Leukotriene receptors in atherosclerosis

Abstract

Leukotriene‐forming enzymes are expressed within atherosclerotic lesions and locally produced leukotrienes exert pro‐inflammatory actions within the vascular wall by means of cell surface receptors of the BLT and CysLT receptor subtypes. The migration and accumulation of inflammatory cells that follow leukotriene receptor activation have been implicated in atherosclerosis initiation and progression. Leukotriene receptors are in addition expressed on endothelial and vascular smooth muscle cells, associated with intimal hyperplasia in early atherosclerosis and restenotic lesions after angioplasty. Taken together, recent evidence suggests that leukotriene receptors may be a potential target in the treatment of atherosclerosis and in the prevention of restenosis after coronary interventions.

• Atherosclerosis
• angioplasty
• BLT receptors
• CysLT receptors
• eicosanoids
• inflammation
• lipoxygenase

Introduction

Although there is an established notion of inflammatory reactions in atherosclerosis 1, the role of the leukotrienes may have been overlooked for a long time. Leukotrienes are inflammatory mediators derived from the 5‐lipoxygenase pathway of arachidonic acid metabolism 2. Medications targeting the leukotriene pathway have been introduced in the treatment of asthma 3,4, and recent studies have suggested that the leukotriene pathway may represent a common link between asthma and atherosclerosis. A key role in both diseases is played by the enzyme 5‐lipoxygenase (5‐LO) and the 5‐LO‐activating protein (FLAP), which together catalyze the metabolism of endogenous arachidonic acid from membrane phospholipids, leading to formation of the unstable precursor leukotriene (LT) A₄5. Subsequent metabolism of LTA₄ can be continued either in the 5‐LO‐expressing cell or by transcellular metabolism after transfer of LTA₄ to neighboring leukocytes, smooth muscle cells, endothelial cells or platelets 6. Leukotriene synthesis downstream of 5‐LO follows two distinct pathways, either hydrolyzation into leukotriene B₄ (LTB₄) or conjugation with glutathione to form the cysteinyl‐leukotrienes. The latter leukotrienes, i.e. LTC₄, LTD₄ and LTE₄, make up the bioactivity previously referred to as ‘slow reacting substance of anaphylaxis’, SRS‐A 5,6.

The leukotrienes exert their actions via four subclasses of 7‐transmembrane G‐protein‐coupled cell surface receptors, BLT₁ and BLT₂, representing the high and low affinity receptor for LTB₄, respectively, and CysLT₁ and CysLT₂ activated by the cysteinyl‐leukotrienes 7,8.

Leukotriene‐forming enzymes are expressed within human atherosclerotic lesions (Figure 1) and the notion of local leukotriene formation in atherosclerosis has received support from measurements of LTB₄ formation from ex vivo stimulated atherosclerotic lesions 9–11.

Figure 1. Immunoperoxidase‐staining for the leukotriene‐synthesizing enzyme 5‐lipoxygenase (brown) in atherosclerotic lesion from human carotid endarterectomy.

Macrophages represent one major source of 5‐LO 10–12. In human atherosclerotic lesions, 5‐LO expression has been localized to a subset of CD68‐positive macrophages 12. There is in addition a correlation between macrophage content and 5‐LO protein expression in carotid endarterectomies from patients with recent symptoms of cerebral ischemia 10. However, increased LTB₄ formation is also detected in isolated macrophages from patients with recent symptoms compared with those isolated from asymptomatic patients 10, supporting an increased LTB₄‐synthesizing capacity in vulnerable plaques. The notion of a role for the leukotrienes in plaque instability was recently extended to FLAP and LTA₄ hydrolase transcripts, but not to LTC₄ synthase 11. However, increased cysteinyl‐leukotriene concentrations are detected in urine samples from patients with unstable angina and myocardial infarction 13, as well as before and after coronary artery bypass surgery 14. In further support of a role of cysteinyl‐leukotriene‐signaling in atherosclerosis, we recently reported that subjects with high levels of cysteinyl‐leukotrienes in gingival crevicular fluid had an increased carotid artery wall thickness, regardless of their dental status 15. Taken together, these data support the existence of a functional leukotriene‐synthesizing pathway in atherosclerosis that can potentially activate para‐ and autocrine leukotriene receptor signaling.

