Review

Adipokines, Vascular Wall, and Cardiovascular Disease: A Focused Overview of the Role of Adipokines in the Pathophysiology of Cardiovascular Disease

Angiology 2015, Vol. 66(1) 8-24 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0003319713520463 ang.sagepub.com

Fabio Maresca, MD1, Vito Di Palma, MD1, Michele Bevilacqua, MD1, Giuseppe Uccello, BS1, Vittorio Taglialatela, BS1, Alessandro Giaquinto, BS1, Giovanni Esposito, MD, PhD1, Bruno Trimarco, MD1, and Plinio Cirillo, MD, PhD1

Abstract Epidemiological evidence has shown that abdominal obesity is closely associated with the development of cardiovascular (CV) disease, suggesting that it might be considered as an independent CV risk factor. However, the pathophysiological mechanisms responsible for the association between these 2 clinical entities remain largely unknown. Adipocytes are considered able to produce and secrete chemical mediators known as ‘‘adipokines’’ that may exert several biological actions, including those on heart and vessels. Of interest, a different adipokine profile can be observed in the plasma of patients with obesity or metabolic syndrome compared with healthy controls. We consider the main adipokines, focusing on their effects on the vascular wall and analyzing their role in CV pathophysiology. Keywords acute coronary syndrome, adipokines, atherosclerosis, cardiovascular disease, inflammation, obesity, vascular wall

Introduction Obesity is rapidly becoming more prevalent. The World Health Organization estimates that more than 700 million of adults, aged 20 years, are obese in the world.1 Obesity predisposes to developing several pathologies, including hypertension, type 2 diabetes mellitus (T2DM), and atherosclerosis; one consequence of these obesity-related comorbidities is a shorter life expectancy. In fact, worldwide, 2.8 million people die each year as a result of being overweight,2 and it was shown that the median survival rate is reduced by 8 to 10 years in obese patients with a body mass index (BMI) in a range of 40 to 45 kg/m2 (compared with those with a normal BMI), mainly because of increased cardiovascular (CV) mortality.3 Epidemiological evidence has shown that abdominal obesity is closely associated with the development of CV disease (CVD).4,5 Specifically, patients in whom the adipose tissue is extensively represented have a higher incidence of other CV risk factors, including a thrombophilic risk 1.5 to 2.5 times higher than nonobese ones.6 Taken together, these observations suggest that obesity might be considered as an independent CV risk factor.7 However, despite these clinical observations, the pathophysiological mechanisms responsible for the association between obesity and CVD remain largely unknown. In this

regard, it is to be considered that the cells of adipose tissue, the adipocytes, are no longer considered only as fat storage cells; in fact, they are able to produce and secrete several substances with biological activity, known as ‘‘adipokines’’ that may exert an endocrine, paracrine, and autocrine action.8 These molecules are considered as a possible link between obesity and CVD. It is known that different adipokine profiles can be observed in the plasma of patients with obesity,9 metabolic syndrome (MetS),10 or T2DM11 compared with healthy controls, suggesting that these adipocyte-derived substances might be considered as novel biomarkers for CV risk in these patients. However, a more recent point of view supported the hypothesis that these molecules might be considered as active participants in the pathogenesis of CVD and not just disease markers. In line with this hypothesis, it has been demonstrated that weight 1

Department of Advanced Biomedical Sciences, Division of Cardiology, University of Naples, Naples, Italy

Corresponding Author: Plinio Cirillo, Department of Advanced Biomedical Sciences, Division of Cardiology, University of Naples ‘‘Federico II’’, via Sergio Pansini, 5, 80131 Naples, Italy. Email: [email protected]

Maresca et al loss, exercise, and several drugs such as fibrates, statins, antihypertensive, and hypoglycemic drugs as well as antiplatelet agents could influence the adipokine profile.12-15 We provide an overview on the molecules produced by adipose tissue, which have been investigated for their activity on the vascular wall and, that, consequently, might be involved in CV pathophysiology.

