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Keywords:

  • Arterial calcification;
  • ATP ;
  • purinergic system

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Overview of the ATP-purinergic system
  5. ATP metabolism and the generation of pyrophosphate – ENPP1 and ANK
  6. Roles played by other ecto-nucleotidases
  7. Purinergic receptor-mediated calcification
  8. Conclusions
  9. Acknowledgements
  10. Address
  11. References

Background

Arterial calcification (AC) is a major health problem associated with extreme morbidity and a shortened survival. It is currently without any effective treatment. ATP and the purinergic system in general are now emerging as being important in the pathogenesis of AC and potentially provide a new focus for novel therapies.

Methods

This review systematically analyses and discusses the current literature examining the relevance of the purinergic system to AC. Particular emphasis is given to the enzymes associated with ATP metabolism and their role in maintaining a balance between promotion and inhibition of arterial mineralization. Points of controversy are highlighted, and areas for future research are suggested.

Conclusion

The potential roles of ATP and the purinergic system in AC are beginning to be elucidated. While further work is necessary, current knowledge suggests that several components of the purinergic system could be targeted to develop new treatments for AC.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Overview of the ATP-purinergic system
  5. ATP metabolism and the generation of pyrophosphate – ENPP1 and ANK
  6. Roles played by other ecto-nucleotidases
  7. Purinergic receptor-mediated calcification
  8. Conclusions
  9. Acknowledgements
  10. Address
  11. References

Arterial calcification (AC) is prevalent in the elderly and in patients with diabetes and chronic kidney disease (CKD). Calcium deposition can occur in the intimal or medial layers of the vessel wall, although medial calcification is more specific to patients with diabetes and CKD and is the focus of this review. AC is linked to adverse health outcomes, and numerous studies have shown that the severity of AC predicts survival, with higher calcification scores associated with increased cardiac mortality [1, 2]. AC leads to decreased arterial compliance and an associated increase in afterload, which may explain this increase in cardiovascular risk [3]. Furthermore, in patients with end-stage renal disease, AC of the iliac vessels frequently precludes successful kidney transplantation, because of the difficulty posed in forming donor-recipient arterial anastomoses. Similarly, creation of arterio-venous fistulae can be difficult in patients requiring haemodialysis. In addition, AC contributes to the devastating and often fatal condition calciphylaxis (calcific uraemic arteriolopathy).

Traditionally, the development of AC was considered to involve the passive deposition in the walls of arteries of calcium and phosphate due to their high circulating concentrations and consequent ‘supersaturation’ in serum. However, AC is seen in patients with ‘normal’ circulating levels of these ions, and not all patients with hyperphosphataemia develop AC. Therefore, this simple model of AC has begun to be challenged. There is now overwhelming evidence showing that the evolution of AC can be due to gene defects and is a cell-mediated, regulated process. A number of mechanisms have been implicated in its pathogenesis which have been reviewed in detail elsewhere [4, 5] and so are only briefly outlined here.

Although a number of cell types have been shown to contribute to AC [4], the vascular smooth muscle cell (VSMC) has emerged as the ‘key player’ in this process. VSMCs demonstrate a continuum of plasticity in their phenotype, ranging from a contractile state at one end to a synthetic entity at the other. This latter state is characterized by the ability of the cell to proliferate and to secrete growth factors and extracellular matrix (ECM) [6]. In the context of AC, VSMCs, when exposed to a number of stimuli, including inflammatory cytokines [7] and elevated concentrations of calcium and phosphate [8], undergo a change in phenotype, moving away from the contractile state to take on characteristics more typically associated with bone-forming, hence synthetic, osteoblast-like cells. Moreover, other biological processes, including VSMC apoptosis [9], loss of calcification inhibitors [10] and release of matrix vesicles capable of mineralization [11], all seem to occur in association with this phenotypic switch in a complicated network of integrated and overlapping pathways, ultimately leading to mineralization of the ECM with hydroxyapatite. Hydroxyapatite is a crystallized, inorganic compound containing calcium and phosphate, and is the primary mineral component of bone. Many parallels can therefore be drawn between AC and bone formation.

In vitro models used to study AC frequently involve the culture of VSMCs exposed to an increased concentration of phosphate, often with additional calcium supplementation. However, it should be noted that the final concentrations of these two ions used in culture media vary markedly throughout the literature, as do the species from which the cells are derived, and the addition of calcification promoters and inhibitors. Therefore, direct comparisons between studies can be challenging. As a model of AC, this in vitro system is somewhat limited in that the use of serum and associated growth factors, along with variations in cell passage number, can influence VSMC phenotype and may promote proliferation [12, 13], thus representing a very different environment from that seen in vivo. In addition, these cells in culture lack their normal ECM. In particular, elastin is absent which in vivo is the initial site of calcification within the arterial wall [14]. Ex vivo aortic ring culture models offer an alternative approach and overcome most of these issues [14], although final phosphate concentrations in the culture medium still differ between laboratories. In man, healthy individuals have serum phosphate levels of around 1 mm, and this can occasionally rise to as much as 3 mm in extreme disease states, usually in the context of CKD [15].

The potential role of ATP and the purinergic system in the AC process has, until recently, been largely unrecognized; however, the last few years have seen a growth of interest in this area. There is now increasing evidence indicating that multiple enzymes that regulate the metabolism of ATP and its breakdown products, and a large family of cell surface receptors for ATP and its metabolites, are important in the pathogenesis of this condition. This review outlines what is already known and highlights areas of future study.

