Ferryl Hemoglobin Inhibits Osteoclastic Differentiation of Macrophages in Hemorrhaged Atherosclerotic Plaques

Intraplaque hemorrhage frequently occurs in atherosclerotic plaques resulting in cell-free hemoglobin, which is oxidized to ferryl hemoglobin (FHb) in the highly oxidative environment. Osteoclast-like cells (OLCs) derived from macrophages signify a counterbalance mechanism for calcium deposition in atherosclerosis. Our aim was to investigate whether oxidized hemoglobin alters osteoclast formation, thereby affecting calcium removal from mineralized atherosclerotic lesions. RANKL- (receptor activator of nuclear factor kappa-Β ligand-) induced osteoclastogenic differentiation and osteoclast activity of RAW264.7 cells were studied in response to oxidized hemoglobin via assessing bone resorption activity, expression of osteoclast-specific genes, and the activation of signalization pathways. OLCs in diseased human carotid arteries were assessed by immunohistochemistry. FHb, but not ferrohemoglobin, decreased bone resorption activity and inhibited osteoclast-specific gene expression (tartrate-resistant acid phosphatase, calcitonin receptor, and dendritic cell-specific transmembrane protein) induced by RANKL. In addition, FHb inhibited osteoclastogenic signaling pathways downstream of RANK (receptor activator of nuclear factor kappa-Β). It prevented the induction of TRAF6 (tumor necrosis factor (TNF) receptor-associated factor 6) and c-Fos, phosphorylation of p-38 and JNK (c-Jun N-terminal kinase), and nuclear translocation of NFκB (nuclear factor kappa-Β) and NFATc1 (nuclear factor of activated T-cells, cytoplasmic 1). These effects were independent of heme oxygenase-1 demonstrated by knocking down HO-1 gene in RAW264.7 cells and in mice. Importantly, FHb competed with RANK for RANKL binding suggesting possible mechanisms by which FHb impairs osteoclastic differentiation. In diseased human carotid arteries, OLCs were abundantly present in calcified plaques and colocalized with regions of calcium deposition, while the number of these cells were lower in hemorrhagic lesions exhibiting accumulation of FHb despite calcium deposition. We conclude that FHb inhibits RANKL-induced osteoclastic differentiation of macrophages and suggest that accumulation of FHb in a calcified area of atherosclerotic lesion with hemorrhage retards the formation of OLCs potentially impairing calcium resorption.


Introduction
Cardiovascular disease is the leading cause of death worldwide [1] and vascular calcification is one of the independent risk factors associated with such morbidity and mortality [2][3][4][5]. Pathogenesis of vascular calcification is an active, finely tuned process with many similarities to the mechanism of skeletal bone formation [6]. In the bone, mineral deposition by osteoblasts and bone resorption by osteoclasts (OCs) are synchronized processes [7]. Disruption of the balance between osteoblast and OC activity may have pathological consequences such as osteoporosis or osteopetrosis [8,9].
RANKL and M-CSF are both expressed in calcifying vessels [23,24]. In atherosclerotic plaques, calcification and bone formation are common phenomena characterized by the presence of vascular smooth muscle cells (VSMCs), osteoblasts, and osteoclast-like cells (OLCs) [25]. OLCs differentiate from infiltrating macrophages and colocalize with cholesterol deposition and mineralization [26]. An intriguing model has been raised by Doherty and coworkers suggesting that arterial calcium deposits represent a unique scenario which might favor the formation of OLCs from hematopoietic precursors possibly limiting calcification in atherosclerosis [27]. This hypothesis is further supported by the fact that Runx2, a key transcription factor that induces transition of VSMCs to osteoblast-like cells, directly binds to the promoter of RANKL and activates its expression leading to mineral deposition by VSMCs-derived osteoblasts and mineral resorption by OLCs [28]. Within the intramural compartment of the arteries, OLCs might degrade mineral deposits, thereby attenuating calcification and counterbalancing the activity of VSMC-derived osteoblasts [26]. The imbalance between bone formation by VSMC-derived osteoblasts and bone resorption by OLCs triggers pathological calcification process in the vessel walls.
Li and coworkers have described that complicated plaques with hemorrhage are characterized by a highly oxidative scenario creating a "death zone" for red blood cells (RBCs) [29]. RBCs in these death zones are lysed, and free Hb is subjected to rapid oxidation forming met-hemoglobin (MetHb, Fe 3+ ) and ferryl hemoglobin (FHb, Fe 4+ = O 2− ). Importantly, oxidation of Hb also leads to the release of heme moieties [30]. A significant body of evidence suggests that MetHb and FHb are present in hemorrhagic complicated plaques [31]. Our research group previously reported that FHb is a potent proinflammatory agonist in endothelial cells that induces morphological changes [32], increases monolayer permeability, and enhances monocyte adhesion [33]. These data suggest that oxidized Hb forms are involved in the pathogenesis of atherosclerosis.
The massive Hb content of hemorrhagic atheromas prompted us to examine whether the compensatory effect of OLCs in vascular calcification is influenced by products of Hb oxidation. The purpose of this study was to investigate the role of oxidized Hb in OC formation and resorption of calcium in calcified atheromas. 2.3. In vitro Osteoclastogenesis. Cells (2 × 10 4 /cm 2 ) were seeded onto 24-well plates and cultured in growth medium supplemented with RANKL (50 ng/mL) (osteoclastogenic medium) in the presence or absence of Hb (10 μmol/L heme group), MetHb (10 μmol/L heme group), and FHb (10 μmol/L heme group) as indicated. The medium was changed every 2-3 days.

