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Purinergic signaling in atherosclerosis Davide Ferrari1, Laura Vitiello2, Marco Idzko3, and Andrea la Sala2 1
Department of Life Sciences and Biotechnology, Biotechnology Centre, University of Ferrara, 44121 Ferrara, Italy Laboratory of Molecular and Cellular Immunology, Istituto Di Ricovero e Cura a Carattere Scientifico (IRCCS), San Raffaele Pisana, 00166 Rome, Italy 3 Department of Pneumology, Freiburg University Medical Center, Albert-Ludwigs-University, Freiburg, Germany 2
Cell surface expression of specific receptors and ectonucleotidases makes extracellular nucleotides such as ATP, ADP, UTP, and adenosine suitable as signaling molecules for physiological and pathological events, including tissue stress and damage. Recent data have revealed the participation of purinergic signaling in atherosclerosis, depicting a scenario in which, in addition to some exceptions reflecting dual effects of individual receptor subtypes, adenosine and most P1 receptors, as well as ecto-nucleotidases, show a protective, antiatherosclerotic function. By contrast, P2 receptors promote atherosclerosis. In consideration of these findings, modulation of purinergic signaling would represent an innovative and valuable tool to counteract atherosclerosis. We summarize recent developments on the participation of the purinergic network in atheroma formation and evolution. Purinergic signaling The nucleotides ATP, ADP, UTP, UDP, the nucleoside adenosine are present at elevated concentrations inside living cells where they play several roles as building elements for nucleic acids, modulators of enzyme activity, and intermediates of energy transfer. Their role is not merely restricted to the intracellular compartments, however, because they are also released into the extracellular space where they behave as mediators of cell–cell communication [1]. Vascular and circulating cells are able to release ATP by vesicular exocytosis, plasma membrane F(1)/F(0)-ATP synthase, ATP-binding cassette (ABC) transporters, connexin hemichannels, and pannexin channels [2] (Figure 1). Although F(1)/F(0)-ATP synthase has been linked to extracellular ATP production, its main activity may be ATP hydrolysis into ADP, thereby contributing to generation of this extracellular agonist [3]. The knowledge that extracellular molecules based on purines (ATP, ADP, and adenosine) or pyrimidines (UTP and UDP) are secreted and act as signaling molecules has prompted a thorough investigation of the network of nucleotide-activated receptors and nucleotide-transforming enzymes. Receptors for extracellular nucleotides have been grouped into P1 (P1Rs) and P2 (P2Rs) receptors. The first group, P1Rs, have the ATP metabolite adenosine as a common agonist; while the Corresponding author: Ferrari, D. ([email protected]). Keywords: atherosclerosis; extracellular ATP; ecto-nucleotidases; adenosine; P1 receptors; P2 receptors. 1471-4914/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2014.12.008
second group, P2Rs, is composed of membrane receptors activated by ATP and/or other nucleotides [4]. P1Rs include four receptors types: Adora 1 (A1R), Adora 2 (A2AR), Adora 2B (A2BR), and Adora 3 (A3R); while P2Rs have been distinguished in two subgroups: P2XRs (of which there are seven cloned human subtypes) and P2YRs (of which there are eight cloned human subtypes). Adenosine, as well as ADP or AMP, can result from the activity of CD39 (an ectonucleoside triphosphate diphosphohydrolase that converts ATP/ADP to AMP) and CD73 (an ecto-50 -nucleotidase that converts AMP to adenosine) [5]. Ecto-adenylate kinase and ecto-nucleoside diphosphate kinase (NDPK) are also involved in regulating extracellular levels of nucleotides [6]. Extracellular nucleotides have been classified as ‘damage-associated molecular patterns’ (DAMPs) (see Glossary) and can start and perpetuate the immune response, even
Glossary ATP-binding cassette (ABC) transporters: membrane proteins that couple energy derived from ATP hydrolysis to the efflux of a wide variety of compounds across the plasma membrane. Cholesterol: lipid molecule mainly produced by the liver. It is a fundamental constituent of the plasma membrane of animal cells and a precursor for the biosynthesis of vitamin D, bile acids, and steroid hormones. Cholesterol in animal fats consumed in the diet contributes to increase blood cholesterol concentration. Cholesterol 27-hydroxylase: enzyme involved in the conversion of cholesterol to oxysterols, thereby enhancing cholesterol efflux from the cell and its elimination by the liver. Damage-associated molecular patterns (DAMPs): molecules of the host cell that, once modified or released into the extracellular milieu upon cell stress or damage, can promote and perpetuate an immune response in non-infectious conditions. Foam cell: macrophage-derived cell filled with lipids. Hypoxia-inducible factors 1 and 2 (HIF1 and 2): a family of transcription factors transactivating a large number of genes in response to hypoxic conditions. Low-density lipoprotein (LDL): a plasmatic lipoprotein carrying cholesterol in the bloodstream. It contains the apolipoprotein apoB100. High LDL concentrations are associated with increased risk of atherosclerosis and coronary artery disease. Metabolic syndrome: the complex of physiological, biochemical, clinical, and metabolic factors that directly increases the risk of atherosclerotic cardiovascular disease. The main features of metabolic syndrome are hypertension, atherogenic dyslipidemia, glucose intolerance, and proinflammatory and prothrombotic conditions. Pathogen-associated molecular patterns (PAMPs): microbe-associated molecular motifs present in specific RNA, polypeptides, and sugar molecules that are rare or absent in host cells. These motifs are recognized by specific host receptors (pattern recognition receptors, PRRs) thus triggering an appropriate immune response. Reverse cholesterol transport (RCT): a process whereby excess cholesterol from peripheral tissues is transported by high-density lipoprotein (HDL) particles to the liver for further metabolism and biliary excretion. Scavenger receptor class A (SR-A): a subclass of pattern recognition receptors composed of five proteins able to bind modified lipoproteins. Shear stress: the force generated by the flowing blood acting on the endothelial wall of a vessel.
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Extracellular
ADO
AMP
ATP
ADP
G
G P2XRs Intracellular
P2YRs
F1-ATPase
CD39
CD73
P1Rs
ENT1 ENT2
Vesicular release ABC transporters Connexins Pannexins P2X7R TRENDS in Molecular Medicine
Figure 1. The purinergic network at the cell membrane. ATP is released in a regulated manner by exocytosis, in ATP-containing vesicles, and through different plasma membrane proteins such as connexins, pannexins, ATP-binding cassette (ABC) transporters, and P2X7R. Nucleotide release also occurs as a consequence of cellular stress or death, for example during infection, ischemia and reperfusion, or inflammation. ATP functions as a signaling molecule via activation of P2XR and P2YR subtypes. P2XRs are multimeric plasma membrane channels selective for monovalent and divalent cations (Na+, K+, Ca2+); their activation induces transmembrane ion fluxes, plasma membrane depolarization, caspase activation, nuclear factor transcription, and cytokine secretion. P2YRs are G protein-coupled receptors with seven transmembranespanning motifs; their activation induces calcium release from the intracellular stores via phospholipase C/inositol-1,4,5-triphosphate production or activation/inhibition of adenylate cyclase depending on subtype. P1Rs have been linked to the activation of different intracellular signaling pathways, the most common involving modulation of adenylyl cyclase with consequent decrease (A1R) or increase (A2R, A3R) in intracellular cAMP concentrations. In the extracellular space, ATP is also metabolized by enzymatic phosphohydrolysis in a two-step process via CD39 conversion of ATP (or ADP) to AMP, and CD73 phosphohydrolysis of AMP to adenosine (ADO), which is the agonist of P1Rs. P1R-mediated signaling is terminated by cellular uptake of adenosine by equilibrative nucleoside transporter 1 (ENT1) and ENT2.
in the absence of pathogen-associated molecular patterns (PAMPs; i.e., foreign molecules belonging to invading microorganisms) [7]. Although a moderate and controlled release of ATP or UTP exerts beneficial effects for the damaged tissue by activating reparative homeostatic responses, large and uncontrolled ATP release due to intense mechanical stress, trauma, allergen inhalation, or infection causes excessive activation of P2R-expressing immune and non-immune cells. These events are followed by an abnormal production of proinflammatory cytokines and inflammatory mediators such as prostaglandins, leukotrienes, and reactive oxygen intermediates (ROIs) that, besides causing tissue damage, induce a massive recruitment of immune cells leading to the establishment of chronic inflammatory conditions. The atherosclerotic process Atherosclerosis is the leading cause of cardiovascular disease, which accounts for approximately 40% of all deaths annually in the United States [8]. It has become clear that atherosclerosis is a chronic inflammation of arteries, where activated immune cells are abundant and play a major role in plaque progression and eventual rupture [9,10]. Atherosclerosis is characterized by a typical lesion called an atheroma (Figure 2). Modification of the artery endothelium begins with intima infiltration of circulating monocytes in response to different mediators, mainly lipids [11,12]. Cholesterol, transported in the bloodstream by low-density lipoproteins (LDLs), accumulates in the intima and triggers a deleterious cascade of events [13], culminating in the formation of the atheromatous plaque surrounded 2
by smooth muscle cells and a fibrous cap. The increasing volume of the plaque reduces the vasal lumen, with a heavy impact on blood flow. In late stages, lesional calcification can lead to plaque instability, and accumulation of platelets can result in the formation of a thrombus over the plaque, further interrupting blood flow. Upon accumulation of lipid molecules, macrophages change their phenotype and become foam cells, the prototypical cells of the atherosclerotic plaque. This event occurs when cholesterol inflow into the cell exceeds the outflow, and reverse cholesterol transport mediated by high-density lipoprotein (HDL) is insufficient to remove the excess cholesterol into the bloodstream. In addition to macrophages, intimal smooth muscle cells can also accumulate considerable amounts of cholesterol, contributing to the atherosclerotic process [14]. Oxidized LDL (oxLDL) prompts an immune reaction by activating endothelial cells (ECs) that upregulate adhesion molecules and secrete chemokines that attract T and B cells, macrophages, dendritic cells (DCs), and natural killer (NK) cells into the sub-endothelial region [15]. Atherosclerosis is reduced by inhibition of the T cell response to native LDL [16]. Most of the T lymphocytes found in atheromas are CD4+ with an effector memory phenotype and a helper T cell type 1 (TH1) profile, while the number of circulating regulatory T cells (Tregs) is reduced in patients with acute coronary syndrome and in apolipoprotein E-deficient mice (ApoE/) [17,18]. Depletion of FOXP3+ regulatory T cells favors hypercholesterolemia and atherosclerosis [19]. The most severe clinical complications of atherosclerosis arise from rupture of the plaque, which leads to exposure of pro-thrombotic substrates of the plaque core, thus causing thrombotic
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Vasal lumen
Aggregated (acvated) platelets ADP
ATP
T cell T cell
P2X1R P2Y1R P2Y11R P1Y12R
Circulang (resng) platelets
P-selecn PF4
Monocyte
T cell LDL
TNF-α
HDL F1-ATPase/P2Y12
Monocyte
ATP
DCs
P2Y2R P2Y1R P2Y2R
A2BR
Panx1 Conn. ABC t.