Overview of the ATP-purinergic system

  1. Top of page
  2. Abstract
  3. Introduction
  4. Overview of the ATP-purinergic system
  5. ATP metabolism and the generation of pyrophosphate – ENPP1 and ANK
  6. Roles played by other ecto-nucleotidases
  7. Purinergic receptor-mediated calcification
  8. Conclusions
  9. Acknowledgements
  10. Address
  11. References

The ability of ATP to exert physiological effects in the mammalian heart, in addition to its role as the main cellular energy source, was first recognised almost a century ago in the seminal work of Drury and Szent-Gyorgi [16]. The extracellular ATP receptor system, known collectively as the purinergic system, was first predicted by Burnstock in the 1970s and is now well characterised (see [17] for a historical review). It has been implicated in a number of biological processes and disease states, well illustrated by the therapeutic role of the P2Y12 antagonist, clopidogrel, in the treatment for myocardial infarction [18].

Two broad categories of purinergic receptors have been identified on a wide range of cells. P1 receptors are G protein-coupled and respond primarily to adenosine. Four subtypes have been described to date, A1, A2A, A2B and A3. P2 receptors are principally activated by tri- and dinucleotides and are classified as either P2X or P2Y. P2X receptors are ion-gated channels consisting of three subunits, seven subtypes are currently recognised. P2Y receptors are G protein-coupled with eight subtypes currently known. The expression of P2 receptors in VSMCs has been investigated, and the presence of P2X1, P2X4 and P2X7 has been demonstrated, along with P2Y2, P2Y4, P2Y6 and P2Y12 [19-22] (Fig. 1).

image

Figure 1. The purinergic system and its regulation of arterial calcification in vascular smooth muscle cells (VSMCs). ATP and other ligands for P2X and P2Y receptors gain access to the extracellular space in a number of ways (see text). ATP is hydrolysed by either CD39 cells (ecto-nucleoside triphosphate diphosphohydrolases (NTPDases)) into AMP plus 2 phosphates (Pi) or by ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPPs) into AMP plus inorganic pyrophosphate (PPi). AMP is hydrolysed into Adenosine (Ad) and Pi by CD73 (ecto-5′-nucleotidase). PPi can also reach the extracellular space from within the cell through the putative membrane transporter ANK (ankylosis) protein. PPi is subsequently hydrolysed into Pi by tissue nonspecific alkaline phosphatase (TNAP). PPi is a potent inhibitor of calcification; however, it is also the substrate for TNAP generation of Pi and subsequent hydroxyapatite formation. The balance between ENPP1 and TNAP activity is therefore critical in determining whether calcification pursues. Autosomal recessive mutations in ENPP1 cause arterial calcifications in infancy, and autosomal recessive mutations in CD73 cause arterial calcifications in adulthood. P2X receptors are ion channels composed of three subunits, and P2Y receptors are G protein-coupled. The expression of P2X receptors 1, 4 and 7 and P2Y receptors 2, 4, 6 and 12 has been demonstrated on VSMCs, and the preferred agonists for these receptors are shown. Additionally, uridine adenosine tetraphosphate (Up4A) has recently been implicated to promote arterial calcification via signalling through P2Y2 and P2Y6 receptors.

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The availability of extracellular nucleotides for activation of these receptors is tightly regulated by a number of enzymes, the ecto-nucleotidases, which catalyse the metabolism of ATP and related molecules (see Zimmerman [23] for in-depth review). There are four broad categories of enzymes in this class. Ecto-nucleoside triphosphate diphosphohydrolases (NTPDases), of which there are eight, including CD39, hydrolyse ATP to yield AMP plus 2 phosphates. In contrast, the ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPPs), of which seven have been described and ENPP1 is the best characterized and studied [24], generate AMP plus inorganic pyrophosphate (PPi) from hydrolysis of ATP. This family of enzymes can alternatively hydrolyse ATP to generate ADP and subsequent AMP with phosphate side-products, although this reaction occurs much less readily than that of PPi generation. PPi is a potent inhibitor of hydroxyapatite formation, and thus tissue calcification, but can be converted to phosphate by the action of the enzyme tissue nonspecific alkaline phosphatase (TNAP), which can thereby promote mineralization. Intracellular PPi may also be secreted via the ankylosis protein (ANK). AMP is converted to adenosine by the action of ecto-5′-nucleotidase (CD73), which also generates one phosphate molecule (Fig. 1).

ATP release from erythrocytes [25] and perivascular nerves [26] has been described, and more recent work has shown that endothelial [27] and VSMCs [28] also seem able to contribute directly to ATP extracellular levels. Proposed mechanisms of release from these cells include vesicular exocytosis, ATP-binding cassettes, and connexin and pannexin hemi-channels. In addition, direct cell surface synthesis of ATP by F1/F0-ATP synthase has been proposed (see Lohman et al. [29]).

ATP metabolism and the generation of pyrophosphate – ENPP1 and ANK

  1. Top of page
  2. Abstract
  3. Introduction
  4. Overview of the ATP-purinergic system
  5. ATP metabolism and the generation of pyrophosphate – ENPP1 and ANK
  6. Roles played by other ecto-nucleotidases
  7. Purinergic receptor-mediated calcification
  8. Conclusions
  9. Acknowledgements
  10. Address
  11. References

Data implicating direct signalling at purine receptors in the process of AC is just beginning to emerge and is discussed below. However, more is known about the roles played by the enzymes involved in the hydrolysis of ATP and the associated metabolites, in particular PPi. The ability of PPi to inhibit ectopic calcification was demonstrated almost half a century ago using subcutaneous injections in rats exposed to high doses of vitamin D [30]. The mechanism whereby this is achieved seems to be the chemisorption of PPi onto the surface of hydroxyapatite, preventing attachment of further calcium and phosphate, and thus inhibiting crystal growth [31]. More recent work has provided further experimental evidence about the importance of this molecule in preventing AC. For example, cultured rodent aortic rings exposed to an elevated phosphate concentration will calcify following PPi removal using alkaline phosphatase [14]. Treatment with intraperitoneal PPi also reduces AC induced in uraemic rats without adverse effects on bone architecture [32], and also in apo E knockout (KO) mice [33]. In the setting of CKD in man, PPi levels inversely correlate with the degree of AC [34], and reduced levels are seen in dialysis patients irrespective of dialysis modality, inflammation or nutritional status, when compared with control subjects [35].

