Etidronate prevents dystrophic cardiac calcification by inhibiting macrophage aggregation

Cardiovascular calcification is associated with high risk of vascular disease. This involves macrophage infiltration of injured vascular tissue and osteoclast-related processes. Splenic monocytes from mice, that are predisposed (C3H) or resistant (B6) to calcification, were isolated and differentiated in vitro with M-CSF to generate macrophages, which aggregate to form multinucleated (MN) cells in the presence of RANKL. MN cell formation was significantly decreased in monocytes from resistant compared with calcifying mice. Conditioned media from C3H macrophages strongly induced calcification in vitro. However, medium from B6 macrophages inhibited calcification. An increase in ICAM-1 was detected in conditioned media from C3H macrophages compared with B6, suggesting a key role for this molecule in calcification processes. Due to natural genetic loss of Abcc6, the causal gene for cardiac calcification, C3H mice have reduced plasma levels of inorganic pyrophosphate (PPi), a potential calcification inhibitor. Supplementation of C3H mice with PPi or Etidronate prevented but did not completely reverse cardiac calcification. Our data provide strong evidence of the pathogenesis of macrophages and MNs during tissue calcification and suggest PPi or its analogue Etidronate as a potential inhibitor of MN formation and calcification. Furthermore, the adhesion molecule ICAM-1 was shown to play a key role in calcification.

SCIenTIfIC REPORTS | (2018) 8:5812 | DOI: 10.1038/s41598-018-24228-y Treatment of macrophages with platelet-free mouse plasma or PPi. To obtain platelet-free plasma, whole mouse blood was collected from mice in 1.5 ml CTAD anti-coagulation tubes and depleted of platelets by filtration through a Centrisart 300,000 kDa cut-off filter (Sartorius, Göttingen, Germany). Samples were stored at −20 °C until use.
Monocyte-derived macrophages were stimulated for 6 days with cell culture medium containing M-CSF/ RANKL. Macrophages were then treated with the indicated concentrations of PPi (Sigma-Aldrich, St. Louis, MO, USA) or platelet-free plasma from DCC-susceptible or -resistant mice. The medium was replaced on day 9, with the addition of fresh plasma. On day 12, cells were fixed and stained using the TRAP kit to detect MN cells. TRAP-positive cells with more than three nuclei were counted under a microscope.

Collection of conditioned media from MN cells.
Monocytes were isolated from the spleens of C3H and B6 mice and differentiated into macrophage-derived MN cells, as described above. After that, cells were washed three times in 1 × PBS and placed in fresh medium without M-CSF or RANKL. The supernatant was collected after 24 hours and used for PPi measurement or in calcification assays with C3H/10T1/2 mesenchymal stem cells, as described below.
Induction of calcification in C3H10T1/2 cells using inorganic phosphate (Pi). C3H/10T1/2 cells were seeded in 48-well plates (0.25 × 10 5 cells/well), and calcification was induced using 2.6 mM inorganic phosphate (Pi). Medium was changed every 3 days until visible calcification occurred. Calcification was determined by calcein staining and quantified using the Randox calcium kit. qPCR analysis. Total RNA was isolated from myocardial tissue or cultured cells using the RNeasy kit (Qiagen, Valencia, CA, USA). After reverse transcription into cDNA, mRNA levels were determined by relative quantitative RT-PCR and analyzed by the ΔΔC T method, as described previously 11,14 . The primers used for gene expression studies are listed in Table 1. β-actin was used as an internal standard.
ICAM-1 ELISA. ICAM-1 levels in blood plasma and conditioned macrophage media were quantified according to the manufacturer's instructions using the mouse ICAM-1/CD54 Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA).
PPi measurement. Blood samples for PPi measurement were collected via cardiac puncture (using 27 G needles). Blood was kept on ice and centrifuged for 10 min at 1000 g at 4 °C. Plasma was transferred into separation tubes (Sartorius Centristart 1, 300,000 MW, 13279E) and filter-centrifuged at 2000 g for 25 min at 4 °C. Filtered plasma samples were stored at −80 °C until analysis.
For PPi measurement monocytes were seeded and differentiated in 24-Well plates (1.5 × 106 cells/cm 2 ). At day 7, 300 µl FBS-free cell culture medium was added to each well. The conditioning media was collected after 24 hours.
Basal ATP level was also measured in plasma samples and was found negligible. Statistical analysis. Data are presented as the mean ± SEM. Unpaired t-tests were performed using GraphPad. P < 0.05 was considered statistically significant. For all tests: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; and n.s., not significant.

