Updates on the Mechanisms and the Care of Cardiovascular Calcification in Chronic Kidney Disease

Updates on the Mechanisms and the Care of Cardiovascular Calcification in Chronic Kidney Disease Lucie Hénaut, PhD,* Jean-Marc Chillon, PhD, PharmD,*,† Saïd Kamel, PhD, PharmD,*,‡ and Ziad A. Massy, PhD, MD§,||

Summary: In chronic kidney disease (CKD), the progressive decrease in renal function leads to disturbances of mineral metabolism that generally cause secondary hyperparathyroidism. The increase in serum parathyroid hormone is associated with reduced serum calcium and calcitriol levels and/or increased serum fibroblast growth factor-23 and phosphate levels. The resulting CKD-associated disorder of mineral and bone metabolism is associated with various other metabolic dysregulations such as acidosis, malnutrition, inflammation, and accumulation of uremic toxins. It favors the occurrence of vascular calcification, which results from an imbalance between numerous inhibitors and promoters of soft-tissue mineralization. This review provides an overview of the most recent state of knowledge concerning the mechanisms that lead to the development of vascular calcification in the CKD setting. It further proposes directions for potential new therapeutic targets. Semin Nephrol 38:233-250 C 2018 Elsevier Inc. All rights reserved. Keywords: Chronic kidney disease, vascular calcification, promoters, inhibitors, treatments

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ascular calcification is a degenerative process characterized by the accumulation of calcium and phosphate salts within the arterial wall. It is observed in nearly all arterial beds and can develop in the media, the intima, or both. Calcification of the intimal layer usually occurs as a consequence of atherosclerosis and may be responsible for coronary ischemic events. Conversely, medial calcification is nonocclusive and preferentially develops along elastic fibers. As a consequence, medial calcification increases *

Laboratory MP3CV, Centre Universitaire de Recherche en Santé, Amiens, France. † Direction de la Recherche Clinique et de l'Innovation, Amiens University Hospital, Amiens, France. ‡ Laboratory of Biochemistry, Amiens University Hospital, Amiens, France. § Division of Nephrology, Ambroise Paré University Hospital, Assistance publique – Hôpitaux de Paris, Boulogne-Billancourt/Paris, France. || Inserm U1018, Team 5, Centre de recherche en Epidémiologie et Santé des Populations, Université de Versailles Saint-Quentin-enYvelines, Paris-Saclay University, Villejuif, France. Financial support: Supported by the Recherche hospitalo-Universitaire project Search Treatment and improve Outcome of Patients with Aortic Stenosis (L.H.). Conflict of interest statement: Z. A. Massy has received grants for CKD REIN and other research projects from Amgen, Baxter, Fresenius Medical Care, GlaxoSmithKline, Merck Sharp and Dohme-Chibret, Sanofi-Genzyme, Lilly, Otsuka, and the French government, as well as fees and grants to charities from Amgen, Bayer, and Sanofi-Genzyme. These sources of funding are not necessarily related to the content of the present article. Address reprint requests to Z. A. Massy, MD, PhD, Division of Nephrology, Ambroise Paré University Hospital, 9 Ave Charles de Gaulle, F-92104 Boulogne Billancourt Cedex, France. E-mail: [email protected] 0270-9295/ - see front matter & 2018 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.semnephrol.2018.02.004

Seminars in Nephrology, Vol 38, No 3, Month 2018, pp 233–250

vessel stiffness, arterial pulse-wave velocity, systolic blood pressure, and pulse pressure, favoring the development of cardiac failure, left ventricular hypertrophy, and diastolic dysfunction. Vascular calcification is a complex process that involves not only the precipitation of minerals (ie, mineral step) but also is a tightly regulated, cell-mediated process, similar to bone formation (ie, cellular step). Despite significant recent growth of knowledge about vascular calcification, the order of appearance and time course of these two steps and of the initiating pathogenic events is still subject to debate. It is extremely difficult, if not impossible, to assess this in patients with chronic kidney disease (CKD).1 Of interest, a recent time course study in uremic rats showed that an increase in tissue nonspecific alkaline phosphatase (TNAP) and Wnt inhibitor Dkk1 expression in the aorta preceded initial calcium deposition, and that this increase was preceded only by increases in circulating fibroblast growth factor (FGF)23 and activin A.2 The presence of vascular calcification in the general population is predicted by traditional Framingham risk factors such as age, sex, family history, hypertension, tobacco use, diabetes, and dyslipidemia. In patients with CKD, vascular calcification is more prevalent and more severe than in the general population. Although CKD patients generally have a high prevalence of the traditional risk factors, vascular calcification in this population also is associated with several nontraditional risk factors. They include the CKD-associated disorder of bone and mineral metabolism (CKD-MBD), inflammation, oxidative stress, and the accumulation of uremic toxins, and predispose to earlier and accelerated vascular calcification as well as an excess cardiovascular morbidity and mortality. The present review has two goals. First, we offer an overview of the current knowledge concerning a 233

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Figure 1. Development of vascular calcification in CKD: a schematic view. CaxPi, calcium phosphate product; IS, indoxyl-sulfate; OPN, osteopontin; Ox. Stress, oxidative stress; PPi, pyrophosphate; uc-dp MGP, uncarboxylateddephosphorylated matrix gla protein; Vit D, vitamin D; Vit K, vitamin K.

variety of mechanisms by which vascular calcification develops in the CKD setting. The main focus is on recently identified mechanisms. Because the implications of parathyroid hormone (PTH) and vitamin D have been discussed in numerous previous reviews, these important factors will not be addressed here in any detail. Second, we present an in-depth examination of potential new therapeutic targets, based on novel pathogenic insights.

UREMIC SYNDROME AND VASCULAR CALCIFICATION Under physiological conditions, blood vessels are protected from supersaturated concentrations of serum calcium (Ca) and phosphate (P) by a number of active inhibitors. Among them, pyrophosphate, matrix Gla protein (MGP), and fetuin-A have been shown to prevent the transformation of soluble, amorphous calcium phosphate (Ca/P) complexes into harmful, stable hydroxyapatite crystals. In the CKD population, a decrease in levels of active inhibitors and a

simultaneous increase in levels of active inducers of calcification are responsible for the extremely high prevalence of intimal and medial vascular calcification3–5 (Fig. 1). Disturbances of mineral metabolism are key inducers of vascular calcification in these patients. Particularly, increased Ca and P levels are associated with vascular calcification in patients with CKD and may directly promote vascular calcification.6–9 In addition, chronic low-grade inflammation, malnutrition, and the gradual accumulation of uremic retention solutes, such as advanced-glycation end products (AGEs) or indoxyl sulfate, promote various types of vascular damage that impact vascular calcification. Disturbances of Calcium and Phosphate Metabolism Although hyperphosphatemia is considered the main risk factor for the development of vascular calcification in CKD patients,9 evidence also implicates a pivotal role for increased serum Ca levels10 and increased Ca × P product.11,12 It is noteworthy that vascular calcification in CKD generally develops before measurable

