Ferritin Prevents Calcification and Osteoblastic Differentiation of Vascular Smooth Muscle Cells

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Abolfazl Zarjou,* Vikto´ria Jeney,* Paolo Arosio,† Maura Poli,† Pe´ter Antal-Szalma´s,‡ ʈ Anupam Agarwal,§ Gyo¨rgy Balla, and Jo´zsef Balla*

ʈ Departments of *Medicine, ‡Clinical Biochemistry and Molecular Pathology, and Pediatrics, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary; †Dipartimento Materno Infantile e Tecnologie Biomediche, University of Brescia, Brescia, Italy; and §Department of Medicine, Nephrology Research and Training Center and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama

ABSTRACT Vascular calcification plays a role in the pathogenesis of atherosclerosis, diabetes, and chronic kidney disease. Human aortic smooth muscle cells (HSMCs) undergo mineralization in response to elevated levels of inorganic phosphate (Pi) in an active and well-regulated process. This process involves increased activity of alkaline phosphatase and increased expression of core binding factor ␣-1, a bone-specific transcription factor, with the subsequent induction of osteocalcin. Mounting evidence suggests an essential role for the heme oxygenase 1 (HO-1)/ferritin system to maintain homeostasis of vascular function. We examined whether induction of HO-1 and ferritin alters mineralization of HSMCs provoked by high Pi. Upregulation of the HO-1/ferritin system inhibited HSMC calcification and osteoblastic differentiation. Of the products of the system, only ferritin and, to a lesser extent, biliverdin were responsible for the inhibition. Ferritin heavy chain and ceruloplasmin, which both possess ferroxidase activity, inhibited calcification; a site-directed mutant of ferritin heavy chain, which lacked ferroxidase activity, failed to inhibit calcification. In addition, osteoblastic transformation of HSMCs provoked by elevated Pi (assessed by upregulation of core binding factor ␣-1, osteocalcin, and alkaline phosphatase activity) was diminished by ferritin/ferroxidase activity. We conclude that induction of the HO-1/ferritin system prevents Pi-mediated calcification and osteoblastic differentiation of human smooth muscle cells mainly via the ferroxidase activity of ferritin.

J Am Soc Nephrol 20: 1254–1263, 2009. doi: 10.1681/ASN.2008070788

Vascular calcification occurs in many pathologic tion follows two distinct patterns: (1) Intimal calci- conditions and can lead to devastating clinical con- fication that occurs with atherosclerotic plaques sequences. For example, it has been related to in- and (2) medial calcification, which is characterized creased risk for cardiovascular morbidities and by diffuse calcification of the media, particularly at complications such as atherosclerotic plaque burden,1–3 myocardial infarction,4,5 coronary artery Received July 26, 2008. Accepted January 22, 2009. disease,6,7 postangioplasty dissection,8 and increased ischemic episodes in peripheral vascular Published online ahead of print. Publication date available at www.jasn.org. disease.9 Studies also have indicated that coronary calcification may be predictive of or associated with A.Z. and V.J. contributed equally to this work. sudden cardiac death.10,11 Indeed, coronary calcifi- G.B. and J.B. contributed equally to this work. cation score measured by electron beam computed Correspondence: Dr. Jo´zsef Balla, Pf. 19, Nagyerdei krt. 98, tomography has been shown to have a prognostic 4012 Debrecen, Hungary. Phone/Fax: 36-52-413-653; E-mail: value for cardiovascular events comparable to that [email protected] of the Framingham risk index.11 Vascular calcifica- Copyright ᮊ 2009 by the American Society of Nephrology