Results
Elevated MN cell formation in mice predisposed to calcification. Monocytes from the spleens of C3H/He and C57BL/6 mice were isolated and differentiated in vitro, first into macrophages using M-CSF, and then into MN cells by treatment with RANKL. MN cell formation was compared between macrophages from calcification-susceptible and -resistant mice. More MN cells formed from macrophages from C3H mice than from macrophages from B6 mice (Fig. 1A,B). Abcc6 (which encodes the Mrp6 protein) was previously identified as a gene responsible for calcification in predisposed mice 10 . Specifically, a polymorphism in Abcc6 reduces the levels of Mrp6, rendering the mice prone to calcification 11 . To determine whether the observed differences in MN formation were due to variations in Abcc6 deficiency, MN formation was analyzed in macrophages from B6.C3H Dyscalc1 (Cg1) mice, congenic mice containing the Abcc6 locus from DCC-susceptible C3H mice on a DCC-resistant B6 genetic background 13,14 . More MN cells formed from Cg1 than from C57BL/6 macrophages (Fig. 1A,B).
To confirm this finding at the molecular level, levels of the osteogenic markers Trap, Ctsk and Runx2 were measured. All three markers were more highly expressed in macrophage-derived MN cells from mice predisposed to calcification than in those from the DCC-resistant B6 mice (Fig. 1C).

Identification of MN cells at sites of calcification after injury in calcification-prone
mice. DCC-susceptible C3H and DCC-resistant B6 mice were used as models for calcification in vivo. Our group previously developed a freeze-thaw injury model of acute calcification in mice. On day 1, monocytes/ macrophages could be detected infiltrating the injury site in the area between necrotic and healthy tissue. On day 3, macrophages had infiltrated most of the necrotic tissue, as shown by immunofluorescence staining for CD68 ( Fig. 2A,B). As early as day 3, calcium phosphate deposits could be visualized by calcein staining, as reported previously 14 . Close inspection of the cell morphology revealed macrophage aggregations within the calcified regions of the cardiac tissue in C3H mice (Fig. 2B, circles), but not in the non-calcified cardiac tissue in B6 mice ( Fig. 2A). Thus, macrophages from C3H mice are prone to aggregation and show osteogenic features, as reported previously by our group 14 .
Calcification-prone mice are prone to macrophage polarization. To characterize the phenotype of the macrophages that mediate DCC, the expression of CD68, a typical marker for the macrophage lineage, was evaluated in spleen-derived macrophages along with the expression of inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1), markers of M1 and M2 macrophages, respectively (Fig. 3). No differences in the expression of CD68 were observed between monocyte-derived macrophages from B6 and C3H mice (Fig. 3A). However, C3H macrophages were more prone to polarization and expressed higher levels of M1 and M2 markers than B6 macrophages (Fig. 3B,C). Expression of Arg-1 was detected only in macrophages derived from splenic monocytes from C3H mice (Fig. 3C).

