ATP Inside and Out: Understanding Atherosclerosis Through Energy Metabolism and Purinergic Signaling

Abstract

Adenosine triphosphate (ATP) is well known as the major intracellular energy carrier. It also functions as a potent extracellular signaling molecule. In atherosclerosis, both intracellular and extracellular ATP have been increasingly implicated in the disease pathophysiology. The intracellular ATP levels are closely regulated by the balance among the metabolic pathways, including glycolysis and oxidative phosphorylation (OXPHOS), and they play diverse and cell type-specific roles in atherosclerotic plaques. In contrast, the extracellular ATP released from these cells binds to purinergic P2 receptors and activates downstream signaling cascades primarily in an autocrine or paracrine manner, thereby regulating various cellular functions during disease progression. These two contrasting ATP-associated pathways are involved in a wide range of atherosclerosis-related processes including inflammation, vascular tone and barrier function, cell proliferation, migration and differentiation, and calcification, some of which are shared or highly context-dependent. This review summarizes the fundamental aspects of ATP biology, discusses the current approaches for measuring ATP concentrations in atherosclerotic plaques, and highlights the functions of intra- and extracellular ATP in major atherosclerosis-related cell types, including endothelial cells, macrophages and vascular smooth muscle cells (VSMCs), to clarify these intricately intertwined pathophysiological processes. Finally, this overview provides insights into the current challenges in ATP biology research and outlines therapeutic opportunities targeting intra- or extracellular ATP in atherosclerotic disease.

Introduction

Atherosclerosis is a form of vascular aging characterized by stiffening and thickening of the vascular wall due to the formation of atherosclerotic plaques. As the disease progresses, it leads to vascular stenosis and occlusion, resulting in fatal events such as myocardial and cerebral infarction ¹⁾ . Over the past several decades, considerable progress has been made in the management of major risk factors for atherosclerosis, including hypertension, dyslipidemia, and diabetes mellitus, and new treatment modalities, such as stent implantation, have been developed. However, atherosclerotic disease remains one of the leading causes of death worldwide ²⁾ .

Atherosclerosis is initiated by the subendothelial retention of apolipoprotein B-containing lipoproteins, including low-density lipoprotein (LDL), in the arterial intima. Retained LDL undergoes oxidative and other atherogenic modifications, which activate endothelial cells and vascular smooth muscle cells (VSMCs) and induce the expression of adhesion molecules, chemokines, and growth factors. These changes promote the recruitment of circulating monocytes to the intima. Monocyte-derived macrophages or resident intimal macrophages take up modified lipoproteins and become foam cells, thereby contributing to the formation and progression of atherosclerotic plaques. As lesions advance, smooth muscle cell migration, proliferation, and extracellular matrix remodeling shape the plaque architecture and stability. Progressive luminal narrowing can cause stable angina, whereas plaque rupture or erosion triggers thrombus formation, which can cause acute arterial occlusion and downstream ischemic injury ^{1 , 3 - 5)} .

Adenosine triphosphate (ATP) is a nucleoside triphosphate consisting of adenosine and three phosphate groups linked to the 5’ carbon of ribose. ATP contains two phosphoanhydride bonds, and its hydrolysis provides free energy to support a wide range of cellular processes. For this reason, ATP is commonly described as the universal energy currency of living cells. Under standard biochemical conditions, the hydrolysis of ATP to adenosine diphosphate (ADP) and inorganic phosphate is associated with a free-energy change of approximately −30.5 kJ/mol, although the actual change in cells is typically more negative depending on cell conditions. ADP can be subsequently rephosphorylated to regenerate ATP. Cellular ATP is generated mainly by glycolysis in the cytosol and oxidative phosphorylation (OXPHOS) in the mitochondria, with the latter producing most ATP in many cell types ⁶⁾ . The end product of glycolysis, pyruvate, can be converted to acetyl-CoA, which enters the tricarboxylic acid (TCA) cycle and in turn supports OXPHOS. In addition, metabolites derived from fatty acid β-oxidation and amino acid catabolism can also fuel the TCA cycle and support OXPHOS. Notably, however, some cell types, such as endothelial cells, depend predominantly on glycolysis for ATP production ⁷⁾ .