Clinical manifestations of genetic mutations in ENPP1 - generalized arterial calcification of infancy and autosomal recessive hypophosphataemic rickets type 2

As previously discussed, ENPP1 generates PPi (and AMP) following the hydrolysis of ATP, and alterations in this reaction are clinically relevant. One of the earliest and most significant discoveries in this respect was that autosomal recessive mutations leading to inactivation of ENPP1 result in the condition of generalized arterial calcification of infancy (GACI) [36]. This disorder is characterized by increased hydroxyapatite deposition within the elastic laminae of arteries, with associated stenosis and mortality, presumably due to reduced PPi production and availability. Indeed, treatment with bisphosphonates, which are analogues of PPi, has been shown to improve the prognosis of the condition [37]. Subsequent genetic studies have demonstrated that inactivating mutations of the Enpp1 gene can also cause autosomal recessive hypophosphataemic rickets type 2 (ARHR2) [38, 39]. Moreover, the Enpp1 inactivating K121Q (rs1044498) polymorphism is associated with increased arterial calcification scores in dialysis patients [40].

Murine models of ENPP1 deficiency - correlation with human disease

The potential importance of ENPP1 in disorders of calcification was initially discovered from work studying the ‘tiptoe walking’ (ttw/ttw) mouse. This mouse, originally developed in Japan in 1978 [41], is characterized by ectopic calcification most marked in peripheral and intervertebral joints [42], leading to progressive ankylosis. Its phenotype was shown to result from a nonsense mutation in the Enpp1 gene [43]. Subsequently, an Enpp1 KO mouse was developed [44], the phenotype of which has been investigated in detail. These animals show spontaneous calcification of the coronary arteries and aorta [45], and markers of bone formation are demonstrable in the aortic media [46]. Decreased serum calcium and phosphate concentrations are present compared with wild-type mice [45]. Of interest, these mice also show increased levels of the phosphaturic hormone fibroblast growth factor 23 (FGF-23) [45], as has also been reported in ARHR2 [38, 39]: FGF23 has been linked to AC in patients with CKD [47] and FGF23 mutations that resist degradation are the cause of autosomal dominant hypophosphataemic rickets (ADHR) [48]. The bones of Enpp1 KO mice show abnormal mineralization and architecture, probably due to decreased PPi availability for conversion to inorganic phosphate by TNAP [45, 49]. It is interesting to note that while the Enpp1 KO mouse biochemical and bone phenotype is similar to that of patients with ARHR2, these patients do not seem to develop AC. Conversely, patients with GACI, while exhibiting the same vascular changes as Enpp1 KO mice, do not tend to show the same skeletal and biochemical abnormalities. Further studies are needed to address the reasons for these differences.

ENPP1 regulation at the cellular level

Limited information is available on the regulation of ENPP1 expression in VSMCs. Using early passage rat VSMCs, Prosdocimo et al.[50] were able to show a decrease in ENPP1 mRNA expression under calcification conditions in vitro, growing cells in 5 mm phosphate and 1 uM forskolin (used to activate adenylate cyclase and thus elevate cAMP) for 10 days. Of note, the authors were not able to demonstrate a significant decrease in mRNA expression when cells were grown in 5 mm phosphate alone, implicating an important role for cAMP in the calcification pathway. These authors went on to assess ENPP1 protein expression and functional activity, and suggested that while both 5 mm phosphate and 1 uM forskolin could independently reduce protein expression by 10 days, functional activity was the same at baseline, as shown by no change in MeATP metabolism, a specific substrate. However, in contrast to this study, Huang et al.[51], using later passage murine aortic VSMCs, showed an increase in ENPP1 expression following exposure to 25 uM forskolin and 5 mm β-glycerolphosphate after 7 days in culture. In addition, Mathieu's group in their work using human valve cells found an increase in ENPP1 expression with increasing concentrations of phosphate in the culture medium [52]. Furthermore, this group generated data suggesting that nonspecific inhibition of ectonucleotidases could attenuate AC in rats treated with the vitamin K inhibitor warfarin [53]. This is a recognised model of AC due to the fact that matrix Gla-protein, a calcification inhibitor, requires the presence of vitamin K to be biologically active. However, it should be noted that this group used a different strain of rodent compared with Prosdocimo et al., and the subtypes of ectonucleotidases detected in aortas were markedly different between the two laboratories. The regulation of ENPP1 expression and activity is clearly complex and negative feedback mechanisms involving TNAP-regulated ENPP1 expression and activity, described in experiments using bone cells [54] is an added complication. Therefore, further research is required to define the regulation of ENPP1, especially by those factors already known to influence AC.

