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Leptin and atherosclerosis Jerzy Beltowski ∗ Department of Pathophysiology, Medical University, ul. Jaczewskiego 8, 20-090 Lublin, Poland Received 20 September 2005; received in revised form 22 February 2006; accepted 3 March 2006
Abstract Leptin, a 167-amino acid peptide hormone produced by white adipose tissue, is primarily involved in the regulation of food intake and energy expenditure. Leptin receptors are expressed in many tissues including the cardiovascular system. Plasma leptin concentration is proportional to body adiposity and is markedly increased in obese individuals. Recent studies suggest that hyperleptinemia may play an important role in obesity-associated cardiovascular diseases including atherosclerosis. Leptin exerts many potentially atherogenic effects such as induction of endothelial dysfunction, stimulation of inflammatory reaction, oxidative stress, decrease in paraoxonase activity, platelet aggregation, migration, hypertrophy and proliferation of vascular smooth muscle cells. Leptin-deficient and leptin receptor-deficient mice are protected from arterial thrombosis and neointimal hyperplasia in response to arterial wall injury. Several clinical studies have demonstrated that high leptin level predicts acute cardiovascular events, restenosis after coronary angioplasty, and cerebral stroke independently of traditional risk factors. In addition, plasma leptin correlates with markers of subclinical atherosclerosis such as carotid artery intima-media thickness and coronary artery calcifications. Inhibition of leptin signaling may be a promising strategy to slow the progression of atherosclerosis in hyperleptinemic obese subjects. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Leptin; Atherosclerosis; Obesity; Oxidative stress; Nitric oxide; Endothelial dysfunction; Paraoxonase
Contents 1. 2. 3. 4.
5. 6. 7.
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biology of leptin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leptin and atherogenesis—an overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of proatherogenic effect of leptin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Endothelial dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Lipid metabolism in the vascular wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Proinflammatory effect of leptin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Oxidative stress and oxidative modification of plasma lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Leptin and paraoxonase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Platelet activity and hemostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. Vascular smooth muscle cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leptin and atherosclerosis in animal in vivo studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leptin as a risk factor of atherosclerosis in clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Obesity (body mass index, BMI > 30 kg/m2 ) and overweight (BMI: 25–30 kg/m2 ) are among the most important health problems in industrialized countries and also an increasing problem in developing countries. It is estimated that worldwide about 1 billion people are overweight and 300 millions are obese . Obesity is now recognized as only one among many components of the metabolic syndrome. According to the International Diabetes Federation (IDF) , the metabolic syndrome is present in persons having central obesity defined as a waist circumference ≥94 cm in men or ≥80 cm in women (these cut-offs may differ depending on the ethnic group), and meeting at least two of the following criteria: (1) fasting serum triglycerides ≥1.7 mmol/L (150 mg/dL), (2) HDL-cholesterol <1 mmol/L (40 mg/dL) in men or <1.3 mmol/L (50 mg/dL) in women, (3) blood pressure >130/85 mmHg or treatment of previously diagnozed hypertension, (4) fasting plasma glucose >5.6 mmol/L (100 mg/dL) or treatment of previously diagnozed type 2 diabetes. Other components of the metabolic syndrome include insulin resistance/hyperinsulinemia, proinflammatory and prothrombotic state, oxidative stress, hyperhomocysteinemia and hyperuricemia. Most components of the metabolic syndrome are independent risk factors of atherosclerosis. Therefore, it is not surprising that the prevalence of cardiovascular diseases is markedly increased in obese individuals [3–6]. Initially, it was suggested that insulin resistance and hyperinsulinemia are responsible for the development of all components of the metabolic syndrome, as well as for the proatherogenic effect of obesity . However, more recent studies suggest that the role of insulin is less important than initially appreciated , and accumulating evidence indicate that adipose tissue hormones (“adipokines”) are also involved. In this article I will review experimental and clinical studies suggesting that leptin – the first adipose tissue hormone identified – is an important pathogenetic link between obesity and atherosclerosis.
2. Biology of leptin Leptin was identified in 1994 by positional cloning of the ob gene which determines the development of obesity in ob/ob mice . Due to inherited lack of leptin, these animals develop morbid obesity associated with insulin resistance, hyperinsulinemia, diabetes mellitus, stimulation of hypothalamo-pituitary adrenal axis and, in the homozygous form, infertility. Supplementation of leptin results in reduction of body weight and correction of metabolic abnormalities in ob/ob mice . In wild-type animals leptin is secreted by white adipose tissue in direct proportion to body fat stores and acts on hypothalamic centres to decrease food intake and increase energy expenditure . Leptin acts on target cells through plasma membrane receptors which exist in at least six isoforms, Ob-Ra through Ob-Rf. All of them are encoded by
a single gene and receptor heterogeneity results from alternative splicing of one mRNA molecule . Consequently, all leptin receptors share the common extracellular and (except Ob-Re) transmembrane domains but differ in the length of their intracellular domains. Ob-Rb, also referred to as the “long” isoform, is highly expressed in the hypothalamus and mediates the anorectic effect of leptin. Ob-Rb contains the longest intracellular domain which, upon ligand binding, activates protein tyrosine kinases belonging to the Janus kinase (JAK) family. When activated, JAKs phosphorylate signal transducers and activators of transcription (STAT). Phosphorylated STAT proteins translocate to the nucleus where they regulate the expression of target genes. Other receptor isoforms including Ob-Ra, Ob-Rc, Ob-Rd and Ob-Rf (“short isoforms”) are unable to activate the JAK-STAT pathway but may perform signal transduction through other mechanisms such as mitogen activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K). Short isoforms of the leptin receptor are expressed in many peripheral tissues. Indeed, leptin has multiple activities on carbohydrate and lipid metabolism, reproductive system, inflammatory and immune reactions, etc. . Ob-Re lacks the intracellular and transmembrane domains and is a soluble circulating leptinbinding protein. Although single cases of leptin deficiency in humans have been described, most obese subjects as well as the animals with dietary-induced obesity are characterized by markedly elevated plasma leptin level, which reflects increased amount of adipose tissue and hypothalamic leptin resistance . Leptin resistance may also be determined by genetic factors. The prototypical model of inherited leptin resistance is db/db mice which bear the missense mutation within that part of the leptin receptor gene which encodes the intracellular domain of Ob-Rb. Consequently, db/db mice do not synthesize Ob-Rb but have intact other receptor isoforms. Obese Zucker fatty rats (fa/fa) have the amino acid substitution within the extracellular portion of the leptin receptor, which results in reduced affinity for leptin and impaired ability to initiate signal transduction by all receptor isoforms. Both db/db mice and fa/fa rats are markedly obese and hyperleptinemic. In humans, only few cases of leptin receptor gene mutations have been described to date .