Several pathophysiological processes involved in atherosclerosis progression are energetically demanding and depend on an adequate intracellular ATP supply. In endothelial cells, ATP supports the metabolically active state required for inflammatory activation, adhesion molecule expression, cytoskeletal remodeling, and barrier regulation ^{8 - 9)} . During monocyte recruitment, intracellular ATP is likely required for chemotaxis and for the integrin- and actin–myosin-dependent cytoskeletal dynamics necessary for migration and transendothelial migration ^{9 - 10)} . In macrophages, ATP contributes to lipid handling, including ATP-dependent fatty acid activation and ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux ^{11 - 12)} . Therefore, intracellular ATP homeostasis and cellular energy metabolism are likely to be important in atherosclerosis development.

Extracellular ATP plays a distinct role as a signaling molecule, in contrast to the intracellular role of ATP as an energy carrier. Various stimuli, including tissue injury, inflammation, and hypoxia, induce the release of ATP from cells. Once released, extracellular ATP activates purinergic P2 receptors on neighboring cells or on the cells themselves. It is then rapidly hydrolyzed by ectonucleotidases, mainly CD39 and CD73, which ultimately generates adenosine. Therefore, extracellular ATP primarily functions in an autocrine or paracrine manner ^{13 - 15)} . Given these differences, intracellular and extracellular ATP likely exert distinct effects on atherosclerosis development.

The Concentration of ATP in Atherosclerotic Plaques

Because intracellular ATP levels can change rapidly during tissue harvest and processing, an accurate assessment of plaque ATP levels in vivo is technically challenging. Levin et al. developed a bioluminescence metabolic imaging technique that enables the mapping of local ATP concentrations in snap-frozen cryosections of arterial tissue and atherosclerotic plaques ¹⁶⁾ . Using this approach, they demonstrated ATP depletion in the deep, macrophage-rich core of severe rabbit atherosclerotic plaques. In ex vivo experiments, hyperoxic incubation of aortic rings containing severe plaques reversed ATP depletion in viable areas of the plaque core, suggesting that insufficient oxygen supply contributes to plaque ATP depletion, although limited glucose availability may also play an important role ^{17 )} . In a subsequent human study using carotid endarterectomy specimens, the ATP concentrations were markedly lower in advanced plaques than in intermediate plaques. Moreover, within advanced plaques, the ATP concentrations were significantly lower in the perinecrotic zone than in the luminal zone ¹⁸⁾ .

The measurement of ATP concentrations in vivo is technically more challenging. Recently, Ohnishi et al. developed the AVID mouse, a genetically engineered mouse that ubiquitously expresses the FRET-based ATP biosensor GO-ATeam2, thus enabling real-time in vivo ATP imaging ¹⁹⁾ . To measure ATP levels in atherosclerotic lesions, we crossed these mice with Apoe^−/− mice and fed them a western diet to induce atherosclerotic plaques. Under anesthesia, the common carotid artery was surgically exposed, and ATP signals in plaque lesions were assessed by measuring the FRET/GFP ratio. Using this approach, Kyoto University Substance (KUS) 121, a selective modulator of valosin-containing protein (VCP) ATPase activity, was shown to maintain higher ATP signals in plaque lesions than in control-treated mice ²⁰⁾ . However, this approach does not allow for the precise determination of absolute ATP concentrations or detailed spatial ATP distribution within atherosclerotic plaques. In addition, conditional GO-ATeam2 mice have recently been generated, enabling ATP imaging in selected cell populations in vivo by crossing GO-ATeam2^flox/＋ mice with cell type-specific Cre lines ²¹⁾ .

Taken together, although ATP measurement in atherosclerosis remains challenging, recent technical advances have shown that ATP is depleted in the deep regions of atherosclerotic plaques. Furthermore, mice carrying cell type-specific ATP biosensors may enable the monitoring of ATP levels in individual cell populations, such as endothelial cells, macrophages, and smooth muscle cells, within atherosclerotic plaques.