The role of ankylosis protein

Another important player implicated in the generation of extracellular PPi is the progressive ankylosis protein (ANK), a transmembrane protein that may mediate intracellular PPi efflux from cells [55]. Autosomal dominant inherited mutations in the gene encoding ANK have been demonstrated to cause two different calcification disorders: craniometaphyseal dysplasia [56] and autosomal dominant familial calcium pyrophosphate dihydrate deposition [57]. Furthermore, aortas taken from Ank KO mice calcify more readily than wild-type when exposed to an elevated phosphate concentration in vitro [58] and Ank KO mice fed a high phosphate diet develop AC [59]. Intriguingly, the paper by Villa-Bellosta et al. [58] also suggested that ANK might be able to transport ATP from the intra- to the extracellular compartment. Interesting insights into ANK regulation have come from the work of Zhao et al.[60]. Using both human and animal tissue, they showed that the transcription factor nuclear factor kappa B (NFkB), induced by tumour necrosis factor, led to a decrease in ANK expression and subsequent extracellular PPi concentrations. These findings were associated with an increase in AC, which was reversed by either ANK rescue or NFkB inhibition.

Roles played by other ecto-nucleotidases

  1. Top of page
  2. Abstract
  3. Introduction
  4. Overview of the ATP-purinergic system
  5. ATP metabolism and the generation of pyrophosphate – ENPP1 and ANK
  6. Roles played by other ecto-nucleotidases
  7. Purinergic receptor-mediated calcification
  8. Conclusions
  9. Acknowledgements
  10. Address
  11. References

Tissue nonspecific alkaline phosphatase

The availability of PPi is not only dependent on its generation, but also on its metabolism which is controlled by the enzyme TNAP. This protein is present on most cell types, including VSMCs, and catalyses the conversion of PPi to inorganic phosphate, thereby promoting mineralization. The human condition hypophosphatasia is caused by missense loss-of-function mutations in the TNAP gene. This inherited trait is considered to be an autosomal dominant negative, although recessive inheritance patterns have also been described [61]. TNAP serum levels are reduced in these patients, PPi concentrations increased and bone mineralization impaired [62]. TNAP KO mice die before weaning and exhibit a similar phenotype to the human disease [63]. TNAP has been linked to AC. Elegant experiments from O'Neill's group [64] used two rodent CKD models to demonstrate that both the expression and activity of TNAP are increased in uraemic conditions, suggesting this as a potential mechanism for the increased prevalence of AC in patients with CKD. While the exact pathway mediating the observed increase in TNAP was not elucidated, mRNA expression was not increased, but when aortic rings were isolated and incubated with uraemic serum, an increase in TNAP activity was detected, indicating that an as yet unidentified circulating factor may be responsible for up-regulating enzyme activity. The authors suggest that this factor is probably not phosphate or PTH, because of the low concentrations likely to have been present in vitro. Other work has demonstrated that VSMCs from Enpp1 and Ank KO mice have increased TNAP expression and enhanced calcification in vitro. This calcification was reduced by TNAP inhibitors [65], thus emphasizing the complex interaction between these three proteins in the regulation of AC.

Mutations in NT5E in patients with calcification of arteries and joints

Another significant finding arose recently from genetic studies of three families exhibiting extensive calcification of lower limb vessels in adulthood [66]. These patients showed autosomal recessive homozygous or compound heterozygous mutations in the NT5E gene. In-depth investigation revealed mutations in the NT5E gene, which encodes the CD73 protein, an enzyme responsible for the conversion of AMP to adenosine. To investigate the potential mechanism behind these findings, the investigators examined skin fibroblasts from affected individuals and demonstrated a marked increase in TNAP activity and calcification when cells were incubated with β-glycerolphosphate compared with healthy controls. These effects in vitro could be reversed by administration of either adenosine or rescue with a CD73-encoding vector. The authors hypothesize that the calcification seen in individuals with CD73 mutations is due to decreased inhibition of TNAP secondary to reduced levels of adenosine. However, as pointed out by some commentators [67], the expression of ectonucleotidases on fibroblasts is different to that on VSMCs; moreover, CD73 KO mice do not seem to exhibit ectopic calcification. Therefore, the exact mechanism underlying these intriguing observations remains a subject for further investigation. However, the phenotype seems linked to disrupted extracellular ATP metabolism, because this enzyme is in the same pathway as ENPP1.

The potential involvement of CD39 in arterial calcification

CD39 or NTPDase 1 has not, at present, been linked to AC directly, despite being highly expressed by VSMCs [68]. It is of interest that this enzyme, in its nucleotide hydrolysing capacity, has been linked to other important roles within the vasculature, including modulation of thrombosis and inflammation [69] and regulation of vascular tone [70]. In addition, of all the ectonucleotidases, CD39 has the most evidence linking it directly to regulation of signalling through purine receptors [71]. CD39 could potentially limit the availability of ATP for conversion to PPi by ENPP1 and so it will be of interest to examine the expression and regulation of this enzyme in the context of AC.

Purinergic receptor-mediated calcification

  1. Top of page
  2. Abstract
  3. Introduction
  4. Overview of the ATP-purinergic system
  5. ATP metabolism and the generation of pyrophosphate – ENPP1 and ANK
  6. Roles played by other ecto-nucleotidases
  7. Purinergic receptor-mediated calcification
  8. Conclusions
  9. Acknowledgements
  10. Address
  11. References

The potential of the P2Y receptor as a therapeutic target for a number of vascular pathologies has recently been reviewed [72], and it has been directly implicated in the process of AC. Schuchardt et al.[73] were able to show enhanced calcification of both cultured rat VSMCs and the medial layer of rodent aortic rings on exposure to calcification medium (culture medium supplemented with 10 mm β-glycerolphosphate) with the addition of uridine adenosine tetraphosphate (Up4A). This molecule, released from endothelial cells under conditions such as hypoxia or exposure to shear stress [21], is able to activate a number of P2X and P2Y receptors [74]. The authors went on to identify P2Y2 and P2Y6 as potential mediators of AC in experiments using VSMCs exposed to specific receptor antagonists and also aortic rings from P2Y2 KO mice. Furthermore, this study found that Up4A is elevated in patients with CKD.

