Published online before print March 26, 2009, doi: 10.1161/CIRCRESAHA.108.189001
© 2009 American Heart Association, Inc.
Molecular Medicine |
RANKL Increases Vascular Smooth Muscle Cell Calcification Through a RANK-BMP4–Dependent Pathway
From the Department of Medicine (S.P., A.C., E.P., P.V.), University of Lleida; Research Laboratory (S.P., A.C., M.E., E.P., P.V., J.M.V.), Hospital Universitari Arnau de Vilanova; Research Unit and Nephrology Section (S.L.-O.), Hospital Universitario Príncipe de Asturias, Alcala de Henares, Madrid; and Unitat de Diagnòstic i Tractament de Malaties Aterotrombòtiques (UDETMA) (B.C., E.F., J.M.V.), Nephrology Department, Hospital Universitari Arnau de Vilanova, Institut de Recerca Biomèdica de Lleida (IRBLLEIDA), Spain.
Correspondence to Dr Jose M. Valdivielso, Laboratorio de Investigación HUAV-UDL, Hospital Universitari Arnau de Vilanova, Rovira Roure 80, 25198 Lleida, Spain. E-mail Valdivielso{at}medicina.udl.es
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Abstract |
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Vascular calcification commonly associated with several pathologies and it has been suggested to be similar to bone mineralization. The axis RANKL-OPG (receptor activator of nuclear factor
B ligand–osteoprotegerin) finely controls bone turnover. RANKL has been suggested to increase vascular calcification, but direct evidence is missing. Thus, in the present work, we assess the effect of RANKL in vascular smooth muscle cell (VSMC) calcification. VSMCs incubated with RANKL showed a dose-dependent increase in calcification, which was abolished by coincubation with OPG. To test whether the effect was mediated by signaling to its receptor, knockdown of RANK was accomplished by short hairpin (sh)RNA. Indeed, cells lacking RANK showed no increases in vascular calcification when incubated with RANKL. To further elucidate the mechanism by which RANK activation increases calcification, we blocked both nuclear factor (NF)-B activation pathways. Only IKK
inactivation inhibited calcification, pointing to an involvement of the alternative NF-B activation pathway. Furthermore, RANKL addition increased bone morphogenetic protein (BMP)4 expression in VSMCs, and that increase disappeared in cells lacking RANK or IKK. The increase in calcification was also blunted by Noggin, pointing to a mediation of BMP4 in the calcification induced by RANKL. Furthermore, in an in vivo model, the increase in vascular calcium content was parallel to an increase in RANKL and BMP4 expression, which was localized in calcified areas. However, blood levels of the ratio RANKL/OPG did not change. We conclude that RANKL increases vascular smooth muscle cell calcification by binding to RANK and increasing BMP4 production through activation of the alternative NF-B pathway.
Key Words: vascular calcification • RANKL • BMP4 • NF-B
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Introduction |
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Vascular calcification is a well recognized and common complication of a variety of pathological conditions like chronic kidney disease (CKD), diabetes mellitus, and atherosclerosis.1 There are 2 main types of vascular calcification, depending on whether the calcium deposits are located in the intima or in the medial layer.2 Intimal calcification is found in atherosclerotic plaques and is associated with a higher likelihood of adverse events such as myocardial infarction and coronary death.3 Atherosclerotic disease involves a complex interplay among several factors like inflammation, thrombosis, and lipid metabolism and different cell types such as endothelial cells, vascular smooth muscle cells (VSMCs), and macrophages. Medial calcification is usually associated with age and CKD patients. Medial calcification generates increased vascular stiffness and reduced vascular compliance, which are associated with increases in systolic blood pressure, pulse pressure, and pulse wave velocity. All of these complications lead to altered coronary perfusion and left ventricular hypertrophy.4 Furthermore, medial calcification of skin arterioles causes calciphylaxis, which is associated with thrombotic cutaneous ischemia, necrotic skin ulceration, and a high mortality rate. In this case, the main cell type involved is the VSMCs.
In the past, arterial calcification was regarded as a passive process. Thus, increases in calcium and phosphate levels over its solubility threshold would induce calcium mineral deposition in soft tissues. Nevertheless, accumulating evidence suggest that arterial calcification is the result of organized and regulated processes similar to bone formation.5 Bone remodeling is a lifelong coordinated process of bone formation and resorption that renews and adapts the skeleton.6 Thus, the balance between bone resorption and formation is finely regulated, and imbalances on one side or the other can cause bone disease. The regulation of that balance is achieved by a combination of hormones and the local cytokine milieu within the bone microenvironment.7 Among the hormones that regulate bone remodeling, the discovery of the RANK-RANKL-OPG system (receptor activator of nuclear factor B [RANK]–RANK ligand–osteoprotegerin) provided a major breakthrough on the understanding of bone remodeling mechanisms.