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the level of the internal elastic lamina, that does not necessarily accompany atherosclerosis. This pattern is commonly seen in patients with chronic kidney disease (CKD), who commonly exhibit hyperphosphatemia. The mechanism of vascular calcification is not completely understood, although abnormalities in mineral metabolism are considered important risk factors. Many studies have demonstrated the role of high extracellular inorganic phosphate (Pi) to induce calcification of vascular cells in vitro12–16 in a process mediated by a sodium-dependent phosphate co-transporter that facilitates entry of Pi into vascular cells.17 This induces transition of human aortic smooth muscle cells (HSMCs) into osteoblast-like cells through a process that is accompanied by increased expression of core binding factor ␣-1 (Cbfa-1), which is an osteoblast- specific transcription factor required for osteoblast differentiation, bone matrix gene expression, and, consequently, bone mineralization.18 There is also an upregulation of alkaline phosphatase (ALP), an important enzyme in early osteogenesis and osteocalcin, a major noncollagenous protein found in bone matrix that is believed to regulate mineralization.19 Heme is a ubiquitous iron-containing molecule that is an absolute necessity for aerobic life. Current evidence suggests that heme can be pro-oxidant and potentially toxic.20–22 Heme induces the synthesis of heme oxygenase 1 (HO-1), the rate- limiting enzyme in the catabolism of heme.23 HO cleaves the porphyrin ring at the ␣-methene bridge to form biliverdin and carbon monoxide and releases free redox active iron. Biliver- Figure 1. Heme inhibits HSMC calcification induced by elevated din is then converted to bilirubin by biliverdin reductase. Pi in a dosage-dependent manner. (A) HSMCs were cultured in Ferritin is another molecule strongly inducible by heme and GM (I) or in calcification medium in the absence (II) or presence of iron. This is an iron storage protein that exhibits antioxidant heme (50 ␮mol/L; III) for 9 d. Von Kossa staining of cells was properties, and it was shown to protect the endothelium performed as described in the Concise Methods section. Repre- against the damaging effects of heme and oxidants.24 Ferritin is sentative picture of three separate experiments. (B) HSMCs were a large (450 kD), spherical shell that can store up to 4500 Fe cultured in GM or in calcification medium alone or supplemented ␮ atoms in a safe, nontoxic form. It is made of 24 subunits of two with NaOH (Vehicle, 1 mmol/L) or 5, 25, and 50 mol/L heme types (heavy [H] and light [L] chain) whose proportion de- (dissolved in NaOH at a final concentration of 1 mmol/L in each group). Calcium contents of cells were measured after 3 (Ⅺ),6(u), pends on the iron status of the cell, the tissue, and the organ.25 and9d(f) of culture as described in the Concise Methods The H-chain has ferroxidase activity that is important not only section and were normalized by protein content. Data are for iron incorporation but also in controlling the potentially means Ϯ SD of three independent experiments performed in toxic Fe (II) ions, thereby reducing oxidative damage.26 duplicate. (C) HSMCs were cultured in GM alone or supple- In our investigations, we tested the role that heme may play mented with 2, 3, or 4 mmol/L Pi (F). The media containing in the process of extracellular calcification, and we observed different amounts of Pi was supplemented with heme (50 ␮mol/L; that heme decreases extracellular matrix calcification in a dos- Œ) or with NaOH (vehicle, 1 mmol/L; ‚). Calcium deposition was age-responsive manner. These observations prompted us to measured at day 9, and results were normalized by protein con- hypothesize that one or more products of heme catabolism tent of the cells. Data show the average of three separate exper- Ͻ Ͻ may inhibit HSMC mineralization. iments performed in duplicate. *P 0.05; **P 0.01. Magnifi- cation, ϫ100.

RESULTS 1AI). Intriguing, we found that addition of heme (50 ␮mol/L, 9 d) to the calcification medium inhibited calcium deposition Heme Decreases HSMC Calcification in a Dosage- as shown by von Kossa staining (Figure 1AIII). Extracellular Responsive Manner calcium measurements showed that the inhibitory effect of To develop an in vitro model, we cultured HSMCs in calcifica- heme on extracellular calcification is dosage dependent, with a tion medium. Granular deposits developed in HSMCs grown highly significant (P Ͻ 0.01) suppression at a dosage of 25 in calcification medium for 9 d (Figure 1AII) but not in the ␮mol/L (Figure 1B). control culture grown in normal growth medium (GM; Figure Heme is a strong inducer of HO-1, and, as expected, we

J Am Soc Nephrol 20: 1254–1263, 2009 Ferritin Prevents Calcification 1255 BASIC RESEARCH www.jasn.org found that HO-1 mRNA, protein, and HO activity were elevated in the cells cultured in heme-containing medium. Pi level of the medium did not affect this heme-mediated induction of HO-1 (Figure 2, A, C, and D). In addition, we found that heme did not significantly alter HO-2 expression (Figure 2B), and it induced expression of ferritin regardless of Pi level of the medium (Figure 2E).