The Role of ATP in Major Cell Types in Atherosclerosis

1. Endothelial Cells

Endothelial cells are metabolically distinct in that most of their ATP is generated through glycolysis (approximately 80-85%) rather than through OXPHOS ⁷⁾ . Low or disturbed shear stress, an atherogenic hemodynamic condition, induces hypoxia-inducible factor 1α (HIF-1α) in endothelial cells and promotes endothelial proliferation and inflammation at least in part through glycolytic activation ²²⁾ . Consistent with this concept, endothelial cell-specific haplodeficiency of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key regulator of glycolytic flux, attenuates atherosclerosis progression ²³⁾ . Because phosphofructokinase-1 (PFK-1) catalyzes the first committed step of glycolysis, namely the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate, and is allosterically inhibited by ATP, preservation of intracellular ATP levels under ATP-depleting conditions may restrain glycolytic flux in endothelial cells. These observations raise the possibility that the maintenance of intracellular ATP may suppress atherogenic endothelial responses by attenuating glycolytic flux. Adequate intracellular ATP is also necessary to maintain endothelial barrier function. The depletion of intracellular ATP by the combined inhibition of oxidative and glycolytic ATP production with KCN and 2-deoxy-D-glucose (2-DG) increases endothelial permeability, accompanied by the disintegration of the actin cytoskeleton, opening of intercellular gaps, rapid loss of cytosolic Ca^2＋, and F-actin fragmentation ^{24 - 25)} . Nitric oxide (NO) is a crucial vasodilating mediator and it is produced predominantly by endothelial cells. AMP-activated protein kinase (AMPK) is a cellular energy sensor that is activated by an increase in the cellular AMP/ATP ratio. In human aortic endothelial cells, the direct activation of AMPK, as well as rosiglitazone-induced increases in the ADP/ATP ratio, stimulates endothelial NO synthase (eNOS)-dependent NO production ^{26 - 27)} . Moreover, mitochondrial ATP production has also been reported to be important for endothelial control of vascular tone ²⁸⁾ . During atherosclerosis development, endothelial cells can acquire fibroblast-like and, to a lesser extent, smooth muscle-like phenotypes in a process known as endothelial to mesenchymal transition (EndMT). EndMT is thought to contribute to atherosclerosis progression and plaque vulnerability ²⁹⁾ . Although direct evidence linking intracellular ATP levels to EndMT remains limited, a high-glucose-induced EndMT model showed reduced intracellular ATP levels in EA.hy926 endothelial cells ³⁰⁾ . In addition, TGF-β-induced EndMT is accompanied by metabolic reprogramming, including enhanced glycolysis ³¹⁾ . These findings suggest that metabolic dysregulation in EndMT is associated with intracellular ATP depletion, although whether intracellular ATP depletion is a driver of EndMT or a secondary consequence of this process remains unclear.

As briefly mentioned above, KUS121 is a compound specifically designed to mildly inhibit the ATPase activity of VCP. VCP, which belongs to the AAA (ATPases Associated with diverse cellular Activities) family, is a ubiquitously expressed protein found across essentially all cell types. Therefore, KUS121 can reduce intracellular ATP consumption ³²⁾ . In Apoe^−/− mice fed a western diet, daily administration of KUS121 reduced atherosclerotic lesion burden by approximately 40–50%, without major changes in serum lipid profiles. Notably, C/EBP homologous protein (CHOP) expression was found predominantly in plaque endothelium, suggesting that endothelial cells are a major site of endoplasmic reticulum (ER) stress in this model. In EA.hy926 endothelial cells, KUS121 preserved intracellular ATP levels under tunicamycin-induced ER stress and attenuated ER stress responses. KUS121 also reduced ER stress-associated apoptosis and inflammatory signaling. Importantly, methyl pyruvate (MePyr) similarly increased the intracellular ATP levels and attenuated ER stress-induced apoptotic and inflammatory pathways, supporting a mechanistic role of intracellular ATP preservation in endothelial cells. Moreover, even in lipopolysaccharide (LPS)-stimulated endothelial cells without overt ER stress, KUS121 restored the intracellular ATP levels, reduced lactate production, and suppressed inflammatory gene expression, suggesting that ATP preservation can also dampen endothelial inflammation by inhibiting glycolytic flux. Collectively, these findings suggest that the maintenance of intracellular ATP in endothelial cells may attenuate atherosclerosis through the suppression of both ER stress and glycolysis ²⁰⁾ .

