Chondrogenesis Mediated by PPi Depletion Promotes Spontaneous Aortic Calcification in NPP1–/– Mice
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动脉硬化血栓血管生物学 2005年第4期
From Rheumatology/Medicine, Veterans Affairs Medical Center/University of California at San Diego, School of Medicine.
Correspondence to Robert Terkeltaub, 111K, Veterans Affairs Medical Center, 3350 La Jolla Village Dr, San Diego, CA 92161. E-mail rterkeltaub@ucsd.edu
Abstract
Objective— We recently linked human arterial media calcification of infancy to heritable PC-1/nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) deficiency. NPP1 hydrolyzes ATP to generate PPi, a physicochemical inhibitor of hydroxyapatite crystal growth. But pathologic calcification in NPP1 deficiency states is tissue-restricted and in perispinal ligaments is endochondral differentiation–mediated rather than simply a dystrophic process. Because ectopic chondro-osseous differentiation promotes artery calcification in atherosclerosis and other disorders, we tested the hypothesis that NPP1 and PPi deficiencies regulate cell phenotype plasticity to promote artery calcification.
Methods and Results— Using cultured multipotential NPP1–/– mouse bone marrow stromal cells, we demonstrated spontaneous chondrogenesis inhibitable by treatment with exogenous PPi. We also demonstrated cartilage-specific gene expression, upregulated alkaline phosphatase, decreased expression of the physiological calcification inhibitor osteopontin, and increased calcification in NPP1–/– aortic smooth muscle cells (SMCs). Similar changes were demonstrated in aortic SMCs from ank/ank mice, which are extracellular PPi–depleted because of defective ANK transmembrane PPi transport activity. Moreover, NPP1–/– and ank/ank mice demonstrated aortic media calcification by von Kossa staining, and intra-aortic cartilage-specific collagen gene expression was demonstrated in situ in NPP1–/– mice.
Conclusions— NPP1 and PPi deficiencies modulate phenotype plasticity in artery SMCs and chondrogenesis in mesenchymal precursors, thereby stimulating artery calcification by modulating cell differentiation.
Human "idiopathic" infantile arterial media calcification is linked to deficient PPi-generating PC-1/nucleotide pyrophosphatase phosphodiesterase 1 (NPP1). We demonstrate that NPP1 and extracellular PPi deficiencies promote chondrogenic differentiation in mesenchymal precursors and vascular smooth muscle cells. Therefore, NPP1 and PPi deficiencies promote active rather than simply dystrophic artery calcification, mediated partly by primary alterations in cell differentiation.
Key Words: nucleotide pyrophosphatase phosphodiesterase 1 ? osteopontin ? vascular smooth muscle cells ? ank/ank
Introduction
Arterial calcification frequently develops in association with atherosclerosis, aging, end-stage renal failure, and diabetes mellitus1,2 and is associated with an increased risk of ischemic cardiovascular events.3,4 Arterial calcification promotes decreased arterial compliance, and induction of cytokines and matrix metalloproteinases by deposited hydroxyapatite in atherosclerotic plaques could affect plaque stability.3,4
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Arterial calcification in atherosclerosis, which is primarily intimal, is driven by inflammation, stimulated by low-density lipoprotein oxidation, and suppressed by high-density lipoprotein.1 In contrast, artery calcification in aging, diabetes mellitus, and renal failure (M?nckeberg’s sclerosis) is predominantly limited to tunica media, concentrated mainly at the internal elastic lamina and not directly localized with atherosclerotic lesions.1,2 Wide distribution of media calcification in the arterial tree likely reflects systemic alterations of regulators of calcification, exemplified by hyperparathyroidism and hyperphosphatemia in renal failure.1,2 But both artery intima and media calcification appear to be mediated by ectopic cartilage and bone formation, a consequence of the well-characterized transdifferentiation potential of vascular smooth muscle cells (VSMCs) and the multilineage potential of pericytes.5–7 Intra-arterial differentiation to bone-forming chondrocytic or osteoblastic or cells associated with artery calcification is illustrated by expression of tissue-nonspecific alkaline phosphatase (AP) and certain matrix proteins (eg, osteocalcin, osteopontin [OPN]) characteristic of mature osteoblasts and of terminally differentiated chondrocytes.1,2