Adipokines White adipose tissue (WAT) consists of a heterogeneous mixture of adipocytes, preadipocytes, endothelial cells (ECs), fibroblasts, macrophages, and leukocytes as well as the blood vessels and nerves. This complex composition allows the integration of multiple metabolic processes, which is the basis of the endocrine function of this tissue.16 In this complex network, adipokines are produced by adipocytes and by the inflammatory cells localized in WAT, like macrophages, leukocytes, and fibroblasts.17,18 In obese patients, the increase in both adipocyte number and their volume can be observed.19 It is believed that adipocyte hypertrophy is associated with oxidative stress,20 hypoxia,21 mechanical trauma,22 and local inflammation.23 These factors are both the cause and the consequence of a dysregulation of the endocrine function of adipose tissue. The biological functions of adipokines are still partially unknown; however, they seem to be involved in the regulation of many physiological processes such as appetite and energy balance, lipid metabolism, blood pressure, insulin sensitivity, hemostasis, angiogenesis, and inflammation.24 These observations led to the suggestion that an ‘‘inflammatory milieu’’ is responsible for the metabolic disorders and CVD reported in obese patients. Recently, an emerging role in the pathophysiology of CVD has been attributed to the epicardial adipose tissue (EAT). The EAT is located along the large coronary arteries and on the surface of ventricles and the apex of the heart. As the WAT, the EAT might produce and secrete several bioactive molecules that can act via paracrine manner. Thus, it has been postulated that the ‘‘local’’ molecular cross-talk between EAT and the heart might be involved in the pathophysiology of CV disorders observed in patients with obesity, diabetes, and MetS,25 such as coronary artery disease (CAD) and acute coronary syndromes.26-34 Under normal physiological conditions, EAT has great flexibility in the storage or release of fatty acids compared with other fat depots. Thus, it can help to fulfill the energy needs of the arterial wall as well as the heart muscle and, moreover, it avoids lipotoxicity. In contrast, in patients with MetS, the EAT expands and there is an increase in macrophage infiltration and T-cell accumulation.35,36 Although EAT has been reported to be a source of both proinflammatory and anti-inflammatory substances, mounting evidence suggest that, in patients with severe coronary disease, the infiltration of macrophages and T cells together with a secretion of proinflammatory cytokines is predominant.37-41 Therefore, due to its anatomical and functional proximity to the coronary circulation, EAT might be considered an even more direct CVD risk

9 marker than central adiposity but this issue remains to be established. To date, the molecules derived from adipose tissue can be classified by both the cell population mainly responsible for their production by WAT and the biological activity exerted. However, some of them can be placed across multiple categories, so that, in our opinion, any attempt at classification is redundant. In addition, only some of these molecules appear to play an active role in CV pathophysiology with effects that may be beneficial and/or detrimental on the CV system.26-41

Adiponectin Adiponectin is a 247-amino acid (aa) protein with a globular carboxyl-terminal domain and an aminoterminal collagenlike domain. This adipocytokine has a similar structure with complement factor 1q.42 In humans, the adiponectin gene is located on chromosome 3q27.43 This adipokine is synthesized predominantly by adipocytes and also by skeletal muscle, ECs, and cardiomyocytes. It represents 0.01% of plasma proteins (3-30 mg/mL).44 Broadly, this adipokine seems to exert protective effects on the CV system. In fact, patients with high atherosclerotic burden have low plasma levels of adiponectin.45 Moreover, it has been demonstrated that low plasma levels of this adipokine are closely related to the progression of coronary atherosclerosis in patients with angina pectoris.46 Furthermore, it has been observed that women with low plasma levels of adiponectin have impaired coronary flow47 and that adiponectin levels appear to decrease after myocardial infarction (MI).48 Interestingly, increased plasma adiponectin correlates with a reduced risk of MI in men49 and a lower risk of coronary heart disease in diabetics.50 Although adipocytes are the main source of adiponectin, patients in which adipose tissue is largely represented, such as obese and those affected by MetS or diabetes mellitus, have low measurable plasma levels of this adipocytokine.51 Three different oligomers of adiponectin have been isolated in the plasma, each one with a specific biological function.52 We can identify (1) low-molecular-weight (LMW) oligomer, constructed by 3 molecules of adiponectin; (2) middle-molecular-weight (MMW) oligomer, formed by 6 adiponectin fractions, and (3) high-molecular-weight (HMW) oligomer constituted by 12 to 18 molecules of adiponectin.53 Another recently isolated oligomer is composed of 3 molecules of adiponectin bound to albumin LMW.54 In humans, MMW and LMW adiponectin represent 25% while HMW adiponectin represents 50% of the circulating adiponectin.54 Since plasma levels of HMW appear to be closely related to insulin sensitivity, it has been suggested that HMW is biologically active.55,56 In vivo, adiponectin increases energy consumption and oxidation of fatty acids in the liver and muscles. These phenomena contribute to reduced triglycerides levels in these tissues and improved insulin sensitivity.57 Adiponectin mediates its actions via 3 receptors: AdipoR1 (expressed ubiquitously, but