RANKL is a member of the tumor necrosis factor superfamily and is expressed mainly by osteoblasts and its immature precursors.8 RANKL activates its receptor (RANK), which is expressed in osteoclasts and its precursors, promoting osteoclast formation and activation and prolonging osteoclast survival by suppressing apoptosis.9 The final step in RANK activation is the nuclear translocation of nuclear factor (NF)-B, which is controlled by 2 main pathways, the classic and the alternative NF-B pathways, which are also controlled by different kinases (IKKβ and IKK, respectively). OPG is a decoy receptor for RANKL, which directly counters all the RANKL-mediated actions.10 Thus, the RANKL/OPG ratio is critical to determining bone remodeling and bone mass, and imbalances in this ratio or in RANK signaling underlie the pathology of many disorders exhibiting excessive bone loss.7,11 In fact, vascular calcification is associated with osteoporotic bone loss, but the reasons for this are unclear. The discovery that mice lacking OPG had severe osteoporosis and arterial calcification provided the first clue that the OPG-RANK-RANKL axis could be an important autocrine/paracrine axis on vascular calcification.12 Furthermore, the fact that RANKL expression increases in calcified arterial tissue13,14 added new evidence to a possible role of RANKL on vascular calcification. However, direct evidence of a role of RANKL on vascular calcification is missing. In the present work, we analyze the role of RANKL in vascular calcification in vitro and in an in vivo model of vascular calcification.
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Materials and Methods |
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All the experiments performed in this study followed the NIH Guide for the Care and Use of Laboratory Animals.
In Vitro Studies
Cell Cultures
Primary rat aortic VSMCs of Sprague–Dawley rats were obtained as described previously15 and maintained in DMEM (GIBCO) containing 10% FBS.
Cells were plated (105 cells/plate) in 100-mm plates. When the cell confluence was 
80%, VSMCs were shifted to calcification media, DMEM containing 15% FBS, 10 mmol/L sodium pyruvate, and 10 mmol/L β-glycerophosphate (Sigma). The effect of RANKL (1, 100, 500, 1000 pmol/L), OPG (100 pmol/L), and noggin (100 pmol/L) (all 3 from Sigma) on the calcification levels was tested.
We used cells between passage 2 and 8. All of the experiments were performed in triplicate. In each experiment, 3 plates were used per condition.
Determination of VSMC Calcification
In all of the calcification experiments, the calcium levels were measured 5 days after the addition of the treatments. First, we measured the rate of calcium incorporation of VSMCs incubated with increasing doses of RANKL. Moreover, we tested the effect of OPG and noggin in the calcification level induced by 100 pmol/L RANKL. Quantification of calcium deposits and von Kossa staining were performed as previously described.16 Alkaline phosphatase (ALP) activity was determined by the para-nitrophenyl phosphate detection (BioAssay Systems).
Lentiviral Production and Infection
Lentiviral-based vectors for RNA interference-mediated gene silencing (FSVsi) consisted of a U6 promoter for expression of short hairpin (sh)RNAs and the Venus variant of yellow fluorescent protein under the control of an SV40 promoter for monitoring transduction efficiency. Oligonucleotides to produce shRNA were annealed in buffer (150 mmol/L NaCl; 50 mmol/L Tris, pH 7.6) and cloned into the AgeI-BamHI sites of FSVsi. shRNA target sequence to RANK was TTAGCTGAGGATGCTGAGGAT. shRNAs to IKK and IKKβ were a generous gift of Dr X. Dolcet.17 Negative controls consisted on scrambled sequences. To produce infective lentiviral particles, 293T cells were cotransfected by the polyethylenimine method with the virion packaging elements (VSV-G and 
8.9) and the shRNA-producing vector (FSVsi-RANK or FSVsi as a control). 293T cells were allowed to produce lentiviral particles during 3 to 4 days in the same culture media used for VSMCs. Culture media was collected and centrifuged for 5 minutes at 1000g, and the supernatant was added to growing VSMCs overnight. After this period, media were replaced with fresh media, and cells were incubated for 4 additional days to allow endogenous gene knockdown. Western blot and/or real-time PCR were performed to check the gene knockdown.