Of interest, Erlinge et al.[75] have linked P2Y2 receptors to a switch in VSMC phenotype from contractile to synthetic in experiments showing that the expression of mRNA for this receptor is up-regulated in rodent VSMCs in long-term culture (representing cells in the synthetic state) compared with rodent aortic medial cells (VSMCs in the contractile state). P2Y2 receptor agonists have also been shown to increase TNAP activity in human heart valve interstitial cells [76], which was interpreted by the authors to indicate a pro-mineralizing function.

However, the situation is clearly complex, because in contrast to these findings, experiments by Côté et al.[52] provoked cultured human aortic valve interstitial cells to calcify using medium containing 2–5 mm phosphate and exposed them to either suramin (a general purinergic receptor antagonist), isoPPADS (P2X receptor antagonist) or CGS15943 (adenosine receptor antagonist). They found that only suramin increased mineralization, whereas the other agents had no effect. They concluded that this was consistent with an inhibitory effect of P2Y on valve calcification. They went on to show that treatment for cells with 2-thioUTP, a P2Y2 agonist, reduced mineralization and that P2Y2 knockdown increased mineral deposition. With further experiments, the authors were able to implicate inhibition of apoptosis by P2Y2 as the underlying mechanism that seemed to protect against calcification.

Because the pathogenesis of AC shares many similarities with that of bone formation, including the up-regulation of transcription factors such as Runx2 and ultimately hydroxyapatite deposition, it is intriguing to speculate how the purine receptors which are important for normal skeletal homoeostasis may influence the development of AC. In keeping with a possible protective role of P2Y2 against developing AC is the observation that P2Y2 KO mice have increased bone density [77]. In vitro studies have also suggested an inhibitory role for P2Y2 in bone formation (see Orriss et al. [78] for in-depth review). The role played by the P2X7 receptor in bone biology has also generated interest, with recent work suggesting that this receptor may enhance bone formation [79]. Of interest, loss-of-function single nucleotide polymorphisms of the P2X7 receptor have been associated with an increased risk of developing osteoporosis [80].

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Overview of the ATP-purinergic system
  5. ATP metabolism and the generation of pyrophosphate – ENPP1 and ANK
  6. Roles played by other ecto-nucleotidases
  7. Purinergic receptor-mediated calcification
  8. Conclusions
  9. Acknowledgements
  10. Address
  11. References

AC is a highly complex process. Despite major advances in recent years, our understanding of the pathogenesis is far from complete. Moreover, this condition is currently without any effective treatment. Recent work has provided evidence for an important part played by the purinergic system, in particular its links to the calcification inhibitor PPi. While further investigation is required to define more clearly the role(s) of the purinergic system in AC, the understanding we have to date is enough to suggest that many of its components could provide potential future therapeutic targets for managing this major clinical problem.

Address

  1. Top of page
  2. Abstract
  3. Introduction
  4. Overview of the ATP-purinergic system
  5. ATP metabolism and the generation of pyrophosphate – ENPP1 and ANK
  6. Roles played by other ecto-nucleotidases
  7. Purinergic receptor-mediated calcification
  8. Conclusions
  9. Acknowledgements
  10. Address
  11. References