10 especially in skeletal muscle), AdipoR2 (predominantly in the liver),58 and T-cadherin.59 All 3 receptors are expressed in cardiac tissue.60,61 The binding of adiponectin to the AdipoR2 receptor, in addition to the increase in energy consumption and the improvement in fatty acid oxidation, as already mentioned, significantly inhibits proatherosclerotic processes such as oxidative stress and inflammation.62 Plasma adiponectin has been correlated to endothelium-dependent vasorelaxation in humans63 and increase in nitric oxide (NO) production as well as NO-mediated and potassium channel-mediated vasorelaxation in rats.64,65 This NO release is likely stimulated by adiponectin’s binding to either the AdipoR2 or the T-cadherin receptors on the endothelial surface.66 So, the increased NO production inhibits platelet aggregation, leukocyte adhesion to ECs, and vascular smooth muscle cell (VSMC) proliferation, improving endothelial function; this has been proven in genetically modified mice to develop hyperlipidemia and atherosclerosis where adiponectin inhibits oxygen free radical production and ameliorates endothelial function.67 From these observations it is evident that adiponectin can play a protective role for the CV system since it is able to interfere with the early steps of atherosclerotic disease. In particular, it has been shown that adiponectin deficiency increases leukocyte–endothelium interactions via upregulation of endothelial c adhesion molecules (CAMs) in vivo.68 Conversely, the expression of adhesion molecules is reduced in the presence of increased adiponectin levels.69 Moreover, adiponectin suppresses smooth muscle cell proliferation70 and inhibits lipopolysaccharide-induced adventitial fibroblast migration and transition to myofibroblasts via the AdipoR1—adenosine monophosphate-activated protein kinase (AMPK)—inducible NO synthase pathway.71 Again, adiponectin suppresses lipid accumulation and class A scavenger receptor expression in human monocyte-derived macrophages.72 Finally, this adipocytokine reduces lipid accumulation in macrophage foam cells,73 and it is able to reduce atherosclerosis in apolipoprotein E (ApoE)-deficient mice.74 Of note, adiponectin seems to play an important role in modulating the inflammatory network involved in the pathophysiology of CVD as it regulates the expression of several important chemokines such as the ‘‘interferon (IFN) g-induced protein 10,’’ the ‘‘monokine induced by IFN-g,’’ and the ‘‘the IFN-inducible T-cell chemoattractant,’’ which bind to the ‘‘chemokine (C-X-C motif) receptor 3,’’ an important regulator of chemotaxis of lymphocytes within the atherosclerotic plaque.75 Adiponectin’s protective role has also been confirmed by recent studies showing that adiponectin receptors AdipoR1 and AdipoR2 are detectable on platelets, thus suggesting that this adipocytokine acts as an endogenous antithrombotic factor.76 It has also been reported that decreased levels of adiponectin are associated with hypertension through various mechanisms including the renin–angiotensin system and sympathetic nervous system (SNS) hyperactivity, endothelial dysfunction, and renal pressure natriuresis impairment.77 Adults with hypertension had lower adiponectin levels than normotensive adults; every 1 mg/mL increase in adiponectin levels was associated with 6% reduced risk of hypertension.78

Angiology 66(1) Moreover, adiponectin appears to play an active role also in myocardial muscle. Indeed, in animal studies, adiponectin deficiency seems to be linked to myocardial damage, heart failure, and cardiac hypertrophy.79-81 Adiponectin is also active in ischemia–reperfusion (I/R) injury, via activation of cyclooxygenase 2 (COX-2) and AMPK and tumor necrosis factor a (TNF-a) suppression and in cardiac remodeling, by limiting the extent of myocardial hypertrophy.82,83 From that mentioned earlier, it is evident that adiponectin is widely involved in the CV pathophysiology.

Leptin Leptin is a polypeptide consisting of 167 aas, encoded by the ob gene.84 The leptin (ob) gene is located on chromosome 7 in humans.85 Leptin acts through appetite suppression and increased energy expenditure.84 Leptin-deficient mice and humans display increased appetite, which is reversed upon leptin administration.86 The main site of action of leptin is the arcuate nucleus of the hypothalamus where it decreases the messenger RNA (mRNA) of the orexigenic neuropeptide Y and increases the expression of the anorexigenic proopiomelanocortin.85 Moreover, there are several mechanisms through which leptin increases energy expenditure: by increasing locomotor activity, thyroid hormone synthesis, and SNS output, which together lead to a more active basal metabolic rate and promote lipolysis.87,88 Subsequently, leptin was considered as a signal of the body’s nutritional status to the reproductive system, immune system, CV system, skeletal system, and thyroid hormone axis.89 But the initial hopes of having found a possible solution to the obesity epidemic failed because the majority of the obese population was leptin resistant rather than leptin deficient.90 Leptin acts on target cells through plasma membrane receptors that include at least 6 isoforms, Ob-Ra through Ob-Rf (Ob-Rb the predominant). The actions of leptin are mediated via Ob-R modulation of the Janus kinase and signal transducer and activator of transcription91 and AMPK pathways92 and inhibited by suppressor of cytokine signaling 3 (SOCS-3).93,94 In addition to these pathways, leptin also mediates its actions via phosphatidylinositide 3 kinases (PI3Ks)—Akt and mitogen-activated protein kinases pathways.95 Leptin and its receptors have been shown to be present even in the CV system. Leptin levels increase in parallel to adipose tissue mass, and also insulin and acute inflammatory mediators and markers such as glucocorticoids, TNF-a, and interleukin (IL) 1 can increase serum leptin. Conversely, factors that decrease serum leptin are cold exposure, fasting, exercise, growth hormone, somatostatin, cigarette smoking, and androgens.96 Obesity is frequently associated with elevated circulating leptin levels, and clinical studies found that hyperleptinemia is correlated not only with the grade of adiposity but also with the circulating biomarkers of metabolic and CV risk or surrogate markers of subclinical atherosclerosis.97,98 In obese patients, despite the high levels of leptin, the expected benefits from its physiological functions are not