Real-Time PCR
Total cellular RNA was isolated from VSMC control, RANK knockdown, IKK and -β knockdown, and tissue samples by the TRIzol method. In the in vitro experiments, isolation of RNA was performed 48 hours after the addition of treatments. Reverse transcription was performed with the first-strand DNA synthesis kit for RT-PCR (Roche Diagnostics). We used TaqMan real-time PCR amplification with gene-specific primer for RANKL, RANK, OPG, bone morphogenetic protein (BMP)2, or BMP4 (Gene Expression Assays from Applied Biosystems), using rat glyceraldehid-3-phosphate-dehydrogenase (GAPDH) as a reference with an ABI Prism 7000 Sequence Detection System (Applied Biosystems). The relative RNA amount was calculated by standard formulae. Average and standard error from 3 experiments were calculated.
Preparation of Nuclear and Cytoplasmic Protein Extracts
VSMCs were treated with 100 pmol/L RANKL for 0, 10, and 30 minutes and 1, 2, and 4 hours. After each time point, VSMCs were washed with cold PBS. Cytoplasmic and nuclear protein fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction KIT (Pierce).
Western Blot Analysis
Western blot analysis was performed as described previously, 48 hours after the addition of treatments.18 After blotting, the membrane was incubated overnight with anti-RANK antibody, anti–active caspase 3 antibody (1:1000. Cell Signaling), anti-IKK, and anti-IKKβ (1:1000; Calbiochem), anti-RELB (1:1000. Santa Cruz Biotechnology), anti–histone 1 (1:500. Santa Cruz Biotechnology), anti–lactic dehydrogenase (1:1000; Rockland), and anti-tubulin (1:10 000 Sigma). Secondary antibody binding was detected with the ECL Advance Western Blotting Detection Kit (Amersham Biosciences) and the VersaDoc Imaging system Model 4000 (Bio-Rad).
Enzyme-Linked Immunosorbent Assay
Levels of BMP2, BMP4, RANKL, and OPG were determined in cell culture supernatant, and rat plasma was determined by a commercially available ELISA (Quantikine, R&D Systems, Minneapolis, Minn, and Biomedica, Vienna, Austria).
In Vivo Studies
Experimental Animals
Sprague–Dawley rats (200 to 225 g) underwent 5/6 nephrectomy by previously described procedures19 and were divided in 2 groups. One group received calcitriol (1 µg/kg 3 times a week for 8 weeks; n=9), whereas the second group received a vehicle injection (n=9). Moreover, 2 more groups of sham-operated rats were used (control, n=9; injected with the same dose of calcitriol, n=9). At euthanasia, a blood sample was extracted and abdominal aortas were collected and divided into 3 pieces. One was fixed, included in paraffin, and sliced; another piece was used to determine calcium content; and the last was used to isolate RNA.
Aortic Calcification
To study the changes in the aortas of the animals, we performed von Kossa staining as described in the section Determination of VSMC Calcification above. Furthermore, we measured total calcium content as described in the same section.
Immunohistochemistry
Sequential slides were used for immunohistochemistry and von Kossa staining as previously described.16 Sections were incubated in 1:50 anti-RANKL (Imgenex), anti-BMP4 (Abcam), anti-TRAP (Santa Cruz Biotechnology) polyclonal antibodies or nonimmune serum (negative controls) overnight at 4°C. After washing, the sections were incubated with 1:200 biotinylated secondary antibody and Vectastain ABC and DAB substrate kits (Vector Labs).
Biochemistry Data
Blood obtained at the end of the experiment was analyzed for calcium and phosphate using a multichannel autoanalyzer (Roche/Hitachi Modular Analytics).
Statistical Analysis
Differences between groups were assessed by ANOVA followed by Dunnett’s post hoc test. A value of P<0.05 was considered statistically significant.
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Results |
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In Figure 1, we show the effect of adding RANKL to VSMCs cultured with calcification media. RANKL increased VSMC calcification measured as VSMC calcium levels (Figure 1A) and ALP activity (Figure 1B) in a dose-dependent manner and starting at concentrations of 100 pmol/L. The calcification induced by RANKL was also visualized by von Kossa staining, as we show in Figure 2B. In this case, we can see that incubation of VSMCs with RANKL increased the brown staining that marks calcified areas.
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In Figure 2, we show the effect of coincubation of RANKL with OPG in the calcification of VSMCs. The increase in calcium levels induced by RANKL was inhibited by OPG (Figure 2B, 2D, and 2E).