Ferritin and Ferroxidase Activity Attenuate HSMC Calcification Heme induces HO-1 and ferritin; therefore, it was of interest to analyze which of the two had a major effect on calcification. We also analyzed the role of the end products of HO catalyzed heme degradation by adding them exogenously to the calcification medium. We found that iron, regardless of its ferric or ferrous state (50 ␮mol/L), completely inhibits calcification (the ferrous state, data not shown). Biliverdin at the concentration of 50 ␮mol/L provided a little but significant (P Ͻ 0.05) decrease in calcification (Figure 3A). Addition of CO (1%) or bilirubin (50 ␮mol/L) did not influence calcification (Figure 3A). Conversely, addition of apoferritin (2 mg/ml) or recombinant H-chain ferritin to the calcification medium abolished calcification (Figure 3B). These two ferritin types have ferroxidase activity; therefore, we tested another protein with ferroxidase activity, ceruloplasmin. Ceruloplasmin was found to mimic the effect of ferritins at a concentration of 4 mg/ml. The protective effect of L-ferritin was minor compared with that of H-ferritin and ceruplasmin. This may have the following ex- planation. The L-ferritin chains taken up by the cells may coas- semble with the endogenous ferritin and thus expand the pool of active ferritins. The H-mutant 222 ferritin, which lacks both ferroxidase activity and iron-storing capability, was not protective at all against mineralization of HSMCs. To confirm the protective role of ferritin, we inhibited HO using tin protoporphyrin (SnPP), a widely known inhibitor of HO activity, and also transfected the cells with small interfer- ing RNA (siRNA) specific for HO-1. We confirmed the effi- ciency of siRNA and observed an approximately 70% decrease of HO-1 protein expression for up to 4 d after transfection (Figure 4D). In fact, cells treated with heme in the presence of SnPP or siRNA showed very low HO enzyme activity (Figure 4B). Treatment with SnPP or siRNA did not affect the heme- Figure 2. Heme induces HO-1 and ferritin in HSMCs. HSMCs mediated ferritin induction (Figure 4C) and, more important, were cultured in GM or in calcification medium in the absence or did not influence heme-mediated inhibition of calcification presence of heme (50 ␮mol/L) for 24 h. (A through E) HO-1 mRNA (Figure 4A), indicating the paramount role of ferritin in this levels (A), HO-2 mRNA levels (B), HO-1 protein expression (C), protection. HO activity (D), and ferritin expression (E) were measured as To confirm further the function of ferritin in the heme- described in the Concise Methods section. Western blot was or iron-induced inhibition of calcification, we selectively stripped and probed for glyceraldehyde-3-phosphate dehydro- downregulated heme- or iron-induced ferritin synthesis by genase (GAPDH) and shown as a representative of three experi- the iron chelator deferoxamine (DFO). Treatment of the ments. Data are means Ϯ SD of three to five independent exper- Ͻ cells with DFO together with equimolar amount of heme or iments each performed in duplicate. **P 0.01. Fe resulted in a complete block of heme- or Fe-induced ferritin synthesis of both H- and L-chains as shown by West- iron (Figure 5A). Moreover, co-treatment with heme and ern blot (Figure 5B). Downregulation of ferritin synthesis DFO resulted in downregulation of both chains of ferritin by DFO led to complete loss of inhibition of calcification by but not of HO-1 (Figure 5B), which was accompanied by