Genetic targeting of 5‐LO in atherosclerosis prone mouse strains has been reported to reduce lesion size 16,17 and hyperlipidemia‐induced aortic aneurysm formation 18. Studies of genetic and pharmacological targeting of the BLT₁ receptor in atherosclerotic mouse strains have further supported the involvement of leukotriene‐signaling in atherosclerosis, by showing reduced plaque burden particularly in younger animals with smaller, less complex lesions 19–21.

In humans, genetic studies have generated support to the notion that variations in the leukotriene pathway can trigger atherosclerosis. Variants in the promoter of the 5‐lipoxygenase gene are associated with an increased carotid artery wall‐thickening in healthy men and women 22. In addition, a haplotype of the FLAP gene has been identified as a common gene associated with increased risk of stroke and myocardial infarction 23,24. Recently, also genetic variations in the gene encoding LTA₄ hydrolase, the enzyme that converts LTA₄ to LTB₄, were shown to confer a modestly increased risk for myocardial infarction, associated with increased LTB₄ formation 25.

Although these studies have brought leukotrienes to the attention in the context of atherosclerosis, the mechanisms by which the leukotrienes exert their actions in atherogenesis and plaque rupture remain largely unknown. In the present review, we will aim to address the potential mechanisms by which leukotrienes could act as effectors in atherosclerosis, by focusing on the events following leukotriene receptor activation in some of the cell types associated with atherosclerosis (Figure 2).

Figure 2. Leukotriene(LT) receptor expression in some of the target cells for leukotrienes in atherosclerosis.

Key messages

• Expression of leukotriene‐synthesizing enzymes within atherosclerotic lesions leads to auto‐ and paracrine leukotriene signaling.

• Activation of leukotriene receptors on cells associated with atherosclerosis induces enhanced inflammatory responses as well as structural alterations of the vascular wall.

• Targeting leukotriene receptors may represent a putative therapeutic strategy in the treatment of atherosclerosis and prevention of intimal hyperplasia after angioplasty.

BLT receptor signaling in atherosclerosis

LTB₄ is one of the most potent leukocyte chemoattractants produced in the atherosclerotic lesion. Chemotaxis of neutrophils takes place at nanomolar concentrations of LTB₄, whereas the release of lysosomal enzymes is stimulated at micromolar LTB₄ concentrations 26. Binding studies have in addition revealed both high‐affinity and low‐affinity binding sites for LTB₄ in human neutrophils 27. Subsequent studies supported this notion by the cloning of cDNA encoding both high‐ and low‐affinity receptors for LTB₄28–32, which are referred to as BLT₁ and BLT₂ receptors, respectively 7. Cells transfected with the human BLT₁ receptor display a bell‐shaped dose dependency for LTB₄‐induced chemotaxis with an optimum concentration of 1–10 nM, whereas the corresponding concentration of LTB₄ for BLT₂‐mediated chemotaxis is higher 30,32–34. In addition, while cells expressing a single receptor only respond toward a narrow range of LTB₄ concentrations, coexpression of both BLT receptors makes cells migrate towards both very low and high concentrations of LTB₄ (1 nM to 10 µM) 34.

Monocyte recruitment to atherosclerotic lesion can be stimulated by LTB₄ through both chemotaxis 34 and by effects on adhesion molecules 35. BLT₁ receptor antagonism reduces the surface expression of the β2‐integrin CD11b, with a corresponding reduced monocyte infiltration in apolipoprotein E (ApoE) and low‐density lipoprotein receptor (LDLR) knockout mice 19. In addition, LTB₄ can also promote monocyte infiltration by enhancing other pro‐inflammatory pathways. For example, LTB₄ increases the expression of monocyte chemoattractant protein (MCP)‐1 and CD36 20,36, both of which have been implicated in atherosclerosis 37–39. In support of a major role of the LTB₄‐induced MCP‐1 expression it has been shown that a BLT₁ receptor antagonist has no additional beneficial effects on lesion size in ApoE^‐/‐ mice with a targeted MCP‐1 gene 19.