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increases in serum phosphate. Increasing data obtained from cell and animal models have shown that increased extracellular Ca or P concentrations induce vascular smooth muscle cell (VSMC) calcification independently and synergistically. Within the vascular wall, increased Ca and P levels lead to the conversion of VSMCs into osteochondrogenic cells, characterized by the loss of VSMC contractile markers and increased expression of bone-promoting genes such as bmp2, runx2, and tnap. This phenomenon is associated with the secretion of a procalcifying matrix rich in type I collagen and the production of matrix metalloproteinases 2 and 9, known to promote elastin degradation and the subsequent release of elastin peptides highly prone to calcify. These procalcifying conditions also favor the release of VSMC-derived matrix vesicles as well as apoptotic bodies able to nucleate hydroxyapatite. According to recent studies, free DNA, present in the dead arterial tissue, also may represent a molecular platform able to initiate Ca/P precipitation and crystal formation.13 Of interest, overexpression of TNAP, which promotes the hydrolysis of the calcification inhibitor pyrophosphate, recently was reported to precede Ca deposition in aortas from uremic animals.2 In this study, bmp2 overexpression was concomitant with the initial deposits and runx2 overexpression appeared even later, suggesting that VSMC osteogenic transition is not the primary but only a secondary event that appears as a consequence of initial Ca deposition.1 In line with this hypothesis, it has been reported that the increased number of Ca/P nanocrystals formed in response to high extracellular Ca and inorganic phosphate (Pi) concentrations directly promotes VSMC osteochondrogenic conversion14 and stimulates the production of proinflammatory cytokines by resident macrophages, and thereby worsens vascular calcification.15 These nanocrystals also may undergo lysosomal degradation by VSMCs, leading to high intracellular Ca levels and, ultimately, cell death.16 The resulting apoptosis further promotes calcification. Interestingly, a recent study showed that Pi can induce unpolarized macrophages to adopt a phenotype closely resembling that of alternatively activated M2 macrophages.17 These macrophages show an anticalcifying action mediated by increased availability of extracellular adenosine triphosphate and pyrophosphate, which suggests the existence of a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification. Abnormal FGF-23 and α-Klotho Levels α-Klotho deficiency and FGF-23 excess are associated with various complications and poor outcomes in CKD patients as well as in the general population. Because α-Klotho–deficient mice and FGF-23 null mice showed

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soft-tissue calcification, it is widely admitted that a dysregulation of the FGF-23–α-Klotho axis favors the development of vascular calcification on the one hand. On the other hand, transgenic uremic mice with α-Klotho overexpression show enhanced phosphaturia, preserved renal function, and less calcification than wild-type uremic mice,18 suggesting that the beneficial effect of α-Klotho on vascular calcification could result from an improvement of renal function and better control of serum P. α-Klotho is a membrane-bound protein highly expressed in kidneys and parathyroid glands that can be processed and released into the circulation as a soluble form. Although the expression of α-Klotho in the vascular wall remains a matter of debate, several studies have examined the possibility of a protective direct role of soluble and membrane-bound α-Klotho against vascular calcification development. An in vitro study showed that VSMC exposure to soluble α-Klotho suppressed high-Pi–induced VSMC osteogenic transition and subsequent mineralization,18 and in another study α-Klotho knockdown was found to potentiate the development of VSMC calcification.19 Moreover, secreted α-Klotho was shown to attenuate Pi-induced calcification of human bone marrow– derived mesenchymal stem cells in vitro via inactivation of the FGF-Receptor isoform 1 (FGFR1)/extracellular signal-regulated kinase (ERK) signaling pathway.20 In vivo, inhibition of the mammalian target of rapamycin (mTOR) signaling by rapamycin suppressed vascular calcification in CKD via up-regulation of membranebound vascular α-Klotho.21 In line with these observations, the up-regulation of membrane-bound α-Klotho in the vessel wall in response to intermedin 1–53 also was found to attenuate vascular calcification in CKD rats.22 Collectively, these results suggest that both soluble and membrane-bound α-Klotho are potential therapeutic targets for the treatment and prevention of vascular calcification in CKD, independent of FGF-23. The Klotho/FGFR-1 complex forms a specific receptor for FGF-23 signaling, which initially was suggestive of a direct protective role of FGF-23 against vascular calcification.19 However, whether FGF-23 directly promotes vascular calcification remains a controversial issue. In CKD, high circulating FGF-23 levels were found to be associated with atherosclerosis, vascular calcification, and cardiovascular death. Initially, it was hypothesized that these effects might be a marker of α-Klotho deficiency. In line with this hypothesis, exposure to exogenous FGF-23 was reported to exert a protective effect on vascular calcification in cultured VSMCs,23 and α-Klotho knockdown was shown to abolish such a FGF-23– mediated protection in vitro.19 Furthermore, complete neutralization of FGF-23 in CKD rats was found to accelerate vascular calcification and increase mortality.24 This suggested the existence of vascular

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resistance to FGF-23 in CKD because of a concomitant vascular α-Klotho deficiency. However, conflicting data by Jimbo et al25 showed that exposure to FGF23 enhanced Pi-induced vascular calcification by promoting osteoblastic transdifferentiation in aortic rings removed from uremic rats. More confusingly, Scialla et al26 subsequently showed that exposure to FGF-23 had no effect on Pi uptake or Pi-induced calcification of VSMCs. In their study, no effect on mouse aortic ring calcification was observed either in the presence or absence of soluble α-Klotho. Similarly, Lindberg et al27 showed that FGF-23 did not affect β-glycerophosphate–induced calcification of bovine VSMCs in vitro. It is noteworthy in this context that FGF-23 knockout mice express a vascular calcification phenotype.28 Clearly, additional studies are needed to elucidate the precise role of FGF-23 in the pathogenesis of vascular calcification in CKD.

deleterious effects of proinflammatory cytokines, to our knowledge, to date no ongoing trial targeting inflammation has been designed to decrease vascular calcification in CKD. Post hoc analyses of CKD patients receiving inflammation-targeted biologicals may provide clues on the role of inflammation in the clinical management of cardiovascular calcification. In this context, FGFR isoform 4 (FGFR4) might be a promising target. Singh et al44 recently showed that FGF-23 stimulated the hepatic secretion of IL6 and C-reactive protein by activating this receptor, and that administration of an isoform-specific FGFR4 blocking antibody reduced hepatic and circulating levels of C-reactive protein in their 5/6 nephrectomy rat model of CKD. Therefore, FGFR4 blockade might have therapeutic anti-inflammatory effects in CKD and in particular decrease the vessel wall inflammation preceding arterial calcification.