3. Leptin and atherogenesis—an overview If one considers the possible role of leptin in atherosclerosis, several facts have to be taken into account. First, although most cases of human obesity are associated with hypothalamic leptin resistance, little is known about peripheral effects of leptin in obese individuals. The concept of selective leptin resistance has been proposed . According to this hypothesis, only anorectic effect of leptin is impaired, whereas its other activities are maintained in obese subjects. Because many potentially proatherogenic effects of leptin have been
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Fig. 1. Two possible explanations of the relationship between leptin and atherosclerosis: (1) obesity is associated with hyperleptinemia due to increased amount of adipose tissue and therefore proatherogenic effects of leptin are enhanced (left), (2) obesity is associated with leptin resistance at both hypothalamic and peripheral levels, and resistance to “beneficial” effects of leptin contributes to atherogenesis. PON1: paraoxonase 1, SMC: smooth muscle cells.
described (see Section 4), hyperleptinemia could contribute to atherogenesis in obese individuals (Fig. 1, left). The observation that leptin-deficient ob/ob mice are protected from arterial thrombosis or neointimal hyperplasia induced by arterial injury (see below) is consistent with this hypothesis. However, some examples of peripheral leptin resistance have been described, which suggests that leptin resistance may have a more generalized nature. Thus, it is not completely clear which of the proatherogenic effects of leptin presented on the left panel of Fig. 1 are indeed enhanced, and which are rather impaired in obese subjects. An alternative possibility is that hyperleptinemia is not causally linked to atherogenesis but only reflects the state of leptin resistance . Moreover, leptin resistance rather than hyperleptinemia could contribute to atherosclerosis due to the impairment of beneficial effects of leptin (Fig. 1, right). The correlation between elevated leptin and atherosclerosis observed in humans does not allow differentiating between these two possibilities. In experimental studies, it is possible to investigate the effect of hyperleptinemia with intact leptin signaling by administering exogenous leptin to nonobese animals. Leptin secretion by adipocytes is stimulated by insulin and plasma leptin significantly correlates with plasma insulin [16,17]. It is not possible to exclude that leptin itself is not atherogenic but simply reflects insulin resistance and hyperinsulinemia. Again, studying the effect of hyperleptinemia induced in lean animals may be helpful since leptin improves insulin sensitivity and usually reduces plasma insulin in such experiments.
Finally, much data about the role of leptin have been obtained by studying its effect in ob/ob mice. These results cannot be directly extrapolated to humans for several reasons. First, the effect of physiological level of leptin (as obtained by leptin supplementation in ob/ob mice) may differ from that of supraphysiological “obese range” concentration, which is sometimes many-fold higher. Second, due to morbid obesity, many metabolic abnormalities are observed in ob/ob mice and leptin therapy corrects these disturbances. Therefore, leptin may tend to exert “beneficial” effects in these animals, although this is not evidence that leptin per se protects against atherosclerosis. It should also be kept in mind that unlike obese humans, ob/ob mice are characterized by enhanced rather than impaired sensitivity to leptin. Leptin may contribute to the development of classic risk factors of atherosclerosis such as arterial hypertension and diabetes mellitus. In particular, leptin stimulates sympathetic nervous system and, if administered chronically, increases blood pressure in experimental animals. In some studies a significant relationship between plasma leptin and blood pressure independent of body weight was observed in both normotensive and hypertensive humans. These aspects of leptin pathophysiology have been extensively described in recent excellent reviews [18–20] and will not be discussed here. In this article, I focus on direct atherogenic effects of leptin such as regulation of endothelial function, oxidative stress, platelet activity, proliferation of smooth muscle cell, etc.
