated that macrophage infiltration into adipose tissue in obesity could be integral to these inflammatory changes [68], [69]. Both studies address the possibility that some inflammatory responses took place outside adipocytes in macrophages infiltrating the expanding adipose tissue. Still, critical questions include the mechanisms by which the inflammatory response is triggered and maintained in obesity: are adipocytes themselves the antigens? Or the inflammatory response takes place without the classic antigen–antibody reaction? Or it is the physical damage to the endothelium produced by the several risk factors for CVD? Whatever the initial stimulus, proinflammatory cytokines negatively affect the endothelium. TNFα, a 26-kDa transmembrane protein that is cleaved into a 17-kDa biologically active protein that exerts its effects via type I and type II TNFα receptors, not only induces insulin resistance but deeply affects endothelial function. TNFα stimulates nuclear transcription factor-kappa B (NF-κB) activation; NF-κB plays a critical role in mediating inflammatory responses and apoptosis: it also regulates the expression of growth factors, proinflammatory cytokines and adhesion molecules [70]. Many products of the genes regulated by NF-κB also, in turn, activate NF-κB (e.g., TNFα). Through this activation, TNFα induces oxidative stress, which exacerbates pathological processes leading to endothelial dysfunction, and atherogenesis. Exaggerated production of TNFα has been shown to increase the activity of inducible NOS, i.e., the enzyme which produced NO in large amount and which is cardiotoxic and promotes apoptosis [71]. It has also been recently shown that TNFα mediates the increased endothelial permeability by activating NADPH oxidase [72]. Finally, TNFα inhibits transcriptional, as well as post-transcriptional, eNOS gene expression an effect this that can account for the endothelial dysfunction [73]. Aljada et al. [74] have clearly demonstrated that TNFα inhibits insulin-induced increase in e-NOS expression in human aortic endothelial cells. Through this mechanism, TNFα may contribute to the inability of insulin to cause vasodilatation in obesity and in type 2 diabetes mellitus.

IL-6 is another cytokine associated with obesity and insulin resistance [75]. IL-6 circulates in multiple glycosylated forms ranging from 22 to 27 kDa in size. IL-6 and IL-6 receptor are expressed by adipocytes and adipose tissue matrix. IL-6 circulates at high levels in the bloodstream and as much as one third of circulating IL-6 originates from adipose tissue [37]. It has been shown that plasma IL-6 concentrations predict the development of cardiovascular disease [76].

IL-6 negatively affects endothelial function; it is an important mediator of increased endothelial permeability via alterations in the ultrastructural distribution of tight junctions and morphologic changes in cell shape. Protein kinase C (PKC) has been shown to be a critical intracellular messenger in these IL-6-mediated changes [77]. It has also been shown that IL-6 can induce endothelial dysfunction by upregulating the angiotensin II receptor AT1: this effect may contribute also to reverberate the oxidative stress caused by pro-inflammatory cytokine in obesity [78].

Yudkin et al. have shown that an increased plasma C reactive protein (CRP) concentration, a marker of a low level of chronic inflammation, is related to the metabolic syndrome [79]. Another study reported an independent association between waist girth and CRP levels; since abdominal fat depot is a source of IL-6 which potently stimulates CRP synthesis by the liver, abdominal obesity is an important factor that helps to explain the inflammatory reaction in obesity [80]. This hypothesis has been substantiated by the group of Despres and colleagues who found a significant relationships between plasma CRP levels and all indices of adiposity, such as BMI, total body fat mass and waist girth [81]. An increased concentration of CRP is important since recent studies suggest that CRP, besides being a marker of inflammation, may also directly contribute to endothelial dysfunction [82]. Exposure of endothelial cells to CRP decreases endothelial NO production and downregulates eNOS expression due to decreased eNOS mRNA stability [83]. Thus, it appears that an increased cytokine production, arising from expanded abdominal fat, could be responsible, not only for the metabolic abnormalities associated with the insulin resistance syndrome, but also for the increased CVD risk observed in abdominally obese patients.