Inflammation

Advanced Glycation End Products

Experimental cell culture and animal data suggest a causal link between inflammation and vascular calcification.29 In vitro, tumor necrosis factor-α (TNF-α) promotes Pi-induced VSMC mineralization and osteogenic transition,30,31 apoptosis,32 endoplasmic reticulum stress,33 and Pi entry into VSMCs.33 TNF-α also decreases the availability of pyrophosphate34 and VSMC expression of α-Klotho.35 Similarly, interleukin 1β (IL1β) has been shown to play a key role in βglycerophosphate–induced VSMC calcification,36 and to favor by itself the osteogenic transition and subsequent calcification of VSMCs.37 In procalcific conditions, neutralization of IL6 in VSMCs cultured in vitro reversed receptor activator of nuclear factor-κB ligand (RANKL)-dependent osteogenic transition.38 In addition, systemic inflammation has effects on distant organs that facilitate vascular calcification. Thus, proinflammatory cytokines such as IL6 decrease the hepatic production of fetuin-A, an agent known to prevent Ca × Pi precipitation by the transient formation of soluble particles containing fetuin-A, Ca, and Pi termed calciprotein particles, and TNF superfamily cytokines reduce kidney tissue expression of antiinflammatory and phosphaturic α-Klotho. In CKD patients, chronic low-grade systemic inflammation is associated with increased prevalence, severity, and progression of vascular calcification.39,40 Recently, Benz et al41 reported that early stages of CKD in fact already are associated with local up-regulation of proinflammatory and pro-osteogenic molecules in the vascular wall and calcification of the aortic media. Of note, in hemodialysis patients with aortic intimal and medial calcification, serum IL6 levels are increased42 and are predictive of death.43 However, among the numerous recent clinical trials that aimed to inhibit the

AGEs are proteins that become glycated as a result of exposure to sugars. Local and circulating AGE levels are increased markedly in both diabetic and nondiabetic patients with CKD.45,46 This increase presumably is owing to an increase in endogenous generation secondary to oxidative stress, increased dietary intake, and/or impaired renal clearance of these compounds.46–49 In vitro, binding of AGE to their receptor RAGE accelerates VSMC osteoegenic transition and subsequent calcification through p38/mitogen-activated protein kinase (MAPK) and Wnt/β catenin signaling.50,51 In a high glucose environment, RAGE-triggered inflammation induces RANKL production by osteoblasts, favoring bone degradation and a subsequent increase in blood Ca and Pi, which, in turn, drives osteogenic differentiation of vascular cells.52 Furthermore, AGEs contribute to several uremiarelated disturbances, including increased synthesis of inflammatory mediators (IL1, TNF-α, and IL6), increased oxidative stress, disturbed endothelial cell function, and vessel wall thickening,53,54 which might indirectly impact vascular calcification development. As a consequence, optimization of AGE clearance and dietary restriction of AGE intake may be important considerations in the care of vascular calcification. At present, renal transplantation remains the best therapeutic modality to normalize AGE levels. Indoxyl Sulfate Indoxyl sulfate is a metabolite of tryptophan derived from dietary protein. It is synthesized in the liver from indole that is produced by intestinal flora including Escherichia coli. In CKD, indoxyl sulfate accumulates after the reduction in renal clearance. Its accumulation

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has been found to be associated with an increased risk of cardiovascular events and mortality in one study,55 although not in another study.56 Evidence from animal and in vitro studies has suggested that indoxyl sulfate may act as a vascular toxin. Indeed, in Dahl saltsensitive hypertensive rats, indoxyl sulfate administration induced aortic wall thickening and aortic calcification with expression of osteoblast-specific protein.57 In vitro, indoxyl sulfate increased Pi-induced VSMC calcification and osteogenic transition through increased Pit-1 expression,58 oxidative stress,59 and senescence.60 Indoxyl sulfate also increased methylation and subsequent transcriptional suppression of the vascular α-klotho gene, which promoted vascular calcification both in VSMCs cultured in vitro and in 5/6-nephrectomized Sprague Dawley rats.57 Moreover, indoxyl sulfate has been reported to suppress the messenger RNA and protein expression of fetuin-A in a concentration- and time-dependent manner in cultured human hepatoma HepG2 cell line,61 suggesting that it could affect fetuin-A deficiency-mediated cardiovascular damage in addition to direct toxic effects on the vascular wall. In CKD patients, serum indoxyl sulfate levels were found to be correlated positively with aortic calcification.55 In this context, it is noteworthy that AST-120 (kremezin; Kureha Chemical, Tokyo, Japan), an orally administered intestinal sorbent, currently is used in Japan, Korea, Taiwan, and the Philippines to adsorb indole in the gut lumen, thereby reducing serum and urinary levels of indoxyl sulfate. In a retrospective study in predialysis CKD patients, the use of AST-120 was found to be associated independently with less aortic calcification than no such treatment,62 suggesting a possible role of the pharmaceutical neutralization of uremic toxins in the management of vascular calcification. However, large trials are needed to show a link of causality between indoxyl sulfate accumulation and vascular calcification in CKD patients.

THERAPEUTIC APPROACHES FOR VASCULAR CALCIFICATION IN CKD In light of the key regulators of vascular calcification in CKD reviewed earlier, a wide variety of treatments has been and continues being evaluated for the prevention or reversal of vascular calcification. These approaches, which essentially consist of existing treatments for related conditions,63 such as those that aim to slow the progression of CKD and cardiovascular disease, are discussed later (Table 1). Calcimimetics The calcimimetic cinacalcet, an allosteric modulator of the calcium-sensing receptor (CaSR) expressed in