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Figure 3. Ferritin/ferroxidase activity is responsible for the inhibition of phosphate-induced HSMC calcification. (A) HSMCs were cultured in GM or in calcification medium alone or in the presence of heme (50 ␮mol/L), biliverdin (BV; 50 ␮mol/L), bilirubin (BR; 50 ␮mol/L), CO (1%), or iron (50 ␮mol/L) for 9 d. Calcium content of cells was measured and normalized by cellular protein content. (B) HSMCs were cultured in calcification medium alone or supplemented with apoferritin (2 mg/ml), H-ferritin (2 mg/ml), mutant 222 ferritin (2 mg/ml), ceruloplasmin (4 mg/ml), or L-ferritin (2 mg/ml). After 9 d, calcium deposition was measured as described Figure 4. Ferritin induced by heme mediates the inhibition of Ϯ in the Concise Methods section. Graphs show means SD of phosphate-provoked HSMC calcification. When applied, cells Ͻ Ͻ three separate experiments. *P 0.05; **P 0.01. (C) For inves- were transfected with siRNA for HO-1 or negative control siRNA tigation of whether any of the compounds cause significant tox- (NC) 24 h before the experiment. HSMCs were cultured in calci- icity, an MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazo- fication medium in the presence of heme (50 ␮mol/L) or heme lium-bromide] assay was performed after9dofincubation. Data and SnPP (50 ␮mol/L each) for 4 d. (A through C) Calcium dep- Ϯ Ͻ are means SD of five separate experiments. **P 0.01. osition (A), HO enzyme activity (B), and ferritin expression (C) were measured. (D) A typical Western blot shows efficacy of HO-1 knockdown by siRNA. Four days after transfection, cells were substantial decrease in inhibition of calcification (Figure ␮ Ͻ treated with heme (50 mol/L) for 24 h, and level of HO-1 protein 5A). Mild but significant (P 0.05) inhibition of calcifica- was determined. Data are means Ϯ SD of three independent tion that may be attributed to biliverdin derived from HO- experiments each performed in duplicate. **P Ͻ 0.01. mediated heme degradation was noted. ferritin and its ferroxidase activity solely inhibit mineralization Ferritin Inhibits Osteoblastic Differentiation of HSMCs or suppress the phenotype transition of HSMCs into osteo- It has been shown that vascular calcification in vivo shares sim- blast-like cells. We examined the activity of alkaline phospha- ilarities with bone mineralization; therefore, we asked whether tase (Figure 6). HSMCs maintained in calcification medium

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Figure 5. Downregulation of ferritin synthesis by DFO decreased heme- or iron-mediated inhibition of calcium deposition. (A) Figure 6. Ferritin attenuates ALP activity induced by elevated Pi. HSMCs were cultured in GM or in calcification medium alone or in (A) HSMCs were cultured in GM or in calcification medium alone the presence of heme (50 ␮mol/L) or iron (50 ␮mol/L), with or or in the presence of heme (50 ␮mol/L), heme ϩ SnPP (50 ␮mol/L without equimolar amount of DFO for 9 d. Calcium content of each), heme ϩ DFO (50 ␮mol/L each), biliverdin (BV; 50 ␮mol/L), cells was measured and normalized by cellular protein content. (B) bilirubin (BR; 50 ␮mol/L), CO (1%), or iron (50 ␮mol/L) for 9 d. (B) Representative Western blots show expression of ferritin H- and HSMCs were cultured in calcification medium alone or supple- L-chains, HO-1, and GAPDH of cells treated as described in A. mented with apoferritin (2 mg/ml), H-ferritin (2 mg/ml), mutant Data are means Ϯ SD of three separate experiments. *P Ͻ 0.05; 222 ferritin (2 mg/ml), ceruloplasmin (4 mg/ml), or L-ferritin (2 **P Ͻ 0.01. mg/ml) for 9 d. ALP activity of cells was measured as described in the Concise Methods section. Data are means Ϯ SD of five independent experiments each performed in duplicate. *P Ͻ for 9 d showed an approximately seven-fold increase in ALP 0.05; **P Ͻ 0.01. activity compared with control. Supplementation with heme provided a decrease in ALP activity. Similarly, exposures of duced osteocalcin expression (Figure 7A). Apoferritin, cells to iron (50 ␮mol/L) abolished high Pi-induced ALP ac- H-ferritin, and ceruloplasmin abolished expression of osteo- tivity. Biliverdin (50 ␮mol/L) caused some inhibition (P Ͻ calcin, whereas H-mutant 222 had no effect at all (Figure 7B). 0.05), whereas other end products of HO-mediated heme deg- Finally, to explore the mechanism underlying the inhibition radation—bilirubin (50 ␮mol/L) and CO (1%)—failed to de- of mineralization, we examined the level of Cbfa-1, the “master crease ALP activity. Co-treatment of the cells with heme and gene” of osteoblast differentiation, in our in vitro model. Cul- SnPP demonstrated similar changes in ALP activity as heme turing HSMCs in calcification medium for 48 h resulted in a alone; conversely, co-treatment with heme and DFO did not 1.8-fold increase in Cbfa-1 mRNA level compared with cells affect the increased ALP activity. Importantly, apoferritin, H- maintained in normal GM. Heme inhibited induction of ferritin, and ceruloplasmin also decreased the activity of ALP Cbfa-1 mRNA (P Ͻ 0.05). Accordingly, apoferritin also signif- to the level seen in controls, but the H-mutant 222 ferritin was icantly suppressed this Cbfa-1 induction (Figure 8A). totally ineffective. We also tested the intracellular levels of Pi (Figure 8B), and Next we investigated the presence of another bone-specific our results indicate neither apoferritin nor ceruloplasmin al- protein, osteocalcin, in the extracellular matrix. Maintaining ters intracellular Pi levels after 24 h. Iron causes an approxi- of HSMCs in calcification medium for 9 d resulted in a Ͼ10- mately 25% decrease in the level of intracellular Pi that must be fold increase in osteocalcin content compared with control attributed to its phosphate-binding capacity; therefore, expo- (Figure 7A). Heme decreased upregulation of osteocalcin, and sure of cells to iron inhibits osteoblastic differentiation via two SnPP did not alter this effect. In contrast, co-treatment of the mechanisms: (1) Increasing intracellular H-ferritin and (2) de- cells with heme and DFO led to the loss of osteocalcin down- creasing extracellular phosphate. Furthermore, we also exam- regulation by heme. Iron inhibited upregulation of osteocalcin ined the role of aluminum, which is both a trivalent cation and similarly to heme. In addition, biliverdin had a mild but signif- a strong phosphate binder. Although there was some inhibi- icant effect (P Ͻ 0.05), whereas other products of HO reaction of calcification, the extent was one third of that observed tion—bilirubin and CO—failed to downregulate high Pi-in- with heme or iron (data not shown).