Materials and Methods
2.4. Immunohistochemistry. Carotid artery specimens were fixed with PBS formaldehyde (4%) solution at pH 7.4 for 1 to 3 days based on the size of the sample. After fixation, calcified samples were decalcified with 1.0 mol/L EDTA/Tris buffer. Paraffin-embedded 5-μm-sections were deparaffanized in xylenes, rehydrated in a series of ethanol rinses from 100% to 70%, then washed with distilled water. Antigen retrieval was performed in RE7119 buffer (Leica, Wetzlar, Germany) at 95°C for 30 minutes. Sections were allowed to cool slowly, washed in distilled water, and incubated in 0.5% H 2 O 2 for 10 minutes. For immunohistochemistry, samples were incubated with Dako EnVision FLEX Peroxidase-Blocking Reagent (Dako, Glostrup, Denmark) for 5 min in a wet chamber. Slides were then washed with EnVisionTM FLEX Wash Buffer, Tris-buffered saline solution containing Tween 20, and pH 7.6 (±0.1). Serial sections slides were incubated with antibodies against TRAcP (Roche, Mannheim, Germany, ready-to-use) or cathepsin K (Abcam, Cambridge, UK) at a dilution of 1 : 200, or CD68 (Roche, ready-to-use) or anti-FHb polyclonal antibody at a dilution of 1 : 50 using the ultraview universal DAB detection kit following the manufacturer's instructions. The intensity and distribution of 2.12. Cytoplasmic and Nuclear Protein Extraction. Cells were grown in 6-well plates in growth medium or osteoclastogenic medium in the presence or absence of FHb for indicated time. Cells were washed three times with PBS and lysed with Harvest buffer (10 mM HEPES pH 7.9, 50 mM NaCl, 0.5 M sucrose, 0.5% Triton X-100, and protease inhibitors). After 10 min of incubation on ice, samples were spinned at 1000 × g for 5 min and supernatants were collected as cytosolic fraction. Pellets containing the nuclear fraction were washed three times with wash buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1% NP-40, and protease inhibitors) and solubilized in nuclear protein extraction buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors).
2.13. Immunofluorescence Staining. Cells were treated as described above with RANKL in the presence or absence of FHb. Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) pH 7.4 for 15 minutes. Coverslips were washed with PBS and samples were blocked with 5% goat serum in PBS supplemented with 0.3% Triton X-100 for 60 min. Samples were then incubated with primary antibody against NFATc1 (Novus Biologicals, Littleton, CO, USA) at a 1 : 250 dilution overnight at 4°C in antibody dilution buffer (1% BSA in PBS supplemented with 0.3% Triton X-100). The secondary antibody was a goat anti-mouse IgG conjugated to Alexa Fluor® 488 (Thermo Scientific) used at a 1 : 500 dilution in antibody dilution buffer and incubated for 60 min at room temperature. Nuclei were visualized with Hoechst. Nuclear translocation was investigated with TCS SP8 STED microscope using the Leica Application Software X (Leica, Mannheim, Germany).
2.15. HO Activity Assay. Cells grown on 6-well plates were washed twice with Hank's Balanced Salt Solution (HBSS), and scraped and centrifuged at 2000 × g for 15 min at 4°C. Cells were re-suspended in 300 μL of potassium phosphate (100 mmol/L (pH 7.4)) buffer containing 2 mmol/L MgCl 2 , frozen and thawed three times, and sonicated and centrifuged at 18,000 × g for 10 min at 4°C. Supernatants containing cell microsomes were used to measure HO activity as described previously [36]. HO activity is expressed as pmol bilirubin formed/mg cell protein per 60 min.
2.16. HO-1 Short-Interfering RNA (siRNA) Transfection. Small interfering RNA (siRNA) specific to HO-1 and negative control siRNA were obtained from Ambion (Austin, TX, USA). Transfection of siRNA into RAW264.7 cells was performed using the Oligofectamine Reagent (Invitrogen, Carlsbad, CA, USA). Briefly, cells were plated in antibioticfree DMEM and cultured for 6 h. HO-1 siRNA at 40 nmol/L and transfection reagent complex were added to the cells in serum-free medium OptiMEM (Gibco, Thermo Scientific) for 16 h. Fresh normal growth medium was added then and the cells were incubated for another 8 h.
2.17. Detection of Crosslinked Hb by Western Blot. The detection of crosslinked Hb in three healthy carotid arteries, three atheromas, and three complicated carotid lesions with hemorrhage by Western blot was performed as described in our previous study [31] using HRP-conjugated goat antihuman Hb polyclonal antibody (ab19362-1 Abcam, Cambridge, UK) at a dilution of 1 : 15000.