Maresca et al observable. This paradox may be explained, as mentioned earlier, in part by the leptin resistance that is able to reduce the appetite suppression via leptin signal to the hypothalamus. This resistance is probably due to an increase in the levels of SOCS3, an inhibitor of leptin signaling.99 However, the exact mechanism of how resistance develops is not clear being able to involve a variety of defects along the leptin’s pathway. Among these are (1) mutations in the leptin receptor gene, db; (2) a defect in the transport of leptin across the blood–brain barrier to the hypothalamus; (3) defects in the long-receptor isoform or its signaling pathway, and (4) downregulation of receptors.100 In this context, the relationship between obesity, elevated plasma levels of leptin, and CVD appears of particular interest. Several clinical studies have shown that patients with increased plasma concentrations of leptin are at a high risk of developing MI101 and stroke.102 In addition, elevated serum levels of leptin were measured in patients with MI having ST-segment elevation.103 Finally, a large prospective study on leptin and CV risk, the West of Scotland Coronary Prevention Study, supported that leptin is an independent predictor of coronary events.104 This adipokine has been recently identified as a good prognostic marker of future CV events in patients with angiographically proven atherosclerosis105; elevated baseline leptin levels are associated with increased risk of cardiac death, new MI, stroke, and coronary revascularization, even in patients without diabetes.105 Hyperleptinemia is associated with in-stent restenosis.106 The leptin–adiponectin ratio also seems to be directly correlated with the magnitude of the intima–media thickness (IMT) of common carotid artery, a good index of subclinical atherosclerosis.107 Moreover, the results of a recent study demonstrate that leptin gene expression increases prominently in the EAT, whereas adiponectin gene expression decreases significantly in EAT in patients with MetS having CAD (compared with the control group).39 Finally, it has been suggested that leptin, by stimulating the SNS, might also play an important role in the pathophysiology of hypertension.108 These clinical observations have been supported by experimental studies that have strongly reinforced the hypothesis that leptin is involved in the pathophysiology of CVD, with specific effects on the vascular wall. Under physiological conditions, leptin induces endothelium-dependent vasorelaxation by stimulating NO and endothelium-derived hyperpolarizing factor (EDHF),109,110 but in pathological conditions, such as obesity and MetS, the NO-mediated vasodilatory effect of leptin is impaired. In short-lasting obesity, impaired leptin-induced NO production is compensated by EDHF; however, in advanced MetS, the contribution of EDHF becomes inefficient. This may contribute to the development of hypertension and may be implicated in the endothelial dysfunction observed in obese patients.111 As reported in a recent review, leptin promotes other processes involved in atherogenesis, such as (VSMC) migration, hypertrophy, proliferation, osteogenic differentiation, and metalloproteinase expression; moreover, it upregulates the synthesis of angiotensin and endothelin hormones as well as

11 their receptors.112 In contrast, it is been shown that leptin can increase NO production in VSMC exerting a vasodilator effect with an impairment of the proliferative and vasoconstrictor actions of angiotensin II. Notably, both the potentially harmful and the potentially beneficial effects exerted by leptin on VSMCs are lost in VSMCs from animal models of genetically determined leptin resistance.112 Taken together, the net effect of leptin at the level of VSMC is not yet completely understood. Furthermore, leptin seems able to modulate platelet aggregation113 and arterial thrombosis.114,115 It has been demonstrated that leptin, at concentrations usually measurable in the plasma of patients with ACS, induces a proatherothrombotic phenotype in human coronary ECs through the expression of tissue factor (TF) and CAMs.116 Moreover, leptin can induce the expression of TF in human peripheral blood mononuclear cells.117 Leptin has been shown to upregulate various mediators of vascular inflammation like TNF-a, IL-2, IL-6, monocyte chemotactic protein 1 (MCP-1), reactive oxygen species, Th1-type cytokines from ECs, and peripheral blood mononuclear cells.118 Clinical studies have shown a positive correlation between leptin and plasminogen activator inhibitor 1 (PAI-1), von Willebrand factor (vWF), tissue plasminogen activator, and plasma fibrinogen levels and an inverse relationship with protein C and TF pathway inhibitor levels.119 Interestingly, it has been demonstrated that leptin stimulates the production of C-reactive protein (CRP) in human coronary ECs.120 Since it has been demonstrated that CRP is able to promote a prothrombotic phenotype in these vascular cells,121 it might be postulated that this is another potential mechanism by which leptin promotes coronary thrombosis. Therefore, leptin might modulate the progression of atherosclerotic plaques. In fact, the treatment of ApoE-deficient mice with leptin causes faster progression of vessel atherosclerosis and increases the amount of calcium in the vessel wall.122 In addition, treatment of hyperlipidemic mice with recombinant leptin increases the atherosclerotic burden and promotes faster thrombus formation115 while leptin deficiency suppresses progression of atherosclerosis in ApoEdeficient mice.123 Interestingly, in vivo studies on animals have shown that db/db (leptin resistant) and ob/ob are resistant to atherosclerosis,108 supporting a pathological role of leptin in atherosclerosis. Taken together, these observations indicate that leptin might promote the development of the atherosclerotic disease and be involved in the pathophysiology of ACS. Although it remains unclear whether, in obese patients, the peripheral cells are resistant or less to leptin, the relative abundance of its receptor isoforms allows for possible mechanisms of localized resistance in certain cell populations. In addition to these widely studied activities, leptin also appears to have effects on cardiac inotropism,124,125 cardiac remodeling and hypertrophy,126-128 I/R injury, and infarct size,129-132 but the discussion of these other modulatory activities are beyond the scope of this work, considering that the heart itself produces leptin, suggesting that it may also act locally to mediate physiological effects.81,133