Higher C3a and C3ar expression in MN precursor cells from DCC-susceptible mice. Complement
factors C3a and C5a play key roles in osteogenesis 23 . Accordingly, the expression of both factors and their corresponding receptors was evaluated during the differentiation of MN from monocytes of both B6 and C3H mice (Fig. 4). Monocytes and macrophages from C3H mice expressed higher levels of C3a and C3ar than those from B6 mice (Fig. 4A), but C3a and C3ar expression in MN cells did not differ between the strains (Fig. 4B). No expression of C5a could be detected in either group (Fig. 4C), although the MN cells of B6 mice tended to express higher levels of C5ar (Fig. 4B).
Higher release of ICAM-1 by macrophages of C3H mice. Splenic monocytes from B6 and C3H mice were differentiated into macrophages with M-CSF. The medium was then changed, and culture supernatants were collected 2 days later. The intercellular adhesion molecule ICAM-1 plays a key role in cell-cell adhesion and aggregation as well as in inflammation and osteoclastogenesis. The level of ICAM-1 in culture supernatants from B6 and C3H macrophages was compared (Fig. 4D). A high level of ICAM-1 was detected in culture supernatants from C3H monocyte-derived macrophages, while very little ICAM-1 was released by B6 macrophages.  systemic disorder, and circulating factors can regulate the initiation and development of the disease. Accordingly, we investigated whether circulating factors could influence MN cell formation in vitro. Splenic monocyte-derived macrophages were treated with serum from calcification-prone C3H and calcification-resistant B6 mice. The serum of B6 mice inhibited the formation of MN cells from macrophages of C3H mice (Fig. 5A,B). However, serum from DCC-susceptible C3H mice had no effect on the formation of MN cells from macrophages of B6 mice (Fig. 5B).
Reduced PPi levels and increased ICAM-1 expression in C3H mice. Jansen et al. showed recently that ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into PPi, a potent inhibitor of calcification 12 . In addition, they also reported reduced plasma PPi levels in Abcc6-deficient mice as well as in human PXE patients. Here, we tested the levels of plasma PPi in C3H and B6 mice. C3H mice, which are prone to calcification, had reduced plasma PPi compared with B6 mice (Fig. 6A).
Due to the high level of ICAM-1 released in vitro by macrophages from calcification-prone C3H mice, the differential expression of this gene was evaluated in vivo, in necrotic myocardial tissue from DCC-susceptible C3H and DCC-resistant B6 mice. Although necrotic tissue and macrophage infiltration were observed in both strains of mice, a marked increase in ICAM1 expression was observed at day 3 after injury in the calcification-susceptible C3H mice (Fig. 6B). PPi inhibits MN cell formation. Very recently, Abcc6 was linked to extracellular production 29 of PPi, which inhibits calcification in smooth muscle cells in vitro 30 . Consistent with this, DCC-resistant wild-type C57BL/6 mice had higher plasma PPi levels than DCC-susceptible Abcc6-deficient mice 7 . We therefore investigated whether PPi could inhibit MN cell formation. A range of PPi concentrations, from 3 to 100 μM, were tested for potential inhibition of MN cell formation from C3H macrophages. PPi significantly decreased MN cell formation in a dose-dependent manner (Fig. 7A,B). Moreover, Ctsk and Trap were downregulated following PPi treatment (Fig. 7C).

Effect of conditioned media from macrophages and MN cells on calcification.
To test whether macrophages or MN cells from C3H mice secreted factors that could induce osteogenesis, conditioned media from macrophages and MN cells from DCC-susceptible C3H and DCC-resistant C57BL/6 mice was collected. This conditioned media was used together with Pi, which induces calcification in C3H/10T1/2 cells. Conditioned media from monocyte-derived macrophages from B6 mice dramatically inhibited calcification of C3H/10T1/2   (Fig. 8A). Calcification was increased when C3H/10T1/2 cells were treated with 75% conditioned media from C3H MN cells compared with medium from B6 MN cells. No effect on calcification was observed when 50% conditioned media was used (Fig. 8B). Conditioned media from B6 macrophages showed a stronger inhibitory effect on calcification than conditioned media from B6 MN cells (Fig. 8A,B). In addition PPi-concentration was measured (Fig. 8C). PPi was found reduced in the conditioned media of macrophages from C3H mice.

PPi inhibits calcification in both C3H and Abcc6 −/− mice in vivo. The in vitro data described above indicate that PPi is a potential inhibitor of MN cell formation, and thus may also inhibit calcification in vivo.
To test this hypothesis, DCC-susceptible Abcc6 −/− mice and C3H mice were subjected to freeze-thaw injury to enhance calcification. For Abcc6 −/− mice, two different concentrations of PPi were used (12 and 25 mg/kg/ day) (Fig. 9A). For C3H mice, a dose of 20 mg/kg/day was used (Fig. 9B). PPi was administered by i.p. injection 1 day before freeze-thaw injury, and then daily for a period of 7 days. At that time, the hearts were excised, and calcium phosphate deposition was assessed. It is known that C3H mice are more predisposed to calcification than Abcc6 −/− mice because additional loci are associated with DCC in mice 1 . As expected, calcification was elevated in the hearts of both strains of mice following freeze-thaw injury, especially in necrotic myocardium as opposed to healthy tissue (Fig. 9B). However, supplementation with PPi inhibited calcification in both Abcc6 −/− mice and C3H mice (Fig. 9A,B).

Etidronate prevents but does not completely reverse DCC.
To determine whether etidronate could prevent or reverse DCC, C3H mice in which cardiac calcification had been induced by freeze-thaw injury were treated with 8 µmol/kg etidronate. Treatment was either initiated immediately after freeze-thaw injury and continued for 3 or 7 days, or started at day 4 post-injury, after the initiation of calcification (Fig. 9C). Treatment with etidronate after freeze-thaw injury and before the initiation of calcification prevented the development of DCC, but treatment after the initiation of calcification did not fully reverse calcium deposition. A single injection of etidronate during the macrophage infiltration and aggregation phase at day 3 after injury dramatically inhibited DCC progression.