Endothelial cells express several purinergic P2 receptors, and extracellular ATP signaling has been implicated in atherosclerosis-related endothelial responses. Oxidized LDL (oxLDL) rapidly induces ATP release from EA.hy926 endothelial cells. The released nucleotides activate the P2Y2 receptor (P2Y2R), leading to the upregulation of the receptor for advanced glycation end products (RAGE) and subsequent RAGE-dependent activation of NADPH oxidase and reactive oxygen species (ROS) production. This signaling cascade increases the expression of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), thereby promoting THP-1 monocyte migration toward and adhesion to endothelial cells ³³⁾ . P2Y2R activation also enhances mitochondrial ROS generation and promotes mitochondrial DNA expression and cytosolic release, which in turn increases caspase-1 activity and interleukin-1β (IL-1β) production ³⁴⁾ . In parallel, the atheroprone flow, characterized by low and oscillatory shear stress, renders endothelial cells more responsive to extracellular ATP by increasing the expression of P2X7 receptor (P2X7R). Under these conditions, P2X7R stimulation enhances extracellular calcium influx, leading to p38 phosphorylation, E-selectin upregulation, and IL-8 secretion. Consistently, en face staining of the murine aorta demonstrated increased endothelial P2X7R expression in atheroprone regions ³⁵⁾ . Conversely, shear stress can induce ATP release from endothelial cells. The released ATP stimulates NO production in endothelial cells mainly through P2Y2R- or P2X4 receptor (P2X4R)-mediated Ca^2＋ signaling and eNOS activation, thereby promoting vasodilation via adjacent VSMCs ^{36 - 37)} .

Consistent with these data, while the supplementation of intracellular ATP by MePyr attenuated apoptosis and inflammatory responses under tunicamycin-induced ER stress, stimulation with extracellular ATP tended to enhance these responses in endothelial cells ²⁰⁾ . Collectively, ATP may play a dual role in endothelial cells during atherogenesis, depending on its intracellular or extracellular localization. While extracellular ATP functions as a pro-atherogenic signal that promotes inflammatory activation, the maintenance of intracellular ATP may suppress endothelial stress responses and glycolytic inflammation ( Fig.1 ) .

Fig.1.

Roles of Intracellular and Extracellular ATP in Endothelial Cells During Atherosclerosis

2. Macrophages

ATP production in macrophages is highly context-dependent. Under homeostatic conditions, many macrophage populations predominantly rely on OXPHOS for ATP generation, although their metabolic phenotype varies according to their tissue environment and activation state ³⁸⁾ . In contrast, macrophages increase their reliance on glycolysis in response to inflammatory stimuli. In atherosclerotic plaques, macrophages reside in a complex microenvironment characterized by inflammatory cytokines, oxidized lipids, hypoxia, and other metabolic stresses. Under these conditions, proatherogenic mediators such as oxLDL, cytokines, and GM-CSF increase both glycolytic flux and proinflammatory activation, and these responses are further potentiated by hypoxia ^{39 - 41)} . HIF-1α and PFKFB3 are key mediators of this metabolic adaptation. Importantly, activated macrophages appear to require glycolytic upregulation for survival in some settings, as inhibition of HIF-1α or PFKFB3 suppresses glycolytic flux and decreases viability under proinflammatory conditions ⁴¹⁾ . Therefore, intracellular ATP depletion appears to be closely linked to inflammatory responses and macrophage survival.