12

Resistin Resistin is a cysteine-rich protein of 12.5 kDa, consisting, in humans, of 108 aas: 17 form the N-terminal signal sequence, 37 the variable portion, and the remaining the constant area at the C terminal. Its gene is located on chromosome 19.134 Recently, a family of resistin-like molecules (RELMs) have been described.135 These polypeptides of 105 to 114 aas are composed of 3 domains: a signal sequence at the N-terminal, a variable central portion, and a highly conserved C terminal. The RELM-a is mainly secreted by adipose tissue while RELM-b is expressed only in the gastrointestinal tract and in neoplastic cells, suggesting a possible role in cell proliferation. The RELM-g, recently discovered, is found in hematopoietic tissue where it is thought to exert cytokine-like activity. In rodents, adipocytes are the main source of resistin while in humans macrophages mainly resident in the adipose tissue have this function.136 Initially, this adipokine was proposed as a potential link between obesity and diabetes by modulating the mechanisms responsible for insulin resistance.137 Then, experimental evidence in vivo and in vitro has shown that resistin is able to trigger the mechanisms involved in inflammation.136 In addition, plasma levels of resistin appear to be closely correlated with other markers of inflammation such as TNF-a, type 2 soluble receptor for TNF-a, and IL-6.138-140 There is a degree of cross-talk between resistin and other adipokines. There is a functional link between leptin and resistin: leptin administration suppresses resistin’s mRNA expression and protein levels in ob/ob mice along with reductions in glucose and insulin.141 Other studies confirm that certain actions of resistin are dependent on cross-talk with leptin (presence/absence of) with respect to glucose metabolism and energy regulation.142 However, high plasma levels of resistin correlate with proatherogenic inflammatory markers,143 increased CV risk, unstable angina (UA), poor prognosis in CAD, and MetS.10,144,145 Of note, patients with ACS have resistin plasma levels significantly higher than patients with stable angina and healthy controls.146,147 It was also shown that resistin might be an independent predictor of coronary atherosclerosis in humans,138-140 and it is associated with myocardial injury in patients with ACS.148 Interestingly, it has been recently demonstrated that EAT of patients with ACS has a significant increase in resistin gene expression as well as in its protein secretion when compared to patients with stable CAD.149 It was also reported that, in patients with atherothrombotic strokes, high resistin levels are associated with an elevated risk of the 5-year mortality.150 Recent experimental evidence has indicated that resistin promotes endothelial dysfunction. In fact, it induces expression of adhesion molecules and cytokine in human ECs.151 In addition, it influences the release of PAI-1, vWF, and endothelin and inhibits the expression of endothelial-NO synthases.152 Finally, recent studies have shown that this molecule is able to promote proliferation and migration of smooth muscle cells through the activation of the extracellular signal-regulated kinases and PI3K.153,154 In a

Angiology 66(1) recent in vitro study, it has also been shown that resistin induces the synthesis and expression of active TF in human coronary artery cells.155

Visfatin Visfatin, identified in 2004, is composed of 491 aas and it has a molecular weight of 52 kDa.156 Visfatin gene corresponds to the gene of the pre-B cell colony-enhancing factor (PBEF), described in 1994157 as a cytokine produced by lymphocytes, involved in the regulation of inflammatory mechanisms. Moreover, visfatin displays intrinsic enzymatic activity as a nicotinamide phosphoribosyl-transferase (NAMPT).158 However, in the current literature, we find indistinctly the terms visfatin/NAMPT/PBEF to indicate this adipokine. The term visfatin refers to visceral fat since it was initially suggested that visfatin was mainly produced in visceral fat compared with subcutaneous fat in both mice and humans.156 Nevertheless, other groups have later reported similar visfatin levels in human subcutaneous and visceral fat tissue.159,160 Importantly, visfatin is also found in other fat depots such as perivascular and epicardial fat, where it might exert a paracrine CV effect.38,161 Interestingly, this adipokine is produced by macrophages resident in adipose tissue and not directly by adipocytes. In this regard, the levels of visfatin are believed to be the expression of macrophages infiltrating the adipose tissue, where they produce it in response to inflammatory signals.162 However, circulating levels of visfatin are closely correlated with WAT accumulation.163 The relationship between circulating visfatin levels, anthropometric, and metabolic parameters of obesity and a possible role in CVD is still not completely understood. It has been shown that in both patients with MetS and patients with T2DM, visfatin levels are associated with advanced carotid atherosclerosis, estimated as the IMT.164,165 So enhanced circulating visfatin levels have been proposed as a marker of atherosclerosis by several groups. Visfatin levels positively correlate with circulating levels of inflammatory markers such as IL-6 and MCP-1 in CAD, and more specifically in ACS.166 In patients with CAD and acute myocardial infarction (AMI), a positive association between visfatin expression and unstable atherosclerotic lesions has been established. In fact, a higher expression of visfatin has been found in the smooth muscle and foam cells from unstable plaques of patients that had an AMI. So a role of visfatin in atherosclerotic plaque destabilization has been proposed.167,168 In this context, in patients with ST-segment elevation myocardial infarction (STEMI), elevated plasma levels of visfatin in the culprit plaques and enhanced visfatin expression in macrophages present therein were recently reported.169 At the molecular level, visfatin has multiple functions in the vasculature. It stimulates the growth of VSMCs161 and endothelial angiogenesis via upregulating vascular endothelial growth factor (VEGF) and matrix metalloproteinases.170 Visfatin can also directly affect vascular contractility and it has been shown to induce endothelium-dependent vasorelaxation.171 On the other hand, endothelial dysfunction has been