oxldl
VEGF
ATP
IL-8 P2Y6R
Dying foam cell VCAM-1 ICAM-1 P-selecn
ATP
P2X7R
IL-1β
A2BR IL-6 Macrophage
A2AR
ATP CD39
CD73
ADO
Foam cell
ATP CD73 CD39
CD39
CD73
ADO
ADO
A2BR SMCs
A2BR Fibroblasts TRENDS in Molecular Medicine
Figure 2. Putative schematic involvement of purinergic signaling in mouse models of atherogenesis. (Left) Genesis of the atherosclerotic plaque. Atheroma begins with a fatty streak almost exclusively populated by infiltrating monocytes that transmigrate through the endothelium. P2Y2R expression increases on endothelial cells (ECs), and its activation induces expression of adhesion molecules [vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and P-selectin] that favor the adhesion and extravasation of immune cells. P2Y2R expression on monocytes also enhances their migration across the endothelium. Once through the endothelium, monocytes differentiate into macrophages and then become foam cells. ATP is released by connexin hemichannel (Conn.), ABC transporter (ABC t.), and Pannexin 1 (Panx1). Released ATP activates P2X7R on macrophages, stimulating the secretion of IL-6 and IL-1b. ATP is also converted to ADP, AMP, and adenosine (ADO) by CD39 and CD73, which are expressed by different cell types including smooth muscle cells (SMCs). ADO activates A2BR on macrophages, which also acts to promote their transformation into foam cells. (Centre) Atheroprogression. Cell surface F1-ATPase/P2Y12R on ECs play an atheroprotective role by favoring high-density lipoprotein (HDL) transcytosis. SMCs and fibroblast proliferation is enhanced by ADO-mediated activation of A2BR. ADO also induces secretion of vascular endothelial growth factor (VEGF) and IL-8 by macrophages. ATP concentrations are increased by release from ECs, as well as from damaged and dying cells present in the atheroma. (Right) Plaque destabilization. Ruptures in the artery wall activate platelets to release of high amounts of ADP and ATP, leading to shape changes/aggregation and thrombi/emboli formation. ATP acts on proinflammatory receptors including P2X7R, which is expressed by macrophages, dendritic cells (DCs) and T lymphocytes, exacerbating the inflammatory conditions. Abbreviations: LDL, low-density lipoprotein; oxLDL, oxidized LDL; PF4, platelet factor 4; TNF-a, tumor necrosis factor a.
occlusion of the artery. Inflammatory cells also participate in plaque rupture, mainly by producing proteolytic enzymes that weaken the cap structure. In particular, macrophages secrete matrix metalloproteinases (MMPs) that cleave collagen to promote plaque rupture [20]. Purinergic signaling is putatively involved in all stages of atherosclerosis and can either foster or counteract disease development, depending on the type of purinergic receptors involved (Table 1). Purinergic signaling and atherosclerosis Purinergic signaling in atherogenesis Both cell associated and soluble mediators released from the cells into the fluid phase are involved in atherosclerosis. Among soluble mediators, extracellular nucleotides
play an important role. Most of the experimental evidence of the involvement of purinergic signaling in atherogenesis has been obtained from double-knockout animal models, for example ApoE/ mice lacking specific P1R, P2R subtypes, or ecto-nucleotidases. However, it is crucial to note that the results of studies employing these models are not immediately transferable to human disease because of existing differences in lipid metabolism between humans and mice. For example, mice do not develop atherosclerosis unless they are genetically modified and, moreover, the commonly used high-fat diet can elicit blood cholesterol concentrations as high as 1000 mg/dL, which far exceeds the values observed in the most severe forms of familial hypercholesterolemia in humans. In addition, mice differ 3
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Table 1. Pro- or anti-atherogenetic effects of P2Rs, P1Rs, CD39, and CD73a Receptor A1R
Agonist Adenosine
Cell/tissue Vessel wall
A2AR
Adenosine
Macrophages Macrophages
A2BR
Adenosine
Macrophages
A3R
Adenosine
VSMC Hepatocytes Macrophages
P2X7R
ATP
ECs
P2Y1R
ADP
DCs Vessel wall ECs
P2Y2R
ATP
ECs
P2Y6R
UDP
P2Y12R
ADP
P2Y13R CD39 CD73
ADP
Monocytes Macrophages ECs Platelets ECs Hepatocytes VSMCs Plaque infiltrating cells
Effect Increases proliferation and IL-5, IL-6, and IL-13 secretion HIF-1a accumulation HIF-1a accumulation Protection from apoptosis HIF-1a accumulation, VEGF and IL-8 secretion, FC formation Inhibition of proliferation Regulation of lipid levels HIF-1a accumulation, VEGF secretion, FC formation IL-1b release IL-1RA release TF release Enhanced gelatinase activity P-selectin, ICAM and VCAM upregulation Leukocyte rolling ROS production Upregulation of ICAM and VCAM Increased migration Increased IL-6 production and iNOS activation Enhanced response to proinflammatory stimuli Platelet aggregation, release of PF4, P-selectin expression uptake and transport of HDL Increased reverse cholesterol transport Neointima formation, VSMC migration Reduced atherosclerotic progression
Atherogenic +
Atheroprotective
+ + + +
Refs [39] [14] [14] [29] [14]
+ + + +
[60] [28] [14]
+ + +
[61] [61] [68] [38] [21]
+
[21]
+ + + +
[21] [37] [36] [24]
+
+ + + +
[70] [6,27,29,30] [69] [33]
a
Abbreviations: DCs, dendritic cells; ECs, endothelial cells; FC, foam cells; HIF, hypoxia inducible factor; ICAM, intercellular adhesion molecule; iNOS, inducible nitric oxide synthase; PF4, platelet factor 4; TF, tissue factor; UDP, uridine diphosphate; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor; VSMC, vascular smooth muscle cells.
from humans in vascular structure, hemostatic function, and the expression of some purinergic receptors (e.g., P2Y11R is not expressed in rodents). Cholesterol levels are a key factor in the initiation of atherosclerosis, and intracellular levels are regulated by HDL-mediated reverse cholesterol transport, by which cholesterol is discharged into the blood and transported to the liver. When cholesterol accumulates in the intima, monocytes are recruited into the vessel wall with the participation of the P2Y2R, which is upregulated in animal models as well as in human monocytes and ECs [21]. Notably, oxLDLs caused the release of ATP and reactive oxygen species (ROS) from in vitro stimulated ECs [22]. Costimulation of ECs with oxLDL and nucleotides induces P2Y1R- and P2Y2R-mediated increases in ICAM-1, VCAM-1, and P-selectin expression, potentially increasing leukocyte rolling and extravasation [21]. Hence, deletion of the P2Y1R in ApoE-deficient mice (P2y1//ApoE/) results in reduced vascular inflammation and diminished plaque size in the aorta as a result of decreased macrophage infiltration and smooth muscle cell proliferation [23]. Similarly, lack of P2Y12R expression in P2y12// ApoE/ mice reduced lesional areas and decreased monocyte/macrophage infiltration, while the fibrous content of the plaque was increased [24]. Further analysis showed that P2Y12R expressed by platelets was responsible for platelet factor 4 secretion and P-selectin expression, thereby inducing monocyte recruitment and infiltration [21]. Interestingly, P2Y13R expressed in mouse hepatocytes played a crucial role in hepatic HDL endocytosis, and the 4
P2Y13R agonist cangrelor (used as an anti-thrombotic drug) strongly activated P2Y13R-mediated HDL endocytosis [23–30]. The same effect was obtained by inhibiting the proteasome, which resulted in increased plasma membrane expression of the receptor [31]. These findings were later confirmed when the specific P2Y13R agonist CT1007900 was shown to promote in vitro cholesterol mobilization and to decrease plaque size in the aorta and carotids of ApoE/ mice [27]. Of note, P2Y13R-mediated HDL endocytosis is also active in human hepatocytes and is regulated by cell surface adenylate kinase [6]. P1R-mediated signaling also plays a role in response to plasma cholesterol and lipids. A2BR is able to regulate hyperlipidemia, exerting an indirect protective effect on the vessel wall. A2BR was upregulated in the liver as a consequence of a high-fat/cholesterol diet and its activation with the specific agonist BAY 60-6053 (Table 2) decreased cholesterol and triglyceride plasma levels, and reduced lesion formation [32]. In one report, lack of A2AR expression was associated with protecting ApoE-deficient mice from atherosclerosis [33]. However, signaling events triggered by A2AR engagement upregulated the expression of cholesterol 27-hydroxylase and ABCA1, two proteins of the reverse cholesterol transport system, thus exerting antiatherosclerotic and anti-inflammatory effects [32–36]. ABCA1 plays an anti-atherosclerotic function by promoting cholesterol efflux from the cell, thus reducing transformation of macrophages into foam cells. Interestingly, lack of expression of ABCA1 and G1 in macrophages increased inflammation and accelerated atherosclerosis in mice
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Table 2. P2R antagonists and P1R agonists available for preclinical and clinical trials Receptor Compound Effect Inhibition of channel opening All P2XR PPADS Suramin Inhibition of channel opening NF279 P2X1R NF449 NF023 NF-864 TNP-ATP PSB-10211 Inhibition of channel opening P2X2R NF770 NF778 Inhibition of channel opening A-317491 MK3901 P2X3R RO-3 RO-4 TNP-ATP AF 353 Inhibition of channel opening P2X3R; P2X2/3R PSB-12062 Inhibition of channel opening P2X4R Paroxetine 5-BDBD BBG P2X5R AZ D9056 P2X7 blockade P2X7R oATP MRS2427 RN-6189 AZ 10606120 JNJ-42253432 GSK1482160 Suppression of ATP induced IL-1b release P2X7R A-804598 AACBA Brilliant blue G (BBG) Inhibition of channel opening, P2X7R KN-62 inhibition of inflammasome activation, A-438079 suppression of ATP-induced IL-1b release, inhibition of IL-6 and CCL2 secretion Inhibition of platelet aggregation and of Ca2+ increase MRS2179 P2Y1R MRS2500 Inhibition of platelet aggregation P2Y1R AR-C 126313 Receptor inhibition P2Y2R Receptor inhibition MRS2577 P2Y4R MRS2578 Receptor inhibition P2Y6R NF157 Receptor inhibition P2Y11R NF340 Cangrelor Inhibition of platelet aggregation P2Y12R Clopidogrel Ticagrelor AR-C 66096 Receptor inhibition MRS2211 P2Y13R MRS2603 Receptor activation CT1007900 Partial agonist Cangrelor PPTN Receptor inhibition P2Y14R CHA Receptor activation, HIF-1a induction A1R Receptor activation CPPA CGS-21680 Receptor activation, increase in cholesterol efflux from macrophages A2AR 2HE-NECA Receptor activation CVT-3146 Receptor activation BAY 60-6053 Receptor activation, decreases lipids and atherosclerosis A2BR Compound 24 Receptor activation, HIF-1a induction Cl-IB-MECA Receptor activation, HIF-1a induction A3R IB-MECA MRS5698 Receptor activation
Human use a Refs N [61] N
[61]
N
[61]
N
[61]
Y
[73]
N
[74]
Y N
[75]
Y N
[76] [61]
Y N
[77,78] [77]
N N
[77] [77]
Y
[26,66]
N
[79]
N Y N
[28] [29] [80] [39]
N
[81]
N N N
[32] [39] [39]
a
Compounds already used in humans are indicated with (Y) while those employed in animals are indicated with (N).