To determine whether the effect of RANKL was mediated by activation of RANK, we designed shRNA to decrease RANK protein levels. In Figure 3, we can see that levels RANK (Figure 3A) were decreased in the cells infected with FSVsi-RANK. The incubation of those cells with 100 pmol/L RANKL showed that the elimination of RANK blunted the increase in calcification (Figure 3B), suggesting that the effect of RANKL in calcification is mediated by binding to RANK. To determine whether or not part of the effect of OPG was mediated by inhibition of TRAIL (tumor necrosis factor–related apoptosis-inducing ligand), and thus inhibiting apoptosis, we also checked the effect of RANKL and coincubation of RANKL and OPG on the levels of active caspase 3. Incubation of VSMCs with RANKL or RANKL plus OPG for 48 hours did not modify the levels of active caspase 3 (data not shown). To further elucidate the pathway, we infected cells with shRNA for both kinases involved in the classic and the alternative pathways of NF-B activation (Figure 3A). The disruption of the alternative pathway (but not the classic pathway) blunted the increase in calcification (Figure 3B). Accordingly, incubation of VSMCs with RANKL increased the nuclear levels of RelB (Figure 3C), confirming the activation of the alternative pathway of NF-B activation.
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In Figure 4 we show the effect of adding RANKL in the mRNA (Figure 4A) and protein (Figure 4B) levels of BMP4. RANKL increased BMP4 levels in VSMCs. No effect of RANKL on BMP2 levels was detected either by real-time PCR or ELISA (data not shown). Elimination of RANK or IKK (but not IKKβ) blunted the effect of RANKL on BMP4, pointing again to the alternative pathway of activation of NF-B as the responsible of the increase in BMP4 induced by RANKL. In Figure 5A and 5B, we show the effect of adding Noggin (a BMP4 inhibitor) in the calcification induced by RANKL. The addition of 100 pmol/L Noggin blunted the increase in calcification induced by RANKL, suggesting that this increase is mediated by a BMP.
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Figure 6 shows the results obtained in the in vivo model of vascular calcification. Arteries obtained from animals with 5/6 nephrectomy showed a significant increase in vascular calcification, measured as calcium content, which was exacerbated by treatment with calcitriol for 8 weeks (Figure 6A). Blood levels of RANKL were not modified by the treatment but OPG levels increased in uremic animals, leading to a tendency to decrease the RANKL/OPG ratio (Figure 6B). However, vascular expression of RANKL in those animals increased with no changes in OPG (Figure 6C) and was colocalized with the calcified areas (Figure 6E and 6F). In addition, BMP4 expression (Figure 6D) also increased in arteries from uremic rats and uremic rats treated with calcitriol and was immunolocalized in heavily calcified areas (Figure 6E and 6G). No staining for TRAP was detected in areas expressing BMP4 or RANKL (Figure 6H).
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Uremia induced a significant increase in serum calcium (Ca) but not in phosphorus (P) levels (control Ca: 10.42±0.24 mg/dL; control P: 5.81±0.24 mg/dL; uremia Ca: 11.02±0.14 mg/dL; uremia P: 6.06±0.27 mg/dL; P<0.01). Administration of vitamin D further increased both calcium and phosphorus levels in uremic animals (uremic+vitamin D Ca: 12.14±0.37 mg/dL; uremic+vitamin D P: 6.68±0.34 mg/dL; P<0.01), whereas treatment of control animals with vitamin D did not change control Ca nor P blood levels (control+vitamin D Ca: 10.78±0.07 mg/dL; control+vitamin D P: 5.7±0.08 mg/dL).
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Discussion |
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To our knowledge, this is the first report showing a direct effect of RANKL increasing VSMC calcification. The possible effect of RANKL on VSMC calcification has been suggested by several authors20,21 mainly based on indirect experimental results found in the literature. On the one hand, the expression of RANKL increases in calcified areas in arteries from experimental animals and from patients. In the OPG knockout mice, a model that shows osteoporosis and vascular calcification, Min et al14 showed that RANKL mRNA could be found near the calcified arterial lesions. In addition, the presence of RANKL has been also found in association with extracellular matrix surrounding calcium deposits in human atherosclerotic plaques13 and in calcified aortic valves.22 On the other hand, the expression of OPG as a transgene on an OPG-null background prevented the apparition of vascular calcification. However, the administration of OPG to adult knockout mice did not inhibit vascular calcification, indicating a possible effect of RANKL in the onset of calcification.14 Furthermore, Price et al23 showed that administration of OPG could inhibit the vascular calcification induced by warfarin and vitamin D in experimental animals. A recent report of Morony et al24 also shows that OPG can inhibit vascular calcification without affecting atherosclerosis in an animal model. Our results show that RANKL added to VSMCs incubated in calcification media increased both calcium content and ALP activity, together with an increase in von Kossa staining. Thus, we have shown a direct effect of RANKL inducing VSMC-mediated matrix mineralization.