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many different tissues including the parathyroid glands, is one of the most effective drugs in treating secondary hyperparathyroidism. By enhancing the receptor's sensing capacity of extracellular Ca, cinacalcet reduces serum PTH, Ca, and P concentrations, allowing better control of secondary hyperparathyroidism and, more generally speaking, of CKD-MBD.64 The CaSR is expressed in VSMCs,64 with lower expression in aortic tissue of CKD patients than healthy subjects.65 Of interest, stimulation of the VSMC receptor by calcimimetics delays both Pi- and Ca-induced mineralization in cellular models in vitro and also in animal models in vivo.66–68 In particular, CaSR activation can protect VSMCs from osteogenic transition and collagen secretion and favor MGP synthesis.69,70 In animal models, calcimimetics were able to prevent vascular calcification even in the absence of changes in serum PTH.66 Given their systemic effects on the Ca × P product and their local effects on VSMC mineralization, it was predicted that the use of calcimimetics in the clinic would slow the progression of vascular calcification. Following this hypothesis, the ADVANCE study (A Randomized Study to Evaluate the Effects of Cinacalcet Plus Low Dose Vitamin D on Vascular Calcification in Subjects With Chronic Kidney Disease (CKD) Receiving Hemodialysis) was performed to assess the progression of vascular and cardiac valve calcifications in hemodialysis patients with secondary hyperparathyroidism in response to cinacalcet-HCl. Although the primary end point of aortic calcification failed to reach the level of significance based on Agatston scoring, the progression of aortic calcification assessed by a prespecified volume score, as well as the progression of vascular calcification at other sites such as the cardiac valves, were slowed, indicating a possible role of CaSR modulation in the prevention of vascular calcification in CKD patients.71 Regarding hard clinical outcomes, there is no definitive evidence that cinacalcet treatment is superior to placebo based on the results of the EVOLVE (EValuation Of Cinacalcet Hydrochloride (HCL) Therapy to Lower CardioVascular Events) study. In this randomized controlled trial, cinacalcet did not significantly reduce the risk of death or major cardiovascular events in dialysis patients with moderate-to-severe secondary hyperparathyroidism per standard intention-to-treat analysis, although there was a nominally significant improvement in hard patient outcomes when using prespecified lag-censoring analysis.72 The development of new approaches such as more active or better-tolerated calcimimetics may improve the treatment of vascular calcification and possibly its prevention in the future. In this context, etelcalcetide (AMG416), a novel third-generation, intravenous, long-acting selective peptide agonist of the CaSR, shows promise. It recently was shown to be noninferior to cinacalcet in the control of secondary hyperparathyroidism.73 Future studies need to examine whether etelcalcetide is superior to cinacalcet

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Table 1. Impact of CKD and Cardiovascular Therapies on the Development of Vascular Calcification Treatments

Phosphate binders

Modulated parameters

Consequences for the development of VC

Protective effects against VC

Harmful effetcs for VC

Basic research

Clinical setting

↓Pi ↓Hypomagnesemia ↓Circulating IS and PCS

↑Serum calcium (if Ca-based phosphate binders)

↓VSMC calcification in vitro ↓VC in vivo both in uremic mice and uremic rats

↓VC in HD patients

No data available

↓VSMC calcification in vitro ↓VC in uremic rats in vivo

VitaVask trial ongoing (HD patients)

Supplementation Vitamin K ↓uc-MGP

↓VSMC osteogenic transition ↓miR30b, miR133a, miR143a ↑BMP-7 and MGP ↑CaSR

No data available

↓VSMC calcification in vitro ↓VC in vivo in uremic rats

Small interventional studies: ↓CAC progression in CKD patients ↓Peripheral arterial calcification in CKD patients MAGiCAL-CKD trial ongoing

Vitamin D

Optimal concentration: ↓sHPT ↑α-Klotho ↑CaSR ↓Inflammation ↓VSMC osteogenic transition

Above optimal concentration: ↑Hypercalcemia ↑VSMC osteogenic transition

Optimal concentration: ↓VSMC calcification in vitro ↓VSMC calcification in uremic mice in vivo Above optimal concentration: ↑VSMC calcification in vitro ↑VC in vivo both in rats and mice

Optimal concentration: ↓VC in pediatric dialysis patients Above optimal concentration: ↑VC in HD patients

Statins

↓LDL cholesterol ↓VSMC apoptosis ↓Protein farnesylation

↓Vitamin K2 ↑Uc-MGP ↓Kidney fuinction

↓VC in vivo in uremic mice and rats

Clinical effects of statins on VC are still debated, needs clarification

Farnesyltransferase inhibitors

↓Circulating lipoproteins ↓Globulins ↑Fetuin A ↓Atherosclerotic plaque ↓Collagen I ↓Oxydative stress

No data available

↓VSMC calcification in vitro ND ↓Intimal and medial VC in vivo in uremic mice

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Magnesium

IS, indoxyl-sulfate; LDL, low-density lipoprotein; ND, no data; OPN, osteopontin; PCS, paracresyl-sulfate; sHPT, secondary hyperparathyroidism; uc-MGP, uncarboxylated MGP; VC, vascular calcification.

ND ↓Uremic toxins (Pi, indoxyl- ↑Serum calcium sulfate, …) ↑AGE ↓Antioxydants ↓Ppi Hemodialysis treatment

ND

↓PTH ↓Ca x Pi ↑CaSR ↓Collagen I ↓α-actin ↑MGP Calcimimetics

No data available

↓VSMC calcification in vitro ↓VC in vivo in uremic mice

In HD patients: ↓Cardiac valve calcification No effects on VC

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in slowing the progression of vascular calcification and improving cardiovascular morbidity and mortality. Phosphate Binders Numerous studies have reported an association between increased serum phosphorus levels and greater prevalence of vascular calcification in patients with advanced CKD. This has led to the hypothesis that phosphorus-lowering therapy might reduce the progression of vascular calcification. Ca-based phosphate binders (calcium carbonate, calcium acetate) have proven to be efficient in decreasing serum phosphate, but their use has been associated with an increased risk of hypercalcemia and less prevention of vascular calcification than the Ca-free binders.74 Ca-free binders such as sevelamer hydrochloride, sevelamer carbonate, and lanthanum carbonate are equally or slightly less effective than Ca-containing compounds in decreasing phosphorous levels. However, they do not induce hypercalcemia and therefore should be more effective in treating vascular calcification. In line with this hypothesis, sevelamer hydrochloride and lanthanum carbonate were reported to decrease the development and/or progression of vascular calcification in CKD rats fed with a high phosphate diet75 and in uremic apolipoprotein E (ApoE)-/- mice.76,77 However, in hemodialysis patients the claimed advantage of Cafree binders in the progression of coronary artery calcification (CAC) remains a matter of debate.78–82 Differences in study design, especially the lack of a placebo arm, study population characteristics, dialysate Ca concentration, and degree of PTH control may account for the reported discrepancies. In patients on dialysis, a negative relationship exists between iron administration and serum intact FGF-23 level,83 suggesting that iron therapy may have a beneficial effect on cardiovascular events in this setting. In this context, several iron-based phosphate binders have undergone testing in clinical trials. Among them, sucroferric oxyhydroxide PA21 was reported to be effective in decreasing serum phosphorous levels in hemodialysis patients.84 In rats with adenine-diet–induced CKD, PA21 treatment significantly decreased serum intact parathyroid hormone and FGF-23 levels and improved vascular calcification scores compared with rats treated with calcium carbonate.85 However, in the same model, PA21 was not more efficient than lanthanum carbonate and sevelamer carbonate in controlling vascular calcification, despite better control of FGF23 levels.86 Very recently, ferric citrate underwent clinical evaluation in patients with CKD. This compound not only binds phosphate in the gut but also favors the absorption of iron. Two slightly different brands have been studied, ferric citrate in the United States, and ferric citrate hydrate (JTT-751) in Japan.