Discussion
Soft tissue calcification shares common features with bone calcification. Loss of Abcc6 is sufficient to cause calcification, and Abcc6 deficiency leads to a lack of PPi 7,10-12 . The cellular component of calcification is mostly due to osteogenic differentiation of infiltrating monocyte-derived macrophages; smooth muscle cells and pericytes also contribute [31][32][33] . Some infiltrating macrophages fuse together to form MN cells. The role of these cells in DCC is not well understood. In this study, we showed that macrophages from calcification-susceptible mice express higher levels of inflammatory markers and the adhesion molecule ICAM-1 than macrophages from calcification-resistant mice, which leads to an increase in the differentiation of MN cells towards an osteogenic phenotype, significantly contributing to calcium phosphate deposits at the site of injury. Administration of PPi or etidronate prevents but does not reverse the calcification process in vivo.
We showed previously that DCC is caused by mutation of Abcc6 in C3H and other inbred strains of mice 11 . Four loci, Dyscalc1-4, were initially shown to be associated with DCC. Subsequently, a major locus containing Abcc6 was fine-mapped and determined to be causal for DCC. The three other loci act as modifiers, and influence the severity of DCC 1 . Higher levels of calcium phosphate deposits were observed in C3H mice with all Dyscalc1-4 loci than in Abcc6 −/− B6 mice or in congenic B6.C3H Dyscalc1 mice that only lack Abcc6 within the Dyscalc1 locus.
In mice and PXE patients, the effect of Abcc6 in ectopic calcification is largely due to systemic PPi deficiency 12,25 . Reduced plasma PPi levels are associated with the mineralization phenotype of several heritable or acquired conditions, including DCC [34][35][36][37][38][39] . In the present study, we confirmed that the calcification-susceptible C3H/He mouse model has reduced plasma levels of PPi. In several animal models, PPi supplementation can attenuate pathological calcification of various etiologies 40,34,37 . Because PPi has a short serum half-life, bisphosphonates (BiPs), stable non-hydrolyzable analogues of PPi, were developed for therapeutic use in osteoporosis and bone metastasis 41 . Two BiPs were tested in Abcc6 −/− mice as a model for PXE, with mixed results 42 . Alendronate had no effect, but a high dose of etidronate modestly decreased calcification in the vibrissae of Abcc6 −/− mice.
In a recent review, Orriss and co-workers described a role for PPi in osteogenic/OC differentiation as a potent inhibitor of osteoclastogenesis 43 . Indeed, the presence of osteogenic markers such as RUNX2, bone morphogenic Monocytes from DCC-susceptible C3H mice were isolated and subjected to MN differentiation using M-CSF and RANKL. At day 6, conditioned media from the macrophages was collected and PPi-concentration was measured (C). At day 12, after MN cell differentiation, cells were washed with PBS and fresh medium was added, and conditioned media from MN cells was collected 1 day later. Calcification was induced in C3H/10T1/2 mesenchymal stem cells with 2.6 mM Pi in the presence of conditioned media from macrophages or MN cells from C3H or B6 mice (diluted 50% or 75%). Calcium deposits were quantitated and compared. Nuclei are shown by DAPI staining, in blue, and calcified deposits are shown by calcein staining, in green.
Here, we demonstrated in two independent mouse models (C3H/He and B6.C3H Dyscalc1 ) that macrophage aggregates exhibit both OC and OB characteristics: they express the OC markers Trap and Ctsk, as well as OB markers such as Runx2, indicating that these cells are incompletely differentiated and appear in lesions before mature calcification. We used conditioned media from macrophages derived from both calcification-prone and calcification-resistant mice and demonstrated that ICAM-1 released from macrophages of calcification-prone mice can increase the calcification of mesenchymal stem cells.
Macrophages from C3H mice were more prone to polarization and expressed higher levels of M1 and M2 markers than those from B6 mice. Expression of Arg-1, an M2 marker, was observed only in macrophages derived from splenic monocytes of C3H mice. Similarly, the complement factors C3a and C3ar were expressed at higher levels in monocytes and macrophages from C3H mice than in those from B6 mice. OCs seem to influence OB differentiation via C3a, which activates the complement system 46 . C3-and C5-deficient mice exhibit reduced bone formation in the early healing phase 47 . Furthermore, ICAM-1, which is chemotactic for a wide variety of immune cells, was released from C3H macrophages in vitro, suggesting that macrophage aggregation in the injured heart tissue of C3H mice may occur in response to ICAM-1 release. ICAM-1 is expressed in mature osteoblasts inducing RANKL-secretion, adhesion of osteoclast precursors and thus osteoclast maturation 48 . The role of ICAM-1 in osteoclastogenesis has been reported previously [49][50][51] . In particular, Suzuki and co-workers demonstrated that neutralization of ICAM-1 inhibited osteoclastogenesis of SW982 cells in vitro 51  Thus, future studies will target ICAM-1 in vivo, to determine whether this molecule protects against cardiac calcification.