The fact that OPG inhibits vascular calcification in experimental models has been attributed in the past to 2 possible mechanisms. One was explained by the fact that OPG inhibits bone resorption, and some authors link an increase in bone resorption with vascular calcification. Thus, it has been proposed that an imbalance in calcium allocation allows its movement from bone to vascular wall via mechanisms that involve OPG.25 This hypothesis was supported by results that link arterial calcification with diseases with a high bone resorption rate12,26 and also because treatments that inhibit bone resorption can inhibit vascular calcification in experimental models.26 However, in patients with chronic renal failure, high levels of OPG seem to be unable to protect against vascular calcification.1,27 Nonetheless the significance of those results is limited because of the fact that in those studies, RANKL levels were not measured, and, thus, RANKL/OPG ratios were not determined. The other mechanism is by acting directly on cells in the artery inhibiting the effect of calcification stimulators. OPG is a decoy receptor for RANKL and also for TRAIL.28 VSMCs express both OPG29 and TRAIL.30 The binding of TRAIL to its receptor induced apoptosis, which has also been related to vascular calcification. In vitro models of calcifying VSMCs have shown that a mineral imbalance induces VSMC apoptosis and vesicle release31 and that these apoptotic bodies and vesicles form a nidus for the deposition of calcium phosphate.31 Thus, inhibition of TRAIL could inhibit also vascular calcification. However, no direct evidence of apoptosis has been found in models of vascular calcification in which OPG treatment was able to inhibit it.23 There is a third possibility, which is that OPG inhibits vascular calcification by directly inhibiting RANKL. Our results clearly show that coincubation with OPG inhibits RANKL-induced VSMC calcification. However, it could be hypothesized that incubation with RANKL will increase VSMC calcification by depleting endogenously produced OPG and, thus, leaving TRAIL free to induce apoptosis. Our experimental results show that incubation of VSMc with RANKL did not increase the level of active caspase 3, which is involved in TRAIL-induced apoptosis. Furthermore, we performed experiments in which we inhibited RANK expression by shRNA. In those cells, incubation with RANKL did not increase VSMC calcification, pointing to a direct role of RANKL increasing VSMC calcification by binding to RANK.
The binding of RANKL to its receptor RANK activates both the canonical and the alternative NF-B pathways.32,33 We further investigated how activation of RANK induced vascular calcification by inhibiting either pathway in the NF-B cascade. The results showed that inhibition of the canonical pathway did not affect vascular calcification, whereas the use of shRNA for the main kinase involved in the alternative pathway totally blunted RANKL-induced VSMC calcification. Accordingly, incubation with RANKL induced an increase in nuclear translocation of RelB, proving an activation of the alternative NF-B pathway. Furthermore, activation of RANK increased the production of BMP4. BMP4 is a member of the BMP family, a group of signaling molecules that belong to the transforming growth factor β superfamily and were initially identified by their capacity to induce endochondral bone formation.34 In addition, BMP4 has been involved in the osteogenic transition of VSMCs, leading to vascular calcification.35 It has also been described that BMP4 increases in vitro VSMC calcification36 and is upregulated in calcified atherosclerotic lesions.13 Our results further show that parallel to a decrease in vascular calcification, inhibition of the alternative NF-B activation pathway also decreased BMP4. Furthermore, the addition of noggin (a pharmacological inhibitor of the BMPs) to the incubation media also inhibited the RANKL-induced VSMC calcification, suggesting that it is mediated by an increase in BMP4 expression.
We also tested our results in an in vivo model of vascular calcification. In that model, subtotally nephrectomized rats were treated with high doses of vitamin D to intensify vascular calcification. This model has been used before, and it has been shown that administration of OPG was able to decrease the vascular calcification.23 The results show that administration of calcitriol to normal animals does not increase either the expression of RANKL/OPG or BMP4 in arteries or in blood. Those animals showed no increases in vascular calcification. In addition, uremia induces an increase in the RANKL/OPG ratio and in BMP4 expression in arteries. Consistently, the levels of vascular calcification are increased in those animals. The increase in vascular calcification is higher in the uremic animals treated with calcitriol, together with a tendency to increase arterial RANKL and BMP4 expression. This finding suggests that there are other factors that have an effect on the degree of vascular calcification in vivo that can be influenced by calcitriol treatment. For instance, uremic animals treated with calcitriol show higher levels of phosphorus in blood, a parameter that has been shown to increase vascular calcification.37 In addition, the expression of RANKL and BMP4 were localized in areas of medial calcification, supporting the role of RANKL in promoting VSMC calcification. The staining for TRAP was negative in those areas and agrees with previous reports suggesting that, contrary to atherosclerotic plaque calcification, in medial calcification macrophage infiltration is not involved.38 The clinical implications of our in vivo model to human pathology are relative, because vitamin D intoxication in uremic patients is currently a rare phenomenon.