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Similar to other phosphate binders, both types of ferric citrate allow phosphate control using relatively low, well-tolerated doses, and both improve iron-deficiency anemia.87 Of note, JTT-751 has been shown further to prevent the progression of aorta calcification in rats with adenine-induced CKD.88 Therefore, their use in the clinical setting appears promising. However, whether they display favorable effects on vascular calcification in patients with CKD has not been investigated to date. Vitamin K2 Supplementation Vitamin K is essential to turn uncarboxylated MGP into carboxylated MGP, which enables the protein to gain its inhibitory function against BMP2 and prevent vascular calcification.89 Most hemodialysis patients show subclinical vitamin K deficiency.90 In these patients, vitamin K–dependent proteins, including the MGP, largely are undercarboxylated or even totally uncarboxylated, indicating that functional vitamin K deficiency may contribute to uremic vascular calcification. In uremic rats, pharmacologic vitamin K supplementation was found to restore plasma carboxylated MGP concentrations and slow the development of aortic calcification.91,92 Similarly, in chronic hemodialysis patients, daily vitamin K supplementation for 6 weeks significantly improved MGP bioactivity,93,94 however, to date there is no clinical evidence to show that vitamin K supplementation can attenuate the progression of vascular calcification in patients with CKD. To test this hypothesis, two prospective randomized studies with vitamin K currently are ongoing in hemodialysis patients: a German trial, vitamin K1 to slow vascular calcification in haemodialysis patients (VitaVasK trial) and a Canadian trial, Inhibiting the Progression of Arterial Calcification with vitamin K in Hemodialysis patients (iPACK-HD trial).95,96 Vitamin K2, which might be more efficacious than vitamin K1, was not available at the trial start in either country. Of note, a trial with menaquinone-7 (vitamin K2) in nonuremic patients with coronary artery calcification is ongoing in The Netherlands.97 The goal of all of these trials is to show that vitamin K supplementation slows the progression of vascular calcification. In case of positive outcomes this may lead to the introduction of vitamin K in the clinic as a cost-effective and safe nutritional strategy to prevent vascular calcification progression in hemodialysis patients. Magnesium Supplementation Clinical studies have shown that low serum magnesium (Mg) levels were associated with a high prevalence of vascular calcification98–100 in patients with

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end-stage renal disease (ESRD). These findings have stimulated interest in understanding Mg’s impact on vascular calcification development in CKD. In experiments in vitro, MgCl2 prevented Pi-induced VSMC osteogenic differentiation and calcification through the Mg transporter transient receptor potential cation channel subfamily M member 7 (TRPM7).101 These effects could be linked to the Pi-induced down-regulation of miR-30b, miR-133a, and miR-143 in VSMCs.102 Furthermore, addition of MgCl2 to the incubation milieu enhanced the expression of calcification-inhibitory proteins (including osteopontin, BMP-7, and MGP) and decreased apoptosis. In another study, MgCl2 was shown to prevent VSMC calcification in vitro through CaSR activation.103 Of note, earlier reports showed that Mg could interfere directly with the process whereby Ca and P crystallize into hydroxyapatite.104,105 However, in vitro, MgCl2 failed to prevent Pi-induced Ca/P deposition on fixed cells, which suggests that cells must be alive for Mg ions to exert a protective effect.106 In male Sprague-Dawley rats with adenine-induced CKD, Mg supplementation attenuated the severity of calcitriol-induced calcification in the abdominal aorta (by 51%), the iliac artery (by 44%), and the carotid artery (by 46%).107 In line with these data, a recent study showed that increased dietary Mg intake in 5/6 nephrectomized rats resulted in a marked reduction in vascular calcification, together with improved mineral metabolism and renal function. The protective effect of Mg on vascular calcification was not limited to its P binding action because Mg administered intraperitoneally also decreased vascular calcification.108 Interestingly, in uremic rats with established vascular calcification, the increase in dietary Mg significantly reversed vascular calcification, an effect that was associated with a reduced mortality rate.108 Whether the protective effects of Mg on vascular calcification are the result of local actions on cardiovascular tissues or of indirect systemic actions on inflammation,109,110 vitamin D metabolism,111 or CKD-MBD112 cannot be determined in the in vivo setting and needs further investigations. In line with these data, small interventional studies in patients with CKD have suggested that Mg supplementation may slow CAC progression113 and peripheral artery calcification.114,115 Data from well-designed, randomized, controlled trials will be needed to confirm a possible clinical benefit of Mg supplementation with regard to vascular calcification in the CKD setting. In this context, the ongoing multicenter, double-blind, placebo-controlled, randomized MAGiCAL-CKD trial is assessing whether oral Mg supplementation (ie, treatment with either slow-release Mg hydroxide 30 mmol/d or matching placebo in a 1:1 ratio) can prevent the progression of CAC in subjects with predialysis CKD.116

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Statins CKD patients show increased risks of hypercholesterolemia, atherosclerosis, and cardiovascular events.117 In this context, the use of 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitors (statins), which effectively lower low-density lipoprotein cholesterol levels, was expected to slow down atherosclerosis, intimal calcification, and cardiovascular events. In line with this hypothesis, simvastatin was shown to reduce vascular calcification in uremic ApoE knockout (KO) mice,118 and pravastatin and olmesartan synergistically ameliorated vascular calcification in uremic rats.119 Pravastatin and olmesartan also were reported to reduce Pi-induced VSMC apoptosis and calcification in vitro, suggesting that statins may show direct pleiotropic effects on the vascular wall. Apart from their lipid-lowering effects, statins also decrease protein farnesylation, which is critical for activation of Ras family small G proteins, which play a major role in signaling, differentiation, proliferation, adhesion, migration, cytokine production, and apoptosis. In this context, the recent development of farnesyltransferase inhibitors offered the opportunity to test the influence of statins' systemic (cholesterol lowering) and local (protein prenylation) actions on vascular calcification. In ApoE KO mice, inhibition of farnesylation reduced the aortic expression of Ras protein as well as oxidative stress and decreased aortic plaques size.120 Moreover, administration of the farnesyltransferase inhibitor R115777 to uremic ApoE KO mice decreased the extent of atheromatous lesions and intimal and medial vascular calcification together with a reduction in serum lipoprotein and fetuin A levels.121 Inhibition of the Ras/Raf pathway by R115777 also directly reduced VSMC calcification in vitro.121 Despite these encouraging data, the clinical benefits of statins regarding vascular calcification and cardiovascular events have been disputed recently.122,123 Indeed, in patients with normal kidney function the use of statins was found to increase CAC progression.124–126 However, the increased progression was not associated with a greater risk of cardiovascular events, which could imply that more intensively calcified plaques are more stable and less prone to rupture. Chen et al127 recently observed that treatment of ESRD patients with lipophilic statins (simvastatin and atorvastatin) was associated with a higher baseline CAC score as well as a more rapid CAC score progression, independently of age, sex, and diabetes. Because statins impaired vitamin K2 synthesis in VSMCs in vitro, the investigators hypothesized that statin-induced accumulation of uncarboxylated MGP might be one factor whereby statins could mitigate any beneficial effect on cardiovascular outcomes in patients with ESRD. The major difference between preclinical studies and clinical