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4. Mechanisms of proatherogenic effect of leptin 4.1. Endothelial dysfunction Endothelial dysfunction, although usually defined as the impaired vasorelaxation in response to endotheliumdependent vasodilators such as acetylcholine, is also associated with the loss of antiplatelet, profibrinolytic, antiinflammatory and antiproliferative properties of healthy endothelium, which are mainly mediated by nitric oxide (NO) . Thus, dysfunctional endothelium drives the growth of atherosclerotic plaque. Endothelial dysfunction precedes the development of structural lesions in the vessel wall, extends to areas of vasculature free of atherosclerotic plaques and predicts acute cardiovascular events . Functional leptin receptors have been identified on endothelial cells; however, the effect of leptin on endothelial function remains controversial. In vitro studies have demonstrated that leptin at high concentrations may elicit endothelium-dependent NO-mediated vasorelaxation [22–24]. Consistently with this, leptin administered acutely at pharmacological doses increases plasma concentration of NO metabolites and of its second messenger, cGMP [25–28]. However, in vivo studies do not allow identifying the source of leptin-induced NO and some reports suggest that leptin may stimulate NO production by nonvascular tissues . In addition, leptin may upregulate the inducible NO synthase (iNOS), and large amounts of NO generated by iNOS may actually impair endothelial function and may be atherogenic by inducing oxidative stress . Moreover, studies performed in conscious animals suggest that leptin-induced endothelial NO plays little, if any, hemodynamic role . Recently, it has been demonstrated that although very high concentrations of leptin (never observed even in the obese state) induce NO-dependent vasorelaxation of isolated canine coronary artery, leptin has no effect on coronary blood flow when infused into the intact animals . On the other hand, leptin at pathophysiologically relevant “obese-range” concentrations (but not at low physiological concentrations) impaired NO-dependent vasorelaxation induced by acetylcholine both in vitro and in vivo . Because obesity is associated with chronic hyperleptinemia, long-term effect of leptin on endothelial function is of special interest. In ob/ob mice endothelial function is impaired and leptin replacement therapy improves NOdependent relaxation of isolated aorta . Although pairfed group, in which food intake is restricted to the level observed in leptin-treated group, was not included in that study, it seems that the beneficial effect of leptin resulted from the improvement of obesity-associated metabolic abnormalities because leptin was administered for 2 weeks and produced a marked decrease in body weight. Zanetti et al.  have demonstrated that leptin administered to lean rats at a dose of 0.4 mg/kg/day for 7 days improved acetylcholine-
induced NO-dependent relaxation of the thoracic aorta, the expression of eNOS, and the release of NO metabolites from the vascular tissue. However, these effects were reproduced by pair-feeding, suggesting that they were accounted for by caloric restriction whereas leptin per se had no effect on endothelial function. In contrast, we observed that leptin administered at a dose of 0.5 mg/kg/day for 7 days decreased whole-body NO production in the rat, and these effects were not reproduced by pair-feeding . As recently discussed , chronic hyperleptinemia may modulate endothelial NO generation through multiple mechanisms including increase in plasma fatty acids, improvement of insulin sensitivity, reduction of ghrelin secretion, etc. Some of these effects tend to increase whereas others to reduce NO generation and the net effect of leptin may be determined by the background metabolic state of the animals. Several studies suggest that leptin may contribute to endothelial dysfunction or damage in humans. For example, plasma leptin inversely correlated with adenosine-induced (NO-dependent) coronary vasorelaxation in healthy obese males . Leptin was positively correlated with plasma level of soluble thrombomodulin (sTM) and vascular cell adhesion molecule (VCAM-1), two markers of endothelial activation/damage, in obese women . This relationship was independent of BMI, waist-to-hip ratio (WHR), C-reactive protein and insulin sensitivity. In addition, decline in plasma leptin during weight loss program correlated with the reduction of sTM and VCAM-1 . The significant positive correlation between leptin and sTM was also observed in renal failure patients . Plasma leptin negatively correlated with NO production in those patients with ischemic heart disease who develop restenosis after coronary angioplasty . In contrast, no correlation between leptin and endothelial function, measured as the decrease in forearm blood flow following administration of NO synthase inhibitor, was observed in healthy males . Similarly, no relationship between plasma leptin and flow-mediated dilatation of the brachial artery (which is mainly NO-mediated) was observed in healthy adolescents  and in normolipidemic healthy obese women . Thus, the role of leptin in regulating endothelial function in humans remains controversial at present. Interestingly, in contrast to healthy dogs, leptin did not attenuate acetylcholine-induced coronary vasorelaxation in animals with obesity induced by high-fat diet . Obese animals were hyperleptinemic; however, the level of Ob-Rb transcript in the coronary arteries was normal, suggesting that leptin resistance is accounted for by postreceptor mechanisms. 4.2. Lipid metabolism in the vascular wall Leptin stimulates lipoprotein lipase (LPL) secretion by cultured human and murine macrophages . In contrast to endothelium-attached LPL which is involved in
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the metabolism of VLDL and chylomicrons, macrophagederived LPL is proatherogenic because it promotes accumulation of lipoproteins in subendothelial space. Leptin increases the accumulation of cholesterol esters in foam cells, especially at high glucose concentrations . This effect results from the upregulation of acyl-CoA:cholesterol acyltransferase and downregulation of cholesterol esterase. In contrast, under normoglycemic conditions leptin protects macrophages from cholesterol overload . These studies suggest that leptin might unfavorably affect local cholesterol balance especially in diabetic patients. Several studies have demonstrated the inverse relationship between leptin and HDL-cholesterol and/or apolipoprotein AI in humans [47,48]. Ob/ob mice have high HDL , and a series of elegant studies has demonstrated that leptin promotes hepatic HDL clearance by upregulating scavenger receptor type B1, and decreases plasma HDL level in these mice [50–52]. Thus, leptin may impair cholesterol removal from peripheral tissues by lowering HDL. 4.3. Proinﬂammatory effect of leptin Inflammatory reaction in the vascular wall plays an important role in the development of atherosclerosis and in plaque destabilization and rupture [53,54]. Consequently, systemic markers of inflammation, in particular C-reactive protein (CRP), are independent risk factors of cardiovascular events [55,56]. CRP exerts many proatherogenic effects including impairment of endothelial NO production, activation of vascular smooth muscle cells, stimulation of monocyte adhesion to endothelial surface, etc. . Proinflammatory and immunostimulatory effects of leptin have been extensively reviewed recently [58–61]. Below are briefly discussed only those aspects which are directly associated with atherosclerosis. Plasma leptin has been demonstrated to correlate with acute phase reactants, CRP and serum amyloid A (SAA) protein, both in normal weight and in obese subjects [62–64]. Interestingly, in subjects with BMI < 25 kg/m2 , CRP correlated with leptin but not with BMI . In addition, acute administration of exogenous leptin markedly increases plasma CRP in normal-weight humans. In contrast, leptin has no effect or causes only a slight elevation of CRP in obese subjects . This suggests that obese patients may be resistant to proinflammatory effect of leptin. Decrease in plasma leptin during weight loss correlates with the decrease in CRP . In addition, in obese subjects both leptin and CRP concentrations are reduced by short-term fasting before any significant changes in body weight. Leptin, administered to fasting obese subjects not only at pharmacological doses  but also at low doses which prevent fasting-induced hypoleptinemia but do not elevate plasma leptin to supraphysiological level , markedly increases plasma CRP concentration. Taken together, these data suggest that leptin might contribute to proinflammatory state associated with obesity.