[36]. This pathway is modulated by apelin-13-mediated activation of PKCa, which phosphorylates ABCA1 and inhibits its calpain-mediated proteolysis [37,38]. By contrast, purinergic signaling may also be involved in foam cell generation because blocking adenosine A2BR and A3R on macrophages reduces the formation of foam cells in vitro [39]. The removal of extracellular ATP and increases in adenosine concentration has an overall protective effect
against atherosclerosis. Cd73//ApoE/ mice have significantly elevated plasma triglycerides and atherosclerotic lesion size is increased by 50% compared to ApoE/ animals [40]. Purinergic signaling in atheroprogression Numerous reports have described an association between atherosclerosis and infection with pathogens such as 5
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Review Chlamydia pneumoniae, Helicobacter pylori, Epstein–Barr virus, or cytomegalovirus [41,42]. Moreover, T lymphocytes specific for different pathogens have been isolated from atherosclerotic lesions. It has recently been shown that bacterial lipopolysaccharide (LPS) increases the uptake of oxidized LDL by upregulating lectin-like oxidized LDL receptor-1 in mouse and human macrophages [43]. LPS also upregulates P2Y6R in ECs [44]. This receptor subtype is highly expressed in atherosclerotic aorta segments of ApoE/ mice, and blockade with suramin or pyridoxalphosphate-6-azophenyl-20 ,40 -disulfonic acid (PPADS) reduces the severity of atherosclerosis [45]. Engagement of the P2Y6R, as well as of the proinflammatory P2X7R (a receptor which is highly represented in the majority of human carotid artery plaques, luminal thrombi, and small plaque ulcerations) results in increased production of interleukin 6 (IL-6), a central cytokine in atherogenesis [46,47]. Deficiency or inhibition of A1R also reduces plasma concentrations of the proinflammatory cytokines IL-5, IL-6, and IL-13, and consequently reduces atherosclerotic lesions in ApoE-deficient mice [48]. Interferon-g (IFN-g) is also present in atherosclerotic lesions and its contribution in the genesis of atheroma has been well established [49]. This cytokine upregulates expression of the P2X7R, thus increasing the proinflammatory properties of macrophages; P2X7R engagement leads to the release of ATP and NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome activation, with consequent IL-1b secretion [50,51]. Notably, lack of IL-1b reduces atherosclerosis severity in ApoE-deficient mice [52], while another study has shown a prominent role for IL-1a in atherosclerosis [53]. However, convincing data have recently shown that activation of NLRP3 inflammasome by extracellular ATP or cholesterol crystals enhances macrophage lipid deposition and migration, suggesting a role of this complex in atherogenesis [54,55]. T cells are also present in atheroma. P2X4R and P2X7R stimulation induces T cell activation and proliferation through IL-2 secretion [56]. Activated T cells also secrete ATP, and Treg cells are sensitive to its cytotoxic effects [57]. This may explain the low numbers of Treg cells found in atherosclerotic plaques. Along with the detrimental effects described above, ATP secretion could have a positive effect because it downmodulates NK chemotaxis and killing capacity via P2Y11R activation [56]. Purinergic signaling in plaque destabilization A detailed description of the mechanisms underlining plaque destabilization is still missing, mainly owing to the lack of appropriate experimental models. The most evident event, however, is erosion and rupture of the fibrous cap surrounding the plaque as a result of the activity of MMPs secreted by macrophages [20]. So far, there is no direct evidence of participation of purinergic signaling in this process. However, P2X7R could be involved because its stimulation has been linked to release of MMPs [58]. Following plaque rupture, pro-thrombotic substrates are exposed, and high amounts of ATP and ADP are secreted from platelets, inducing thrombi formation. The development of transplant arteriosclerosis in 6
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P2y12/ mice further implicates this receptor in cell recruitment and vasal occlusion during atherosclerosis [59]. Hypoxic conditions in the plaque are caused by the absence of neo-vascularization and the presence of a surrounding cap. In vitro experiments under hypoxic conditions have shown activation of A2BR and A3R, inducing hypoxia-inducible factor 1a (HIF-1a)-dependent secretion of vascular endothelial growth factor (VEGF) and IL-8 secretion in an extracellular signal-regulated kinase (ERK) 1/2-, p38-, and Akt kinase-dependent manner [39]. Removal of dead cells from the plaque is also an important issue. Activation of the ATP channel pannexin 1 (PANX1) stimulates macrophage chemotaxis. PANX1 has a cleavage site for caspase 3 and caspase 7, and expression of truncated PANX1 protein results in a constitutively open channel able to continuously release ATP, thus stimulating phagocytosis of apoptotic cells [60]. At present it is not known whether this mechanism is involved in the elimination of apoptotic cells in the plaque. Potential therapeutic implications in cardiovascular diseases As cell-to-cell mediators, purines act as pro- or anti-atherosclerotic factors depending on the receptors engaged and the downstream signaling networks. It is particularly interesting that genetic deletion of specific purinergic receptors in mice results in decreased artery inflammation and reduced plaque size. This is true for those receptors whose responses are proinflammatory: A1R among adenosine receptors, P2X4R and P2X7R for P2X receptors, and P2Y1R, P2Y2R, and P2Y12R for the P2YR subgroup. Therefore, pharmacological inhibition of these receptors would likely dampen atherosclerotic inflammation and improve the disease state. This concept is strengthened by recent observations on therapies using reductase inhibitors or statins that may in part inhibit IFN-g-dependent macrophage activation in the atheromatous plaque. Owing to the fact that P2X7R is highly expressed in macrophages and is upregulated by IFN-g, it would be a candidate target for clinical trials. P2XR-specific antagonists have already been used in clinical trials for different pathologies, such as for pain, osteoporosis, spinal cord injury, bladder dysfunction, and cough [61] (Table 2). Another way to reduce atherosclerotic inflammation could be through the modulation of nucleotide-degrading enzymes. In several acute disease models, ecto-nucleotidases attenuate inflammation, prevent intimal hyperplasia, and preserve vascular function [62–64]. Moreover, it has to be underlined that activity of CD39 and CD73 could be lower in atherosclerotic human tissues, as for example in thoracic aortas, lymph nodes, spleen, and serum of young and mature ApoE/ mice where nucleoside triphosphate diphosphohydrolase (NTPDase) activity decreases up to 50%, favoring local accumulation of ATP and ADP, and eventually exacerbating atherosclerosis [65]. In mice, stimulation of A2AR or activation of P2YR13 upregulates HDL-c metabolism by promoting reverse cholesterol transport, decreasing macrophage transformation into foam cells [26,34,35,65]. This may also provide an opportunity for intervention in humans. Lesions of the coronary and carotid arteries contribute to stroke and
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myocardial infarction by the formation of thrombi and emboli, occluding vessels and resulting in ischemia. Very recent data show that the P2Y12R inhibitor clopidogrel, commonly used as an anti-thrombotic drug, also reduced lipid peroxidation, systemic inflammation, and aortic expression of inflammatory markers [66,67]. Precisely defining the role of the purinergic signaling network in the atherogenic process is an urgent issue, particularly for those receptor subtypes such as P2Y1R that can have both pro- or anti-atherogenic activity depending on the cell type. P2Y1R stimulates human endothelial cell migration via mitogen-activated protein kinase pathways but also coronary vasodilation via an endothelium/nitric oxide-dependent mechanism [68,69]. Similarly, P2Y12R function is pro-atherogenic in platelets and SMCs by stimulating vasoconstriction, proliferation, and inflammation; further investigation will be necessary to establish whether P2Y12R-stimulated transcytosis of HDL through the endothelium decreases cholesterol levels in the subendothelium region [70]. Therefore, clarification of the roles played by single receptors is highly desirable and will be instrumental for the design of innovative therapeutic interventions to counteract atherosclerosis (Box 1). Concluding remarks and future perspectives Atherosclerosis can be reduced by improved diet and regular physical exercise, steps that represent the most successful approach for preventing and counteracting the effects of metabolic syndrome [8]. However, recent findings suggest that formation of the atherosclerotic lesions is prompted or facilitated by concurring secondary causes; hence, proinflammatory responses by immune cells contribute to the subclinical inflammatory status characterizing atheromasic lesion-bearing patients, increasing their cardiovascular risk [8]. Atherosclerotic inflammation can occur even in the absence of microbes, as shown by studies using germ-free animals, which suggests that one or more endogenous substrates are endowed with the ability to start atherosclerotic inflammation. Autoimmune reactions towards heat-shock protein 60 (HSP60) and HSP90, highly expressed in the cytoplasm of eukaryotic cells under nonstress conditions, and that are secreted by vascular smooth muscle cells in response to oxidative stress, has been indicated as concurring cause of atherosclerosis [71,72]. Cholesterol crystals can trigger inflammation by activating the NLRP3 inflammasome [53], and dying cells of the necrotic centre release nucleotides acting as DAMP signals for P2R-expressing immune cells [7]. In this context, in vivo trials of compounds able to positively or negatively modulate the molecular components of the purinergic network
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according to their main activity in atherogenesis may produce valuable information. P2R antagonists, as well as new atheroprotective pharmacological P1R agonists, may be useful to block the initial atherosclerotic inflammation and counteract its transition to chronic disease. It is extremely urgent to exploit the enormous potential of the purinergic signaling to find new treatments for atherosclerosis. Collaborations between purinologists, immunologists, and cardiologists will likely result in the design of new drugs targeting proteins of the purinergic signaling network for treating the disease. Acknowledgments This work is supported in part by the Commission of European Union (2012-2014, ERA-NET NEURON program, Role of Danger Signals in Stroke and Therapeutic Targeting by Nanobodies). We apologize to all whose work we could not cite owing to space limitations.
