Role of leptin in atherogenesis

Abstract

The pathogenesis of atherosclerosis involves multiple cellular events, including endothelial cell dysfunction, inflammation, proliferation of vascular smooth muscle cells and matrix alteration that is subsequently characterized by hardening, thickening, loss of elasticity and, finally, a reduction in the vessel’s lumen. Leptin, a peptide hormone, is produced by adipocytes, and the majority of obese individuals have high plasma leptin concentrations. Leptin regulates food intake as well as metabolic function. Originally thought to be a satiety factor, leptin is a pleiotropic molecule. In addition to its metabolic effects, leptin regulates the production of several pro- and anti-inflammatory cytokines by activating immune cells. It is associated with increased plasma C-reactive protein concentrations, vascular proliferation, calcification and decreased arterial distensibility. Leptin also increases oxidative stress. Moreover, leptin contributes to increases in blood pressure, and thus, probably plays an important role in the initiation and progression of atherosclerosis. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) lower lipid concentrations and also decrease endothelial apoptosis, inhibit smooth muscle cell proliferation, and lower concentrations of C-reactive protein and proinflammatory cytokines; moreover, it is now known that statins can inhibit leptin release by adipocytes. Therefore, statins have been shown to be beneficial in atherosclerosis. The present review mainly focuses on the various evidence that suggest a potential atherogenic mechanism of leptin, and also briefly addresses the beneficial role of statins in atherosclerosis.

Atherosclerosis is a chronic disease of the vessel wall that affects various vascular beds (1). The response-to-injury hypothesis is the most conventional hypothesis for the pathophysiology of atherosclerosis; that is, atherosclerotic lesion formation is generally initiated by endothelial cell damage, and endothelial cell dysfunction is characterized by concomitant disturbance of the vascular wall permeability that follows inflammatory cell infiltration, accumulation of lipids, smooth muscle cell migration and proliferation, production of extracellular matrix and neovascularization.

Leptin (from the Greek ‘leptos’, meaning thin) is a 16 kDa peptide hormone that is encoded by the Ob gene and plays a central role in the regulation of body weight. Leptin regulates feeding and stimulates thermogenesis by acting on the hypothalamus (2). The concentration of leptin is directly proportional to total body fat (3), and thus, obese humans have higher leptin concentrations than nonobese humans. However, not all obese persons have increased concentrations of leptin; in fact, leptin deficiency and resistance to the effects of leptin are both associated with weight gain. Leptin resistance, which leads to hyperleptinemia, is much more common than leptin deficiency in human obesity (4). The three-dimensional structure of leptin shows a composition of four alpha-helices and two short beta-strands, and therefore, leptin has a similar structure to the interleukin (IL)-6 family of cytokines (5). Leptin receptor (ObR) is encoded by the db gene and is a member of the class I cytokine receptor superfamily. The functional long form of ObR (ObRb) has a long cytoplasmic tail, and is mainly expressed in the hypothalamus, and in endothelial and immune system cells (6–8). ObRb is considered to be of major importance for leptin signalling, with full signalling capacity being achieved via activation of the mitogen-activated protein (MAP) kinase and Janus kinase/signal transducer and activator of transcription signalling pathways (9,10). The short form of ObR (ObRa) lacks most of the cytoplasmic domain of the receptor. The functional capacity of ObRa is not fully established, although it may have signalling capabilities through MAP kinase, but not through signal transducer and activator of transcription 3 (9). ObRa is expressed ubiquitously.