Our in vitro data provide further evidence that exogenous PPi inhibits calcification in C3H mice by influencing the differentiation of MN cells. The formation of MN cells from B6 macrophages did not increase in the presence of serum from C3H mice. These results indicate that the DCC-resistant B6 mice are genetically resistant to MN formation and calcification. However, C3H mice, in which Abcc6 function is altered, are genetically predisposed to MN cells formation, and thus initiate calcification after injury. Thus, we hypothesized that targeting MN cells formation with PPi or other drugs may be an effective therapeutic approach to prevent calcification in C3H mice.
We next showed that administration of PPi inhibited cardiac calcification in response to freeze-thaw injury in vivo, in two different calcification-prone mouse models (Abcc6 −/− B6 mice and C3H mice). Thus, BiPs, analogues of PPi, may have potential as inhibitors of soft tissue calcification. Cardiac calcification is not an uncommon pathology [52][53][54][55][56] . In light of our findings and in parallel with this work, we very recently demonstrated that . Control mice were injected with NaCl or PBS. One week after injury, hearts were collected. Whole heart (Panel A and C) or Healthy myocardium and necrotic myocardium (Panel B) from the same heart were excised, and calcium phosphate deposits were assessed. PPi-and etidronate-treated mice were compared with PBS-or NaCl-treated (control) mice.
SCIenTIfIC REPORTS | (2018) 8:5812 | DOI:10.1038/s41598-018-24228-y etidronate prevents cardiac calcification 25 . A partial inhibition of cardiac calcification was observed in mice treated with PPi, but a strong inhibition was seen in mice treated with etidronate. To further test whether calcification is reversible, mice were treated with etidronate after calcification had begun, at day 4 post-injury. In this experiment, no dramatic inhibition of calcification was observed. Interestingly, a single injection of etidronate on day 3, when macrophage infiltration and aggregation is evident in injured tissue, reduced calcification dramatically. This finding demonstrates the key role of macrophage infiltration, aggregation, and fusion in the calcification process. A single dose treatment at defined time of macrophage infiltration may be proposed as therapeutic approach rather than the daily treatment, if one takes in account the bad side effect of BiPs.
In summary, these data demonstrate that mice predisposed to calcification are deficient in circulating PPi and exhibit higher macrophage secretion of ICAM-1, leading to further macrophage aggregation and calcium phosphate deposition (Fig. 10). Exogenous administration of PPi or agents targeting ICAM-1 may be possible therapeutic approaches to prevent calcification. Figure 10. Proposed model: Abcc6 prevents DCC by inhibiting osteoclastogenesis. In our mouse model, tissue damage in the cardiovascular system in response to freeze-thaw injury of the heart induces an inflammatory response involving cross-talk between organs, mainly the liver and heart, mediated by the circulating blood. Hematopoietic stem cells in the bone marrow are mobilized and circulate to the spleen, which serves as a monocyte reservoir and site of monocyte differentiation (1). Circulating monocytes infiltrate and accumulate in necrotic heart tissue and differentiate into macrophages under the influence of local inflammatory mediators such as ICAM-1 (2). Subsequently, macrophages aggregate and fuse to form multinucleated (MN) cells, which are potentially relevant in calcification processes leading to DCC (3). DCC-susceptible mice exhibit elevated formation of MN cells expressing osteoclastic (OC) and osteoblastic (OB) markers. MN cells generate calcium deposits involving cardiac cells. Abcc6, which is mainly expressed in hepatocytes, encodes a membrane transporter and is the causal gene for DCC in mice. The functional Abcc6 transporter promotes the release of ATP, which is converted into AMP and inorganic pyrophosphate (PPi) via Enpp1 (ectonucleotide pyrophosphatase/phosphodiesterase). PPi is a potent inhibitor of osteoclastogenesis and smooth muscle cell calcification. High circulating levels of PPi in DCC-resistant mice or supplementation of DCC-susceptible mice with PPi inhibits macrophage differentiation into MN cells, and thus prevents formation of calcium deposits (3).