Expression of Recombinant Mouse RANKL with 6× His
Tag. For in vitro interaction assay, recombinant mouse RANKL with N-terminal 6 × His tag was expressed in E. coli Rosetta 2. Total mRNA was isolated from the lung tissue of C57BL mice, reverse transcribed, cloned into pTriex-4 Neo, and verified by sequencing. To express His-tagged RANKL, E. coli Rosetta 2 cells were transformed and cultured in Luria-Bertani medium containing 100 μg/mL carbenicilllin at 30°C 250 rpm until OD600 = 0:5, then protein expression was induced with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) followed by shaking at 250 rpm at 30°C for 3 h. Cells were then pelleted at 3990 × g at room temperature for 15 min and lysed with cold lysis buffer pH 8.0 (50 mM NaH 2 PO 4 , 300 mM NaCl, 1% Triton X-100, protease inhibitors, and 1 mg/mL lysozyme). His-tagged RANKL was purified using Protino Ni-TED 150 packed columns according to the manufacturer's guide. Endotoxin contamination was removed using Pierce™ High Capacity Endotoxin Removal Resin (Thermo). The quality of recombinant RANKL was analyzed by Coomassie staining and immunoblot. Recombinant His-tagged RANKL was effective to induce OC formation demonstrated by TRAP staining.

In Vitro RANK-RANKL Interaction Assay.
To study the inhibitory effect of FHb on RANK-RANKL interaction in a test tube experiment, RANK was purified from RAW cells using immunoprecipitation. Briefly, cells were lysed with cold lysis buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 2× protease inhibitor cocktail, and 1% Triton X-100; incubated on ice for 10 min; and clarified by centrifugation at 14000 × g for 10 min at 4°C. Supernatants were then gently rocked at 4°C with 15 μg of RANK antibody overnight, then antigen-antibody complexes were coincubated with prewashed protein A/G magnetic beads (Thermo Scientific) for 60 min at room temperature. Beads were then washed three times with cold wash buffer containing detergent (50 mM Tris pH 7.5, 150 mM NaCl, 0.05% Igepal CA630, and protease inhibitors), then three times with wash buffer without detergent (50 mM Tris pH 7.5, 150 mM NaCl), and coincubated with 1 μg RANKL or 1 μg RANKL and 10 μM FHb at room temperature for 60 min. Beads were then washed three times with 50 mM Tris pH 7.5 and 150 mM NaCl, and samples were eluted with 2 × SDS sample buffer without reducing agent at 50°C for 10 min, supplemented with 100 mM DTT and subjected to immunoblot analysis.
2.20. Statistical Analysis. Statistical analysis was performed with GraphPad Prism 5 by one-way ANOVA test followed by post hoc Bonferroni's Multiple Comparison test or t test. A significant value of p < 0:05 was marked with * , p < 0:01 with * * , and p < 0:001 was marked with * * * . Nonsignificant (ns) differences were also marked. Data are shown as mean ± SEM.