Obesity is an important determinant of atherosclerosis, but the mechanism behind it is only partially understood. Obese persons have high concentrations of circulating leptin, and there is widespread peripheral distribution of ObRs in, for example, immune cells, vascular smooth muscle cells and endothelial cells, as well as in atherosclerotic plaques. Therefore, it is now thought that leptin may be one mechanism by which body fatness is linked to cardiovascular disease (CVD). Obesity is one component of the metabolic syndrome, which is characterized by overall and central obesity, elevated blood pressure, hyperinsulinemia, reduced high density lipoprotein cholesterol and hypertriglyceridemia (11), and each component of the metabolic syndrome may contribute to an increased risk of CVD. Hyperleptinemia is associated with insulin resistance and has been suggested to play a central role in the metabolic syndrome (12). It has been reported that plasma leptin concentrations correlate with body mass index, and are three to four times higher in cases of obesity and diabetes (13,14), both of which are major risk factors for atherosclerosis. Recently, Bodary et al (15) showed that direct administration of leptin in apolipoprotein E-deficient mice results in increased atherosclerosis. The prospective West of Scotland Coronary Prevention Study (WOSCOPS) (16) also showed that leptin moderately but independently increases the relative risk of coronary artery disease. Leptin’s role in atherosclerosis is supported by findings in ob/ob mice that lacked a functioning leptin gene and were resistant to atherosclerosis despite being grossly obese and diabetic; moreover, leptin administration in these mice caused atherosclerotic changes (17). Leptin contributes to the pathogenesis of atherosclerosis by several mechanisms. Human leptin has been shown to enhance cytokine production in murine peritoneal macrophages and human circulating monocytes (18), and in a concentration-dependent manner, to enhance the proliferation and activation of human circulating T lymphocytes, as well as stimulating the production of inflammatory cytokines (19). Leptin has angiogenic activity (20), causes increased oxidative stress in endothelial cells (21), promotes vascular smooth muscle cell migration and proliferation (22), decreases arterial distensibility (23) and contributes to obesity-associated hypertension. All of these effects are inversely related to vascular health and are strongly involved in the pathophysiology of atherosclerosis. It has also been reported that leptin is independently associated with serum C-reactive protein (CRP) concentration, which is not only a potential inflammatory marker but also a direct cause of CVD. These experimental results strongly suggest that leptin may contribute to the pathophysiology of atherogenesis by promoting vascular inflammation, stiffness, calcification and proliferation by increasing oxidative stress and having effects on blood pressure (Figure 1).

Figure 1.

Atherogenic mechanisms of leptin. CRP C-reactive protein; BP Blood pressure; SMC Smooth muscle cell

ATHEROGENIC MECHANISMS OF LEPTIN

Leptin and immune cells

Immune mechanisms play a pivotal role in atherogenesis. Atherosclerotic lesions are filled with immune cells that can coordinate and effect inflammatory responses. The common components of atherosclerotic lesions are macrophages and T lymphocytes, and may also contain mast cells and dendritic cells (DCs) (24). Macrophages are seen in all phases of atherosclerosis (25), whereas both types of T helper (Th) cells (CD4⁺ and CD8⁺) have been detected in human atheromas and have been shown to be immunologically activated (26). Different cytokines are known to play roles in the immunological cascade that leads up to atherosclerosis. IL-2, IL-6, IL-12 and IL-18, as well as tumour necrosis factor-alpha (TNF-α) and interferon-gamma, have been shown to be potentially atherogenic. On the other hand, cytokines such as IL-10 and IL-4 have been shown to play a protective role against developing cardiovascular events (Table 1) (27–45). Many studies have shown that leptin can stimulate various immune cells and regulate the production of several pro- and anti-inflammatory cytokines (Figure 2). In turn, these proinflammatory cytokines can increase systemic leptin concentrations in vivo (46). Human leptin enhances cytokine production by murine peritoneal macrophages and also, in a concentration-dependent manner, stimulates the proliferation and activation of human circulating monocytes in vitro by inducing the production of cytokines, such as TNF-α, IL-6, IL-12 and IL-18, when these cells are treated with lipopolysaccharide (18,47,48). In addition, it has been shown that macrophages from leptin-deficient mice have impaired phagocytic functions because they cannot clear and kill circulating Escherichia coli as efficiently as normal mice (47). Therefore, leptin enhances phagocytosis and pro-inflammatory cytokine production in cultured macrophages treated with lipopolysaccharide. It has been shown that when coadministered with other nonspecific immunostimulants, leptin causes the induction of early (CD69) and late (CD25 and CD71) activation markers in both CD4⁺ and CD8⁺ lymphocytes, and enhances, in a concentration-dependent manner, the proliferation and activation of T lymphocytes in human peripheral blood (19). Some researchers have shown that leptin polarizes Th cells toward a Th1 phenotype, and stimulates the production of the proinflammatory cytokines IL-2 and interferon-gamma, whereas it suppresses production of the anti-inflammatory cytokines IL-4 and IL-10 in Th2 cells, in both human and animal models (6,19). Recently, it was reported that mature and immature human DCs express ObRb, and leptin can promote their maturation and make them more competent antigen-presenting cells (APCs) (49). This study also supports leptin’s role in immune systems because DCs are professional APCs that have a unique ability to activate naïve T cells and stimulate the production of inflammatory cytokines from T cells. It has also been reported that leptin upregulates the proinflammatory cytokines IL-6, IL-12 and TNF-α, and downregulates the anti-inflammatory cytokine IL-10 in both mature and immature human DCs (49). These results indicate that leptin is involved in the regulation of immune function and cytokine secretion, by which leptin could promote endothelial dysfunction and atherogenesis.