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Figure 8. (A) Both heme and apoferritin inhibit elevated Pi- Figure 7. Ferritin attenuates the upregulation of osteocalcin induced increase in Cbfa-1 mRNA level. HSMCs were cultured in induced by elevated Pi. (A and B) HSMCs were treated as de- GM or in calcification medium alone or in the presence of heme scribed at Figure 6, and osteocalcin levels were determined as (50 ␮mol/L) or apoferritin (2 mg/ml) for 48 h. Cbfa-1 mRNA levels Ϯ described in the Concise Methods section. Data are means SD were determined by quantitative reverse transcription–PCR as of three independent experiments each performed in duplicate. described in the Concise Methods section. Data are means Ϯ Ͻ Ͻ *P 0.05; **P 0.01. SEM of five independent experiments performed in triplicate. *P Ͻ 0.05; **P Ͻ 0.01. (B) Intracellular Pi concentrations are not DISCUSSION affected by apoferritin or ceruloplasmin. HSMCs were cultured in GM and calcification medium supplemented by iron (50 ␮mol/L), CKD often translates to deranged metabolism of both phos- apoferritin (2 mg/ml), or ceruloplasmin (4 mg/ml) for 24 h. Cell lysates were used to measure Pi levels. Data are means Ϯ SEM of phate and iron. Accumulation of phosphate starts relatively Ͻ early in kidney disease, but overt hyperphosphatemia does not three independent experiments performed in duplicate. *P 0.05; **P Ͻ 0.01. develop until the later stages of CKD. Evidence from clinical, animal, and in vitro studies indicate that such elevated phosphate level is an important inducer of vascular calcification. leads to internalization and degradation of ferroportin. The Analyzing data from hemodialysis patients reveals that the ex- corresponding sequestration of iron within the macrophages tent of elevated serum phosphate is positively correlated with limits iron availability to all cells. On the basis of our observa- mortality,27–29 and cardiovascular events are the major cause of tions, we suggest that such derangements in iron metabolism mortality in this group of patients. In particular, development may facilitate Pi-induced vascular calcification; therefore, par- of calciphylaxis, which is a syndrome of vascular calcification enteral iron administration may be considered not only to re- and skin necrosis, is almost exclusively seen in patients with plete iron and correct anemia but also to prevent vascular cal- stage 5 CKD and correlates with extremely high fatal rates. cification via increasing intracellular ferritin expression and Moreover, in patients with CKD, there is an accumulation of decreasing extracellular Pi level, especially when the inflamma- iron in reticuloendothelial cells that is accompanied by higher tion is well controlled. levels of plasma ferritin; however, this increase largely results Previous studies indicated that elevated phosphate could because most of such iron is sequestered by reticuloendothelial induce SMC calcification as well as an osteochondrogenic phe- cells and its availability to other cells is significantly reduced. notypic change. Evidence suggests a highly regulated cellular This translates to depletion of intracellular ferritin and subse- process whereby many different inducers and inhibitors of os- quent anemia of chronic disease. In inflammatory diseases teoblast differentiation have been recognized.31 such as CKD, cytokines released by activated leukocytes and Growing evidence indicates the importance of the HO-fer- other cells exert multiple effects.30 These contribute to the re- ritin system in vascular homeostasis. Upregulation of HO-1 duction in hemoglobin levels and increased hepatic synthesis and ferritin occurs in the early phase of progression of athero- of hepcidin that in turn binds to ferroportin, the transporter sclerotic lesions,32–34 possibly reflecting cellular response to that allows egress of iron from reticuloendothelial macro- heme and/or heme-iron–generated lipid peroxidation prod- phages and from intestinal epithelial cells. Binding of hepcidin ucts. There is growing evidence that induction of the HO/fer-