ApoE and BLT₁ receptor double‐knockout mice display a decreased monocyte infiltration 21 and a decreased lesion size in early atherosclerosis. However, the effect of BLT₁ receptor‐targeting is less pronounced in advanced, more complex lesions 20. We have detected expression of both BLT₁ and BLT₂ receptor proteins in macrophage‐rich areas within human atherosclerotic lesions 40. In human monocytes in vitro, BLT₂ receptor mRNA is expressed at higher levels compared with BLT₁ receptor mRNA 34. Interestingly, whereas integrin‐dependent adhesion and MCP‐1 production are mediated through the BLT₁ receptor 20,35,36, macrophages from BLT₁^‐/‐ mice display chemotaxis towards LTB₄ by activation of the BLT₂ receptor 20. Taken together, these findings suggest that monocyte low‐affinity BLT₂ receptors may represent an additional target for LTB₄ in atherosclerosis, and that BLT₂ receptor‐signaling could offer a possible explanation as to the limited effects of either pharmacological or genetic targeting of the BLT₁ receptor on advanced lesions in different mouse models of atherosclerosis 19,20.

Since macrophages represent a major source of LTB₄ production 12, BLT receptor driven monocyte recruitment may act in a vicious circle to further increase the inflammatory activity at sites of atherosclerotic lesions. In the vicinity of 5‐LO‐positive macrophages there is also an accumulation of T cells 12,18. Human T cells express both BLT₁ and BLT₂ receptor mRNA, but BLT₂ receptor expression has been reported to decrease upon stimulation of the cells 34. Still, BLT₁ receptor‐positive T cells are rare populations in healthy individuals 41. However, in vitro priming transiently upregulates BLT₁ receptor expression on human effector T cells, with a maximum at day 8, after which the BLT₁ receptor is downregulated 41. LTB₄ stimulation of T cells induces migration 42 and LTB₄ is involved in early effector T cell recruitment via activation of the BLT₁ receptor 43–45. Human T cells increase the secretion of metalloproteinases (MMP) 2, 3 and 9 in response to LTB₄42. Atherosclerotic lesions in BLT₁ ApoE double‐knockout mice display a significant decrease in T cell accumulation compared with ApoE^‐/‐ controls 21. Taken together, LTB₄‐signaling via the BLT₁ receptor on T cells may provide a potential link between innate and adaptive immunological effector responses in atherosclerosis.

BLT receptors within the vascular wall

Although the BLT receptors mainly have been associated with leukocytes, also vascular cells are activated by LTB₄. Initial studies failed to demonstrate an LTB₄‐induced vasoconstriction in human pulmonary 46 and systemic 47 vessels, as well as aorta from normal and spontaneously hypertensive rats 48. In the latter studies, potential LTB₄‐induced effects may have been masked by the rapid desensitization that has been reported for the BLT₁ receptor after ligand binding 49,50. By performing noncumulative concentration response curves to LTB₄ we have unmasked a vasoconstriction in isolated vessels 51,52, which was recently confirmed by other investigators using the same approach 53. The vasoconstriction induced by LTB₄ is however an indirect response mediated by the release of histamine and thromboxane (TX) A₂51,52. Since local LTB₄ formation has been described in atherosclerotic lesions 9–11, release of pro‐thrombotic TXA₂ by LTB₄ may further aggravate the thrombotic response during plaque rupture 52.

Some of the effects of LTB₄ in isolated vessels have been shown to be endothelium‐dependent 52, whereas in others the vasoconstrictive effects of LTB₄ are preserved even after endothelial denudation 51. Interestingly, in human vessels, endothelial expression of BLT₁ receptors is observed only in atherosclerotic and not in healthy arteries, suggesting an induction during atherogenesis 40. Activation of endothelial BLT receptors may also be involved in the LTB₄‐stimulated leukocyte adhesion 54–56. However, stimulation of endothelial cells with LTB₄ does not enhance monocyte arrest under physiological flow conditions 35, and the exact role of endothelial cells in LTB₄‐induced leukocyte recruitment during atherogenesis remains to be established.