Analogous to bone remodeling, arterial calcification is subject to regulation by several physiological inhibitors whose identification has been expedited by analyses of genetically deficient mice.1 For example, fetuin appears to act primarily by limiting hydroxyapatite crystal growth,8 an effect shared by OPN which also promotes hydroxyapatite dissolution.9,10 Requisite physiological inhibition of artery calcification by osteoprotegerin (OPG) may be mediated by effects on macrophage lineage cell differentiation that influence mineral resorption.11 Certain inhibitors of differentiation to bone-forming cells also physiologically suppress artery calcification.1,2 Specifically, targeted gene inactivation of murine matrix Gla protein (MGP) causes spontaneous intra-arterial chondroid metaplasia9 reflecting loss of physiological MGP inhibition of bone morphogenic protein (BMP)-2–induced chondrogenesis.12 Intra-arterial chondroid metaplasia and artery calcification resembling endochondral bone formation also occur in mice with targeted gene inactivation of the BMP signal transduction inhibitor Smad6.13
Recently, we discovered that recessive (or compound heterozygotic) inheritance of mutations that inhibit catalytic activity of PC-1/nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), which generates the potent physiological hydroxyapatite crystal deposition inhibitor PPi,14 cause the majority of cases of human "idiopathic" infantile media calcification, a spontaneous and frequently lethal form of widely disseminated artery media calcification.15,16 Significantly, idiopathic infantile arterial calcification linked to human NPP1 deficiency is associated with concurrent periarticular and aortic calcifications,17 similar to phenotypic features described in NPP1-deficient ttw/ttw mice.18,19
The shared NPP1–/– and ttw/ttw mouse skeletal phenotype of spontaneous bone fusion of periarticular and perispinal soft tissues in early life is remarkably similar to that of the ank/ank mouse.20 The ank/ank mouse is homozygous for a C-terminal cytosolic domain truncation mutation of the multiple-pass transmembrane protein ANK that disables PPi channeling.20 Furthermore, the marked depletion of extracellular PPi and the rapid extensive calcification by both NPP1–/– and ank/ank osteoblasts in culture are corrected by soluble NPP1, which suggests a common pathogenic role of PPi deficiency.21 Shared features of pathologic bony fusion in NPP1-deficient and ank/ank mice include de novo chondrogenesis and endochondral bone formation involving periarticular and perispinal ligaments.18,19,22 Hence, we studied NPP1–/– and ank/ank mice to test the hypothesis that transdifferentiation of VSMCs and intra-arterial chondrogenic differentiation mediated by PPi depletion promotes spontaneous aortic calcification.
Methods
Reagents
All chemical reagents were obtained from Sigma, unless otherwise indicated.
Source and Breeding of Mice
NPP1–/– mice were on a C57BL/6 background21 and the ank/ank mice on a BALB/c X C3H and C57BL/6 hybrid background.22 NPP1 and ANK genotypes were analyzed by polymerase chain reaction, and heterozygote breeders were used to generate distinct litters (NPP1–/– and ank/ank mice and respective littermates).21
Isolation, Culture, and Analyses of Mouse Bone Marrow Stromal Cells
To isolate primary plastic-adherent bone marrow stromal cells, mouse femurs were flushed with 1% FCS-containing DMEM low glucose medium, with hematopoietic cells removed using 1.44 g/L Ficoll density gradient centrifugation. Stromal cells were cultured for 14 days in basal mesenchymal stem cell media (Cambrex, Walkersville, Md) and at confluence transferred to high-density culture conditions in complete serum-free medium (Mediatech, Herndon, Va) for chondrogenesis assays. To assay for multipotential bone marrow stromal precursor cells (colony-forming unit–fibroblastoid cells [CFU-F]), viable washed stromal cells (assessed by trypan blue staining) were resuspended (2.25x105 cells per ml) and plated in 2-cm2 dishes in 0.4 mL DMEM containing 10% FCS and 3.7 g/L HEPES, pH 7.3 (medium B, which was replaced on days 3 and 8). CFU-F were counted on day 13 after fixation and Giemsa staining, with a colony defined as constituting a minimum of 5 cells per group.23
To quantify chondrogenesis by measuring 35S incorporation into sulfated proteoglycans,24 plastic-adherent bone marrow stromal cells in high-density culture were labeled with 1μCi/ml of 35S sulfate and 3H proline for 24 hours. Cellular proteoglycans were extracted with 8 mol/L guanidine HCl, 0.01 mol/L sodium acetate, 0.02 mol/L EDTA, 0.2 mol/L 6-aminocaproic acid, 5 mmol/L benzamidine HCl, 10 mmol/L N-ethylmaleimide, and 0.5 mmol/L phenylmethylsulphonyl fluoride for 24 hours at 4°C for scintillation counting.