We also tested the role of circulating levels of RANKL as a possible marker for vascular calcification in our model. However, whereas the vascular expression of RANKL increased, the circulating levels of RANKL did not change. The association of circulating OPG levels with traditional vascular risk factors has been extensively reported,39–41 but the possible use of RANKL levels is still controversial. Schett et al42 showed that serum RANKL levels were not associated with traditional vascular risk factors. In general, serum RANKL levels appear unaltered, although they have sometimes declined as serum OPG levels increased.20,21,43 Furthermore, in a recent study, Kiechl et al showed in a large scale epidemiological study that serum RANKL levels did not correlate with atherosclerosis, but baseline RANKL levels were shown to be a predictor of vascular risk.44 Thus, the role of serum RANKL levels as predictors of cardiovascular risk is unclear. In our experimental model, we have shown that although vascular levels of RANKL are increased, circulating levels did not change. Thus, changes in circulating levels of RANKL could not reflect changes in vascular levels. Furthermore, and in agreement with previous reports, OPG levels increase in all uremic animals, leading to a tendency to decrease the RANKL/OPG ratios. Nevertheless, the importance of these results in the clinical settings needs further investigation.
In summary, we have shown that RANKL is able to induce VSMC calcification in vitro by binding to RANK. The activation of RANK will increase BMP4 expression by launching the alternative NF-B pathway. These results add new evidence to the role of the OPG-RANK-RANKL system in vascular calcification and confirm RANKL inhibition as a possible target to treat vascular calcification.
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Acknowledgments |
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Sources of Funding
This work was supported, in part, by grants from Fondo de Investigaciones Sanitarias (grants PI06/0010 and PI07/0427) and REDinREN (16/06). J.M.V. and B.C. hold a contract from the Miguel Servet program. M.E. holds a contract from the Ramon y Cajal program. S.P. and P.V. hold a fellowship from Generalitat de Cataluña.
Disclosures
None.
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Footnotes |
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*Both authors contributed equally as senior authors of this work.
Original received October 7, 2008; revision received March 16, 2009; accepted March 17, 2009.
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References |
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1. Vattikuti R, Towler DA. Osteogenic regulation of vascular calcification: an early perspective. Am J Physiol Endocrinol Metab. 2004; 286: E686–E696.
2. London GM, Marchais SJ, Guerin AP, Metivier F. Arteriosclerosis, vascular calcifications and cardiovascular disease in uremia. Curr Opin Nephrol Hypertens. 2005; 14: 525–531.[Medline] [Order article via Infotrieve]
3. Detrano RC, Doherty TM, Davies MJ, Stary HC. Predicting coronary events with coronary calcium: pathophysiologic and clinical problems. Curr Probl Cardiol. 2000; 25: 374–402.[CrossRef][Medline] [Order article via Infotrieve]
4. Blacher J, Guerin AP, Pannier B, Marchais SJ, London GM. Arterial calcifications, arterial stiffness, and cardiovascular risk in end-stage renal disease. Hypertension. 2001; 38: 938–942.
5. Doherty TM, Asotra K, Fitzpatrick LA, Qiao JH, Wilkin DJ, Detrano RC, Dunstan CR, Shah PK, Rajavashisth TB. Calcification in atherosclerosis: bone biology and chronic inflammation at the arterial crossroads. Proc Natl Acad Sci U S A. 2003; 100: 11201–11206.
6. Karsenty G. The complexities of skeletal biology. Nature. 2003; 423: 316–318.[CrossRef][Medline] [Order article via Infotrieve]
7. Hofbauer LC, Schoppet M. Clinical implications of the osteoprotegerin/RANKL/RANK system for bone and vascular diseases. JAMA. 2004; 292: 490–495.
8. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 1998; 93: 165–176.[CrossRef][Medline] [Order article via Infotrieve]
9. Hsu HL, Lacey DL, Dunstan CR, Solovyev I, Colombero A, Timms E, Tan HL, Elliott G, Kelley MJ, Sarosi I, Wang L, Xia XZ, Elliott R, Chiu L, Black T, Scully S, Capparelli C, Morony S, Shimamoto G, Bass MB, Boyle WJ. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci U S A. 1999; 96: 3540–3545.
10. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, RenshawGegg L, Hughes TM, Hill D, Pattison W, Campbell P, Sander S, Van G, Tarpley J, Derby P, Lee R, Boyle WJ. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell. 1997; 89: 309–319.[CrossRef][Medline] [Order article via Infotrieve]
11. Hofbauer LC, Heufelder AE. Role of receptor activator of nuclear factor-kappa B ligand and osteoprotegerin in bone cell biology. J Mol Med. 2001; 79: 243–253.[CrossRef][Medline] [Order article via Infotrieve]
12. Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu WL, Lacey DL, Boyle WJ, Simonet WS. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998; 12: 1260–1268.