Initial studies in animal models proposed smooth muscle cell activation by LTB₄57. However, the LTB₄‐induced effects observed were mainly a result of cyclooxygenase activation 58. Recently, BLT₁ receptor expression was demonstrated in human vascular smooth muscle cells 40, mediating migration and proliferation in response to LTB₄, with a pharmacological profile consistent with BLT₁ receptor activation (Figure 3). Intriguingly, maximal smooth muscle cell migration in the bell‐shaped concentration‐response curve occurs at LTB₄ concentrations of 100 nM 40, i.e. almost 10–100‐fold higher concentrations compared with BLT₁‐mediated leukocyte migration 32. The migration of vascular smooth muscle cells is a key feature of atherosclerosis 1, and atherosclerotic mouse strains with a targeted BLT₁ receptor have fewer smooth muscle cells in their lesions 21. Since the most pronounced effects of disrupted BLT₁‐signaling is observed in mice with early atherosclerotic lesions 19–21, it could be anticipated that LTB₄ exerts its predominant effects in the initiating stages of atherosclerosis, with smooth muscle cells as primary targets.

Figure 3. Migration of human coronary artery smooth muscle cells through an 8‐mm pore filter in the absence or presence of the BLT receptor antagonists U75302 (1 µM) and ONO 4057 (10 µM). *P<0.05 versus negative control. Reprinted from Bäck et al., Proc Natl Acad Sci USA. 2005;102:17501–6.

Migration and proliferation of smooth muscle cells are also key features of intimal hyperplasia that causes restenosis after, for example, coronary interventions 59. Although the use of stents has reduced the incidence of coronary restenosis, this complication remains a substantial clinical problem 59. Interestingly, angioplasty is a stimulus for intracoronary formation of lipoxygenase products 60. We have shown that treatment with a BLT receptor antagonist prevented intimal hyperplasia in a rat model of vascular injury 40. Taken together, targeting leukotriene‐signaling in vascular smooth muscle cells may prevent coronary reocclusion in patients undergoing angioplasty 40. BLT receptor antagonists could represent either an alternative or a complement to the present use of stents coated with rapamycin derivates 59. In this context is it interesting to note that oral treatment with one rapamycin derivate, tacrolimus, decreases leukotriene formation in asthmatics 61.

Transcriptional regulation of BLT receptor expression

Unexpectedly, BLT₁ receptor expression is downregulated by pro‐inflammatory stimuli in human leukocytes, whereas anti‐inflammatory mediators, such as dexamethasone and interleukin (IL) 10, upregulate BLT₁ receptor expression in these cells 62,63. Findings with the all‐trans retinoic acid further support an anti‐inflammatory stimulation of BLT₁ receptor transcription. First, the cloning of the BLT₁ receptor was achieved in retinoic acid‐differentiated human leukemia HL‐60 cells 32. Second, all‐trans retinoic acid upregulates BLT₁ receptor mRNA expression as well as the indirectly mediated vasoconstriction to LTB₄ in isolated aortic preparations 52. The transcriptional regulation of the BLT₁ receptor may, however, be cell‐specific, since the promoter activity differs in cell lines derived from hematopoietic and nonhematopoietic origin 64. Furthermore, while BLT₁ receptor expression is downregulated by lipopolysaccharide (LPS) and interferon (IFN) γ in monocytic cells 62, the effect of these pro‐inflammatory mediators is the opposite in endothelial 65 and vascular smooth muscle cells 21,40. The upregulation of BLT₁ receptors by LPS and IL‐1β in vascular smooth muscle cells is mediated via activation of nuclear factor κB (NF‐κB) 40. In addition, the BLT₁ receptor induction by vascular injury can be prevented by disruption of the NF‐κB pathway 40. These results hence support a pro‐inflammatory enhancement of BLT₁ receptor‐signaling within the vascular wall.

Nuclear receptor for LTB₄

In addition to the cell surface BLT₁ and BLT₂ receptors, LTB₄ has also been reported to be an endogenous activating ligand for the nuclear peroxisome proliferator‐activated receptor (PPAR) α 66. PPARα regulates gene expression of enzymes associated with lipid homeostasis in atherosclerosis and vascular inflammation 67. LTB₄‐induced activation of PPARα leads to oxidative degradation of the ligand, suggesting a possible feedback mechanism that controls the duration of the LTB₄‐induced inflammatory response 66. Interestingly, clinical trials using synthetic PPARα ligands, such as fibric acid, have supported beneficial effects on cardiovascular disease outcome 67. PPARα activation by LTB₄ may hence have beneficial effects in atherosclerosis, and it could be anticipated that cell surface LTB₄ receptors represent a more specific anti‐inflammatory target compared with 5‐lipoxygenase inhibition.