4.4. Oxidative stress and oxidative modiﬁcation of plasma lipoproteins Oxidative stress, in particular oxidative modification of plasma lipoproteins, plays an important role in atherogenesis . Recent animal and human studies demonstrate that obesity is associated with chronic low-grade oxidative stress independently of other known risk factors such as hyperlipidemia, diabetes mellitus and hyperinsulinemia [70–72]. However, little is known about role of leptin in prooxidant effect of obesity. Ob/ob mice are characterized by impaired antioxidant defense as evidenced by reduced activity of catalase, glutathione peroxidase (GSH-Px) and glutathione reductase, and leptin therapy corrects these abnormalities . Impaired antioxidant defense, i.e. reduced activity of GSH-Px in plasma and erythrocytes and decreased activity of erythrocyte superoxide dismutase, was also reported in members of a Turkish family with congenital leptin deficiency . However, the effect of leptin replacement was not studied in these patients. In 1999, Bouloumie et al.  have reported that leptin, at (patho)physiologically relevant concentrations (1–100 ng/mL), increases reactive oxygen species (ROS) generation in cultured human umbilical vein endothelial cells. In that study leptin triggered the generation of H2 O2 rather than superoxide. Stimulation of ROS resulted in increased activity of c-jun N-terminal kinase (JNK), increased DNAbinding activity of two proinflammatory transcription factors, AP-1 and NF-B, and overexpression of monocyte chemoattractant protein-1 (MCP-1)—a chemokine involved in atherogenesis. In bovine aortic endothelial cells, leptin at physiological concentration (10 ng/mL) increased ROS production by stimulating mitochondrial fatty acid oxidation . Later studies revealed prooxidant effect of leptin in other cells such as vascular smooth muscle cells  and macrophages . Less is known about the effect of leptin on oxidative stress in vivo. In mice, leptin administered at a dose of 0.23 mg/kg every alternate day for 15 days had no effect on lipid peroxidation products in the liver, kidney and brain; however, it aggravated prooxidant effect of ethanol . We have investigated the effect of leptin administered at a dose of 0.25 mg/kg (s.c. twice daily for 7 days) in the rat. This protocol resulted in about threefold elevation of plasma leptin level. Leptintreated group demonstrated increase in plasma concentration and urinary excretion of isoprostanes, a sensitive and specific marker of oxidative stress in vivo . In addition, leptin increased plasma concentration of other lipid peroxidation products, malonyldialdehyde (MDA), 4-hydroxyalkenals (4HNE) and lipid hydroperoxides, as well as of protein carbonyl groups which originate following ROS-mediated oxidation of proteins. Furthermore, leptin treatment was associated with the increase in MDA and 4-HNE in renal tissue as well as reduced aconitase activity . Aconitase is inactivated by superoxide and is used as an index of intracellular O2 − generation. Recently, Kutlu et al.  have demonstrated that leptin administered for 5 days at a dose of 1.2 mg/day increases lipid
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peroxide level and decreases the concentration of reduced glutathione in mice brain. Taken together, these data indicate that hyperleptinemia induces oxidative stress in plasma and various tissues of experimental animals. There are several potential sources of ROS in the cardiovascular system including NADPH oxidase, uncoupled endothelial nitric oxide synthase, xanthine oxidase and mitochondrial respiratory chain. Little is known about the mechanism through which leptin increases ROS formation, however, the involvement of mitochondrial respiratory chain  and NADPH oxidase  has been suggested by in vitro studies. Recently, it has been demonstrated that leptin increases NADPH oxidase protein expression and activity in isolated murine cardiomyocytes and this effect is attenuated by endothelin (ET-1) receptor antagonists . Because leptin stimulates ET-1 production in cardiomyocytes  as well as in endothelial cells , these results suggest that leptin may stimulate ROS formation through endothelin and NADPH oxidase-dependent pathway. In addition, iNOS may lead to oxidative stress by generating large amounts of NO, and leptin may increase the expression of iNOS as has been demonstrated e.g. in macrophages . We demonstrated that leptin-induced oxidative stress was abolished by coadministration of O2 âˆ’ scavenger, tempol, or NADPH oxidase inhibitor, apocynin , suggesting for a key role of NADPH oxidase-derived superoxide in leptin-induced oxidative stress in vivo. At least two studies suggest the relationship between leptin and oxidative stress in humans. Porreca et al.  observed a significant correlation between plasma leptin and oxidized LDL in 60 healthy postmenopausal women. In multivariate analysis only leptin, total cholesterol and soluble thrombomodulin but not BMI, WHR, LDL cholesterol, or fasting plasma insulin correlated with oxidized LDL level. In addition, decrease in plasma oxLDL during a 12-week weight-reducing program correlated only with the decrease in plasma leptin . In 259 Japanese Americans, plasma leptin correlated with urinary excretion of isoprostanes and this relationship remained significant after adjustment for age, gender and smoking status . 4.5. Leptin and paraoxonase Paraoxonase 1 (PON1) is synthesized in hepatocytes and secreted to the bloodstream where it circulates attached to HDL. Initially recognized as an enzyme destroying organophosphate insecticide, paraoxon, PON1 plays an important role in the protection against atherosclerosis by preventing oxidative modification of plasma lipoproteins and by hydrolyzing homocysteine thiolactone [88,89]. PON1 activity is reduced in patients with virtually all risk factors of atherosclerosis including hypercholesterolemia, obesity, diabetes mellitus and smoking . PON1 knockout mice are prone to atherosclerosis  and low PON1 activity predicts acute cardiovascular events in human prospective studies . Apart from plasma and liver, PON1 is contained in small