UCL Centre for Nephrology, UCL Medical School, London NW3 2QG, UK (R. S. Fish, E. Klootwijk, R. Kleta, D. C. Wheeler, R. J. Unwin, J. Norman); Imperial College Kidney and Transplant Institute, Hammersmith Hospital Campus, London W12 0HS, UK (Frederick W.K. Tam).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Overview of the ATP-purinergic system
  5. ATP metabolism and the generation of pyrophosphate – ENPP1 and ANK
  6. Roles played by other ecto-nucleotidases
  7. Purinergic receptor-mediated calcification
  8. Conclusions
  9. Acknowledgements
  10. Address
  11. References
  • 1
    London GM, Guerin AP, Marchais SJ, Metivier F, Pannier B, Adda H. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant 2003;18:173140.
  • 2
    Chiu YW, Adler SG, Budoff MJ, Takasu J, Ashai J, Mehrotra R. Coronary artery calcification and mortality in diabetic patients with proteinuria. Kidney Int 2010;77:110714.
  • 3
    Sigrist MK, Taal MW, Bungay P, McIntyre CW. Progressive vascular calcification over 2 years is associated with arterial stiffening and increased mortality in patients with stages 4 and 5 chronic kidney disease. Clin J Am Soc Nephrol 2007;2:12418.
  • 4
    Shanahan CM, Crouthamel MH, Kapustin A, Giachelli CM. Arterial calcification in chronic kidney disease: key roles for calcium and phosphate. Circ Res 2011;109:697711.
  • 5
    Mizobuchi M, Towler D, Slatopolsky E. Vascular calcification: the killer of patients with chronic kidney disease. J Am Soc Nephrol 2009;20:145364.
  • 6
    Rensen SSM, Doevendans PAFM, van Eys GJJM. Regulation and characteristics of vascular smooth muscle cell phenotypic diversity. Neth Heart J 2007;15:1008.
  • 7
    Tintut Y, Patel J, Parhami F, Demer LL. Tumor necrosis factor-α promotes in vitro calcification of vascular cells via the cAMP pathway. Circulation 2000;102:263642.
  • 8
    Jono S, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K et al. Phosphate regulation of vascular smooth muscle cell calcification. Circ Res 2000;87:e107.
  • 9
    Shroff RC, McNair R, Skepper JN, Figg N, Schurgers LJ, Deanfield J et al. Chronic mineral dysregulation promotes vascular smooth muscle cell adaptation and extracellular matrix calcification. J Am Soc Nephrol 2010;21:10312.
  • 10
    Villa-Bellosta R, Millan A, Sorribas V. Role of calcium-phosphate deposition in vascular smooth muscle cell calcification. Am J Physiol Cell Physiol 2011;300:C21020.
  • 11
    Kapustin AN, Davies JD, Reynolds JL, McNair R, Jones GT, Sidibe A et al. Calcium regulates key components of vascular smooth muscle cell-derived matrix vesicles to enhance mineralization. Circ Res 2011;109:e112.
  • 12
    Zeidan A, Nordström I, Dreja K, Malmqvist U, Hellstrand P. Stretch-dependent modulation of contractility and growth in smooth muscle of rat portal vein. Circ Res 2000;87:22834.
  • 13
    Nakano-Kurimoto R, Ikeda K, Uraoka M, Nakagawa Y, Yutaka K, Koide M et al. Replicative senescence of vascular smooth muscle cells enhances the calcification through initiating the osteoblastic transition. Am J Physiol Heart Circ Physiol 2009;297:H167384.
  • 14
    Lomashvili KA, Cobbs S, Hennigar RA, Hardcastle KA, O'Neill WC. Phosphate-induced vascular calcification: role of pyrophosphate and osteopontin. J Am Soc Nephrol 2004;15:1392401.
  • 15
    Kestenbaum B, Sampson JN, Rudser KD, Patterson DJ, Seliger SL, Young B et al. Serum phosphate levels and mortality risk among people with chronic kidney disease. J Am Soc Nephrol 2005;16:5208.
  • 16
    Drury AN, Szent-Gyorgyi A. The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. J Physiol 1929;68:21337.
  • 17
    Burnstock G. Purinergic signalling. Br J Pharmacol 2006;147:S17281.
  • 18
    Sabatine MS, Cannon CP, Gibson CM, López-Sendón JL, Montalescot G, Theroux P et al. Addition of clopidogrel to aspirin and fibrinolytic therapy for myocardial infarction with ST-segment elevation. N Engl J Med 2005;352:117989.
  • 19
    Wang L, Karlsson L, Moses S, Hultgårdh-Nilsson A, Andersson M, Borna C et al. P2 receptor expression profiles in human vascular smooth muscle and endothelial cells. J Cardiovasc Pharmacol 2002;40:84153.
  • 20
    Wang L, Andersson M, Karlsson L, Watson MA, Cousens DJ, Jern S et al. Increased mitogenic and decreased contractile P2 receptors in smooth muscle cells by shear stress in human vessels with intact endothelium. Arterioscler Thromb Vasc Biol 2003;23:13706.
  • 21
    Erlinge D, Burnstock G. P2 receptors in cardiovascular regulation and disease. Purinergic Signal 2008;4:120.
  • 22
    Schuchardt M, Prüfer J, Prüfer N, Wiedon A, Huang T, Chebli M et al. The endothelium-derived contracting factor uridine adenosine tetraphosphate induces P2Y2-mediated pro-inflammatory signalling by monocyte chemoattractant protein-1 formation. J Mol Med 2011;89:799810.
  • 23
    Zimmermann H, Zebisch M, Sträter N. Cellular function and molecular structure of ecto-nucleotidases. Purinergic Signal 2012;8:437502.
  • 24
    Mackenzie NCW, Huesa C, Rutsch F, MacRae VE. New insights into NPP1 function: lessons from clinical and animal studies. Bone 2012;51:9618.
  • 25
    Bergfeld GR, Forrester T. Release of ATP from human erythrocytes in response to a brief period of hypoxia and hypercapnia. Cardiovasc Res 1992;26:407.
  • 26
    Lew MJ, White TD. Release of endogenous ATP during sympathetic nerve stimulation. Br J Pharmacol 1987;92:34955.