Effects on ion channels

In neurons, LTB₄ has been reported to directly activate ligand‐gated ion channels, such as the ryanodine receptor and vanilloid receptor 1 (VR1) 68,69. Ion channel activation after LTB₄ stimulation has also been detected in human coronary artery smooth muscle cells, by measurements of whole cell currents using patch‐clamp 40. However, LTB₄ does not affect membrane currents in isolated membrane patches from those cells, excluding direct effects of LTB₄ on ion channels in vascular smooth muscle cells 40.

CysLT‐signaling in atherosclerosis

The cysteinyl‐leukotrienes activate receptors denoted CysLT₁ and CysLT₂ receptors, which are sensitive and resistant, respectively, to a number of antagonists that have been developed to inhibit leukotriene‐induced bronchoconstriction 4,7. Antagonists of the CysLT₁ receptor have beneficial effects in the treatment of asthma, and zafirlukast (Accolate^™), montelukast (Singulair^™) and pranlukast (Onon^™) are clinically used for this purpose 3,4.

The human CysLT₁ receptor is expressed in B lymphocytes, eosinophils, and monocytes, but not in T cells or neutrophils 70,71. Human mast cells, macrophages and peripheral blood monocytes express both CysLT₁ and CysLT₂ receptors 70,72–74. The calcium‐signaling induced by LTD₄ in macrophages is, however, blocked by montelukast, suggesting a CysLT₁ receptor‐dominant signaling in these cells 74. In addition, priming of either human peripheral blood monocytes or monocyte‐derived macrophages with IL‐4 or IL‐13 increases their CysLT₁ receptor expression leading to an enhanced migration in response to LTD₄75. Furthermore, LTD₄ induces expression of macrophage inflammatory protein (MIP) 1α and MCP‐1 in human monocytic cell lines 18,76.

Although T cells may not express CysLT receptors, adaptive immunological reactions could potentially be modulated by cysteinyl‐leukotriene induced activation of antigen‐presenting cells. In a model of asthma, myeloid dendritic cells were shown to express CysLT₁ receptors as well as cysteinyl‐leukotriene‐synthesizing enzymes 77. LTD₄ stimulation increased production of the immunomodulatory cytokine IL‐10, which was inhibited by treatment with CysLT₁ receptor antagonists 77. Since experimental atherosclerosis in mice is reduced by overexpression of IL‐10 78 and increased by genetic IL‐10 targeting 79, LTD₄‐induced IL‐10 production could have a potential beneficial effect in atherosclerosis. Likewise, IL‐10 inhibits intimal hyperplasia after angioplasty in rabbits 80. Although the role of CysLT‐receptor‐signaling in antigen‐presenting cells has not been investigated in the context of atherosclerosis, cells positive for the dendritic cell marker CD1a express 5‐LO in human atherosclerotic lesions 12. Taken together, these findings suggest that CysLT‐signaling may represent one possible regulator of immunomodulatory functions in atherosclerosis.

CysLT receptor‐induced vascular effects

The first evidence of contractile effects of cysteinyl‐leukotrienes on isolated human vessels was provided by studies of the pulmonary vasculature, which reported a preferential vasoconstriction to cysteinyl‐leukotrienes in human pulmonary veins compared with pulmonary arteries 46,81–86. Likewise, studies of systemic vessels have detected significant contractions in human saphenous veins, whereas arterial preparations such as femoral, gastroepiploic and internal mammary arteries in general are unresponsive to cysteinyl‐leukotrienes 47,87.

In contrast to the airways, contractions of isolated human vascular preparations were initially shown to be resistant to the CysLT₁ receptor antagonists developed for treatment of asthma, but inhibited by the dual CysLT₁/CysLT₂ receptor antagonist BAY u9773 82. However, limited effects of the latter antagonist in some vascular preparations have raised arguments for further CysLT receptor subtypes in the vasculature 8,84,86,88–91.

Despite observations that CysLT₂ receptor mRNA expression can be detected in coronary artery smooth muscle cells and that LTC₄ induces calcium mobilization in vitro92, also this vascular segment is unresponsive to leukotrienes 93,94. In contrast, atherosclerotic coronary arteries contract in response to cysteinyl‐leukotrienes 93,94, suggesting that changes of the vascular reactivity to cysteinyl‐leukotrienes may be induced in atherosclerosis. The reason for this change of reactivity is not completely examined, but it has been noted that the number of leukotriene binding sites are increased in atherosclerotic vessels 93. Interestingly, ApoE^‐/‐ mice display an increased CysLT receptor expression in the aorta compared with nonatherosclerotic mice 11,18.