FHb Inhibits Osteoclastogenesis and OC Bone Resorption
Activity. Macrophages can transform into OCs in response to RANKL [37,38]. To investigate the effect of Hbs on RANKL-induced osteoclastogenesis, murine macrophage RAW264.7 cells were cultured in osteoclastogenic medium containing 50 ng/mL RANKL in the presence or absence of Hb, MetHb, or FHb. First, we analyzed the effect of the different Hb forms on RANKL-induced osteoclastogenesis using TRAP staining (Figure 1(a)). Heme (50 μmol/L), a potent inhibitor of RANKL-induced osteoclastogenesis, was used as positive control for inhibition assays. RANKL-induced OC formation was significantly inhibited either by heme (50 μmol/L) or FHb (10 μmol/L heme group), but not by Hb (10 μmol/L heme group) or MetHb (10 μmol/L heme group). To quantify OC formation, the areas of TRAP-positive cells were measured by the ImageJ software (Figure 1(b)). This analysis revealed that RANKL-induced OC formation was significantly (p < 0:001) impaired either by heme or FHb, but not by Hb or MetHb (Figure 1(b)). Next, we examined whether FHb inhibits bone resorption activity of OCs (Figure 1(c)). Our data suggest that both FHb and heme significantly (p < 0:001) reduced the resorption area of OCs, while neither Hb nor MetHb influenced bone resorptive activity (Figures 1(c) and 1(d)). Next, we examined whether a doseresponse relationship exists between FHb concentration and inhibition of OC differentiation. We showed that FHb inhibited osteoclastogenesis in a dose-dependent manner, and as low as 2.5 μmol/L FHb significantly prevented OC formation as evidenced by TRAP staining (Figure 1(e) upper panel and Figure 1(f)) and bone resorption assay (Figure 1(e) lower panel and Figure 1(g)). To explore whether this inhibitory effect of FHb is associated with its potential cytotoxic effect, we cultured RAW264.7 cells for 4 days in the presence or absence of RANKL and FHb. Cell proliferation and viability were analyzed with MTT assay at various time points (one to four days). Our results showed the proliferation of RAW264.7 cells decreased in response to RANKL as well as to RANKL+FHb compared with untreated cells; however, FHb did not affect proliferation compared with RANKLtreated cells (Supplementary fig. 1A). Next, we explored whether such decreased proliferation was associated with apoptotic cell death. Accordingly, we analyzed caspase-3 cleavage as a marker of apoptosis by immunoblot which showed that apoptotic cell death did not occur during OC differentiation in response to RANKL and FHb (Supplementary fig. 1B).

Inhibitory Effect of FHb on Osteoclastogenesis Is
Independent of HO-1. We have previously described that FHb induces HO-1 in endothelial cells [33] and it has been reported that upregulation of HO-1 by heme inhibits OC formation and bone resorption in vitro [40]. Therefore, we tested whether HO-1 mediates the inhibitory effect of FHb on OC formation. As shown in Figure 3, FHb, similar to heme, significantly induced HO-1 expression both at mRNA and protein levels (Figures 3(a)-3(c)) and increased HO enzyme activity (Figure 3(d)) in RAW264.7 cells.
We knocked down HO-1 expression by HO-1-specific siRNA and analyzed osteoclastogenesis in response to RANKL and FHb. Surprisingly, FHb prevented RANKLinduced OC formation in HO-1-silenced cells similar to the controls that was demonstrated by TRAP staining (Figures 4(a) and 4(c)) and bone resorption assay (Figures 4(b) and 4(d)). Silencing HO-1 expression was confirmed by immunoblotting (Figure 4(e)). To further verify our results at the molecular level, CTR expression-as OC marker-was monitored by q-RT-PCR in HO-1-knocked down cells after RANKL treatment. We showed that irrespective of the degree of HO-1 expression, administration of FHb significantly decreased RANKL-mediated CTR mRNA expression (Figure 4(f)).