In Pam3CSK4-primed bone marrow–derived macrophages (BMDMs), the experimental reduction of intracellular ATP by 2-DG increases IL-1β release through NLRP3 inflammasome and caspase-1 activation. Additionally, the NLRP3 activators, including nigericin and monosodium urate crystals, decrease intracellular ATP levels before robust IL-1β release, suggesting that ATP loss is an upstream event contributing to NLRP3 inflammasome activation ⁴²⁾ . Under sustained inflammatory stress in advanced atherosclerotic lesions, HIF-1α may also promote atherosclerosis by reprogramming inflammatory macrophages toward ATP depletion and necroptotic death, thereby enhancing necrotic core formation ⁴³⁾ . These findings are consistent with the previous observations that ATP is depleted in the perinecrotic regions of atherosclerotic plaques ¹⁸⁾ . On the other hand, lipid handling in foam cells, particularly cholesterol efflux, is an ATP-dependent process. The inhibition of mitochondrial ATP synthase by oligomycin markedly reduces macrophage cholesterol efflux capacity, indicating that adequate mitochondrial ATP production is required for efficient cholesterol removal ⁴⁴⁾ . MicroRNA-33 (miR-33) is known to repress cholesterol efflux-related genes, including ABCA1 and ATP-binding cassette transporter G1 (ABCG1) ^{45 - 47)} , and miR-33-deficient mice crossed with Apoe^−/− mice exhibit increased expression of these transporters, enhanced macrophage cholesterol efflux, and reduced atherosclerosis ⁴⁸⁾ . Beyond this canonical pathway, miR-33 also targets mitochondrial energy metabolism-related genes such as Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), pyruvate dehydrogenase kinase 4 (PDK4) and solute carrier family 25 member 25 (SLC25A25) in macrophages, thereby attenuating mitochondrial respiration and ATP production. Therefore, anti-miR-33 treatment promotes macrophage cholesterol efflux in conjunction with increased ABCA1 and ABCG1 expression in vitro. Notably, anti-miR-33 reduced the atherosclerotic lesion size in Apoe^−/− mice without altering circulating high-density lipoprotein cholesterol (HDL-C) or other major lipid parameters ⁴⁴⁾ . In addition, monocytes and hematopoietic precursors under hypercholesterolemic conditions exhibit increased glycolysis and OXPHOS ^{39 , 49 - 50)} , and monocytes from patients with coronary artery disease show increased mitochondrial ROS production and inflammatory priming ⁵⁰⁾ , suggesting that systemic metabolic rewiring may predispose myeloid cells to energetically demanding inflammatory activation even before plaque entry. In contrast to endothelial cells, KUS121 did not protect macrophages from tunicamycin-induced ER stress or suppress LPS-induced glycolysis and inflammation, which may reflect cell-type-specific differences in VCP-dependent ATP consumption or in the cellular response to ATP maintenance ²⁰⁾ .

Macrophages also express ATP-responsive P2 receptors. The activation of P2X7R in macrophages induces IL-1β secretion through inflammasome activation ⁵¹⁾ . In addition, apoptotic cells release nucleotides, including ATP, as caspase-dependent “find-me” signals that recruit monocytes and macrophages for the clearance of dying cells. P2Y2R on monocytes and macrophages functions as a key sensing receptor in this process, and the disruption of P2Y2 signaling impairs recruitment toward apoptotic cells and reduces apoptotic cell clearance ⁵²⁾ . Thus, extracellular ATP signaling in macrophages can exert distinct effects depending on the context. In atherosclerotic plaques, P2X7R is highly expressed in macrophage-rich areas, and its knockdown in THP-1-derived macrophages suppresses NLRP3 inflammasome activation, thereby reducing caspase-1 activation and IL-1β production ⁵³⁾ . Moreover, systemic P2X7R knockdown by small interfering RNA (siRNA) or genetic deletion attenuates atherosclerosis progression in vivo, although contributions from P2X7R deficiency in cell types other than macrophages cannot be excluded ^{53 - 54)} . P2X4R has also been implicated in atherosclerosis. P2X4R-deficient BMDMs show reduced ATP-induced release of inflammatory cytokines and chemokines, and systemic P2X4R deficiency attenuates atherosclerosis in vivo ⁵⁵⁾ . However, because P2X4R is also highly expressed in plaque endothelium, endothelial P2X4R may also contribute to this phenotype. In addition, the activation of the NLRP3 inflammasome by extracellular ATP has been reported to enhance oxLDL-induced lipid accumulation in macrophages ⁵⁶⁾ . This process may involve P2X7R because P2X7R knockdown attenuates oxLDL-induced foam cell formation in macrophages ⁵³⁾ .