RT-PCR Analyses
Total RNA was isolated using TriZOL (Invitrogen), reverse-transcribed, and amplified for 40 cycles as described.21 Primers were as previously described for Sox9,25 type II collagen,26 SM22,27 osteocalcin, MGP, OPG, cbfa1, and the ribosomal housekeeping gene L30.21 Type I collagen primers were forward 5'-CGA GAA AGG ATC CCC TGG TGC-3 and reverse 5'-TTT ACC GGT CTC ACC ACG GTG A-3'.
Isolation and Analyses of Cultured Murine Aortic VSMCs
Enriched preparations of aortic SMCs from 2-week-old mice were obtained by collagenase and elastase digestion as described.28 After digestion, aortic cells (1x104 cells per mouse aorta) were cultured in DMEM low glucose medium supplemented with 100 U/mL Penicillin, 50 μg/mL streptomycin, 1% glutamine, and 1x nonessential amino acids (medium A) containing 10% FCS.28 After 2 days, in which an approximate doubling of SMC numbers had occurred, cells from animals of the same genotype were pooled in medium A supplemented with 1% FCS. In each case, aortic VSMC preparations were confirmed to have typical VSMC morphology and to be >95% positive for SMC-specific -actin staining (using a monoclonal antibody from Chemicon, Temecula, Calif) and <1% positive for von Willebrand factor staining (using polyclonal antibody from Chemicon). To quantify VSMC matrix calcification, we used a previously described Alizarin red S binding assay21 and analyzed aliquots of 5x103 VSMCs in medium A supplemented with 1% FCS and 1 mmol/L NaPi. BMP-2 was measured in conditioned media by ELISA (R&D Systems) according to manufacturer’s instructions.
PPi concentrations were determined radiometrically and equalized for DNA concentration.21 AP-specific activity was determined colorimetrically.21 To measure OPN by ELISA,21 we used plates coated with monoclonal antibody to native OPN (Chemicon, Temecula, Calif). Recombinant murine OPN was the standard. Washed wells were incubated for 1 hour at 37°C with rabbit anti-OPN (1:1000; Chemicon, Temecula, Calif) followed by detection as described.21
von Kossa Staining and Immunohistochemistry
Aortic specimens were embedded in optimal cutting temperature compound (Tissue Tek). Using a cryostat, 20-μm-thick sagittal frozen sections were stained for calcium phosphate by the von Kossa reaction.9 For immunohistochemical analyses, frozen sections were fixed with cold acetone for 10 minutes and stained using polyclonal antibodies specific for type X collagen (Calbiochem) or types IX/XI collagen (Chemicon) using the HistoMouse staining kit (Zymed).
Statistics
Where indicated, error bars represent SD. Statistical analyses used the Student t test (paired 2-sample testing for means).