amounts in other tissues where it may confer local antioxidant protection. We have demonstrated that leptin administered for 7 days decreases plasma PON1 activity in the rat . In addition, leptin reduced PON1 activity in aorta, renal cortex and medulla but not in the heart, lung or liver . Interestingly, leptin decreased PON1 activity only in tissues in which it stimulated oxidative stress. Moreover, coadministration of tempol together with leptin normalized tissue but not plasma PON1 activity . These data suggest that leptin-induced decrease in PON1 in tissues results from excessive ROS production, consistently with a well-known inactivation of the enzyme by oxidants. However, the effect of leptin on plasma PON1 is independent of oxidative stress. Platelet-activating factor acetylhydrolase (PAF-AH) is another enzyme contained in plasma lipoproteins . In contrast to PON1, the majority of PAF-AH is attached to LDL in humans and only a minor portion is carried by HDL. The role of PAF-AH in atherogenesis is unclear, however, it is generally accepted that LDL-bound PAF-AH is proatherogenic whereas HDL-attached enzyme is protective. In rodents in which LDL level is low, most PAF-AH in plasma is bound to HDL. We observed that in contrast to PON1, leptin administration did not modify PAF-AH activity . However, PAF-AH cannot compensate for PON1 deficiency as evidenced by augmented oxidative stress and atherosclerosis in PON1 knockout mice in which PAF-AH activity is normal . PON1 activity is depressed in obese women in comparison to normal-weight controls and inversely correlates with plasma leptin . In addition, in 40 morbidly obese subjects decrease in plasma leptin after gastric banding strongly correlated with the increase in PON1 activity . These data suggest that hyperleptinemia might lead to PON1 deficiency in humans. 4.6. Platelet activity and hemostasis Nakata et al.  first demonstrated that the long isoform of leptin receptor is expressed in human platelets. Although leptin itself had no effect on platelet function, it augmented ADP-induced platelet aggregation if applied at concentrations found in obese subjects (>30 ng/mL). This study suggested that leptin might contribute to platelet hyperaggregability only when plasma level of this hormone is supraphysiological. These findings were subsequently confirmed by Corsonello et al. . Intracellular mechanism through which leptin enhances platelet aggregation was partially elucidated . It was demonstrated that inhibitors of phospholipase C, protein kinase C (PKC) and phospholipase A2 abolished platelet-activating effect of leptin. In addition, leptin increased intracellular Ca2+ concentration in human platelets. These data suggest that leptin stimulates the breakdown of membrane phospholipids by phospholipase C to yield diacylglycerol which activates PKC, and inositol triphosphate which stimulates Ca2+ release from intracellular stores .
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In addition, leptin increased the expression of P-selectin on human platelets in vitro . The study of Elbatarny and Maurice  has demonstrated that leptin-induced platelet activation requires JAK2, insulin receptor substrate1 (IRS-1), PI3K, protein kinase B/Akt and cAMP-specific phosphodiesterase-3 (PDE-3). This study suggests that leptin activates PI3K in JAK2 and IRS-1-dependent manner, with subsequent activation of PKB/Akt, increased phosphorylation and activity of PDE-3, and fall in intracellular cAMP, which is a well-known inhibitor of platelet activity . In vivo studies confirm the prothrombotic effect of leptin. Thrombus formation in the carotid artery after FeCl3 -induced injury was impaired in ob/ob and db/db mice in comparison to their wild-type controls . Single i.p. leptin injection at a dose of 0.06 mg/kg before arterial injury normalized thrombus formation in ob/ob but not in db/db mice. Moreover, higher dose of leptin (0.6 mg/kg) enhanced injury-induced arterial thrombosis not only in ob/ob but also in wild-type animals. Since a single dose of leptin could not ameliorate metabolic abnormalities observed in ob/ob mice, impaired thrombus formation in these animals results from leptin deficiency rather than from obesity itself. In addition, leptin at concentrations >10 ng/mL augmented the stimulatory effect of ADP on the aggregation of platelets obtained from wildtype and ob/ob mice but not from db/db mice, which is consistent with the Nakata’s study on human platelets . Similarly, thrombus formation after photochemical injury of the carotid artery induced by the infusion of photochemical rose bengal and its activation by locally applied green laser light was also impaired in leptin- or leptin receptor-deficient mice . Again, leptin administered at a single dose just before arterial injury normalized thrombus formation in ob/ob animals. Moreover, bone marrow transplantation from db/db to wild type mice resulted in impaired thrombosis indicating that long isoform of the leptin receptor expressed on marrow-derived cells (presumably platelets) is required for normal arterial thrombosis. Furthermore, injection of leptinneutralizing antibody to block endogenous leptin in wild-type mice impaired arterial thrombosis after FeCl3 -induced injury, indicating that leptin even at physiological level contributes to platelet activity in vivo . Interestingly, enhancing effect of leptin on ADP-induced aggregation was attenuated in platelets obtained from overweight or obese individuals in comparison to normal-weight subjects . This indicates that, in contrast to the resistance to anorectic and weight-reducing effect of leptin, resistance to some peripheral effects of this hormone might be beneficial. Contrary to these studies, Ozata et al.  observed no effect of leptin at concentrations up to 500 ng/mL on either spontaneous or ADP-induced aggregation of platelets isolated from normal-weight or obese subjects. In addition, these authors observed that although platelet aggregability was higher in obese patients than in normal-weight individuals, it was similar in hyperleptinemic patients with simple obesity and in those with inherited leptin deficiency, suggesting that factors other than leptin are responsible for platelet