Maresca et al described in patients with elevated plasma levels of visfatin.172 In fact, visfatin is able to increase the expression of adhesion molecules173 and TF174 in ECs by activating the ‘‘nuclear factor kappa B (NF-kB) light-chain enhancer of activated B cells.’’ Visfatin also seems to mediate inflammatory responses in monocytes by the induction of proinflammatory cytokines IL-1B, IL-6, and TNF-a. However, higher concentrations of visfatin also augment the expression of anti-inflammatory cytokines, for example, IL-10.175 Finally, Lim et al176 have demonstrated direct cardioprotective effects of visfatin in an in vivo I/R model with a reduction in the infarct size following a single intravenous bolus dose of visfatin. Taken together, these data suggest that visfatin might play a detrimental role in CVDs, despite the existence of some evidence in favor of a protective role.

Apelin In 1992, a gene for a receptor with marked similarities to the angiotensin receptor 1 (AT1) was identified. At that time, there was no known ligand and it was named APJ receptor (also known as angiotensin-like 1 receptor).177 It was known as an ‘‘orphan’’ receptor until 1998 when apelin was isolated from bovine stomach extracts.178 The human apelin (Apln) gene is located on the long arm of the X chromosome and encodes a 77-aa preproapelin protein that is proteolytically cleaved to yield bioactive peptides of 36, 17, and 13 aas. Each of these peptides contains the C-terminal 13 residues of the precursor protein, and most bioactivity is thought to reside in this segment.179 The sequence of the apelin 13 peptide is the same in most vertebrates, suggesting evolutionary conservation of a critical function.180 Apelin is present in adipose tissue and in the bloodstream, and it is produced and secreted by adipocytes, stromal vascular fraction, and CV tissues.181 Apelin plasma levels are increased in obesity. Conversely, both circulating apelin and its expression in adipose tissue were reduced after weight loss consecutive to a hypocaloric diet in obese women.182 There are high concentrations of APJ receptors in the heart, and it is expressed on a number of cell types including ECs, smooth muscle cells, and myocytes.183 Apelin immunoreactivity is also found in ECs of human vascular tissue.184 The contribution of the apelin-APJ system to atherogenesis is generally accepted. However, the mechanisms underlying this effect remain to be elucidated, but, probably, the apelin-APJ system is involved in the initiation of atherosclerosis through endothelial inflammation-related pathways. Recently, it has been shown that high plasma apelin concentrations are associated with obesity in humans and hyperinsulinemic obese mice,185 suggesting a link between apelin and feeding. Conversely, apelin concentrations were considerably lower in patients with CAD with respect to the control group. Most importantly, patient with UA and AMI had even lower apelin levels compared with patients with asymptomatic CAD.186 It has also been reported that plasma apelin levels are reduced early after AMI, elevated significantly over time, but still remained significantly lower than in the healthy control