Results
Spontaneous Chondrogenesis in NPP1–/– Multipotential Bone Marrow Stromal Cells
We placed NPP1–/– plastic-adherent monocyte-depleted primary bone marrow stromal cells in high-density culture. By avoiding standard addition of BMP-2 or transforming growth factor ? to stimulate chondrogenesis,24 we tested for emergence of "spontaneous" chondrogenesis from a mixed cell population enriched in multipotential cells. We used noncalcifying conditions to avoid secondary effects due to matrix calcification. We observed increased 35S incorporation into sulfated proteoglycans in NPP1–/– bone marrow stromal cells relative to littermate NPP1+/+ cells (Figure 1A). NPP1–/– bone marrow stromal cells demonstrated upregulated expression of the chondrogenic master transcription factor Sox9 and of cartilage-selective type II collagen, which were inhibited by regular media supplementation with 5 pmol of exogenous PPi (Figure 1B). Significantly, upregulated chondrogenesis was not caused by altered multipotential bone marrow stromal precursor cell (CFU-F) pool size (Figure 1C).
Figure 1. Spontaneous emergence of chondrogenesis in NPP1–/–mouse plastic-adherent bone marrow stromal cells. A, 35S incorporation into sulfated proteoglycans. Plastic-adherent bone marrow stromal cells from euthanized 3-month-old NPP1–/– and littermate NPP1+/+ mice were studied in high-density culture in complete serum-free medium as described in Methods. Cells were labeled with 1μCi/ml of 35S sulfate and 3H proline for 24 hours before collection. Washed cell extracts were collected on the days indicated for analysis of incorporation of 35S into sulfated proteoglycans. Data were pooled from 3 mice of each genotype, each studied in 3 separate experiments. *P<0.05. B, RT-PCR analyses for chondrogenic gene expression in NPP1–/– bone marrow stromal cells. Sox9 and collagen II expression were determined by RT-PCR of bone marrow stromal cells in high-density cultures on the days indicated as described in Methods. Where indicated, NPP1–/– cells were treated with 5 pmol sodium PPi at time 0 and every 72 hours thereafter to raise the extracellular concentration of PPi by 2.5 nM. Results shown are from cultures pooled from 5 mice of each genotype. C, Lack of significant change in multipotential bone marrow stromal precursor cells (CFU-F) from NPP1–/– mice. Bone marrow stromal cell preparations were harvested as described above from 4-month-old mice and aliquots of viable cells studied during 13 days in culture as described in Methods. CFU-F were counted on day 13 after fixation and Giemsa staining, with a colony defined as constituting a minimum of 5 cells per group. Results shown are from cultures pooled from the marrow of 5 mice of each genotype.
Altered Differentiation and Calcification in Cultured Aortic NPP1–/– VSMCs
Type II collagen expression was consistent with chondrogenic differentiation in preparations of enriched aortic VSMCs of NPP1–/– mice but not littermate NPP1+/+ mice (Figure 2). Under these conditions, both NPP1–/– and NPP1+/+ VSMC preparations demonstrated expression of type I collagen and the SMC-specific marker SM22 as well as MGP and OPG (Figure 2). Parallel experiments revealed similarly upregulated collagen II expression in aortic ank/ank VSMCs (not shown). Cultured aortic SMC preparations demonstrated significantly decreased extracellular PPi and OPN and increased AP activity and calcification in NPP1–/– and ank/ank mice cells relative to normal littermates (Figure 3).
Figure 2. RT-PCR analyses for chondro-osseous gene expression in cultured aortic SMC preparations from NPP1–/– mice. Aortic SMCs from euthanized 2-week-old NPP1–/– mice and littermate control NPP1+/+ mice were harvested by elastase and collagenase digestion as described in Methods. We studied expression of the indicated mRNAs by RT-PCR in aliquots of 5x104 primary cells cultured for 9 days as described in Methods. Representative of RNA isolated from 4 individual animals of each genotype are shown.