References 1 Burnstock, G. (2012) Purinergic signalling: its unpopular beginning, its acceptance and its exciting future. Bioessays 34, 218–225 2 Lohman, A.W. et al. (2012) Mechanisms of ATP release and signalling in the blood vessel wall. Cardiovasc. Res. 95, 269–280 3 Vantourout, P. et al. (2010) Ecto-F1-ATPase: a moonlighting protein complex and an unexpected apoA-I receptor. World J. Gastroenterol. 16, 5925–5935 4 Burnstock, J. (1978) A basis for distinguishing two types of purinergic receptor. In Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach (Straub, R.W. and Bolis, L., eds), pp. 107–118, Raven Press 5 Zimmermann, H. et al. (2012) Cellular function and molecular structure of ecto-nucleotidases. Purinergic Signal. 8, 437–502 6 Fabre, A.C. et al. (2006) Cell surface adenylate kinase activity regulates the F(1)-ATPase/P2Y (13)-mediated HDL endocytosis pathway on human hepatocytes. Cell. Mol. Life Sci. 63, 2829–2837 7 Idzko, M. et al. (2014) Nucleotide signalling during inflammation. Nature 509, 310–317 8 Jamkhande, P.G. et al. (2014) Therapeutic approaches to drug targets in atherosclerosis. Saudi Pharm. J. 22, 179–190 9 Hansson, G.K. and Hermansson, A. (2011) The immune system in atherosclerosis. Nat. Immunol. 12, 204–212 10 Ross, R. (1999) Atherosclerosis – an inflammatory disease. N. Engl. J. Med. 340, 115–126 11 Mullick, A.E. et al. (2008) Increased endothelial expression of Toll-like receptor 2 at sites of disturbed blood flow exacerbates early atherogenic events. J. Exp. Med. 205, 373–383 12 Flood, C. et al. (2004) Molecular mechanism for changes in proteoglycan binding on compositional changes of the core and the surface of low-density lipoprotein-containing human apolipoprotein B100. Arterioscler. Thromb. Vasc. Biol. 24, 564–570 13 Shi, Y. et al. (2011) Oxidized low-density lipoprotein activates p66Shc via lectin-like oxidized low-density lipoprotein receptor-1, protein kinase C-beta, and c-Jun N-terminal kinase kinase in human endothelial cells. Arterioscler. Thromb. Vasc. Biol. 31, 2090–2097 14 Allahverdian, S. et al. (2014) Contribution of intimal smooth muscle cells to cholesterol accumulation and macrophage-like cells in human atherosclerosis. Circulation 129, 1551–1559 15 Nakashima, Y. et al. (1998) Upregulation of VCAM-1 and ICAM-1 at atherosclerosis-prone sites on the endothelium in the ApoE-deficient mouse. Arterioscler. Thromb. Vasc. Biol. 18, 842–851 16 Hermansson, A. (2010) Inhibition of T cell response to native lowdensity lipoprotein reduces atherosclerosis. J. Exp. Med. 2075, 1081–1093 17 Buono, C. et al. (2005) T-bet deficiency reduces atherosclerosis and alters plaque antigen-specific immune responses. Proc. Natl. Acad. Sci. U.S.A. 102, 1596–1601 18 Liu, Z.D. (2012) Increased Th17 cell frequency concomitant with decreased Foxp3+ Treg cell frequency in the peripheral circulation of patients with carotid artery plaques. Inflamm. Res. 61, 1155–1165 7
TRMOME-1011; No. of Pages 9
Review 19 Klingenberg, R. (2013) Depletion of FOXP3+ regulatory T cells promotes hypercholesterolemia and atherosclerosis. J. Clin. Invest. 123, 1323–1334 20 Newby, A.C. (2008) Metalloproteinase expression in monocytes and macrophages and its relationship to atherosclerotic plaque instability. Arterioscler. Thromb. Vasc. Biol. 28, 2108–2114 21 Eun, S.Y. et al. (2014) P2Y2R activation by nucleotides released from oxLDL-treated endothelial cells (ECs) mediates the interaction between ECs and immune cells through RAGE expression and reactive oxygen species production. Free Radic. Biol. Med. 69, 157–166 22 Seye, C.I. et al. (2002) Functional P2Y2 nucleotide receptors mediate uridine 50 -triphosphate-induced intimal hyperplasia in collared rabbit carotid arteries. Circulation 106, 2720–2726 23 Zerr, M. et al. (2011) Major contribution of the P2Y1 receptor in purinergic regulation of TNFa-induced vascular inflammation. Circulation 123, 2404–2413 24 Hechler, B. et al. (2008) Reduced atherosclerotic lesions in P2Y1/ apolipoprotein E double-knockout mice: the contribution of nonhematopoietic-derived P2Y1 receptors. Circulation 118, 754–763 25 Li, D. et al. (2012) Roles of purinergic receptor P2Y, G protein-coupled 12 in the development of atherosclerosis in apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. 32, e81–e89 26 Jacquet, S. et al. (2005) The nucleotide receptor P2Y13 is a key regulator of hepatic high-density lipoprotein (HDL) endocytosis. Cell. Mol. Life Sci. 62, 2508–2515 27 Fabre, A.C. et al. (2010) P2Y13 receptor is critical for reverse cholesterol transport. Hepatology 52, 1477–1483 28 Goffinet, M. et al. (2014) P2Y13 receptor regulates HDL metabolism and atherosclerosis in vivo. PLoS ONE 9, e95807 29 Serhan, N. et al. (2013) Chronic pharmacological activation of P2Y13 receptor in mice decreases HDL-cholesterol level by increasing hepatic HDL uptake and bile acid secretion. Biochim. Biophys. Acta 1831, 719–725 30 Lichtenstein, L. et al. (2013) Lack of P2Y13 in mice fed a high cholesterol diet results in decreased hepatic cholesterol content, biliary lipid secretion and reverse cholesterol transport. Nutr. Metab. (Lond.) 10, 67 31 Pons, V. et al. (2014) Role of the ubiquitin-proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake. Cell. Mol. Life Sci. 71, 1775–1788 32 Koupenova, M. et al. (2012) A2b adenosine receptor regulates hyperlipidemia and atherosclerosis. Circulation 125, 354–363 33 Wang, H. et al. (2009) Inactivation of the adenosine A2A receptor protects apolipoprotein E-deficient mice from atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 29, 1046–1052 34 Bingham, T.C. et al. (2010) A2A adenosine receptor stimulation decreases foam cell formation by enhancing ABCA1-dependent cholesterol efflux. J. Leukoc. Biol. 87, 683–690 35 Bingham, T.C. et al. (2012) Cholesterol 27-hydroxylase but not apolipoprotein apoE contributes to A2A adenosine receptor stimulated reverse cholesterol transport. Inflammation 35, 49–57 36 Westerterp, M. et al. (2013) Deficiency of ATP-binding cassette transporters A1 and G1 in macrophages increases inflammation and accelerates atherosclerosis in mice. Circ. Res. 112, 1456–1465 37 Martinez, L. et al. (2003) Phosphorylation of a pest sequence in ABCA1 promotes calpain degradation and is reversed by ApoA-I. J. Biol. Chem. 278, 37368–37374 38 Liu, X.Y. et al. (2013) Apelin-13 increases expression of ATP-binding cassette transporter A1 via activating protein kinase C a signalling in THP-1 macrophage-derived foam cells. Atherosclerosis 226, 398–407 39 Gessi, S. et al. (2010) Adenosine modulates HIF-1a, VEGF, IL-8, and foam cell formation in a human model of hypoxic foam cells. Arterioscler. Thromb. Vasc. Biol. 30, 90–97 40 Buchheiser, A. et al. (2011) Inactivation of CD73 promotes atherogenesis in apolipoprotein E-deficient mice. Cardiovasc. Res. 92, 338–347 41 Ayada, K. (2009) Chronic infections and atherosclerosis. Clin. Rev. Allergy Immunol. 37, 44–48 42 van der Meer, J.J. (2008) Multiple bacteria contribute to intraplaque T-cell activation in atherosclerosis. Eur. J. Clin. Invest. 38, 857–862 43 Hossain, E. et al. (2014) Lipopolysaccharide augments the uptake of oxidized LDL by up-regulating lectin-like oxidized LDL receptor-1 in 8
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macrophages. Mol. Cell. Biochem. Published online October 28, 2014. http://dx.doi.org/10.1007/s11010-014-2259-0 Riegel, A.K. et al. (2011) Selective induction of endothelial P2Y6 nucleotide receptor promotes vascular inflammation. Blood 117, 2548–2555 Guns, P.J. et al. (2010) P2Y receptors and atherosclerosis in apolipoprotein E-deficient mice. Br. J. Pharmacol. 159, 326–336 Piscopiello, M. et al. (2013) P2X7 receptor is expressed in human vessels and might play a role in atherosclerosis. Int. J. Cardiol. 168, 2863–2866 Hartman, J. and Frishman, W.H. (2014) Inflammation and atherosclerosis: a review of the role of interleukin-6 in the development of atherosclerosis and the potential for targeted drug therapy. Cardiol. Rev. 22, 147–151 Teng, B. et al. (2014) A1 adenosine receptor deficiency or inhibition reduces atherosclerotic lesions in apolipoprotein E deficient mice. Cardiovasc. Res. 102, 157–165 Leon, M.L. and Zuckerman, S.H. (2005) Gamma interferon: a central mediator in atherosclerosis. Inflamm. Res. 54, 395–411 Pellegatti, P. et al. (2005) A novel recombinant plasma membranetargeted luciferase reveals a new pathway for ATP secretion. Mol. Biol. Cell 16, 3659–3665 Ferrari, D. et al. (2006) The P2X7 receptor: a key player in IL-1 processing and release. J. Immunol. 176, 3877–3883 Kirii, H. et al. (2003) Lack of interleukin-1beta decreases the severity of atherosclerosis in ApoE-deficient mice. Arterioscler. Thromb. Vasc. Biol. 23, 656–660 Freigang, S. et al. (2013) Fatty acid-induced mitochondrial uncoupling elicits inflammasome-independent IL-1a and sterile vascular inflammation in atherosclerosis. Nat. Immunol. 14, 1045–1053 Duewell, P. et al. (2010) NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 464, 1357–1361 Li, X. et al. (2014) Activation of Nlrp3 inflammasomes enhances macrophage lipid-deposition and migration: implication of a novel role of inflammasome in atherogenesis. PLoS ONE 9, e87552 Gorini, S. et al. (2010) ATP secreted by endothelial cells blocks CXCL 1elicited natural killer cell chemotaxis and cytotoxicity via P2Y11R. Blood 116, 4492–4500 Trabanelli, S. et al. (2012) Extracellular ATP exerts opposite effects on activated and regulatory CD4+ T cells via purinergic P2 receptor activation. J. Immunol. 189, 1303–1310 Gu, B.J. and Wiley, J.S. (2006) Rapid ATP-induced release of matrix metalloproteinase 9 is mediated by the P2X7 receptor. Blood 107, 4946–4953 Harada, K. et al. (2011) Adenosine diphosphate receptor P2Y12mediated migration of host smooth muscle-like cells and leukocytes in the development of transplant arteriosclerosis. Transplantation 92, 148–154 Chekeni, F.B. et al. (2010) Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis. Nature 467, 863–867 North, R.A. and Jarvis, M.F. (2013) P2X receptors as drug targets. Mol. Pharmacol. 83, 759–769 Marcus, A.J. et al. (2003) Metabolic control of excessive extracellular nucleotide accumulation by CD39/ecto-nucleotidase-1: implications for ischemic vascular diseases. J. Pharmacol. Exp. Ther. 305, 9–16 Deaglio, S. and Robson, S.C. (2011) Ectonucleotidases as regulators of purinergic signaling in thrombosis, inflammation, and immunity. Adv. Pharmacol. 61, 301–332 Kaczmarek, E. and Koziak, K. (2011) Targeting stenosis with nucleotide-hydrolyzing enzymes. Curr. Pharm. Biotechnol. 12, 1799– 1804 Voloshyna, I. et al. (2013) Adenosine A(2A) receptor activation supports an atheroprotective cholesterol balance in human macrophages and endothelial cells. Biochim. Biophys. Acta 1831, 407–416 Angiolillo, D.J. (2012) Pharmacodynamic effects of cangrelor and clopidogrel: the platelet function substudy from the cangrelor versus standard therapy to achieve optimal management of platelet inhibition (CHAMPION) trials. J. Thromb. Thrombolysis 34, 44–55 Hadi, N.R. et al. (2013) Antiatherosclerotic potential of clopidogrel: antioxidant and anti-inflammatory approaches. Biomed. Res. Int. 2013, 790263
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Review 68 Shen, J. and DiCorleto, P.E. (2008) ADP stimulates human endothelial cell migration via P2Y1 nucleotide receptor-mediated mitogen-activated protein kinase pathways. Circ. Res. 102, 448–456 69 da Silva, C.G. et al. (2009) Mechanism of purinergic activation of endothelial nitric oxide synthase in endothelial cells. Circulation 119, 871–879 70 Cavelier, C. et al. (2012) The b-chain of cell surface F(0)F(1) ATPase modulates apoA-I and HDL transcytosis through aortic endothelial cells. Arterioscler. Thromb. Vasc. Biol. 32, 131–139 71 Benagiano, M. et al. (2005) Human 60-kDa heat shock protein is a target autoantigen of T cells derived from atherosclerotic plaques. J. Immunol. 174, 6509–6517 72 Businaro, R. (2009) Heat-shock protein 90: a novel autoantigen in human carotid atherosclerosis. Atherosclerosis 207, 74–83 73 Gever, J.R. et al. (2010) AF-353 a novel potent and orally bioavailable P2X3/P2X2/3 receptor antagonist. Br. J. Pharmacol. 160, 1387–1398 74 Hernandez-Olmos, V. et al. (2012) N-substituted phenoxazine and acridone derivatives: structure-activity relationships of potent P2X4 receptor antagonists. J. Med. Chem. 55, 9576–9588
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75 Keystone, E.C. (2012) Clinical evaluation of the efficacy of the P2X7 purinergic receptor antagonist AZD9056 on the signs and symptoms of rheumatoid arthritis in patients with active disease despite treatment with methotrexate or sulphasalazine. Ann. Rheum. Dis. 71, 1630–1635 76 Ali, Z. et al. (2013) Pharmacokinetic and pharmacodynamic profiling of a P2X7 receptor allosteric modulator GSK1482160 in healthy human subjects. Br. J. Clin. Pharmacol. 75, 197–207 77 Burnstock, G. and Ralevic, V. (2013) Purinergic signaling and blood vessels in health and disease. Pharmacol. Rev. 66, 102–192 78 Dawood, B.B. (2007) Reference curves for aggregation and ATP secretion to aid diagnose of platelet-based bleeding disorders: effect of inhibition of ADP and thromboxane A(2) pathways. Platelets 18, 329–345 79 Kim, Y.C. (2005) Synthesis of pyridoxal phosphate derivatives with antagonist activity at the P2Y13 receptor. Biochem. Pharmacol. 70, 266–274 80 Barrett, M.O. (2013) A selective high-affinity antagonist of the P2Y14 receptor inhibits UDP-glucose-stimulated chemotaxis of human neutrophils. Mol. Pharmacol. 84, 41–49 81 Reiss, A.B. and Cronstein, B.N. (2012) Regulation of foam cells by adenosine. Arterioscler. Thromb. Vasc. Biol. 32, 879–886
9
Review
Purinergic signaling in atherosclerosis Davide Ferrari1, Laura Vitiello2, Marco Idzko3, and Andrea la Sala2 1