FHb
Blocks RANK-RANKL Interaction. The formation of OCs requires RANKL attachment to its receptor RANK [41]. In addition, RANKL directly induces RANK expression [42]. To decipher the molecular mechanism by which FHb inhibits OC formation, we tested whether FHb influences RANKLinduced RANK expression. We demonstrated that FHb significantly attenuated RANK expression in response to RANKL both at mRNA (Figure 5(a)) and protein levels ( Figures 5(b) and 5(c)). To gain a more mechanistic insight into the inhibitory effect of FHb on OC formation, we analyzed whether FHb inhibits the RANK-RANKL interaction by measuring the amount of cell-associated RANKL by immunoblot. We showed that the association of exogenous RANKL to cells was markedly decreased when FHb was present in the experimental medium (Figures 5(d) and 5(e)). To further verify this observation, we developed a recombinant His-tagged RANKL to study RANK-RANKL interaction in test tube experiments. The purity of our in-house Histagged RANKL was validated by Coomassie staining and immunoblot (Supplementary fig. 2A). We demonstrated that His-tagged RANKL effectively induced OC formation in RAW cultures demonstrated by TRAP staining (Supplementary fig. 2B). In the test tube experiments, RANK was These data suggest that the inhibitory effect of FHb on RANK expression and osteoclastic transformation of RAW264.7 cells were mediated by inhibition of the direct interaction between RANK and RANKL.

FHb Inhibits RANKL-Induced Signaling Involved in OC
Differentiation. RANK-RANKL interaction initiates a series of signaling events leading to OC formation from macrophages [43]. This includes TRAF6 which activates downstream pathways, such as NFκB, JNK, p38, c-Fos, and NFATc1, which are all crucial factors in OC differentiation.
Here, we showed that RANKL induced TRAF6 expression (Figure 6(a)), p38 and JNK activation (Figure 6(b)), c-Fos expression (Figure 6(c)), nuclear translocation of NFκB ( Figure 6(d)), and NFATc1 (Figures 6(e)-6(h)). Importantly, the exposure of cells to FHb prevented the induction of TRAF6 and c-Fos, phosphorylation of p38 and JNK, and nuclear translocation of NFκB and NFATc1. These results corroborate our hypothesis that FHb inhibits RANK-RANKL interaction and its subsequent signaling pathways, thereby preventing OC differentiation from macrophages.

Oxidation of Hb Occurs in Calcified Lesions with
Hemorrhage. Our previous studies revealed that complicated lesions with hemorrhage contain oxidized forms of hemoglobin [31] which is also corroborated by our current study. Spectrophotometric analysis of the human vessel samples showed that oxidized Hb was present in the calcified atheromas with hemorrhage, while healthy arteries and calcified lesions did not contain oxidized Hb (Figure 7(a)). We observed the marked accumulation of crosslinked Hb dimers, tetramers, and multimers in hemorrhagic calcified plaques reflecting that Hb oxidation are extensive in these lesions compared with calcified atheromas without hemorrhage or healthy carotid arteries (Figure 7(b)).