  • 27
    Bodin P, Burnstock G. ATP-stimulated release of ATP by human endothelial cells. J Cardiovasc Pharmacol 1996;27:8725.
  • 28
    Katsuragi T, Tokunaga T, Ogawa S, Soejima O, Sato C, Furukawa T. Existence of ATP-evoked ATP release system in smooth muscles. J Pharmacol Exp Ther 1991;259:5138.
  • 29
    Lohman AW, Billaud M, Isakson BE. Mechanisms of ATP release and signalling in the blood vessel wall. Cardiovas Res 2012;95:26980.
  • 30
    Fleisch H, Schibler D, Maerki J, Frossard I. Inhibition of aortic calcification by means of pyrophosphate and polyphosphates. Nature 1965;207:13001.
  • 31
    Francis MD. The inhibition of calcium hydroxyapatite crystal growth by polyphosphonates and polyphosphates. Calcif Tissue Res 1969;3:15162.
  • 32
    O'Neill WC, Lomashvili KA, Malluche HH, Faugere MC, Riser BL. Treatment with pyrophosphate inhibits uremic vascular calcification. Kidney Int 2011;79:5127.
  • 33
    Riser BL, Barreto FC, Rezg R, Valaitis PW, Cook CS, White JA et al. Daily peritoneal administration of sodium pyrophosphate in a dialysis solution prevents the development of vascular calcification in a mouse model of uraemia. Nephrol Dial Transplant 2011;26:334957.
  • 34
    O'Neill WC, Sigrist MK, McIntyre CW. Plasma pyrophosphate and vascular calcification in chronic kidney disease. Nephrol Dial Transplant 2010;25:18791.
  • 35
    Lomashvili KA, Khawandi W, O'Neill WC. Reduced plasma pyrophosphate levels in hemodialysis patients. J Am Soc Nephrol 2005;16:2495500.
  • 36
    Rutsch F, Ruf N, Vaingankar S, Toliat MR, Suk A, Höhne W et al. Mutations in ENPP1 are associated with ‘idiopathic’ infantile arterial calcification. Nat Genet 2003;34:37981.
  • 37
    Rutsch F, Böyer P, Nitschke Y, Ruf N, Lorenz-Depierieux B, Wittkampf T et al. Hypophosphatemia, hyperphosphaturia, and bisphosphonate treatment are associated with survival beyond infancy in generalized arterial calcification of infancy. Circ Cardiovasc Genet 2008;1:13340.
  • 38
    Levy-Litan V, Hershkovitz E, Avizov L, Leventhal N, Bercovich D, Chalifa-Caspi V. Autosomal-recessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene. Am J Hum Genet 2010;86:2738.
  • 39
    Lorenz-Depiereux B, Schnabel D, Tiosano D, Hausler G, Strom TM. Loss-of-function ENPP1 mutations cause both generalized arterial calcification of infancy and autosomal-recessive hypophosphatemic rickets. Am J Hum Genet 2010;86:26772.
  • 40
    Eller P, Hochegger K, Feuchtner GM, Zitt E, Tancevski I, Ritsch A et al. Impact of ENPP1 genotype on arterial calcification in patients with end-stage renal failure. Nephrol Dial Transplant 2008;23:3217.
  • 41
    Hosoda Y, Yoshimura Y, Hiqaki S. A new breed of mouse showing multiple osteochondral lesions – twy mouse. Ryumachi 1981;21(Suppl):15764.
  • 42
    Sakamoto M, Hosoda Y, Kojimahara K, Yamazaki T, Yoshimura Y. Arthritis and ankylosis in twy mice with hereditary multiple osteochondral lesions: with special reference to calcium deposition. Pathol Int 1994;44:4207.
  • 43
    Okawa A, Nakamura I, Goto S, Moriya H, Nakamura Y, Ikegawa S. Mutation in Npps in a mouse model of ossification of the posterior longitudinal ligament of the spine. Nat Genet 1998;19:2713.
  • 44
    Sali A, Favaloro J, Terkeltaub R, Goding J. Germline deletion of the nucleoside triphosphate pyrophosphohydrolase (NTPPPH) plasma cellmembrane glycoprotein-1 (PC-1) produces abnormal calcification of periarticular tissues. In: Vanduffel L, Lemmems R, editors. Ecto-ATPases and Related Ectoenzymes. Maastrich, the Netherlands: Shaker Publishing; 1999: pp 26782.
  • 45
    Mackenzie NCW, Zhu D, Milne EM, van't Hof R, Martin A, Quarles DL et al. Altered bone development and an increase in FGF-23 expression in Enpp1−/− mice. PLoS One 2012;7:e32177.
  • 46
    Zhu D, Mackenzie NCW, Millan JL, Farquharson C, MacRae VE. The appearance and modulation of osteocyte marker expression during calcification of vascular smooth muscle cells. PLoS One 2011;6:e19595.
  • 47
    Desjardins L, Liabeuf S, Renard C, Lenglet A, Lemke HD, Choukroun G. FGF23 is independently associated with vascular calcification but not bone mineral density in patients at various CKD stages. Osteoporos Int 2012;7:201725.
  • 48
    ADHR Consortium. Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet 2000;26:3458.
  • 49
    Anderson HC, Harmey D, Camacho NP, Garimella R, Sipe JB, Tague S et al. Sustained osteomalacia of long bones despite major improvement in other hypophosphatasia-related mineral deficits in tissue nonspecific alkaline phosphatase/nucleotide pyrophosphatase phosphodiesterase 1 double-deficient mice. Am J Pathol 2005;166:171120.
  • 50
    Prosdocimo DA, Wyler SC, Romani AM, O'Neill WC, Dubyak GR. Regulation of vascular smooth muscle cell calcification by extracellular pyrophosphate homeostasis: synergistic modulation by cyclic AMP and hyperphosphatemia. Am J Physiol Cell Physiol 2010;298:C70213.
  • 51
    Huang MS, Sage AP, Lu J, Demer LL, Tintut Y. Phosphate and pyrophosphate mediate PKA-induced vascular cell calcification. Biochem Biophys Res Commun 2008;374:5538.
  • 52
    Côté N, Husseini DE, Pépin A, Guauque-Olarte S, Ducharme V, Bouchard-Cannon P et al. ATP acts as a survival signal and prevents the mineralization of aortic valve. J Mol Cell Cardiol 2012;52:1191202.
  • 53