13. Dhore CR, Cleutjens JPM, Lutgens E, Cleutjens KBJM, Geusens PPM, Kitslaar PJEH, Tordoir JHM, Spronk HMH, Vermeer C, Daemen MJAP. Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 2001; 21: 1998–2003.
14. Min H, Morony S, Sarosi I, Dunstan CR, Capparelli C, Scully S, Van G, Kaufman S, Kostenuik PJ, Lacey DL, Boyle TJ, Simonet WS. Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis. J Exp Med. 2000; 192: 463–474.
15. Cardus A, Parisi E, Gallego C, Aldea M, Fernandez E, Valdivielso JM. 1,25-Dihydroxyvitamin D-3 stimulates vascular smooth muscle cell proliferation through a VEGF-mediated pathway. Kidney Int. 2006; 69: 1377–1384.[Medline] [Order article via Infotrieve]
16. Cardus A, Panizo S, Parisi E, Fernandez E, Valdivielso JM. Differential effects of vitamin D analogues on vascular calcification. J Bone Miner Res. 2007; 22: 860–866.[CrossRef][Medline] [Order article via Infotrieve]
17. Dolcet X, Llobet D, Encinas M, Pallares J, Cabero A, Schoenenberger JA, Comella JX, Matias-Guiu X. Proteasome inhibitors induce death but activate NF-kappaB on endometrial carcinoma cell lines and primary culture explants. J Biol Chem. 2006; 281: 22118–22130.
18. Valdivielso JM, Perez-Barriocanal F, Garcia-Estan J, Lopez-Novoa JM. Role of nitric oxide in the early renal hemodynamic response after unilateral nephrectomy. Am J Physiol Integ Comp Physiol. 1999; 276: R1718–R1723.
19. Perez-Ruiz L, Ros-Lopez S, Cardus A, Fernandez E, Valdivielso JM. A forgotten method to induce experimental chronic renal failure in the rat by ligation of the renal parenchyma. Nephron Exp Nephrol. 2006; 103: e126–e130.[Medline] [Order article via Infotrieve]
20. Schoppet M, Preissner KT, Hofbauer LC. RANK ligand and osteoprotegerin - paracrine regulators of bone metabolism and vascular function. Arterioscler Thromb Vasc Biol. 2002; 22: 549–553.
21. Sattler AM, Schoppet M, Schaefer JR, Hofbauer LC. Novel aspects on RANK ligand and osteoprotegerin in osteoporosis and vascular disease. Calcif Tissue Int. 2004; 74: 103–106.[CrossRef][Medline] [Order article via Infotrieve]
22. Kaden JJ, Bickelhaupt S, Grobholz R, Haase KK, Sartkoc A, Kilic R, Brueckmann M, Lang S, Zahn I, Vahl C, Hagl S, Dempfle CE, Borggrefe M. Receptor activator of nuclear factor kappa B ligand and osteoprotegerin regulate aortic valve calcification. J Mol Cell Cardiol. 2004; 36: 57–66.[CrossRef][Medline] [Order article via Infotrieve]
23. Price PA, June HH, Buckley JR, Williamson MK. Osteoprotegerin inhibits artery calcification induced by warfarin and by vitamin D. Arterioscler Thromb Vasc Biol. 2001; 21: 1610–1616.
24. Morony S, Tintut Y, Zhang Z, Cattley RC, Van G, Dwyer D, Stolina M, Kostenuik PJ, Demer LL. Osteoprotegerin inhibits vascular calcification without affecting atherosclerosis in ldlr(-/-) mice. Circulation. 2008; 117: 411–420.
25. Hofbauer LC, Schoppet M. Osteoprotegerin: a link between osteoporosis and arterial calcification? Lancet. 2001; 358: 257–259.[CrossRef][Medline] [Order article via Infotrieve]
26. Price PA, Faus SA, Williamson MK. Bisphosphonates alendronate and ibandronate inhibit artery calcification at doses comparable to those that inhibit bone resorption. Arterioscler Thromb Vasc Biol. 2001; 21: 817–824.
27. Nitta K, Akiba T, Uchida K, Otsubo S, Takei T, Yumura W, Kabaya T, Nihei H. Serum osteoprotegerin levels and the extent of vascular calcification in haemodialysis patients. Nephrol Dial Transplant. 2004; 19: 1886–1889.
28. Emery JG, McDonnell P, Burke MB, Deen KC, Lyn S, Silverman C, Dul E, Appelbaum ER, Eichman C, DiPrinzio R, Dodds RA, James IE, Rosenberg M, Lee JC, Young PR. Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem. 1998; 273: 14363–14367.