Department of Life Sciences and Biotechnology, Biotechnology Centre, University of Ferrara, 44121 Ferrara, Italy Laboratory of Molecular and Cellular Immunology, Istituto Di Ricovero e Cura a Carattere Scientifico (IRCCS), San Raffaele Pisana, 00166 Rome, Italy 3 Department of Pneumology, Freiburg University Medical Center, Albert-Ludwigs-University, Freiburg, Germany 2
Cell surface expression of specific receptors and ectonucleotidases makes extracellular nucleotides such as ATP, ADP, UTP, and adenosine suitable as signaling molecules for physiological and pathological events, including tissue stress and damage. Recent data have revealed the participation of purinergic signaling in atherosclerosis, depicting a scenario in which, in addition to some exceptions reflecting dual effects of individual receptor subtypes, adenosine and most P1 receptors, as well as ecto-nucleotidases, show a protective, antiatherosclerotic function. By contrast, P2 receptors promote atherosclerosis. In consideration of these findings, modulation of purinergic signaling would represent an innovative and valuable tool to counteract atherosclerosis. We summarize recent developments on the participation of the purinergic network in atheroma formation and evolution. Purinergic signaling The nucleotides ATP, ADP, UTP, UDP, the nucleoside adenosine are present at elevated concentrations inside living cells where they play several roles as building elements for nucleic acids, modulators of enzyme activity, and intermediates of energy transfer. Their role is not merely restricted to the intracellular compartments, however, because they are also released into the extracellular space where they behave as mediators of cell–cell communication [1]. Vascular and circulating cells are able to release ATP by vesicular exocytosis, plasma membrane F(1)/F(0)-ATP synthase, ATP-binding cassette (ABC) transporters, connexin hemichannels, and pannexin channels [2] (Figure 1). Although F(1)/F(0)-ATP synthase has been linked to extracellular ATP production, its main activity may be ATP hydrolysis into ADP, thereby contributing to generation of this extracellular agonist [3]. The knowledge that extracellular molecules based on purines (ATP, ADP, and adenosine) or pyrimidines (UTP and UDP) are secreted and act as signaling molecules has prompted a thorough investigation of the network of nucleotide-activated receptors and nucleotide-transforming enzymes. Receptors for extracellular nucleotides have been grouped into P1 (P1Rs) and P2 (P2Rs) receptors. The first group, P1Rs, have the ATP metabolite adenosine as a common agonist; while the Corresponding author: Ferrari, D. ([email protected]). Keywords: atherosclerosis; extracellular ATP; ecto-nucleotidases; adenosine; P1 receptors; P2 receptors. 1471-4914/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2014.12.008
second group, P2Rs, is composed of membrane receptors activated by ATP and/or other nucleotides [4]. P1Rs include four receptors types: Adora 1 (A1R), Adora 2 (A2AR), Adora 2B (A2BR), and Adora 3 (A3R); while P2Rs have been distinguished in two subgroups: P2XRs (of which there are seven cloned human subtypes) and P2YRs (of which there are eight cloned human subtypes). Adenosine, as well as ADP or AMP, can result from the activity of CD39 (an ectonucleoside triphosphate diphosphohydrolase that converts ATP/ADP to AMP) and CD73 (an ecto-50 -nucleotidase that converts AMP to adenosine) [5]. Ecto-adenylate kinase and ecto-nucleoside diphosphate kinase (NDPK) are also involved in regulating extracellular levels of nucleotides [6]. Extracellular nucleotides have been classified as ‘damage-associated molecular patterns’ (DAMPs) (see Glossary) and can start and perpetuate the immune response, even
Glossary ATP-binding cassette (ABC) transporters: membrane proteins that couple energy derived from ATP hydrolysis to the efflux of a wide variety of compounds across the plasma membrane. Cholesterol: lipid molecule mainly produced by the liver. It is a fundamental constituent of the plasma membrane of animal cells and a precursor for the biosynthesis of vitamin D, bile acids, and steroid hormones. Cholesterol in animal fats consumed in the diet contributes to increase blood cholesterol concentration. Cholesterol 27-hydroxylase: enzyme involved in the conversion of cholesterol to oxysterols, thereby enhancing cholesterol efflux from the cell and its elimination by the liver. Damage-associated molecular patterns (DAMPs): molecules of the host cell that, once modified or released into the extracellular milieu upon cell stress or damage, can promote and perpetuate an immune response in non-infectious conditions. Foam cell: macrophage-derived cell filled with lipids. Hypoxia-inducible factors 1 and 2 (HIF1 and 2): a family of transcription factors transactivating a large number of genes in response to hypoxic conditions. Low-density lipoprotein (LDL): a plasmatic lipoprotein carrying cholesterol in the bloodstream. It contains the apolipoprotein apoB100. High LDL concentrations are associated with increased risk of atherosclerosis and coronary artery disease. Metabolic syndrome: the complex of physiological, biochemical, clinical, and metabolic factors that directly increases the risk of atherosclerotic cardiovascular disease. The main features of metabolic syndrome are hypertension, atherogenic dyslipidemia, glucose intolerance, and proinflammatory and prothrombotic conditions. Pathogen-associated molecular patterns (PAMPs): microbe-associated molecular motifs present in specific RNA, polypeptides, and sugar molecules that are rare or absent in host cells. These motifs are recognized by specific host receptors (pattern recognition receptors, PRRs) thus triggering an appropriate immune response. Reverse cholesterol transport (RCT): a process whereby excess cholesterol from peripheral tissues is transported by high-density lipoprotein (HDL) particles to the liver for further metabolism and biliary excretion. Scavenger receptor class A (SR-A): a subclass of pattern recognition receptors composed of five proteins able to bind modified lipoproteins. Shear stress: the force generated by the flowing blood acting on the endothelial wall of a vessel.
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ADO
AMP
ATP
ADP
G
G P2XRs Intracellular
P2YRs
F1-ATPase
CD39
CD73
P1Rs
ENT1 ENT2
Vesicular release ABC transporters Connexins Pannexins P2X7R TRENDS in Molecular Medicine
Figure 1. The purinergic network at the cell membrane. ATP is released in a regulated manner by exocytosis, in ATP-containing vesicles, and through different plasma membrane proteins such as connexins, pannexins, ATP-binding cassette (ABC) transporters, and P2X7R. Nucleotide release also occurs as a consequence of cellular stress or death, for example during infection, ischemia and reperfusion, or inflammation. ATP functions as a signaling molecule via activation of P2XR and P2YR subtypes. P2XRs are multimeric plasma membrane channels selective for monovalent and divalent cations (Na+, K+, Ca2+); their activation induces transmembrane ion fluxes, plasma membrane depolarization, caspase activation, nuclear factor transcription, and cytokine secretion. P2YRs are G protein-coupled receptors with seven transmembranespanning motifs; their activation induces calcium release from the intracellular stores via phospholipase C/inositol-1,4,5-triphosphate production or activation/inhibition of adenylate cyclase depending on subtype. P1Rs have been linked to the activation of different intracellular signaling pathways, the most common involving modulation of adenylyl cyclase with consequent decrease (A1R) or increase (A2R, A3R) in intracellular cAMP concentrations. In the extracellular space, ATP is also metabolized by enzymatic phosphohydrolysis in a two-step process via CD39 conversion of ATP (or ADP) to AMP, and CD73 phosphohydrolysis of AMP to adenosine (ADO), which is the agonist of P1Rs. P1R-mediated signaling is terminated by cellular uptake of adenosine by equilibrative nucleoside transporter 1 (ENT1) and ENT2.
in the absence of pathogen-associated molecular patterns (PAMPs; i.e., foreign molecules belonging to invading microorganisms) [7]. Although a moderate and controlled release of ATP or UTP exerts beneficial effects for the damaged tissue by activating reparative homeostatic responses, large and uncontrolled ATP release due to intense mechanical stress, trauma, allergen inhalation, or infection causes excessive activation of P2R-expressing immune and non-immune cells. These events are followed by an abnormal production of proinflammatory cytokines and inflammatory mediators such as prostaglandins, leukotrienes, and reactive oxygen intermediates (ROIs) that, besides causing tissue damage, induce a massive recruitment of immune cells leading to the establishment of chronic inflammatory conditions. The atherosclerotic process Atherosclerosis is the leading cause of cardiovascular disease, which accounts for approximately 40% of all deaths annually in the United States [8]. It has become clear that atherosclerosis is a chronic inflammation of arteries, where activated immune cells are abundant and play a major role in plaque progression and eventual rupture [9,10]. Atherosclerosis is characterized by a typical lesion called an atheroma (Figure 2). Modification of the artery endothelium begins with intima infiltration of circulating monocytes in response to different mediators, mainly lipids [11,12]. Cholesterol, transported in the bloodstream by low-density lipoproteins (LDLs), accumulates in the intima and triggers a deleterious cascade of events [13], culminating in the formation of the atheromatous plaque surrounded 2
by smooth muscle cells and a fibrous cap. The increasing volume of the plaque reduces the vasal lumen, with a heavy impact on blood flow. In late stages, lesional calcification can lead to plaque instability, and accumulation of platelets can result in the formation of a thrombus over the plaque, further interrupting blood flow. Upon accumulation of lipid molecules, macrophages change their phenotype and become foam cells, the prototypical cells of the atherosclerotic plaque. This event occurs when cholesterol inflow into the cell exceeds the outflow, and reverse cholesterol transport mediated by high-density lipoprotein (HDL) is insufficient to remove the excess cholesterol into the bloodstream. In addition to macrophages, intimal smooth muscle cells can also accumulate considerable amounts of cholesterol, contributing to the atherosclerotic process [14]. Oxidized LDL (oxLDL) prompts an immune reaction by activating endothelial cells (ECs) that upregulate adhesion molecules and secrete chemokines that attract T and B cells, macrophages, dendritic cells (DCs), and natural killer (NK) cells into the sub-endothelial region [15]. Atherosclerosis is reduced by inhibition of the T cell response to native LDL [16]. Most of the T lymphocytes found in atheromas are CD4+ with an effector memory phenotype and a helper T cell type 1 (TH1) profile, while the number of circulating regulatory T cells (Tregs) is reduced in patients with acute coronary syndrome and in apolipoprotein E-deficient mice (ApoE/) [17,18]. Depletion of FOXP3+ regulatory T cells favors hypercholesterolemia and atherosclerosis [19]. The most severe clinical complications of atherosclerosis arise from rupture of the plaque, which leads to exposure of pro-thrombotic substrates of the plaque core, thus causing thrombotic
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oxldl
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IL-8 P2Y6R
Dying foam cell VCAM-1 ICAM-1 P-selecn
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IL-1β
A2BR IL-6 Macrophage
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ATP CD39
CD73
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Foam cell
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CD73
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A2BR Fibroblasts TRENDS in Molecular Medicine
Figure 2. Putative schematic involvement of purinergic signaling in mouse models of atherogenesis. (Left) Genesis of the atherosclerotic plaque. Atheroma begins with a fatty streak almost exclusively populated by infiltrating monocytes that transmigrate through the endothelium. P2Y2R expression increases on endothelial cells (ECs), and its activation induces expression of adhesion molecules [vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and P-selectin] that favor the adhesion and extravasation of immune cells. P2Y2R expression on monocytes also enhances their migration across the endothelium. Once through the endothelium, monocytes differentiate into macrophages and then become foam cells. ATP is released by connexin hemichannel (Conn.), ABC transporter (ABC t.), and Pannexin 1 (Panx1). Released ATP activates P2X7R on macrophages, stimulating the secretion of IL-6 and IL-1b. ATP is also converted to ADP, AMP, and adenosine (ADO) by CD39 and CD73, which are expressed by different cell types including smooth muscle cells (SMCs). ADO activates A2BR on macrophages, which also acts to promote their transformation into foam cells. (Centre) Atheroprogression. Cell surface F1-ATPase/P2Y12R on ECs play an atheroprotective role by favoring high-density lipoprotein (HDL) transcytosis. SMCs and fibroblast proliferation is enhanced by ADO-mediated activation of A2BR. ADO also induces secretion of vascular endothelial growth factor (VEGF) and IL-8 by macrophages. ATP concentrations are increased by release from ECs, as well as from damaged and dying cells present in the atheroma. (Right) Plaque destabilization. Ruptures in the artery wall activate platelets to release of high amounts of ADP and ATP, leading to shape changes/aggregation and thrombi/emboli formation. ATP acts on proinflammatory receptors including P2X7R, which is expressed by macrophages, dendritic cells (DCs) and T lymphocytes, exacerbating the inflammatory conditions. Abbreviations: LDL, low-density lipoprotein; oxLDL, oxidized LDL; PF4, platelet factor 4; TNF-a, tumor necrosis factor a.