Taken together, intracellular and extracellular ATP play distinct roles in macrophage function during atherosclerosis progression. The depletion of intracellular ATP can promote proinflammatory responses and necroptosis in macrophages. Therefore, maintaining intracellular ATP levels under atherogenic conditions may exert protective effects against atherosclerosis, although preserving intracellular ATP levels in macrophages during disease progression remains technically challenging. In contrast, extracellular ATP predominantly acts as a danger signal that promotes macrophage inflammatory activation, including cytokine and chemokine release, thereby contributing to disease progression ( Fig.2 ) .

Fig.2.

Roles of Intracellular and Extracellular ATP in Macrophages During Atherosclerosis

3. Vascular Smooth Muscle Cells

VSMCs generate ATP through both glycolysis and OXPHOS under basal conditions, although the contribution of each pathway dramatically changes depending on the VSMC phenotype and microenvironment ⁵⁷⁾ . These pathways provide sustained energy production, whereas the phosphocreatine/creatine kinase system serves as a rapid but short-lived ATP buffer. During smooth muscle cell contraction, intracellular ATP is directly consumed by myosin ATPase to generate contractile force and indirectly consumed by ATP-dependent ion pumps, particularly Ca^2＋-ATPases, to restore intracellular Ca^2＋ homeostasis ⁵⁸⁾ . The restoration of Ca^2＋ handling in VSMCs by the forced expression of sarcoplasmic/endoplasmic reticulum Ca^2＋‐ATPase 2a (SERCA2a) inhibits cell proliferation, indicating that a disruption of ATP-dependent Ca^2＋ handling may facilitate the transition from a contractile to a synthetic phenotype ⁵⁹⁾ . The classical synthetic phenotype of VSMCs has been thought to promote atherosclerosis by enhancing lesion growth through proliferation. However, it is now recognized that they can also exert plaque-stabilizing effects by producing extracellular matrix and contributing to fibrous cap formation ⁶⁰⁾ .

OXPHOS appears to decrease in a subset of plaque VSMCs, particularly those in fibrous cap regions, whereas glycolytic activity is increased as a compensatory response. Thus, some plaque VSMCs may adapt to mitochondrial dysfunction by shifting toward glycolysis during disease progression ⁶¹⁾ . Consistent with this idea, glycolysis-associated ATP production appears to contribute to VSMC proliferation and migration. The inhibition of pyruvate kinase M2 (PKM2) or lactate dehydrogenase A (LDHA), both key glycolytic enzymes, reduces intracellular ATP production and suppresses VSMC proliferation and migration ^{62 - 63)} . Notably, the PKM2 inhibitor shikonin reduced atherosclerotic lesion progression in vivo, although plaque stability was not assessed in that study ⁶³⁾ . Moreover, VSMC-specific deletion of PTEN induced kinase 1 (PINK1), a key mitochondrial quality-control kinase, does not significantly affect plaque size, but it is associated with a more vulnerable plaque, characterized by reduced VSMC and extracellular matrix content, including collagen and elastin ⁶⁴⁾ . VSMCs also contribute to vascular calcification, although the metabolic basis of this process is complex. VSMC-specific deletion of thioredoxin-interacting protein (TXNIP), a pro-oxidative regulator, preserves mitochondrial function, including mitochondrial ATP production, and attenuates calcification under treatment with vitamin D3 or inorganic phosphate. VSMC-specific TXNIP-deficient mice exhibit reduced medial vascular calcification in vitamin D3-induced or partial nephrectomy-induced vascular calcification models ⁶⁵⁾ . In contrast, another study reported that the inhibition of glycolysis with 2-DG increased OXPHOS-related gene expression and exacerbated vascular calcification in high phosphate-treated VSMCs in vitro ⁶⁶⁾ . During atherosclerosis progression, VSMCs can also acquire additional phenotypic states, including macrophage-like and mesenchymal-like phenotypes ⁶⁷⁾ . Although the relationship between intracellular ATP availability and these phenotypic transitions remains incompletely understood, a metabolic shift toward glycolysis is required for the Krüppel-like factor 4 (KLF4)-dependent switching of VSMCs to a plasmacytoid dendritic cell (pDC)-like phenotype ⁶⁸⁾ .