TABLE 1.

Actions of cytokines in atherosclerosis

Cytokines	Actions	References
IL-2	Atherogenic: Promotes differentiation and proliferation of Th1 cells, and SMC proliferation	27
IL-4	Atherogenic: Upregulates P-selectin, 15-lipoxygenase and VCAM-1	27,28–30
	Antiatherogenic: Inhibits SMC proliferation, LDL oxygenation and macrophage adhesiveness	31,32
IL-6	Atherogenic: Increases SMC proliferation, CRP production	33
IL-10	Antiatherogenic: Anti-inflammatory. Inhibits inducible nitric oxide synthase. Reduces cell death	34–36
IL-12	Atherogenic: Increases Th1 cell function and decreases Th2 cell function. LDL oxidation. Acts in synergy with IL-18 for the production of IFN-γ by T cells. Augments the cytotoxic activity of natural killer cells	37–39
IL-18	Atherogenic: Induces IFN-γ production	40
IFN-γ	Atherogenic: Activates macrophages. Regulates SMC production. Increases expression of VCAM-1 in endothelial cells	41,42
TNF-α	Atherogenic: Increases permeability of endothelial cells. Promotes monocyte adhesion. Induces macrophage differentiation and promotes foam cell formation	43–45

CRP C-reactive protein; IFN-γ Interferon-gamma; IL Interleukin; LDL Low density lipoprotein; SMC Smooth muscle cell; Th T helper; TNF-α Tumour necrosis factor-alpha; VCAM Vascular cell adhesion molecule

Figure 2.

Regulation of cytokines by different immune cells stimulated by leptin. Leptin induces functional and morphological changes in dendritic cells. Leptin upregulates interleukin (IL)-6, IL-12 and tumour necrosis factor-alpha (TNF-α), and downregulates IL-10. Dendritic cell- and macrophage-produced leptin, IL-12 and IL-18 result in T helper (Th) cell differentiation into the Th1 phenotype, as well as stimulation of Th1 cells and the production of cytokines IL-2 and interferon-gamma (IFN-γ), whereas they result in the suppression of the production of cytokines IL-4 and IL-10 in Th2 cells. In turn, by producing IFN-γ and leptin, Th1 cells activate macrophages and stimulate the production of cytokines TNF-α, IL-6, IL-12 and IL-18

Major cytokines stimulated by leptin and their effects in atherosclerosis

As discussed above, leptin enhances the secretion of different proinflammatory cytokines and suppresses anti-inflammatory cytokine production in both human and animal models. These cytokines are known to play a role in the immunological cascade that leads to atherosclerosis. Their effects in the process of atherogenesis are shown in Table 1.