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studies is the fact that in preclinical studies statin treatment generally was used preventively (ie, before the development of vascular calcification), whereas in clinical studies treatment was initiated in the presence of vascular calcification. Already established vascular calcification may need higher statin doses to be efficacious, but higher doses may be associated with more marked side effects preventing their use in the clinical setting. Farnesyltransferase inhibitors could represent a potentially alternative approach, based on the observation that their use was associated with a decrease in arterial pulse-wave velocity and carotid artery echogenicity (indirect vascular calcification markers) in patients with the Hutchinson-Gilford progeria syndrome.128 Whether farnesyltransferase inhibitors also can be used successfully in CKD patients for the treatment of vascular diseases including vascular calcification needs to be studied.

Clinical Management of Inflammation As mentioned earlier, inflammatory cytokines such as IL6 and TNF-α are strong inducers of vascular calcification. Given the availability of biologicals targeting TNF-α (infliximab, adalinumab, golinumab, certolizumab, and etanercept) and IL6 (siltuximab and otcilizumab), the therapeutic potential of anti-IL6 or anti–TNF-α antibodies for preventing the onset or reducing the progression of vascular calcification should be explored in patients with CKD. In the same vein, the study of CKD patients receiving inflammation-targeted biologicals may provide clues on the possible usefulness of TNF-α and IL6 in treating vascular calcification. In this context it is noteworthy that inflammatory mediators in the 15 to 45 kDa range, such as IL6 and TNF-α, cannot be removed effectively by conventional dialysis membranes.129,130 Exposure of blood to bioincompatible dialysis membranes or less-than-sterile dialysate causes activation of circulating mononuclear cells131 and is regarded as a potentially additional cause of inflammation in CKD patients receiving renal replacement therapy.132 Therefore, ultrapure dialysates and other, more biocompatible dialysis materials have been introduced to improve the inflammatory state. In this context, Girndt et al133 recently reported a dampening effect of high-cutoff dialysis treatment on systemic inflammation compared with high-flux treatment. In particular, treatment with high-cut-off dialysis removed β2-microglobulin, sTNF-RI, factor D, and high molecular AGE significantly better than conventional high-flux membranes.134 Subsequent studies from the same team reported that high-cut-off dialysis serum led to significant reductions of TNF-α and IL6 expression in monocytes cultured in vitro as compared with high-flux membrane treatment.135 Further studies are necessary to evaluate the potential

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Table 2. Newly Discovered Sources of Calcifying Vascular Cells Cell types

Niche

Markers

Homing

Differentiation

Osteogenic Others Gli 1+ MSC

Adventitia

Consequences for vascular calcification Basic research

Clinical setting ↑Gli 1+ cells in calcified arteries of CKD patients

TNAP RunX2

Gli 1+ Media CD34+ Intima Sca1+ PDGFRβ + CD29+ CD105+ Vimentin

VSMC (initially) Osteoblast-lie cells (secondarily)

BMP2 TNAP Collagen type I Osteonectin α-SMA OPN BSP

CD-105 No data + available CD45CD14CD34CD44+ CXCR4+

Osteoblast progenitor cells

Form mineralized nodules when cultured in vitro Form bone after in vivo transplantation in nude mice

EPC

OC

CD34+ CD133KDR+ CD45-

No data available

Mature endothelial cells

EPC isolated from CKD patients calcify Positive correlation between OC when cultured in vitro expression in CD34+ CD133-KDR In vitro, vitamin D prevents the calcification +CD45- EPC and vascular calcium of EPC score, PTH, and phosphate in CKD isolated from CKD patients patients

MCC

BAP OC RunX2 Collagen type 1 Osterix

CD34CD45+ CD14+ CD68+

Atherosclerotic lesions

Osteoblast-like cells

Calcify when cultured in vitro with osteogenic medium Calcified MCC show high BMP2 and RANKL MCC produce MMP1 and 9 as well as TIMP1 and 4 Promote ectopic calcification when implanted in nude mice MCC calcification in vitro is increased by glucose

CCC MSC Bone marrow (initially) Blood (secondarily)

Calcify when cultured in vitro with osteogenic medium ↑Gli 1+ cells in vascular media and neointima of uremic mice

No data available

↑MCC in circulation and atherosclerotic lesions of T2DM patients ↑MCC in blood of nondiabetic individuals with CVD

L. Hénaut et al.

BAP, bone-specific alkaline phosphatase; BSP, bone sialoprotein; CCC, calcifying circulating cells; CMC, calcifying myeloid cells; CVD, cardiovascular disease; EPC, endothelial progenitor cells; MMP, matrix metalloproteinase; MSC, mesenchymal stem cells; OC, osteocalcin; OPN, osteopontin; TIMP, tissue inhibitor of metalloproteinases; T2DM, type 2 diabetes mellitus.

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efficacy of high-cut-off dialyzers in decreasing vascular calcification. A better management of AGEs, which are highly proinflammatory and show direct procalcifying properties on VSMCs,50,51,136 by highly efficient hemodialysis procedures also may improve inflammation-induced vascular calcification in the hemodialysis population.

AGE absorbed in the gut by sevelamer carbonate may be an effective strategy to reduce AGE accumulation and inflammation and consequently slow vascular calcification progression in patients with diabetic nephropathy.

Clinical Management of AGE

Although the majority of investigators agree that the essential cellular stage of vascular calcification is the osteogenic transition of VSMCs, recent evidence has suggested the involvement of other cell types. Among them, Gli1þ mesenchymal stem cells of the adventitia, calcifying circulating cells, and osteoclast-like cells might represent interesting new targets (Table 2).