occlusion of the artery. Inflammatory cells also participate in plaque rupture, mainly by producing proteolytic enzymes that weaken the cap structure. In particular, macrophages secrete matrix metalloproteinases (MMPs) that cleave collagen to promote plaque rupture [20]. Purinergic signaling is putatively involved in all stages of atherosclerosis and can either foster or counteract disease development, depending on the type of purinergic receptors involved (Table 1). Purinergic signaling and atherosclerosis Purinergic signaling in atherogenesis Both cell associated and soluble mediators released from the cells into the fluid phase are involved in atherosclerosis. Among soluble mediators, extracellular nucleotides
play an important role. Most of the experimental evidence of the involvement of purinergic signaling in atherogenesis has been obtained from double-knockout animal models, for example ApoE/ mice lacking specific P1R, P2R subtypes, or ecto-nucleotidases. However, it is crucial to note that the results of studies employing these models are not immediately transferable to human disease because of existing differences in lipid metabolism between humans and mice. For example, mice do not develop atherosclerosis unless they are genetically modified and, moreover, the commonly used high-fat diet can elicit blood cholesterol concentrations as high as 1000 mg/dL, which far exceeds the values observed in the most severe forms of familial hypercholesterolemia in humans. In addition, mice differ 3
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Table 1. Pro- or anti-atherogenetic effects of P2Rs, P1Rs, CD39, and CD73a Receptor A1R
Agonist Adenosine
Cell/tissue Vessel wall
A2AR
Adenosine
Macrophages Macrophages
A2BR
Adenosine
Macrophages
A3R
Adenosine
VSMC Hepatocytes Macrophages
P2X7R
ATP
ECs
P2Y1R
ADP
DCs Vessel wall ECs
P2Y2R
ATP
ECs
P2Y6R
UDP
P2Y12R
ADP
P2Y13R CD39 CD73
ADP
Monocytes Macrophages ECs Platelets ECs Hepatocytes VSMCs Plaque infiltrating cells
Effect Increases proliferation and IL-5, IL-6, and IL-13 secretion HIF-1a accumulation HIF-1a accumulation Protection from apoptosis HIF-1a accumulation, VEGF and IL-8 secretion, FC formation Inhibition of proliferation Regulation of lipid levels HIF-1a accumulation, VEGF secretion, FC formation IL-1b release IL-1RA release TF release Enhanced gelatinase activity P-selectin, ICAM and VCAM upregulation Leukocyte rolling ROS production Upregulation of ICAM and VCAM Increased migration Increased IL-6 production and iNOS activation Enhanced response to proinflammatory stimuli Platelet aggregation, release of PF4, P-selectin expression uptake and transport of HDL Increased reverse cholesterol transport Neointima formation, VSMC migration Reduced atherosclerotic progression
Atherogenic +
Atheroprotective
+ + + +
Refs [39] [14] [14] [29] [14]
+ + + +
[60] [28] [14]
+ + +
[61] [61] [68] [38] [21]
+
[21]
+ + + +
[21] [37] [36] [24]
+
+ + + +
[70] [6,27,29,30] [69] [33]
a
Abbreviations: DCs, dendritic cells; ECs, endothelial cells; FC, foam cells; HIF, hypoxia inducible factor; ICAM, intercellular adhesion molecule; iNOS, inducible nitric oxide synthase; PF4, platelet factor 4; TF, tissue factor; UDP, uridine diphosphate; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor; VSMC, vascular smooth muscle cells.
from humans in vascular structure, hemostatic function, and the expression of some purinergic receptors (e.g., P2Y11R is not expressed in rodents). Cholesterol levels are a key factor in the initiation of atherosclerosis, and intracellular levels are regulated by HDL-mediated reverse cholesterol transport, by which cholesterol is discharged into the blood and transported to the liver. When cholesterol accumulates in the intima, monocytes are recruited into the vessel wall with the participation of the P2Y2R, which is upregulated in animal models as well as in human monocytes and ECs [21]. Notably, oxLDLs caused the release of ATP and reactive oxygen species (ROS) from in vitro stimulated ECs [22]. Costimulation of ECs with oxLDL and nucleotides induces P2Y1R- and P2Y2R-mediated increases in ICAM-1, VCAM-1, and P-selectin expression, potentially increasing leukocyte rolling and extravasation [21]. Hence, deletion of the P2Y1R in ApoE-deficient mice (P2y1//ApoE/) results in reduced vascular inflammation and diminished plaque size in the aorta as a result of decreased macrophage infiltration and smooth muscle cell proliferation [23]. Similarly, lack of P2Y12R expression in P2y12// ApoE/ mice reduced lesional areas and decreased monocyte/macrophage infiltration, while the fibrous content of the plaque was increased [24]. Further analysis showed that P2Y12R expressed by platelets was responsible for platelet factor 4 secretion and P-selectin expression, thereby inducing monocyte recruitment and infiltration [21]. Interestingly, P2Y13R expressed in mouse hepatocytes played a crucial role in hepatic HDL endocytosis, and the 4
P2Y13R agonist cangrelor (used as an anti-thrombotic drug) strongly activated P2Y13R-mediated HDL endocytosis [23–30]. The same effect was obtained by inhibiting the proteasome, which resulted in increased plasma membrane expression of the receptor [31]. These findings were later confirmed when the specific P2Y13R agonist CT1007900 was shown to promote in vitro cholesterol mobilization and to decrease plaque size in the aorta and carotids of ApoE/ mice [27]. Of note, P2Y13R-mediated HDL endocytosis is also active in human hepatocytes and is regulated by cell surface adenylate kinase [6]. P1R-mediated signaling also plays a role in response to plasma cholesterol and lipids. A2BR is able to regulate hyperlipidemia, exerting an indirect protective effect on the vessel wall. A2BR was upregulated in the liver as a consequence of a high-fat/cholesterol diet and its activation with the specific agonist BAY 60-6053 (Table 2) decreased cholesterol and triglyceride plasma levels, and reduced lesion formation [32]. In one report, lack of A2AR expression was associated with protecting ApoE-deficient mice from atherosclerosis [33]. However, signaling events triggered by A2AR engagement upregulated the expression of cholesterol 27-hydroxylase and ABCA1, two proteins of the reverse cholesterol transport system, thus exerting antiatherosclerotic and anti-inflammatory effects [32–36]. ABCA1 plays an anti-atherosclerotic function by promoting cholesterol efflux from the cell, thus reducing transformation of macrophages into foam cells. Interestingly, lack of expression of ABCA1 and G1 in macrophages increased inflammation and accelerated atherosclerosis in mice
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Table 2. P2R antagonists and P1R agonists available for preclinical and clinical trials Receptor Compound Effect Inhibition of channel opening All P2XR PPADS Suramin Inhibition of channel opening NF279 P2X1R NF449 NF023 NF-864 TNP-ATP PSB-10211 Inhibition of channel opening P2X2R NF770 NF778 Inhibition of channel opening A-317491 MK3901 P2X3R RO-3 RO-4 TNP-ATP AF 353 Inhibition of channel opening P2X3R; P2X2/3R PSB-12062 Inhibition of channel opening P2X4R Paroxetine 5-BDBD BBG P2X5R AZ D9056 P2X7 blockade P2X7R oATP MRS2427 RN-6189 AZ 10606120 JNJ-42253432 GSK1482160 Suppression of ATP induced IL-1b release P2X7R A-804598 AACBA Brilliant blue G (BBG) Inhibition of channel opening, P2X7R KN-62 inhibition of inflammasome activation, A-438079 suppression of ATP-induced IL-1b release, inhibition of IL-6 and CCL2 secretion Inhibition of platelet aggregation and of Ca2+ increase MRS2179 P2Y1R MRS2500 Inhibition of platelet aggregation P2Y1R AR-C 126313 Receptor inhibition P2Y2R Receptor inhibition MRS2577 P2Y4R MRS2578 Receptor inhibition P2Y6R NF157 Receptor inhibition P2Y11R NF340 Cangrelor Inhibition of platelet aggregation P2Y12R Clopidogrel Ticagrelor AR-C 66096 Receptor inhibition MRS2211 P2Y13R MRS2603 Receptor activation CT1007900 Partial agonist Cangrelor PPTN Receptor inhibition P2Y14R CHA Receptor activation, HIF-1a induction A1R Receptor activation CPPA CGS-21680 Receptor activation, increase in cholesterol efflux from macrophages A2AR 2HE-NECA Receptor activation CVT-3146 Receptor activation BAY 60-6053 Receptor activation, decreases lipids and atherosclerosis A2BR Compound 24 Receptor activation, HIF-1a induction Cl-IB-MECA Receptor activation, HIF-1a induction A3R IB-MECA MRS5698 Receptor activation
Human use a Refs N [61] N
[61]
N
[61]
N
[61]
Y
[73]
N
[74]
Y N
[75]
Y N
[76] [61]
Y N
[77,78] [77]
N N
[77] [77]
Y
[26,66]
N
[79]
N Y N
[28] [29] [80] [39]
N
[81]
N N N
[32] [39] [39]
a
Compounds already used in humans are indicated with (Y) while those employed in animals are indicated with (N).