Lack of OLCs Is Associated with the Presence of FHb in
Hemorrhagic Calcified Lesions in Human Vessels. To examine whether FHb inhibits OLC formation in patients who underwent carotid endarterectomy, the presence of OLCs was analyzed in healthy carotid arteries, calcified atheromas, and calcified atheromas with hemorrhage (Figure 7(c)). Extracellular calcium deposits were present in calcified atheromas and in calcified atheromas with hemorrhage as evidenced by Von Kossa staining (row B). The presence of FHb was prominent in calcified atheromas with hemorrhage while no positive staining pattern could be seen in carotid arteries from healthy individuals or calcified lesions without hemorrhage (row C). Furthermore, multiple CD68 positive, multinucleated giant cells were detected in calcified lesions while

Discussion
The presence of OLCs in the calcified area of atherosclerotic plaques is well demonstrated [44]. According to the "osteoclast theory," an increased osteoblastic and a reduced osteoclastic activity might contribute to intima calcification [45]. Therefore, disturbances in OLC differentiation and activity in the atherosclerotic plaques might be considered a pathogenic factor in vessel wall calcification.
Vascular calcification is associated with increased cardiovascular morbidity and about one-fifth of the calcified vessels and valves contain trabecular bone [46]. The molecular mechanism of arterial calcification resembles bone mineralization sharing a number of similarities [47]. Under  Figure 2: FHb downregulates OC-specific gene expression in response to RANKL RAW264.7 cells were cultured on 24-well plates in control growth medium or in osteoclastogenic medium (control growth medium supplemented with 50 ng/mL RANKL) or in osteoclastogenic medium with heme (50 μmol/L), Hb (10 μmol/L heme group), MetHb (10 μmol/L heme group), or FHb (10 μmol/L heme group). CTR (a), DC-STAMP (b), and NFATc1 (c) gene expressions were analyzed with quantitative RT-PCR and immunoblot after 5 days for CTR, 4 days for DC-STAMP, and 3 days for NFATc1. qRT-PCR was normalized to β-actin while immunoblot was normalized to GAPDH. Data are expressed as mean ± SE of three independent experiments. 8 Oxidative Medicine and Cellular Longevity homeostatic conditions in bones, mineral deposition by osteoblast and resorption mediated by OCs are strictly coupled resulting in a delicate balance between bone anabolism and catabolism [48,49]. The importance of functional OLCs in vascular calcification has been documented by several studies. Mice lacking carbonic anhydrase II, which is essential for bone resorption activity of OLCs, develop arterial calcification [50]. In addition, the lack of M-CSF, which is necessary for OLC formation, promotes vascular calcification in mice possibly due to the impaired OLC formation [51]. Overall, these findings suggest that functional OLCs are essential for mineral resorption in the vasculature, and that lack of OLC activity promotes vascular calcification. Multinucleated, TRAP-positive cells with typical osteoclastic morphology were identified in atherosclerotic lesions close to the mineralized areas [26]. This observation was also supported by our present results in human atherosclerotic carotid arteries demonstrating the presence of a vast number of OLCs with strong cathepsin K and TRAP positivity in the vessel wall areas where mineralization occurred. However, the number of OLCs was limited in the calcified atheromas with hemorrhage that were characterized by the presence of oxidized Hbs, and this observation supports our hypothesis that FHb abolishes OLCs from hem-orrhagic, calcified carotid tissues resulting in insufficient mineral resorption from the vessel wall.
The formation of OLCs in the vasculature is dependent on RANKL that is abundantly secreted by a number of cell types, such as VSMCs and endothelial cells [52][53][54]. Following interaction with RANK, RANKL induces the activation of downstream signalization, such as tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) [12], c-Jun N-terminal kinase (JNK) [13], p38 [14], receptor activator of nuclear factor-kappa B (NFκB) [15], c-Fos [16], and NFATc1. NFATc1 activates a number of downstream genes such as CTR, cathepsin K, and TRAP [55]. The pivotal function of NFATc1 is highlighted by the significant impairment of osteoclastogenesis in OC-specific conditional NFATc1-deficient mice [56]. In addition, NFATc1-deficient embryonic stem cells are unable to differentiate into OCs in response to RANKL [55]. In our study, we showed that FHb but not MetHb or ferrous Hb inhibited the expression of NFATc1. In addition, the expression of other OC markers, such as CTR and DC-STAMP, was also alleviated by FHb, and, to a lesser degree, by MetHb, but not by ferrous Hb. These data indicate that the inhibitory effect of FHb on osteoclastogenesis is mediated by the suppression of the key genes involved in this process. Furthermore, we demonstrated that this