If conventional hemodialysis reduces the concentration of circulating AGEs by only 20%, the use of new highflux polysulfone membranes makes it possible to optimize extraction performances up to 80%.137 This might constitute an alternative approach in the prevention of vascular calcification. Of note, the clearance of AGEs by peritoneal dialysis is greater than that obtained by hemodialysis.138 However, the high glucose concentrations of the main peritoneal dialysis fluids promote AGE formation in the peritoneal cavity. Moreover, glycoxidation products induced by heat sterilization of peritoneal dialysis fluid also are ideal precursors of AGE formation. In the future, the use of more stable molecules such as icodextrin (glucose polymer), which reduces the formation of glucosederived products during sterilization, may allow a more efficacious reduction of circulating AGEs139 and hopefully of their deleterious effects on vascular calcification development. Because diet-derived AGEs are major contributors to the total body AGE pool, it was postulated that a reduction in dietary AGE intake might reduce the high circulating AGE levels in patients with CKD. Thus, Uribarri et al49 reported that low dietary AGE intake decreased serum N (e)-carboxymethyl-lysine (CML), serum methylglyoxal-derivatives (MG), CML–low-density lipoprotein, CML-apoB, dialysate CML, and dialysate MG output in nondiabetic CKD patients on peritoneal dialysis therapy. High dietary AGE intake increased serum CML, serum MG, CML-, CML-apoB, and dialysate CML output. Serum AGE correlated with blood urea nitrogen, serum creatinine, total protein, albumin, and phosphorus. These observations suggest that dietary restriction of AGE may be an effective method to reduce excess toxic AGE and possibly the cardiovascular mortality associated with increased vascular calcification. Of interest, in patients with type 2 diabetes mellitus and stages 2 to 4 diabetic kidney disease, sevelamer carbonate administration reduced the levels of systemic and cellular AGEs, restored innate defense (including nuclear factor like-2), AGE receptor 1, nicotinamide adenine dinucleotide-dependent deacetylase sirtuin-1, and estrogen receptor α levels, and improved inflammation, compared with calcium carbonate treatment.140 Thus, in conjunction with dietary AGE restriction, reducing the amount of

Novel Cellular Actors of Vascular Calcification as Potential Targets

Gli1þ mesenchymal stem cells The perivasculature (ie, the adventitia and pericytes) represents the in vivo niche for mesenchymal stem cells. Gli1 is a specific marker of these cells in adult tissues.141 In vitro, Gli1þ adventitial stem cells are multipotent and can generate adipocytes, chondrocytes, and osteoblasts.141 In an elegant study, Kramann et al142 used genetic fate tracing to show that CKD significantly increased the number of Gli1þ cells in the media and neointima of ApoE KO mice. In this model, adventitial Gli1þ cells first differentiated toward the VSMC lineage and then into osteoblast-like cells, expressing Runx2 and TNAP. Genetic ablation of Gli1þ mesenchymal stem-like cells before the onset of CKD reduced Runx2 expression and completely abolished vascular calcification. The expression of Gli1 depends on the activation of the Sonic Hedgehog (SHH) pathway. The use of Smoothened agonist (SAG), a co-agonist of SHH's Smoothened co-receptor, stimulated the proliferation of Gli1þ progenitors in vitro and enhanced calcification during their osteogenic differentiation, which suggests a potential role of canonical SHH signaling in Gli1þ cell expansion and calcification. In line with these data, the investigators reported an increase of both Gli1 and SHH expression within the media, adventitia, and plaques in calcified arteries of deceased CKD patients as compared with healthy subjects. These findings implicate Gli1þ mesenchymal stem cells as a major source of osteoblastlike cells during uremic media and intima calcification and suggest that they may be an important therapeutic target to prevent vascular calcification in CKD. Circulating calcifying cells

Circulating cells with in intima osteocalcin

calcifying cells are bone marrow–derived an osteogenic phenotype, participating calcification processes and defined by and bone-specific alkaline phosphatase

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L. Hénaut et al.

Figure 2. New insights into the role of osteoclast-like cells in the development of vascular calcification. (A) Hypothesis of the impact of CKD on the formation of osteoclast-like cells within the vascular wall. Under physiological conditions, the presence of RANKL and M-CSF in the vascular wall favors the differentiation of resident monocyte-macrophages into osteoclast-like cells able to demineralize calcified vascular lesions. In CKD, the increased number of calcifying vascular cells (CVC) inhibits osteoclastic differentiation through increased IL18 secretion. In addition, uremic toxins, such as Pi and indoxyl-sulfate, block monocyte differentiation into osteoclastlike cells. As a consequence, the uremic milieu, while stimulating vascular calcification through increased formation of osteoblast-like cells, inhibits their resorption by blocking osteoclastic differentiation. (B) Novel findings regarding the impact of macrophage polarization on the formation of osteoclast-like cells. Recent studies have suggested that the polarization in vitro of peripheral blood CD14þ monocytes to either classically activated M1 macrophages (by IFN-ɤ) or alternatively activated M2 macrophages (by IL4) leads to formation of morphologically and functionally defective osteoclasts, which show decreased resorption abilities as compared with osteoclast-like cells derived from unpolarized cells. Carb. An., carbonic anhydrase; Cat K, cathepsin K; IS, indoxyl-sulfate; M-CSF, macrophage colony-stimulating factor; M1, classically activated macrophages; M2, alternatively activated macrophages; OPN, osteopontin.

expression.143 PTH and osteoblasts are key regulators of hematopoietic stem/progenitor cell expansion, mobilization, and homing, and, more generally speaking, CKD-MBD may play a role. Therefore, the alterations of bone remodeling in CKD-MBD may impact the development of these osteogenic cell subsets and consequently modulate vascular calcification. The pool of circulating calcifying cells includes mesenchymal stem cell–derived circulating osteoprogenitors, circulating calcifying endothelial progenitor cells, and calcifying myeloid cells.144 Mesenchymal stem cell– derived circulating osteoprogenitors phenotypically resemble bone marrow–derived mesenchymal stem cell and express mesenchymal and osteogenic markers

but not hematopoietic and endothelial markers. These cells can form mineralized nodules in vitro as well as bone in an in vivo transplantation.145–148 According to recent studies, BMP-2 may be one of the factors leading to mesenchymal stem cell mobilization from bone marrow to ectopic sites.147,148 Because BMP2 is highly expressed in calcified-vascular lesions of uremic patients, recruitment of mesenchymal stem cell– derived circulating osteoprogenitors within these lesions is conceivable. In human blood, the frequency of these cells is extremely low (o1/106 cells).145 However, mesenchymal stem cell–derived circulating osteoprogenitors were found to increase after bone fractures.149 Of note, the progression of vascular