[36]. This pathway is modulated by apelin-13-mediated activation of PKCa, which phosphorylates ABCA1 and inhibits its calpain-mediated proteolysis [37,38]. By contrast, purinergic signaling may also be involved in foam cell generation because blocking adenosine A2BR and A3R on macrophages reduces the formation of foam cells in vitro [39]. The removal of extracellular ATP and increases in adenosine concentration has an overall protective effect
against atherosclerosis. Cd73//ApoE/ mice have significantly elevated plasma triglycerides and atherosclerotic lesion size is increased by 50% compared to ApoE/ animals [40]. Purinergic signaling in atheroprogression Numerous reports have described an association between atherosclerosis and infection with pathogens such as 5
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Review Chlamydia pneumoniae, Helicobacter pylori, Epstein–Barr virus, or cytomegalovirus [41,42]. Moreover, T lymphocytes specific for different pathogens have been isolated from atherosclerotic lesions. It has recently been shown that bacterial lipopolysaccharide (LPS) increases the uptake of oxidized LDL by upregulating lectin-like oxidized LDL receptor-1 in mouse and human macrophages [43]. LPS also upregulates P2Y6R in ECs [44]. This receptor subtype is highly expressed in atherosclerotic aorta segments of ApoE/ mice, and blockade with suramin or pyridoxalphosphate-6-azophenyl-20 ,40 -disulfonic acid (PPADS) reduces the severity of atherosclerosis [45]. Engagement of the P2Y6R, as well as of the proinflammatory P2X7R (a receptor which is highly represented in the majority of human carotid artery plaques, luminal thrombi, and small plaque ulcerations) results in increased production of interleukin 6 (IL-6), a central cytokine in atherogenesis [46,47]. Deficiency or inhibition of A1R also reduces plasma concentrations of the proinflammatory cytokines IL-5, IL-6, and IL-13, and consequently reduces atherosclerotic lesions in ApoE-deficient mice [48]. Interferon-g (IFN-g) is also present in atherosclerotic lesions and its contribution in the genesis of atheroma has been well established [49]. This cytokine upregulates expression of the P2X7R, thus increasing the proinflammatory properties of macrophages; P2X7R engagement leads to the release of ATP and NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome activation, with consequent IL-1b secretion [50,51]. Notably, lack of IL-1b reduces atherosclerosis severity in ApoE-deficient mice [52], while another study has shown a prominent role for IL-1a in atherosclerosis [53]. However, convincing data have recently shown that activation of NLRP3 inflammasome by extracellular ATP or cholesterol crystals enhances macrophage lipid deposition and migration, suggesting a role of this complex in atherogenesis [54,55]. T cells are also present in atheroma. P2X4R and P2X7R stimulation induces T cell activation and proliferation through IL-2 secretion [56]. Activated T cells also secrete ATP, and Treg cells are sensitive to its cytotoxic effects [57]. This may explain the low numbers of Treg cells found in atherosclerotic plaques. Along with the detrimental effects described above, ATP secretion could have a positive effect because it downmodulates NK chemotaxis and killing capacity via P2Y11R activation [56]. Purinergic signaling in plaque destabilization A detailed description of the mechanisms underlining plaque destabilization is still missing, mainly owing to the lack of appropriate experimental models. The most evident event, however, is erosion and rupture of the fibrous cap surrounding the plaque as a result of the activity of MMPs secreted by macrophages [20]. So far, there is no direct evidence of participation of purinergic signaling in this process. However, P2X7R could be involved because its stimulation has been linked to release of MMPs [58]. Following plaque rupture, pro-thrombotic substrates are exposed, and high amounts of ATP and ADP are secreted from platelets, inducing thrombi formation. The development of transplant arteriosclerosis in 6
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P2y12/ mice further implicates this receptor in cell recruitment and vasal occlusion during atherosclerosis [59]. Hypoxic conditions in the plaque are caused by the absence of neo-vascularization and the presence of a surrounding cap. In vitro experiments under hypoxic conditions have shown activation of A2BR and A3R, inducing hypoxia-inducible factor 1a (HIF-1a)-dependent secretion of vascular endothelial growth factor (VEGF) and IL-8 secretion in an extracellular signal-regulated kinase (ERK) 1/2-, p38-, and Akt kinase-dependent manner [39]. Removal of dead cells from the plaque is also an important issue. Activation of the ATP channel pannexin 1 (PANX1) stimulates macrophage chemotaxis. PANX1 has a cleavage site for caspase 3 and caspase 7, and expression of truncated PANX1 protein results in a constitutively open channel able to continuously release ATP, thus stimulating phagocytosis of apoptotic cells [60]. At present it is not known whether this mechanism is involved in the elimination of apoptotic cells in the plaque. Potential therapeutic implications in cardiovascular diseases As cell-to-cell mediators, purines act as pro- or anti-atherosclerotic factors depending on the receptors engaged and the downstream signaling networks. It is particularly interesting that genetic deletion of specific purinergic receptors in mice results in decreased artery inflammation and reduced plaque size. This is true for those receptors whose responses are proinflammatory: A1R among adenosine receptors, P2X4R and P2X7R for P2X receptors, and P2Y1R, P2Y2R, and P2Y12R for the P2YR subgroup. Therefore, pharmacological inhibition of these receptors would likely dampen atherosclerotic inflammation and improve the disease state. This concept is strengthened by recent observations on therapies using reductase inhibitors or statins that may in part inhibit IFN-g-dependent macrophage activation in the atheromatous plaque. Owing to the fact that P2X7R is highly expressed in macrophages and is upregulated by IFN-g, it would be a candidate target for clinical trials. P2XR-specific antagonists have already been used in clinical trials for different pathologies, such as for pain, osteoporosis, spinal cord injury, bladder dysfunction, and cough [61] (Table 2). Another way to reduce atherosclerotic inflammation could be through the modulation of nucleotide-degrading enzymes. In several acute disease models, ecto-nucleotidases attenuate inflammation, prevent intimal hyperplasia, and preserve vascular function [62–64]. Moreover, it has to be underlined that activity of CD39 and CD73 could be lower in atherosclerotic human tissues, as for example in thoracic aortas, lymph nodes, spleen, and serum of young and mature ApoE/ mice where nucleoside triphosphate diphosphohydrolase (NTPDase) activity decreases up to 50%, favoring local accumulation of ATP and ADP, and eventually exacerbating atherosclerosis [65]. In mice, stimulation of A2AR or activation of P2YR13 upregulates HDL-c metabolism by promoting reverse cholesterol transport, decreasing macrophage transformation into foam cells [26,34,35,65]. This may also provide an opportunity for intervention in humans. Lesions of the coronary and carotid arteries contribute to stroke and
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Review Box 1. Outstanding questions Would it be possible to pharmacologically target single molecules of the purinergic signaling network to treat atherosclerosis, without impairing positive purinergic-mediated functions in the body? What ATP-releasing molecules (connexins, pannexins, ABC transporters, P2X7R) play a major role in atherosclerosis? Are CD39 and CD73 still active in cells forming the atherosclerotic plaque?
myocardial infarction by the formation of thrombi and emboli, occluding vessels and resulting in ischemia. Very recent data show that the P2Y12R inhibitor clopidogrel, commonly used as an anti-thrombotic drug, also reduced lipid peroxidation, systemic inflammation, and aortic expression of inflammatory markers [66,67]. Precisely defining the role of the purinergic signaling network in the atherogenic process is an urgent issue, particularly for those receptor subtypes such as P2Y1R that can have both pro- or anti-atherogenic activity depending on the cell type. P2Y1R stimulates human endothelial cell migration via mitogen-activated protein kinase pathways but also coronary vasodilation via an endothelium/nitric oxide-dependent mechanism [68,69]. Similarly, P2Y12R function is pro-atherogenic in platelets and SMCs by stimulating vasoconstriction, proliferation, and inflammation; further investigation will be necessary to establish whether P2Y12R-stimulated transcytosis of HDL through the endothelium decreases cholesterol levels in the subendothelium region [70]. Therefore, clarification of the roles played by single receptors is highly desirable and will be instrumental for the design of innovative therapeutic interventions to counteract atherosclerosis (Box 1). Concluding remarks and future perspectives Atherosclerosis can be reduced by improved diet and regular physical exercise, steps that represent the most successful approach for preventing and counteracting the effects of metabolic syndrome [8]. However, recent findings suggest that formation of the atherosclerotic lesions is prompted or facilitated by concurring secondary causes; hence, proinflammatory responses by immune cells contribute to the subclinical inflammatory status characterizing atheromasic lesion-bearing patients, increasing their cardiovascular risk [8]. Atherosclerotic inflammation can occur even in the absence of microbes, as shown by studies using germ-free animals, which suggests that one or more endogenous substrates are endowed with the ability to start atherosclerotic inflammation. Autoimmune reactions towards heat-shock protein 60 (HSP60) and HSP90, highly expressed in the cytoplasm of eukaryotic cells under nonstress conditions, and that are secreted by vascular smooth muscle cells in response to oxidative stress, has been indicated as concurring cause of atherosclerosis [71,72]. Cholesterol crystals can trigger inflammation by activating the NLRP3 inflammasome [53], and dying cells of the necrotic centre release nucleotides acting as DAMP signals for P2R-expressing immune cells [7]. In this context, in vivo trials of compounds able to positively or negatively modulate the molecular components of the purinergic network
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according to their main activity in atherogenesis may produce valuable information. P2R antagonists, as well as new atheroprotective pharmacological P1R agonists, may be useful to block the initial atherosclerotic inflammation and counteract its transition to chronic disease. It is extremely urgent to exploit the enormous potential of the purinergic signaling to find new treatments for atherosclerosis. Collaborations between purinologists, immunologists, and cardiologists will likely result in the design of new drugs targeting proteins of the purinergic signaling network for treating the disease. Acknowledgments This work is supported in part by the Commission of European Union (2012-2014, ERA-NET NEURON program, Role of Danger Signals in Stroke and Therapeutic Targeting by Nanobodies). We apologize to all whose work we could not cite owing to space limitations.