Figure 3. Comparable changes in extracellular PPi and OPN in cellular AP-specific activity and in calcification in primary mouse VSMCs from NPP1–/– and ank/ank mice. Primary VSMCs from NPP1 –/– and ank/ank mice and their respective congenic wild-type littermates at 2 weeks of age were harvested as described above and aliquots of 5x104 cells cultured in medium supplemented with 1 mmol/L sodium phosphate. At the times indicated, conditioned media and cell lysates were collected for determination of the extracellular PPi levels (A), cell AP-specific activity (B), and extracellular OPN (C) as described in Methods. To quantify matrix calcification (D), aliquots of 5x103 cells per well of primary VSMCs from NPP1–/–, ank/ank, and respective wild-type littermates were analyzed by Alizarin red S staining as described in Methods. Each experiment used VSMCs pooled from 8 animals from each genotype, run in triplicate. Data were pooled from 3 experiments. *P<0.05.
Conditioned media BMP-2 levels of the VSMCs of 2-week-old mice at 7 days in culture did not significantly differ between NPP1+/+ and NPP1–/– states (13.44±5.5 pg/mL versus 10.43±4.5 pg/mL, respectively, analyzing cells from 5 mice of each genotype, run in replicates of 3). Aortic cell preparations contained not only typical VSMCs but also cells with irregular borders and ruffled edges that assembled in nodular clusters in the postconfluent state (not shown). But the pattern of matrix calcification was diffuse in these cultured VSMCs preparations, unlike the typical nodular pattern of in vitro calcification by pericytes,21,22 suggesting calcification driven by transdifferentiated PPi-depleted VSMCs.
Aortic Media Calcification and Chondrogenic Differentiation In Situ in NPP1–/– Mice
von Kossa staining demonstrated aortic tunica media calcifications at 21 to 22 weeks of age in NPP1–/– and to a lesser degree in ank/ank mice (Figure 4). Assessment of whole aortic tissue by RT-PCR demonstrated in situ expression of collagen II in NPP1–/– and ank/ank mice (Figure 5). In addition, NPP1–/– and ank/ank mouse aortas demonstrated expression of the chondrocyte hypertrophy and late osteoblast differentiation marker osteocalcin and of cbfa1, a transcription factor centrally involved in both chondrocyte maturation to hypertrophy and osteoblastic differentiation.5 In contrast, expression of SM22, type I collagen, MGP, and OPG were detected in aortas from normal and NPP1–/– and ank/ank mice (Figure 5).
Figure 4. Pathological calcification involving the tunica media of the aorta in NPP1–/– and ank/ank mice. Thoracic aortas were removed from NPP1–/–, ank/ank, and respective wild-type littermates at 21 to 22 weeks of age. We performed von Kossa staining on 20 μmol/L frozen sagittal sections as described in Methods. As shown, black/brown staining calcium phosphate mineral deposition foci were seen in the media of NPP1–/– and ank/ank but not wild-type mice.
Figure 5. Aortic chondro-osseous mRNA expression in situ in both NPP1–/– and ank/ank mice. We extracted total RNA from aortas from 3-month-old mice and pooled RNA from 5 animals of each genotype indicated. After reverse transcription, 40 cycles of polymerase chain reaction amplification were performed for each mRNA analyzed.
Last, we immunohistochemically assessed for ectopic cartilage-specific collagen expression in NPP1–/– mouse aortas in comparison with NPP1+/+ controls. We studied types IX/XI collagen, whose ectopic expression at sites of perispinal ligament calcification was described in NPP1-deficient mice.29 Because chondrocyte maturation to hypertrophy is intimately linked with matrix calcification,30 we also tested for the stereotypic chondrocyte hypertrophy marker type X collagen. We detected aortic foci of expression of types IX/XI collagen in the media and adventitia, and of type X collagen in the media in 21-week-old NPP1–/– mice but not normal littermates (Figure 6).
Figure 6. Immunohistochemical detection of chondrocyte-specific type X and types IX/XI collagens in NPP1–/– aortas. Aortic specimens from 21-week-old NPP1–/– mice and NPP1+/+ littermate controls were immunohistochemically analyzed for chondrocyte-specific type X and types IX/XI collagen expression as described in Methods, using 20-μm thick sagittal frozen sections and using nonimmune rabbit IgG as a control. Brown staining foci of expression of types IX/XI collagen in the media and adventitia and of type X collagen in the media were detected in aortas of NPP1–/– mice but not the normal age-matched littermates. Representative of 4 animals of each genotype are shown. All images are displayed at x160 magnification.