J Am Soc Nephrol 20: 1254–1263, 2009 Ferritin Prevents Calcification 1259 BASIC RESEARCH www.jasn.org ritin system is protective against atherosclerosis.34 Upregula- mild inhibition of calcification may be attributed to biliver- tion of HO-1 and ferritin inhibits cytotoxicity induced by din derived from enhanced HO-mediated heme degrada- oxidized LDL in endothelial cells35 and atherosclerotic lesion tion. formation in LDL receptor knockout mice, whereas inhibition We observed an approximately 25% decrease in the level of of HO enzyme activity by SnPP leads to accelerated atheroscle- intracellular Pi after exposure of cells to iron. Considering that rosis in these mice.36 ferric iron can bind up to five phosphates per mole, the de- In this study, we confirm that growing HSMC in Pi-con- crease of intracellular Pi level resulted from the decrease of taining calcification medium causes mineralization in a time- extracellular Pi level after iron treatment; therefore, inhibition dependent manner, and, in agreement with other findings, we of osteoblastic differentiation by iron occurs via two mecha- observed significant upregulation of specific osteoblast cell nisms: (1) Increasing intracellular H-ferritin and (2) decreas- markers during the culture period, supporting that this transi- ing extracellular Pi level. tion is an active cell-regulated process. Observation of the in- The cellular mechanisms of vascular calcification still re- hibitory effect of heme prompted us to hypothesize that one or main to be elucidated. Increased expression of Cbfa-1 is impli- more products of heme catabolism might regulate HSMC cated in the transition of SMCs into osteoblast-like cells.12,16 mineralization. That heme or apoferritin significantly suppressed Cbfa-1 in- To identify the mediator for inhibition of HSMC calcifica- duction by high Pi indicates that inhibition of mineralization tion and osteoblastic transformation, we first tested the prod- by ferritin might occur via transcription factor Cbfa-1. H-fer- ucts of heme degradation by HO. Iron almost completely at- ritin has been found to localize also in the nucleus, where it tenuated extracellular calcification and upregulation of may participate in the regulation of gene expression, for exam- osteocalcin and ALP. Biliverdin was less effective, whereas bil- ple in the suppression of ␤ globin expression.39 This raises the irubin and CO failed to alter mineralization. Then we analyzed possibility that it might be involved in the regulation of genes the possible role of ferritin that is also strongly upregulated by for HSMC differentiation into osteoblast-like cells, namely heme. We examined whether exogenous ferritin affected min- Cbfa-1. A relationship between calcification and iron metabo- eralization, because the uptake of exogenous apoferritin in a lism has never been explored, although it should be noted that dosage-responsive manner has already been demonstrated.24 most patients who have CKD and are on dialysis have vascular We found that apoferritin caused a dosage-responsive sup- calcification27–29 and deranged iron homeostasis.40 pression of HSMC mineralization. Also exogenous H-ferritin In conclusion, we report for the first time a novel role for and ceruloplasmin—two largely different proteins that share ferritin in the context of HSMC mineralization. These results only ferroxidase activity—showed the same suppression of os- provide new insights into the mechanisms of vascular calcifi- teoblastic differentiation. The importance of ferroxidase activ- cation and uncover the HO/ferritin pathway as a target for new ity in the process was confirmed by the finding that a structur- strategies to prevent vascular calcification. ally analogous molecule to H-ferritin, namely the recombinant H-ferritin mutant 222, which lacks ferroxidase activity and iron storage capability, was ineffectual. These results strongly CONCISE METHODS support the notion that inhibition of mineralization may be attributed to ferritin and its ferroxidase activity. Cell Culture and Reagents Upregulation of H- and L-chains of ferritin in cells ex- HSMCs were obtained from Cambrex Bioscience (Wokingham, posed to heme is driven at the translational level via labile United Kingdom), FBS from Life Technologies (Vienna, Austria), iron provided from heme catalysis by HO.37 In addition, biliverdin from MP Biomedicals (Solon, OH), SnPP from Frontier heme itself enhances ferritin expression by increasing its Scientific (Logan, UT), 1% CO gas from Linde Gas (Repcelak, Hun- translational rate.38 This explains why induction of ferritin gary), and the gas chamber from Billups-Rothenburg (DelMar, CA). is not affected by inhibition of HO activity in cells exposed Unless otherwise mentioned, all other reagents were obtained from to heme, as observed in previous studies.24 Accordingly, in Sigma (St. Louis, MO). Cell cultures were maintained in GM DMEM this study, cells treated with heme in the presence of SnPP or (high glucose) containing 15% FBS, 100 U/ml penicillin, 100 ␮g/ml siRNA for HO-1 exhibited very low HO activity but high streptomycin and neomycin, and 1 mM sodium pyruvate. Cells were ferritin level. Treatment of cells with SnPP or siRNA for grown to confluence and used from passages 4 through 8. Iron was HO-1 did not affect heme-mediated ferritin induction and introduced as ammonium ferric citrate or ferric sulfate as well as did not influence heme-mediated inhibition of mineraliza- ferrous form. To keep the ferrous state, the media were supplemented tion. These results indicate that ferritin alone is capable of with 200 ␮mol/L ascorbic acid. Iron was dissolved in deionized water. preventing HSMC calcification and differentiation after siRNA specific to HO-1 and negative control siRNA were obtained cells are exposed to heme. To confirm further the role of from Ambion (Austin, TX) and were transfected with Oligofectamine ferritin in the heme-induced inhibition of HSMC mineral- Reagent (Invitrogen, Carlsbad, CA) 24 h before the experiment. ization, we selectively downregulated heme-induced fer- Heme, biliverdin, and bilirubin were dissolved in NaOH. Final con- ritin synthesis by the iron chelator DFO, which led to sub- centration of NaOH was kept below 2 mmol/L in all experiments. This stantial loss of inhibition of calcification. The remaining amount of NaOH caused a little change in the pH of the medium (7.40