References 1 Burnstock, G. (2012) Purinergic signalling: its unpopular beginning, its acceptance and its exciting future. Bioessays 34, 218–225 2 Lohman, A.W. et al. (2012) Mechanisms of ATP release and signalling in the blood vessel wall. Cardiovasc. Res. 95, 269–280 3 Vantourout, P. et al. (2010) Ecto-F1-ATPase: a moonlighting protein complex and an unexpected apoA-I receptor. World J. Gastroenterol. 16, 5925–5935 4 Burnstock, J. (1978) A basis for distinguishing two types of purinergic receptor. In Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach (Straub, R.W. and Bolis, L., eds), pp. 107–118, Raven Press 5 Zimmermann, H. et al. (2012) Cellular function and molecular structure of ecto-nucleotidases. Purinergic Signal. 8, 437–502 6 Fabre, A.C. et al. (2006) Cell surface adenylate kinase activity regulates the F(1)-ATPase/P2Y (13)-mediated HDL endocytosis pathway on human hepatocytes. Cell. Mol. Life Sci. 63, 2829–2837 7 Idzko, M. et al. (2014) Nucleotide signalling during inflammation. Nature 509, 310–317 8 Jamkhande, P.G. et al. (2014) Therapeutic approaches to drug targets in atherosclerosis. Saudi Pharm. J. 22, 179–190 9 Hansson, G.K. and Hermansson, A. (2011) The immune system in atherosclerosis. Nat. Immunol. 12, 204–212 10 Ross, R. (1999) Atherosclerosis – an inflammatory disease. N. Engl. J. Med. 340, 115–126 11 Mullick, A.E. et al. (2008) Increased endothelial expression of Toll-like receptor 2 at sites of disturbed blood flow exacerbates early atherogenic events. J. Exp. Med. 205, 373–383 12 Flood, C. et al. (2004) Molecular mechanism for changes in proteoglycan binding on compositional changes of the core and the surface of low-density lipoprotein-containing human apolipoprotein B100. Arterioscler. Thromb. Vasc. Biol. 24, 564–570 13 Shi, Y. et al. (2011) Oxidized low-density lipoprotein activates p66Shc via lectin-like oxidized low-density lipoprotein receptor-1, protein kinase C-beta, and c-Jun N-terminal kinase kinase in human endothelial cells. Arterioscler. Thromb. Vasc. Biol. 31, 2090–2097 14 Allahverdian, S. et al. (2014) Contribution of intimal smooth muscle cells to cholesterol accumulation and macrophage-like cells in human atherosclerosis. Circulation 129, 1551–1559 15 Nakashima, Y. et al. (1998) Upregulation of VCAM-1 and ICAM-1 at atherosclerosis-prone sites on the endothelium in the ApoE-deficient mouse. Arterioscler. Thromb. Vasc. Biol. 18, 842–851 16 Hermansson, A. (2010) Inhibition of T cell response to native lowdensity lipoprotein reduces atherosclerosis. J. Exp. Med. 2075, 1081–1093 17 Buono, C. et al. (2005) T-bet deficiency reduces atherosclerosis and alters plaque antigen-specific immune responses. Proc. Natl. Acad. Sci. U.S.A. 102, 1596–1601 18 Liu, Z.D. (2012) Increased Th17 cell frequency concomitant with decreased Foxp3+ Treg cell frequency in the peripheral circulation of patients with carotid artery plaques. Inflamm. Res. 61, 1155–1165 7
TRMOME-1011; No. of Pages 9
Review 19 Klingenberg, R. (2013) Depletion of FOXP3+ regulatory T cells promotes hypercholesterolemia and atherosclerosis. J. Clin. Invest. 123, 1323–1334 20 Newby, A.C. (2008) Metalloproteinase expression in monocytes and macrophages and its relationship to atherosclerotic plaque instability. Arterioscler. Thromb. Vasc. Biol. 28, 2108–2114 21 Eun, S.Y. et al. (2014) P2Y2R activation by nucleotides released from oxLDL-treated endothelial cells (ECs) mediates the interaction between ECs and immune cells through RAGE expression and reactive oxygen species production. Free Radic. Biol. Med. 69, 157–166 22 Seye, C.I. et al. (2002) Functional P2Y2 nucleotide receptors mediate uridine 50 -triphosphate-induced intimal hyperplasia in collared rabbit carotid arteries. Circulation 106, 2720–2726 23 Zerr, M. et al. (2011) Major contribution of the P2Y1 receptor in purinergic regulation of TNFa-induced vascular inflammation. Circulation 123, 2404–2413 24 Hechler, B. et al. (2008) Reduced atherosclerotic lesions in P2Y1/ apolipoprotein E double-knockout mice: the contribution of nonhematopoietic-derived P2Y1 receptors. Circulation 118, 754–763 25 Li, D. et al. (2012) Roles of purinergic receptor P2Y, G protein-coupled 12 in the development of atherosclerosis in apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. 32, e81–e89 26 Jacquet, S. et al. (2005) The nucleotide receptor P2Y13 is a key regulator of hepatic high-density lipoprotein (HDL) endocytosis. Cell. Mol. Life Sci. 62, 2508–2515 27 Fabre, A.C. et al. (2010) P2Y13 receptor is critical for reverse cholesterol transport. Hepatology 52, 1477–1483 28 Goffinet, M. et al. (2014) P2Y13 receptor regulates HDL metabolism and atherosclerosis in vivo. PLoS ONE 9, e95807 29 Serhan, N. et al. (2013) Chronic pharmacological activation of P2Y13 receptor in mice decreases HDL-cholesterol level by increasing hepatic HDL uptake and bile acid secretion. Biochim. Biophys. Acta 1831, 719–725 30 Lichtenstein, L. et al. (2013) Lack of P2Y13 in mice fed a high cholesterol diet results in decreased hepatic cholesterol content, biliary lipid secretion and reverse cholesterol transport. Nutr. Metab. (Lond.) 10, 67 31 Pons, V. et al. (2014) Role of the ubiquitin-proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake. Cell. Mol. Life Sci. 71, 1775–1788 32 Koupenova, M. et al. (2012) A2b adenosine receptor regulates hyperlipidemia and atherosclerosis. Circulation 125, 354–363 33 Wang, H. et al. (2009) Inactivation of the adenosine A2A receptor protects apolipoprotein E-deficient mice from atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 29, 1046–1052 34 Bingham, T.C. et al. (2010) A2A adenosine receptor stimulation decreases foam cell formation by enhancing ABCA1-dependent cholesterol efflux. J. Leukoc. Biol. 87, 683–690 35 Bingham, T.C. et al. (2012) Cholesterol 27-hydroxylase but not apolipoprotein apoE contributes to A2A adenosine receptor stimulated reverse cholesterol transport. Inflammation 35, 49–57 36 Westerterp, M. et al. (2013) Deficiency of ATP-binding cassette transporters A1 and G1 in macrophages increases inflammation and accelerates atherosclerosis in mice. Circ. Res. 112, 1456–1465 37 Martinez, L. et al. (2003) Phosphorylation of a pest sequence in ABCA1 promotes calpain degradation and is reversed by ApoA-I. J. Biol. Chem. 278, 37368–37374 38 Liu, X.Y. et al. (2013) Apelin-13 increases expression of ATP-binding cassette transporter A1 via activating protein kinase C a signalling in THP-1 macrophage-derived foam cells. Atherosclerosis 226, 398–407 39 Gessi, S. et al. (2010) Adenosine modulates HIF-1a, VEGF, IL-8, and foam cell formation in a human model of hypoxic foam cells. Arterioscler. Thromb. Vasc. Biol. 30, 90–97 40 Buchheiser, A. et al. (2011) Inactivation of CD73 promotes atherogenesis in apolipoprotein E-deficient mice. Cardiovasc. Res. 92, 338–347 41 Ayada, K. (2009) Chronic infections and atherosclerosis. Clin. Rev. Allergy Immunol. 37, 44–48 42 van der Meer, J.J. (2008) Multiple bacteria contribute to intraplaque T-cell activation in atherosclerosis. Eur. J. Clin. Invest. 38, 857–862 43 Hossain, E. et al. (2014) Lipopolysaccharide augments the uptake of oxidized LDL by up-regulating lectin-like oxidized LDL receptor-1 in 8
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macrophages. Mol. Cell. Biochem. Published online October 28, 2014. http://dx.doi.org/10.1007/s11010-014-2259-0 Riegel, A.K. et al. (2011) Selective induction of endothelial P2Y6 nucleotide receptor promotes vascular inflammation. Blood 117, 2548–2555 Guns, P.J. et al. (2010) P2Y receptors and atherosclerosis in apolipoprotein E-deficient mice. Br. J. Pharmacol. 159, 326–336 Piscopiello, M. et al. (2013) P2X7 receptor is expressed in human vessels and might play a role in atherosclerosis. Int. J. Cardiol. 168, 2863–2866 Hartman, J. and Frishman, W.H. (2014) Inflammation and atherosclerosis: a review of the role of interleukin-6 in the development of atherosclerosis and the potential for targeted drug therapy. Cardiol. Rev. 22, 147–151 Teng, B. et al. (2014) A1 adenosine receptor deficiency or inhibition reduces atherosclerotic lesions in apolipoprotein E deficient mice. Cardiovasc. Res. 102, 157–165 Leon, M.L. and Zuckerman, S.H. (2005) Gamma interferon: a central mediator in atherosclerosis. Inflamm. Res. 54, 395–411 Pellegatti, P. et al. (2005) A novel recombinant plasma membranetargeted luciferase reveals a new pathway for ATP secretion. Mol. Biol. Cell 16, 3659–3665 Ferrari, D. et al. (2006) The P2X7 receptor: a key player in IL-1 processing and release. J. Immunol. 176, 3877–3883 Kirii, H. et al. (2003) Lack of interleukin-1beta decreases the severity of atherosclerosis in ApoE-deficient mice. Arterioscler. Thromb. Vasc. Biol. 23, 656–660 Freigang, S. et al. (2013) Fatty acid-induced mitochondrial uncoupling elicits inflammasome-independent IL-1a and sterile vascular inflammation in atherosclerosis. Nat. Immunol. 14, 1045–1053 Duewell, P. et al. (2010) NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 464, 1357–1361 Li, X. et al. (2014) Activation of Nlrp3 inflammasomes enhances macrophage lipid-deposition and migration: implication of a novel role of inflammasome in atherogenesis. PLoS ONE 9, e87552 Gorini, S. et al. (2010) ATP secreted by endothelial cells blocks CXCL 1elicited natural killer cell chemotaxis and cytotoxicity via P2Y11R. Blood 116, 4492–4500 Trabanelli, S. et al. (2012) Extracellular ATP exerts opposite effects on activated and regulatory CD4+ T cells via purinergic P2 receptor activation. J. Immunol. 189, 1303–1310 Gu, B.J. and Wiley, J.S. (2006) Rapid ATP-induced release of matrix metalloproteinase 9 is mediated by the P2X7 receptor. Blood 107, 4946–4953 Harada, K. et al. (2011) Adenosine diphosphate receptor P2Y12mediated migration of host smooth muscle-like cells and leukocytes in the development of transplant arteriosclerosis. Transplantation 92, 148–154 Chekeni, F.B. et al. (2010) Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis. Nature 467, 863–867 North, R.A. and Jarvis, M.F. (2013) P2X receptors as drug targets. Mol. Pharmacol. 83, 759–769 Marcus, A.J. et al. (2003) Metabolic control of excessive extracellular nucleotide accumulation by CD39/ecto-nucleotidase-1: implications for ischemic vascular diseases. J. Pharmacol. Exp. Ther. 305, 9–16 Deaglio, S. and Robson, S.C. (2011) Ectonucleotidases as regulators of purinergic signaling in thrombosis, inflammation, and immunity. Adv. Pharmacol. 61, 301–332 Kaczmarek, E. and Koziak, K. (2011) Targeting stenosis with nucleotide-hydrolyzing enzymes. Curr. Pharm. Biotechnol. 12, 1799– 1804 Voloshyna, I. et al. (2013) Adenosine A(2A) receptor activation supports an atheroprotective cholesterol balance in human macrophages and endothelial cells. Biochim. Biophys. Acta 1831, 407–416 Angiolillo, D.J. (2012) Pharmacodynamic effects of cangrelor and clopidogrel: the platelet function substudy from the cangrelor versus standard therapy to achieve optimal management of platelet inhibition (CHAMPION) trials. J. Thromb. Thrombolysis 34, 44–55 Hadi, N.R. et al. (2013) Antiatherosclerotic potential of clopidogrel: antioxidant and anti-inflammatory approaches. Biomed. Res. Int. 2013, 790263
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Review 68 Shen, J. and DiCorleto, P.E. (2008) ADP stimulates human endothelial cell migration via P2Y1 nucleotide receptor-mediated mitogen-activated protein kinase pathways. Circ. Res. 102, 448–456 69 da Silva, C.G. et al. (2009) Mechanism of purinergic activation of endothelial nitric oxide synthase in endothelial cells. Circulation 119, 871–879 70 Cavelier, C. et al. (2012) The b-chain of cell surface F(0)F(1) ATPase modulates apoA-I and HDL transcytosis through aortic endothelial cells. Arterioscler. Thromb. Vasc. Biol. 32, 131–139 71 Benagiano, M. et al. (2005) Human 60-kDa heat shock protein is a target autoantigen of T cells derived from atherosclerotic plaques. J. Immunol. 174, 6509–6517 72 Businaro, R. (2009) Heat-shock protein 90: a novel autoantigen in human carotid atherosclerosis. Atherosclerosis 207, 74–83 73 Gever, J.R. et al. (2010) AF-353 a novel potent and orally bioavailable P2X3/P2X2/3 receptor antagonist. Br. J. Pharmacol. 160, 1387–1398 74 Hernandez-Olmos, V. et al. (2012) N-substituted phenoxazine and acridone derivatives: structure-activity relationships of potent P2X4 receptor antagonists. J. Med. Chem. 55, 9576–9588
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75 Keystone, E.C. (2012) Clinical evaluation of the efficacy of the P2X7 purinergic receptor antagonist AZD9056 on the signs and symptoms of rheumatoid arthritis in patients with active disease despite treatment with methotrexate or sulphasalazine. Ann. Rheum. Dis. 71, 1630–1635 76 Ali, Z. et al. (2013) Pharmacokinetic and pharmacodynamic profiling of a P2X7 receptor allosteric modulator GSK1482160 in healthy human subjects. Br. J. Clin. Pharmacol. 75, 197–207 77 Burnstock, G. and Ralevic, V. (2013) Purinergic signaling and blood vessels in health and disease. Pharmacol. Rev. 66, 102–192 78 Dawood, B.B. (2007) Reference curves for aggregation and ATP secretion to aid diagnose of platelet-based bleeding disorders: effect of inhibition of ADP and thromboxane A(2) pathways. Platelets 18, 329–345 79 Kim, Y.C. (2005) Synthesis of pyridoxal phosphate derivatives with antagonist activity at the P2Y13 receptor. Biochem. Pharmacol. 70, 266–274 80 Barrett, M.O. (2013) A selective high-affinity antagonist of the P2Y14 receptor inhibits UDP-glucose-stimulated chemotaxis of human neutrophils. Mol. Pharmacol. 84, 41–49 81 Reiss, A.B. and Cronstein, B.N. (2012) Regulation of foam cells by adenosine. Arterioscler. Thromb. Vasc. Biol. 32, 879–886
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