Leptin and CRP

CRP is synthesized by the liver and regulated by cytokines, especially IL-6 (50). CRP has been indicated to be an independent risk factor for a variety of CVDs. It has been shown that CRP may be a causative agent for the progression of atherosclerosis. However, there is controversy about the direct effect of CRP on atherogenesis. Two independent reports have shown that transgenic overexpression of CRP does not affect the development of atherosclerosis in mice (51,52). In contrast, Paul et al (53) have shown that CRP has a proatherogenic role and accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice. More recently, Li and Fang (54) reported that CRP is not only an inflammatory marker, but also a direct cause of CVD, and administration of agents that reduce CRP concentrations may prevent CVD. CRP activates the classic complement cascade, mediates phagocytosis and regulates inflammation. A number of epidemiological studies have shown that even a small rise in serum CRP concentration is associated with a high risk for atherosclerosis (55,56). CRP contributes to vascular disease by directly impairing endothelium-dependent vasodilation by inhibiting nitric oxide synthase (NOS) expression and bioactivity in human coronary artery endothelial cells (57), increasing cell adhesion molecule expression (58) and activating vascular smooth muscle cells (59). CRP plays a crucial role in the chemotaxis of monocytes and foam cell formation in atherosclerotic plaques (60). Both leptin and CRP are increased in women, and in the setting of obesity and inflammation. However, it has been shown that in healthy humans, increased leptin is associated with increased CRP (independently of sex), measures of adiposity and other variables (61). The only source of leptin is adipocytes, and adipocytes are also an important source of circulating IL-6 and TNF-α (62,63). Proinflammatory cytokines, such as IL-1, IL-6 and TNF-α, contribute to the hepatic synthesis of CRP. Thus, adipocytes not only secrete leptin, but also contribute to CRP synthesis. Leptin also stimulates IL-6 and TNF-α production by different immune cells. In addition, ObR has been shown to have signalling capabilities similar to IL-6-type cytokine receptors. Therefore, it is likely that leptin may act via IL-6, or perhaps even via ObR, to upregulate CRP production.

Leptin and oxidative stress in endothelial cells

Studies have shown that the generation of oxidative stress, characterized by enhanced reactive oxygen species (ROS) formation, plays a regulatory role in atherosclerotic events. Leptin increases oxidative stress through multiple mechanisms. Human umbilical vein endothelial cells (HUVECs) express functional receptors to leptin. In HUVECs, leptin increases the generation and accumulation of ROS by activating C-jun-amino-terminal kinase, AP-1 and nuclear factor-kappa B pathways (21). In bovine aortic endothelial cells, leptin causes concentration-dependent increases in the formation of ROS (64). In turn, ROS enhances the expression of adhesion molecules, such as vascular cell adhesion molecule-1 (65), intercellular adhesion molecule-1 (66) and monocyte chemoattractant molecule-1 (MCP-1) (67). MCP-1 selectively promotes the chemotaxis of monocytes. Genetically modified mice lacking MCP-1 or its receptors (chemokine [C-C motif] receptor 2) have delayed and attenuated atheroma formation when crossed with an atherosclerosis-prone hyperlipidemic genetic background (68,69). Oxidative stress may also operate as an indirect factor to increase serum atherogenic factors. By increasing oxidative stress and activating protein kinase C, leptin increases the secretion of atherogenic lipoprotein lipase (LPL) from macrophages in vitro (70). LPL is a key enzyme in lipid metabolism. Contrary to the antiatherogenic effect of plasma LPL, the LPL secreted by macrophages in the arterial wall is proatherogenic (71,72). LPL promotes the retention of lipoproteins in the subendothelial space and favours monocyte adhesion to the endothelium, and stimulate the transformation of macrophages into foam cells.

Leptin and the vessel wall

While the early events in atheroma formation primarily involve altered endothelial function and the recruitment and accumulation of leukocytes, the subsequent evolution of an atheroma into a more complex plaque involves migration of smooth muscle cells from the arterial media into the injured intima, proliferation of smooth muscle cells within the intima and secretion of large amounts of connective tissue by smooth muscle cells. Several growth factors, such as platelet-derived growth factor, insulin-like growth factor-1 and nerve growth factor are involved in vascular smooth muscle cell proliferation and thickening (73,74), and induce vascular smooth muscle cell migration via phosphatidylinositol 3-kinase activation (75). The presence of leptin and ObR in the arterial wall suggest that the physiological or pathological functions of arterial wall cells may be targets of leptin action. ObRa is expressed in vascular smooth muscle cells and, in experimental animal models, leptin promotes vascular smooth muscle cell proliferation and migration by stimulating MAP kinases and phosphatidylinositol 3-kinase activation (22). It has been reported that leptin-treated rat vascular smooth muscle cells increased in number in a concentration-dependent manner. Moreover, leptin induced a concentration-dependent proliferation of HUVECs and elevated concentrations of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (76). MMPs, a family of zinc-dependent proteinases, have been identified in human atherosclerotic lesions (77), and play an important role in the progression of atherosclerosis and plaque rupture by degradation of the extracellular matrix. MMP activity is tightly controlled by tissue inhibitors of metalloproteinases. In experimental animal models, MMPs have also been shown to be important for smooth muscle cell migration from the media to intima, and it has been shown that the administration of MMP inhibitors almost completely inhibits the number of smooth muscle cells migrating to the intima (78). These findings suggest that leptin plays a role in vascular smooth muscle cell proliferation and matrix remodelling.