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versus 7.46), which did not influence calcification and underlying Quantification of Ferritin and Osteocalcin gene expression of HSMCs. Ferritin content of cell lysate was measured with the IMx ferritin enzyme immunoassay (Abbott Laboratories, Abbott Park, Illinois). For Induction of Calcification osteocalcin detection, extracellular matrix of cells grown on six-well ␮ At confluence, cells were switched to calcification medium, which was plates was dissolved in 300 l of EDTA (0.5 mol/L [pH 6.9]). Osteo- prepared by addition of 4 mmol/L Pi to the GM. Both GM and calci- calcin content of the EDTA-solubilized extracellular matrix samples fication medium were changed every 2 d. For time-course experi- was quantified by an ELISA (Bender MedSystems, Burlingame, CA). ments, the first day of culture in calcification medium was defined as day 0. Quantitative Reverse Transcription–PCR Total RNA was isolated and reverse-transcribed, and HO-1 mRNA was Quantification of Calcium Deposition determined as described previously.41 For measurement of mRNA levels, Cells grown on 48-well plates were washed twice with PBS and decal- the 25-␮l reaction mixture contained 5 ␮l of reverse-transcribed sample, cified with 0.6 mol/L HCl for 24 h at 37°C. Calcium content of the 0.3 nmol/L of forward (5Ј-CAGGCAGGCACAGTCTTC-3Ј) and reverse supernatants was determined by the QuantiChrome Calcium Assay primers (5Ј-CAGAGGTGGCAGTGTCATC-3Ј) for Cbfa-1, forward Kit (Gentaur, Brussels, Belgium). After decalcification, cells were sol- (5Ј-GGTGATAGAAGAGGCCAAGACTG-3Ј) and reverse (5ЈGGTGT- ubilized with a solution of NaOH 0.1 mol/L and SDS 0.1%, and pro- CATGGGTCAGCAGCT-3Ј) primers for HO-1, forward (5Ј-GCAAT- tein content of samples was measured with BCA protein assay kit GTCAGCGGAAGTGGAA-3Ј) and reverse (5Ј-AAGTCACCTGAGGT- (Pierce, Rockford, IL). Calcium content of the cells was normalized to GGTAGTT-3Ј) primers for HO-2, and 12.5 ␮l of iQ SYBR Green protein content and expressed as ␮g/mg protein. Mineralization was Supermix (Bio-Rad, Hercules, CA). PCRs were carried out using the iCy- determined by von Kossa staining. cler iQ Real-Time PCR System (Bio-Rad). Results were normalized by glyceraldehyde-3-phosphate dehydrogenase mRNA levels. ALP Activity Assay Cells grown on six-well plates were washed with PBS twice, solubilized Ferritins and Ceruloplasmin with 1% Triton X-100 in 0.9% NaCl, and assayed for ALP activity. Apoferritin and ceruloplasmin were from Sigma. Human recombi- ␮ Briefly, 130 l of Alkaline Phosphatase Yellow Liquid Substrate nant wild-type H- and L-chain ferritins and the H-chain mutant 222 ␮ (Sigma) was combined with 50 g of protein samples and incubated deleted ferroxidase activity were expressed in Escherichia coli and pu- at 37°C for 30 min, and then the kinetics of p-nitrophenol formation rified as described previously.42 Final concentrations of ferritins were was followed for 30 min at 405 nm. Maximum slope of the kinetic 2 mg/ml, which correspond to 4.5 ␮mol/L for apoferritin, 3.95 curves was used for calculation. ␮mol/L for H-ferritin, and 4.19 ␮mol/L for L-ferritin. Final concentration of ceruloplasmin was 4 mg/ml, which corresponds to 32.7 HO Enzyme Activity Assay ␮mol/L. Cells grown on P100 dishes were washed twice with HBSS, scraped, and centrifuged at 2000 ϫ g for 15 min at 4°C. Cells were resuspended ␮ Phosphate Measurement in 300 l of potassium phosphate (100 mmol/L [pH 7.4]) buffer con- Pi content of the cell lysate was determined by the QuantiChrome taining 2 mmol/L MgCl2, frozen and thawed three times, sonicated, phosphate Assay Kit (Gentaur). After 24 h of incubation, cells were ϫ and centrifuged at 18,000 g for 10 min at 4°C. The supernatant washed twice with PBS and solubilized with 1% Triton, and the cell containing cell microsomes was used to measure HO activity as de- lysates were assayed for Pi. Phosphate content of the cells was normal- 24 scribed previously. HO activity is expressed as pmol bilirubin ized to protein content and expressed as ␮m/L per mg of cell protein. formed/mg cell protein per 60 min.