29. Hofbauer LC, Shui CX, Riggs BL, Dunstan CR, Spelsberg TC, O'Brien T, Khosla S. Effects of immunosuppressants on receptor activator of NF-kappa B ligand and osteoprotegerin production by human osteoblastic and coronary artery smooth muscle cells. Biochem Biophys Res Commun. 2001; 280: 334–339.[CrossRef][Medline] [Order article via Infotrieve]
30. Gochuico BR, Zhang J, Ma BY, Marshak-Rothstein A, Fine A. TRAIL expression in vascular smooth muscle. Am J Physiol Lung Cell Mol Physiol. 2000; 278: L1045–L1050.
31. Reynolds JL, Joannides AJ, Skepper JN, McNair R, Schurgers LJ, Proudfoot D, Jahnen-Dechent W, Weissberg PL, Shanahan CM. Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD. J Am Soc Nephrol. 2004; 15: 2857–2867.
32. Chaisson ML, Branstetter DG, Derry JM, Armstrong AP, Tometsko ME, Takeda K, Akira S, Dougall WC. Osteoclast differentiation is impaired in the absence of inhibitor of kappa B kinase alpha. J Biol Chem. 2004; 279: 54841–54848.
33. Novack DV, Yin L, Hagen-Stapleton A, Schreiber RD, Goeddel DV, Ross FP, Teitelbaum SL. The IkappaB function of NF-kappaB2 p100 controls stimulated osteoclastogenesis. J Exp Med. 2003; 198: 771–781.
34. Leboy PS. Regulating bone growth and development with bone morphogenetic proteins. Ann N Y Acad Sci. 2006; 1068: 14–18.[CrossRef][Medline] [Order article via Infotrieve]
35. Hayashi K, Nakamura S, Nishida W, Sobue K. Bone morphogenetic protein-induced MSX1 and MSX2 inhibit myocardin-dependent smooth muscle gene transcription. Mol Cell Biol. 2006; 26: 9456–9470.
36. Mikhaylova L, Malmquist J, Nurminskaya M. Regulation of in vitro vascular calcification by BMP4, VEGF and Wnt3a. Calcif Tissue Int. 2007; 81: 372–381.[CrossRef][Medline] [Order article via Infotrieve]
37. Giachelli CM, Speer MY, Li X, Rajachar RM, Yang H. Regulation of vascular calcification: roles of phosphate and osteopontin. Circ Res. 2005; 96: 717–722.
38. Shanahan CM, Cary NR, Salisbury JR, Proudfoot D, Weissberg PL, Edmonds ME. Medial localization of mineralization-regulating proteins in association with Monckeberg’s sclerosis: evidence for smooth muscle cell-mediated vascular calcification. Circulation. 1999; 100: 2168–2176.
39. Kiechl S, Schett G, Wenning G, Redlich K, Oberhollenzer M, Mayr A, Santer P, Smolen J, Poewe W, Willeit J. Osteoprotegerin is a risk factor for progressive atherosclerosis and cardiovascular disease. Circulation. 2004; 109: 2175–2180.
40. Schoppet M, Sattler AM, Schaefer JR, Herzum M, Maisch B, Hofbauer LC. Increased osteoprotegerin serum levels in men with coronary artery disease. J Clin Endocrinol Metab. 2003; 88: 1024–1028.
41. Hermann-Arnhof KM, Kastenbauer T, Publig T, Novotny P, Loho N, Schwarz S, Koller U, Fitzgerald R. Initially elevated osteoprotegerin serum levels may predict a perioperative myocardial lesion in patients undergoing coronary artery bypass grafting. Crit Care Med. 2006; 34: 76–80.[CrossRef][Medline] [Order article via Infotrieve]
42. Schett G, Kiechl S, Redlich K, Oberhollenzer F, Weger S, Egger G, Mayr A, Jocher J, Xu QB, Pietschmann P, Teitelbaum S, Smolen J, Willeit J. Soluble RANKL and risk of nontraumatic fracture. JAMA. 2004; 291: 1108–1113.
43. Schoppet M, Schaefer JR, Hofbauer LC. Low serum levels of soluble RANK ligand are associated with the presence of coronary artery disease in men. Circulation. 2003; 107: e76.[CrossRef][Medline] [Order article via Infotrieve]
44. Kiechl S, Schett G, Schwaiger J, Seppi K, Eder P, Egger G, Santer P, Mayr A, Xu QB, Willeit J. Soluble receptor activator of nuclear factor-kappa B ligand and risk for cardiovascular disease. Circulation. 2007; 116: 385–391.
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