    Cote N, Husseini DE, Pepin A, Bouvet C, Gilbert LA, Audet A et al. Inhibition of ectonucleotidase with ARL67156 prevents the development of calcific aortic valve disease in warfarin-treated rats. Eur J Pharmacol 2012;689:13946.
  • 54
    Johnson KA, Hessle L, Vaingankar S, Wennberg C, Mauro S, Narisawa S et al. Osteoblast tissue-nonspecific alkaline phosphatase antagonizes and regulates PC-1. Am J Physiol Regul Integr Comp Physiol 2000;279:R136577.
  • 55
    Ho AM, Johnson MD, Kingsley DM. Role of the mouse ank gene in control of tissue calcification and arthritis. Science 2000;289:26570.
  • 56
    Nürnberg P, Thiele H, Chandler D, Höhne W, Cunningham ML, Ritter H et al. Heterozygous mutations in ANKH, the human ortholog of the mouse progressive ankylosis gene, result in craniometaphyseal dysplasia. Nat Genet 2001;28:3741.
  • 57
    Williams CJ, Zhang Y, Timms A, Bonavita G, Caeiro F, Broxholme J et al. Autosomal dominant familial calcium pyrophosphate dihydrate deposition disease is caused by mutation in the transmembrane protein ANKH. Am J Hum Genet 2002;71:98591.
  • 58
    Villa-Bellosta R, Wang X, Millán JL, Dubyak GR, O'Neill WC. Extracellular pyrophosphate metabolism and calcification in vascular smooth muscle. Am J Physiol Heart Circ Physiol 2011;301:H618.
  • 59
    Murshed M, Harmey D, Millán JL, McKee MD, Karsenty G. Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev 2005;19:1093104.
  • 60
    Zhao G, Xu MJ, Zhao MM1, Dai XY, Kong W, Wilson GM et al. Activation of nuclear factor-kappa B accelerates vascular calcification by inhibiting ankylosis protein homolog expression. Kidney Int 2012;82:3444.
  • 61
    Mornet E. Hypophosphatasia. Orphanet J Rare Dis 2007;2:40.
  • 62
    Russell RG, Bisaz S, Donath A, Morgan DB, Fleisch H. Inorganic pyrophosphate in plasma in normal persons and in patients with hypophosphatasia, osteogenesis imperfecta, and other disorders of bone. J Clin Invest 1971;50:9619.
  • 63
    Fedde KN, Blair L, Silverstein J, Coburn SP, Ryan LM, Weinstein RS et al. Alkaline phosphatase knock_out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia. J Bone Miner Res 1999;14:201526.
  • 64
    Lomashvili KA, Garg P, Narisawa S, Millan JL, O'Neill WC. Upregulation of alkaline phosphatase and pyrophosphate hydrolysis: potential mechanism for uremic vascular calcification. Kidney Int 2008;73:102430.
  • 65
    Narisawa S, Harmey D, Yadav MC, O'Neill WC, Hoylaerts MF, Millan JL. Novel inhibitors of alkaline phosphatase suppress vascular smooth muscle cell calcification. J Bone Miner Res 2007;22:170010.
  • 66
    St Hilaire C, Ziegler SG, Markello TC, Brusco A, Groden C, Gill F et al. NT5E mutations and arterial calcifications. N Engl J Med 2011;364:43242.
  • 67
    Robson SC. Role of CD73 and extracellular adenosine in disease. Purinergic Signal 2011;7:36772.
  • 68
    Robson SC, Sevigny J, Zimmermann H. The E-NTPDase family of ectonucleotidases: structure function relationships and pathophysiological significance. Purinergic Signal 2006;2:40930.
  • 69
    Robson SC, Wu Y, Sun XF, Knosalla C, Dwyer K, Enjyoji K. Ectonucleotidases of CD39 family modulate vascular inflammation and thrombosis in transplantation. Semin Thromb Hemost 2005;31:21733.
  • 70
    Kauffenstein G, Fürstenau CR, D'Orleans-Juste P, Sévigny J. The ecto-nucleotidase NTPDase1 differentially regulates P2Y1 and P2Y2 receptor-dependent vasorelaxation. Br J Pharmacol 2010;159:57685.
  • 71
    Kauffenstein G, Drouin A, Thorin-Trescases N, Bachelard H, Robaye B, D'Orleans-Juste P et al. NTPDase1 (CD39) controls nucleotide-dependent vasoconstriction in mouse. Cardiovasc Res 2010;85:20413.
  • 72
    Schuchardt M, Tölle M, van der Giet M. P2Y purinoceptors as potential emerging therapeutical target in vascular disease. Curr Pharm Des 2012;18:616980.
  • 73
    Schuchardt M, Tolle M, Prufer J, Prufer N, Huang T, Jankowski V et al. Uridine adenosine tetraphosphate activation of the purinergic receptor P2Y enhances in vitro vascular calcification. Kidney Int 2012;81:25665.
  • 74
    Matsumoto T, Tostes RC, Webb RC. The role of uridine adenosine tetraphosphate in the vascular system. Adv Pharmacol Sci 2011;2011:435132.
  • 75
    Erlinge D, Hou M, Webb TE, Barnard EA, Möller S. Phenotype changes of the vascular smooth muscle cell regulate P2 receptor expression as measured by quantitative RT-PCR. Biochem Biophys Res Commun 1998;248:86470.
  • 76
    Osman L, Chester AH, Amrani M, Yacoub MH, Smolenski RT. A novel role of extracellular nucleotides in valve calcification: a potential target for atorvastatin. Circulation 2006;114(1 Suppl):I5661572.
  • 77
    Orriss IR, Utting JC, Brandao-Burch A, Colston K, Grubb BR, Burnstock G et al. Extracellular nucleotides block bone mineralization in vitro: evidence for dual inhibitory mechanisms involving both P2Y2 receptors and pyrophosphate. Endocrinology 2007;148:420816.
  • 78
    Orriss IR, Burnstock G, Arnett TR. Purinergic signalling and bone remodelling. Curr Opin Pharmacol 2010;3:32230.
  • 79
    Grol MW, Panupinthu N, Korcok J, Sims SM, Dixon SJ. Expression, signalling, and function of P2X7 receptors in bone. Purinergic Signal 2009;5:20521.
  • 80
    Gartland A, Skarratt KK, Hocking LJ, Parsons C, Stokes L, Jorgensen NR et al. Polymorphisms in the P2X7 receptor gene are associated with low lumbar spine bone mineral density and accelerated bone loss in post-menopausal women. Eur J Hum Genet 2012;20:55964.