13 population at 24 weeks.187,188 Reduced plasma levels of apelin 36 are also found in patients with first STEMI during the first 5 days, independently from the degree of left ventricular dysfunction and prognosis.189,190 Other studies have identified decreased191,192 or unchanged193 levels of apelin in atherosclerosis-related diseases. Apelin levels were found to be increased in atherosclerotic coronary arteries and this peptide localized specifically to the plaque.194 Increased apelin expression within the plaque may contribute to atherogenesis. In fact, apelin stimulates both the VSMC proliferation195 and the division and migration of VSMC into the neointima,196 which indicates that the apelin-APJ system may have detrimental effects on atherosclerosis. Apelin significantly stimulates time- and concentration-dependent expression of the intercellular adhesion molecule 1 (ICAM-1), vascular CAM 1, and MCP-1 in cultured ECs, via c-Jun N-terminal kinase (JNK) and NF-kB signaling.197 Furthermore, a marked reduction in the number of atherosclerotic lesions was detected in APJ and ApoE double-knockout (APJ / ApoE / ) mice that were fed a high-cholesterol diet compared with APJ / ApoE / mice,198 suggesting that APJ deficiency could reduce atherogenesis. On the other hand, apelin may limit atherosclerosis progression by inhibiting the effects of angiotensin II on the vasculature.199 These last observations are in contrast to other findings; so, whether apelin-APJ activation is beneficial or detrimental in atherosclerosis requires further investigation. However, growing evidence supports the role of the apelin-APJ in the modulation of some CV functions such as vasomotion, myocardial contractility, and artery calcification.81,200 Also, regarding the role of apelin in regulating vascular tone, the results are discordant; studies have demonstrated vasodilator and vasoconstrictor effects.201-206 These results are difficult to interpret because of differences in methodology and doses of apelin administered in the various studies. The mechanisms by which apelin can act on vascular tone may involve the NO synthase and NO pathways in ECs.207 The difficulties associated with unraveling the role of apelin in the modulation of vascular tone are compounded by its possible effects on the central regulation of blood pressure; an effect that may be greater than its peripheral effects.208,209 The actions of apelin may also be dependent on fragment size and the type and location of the vascular bed upon which it is acting. Further research is required to determine the precise role of apelin-APJ signaling in controlling vascular tone and blood pressure. As previously mentioned, apelin is also produced by cardiomyocytes. In animal models of heart failure, cardiac apelin is downregulated by angiotensin II while its production is restored after treatment with an AT1 antagonist.210 In rats, the cardiac production of apelin is increased by hypoxia211 and ischemia.212 In spontaneously hypertensive rats, exercise has also been shown to stimulate the production of apelin.213 This molecule also has a positive hemodynamic effect, acting with inotropic mechanism in rats with heart failure as well as in isolated cardiomyocytes.214 Moreover, in the failing heart after experimental infarction, apelin infusion also resulted in an improvement in cardiac contractility making its role in the treatment of heart failure an interesting possibility.215

14 Apelin is thought to exert its inotropic action by increasing the availability of intracellular calcium rather than enhancing the calcium sensitivity of the myofilaments.212,216,217 In humans, low plasma apelin was observed in patients with chronic heart failure218 and in patients with atrial fibrillation.219 In these, cardiac resynchronization therapy is accompanied by increases in the concentrations of apelin.220 In conclusion, the apelin axis is involved in wide range in CV pathophysiology; however, its impact on CVDs is still controversial.

Omentin Omentin is a protein selectively produced in the visceral adipose tissue where it is synthesized by the visceral stromal vascular cells. It has also been found in the human lung, intestine, ovaries, placenta, and heart. It is codified by 2 genes (1 and 2) with omentin 1 predominating as the circulating form of the adipokine. Omentin plasma levels are reduced in obese patients and correlate negatively with BMI, waist circumference, and insulin resistance and positively with the high-density lipoproteins (HDLs) and plasma adiponectin.221 Conversely, elevated levels of omentin are measurable in the plasma of lean patients, with increased levels of adiponectin and HDL.221,222 Omentin increases insulin-stimulated glucose uptake in both omental and subcutaneous adipocytes and promotes Akt phosphorylation.223 Interestingly, it has been shown that low levels of omentin are measurable in patients with severe coronary atherosclerotic disease,224-226 and circulating omentin levels are lower in patients with MetS and atherosclerosis than in patients with MetS but without atherosclerosis.227 Moreover, circulating omentin may also play a role in preventing arterial calcification that contributes to atherosclerotic lesions.228 A negative association of circulating omentin level with arterial stiffness and carotid plaque in patients with diabetes has also been demonstrated.229 Omentin exhibits a vasodilator effect on blood vessels via endothelium-produced NO, and this omentin-derived NO could inhibit TNF-a-mediated COX-2 induction via suppression of JNK activation. Furthermore, in ECs, omentin activates AMPK that could directly inhibit E-selectin induction and subsequent lymphocyte adhesion to vascular ECs.230 Moreover, omentin decreased in vitro migration and angiogenesis in human ECs as well as NF-kB and Akt activation, induced by human sera, CRP, and VEGF.231 So evidence from clinical and in vitro studies suggests that omentin 1 can have cardioprotective properties.232

Vaspin Vaspin is a novel adipokine expressed in the visceral and subcutaneous adipose tissue, involved in the development of obesity and insulin resistance.233-236 Vaspin was first isolated from the visceral adipose tissue of a rat model for human T2DM in 2007.237 Human vaspin contains 415 aas.238 In humans, vaspin concentrations are significantly higher in women; moreover, vaspin levels correlated positively with age and HDL cholesterol and negatively with waist–hip ratio in diabetic and

Angiology 66(1) nondiabetic patients.239 Relationship between vaspin and CVD is still obscure. Patients with UA have reduced plasma and mRNA levels of this adipokine.240 In addition, vaspin serum concentrations were significantly low in patients with atherosclerosis of carotid arteries.241 Vaspin seems to have a protecting role in the vascular wall preventing apoptosis and improving insulin sensitivity at endothelial level242 and reducing vascular cell-derived cytokines involved in insulin resistance and atherosclerosis.238,243,244 Accumulating data suggest a role for vaspin in obesity and metabolic disorders, but it is not known whether the role of vaspin is protective or causative in the development of these conditions.