In addition to smooth muscle cells, endothelial cells also migrate and replicate as plaques develop a microcirculatory network through angiogenesis, characterized by plexuses of newly formed vessels (79). Administration of inhibitors of angiogenesis to mice with experimentally induced atheromas limit lesion expansion (80). Angiogenesis starts by cell-mediated degradation of the basement membrane, followed by migration and proliferation of endothelial cells. In vascular tissue, endothelial cell proliferation and promotion of angiogenesis by leptin have been reported. It has been shown that leptin enhances the formation of capillary-like tubes in vitro and neovascularization in vivo (17,81). Leptin, via activation of endothelial ObR, generates a growth signal, involving a tyrosine kinase-dependent intracellular pathway, and promotes angiogenic processes (81,82).

Immunohistochemical analysis of human atherosclerotic plaques has shown increased expression of ObRs in the neovascularized neointima (81,82). Furthermore, human plasma leptin concentrations are independently associated with the intima-media thickness of the common carotid artery (83) and have also been shown to accelerate vascular calcification in experimental models, both of which are the hallmarks of atherosclerosis (84). It has been reported that there is a strong inverse association between arterial distension and leptin concentration. An elevation of leptin concentrations in adolescents, independent of fat mass and metabolic and inflammatory markers, is associated with decreased arterial distensibility (23), which is known to correlate closely with atherosclerotic risk factors, extent of disease and cardiovascular risk.

Leptin and blood pressure

Hypertension is one of the major risk factors for atherosclerosis. Although the association of obesity and hypertension is well established, information about the underlying mechanism is limited. Leptin acting on the hypothalamus reduces food intake and stimulates thermogenesis (2). The primary mechanism of this thermogenesis is the activation of the sympathetic nervous system by leptin. It has been reported that chronic leptin infusion in rats raised mean arterial pressure and heart rate, despite a marked reduction in food intake (85). Chronic intravenous or intracerebroventricular infusion of leptin increases blood pressure in normotensive rats by increasing sympathetic activity in the kidneys, and in adrenal and brown adipose tissue (86,87). The sympathetic nervous system plays a major role in mediating obesity-associated hypertension (88), but obese mice that are deficient in leptin are not hypertensive and have slightly lower blood pressure than lean mice (89). These observations show that hyperleptinemia may increase sympathetic activity and contribute to obesity hypertension. However, acute leptin administration has little or no effect on arterial pressure despite an increase in sympathetic activity (90). This is because acute leptin administration stimulates endothelial NO production by activating protein kinase B/Akt, which phosphorylates endothelial NOS and increases its activity even at low calcium concentrations (91–93), and the pressor effect of sympathetic activation is counterbalanced by the depressor effect of endothelial-derived NO (94). However, the acute effect of leptin is quite different from the long-term elevation of this hormone. We already mentioned that leptin increases oxidative stress in endothelial cells, and long-term outcomes of oxidative stress include reductions in NO bioactivity and/or synthesis, and thus, the vasodilatory effect of leptin is impaired, leading to unbalanced vasoconstriction and blood pressure elevation. There are a few more observations that help to explain the apparent effect of leptin on blood pressure. Leptin has been shown to stimulate the secretion of endothelin by endothelial cells in vitro, and several studies have shown that the serum concentrations of endothelin-1 are increased in patients with hypertension. Moreover, it has also been shown that the function of the reninangiotensin system may be associated with leptin secretion, and there is a significant positive correlation between hyperleptinemia and plasma renin activity in essential hypertension (95). Therefore, leptin seems to play an important role in the regulation of blood pressure by influencing the activity of the sympathetic nervous system, endothelial function and the reninangiotensin system.