Cardiovascular calcification in CKD

calcification is associated with increased bone loss and fractures in CKD patients.150 Thus, the possibility that mesenchymal stem cell–derived circulating osteoprogenitors might intervene in this context cannot be ruled out and should be investigated. To date, data on the involvement of this type of circulating osteoprogenitors in uremic vascular calcification are lacking. Endothelial progenitor cells are a subgroup of blood mononuclear cells derived from bone marrow, which circulate, proliferate, and differentiate into mature endothelial cells. In 2013, Cianciolo et al151 reported higher osteocalcin expression in a specific endothelial progenitor cell subset (CD34þ/CD133–/KDRþ/ CD45– cells) of CKD patients compared with healthy subjects. They reported a positive association between osteocalcin expression in CD34þ/CD133–/KDRþ/ CD45–endothelial progenitor cells and the calcium score in uremic patients. In vitro, CD34þ/CD133–/ KDRþ/CD45– cells isolated from uremic patients showed a significantly higher potential to calcify than those isolated from healthy subjects. The significant negative association between osteocalcin expression and 25(OH)D serum levels, together with the reduction in calcium deposition after addition of vitamin D– receptor agonists in cell cultures, indicated a protective role of vitamin D against intimal vascular calcification in CKD patients. The presence of circulating endothelial progenitor cells with an osteogenic phenotype provides new insight into the mechanisms by which vascular calcification develops in CKD. In 2011, Fadini et al152 showed that a fraction of circulating monocytes (1% in healthy adults) express bone-specific alkaline phosphatase and osteocalcin driven by Runx2, a master regulator of osteogenesis. Because this fraction of monocytes calcifies when put in culture and promotes ectopic calcification when implanted in nude mice, the investigators proposed the term calcifying myeloid cells for this cell population and reasoned that an excess of these cells in the circulation may cause vascular calcification. They reported that the cells were over-represented in the blood of patients with type 2 diabetes and in atherosclerotic lesions.152 Glycemic control reduced the number of calcifying myeloid cells toward normal whereas exposure to high glucose increased calcifying myeloid cell calcification in vitro. Together, these data suggest that diabetes might increase bone marrow generation and release of this monocyte subfraction, which homes to sites of vascular disease and promotes ectopic calcification. Despite the key role played by CKD-MBD in the disruption of bone microarchitecture and the subsequent perturbation of the functional and anatomic integrity of bone marrow niches, no information currently is available on the presence of calcifying myeloid cells in CKD patients.

245

Osteoclast-like cells

Calcified vascular lesions contain precursors of osteoclasts in the form of monocyte macrophages, as well as osteoblast-like VSMCs able to secrete factors involved in osteoclast differentiation such as RANKL and macrophage colony-stimulating factor.153,154 This suggests that some form of osteoclastogenesis occurs in the calcifying vascular wall. In keeping with this hypothesis, a report from 1998 showed the presence of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated giant cells, morphologically similar to osteoclasts, in close association with calcium deposits in human atherosclerotic lesions.155 These giant cells were shown to be positive for CD68, a macrophage marker, and to strongly express osteoclast-associated antigens cathepsin K, RANK, and osteoprotegerin (OPG).156 Initially, the presence of TRAP and cathepsin K, two enzymes associated with bone resorption, suggested that these osteoclast-like cells might have the ability to demineralize calcified vascular lesions. Recent evidence that mature osteoclasts actively reduce the mineral load of precalcified aortic elastin in vitro157 reinforced this hypothesis. However, the number of osteoclast-like cells within calcified vascular lesions is limited, and their resorption potential appears to be impaired because vascular calcification rarely regresses in vivo. This suggests that some factors perturb the differentiation of monocyte/macrophages into osteoclast-like cells and their activity. In keeping with this line of thought, calcifying vascular cells were shown to inhibit osteoclastic differentiation in vitro through increased IL18 secretion.158 In this context it is interesting to note that uremic toxins, such as Pi and indoxyl sulfate, were reported to block monocyte differentiation into osteoclasts in vitro and to reduce their bone-resorbing capacity.159,160 Thus, the uremic milieu, while stimulating vascular calcification via the formation of osteoblast-like cells, inhibits the resorption of vascular apatite nanocrystals by blocking osteoclastic differentiation (Fig. 2A). Recent evidence further suggests that macrophage heterogeneity also contributes to low osteoclastic activity in human calcified atherosclerotic plaques, increasing the propensity of mineral deposits to stay in place161 (Fig. 2B). Macrophages can be driven to a classically activated phenotype (M1) by stimuli, such as interferon (IFN)-γ, or an alternatively activated phenotype (M2) through factors including IL4 and IL13. In a recent report, we showed that macrophages surrounding calcium deposits of human atherosclerotic plaques expressed the mannose receptor, a marker typically associated with alternatively activated M2 macrophages. These macrophages expressed carbonic anhydrase type II, a molecule associated with osteoclast differentiation and bone resorption, and a relatively

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low level of cathepsin K.162 Monocytes differentiated in vitro with IL4 in addition to RANKL and macrophage colony-stimulating factor showed low cathepsin K expression and low TRAP activity and presented impaired capacities to degrade bone matrix. In an elegant study, Nagy et al163 recently investigated the impact of M1 polarization on the resorbing capacities of cardiac valve monocytes. Treatment of peripheral blood CD14þ monocytes in vitro with IFN-δ led to the formation of morphologically and functionally defective osteoclasts. Data obtained ex vivo showed that the release of IFN-δ from cytotoxic activated T cells suppressed osteoclastogenesis within cardiac valve tissue, with a subsequent increase in valvular calcium content. Collectively, these data indicate that the presence of either a proinflammatory or anti-inflammatory environment renders macrophages surrounding vascular calcium deposits phenotypically defective, and hence unable to resorb calcification. The development of pharmacologic approaches to enhance osteoclastic activity of macrophages may provide exciting opportunities for cell-mediated therapy aimed at resorbing vascular calcification.

CONCLUSIONS Although our understanding of the mechanisms underlying the development of vascular calcification in CKD has improved considerably, it is probably the complexity of the disturbances involved that explains that entirely satisfactory treatments are not yet available. This is particularly true for the still unsuccessful attempts to achieve regression of calcified vascular lesions. The constant improvement of proteomics, metabolomics, and transcriptomics techniques, and the discovery of new actors such as circulating calcifying cells, Gli1þ mesenchymal stem cells, osteoclastlike cells, and microRNAs may enable the emergence of efficient, easily accessible, and inexpensive diagnostic tools. Moreover, this may lead to the identification of novel therapeutic approaches allowing us to halt, and why not regress, vascular calcification in patients with CKD.

ACKNOWLEDGMENTS The authors wish to thank Tilman B. Drüeke, MD, for his critical review of the manuscript.

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