Extracellular ATP released from perivascular sympathetic nerves induces vasoconstriction largely through the P2X1 receptor (P2X1R) on VSMCs. In contrast, ATP released from sensory-motor nerves during antidromic reflex activity can induce relaxation in some vascular beds. Thus, extracellular ATP can act as either a vasoconstrictor or a vasodilator signal in a context-dependent manner ⁶⁹⁾ . Extracellular nucleotides, including ATP, also promote rat aortic SMC migration, largely through the induction of osteopontin via P2Y2R, which in turn drives migration through integrin αvβ3/αvβ5 signaling ⁷⁰⁾ . Because osteopontin has also been implicated in VSMC proliferation ⁷¹⁾ and vascular intimal hyperplasia ⁷²⁾ , extracellular ATP may facilitate the acquisition of a synthetic phenotype albeit in a concentration-dependent manner; specifically, higher extracellular ATP drives a synthetic phenotype including migration and proliferation whereas low basal extracellular ATP has been reported to help maintain the contractile phenotype through P2Y2R ^{73 - 74)} . Furthermore, in the context of vascular calcification, extracellular ATP is thought to suppress VSMC calcification primarily through P2Y2R ^{75 - 76)} . Systemic P2Y2R-deficient mice showed reduced atherosclerosis progression but accelerated arterial intimal calcification when crossed with Apoe^−/− mice ⁷⁶⁾ .

Overall, the functions of intracellular and extracellular ATP in VSMCs are more complex than those in endothelial cells and macrophages. Intracellular ATP levels, which are closely linked to the balance between glycolysis and OXPHOS, are associated with the phenotypic modulation of VSMCs and influence multiple aspects of atherosclerosis, including plaque formation, plaque stability, and calcification. Extracellular ATP also exerts dual effects on VSMC phenotypic modulation in a concentration-dependent manner and has been reported to suppress calcification ( Fig.3 ) .

Fig.3.

Roles of Intracellular and Extracellular ATP in Vascular Smooth Muscle Cells During Atherosclerosis

4. Other Cell Types

Intracellular and extracellular ATP also play important roles in other cell types, including neutrophils and T cells. Beyond their traditional role in infiltrating plaques and releasing inflammatory cytokines and chemoattractants, the discovery of neutrophil extracellular traps (NETs) has highlighted the increasingly pivotal role of neutrophils in the progression of atherosclerosis. Neutrophils regulate their chemotactic orientation by releasing ATP from the leading edge and sensing the released ATP through P2Y2R ⁷⁷⁾ . Extracellular ATP also mediates NLRP3 inflammasome-dependent IL-1β secretion from neutrophils via P2X7R activation ⁷⁸⁾ . Moreover, glycolytic ATP production is required for NET formation ⁷⁹⁾ , which exacerbates atherosclerosis progression through macrophage priming ⁸⁰⁾ and the enhancement of cell adhesion molecule expression and tissue factor production in endothelial cells ⁸¹⁾ .

The importance of T cells in atherosclerosis progression has also become increasingly recognized since single-cell RNA-seq analyses of human atherosclerotic plaques have demonstrated that T cells constitute a major immune cell population within plaques ⁸²⁾ . Evidence from broader T-cell immunometabolism studies suggests that metabolic reprogramming driven by microenvironmental factors, such as lipid and glucose availability, hypoxia, and oxidative stress, shapes the differentiation and function of T cells infiltrating atherosclerotic plaques ⁸³⁾ . Enhanced glycolysis is important for T-cell differentiation into pro-inflammatory Th1 and Th17 cells ^{84 - 85)} . In particular, glycolytic ATP fueling is a key regulator of Th17 differentiation ⁸⁵⁾ . Interestingly, differentiation into anti-inflammatory Th2 cells also requires glycolytic ATP production ^{86 - 87)} . However, excessive glycolysis suppresses the expression of forkhead box P3 (FOXP3), a key transcription factor for the immunosuppressive function of Tregs, which are generally considered atheroprotective, although a certain level of glycolytic activity is indispensable for Treg differentiation and function ^{88 - 89)} . Autocrine extracellular ATP signaling is important for T cell activation, primarily via P2X7R ⁹⁰⁾ . In contrast, purinergic P2X signaling inhibits the differentiation and function of Tregs ⁹¹⁾ . Moreover, CD39 expressed on Tregs hydrolyzes extracellular ATP to AMP, thereby contributing to the attenuation of ATP-related inflammatory effects ⁹²⁾ .