Retinol-Binding Protein 4 The retinol-binding protein 4 (RBP4) gene is located on chromosome 10 (10q23-q24).245 It encodes a protein of 201 aas, with a molecular mass of 21 kDa.246 The RBP4 is the transport protein for vitamin A and it is secreted into the circulation bound to vitamin A and transthyretin.247 The RBP4 is manly produced by liver and adipose tissue.248,249 Specifically, RBP4 is expressed in visceral fat.250,251 Plasma RBP4 levels positively correlate with those of retinol, which, in turn, can influence the circulating RBP4 levels.252 In the peripheral tissues, RBP4 may act directly by binding to cell surface receptors253-255 or, through retinoic acid, on retinoic acid receptors.256 The RBP4 is upregulated in insulin-resistant states associated with obesity and it also provokes insulin resistance.257 Serum RBP4 levels are positively correlated with BMI in obese nondiabetic and diabetic patients.257-259 Moreover, higher waist circumference and waist-to-hip ratio were associated with higher RBP4 levels and markers of systemic inflammation.260 It has been demonstrated that weight loss reduces circulating and/or adipose tissue RBP4 levels.258,261-264 Serum RBP4 levels are increased in patients with impaired glucose tolerance and T2DM and correlated inversely with insulin sensitivity in nondiabetic patients with a family history of T2DM.257,258,265-267 Furthermore, RBP4 is closely related to several markers of low-grade inflammation and it has been postulated that by this mechanism it may cause, at least in part, insulin resistance.259,268,269 A decrease in serum RBP4 levels achieved by exercise training predicts the improvement in insulin sensitivity, with greater specificity than leptin, adiponectin, IL-6, or CRP.258 Patients with MetS have elevated plasma levels of RBP4 as compared with patients without MetS.270-274 Circulating RBP4 levels appear related to subclinical CVD. Specifically, plasma RBP4 levels have been shown to be positively correlated with the carotid IMT and left ventricular wall thickness and negatively correlated with the flow-mediated dilatation and with the grayscale median in IMT.275-278 Consistently, the presence of clinical arteriosclerosis is associated with higher circulating RBP4.279 Similarly, circulating RBP4 has been associated with cerebrovascular disease and any hospitalization for CVD.270 Moreover, a close relationship between circulating RBP4 levels and a second CV event has been demonstrated.280 Interestingly,

Maresca et al RBP4 is associated with endothelial dysfunction since its plasma concentrations reflect those of the soluble adhesion molecules sICAM-1 and sE-selectin.275 However, despite this evidence, the role of RBP4 in the pathophysiology of diabetes mellitus and CVD is still controversial.

Adipokines and Nonalcoholic Fatty Liver Disease Obesity is associated not only with an increased risk of CVD but also with an increased risk of nonalcoholic fatty liver disease (NAFLD), which is due to excessive amount of intrahepatic triglycerides known as steatosis.281 The histological aspects of NAFLD encompass a wide spectrum of liver damage ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) and NASH-related cirrhosis with its complications.282 The NAFLD is considered the hepatic component of the MetS since it is also highly correlated with other features of the MetS beyond obesity, such as insulin resistance and T2DM.283 Adipocytokines are likely to be involved in the pathogenesis of NAFLD because they are secreted not only from adipose tissue but also partially from the liver.284 In fact, reduced adiponectin levels are a common finding in NAFLD285 and, in its inflammatory evolution, the NASH, this reduction is more evident.286,287 Moreover, recent therapeutic strategies, focused on the indirect upregulation of adiponectin, through the administration of various therapeutic agents and/or lifestyle modifications, have been demonstrated to ameliorate liver histology.288 On the other hand, serum levels of leptin were also correlated with severity of steatosis and it is increased in NASH.289 The links between resistin and NAFLD are unclear; in fact, resistin levels, in patients with NAFLD, vary between different studies. Some investigators found raised plasma levels of resistin in 1 group of patients with NAFLD and NASH compared with lean and obese control groups.290 Other ones reported decreased resistin concentrations in patients with NAFLD.291 Moreover, RBP4 might be involved in the NAFLD pathophysiology.284 In fact, RBP4 expression in liver was shown to be increased in patients with NAFLD and correlated with NAFLD severity.292 Interestingly, the association between RBP4 levels and liver fat was shown to be independent of insulin resistance, suggesting a strong relationship between RBP4 levels and liver fat.293,294 Studies investigating other adipokines like visfatin with regard to NAFLD are rare. Patients with NASH exhibited lower visfatin levels than those with simple steatosis or obese controls although, compared with healthy controls, all groups of obese patients had increased visfatin concentrations.295

Conclusions A growing amount of evidence has indicated that adipose tissue is not a ‘‘simple’’ storage organ but that it is actively involved

15 in human pathophysiology. Adipokines represent the chemical mediators responsible for this ‘‘endocrine’’ activity. These molecules are considered as a possible link between obesity and CVD. Interestingly, many adipokines exert direct effects on the vascular wall suggesting that they might be considered as active participants in CVD, including ACS. As new adipokines are discovered and studied, we will achieve a better understanding of the complex relationship between adipose tissue, adipokines, and CVD. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.

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