Stimulation of human endothelial cells with cysteinyl‐leukotrienes leads to an increase in intracellular calcium 74, the release of vasoactive factors 83–85,95 and induction of gene expression 18,96. In cultured human umbilical vein endothelial cells (HUVECs) and coronary artery endothelial cells, the predominantly expressed CysLT receptor is of the CysLT₂ subtype 74,92.

Studies of isolated vessels have suggested that cysteinyl‐leukotriene stimulation is mainly linked to nitric oxide in venous endothelial cells, whereas in arteries, cyclooxygenase metabolites may dominate 83–85,88. For example, LTC₄ and LTD₄ induce an endothelium‐dependent release of prostacyclin that almost completely counteracts their vasoconstrictive effects in the human pulmonary artery 84,86. By overexpression of an endothelial cell‐specific human CysLT₂ receptor in a transgenic mouse model, it has been shown that the release of nitric oxide (NO) in response to LTC₄ is linked to increased vascular permeability and diminished systemic pressor responses 97. However, CysLT receptor stimulation in endothelial cells may also liberate contractile factors 83,88.

CysLT receptor‐signaling in endothelial cells may also play a role in leukocyte recruitment through expression of chemokines, such as MIP‐2 18,96. Furthermore, incubation of cultured HUVECs with either LTC₄ or LTD₄ rapidly induces surface expression of the adhesion molecule P‐selectin, and a concentration‐dependent increase in adherence of anti‐P‐selectin labeled beads 98,99. Also the effects on gene expression are resistant to the class of CysLT₁ receptor antagonists developed for asthma treatment 18,96,99, consistent with a major role for the CysLT₂ receptor in endothelial cells.

Conclusion

Recent studies have provided evidence for a genetic link between the 5‐LO pathway and atherosclerosis 22–25, and local leukotriene formation as well as expression of the specific leukotriene receptors have been detected in atherosclerotic lesions 9,11,12,40. Release of both LTB₄ and cysteinyl‐leukotrienes promotes recruitment of inflammatory cells and reinforces the inflammatory response. In addition, leukotriene receptors on vascular smooth muscle and endothelial cells induce alterations of structure and function within the vascular wall that are associated with atherosclerosis 40.

The present clinical use of leukotriene modifiers in the treatment of asthma and allergy opens up for a rapid transfer of knowledge from bench to bedside in the context of atherosclerosis. Recently, the effect of leukotriene inhibition on biomarkers associated with myocardial infarction was examined in a randomized, prospective, placebo‐controlled, crossover trial 100. All participating subjects were carriers of the variants of the FLAP and/or LTA₄ hydrolase gene that the investigators previously had associated with an increased risk for myocardial infarction 23,25. Reduced serum levels of C‐reactive protein, serum amyloid A, and myeloperoxidase were reported from that short‐term study (2 weeks) 100. However, only modest effects on LTB₄ formation by stimulated leukocytes could be detected in treated patients 100. In addition, cysteinyl‐leukotriene formation, measured as urinary LTE₄, was unexpectedly increased after treatment, hence raising a concern of the specificity of the observed effects. The reason for these unexpected results from this, today, only available clinical study of leukotriene inhibition in atherosclerosis is unclear at present. It is possible that a longer duration of treatment is needed not only to obtain complete inhibition of leukotriene formation, but also to observe effects of abrogated leukotriene signaling. Mechanistic studies have in addition suggested that LTB₄ may have its predominant effects early during atherogenesis 20 and in smooth muscle cell recruitment 40, suggesting that subjects with established atherosclerosis may be less prone to responding to an antileukotriene therapy.

The local formation of leukotrienes within the atherosclerotic lesion and the potent pro‐inflammatory effects of leukotriene receptor activation in target cells of atherosclerosis provide a rationale for a role of leukotrienes in this disease. Further experimental and clinical studies are needed to develop therapeutic strategies of treatments targeting leukotriene‐signaling in atherosclerosis.