Future Perspectives

Much remains to be clarified regarding ATP biology in the pathophysiology of atherosclerosis. In particular, several specific bottlenecks currently limit progress in this field. First, it is still challenging to directly assess plaque or cellular ATP dynamics in vivo. Recently developed AVID mice may serve as a useful tool for this purpose. However, in applying these mice to atherosclerosis research, several technical challenges remain, including limited access to narrow or deep vessels, difficulties in the precise quantification of ATP levels using FRET-based sensors in vivo, and motion artifacts caused by vascular pulsation. The development of mice carrying single-wavelength or fixation-compatible ATP biosensors may help overcome some of these problems. In addition, the noninvasive assessment of ATP dynamics in human atherosclerotic plaques is even more challenging. Second, strategies for modulating intracellular ATP in vivo, especially within atherosclerotic plaques, are currently lacking. In particular, the cell-specific modulation of intracellular ATP in atherosclerosis is currently almost impossible. Therefore, direct in vivo evidence for the biological effects of intracellular ATP modulation is still difficult to obtain. KUS121 may be an option for the direct modulation of intracellular ATP levels, although, as with other currently available approaches, cell-specific targeting remains difficult.

Related to these technical limitations, ATP biology is likely to vary across different regions within plaques, such as the fibrous cap, plaque shoulders, and necrotic core. Furthermore, although we have described, to some extent, differences in ATP metabolism among subpopulations of VSMCs and T cells, aortic endothelial cells and macrophages can also be classified into multiple subpopulations. Thus, further location- or subpopulation-specific analyses of ATP metabolism are required. Time-dependent analyses are also necessary because ATP metabolism is likely to differ across various stages of atherosclerosis progression. Finally, although most previous studies have focused on either intracellular or extracellular ATP metabolism, a more integrated understanding of intra- and extracellular ATP biology is required.

Overall, intracellular ATP depletion and extracellular ATP signaling appear to be proatherogenic in many contexts. Therefore, maintaining intracellular ATP levels or blocking extracellular ATP signaling may be a promising therapeutic approach for atherosclerotic disease. KUS121 may be a novel therapeutic candidate for atherosclerotic disease and has indeed been shown to attenuate atherosclerosis progression in vivo, although comparable protective effects were not evident in macrophages ²⁰⁾ . The inhibition of miR-33 may also represent a potential therapeutic approach for atherosclerosis by enhancing macrophage cholesterol efflux through the upregulation of both mitochondrial energy metabolism-related genes and cholesterol efflux-associated genes ^{44 - 48)} . Several P2X7 antagonists have been developed, and some have been reported to reduce ICAM-1 expression in the brain microvasculature, with concomitant inhibition of leukocyte adhesion, and to downregulate matrix metalloproteinase-9 (MMP-9) expression in activated macrophages ^{93 - 94)} . These findings raise the possibility that P2X7 antagonists may have therapeutic potential in atherosclerotic disease. However, this should be interpreted cautiously because previous clinical trials of P2X7 antagonists in rheumatoid arthritis were unsuccessful, and the trial in Crohn’s disease did not provide any clear evidence of anti-inflammatory efficacy ^{95 - 96)} . Thus, further development and evaluation of therapeutic approaches for atherosclerosis by modulating intracellular ATP homeostasis or extracellular ATP signaling are needed.

Conflicts of Interest

The authors have no conflicts of interest to disclose.