CO Exposure Western Blot to Detect HO-1 and Ferritin H- and CO at a concentration of 1% (10,000 parts per million) in compressed L-Chains air was mixed with compressed air containing 5% CO before being For evaluation of HO-1 protein expression, cell lysate was electropho- 2 delivered into the culture incubator, yielding a final concentration of resed in 12.5% SDS-PAGE. For ferritin H- and L-chain detection, cell 400 parts per million CO. The incubator was humidified and main- lysate was subjected to 8% nondenaturing PAGE. Western blotting tained at 37°C. A CO analyzer was used to determine CO levels in the was performed with a polyclonal anti–HO-1 antibody at 1:2500 dilu- chamber. After the chamber had stabilized, no oscillations were mea- tion (Calbiochem, San Diego, CA) or with mouse anti-human ferritin sured in the CO concentration. H- or L-chain antibodies (from P. Arosio) at 1:1000 dilution followed by horseradish peroxidase–labeled anti mouse IgG antibody. Anti- gen-antibody complexes were visualized with the horseradish perox- Statistical Analysis idase chemiluminescence system (Amersham Biosciences, Little Data are shown as means Ϯ SD. Statistical analysis was performed by Chalfont, United Kingdom). After detection, membranes were ANOVA test followed by post hoc, Newmann-Keuls test for multiple stripped and reprobed for glyceraldehyde-3-phosphate dehydroge- comparisons. P Ͻ 0.05 was considered significant, and P Ͻ 0.01 was nase. considered highly significant.

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