THERAPEUTIC IMPLICATIONS

Studies have shown that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) reduce plasma cholesterol concentrations and improve survival in patients with coronary artery disease. However, several studies have indicated that statins appear to have beneficial effects that are independent of their cholesterol-lowering properties. Recent studies have focused on mechanisms by which statins exert cardioprotective effects beyond cholesterol reduction. Such effects are believed to include anti-inflammatory actions, the ability to reverse endothelial dysfunction by prevention of oxidization of low density lipoprotein and increases in NO bioavailability (96,97). Statins cause a concentration-dependent decrease in smooth muscle cell migration and proliferation, and alter the assembling of atherosclerotic plaques. Studies have shown that statin therapy exhibits excellent inhibition of circulating proinflammatory cytokines (98). Furthermore, statins have been shown to lower CRP concentrations independently of cholesterol concentrations (99,100). Recently, it was reported that leptin promotes the survival and maturation of human DCs, and makes them more competent APCs. However, Yilmaz et al (101) have shown that statins inhibit the maturation and antigen-presenting function of human myeloid DCs, and also reduce the ability of DCs to induce T cell proliferation; thus, this may contribute to their beneficial effects in atherosclerosis. Evidence has suggested that leptin is responsible for the initiation and progression of atherosclerosis. In addition, ob/ob mice, which lack a functioning leptin gene, are resistant to atherosclerosis, despite being grossly obese and diabetic. With increasing evidence of a significant role for leptin in atherogenesis, leptin has become a potential therapeutic target for the treatment of atherosclerosis, and the development of pharmacological leptin antagonists is a very attractive idea and may have implications for the treatment of atherosclerotic disease. Several studies have reported that statins may directly lower serum leptin concentrations. It has been shown that statins can significantly reduce plasma concentrations of leptin in patients with type II diabetes (102), as well as in patients with accelerated atherosclerosis (103). Recently, Zhao and Wu (104) showed that atorvastatin causes concentration-dependent reductions in serum leptin concentrations in rabbits. In that study, it was reported that atorvastatin treatment in hypercholesterolemic rabbits significantly inhibits leptin release and leptin messenger RNA expression in adipocytes compared with the control group. These findings suggest that the inhibition of leptin secretion from adipocytes may be one of the pleiotropic effects of statins by which they can inhibit the atherogenic mechanism and contribute to lowering the morbidity and mortality of CVD.

CONCLUSIONS

The key mechanism in the pathology of atherosclerosis is the alteration of vascular health characterized by damage to the endothelial cells, as well as hardening, thickening, loss of elasticity and, finally, a reduction in the vessel’s lumen. Leptin is produced by adipocytes, and circulating concentrations of leptin reflect the fat stores of the body. Therefore, established obesity is associated with leptin resistance and clear hyperleptinemia. There is a widespread distribution of ObRs in, for example, immune cells, vascular smooth muscle cells and endothelial cells, as well as in atherosclerotic plaques. Leptin affects the function of each of these cell types. Leptin promotes vascular inflammation by activating immune cells, increasing CRP and increasing oxidative stress, and also directly alters vascular health by promoting smooth muscle cell proliferation and migration by causing wall calcification and reducing distensibility. Moreover, by increasing blood pressure, leptin may be indirectly involved in the pathogenesis of atherosclerosis. Thus, leptin mainly exerts adverse effects on vascular health by activating immune cells or by acting on the vascular wall, which may contribute to the pathogenesis of atherosclerosis. Therefore, leptin may be a potential target for the treatment of atherosclerosis. Statins are well-known lipid-lowering agents. However, data indicate that the beneficial role of this drug class does not only depend on its lipid-lowering effect; the so-called pleiotropic effects of statins include anti-inflammatory effects, prevention of oxidization of low density lipoprotein, decreased endothelial apoptosis, inhibition of vascular smooth muscle cell proliferation, lowered CRP concentrations and lowered proinflammatory cytokine concentrations. Thus, these pleiotropic effects have a major role in the prevention and inhibition of atherosclerosis. Furthermore, statins exert a direct inhibitory effect on leptin secretion from adipocytes, reducing plasma leptin concentrations, which plays an important role in atherogenesis. This may be one of the most important pleiotropic effects of statins that is responsible for the inhibition of the initiation and progression of atherosclerosis. In the future, more approaches aimed at reducing plasma leptin concentrations or blocking leptin’s peripheral action on immune cells or on the vascular wall would be useful for the prevention or inhibition of atherosclerotic vascular lesions.