hyperactivity. These discrepancies may be partially explained by the recent study , which has demonstrated that leptin enhances platelet aggregation induced by ADP, collagen or epinephrine only in about 40% of studied persons (“responders”), but has no effect on platelets obtained from the remaining subjects (“non-responders”). In addition, non-responders are characterized by lower expression of the leptin receptor on isolated platelets. These results suggest that leptin might contribute to atherothrombosis only in a selected group of patients. Little is known about the role of endogenous leptin in the regulation of hemostasis in humans. In 44 obese women without conventional cardiovascular risk factors, plasma leptin significantly correlated with urinary excretion of 11-dehydrothromboxane B2 , a stable metabolite of thromboxane A2 and a marker of platelet activity in vivo . Caloric restriction, which reduces leptin level, is associated with reduced platelet activity as reflected by the decrease in plasma concentration of P-selectin. A recent study  has demonstrated that leptin administered at a physiological dose prevents fastinginduced decrease in P-selectin in obese individuals. This suggests that the decrease in platelet activity during caloric restriction results from the reduction of plasma leptin . It was also demonstrated that leptin positively correlated with plasma concentration of plasminogen activator inhibitor-1 (PAI-1), a major endogenous inhibitor of fibrinolysis, in men with ischemic heart disease . The positive correlation between leptin and PAI-1 independent of body adiposity and insulin sensitivity was also observed in premenopausal women . In the Health Professionals Follow-up Study, leptin significantly correlated with fibrinogen and von Willebrand factor (vWF) in 268 adult males without cardiovascular diseases . In a Swedish population-based study, leptin positively correlated with plasma fibrinogen and PAI-1 and inversely correlated with tissue plasminogen activator (tPA) concentration in plasma . Similarly, leptin positively correlated with PAI-1 and inversely with t-PA in 74 mildly hypertensive overweight subjects . In patients with end-stage renal disease treated with continuous ambulatory peritoneal dialysis, plasma leptin was associated with ristocetin-induced platelet aggregation . The inverse relationship between leptin and two inhibitors of coagulation, protein C and tissue factor pathway inhibitor (TFPI), was also noted . In 51 healthy obese women, plasma leptin was positively correlated with vWF and coagulation factor VIIa independently of age, BMI, WHR or lipid profile . Taken together, these data suggest that leptin may contribute not only to platelet hyperactivity but also to unbeneficial shift in the coagulation-fibrinolysis balance observed in the metabolic syndrome. 4.7. Vascular smooth muscle cells Oda et al.  first demonstrated that leptin receptors are expressed in vascular smooth muscle cells (VSMC) and that in vitro leptin concentration-dependently stimulates
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migration and proliferation of VSMC isolated from rat thoracic aorta. Proliferative effect of leptin was also observed in human aortic SMC . In addition, leptin stimulates VSMC hypertrophy as evidenced by increased protein/DNA ratio . The effect of leptin on VSMC migration is mediated by PI3K  and on cell hypertrophy by JAK2 and extracellular signal-regulated kinase . Interestingly, stretching the vascular wall, which is a well-known stimulus for VSMC hypertrophy, induced the expression of both leptin and its receptor in the rabbit portal vein . In addition, leptin-neutralizing antibodies abolished stretch-induced hypertrophy. Exogenous leptin also stimulated VSMC hypertrophy in this preparation as documented by the increase in cell size and leucine incorporation. These data indicate that locally produced leptin may mediate stretch-induced vascular hypertrophy in a paracrine/autocrine manner. Interestingly, leptin stimulated the expression of matrix metalloproteinase-2 (MMP-2) by human vascular smooth muscle cells . Matrix metalloproteinases play an important role in the migration of VSMC from media to intima and also in plaque rupture . Apart from directly modulating VSMC proliferation, leptin may stimulate vascular remodeling by promoting local production of proliferative and profibrotic cytokines. In particular, leptin stimulates synthesis and secretion of endothelin-1 (ET-1) by human umbilical vein endothelial cells . ET-1 is a potent mitogen for vascular cells and its expression in the cardiovascular system is upregulated in obesity . In rabbit portal vein SMC, leptin induced the expression of preproendothelin-1 and endothelin ETA receptor genes . In addition, leptin stimulated angiotensinogen and angiotensin type 1 receptor expression in these cells. Inhibiting ET-1 or angiotensin II synthesis as well as blockade of their receptors abolished the hypertrophic response to leptin . However, the effect of leptin on ET-1 is controversial since in one study  no effect of leptin on ET-1 secretion was observed. Transforming growth factor-␤ (TGF-␤) may be another mediator of proatherogenic effect of leptin. Leptin stimulates TGF-␤ synthesis by endothelial cells  and plasma leptin correlates with TGF-␤ concentration in hypertensive patients . The role of TGF-␤ in atherogenesis is, however, uncertain. TGF-␤ may stimulate VSMC growth and production of extracellular matrix thus contributing to plaque growth, but, on the other hand, it inhibits local inflammatory reaction and stimulates fibrosis and thus might increase plaque stability. Indeed, reduced TGF-␤ signaling has been observed in unstable plaques . Finally, leptin stimulates osteoblastic differentiation and hydroxyappatite production by calcifying vascular cells (CVC), a subset of smooth muscle cells .