10
Oxidative Medicine and Cellular Longevity inhibitory effect of FHb was dose-dependent, occurred even at low micromolar concentrations and was not associated with cell death. To explore the signaling events downstream of RANK in response to RANKL and FHb, we analyzed the key proteins involved in osteoclastogenesis. Previous studies have revealed that TRAF6 is essential in the cytoskeletal organization and resorptive activity of OC [12]. Mitogen-activated protein kinases (MAPKs), such as JNK and p38, are critical for normal osteoclastogenic differentiation and activation [13,14]. NFκB, together with c-Fos, is also important determinants of OC formation [15,16]. Here, we showed that FHb blunted early signaling events of osteoclastogenesis involving MAPK and NFκB activation as well as c-Fos and TRAF-6, thereby blocking the osteoclastogenic reprogramming of macrophages. Previously, it was demonstrated that heme inhibits osteoclastogenesis as well as the expression of OC marker genes such as TRAP, CTR, and DC-STAMP via the induction of HO-1, a key enzyme of heme catabolism [40]. We have reported that MetHb but not ferrous Hb releases its heme   13 Oxidative Medicine and Cellular Longevity prosthetic group that is taken up by endothelial cells followed by the activation of HO-1 [57]. Here, we demonstrated that FHb induced catalytically active HO-1 supposing that its heme group could be responsible for the inhibition of osteoclastogenesis [40]. Furthermore, others have demonstrated that deltamethrin, a pyrethroid pesticide or magnolol, an extract with anti-inflammatory properties isolated from Magnolia officinalis prevents OC formation in a HO-1 dependent fashion [58,59]. These observations underscore the inhibitory effects of HO-1 in osteoclastogenesis. However, our results presented here showed that the inhibitory effect of FHb on OC formation was independent of HO-1. This notion was also corroborated by using murine BMDMs derived from HO-1 −/− that indicated other mechanisms are involved in FHb-mediated inhibition of OC formation that are independent of HO enzyme activity.
RANK and RANKL interaction is essential for OC formation, and RANK expression is vital for the differentiation of myeloid-derived OCs [7,37,54] as evidenced by the lack of OC differentiation in RANK (−/−) mice [60]. Our study provides evidence that FHb can attenuate RANKL-induced RANK expression in RAW264.7 cells suggesting a possible mechanism by which FHb impairs OC differentiation from macrophages.
Osteoprotegerin (OPG) is a decoy receptor for RANKL and plays a regulatory role in bone resorption by inhibiting OC function [61]. As a dimer, OPG competes with RANK for RANKL binding and effectively inhibits RANK-RANKL interaction [62]. Here, by demonstrating the decrease of RANKL interaction with RANK in RAW264.7 cells, we provide an additional potential explanation for the involvement of FHb in the impairment of OC differentiation. This observation was corroborated by our test tube experiments, which support the hypothesis that FHb directly hinders RANK-RANKL interaction. Our results suggest that FHb inhibits OC formation in an OPG-like manner by directly inhibiting RANK-RANKL interaction. The exact mechanism of such interaction requires further investigations.

Conclusions
In summary, we provide evidence for the involvement of FHb in the inhibition of osteoclastogenesis ( Figure 8). This effect of FHb suggests that the presence of FHb in hemorrhagic atheromas might create a unique microenvironment where OLC-mediated resorption of calcium deposits is impaired that blocks the endogenous calcium resorption capability in the vasculature.

Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
The authors confirm that there are no conflicts of interest.

Acknowledgments
The research group of G.B. is supported by the Hungarian Academy of Sciences (11003). This research/project is FHb, which is abundantly present in hemorrhagic calcified lesions, impedes the formation of OCs from macrophages disturbing OC bone resorption activity by downregulating OC-specific gene expression such as NFATc1, DC-STAMP, TRAP, CatK, and CTR. During vascular calcification, smooth muscle cells in the vessel walls undergo osteochondrogenic reprogramming and produce RANKL, which initiates OC formation from macrophages as a potential compensatory mechanism to remove calcium deposits. However, the presence of FHb in hemorrhagic atheromas might create a unique microenvironment where OLCs-mediated resorption of calcium deposits is impaired, thereby blocking the endogenous calcium resorption capability in the vasculature.