Discussion
Human aortic SMCs constitutively express the physiological calcification inhibitor NPP1.17 Moreover, IIAC is linked to NPP1 mutations that decrease catalytic activity for PPi generation.15–17 Here we demonstrated that NPP1–/– mice, previously characterized to have marked extracellular PPi depletion,21 develop aortic media calcification. Chondro-osseous differentiation of cells in the artery wall mediates both atherosclerotic intima and nonatherosclerotic media forms of artery calcification.1,2 Here we discovered that NPP1 deficiency promoted the spontaneous emergence of chondrogenesis from bone marrow stromal cells. Our arterial studies were limited by small yields of VSMC-enriched mixed cell populations from mouse aortas. Nevertheless, it was significant that cultured NPP1–/– aortic SMC preparations and NPP1–/– aortic cells in situ expressed cbfa1, osteocalcin, and chondrocyte-specific collagens. Procalcifying AP specific activity was markedly upregulated in cultured NPP1–/– VSMCs. In contrast, there was no gross alteration in expression of the physiological artery calcification inhibitors MGP and OPG in NPP1–/– mouse arterial cells, consistent with previous results for NPP1–/– versus NPP1+/+ osteoblasts21
NPP1 not only generates PPi but also scavenges ATP and regulates purinergic receptor signaling, N-glycosylation, secretion of glycoproteins, proteoglycans sulfation, and insulin receptor signaling.31 But the capacity of exogenous PPi to correct spontaneous chondrogenesis in NPP1–/– bone marrow stromal cells under noncalcifying conditions suggested that extracellular PPi deficiency directly promoted chondrogenesis. Critical evidence supporting this conclusion was provided by our studies of ank/ank mice, which we observed to develop aortic media calcification. Cultured ank/ank mouse VSMCs demonstrated comparable changes to those in NPP1–/– VSMCs for extracellular PPi levels, OPN expression, AP-specific activity, and matrix calcification. Moreover, collagen II, cbfa1, and osteocalcin were expressed in situ in the ank/ank mouse aorta.
The extent of aortic media calcification was substantially less in ank/ank than in NPP1–/– mice between 4 to 5 months of age. Similar to NPP1–/– mice, we have observed that ank/ank mice also develop perispinal ligament calcification at 2 months of age (our unpublished observations, 2004). It will be of interest to assess whether the lesser extent of aortic media calcification in ank/ank mice relative to NPP1–/– mice persists beyond 21 to 22 weeks of age.
Chondrogenesis is a complex process mediated by precursor cell recruitment, condensation, and transcriptional pathways modulating chondrocyte-specific gene expression, including direct transcriptional regulatory effects of Sox9, effects of Wnt-frizzled receptor signaling, and the interplay between these factors exerted partly at the level of Sox9 interaction with ?-catenin.24,30 In this study, we observed that Sox9 expression was upregulated in NPP1–/– bone marrow stromal cells, an effect reversed by exogenous PPi. Definition of the primary mechanism(s) by which NPP1 and extracellular PPi regulate Sox9 expression and chondrogenesis will be of interest. We did not observe altered expression of BMP-2 by NPP1–/– VSMCs, but this finding did not rule out deficiency of 1 or more BMP inhibitors or changes in BMP signaling in NPP1–/– cells. It will also be of interest to assess whether NPP1 deficiency selectively promotes chondrogenesis in both mice and human arteries or whether NPP1 deficiency also impacts on Msx2-mediated vascular ossification programs that circumvent chondrogenesis.32
Cell surface receptors or active cellular or subcellular uptake mechanisms for PPi have yet to be identified in eukaryotic cells.14 But extracellular inorganic phosphate (Pi) generated partly through tissue-nonspecific AP pyrophosphohydrolysis of PPi is taken up by cells through defined sodium-dependent cotransporters.33 Though Pi uptake can promote chondrogenic differentiation, chondrocyte and osteoblastic maturation, and calcification,33–35 it also appears likely that extracellular PPi by itself can directly regulate differentiation of arterial cells. First, PPi inhibits calcification triggered by experimental artery injury, an effect that is removed by AP.36 Second, nonhydrolyzable bisphosphonate PPi analogues, which suppress artery calcification in states including IIAC,17 clearly regulate cellular signal transduction, gene expression, and viability.14 Third, intracellular PPi is elevated in ank/ank cells but decreased in NPP1–/– skeletal cells,21 pointing to shared critical effects of extracellular PPi depletion on VSMC function and chondrogenic differentiation. Fourth, analogous to effects of Pi,37 we have observed direct effects of extracellular PPi on expression of genes that regulate skeletal remodeling, such as matrix metalloproteinase-13 and OPN.21,38 In this context, the observation of decreased OPN expression by NPP1–/– and ank/ank VSMCs was compelling because OPN promotes atherogenesis.39 Hence, diminished OPN expression by NPP1–/– VSMCs may help explain why inflammation is not observed at IIAC artery lesions.