5. Leptin and atherosclerosis in animal in vivo studies Several in vivo studies strongly suggest that leptin is involved in atherogenesis. It has been known for a long time
that ob/ob mice are resistant to atherosclerosis but it was usually attributed to high HDL in these animals . More recent studies indicate that lack of leptin may be a key protector of these animals. First, hyperleptinemia induced by high-fat diet aggravated neointima formation in the carotid artery after FeC3 injury in wild-type mice . In contrast, ob/ob and db/db mice develop only small lesions after arterial injury even if kept on atherogenic diet despite severe obesity and proatherogenic metabolic profile. Administration of leptin enhances lesion formation in wild-type and ob/ob mice, despite reducing body weight and plasma lipids and improving insulin sensitivity . Wire-induced injury of the femoral artery in mice is used as an experimental model of restenosis after coronary angioplasty. Despite severe obesity, hyperglycemia and hyperinsulinemia, neointimal formation after arterial injury is nearly absent in db/db mice . This is in apparent contrast to a well-known unfavorable effect of diabetes on the risk of restenosis in humans. The only logical explanation of this paradox is that the lack of Ob-Rb protects db/db mice against neointima growth. However, leptin resistant Zucker fa/fa rats are characterized by augmented hyperplastic response of the vascular wall to balloon-induced injury . It is unclear whether this reflects species-specific differences or results from the divergent roles of various leptin receptor isoforms in atherogenesis. In contrast to studies mentioned above, some data indicate that leptin may protect against atherosclerosis in specific animal models. For example, LDL-receptor knockout mice which also lack leptin (LDLR−/− ob/ob) develop more atherosclerotic lesions than LDLR−/− mice with intact leptin system [128,129]. Lack of leptin superimposed on LDL receptor knockout background results in the worsening of the lipid profile and deterioration of carbohydrate metabolism, since LDLR−/− ob/ob mice have higher plasma cholesterol, triglycerides, glucose and insulin than their LDLR−/− counterparts. In addition, PON1 and lecithin:cholesterol acyltransferase (LCAT) activities were lower and the titer of antibodies against oxidized LDL was higher in LDLR−/− ob/ob mice suggesting enhancement of oxidative stress . Thus, accelerated atherosclerosis in these animals most likely results from further metabolic derangement rather than from the lack of direct protective effects of leptin. This is supported by significant reduction of lesions with concomitant improvement of the lipid and carbohydrate metabolism following weight loss in these animals . Apolipoprotein E knockout mice are commonly used as an experimental model of hyperlipidemia. Apo-E−/− mice lacking the long isoform of the leptin receptor (apo-E−/− db/db) are characterized by five-fold higher area of spontaneous atherosclerotic lesions in the aorta than apo-E−/− with intact leptin receptor . Hyperlipidemia is more marked in apo-E−/− db/db mice and, unlike apo-E−/− mice, these animals are hyperglycemic, hyperinsulinemic and obese. Taken together, these data suggest that alterations of leptin signaling
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accelerate atherosclerosis if superimposed on specific genetic background. Interestingly, exogenous leptin administered for 4 weeks aggravates spontaneous atherosclerotic lesions in apo-E−/− mice . How can be explained this paradoxical finding that both leptin  and db mutation  have proatherogenic effect in these animals? First, in contrast to db mutation, exogenous leptin has no major effects on the lipid profile in apo-E−/− mice  and direct atherogenic effect of leptin could thus be unmasked. Second, one could speculate that leptin at a physiological level is protective whereas at supraphysiological level is harmful. Finally, leptin administration results in enhanced signaling through all isoforms of its receptor whereas db/db mice have impaired signaling through Ob-Rb but intact or augmented signaling through other isoforms. The possibility that leptin aggravates atherosclerosis in apo-E−/− mice through the short isoforms of its receptor would be consistent with the proatherogenic impact of both db/db genotype and exogenous leptin, however, this requires further studies.