We did not quantitatively examine for SMC phenotype suppression5 in NPP1–/– aortic cells in this study, but transdifferentiation of VSMCs to calcifying chondro-osseous cells5 appeared central to the observed artery media calcification in NPP1–/– mice. Pericytes6,7 and other arterial cells also likely contributed. For example, we observed ectopic expression of types IX/XI collagen in the NPP1–/– mouse aortic adventitia, a potential source of multipotential cells (stenmark) and migratory fibroblasts capable of transdifferentiation to myofibroblasts, which drive vascular remodeling and fibrosis.40 Interestingly, a marked myointimal fibroproliferative response is a cardinal feature of IIAC associated with NPP1 "loss-of-function" mutations17 but, for undefined reasons, was not seen in NPP1–/– or ank/ank mouse aortas.
In conclusion, NPP1 and PPi physiologically function to prevent IIAC by suppressing artery calcification at the level of cell differentiation and not simply at the level of mineral formation and resorption in the extracellular matrix. Extracellular PPi levels are regulated by both genetic and acquired factors14 such as oxidative stress, which can depress PPi and promote calcification in vitro.26 In addition, cell stimulation by the atherogenesis mediators interleukin-1 or insulin-like growth factor-I depress extracellular PPi, partly through suppression of NPP1 and ANK expression.14 Therefore, acquired decrements in lesion NPP1 and ANK expression and extracellular PPi could contribute to intra-arterial chondro-osseous metaplasia and calcification in atherosclerosis and other conditions.
Acknowledgments
This work was supported by the Department of Veterans Affairs and National Institutes of Health (NIH) grants HL077360, P01AGO7996, and AR049366.
References
Demer LL, Tintut Y. Mineral Exploration: search for the Mechanism of Vascular Calcification and Beyond. The 2003 Jeffrey M. Hoeg Award Lecture. Arterioscler Thromb Vasc Biol. 2003; 23: 1739–1743.
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.
Wayhs R, Zelinger A, Raggi P. High coronary artery calcium scores pose an extremely elevated risk for heart events. J Am Coll Cardiol. 2002; 39: 225–230.
Abedin M, Tintut Y, Demer LL. Vascular calcification: mechanisms and clinical ramifications. Arterioscler Thromb Vasc Biol. 2004; 24: 1161–1170.
Steitz SA, Speer MY, Curinga G, Yang HY, Haynes P, Aebersold R, Schinke T, Karsenty G, Giachelli CM. Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 and downregulation of smooth muscle lineage markers. Circ Res. 2001; 89: 1147–1154.
Tintut Y, Alfonso Z, Saini T, Radcliff K, Watson K, Bostrom K, Demer LL. Multilineage potential of cells from the artery wall. Circulation. 2003; 108: 2505–2510.
Canfield AE, Doherty MJ, Wood AC, Farrington C, Ashton B, Begum N, Harvey B, Poole A, Grant ME, Boot-Handford RP. Role of pericytes in vascular calcification: a review. Z Kardiol. 2000; 89 (suppl 2): 20–27.