6. Leptin as a risk factor of atherosclerosis in clinical studies Sodeberg et al.  first demonstrated that plasma leptin was higher in patients who subsequently develop firstever myocardial infarction (MI) than in control subjects in a population-based case-controlled study. In addition, leptin predicted myocardial infarction independently of traditional risk factors. This finding was later conformed in a larger study . Leptin is also an independent predictor of MI in men and women with arterial hypertension . Plasma leptin is higher in the offspring with paternal history of premature MI than in those without family history of cardiovascular events . Until now, the most convincing evidence of proatherogenic role of leptin in humans was provided by the West of Scotland Coronary Prevention Study (WOSCOPS), a prospective trial designed to examine the effectiveness of pravastatin in primary prevention of ischemic heart disease in men with moderate hypercholesterolemia. It was demonstrated that elevated plasma leptin predicted acute cardiovascular events (combined acute MI, need for revascularization and mortality) during a 5-year follow-up period in a group of >1000 subjects . When the group was divided into quintiles of baseline plasma leptin, the risk of cardiovascular events was two-fold greater in two upper quintiles than in the lowest quintile. Leptin remained a significant predictor after adjustment for BMI, plasma lipids, glucose and CRP. Wolk et al.  examined the relationship between leptin and coronary events in 382 nondiabetic patients with angiographically confirmed coronary artery stenosis (60% with unstable angina and 40% with stable angina). Baseline plasma leptin was measured and patients were followed-up for a median period of 4 years. High baseline leptin was associated
with increased prevalence of combined end-points (cardiac death, new myocardial infarction, cerebrovascular accident and need for coronary revascularization). More importantly, in multivariate analysis only leptin and a number of coronary vessels with >50% stenosis were independent predictors of cardiovascular events. Finally, Piatti et al.  observed that plasma leptin was higher in patients who subsequently developed restenosis after coronary angioplasty than in those who did not develop restenosis. In contrast, leptin did not predict ischemic heart disease in a 5-year follow-up Quebec Cardiovascular Study . However, this study was relatively small (86 cases and 95 controls) and included different end-points (effort angina, acute coronary insufficiency, nonfatal myocardial infarction and cardiac death). It is possible that leptin is a more important predictor of “hard” end-points such as acute coronary syndromes than of stable angina. In another study performed on a group of 207 women with normal glucose tolerance, impaired glucose tolerance or type 2 diabetes, low plasma leptin predicted cardiovascular mortality during a 7-year follow-up period . Since about 50% of a studied population was glucose-intolerant or diabetic, one might suspect that leptin is protective specifically in diabetic patients. Indeed, predictive value of low leptin level was observed if the analysis was confined to subgroups with abnormal glucose tolerance or overt hyperglycemia. Leptin is an independent predictor of hemorhagic and ischemic cerebral stroke [140,141]. Plasma leptin is also higher in patients with acute stroke events admitted to the hospital than in control subjects , however, this could be secondary to acute phase response since similar surge of leptin was observed following acute myocardial infarction . In 120 normal-weight or obese individuals without diabetes, familial hyperlipidemia, hypertension and clinical manifestations of atherosclerosis, the significant correlation between leptin and intima-media thickness (IMT) of a common carotid artery, a marker of subclinical atherosclerosis, was noted . The similar relationship was observed in children and adolescents with type 1 diabetes . In contrast, no correlation between leptin and IMT was noted among 403 elderly males without ischemic heart disease , in healthy obese women , and in children with obesity or type 1 diabetes . In a recent study , a significant relationship between leptin level and coronary artery calcification scores measured by electron beam tomography was observed in patients with type 2 diabetes without clinical manifestations of atherosclerosis. This relationship was independent of traditional risk factors including lipid profile, blood glucose, family history of atherosclerosis, smoking, albuminuria, BMI, waist circumference and CRP. Thus, the relationship between leptin and markers of preclinical atherosclerosis is still controversial. Reduced vascular compliance (increased arterial stiffness) predicts the development of atherosclerosis  and is observed in obese subjects . Singhal et al.  reported
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that brachial artery distention during systolic pulse wave (a marker of arterial compliance) inversely correlated with plasma leptin in healthy adolescents independently of body weight, WHR, plasma lipids, glucose and blood pressure. Finally, Glader et al.  have shown that plasma leptin is higher in patients with valvular aortic stenosis (which demonstrates some structural similarities to atherosclerotic lesions) than in healthy controls.
7. Conclusions As can be concluded from the data presented above, leptin exerts many potentially proatherogenic effects both in vitro and in vivo. Several clinical studies including some prospective ones suggest that high plasma leptin is associated with the development of atherosclerosis and its complications. However, the question if hyperleptinemia is involved in the pathogenesis of atherosclerosis in obese subjects is still unresolved since all the evidence currently available is only indirect. The findings that ob/ob and db/db mice are protected from atherosclerosis indicate that physiological level of leptin is necessary for its development [125,126]. However, this does not necessarily indicate that supraphysiologically elevated leptin further augments atherogenesis, especially if one considers that obesity induces resistance to some proatherosclerotic effects of leptin [43,68,105]. In addition, arterial injury models used in experimental studies resemble but not exactly reproduce human atherosclerotic lesions. Studies in which exogenous leptin is chronically administered to experimental animals suffer from the limitation of intact leptin signaling, although chronic hyperleptinemia may also per se induce leptin resistance . The association between leptin and atherosclerosis in humans does not necessarily prove the causal relationship since leptin might simply correlate with the other proatherogenic factor not examined in a given study or even still not recognized such as other adipokine(s). Finally, few studies suggesting protective effects of leptin [128,129,141], and possibly atherogenic effect of leptin resistance (Fig. 1) should not be completely neglected. It seems that the most convincing evidence of proatherogenic effect of leptin could be obtained by examining the effect of manipulations which reduce leptin signaling in hyperleptinemic obese animals. However, due to limited availability of leptin antagonist, only few studies used this approach. The only one relevant for cardiovascular pathology  examined the short-term effect of leptin-neutralizing antibodies on arterial thrombosis (but not atherosclerosis) and was performed in nonobese animals. Nothing is known about effect of leptin on many important aspects of atherogenesis such as homocysteine metabolism, endogenous nitric oxide synthase inhibitors, etc. Leptin produced locally in the vasculature  might play an important role, not necessarily parallel to that of circulating hormone. The predictive value of measuring plasma leptin should be
studied in different patient groups, e.g. males versus females, diabetics versus nondiabetics, etc. Nevertheless, this effort is worth doing since it may allow developing novel treatment strategies such as ameliorating selective leptin resistance or inhibiting leptin signaling. Interestingly, leptin may also be targeted by some currently used therapeutic interventions. For example, statins , fibrates  and thiazolidinediones  decrease leptin production. Reduction of plasma leptin was also observed in human subjects consuming a diet rich in fish oil, which contains large amounts of n − 3 polyunsaturated fatty acids .
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