Ketteler M, Wanner C, Metzger T, Bongartz P, Westenfeld R, Gladziwa U, Schurgers LJ, Vermeer C, Jahnen-Dechent W, Floege J. Deficiencies of calcium-regulatory proteins in dialysis patients: a novel concept of cardiovascular calcification in uremia. Kidney Int Suppl. 2003; S84–S87.
Speer MY, McKee MD, Guldberg RE, Liaw L, Yang HY, Tung E, Karsenty G, Giachelli CM. Inactivation of the osteopontin gene enhances vascular calcification of matrix Gla protein-deficient mice: evidence for osteopontin as an inducible inhibitor of vascular calcification in vivo. J Exp Med. 2002; 196: 1047–1055.
Steitz SA, Speer MY, McKee MD, Liaw L, Almeida M, Yang H, Giachelli CM. Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification. Am J Pathol. 2002; 161: 2035–2046.
Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu W, Lacey DL, Boyle WJ, Simonet WS. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998; 12: 1260–1268.
B?strom K, Tsao D, Shen S, Wang Y, Demer LL. Matrix GLA protein modulates differentiation induced by bone morphogenetic protein-2 in C3H10T1/2 cells. J Biol Chem. 2001; 276: 14044–14052.
Galvin KM, Donovan MJ, Lynch CA, Meyer RI, Paul RJ, Lorenz JN, Fairchild-Huntress V, Dixon KL, Dunmore JH, Gimbrone MA Jr, Falb D, Huszar D. A role for smad6 in development and homeostasis of the cardiovascular system. Nat Genet. 2000; 24: 171–174.
Terkeltaub RA. Inorganic pyrophosphate generation and disposition in pathophysiology. Am J Physiol Cell Physiol. 2001; 281: C1–C11.
Rutsch F, Ruf N, Vaingankar S, Toliat MR, Suk A, H?hne W, Schauer G, Schnabel D, Epplen JT, Knisely A, Superti-Furga A, McGill J, Roscioli T, Filippone M, Vallance H, Hinrichs B, Smith W, Ferre M, Terkeltaub R, Nürnberg P. Mutations in ENPP1 are associated with "idiopathic infantile arterial" calcification. Nat Genet. 2003; 34: 379–381.
Ruf N, Uhlenberg B, Terkeltaub R, Nürnberg P, Rutsch F. The mutational spectrum of ENPP1 as arising after the analysis of 23 unrelated patients with generalized arterial calcification of infancy (GACI). Hum Mutat. 2005; 25: 98.
Rutsch F, Vaingankar S, Johnson K, Goldfine I, Maddux B, Schauerte P, Kalhoff H, Boisvert WA, Superti-Furga A, Terkeltaub R. PC-1 nucleoside triphosphate pyrophosphohydrolase deficiency in idiopathic infantile arterial calcification. Am J Pathol. 2001; 158: 543–554.
Hosoda Y, Yoshimura Y, Higaki S. A new breed of mouse showing multiple osteochondral lesions: twy mouse. Ryumachi. 1981; 21 (suppl 1): 157–164.
Okawa A, Nakamura I, Goto S, Moriya H, Nakamura Y, Ikegawa S. Mutation in Npps in a mouse model of ossification of the posterior longitudinal ligament of the spine. Nat Genet. 1998; 19: 271–273.
Ho AM, Johnson MD, Kingsley DM. Role of the mouse ank gene in control of tissue calcification and arthritis. Science. 2000; 289: 265–270.
Johnson K, Goding J, van Etten D, Sali A, Hu SI, Farley D, Krug H, Hessle L, Millan JL, Terkeltaub R. Linked deficiencies in extracellular inorganic pyrophosphate and osteopontin expression mediate pathologic calcification in PC-1 null mice. Am J Bone Min Res. 2003; 18: 994–1004.
Krug HE, Mahowald ML, Halverson PB, Sallis JD, Cheung HS. Phosphocitrate prevents disease progression in murine progressive ankylosis. Arthritis Rheum. 1993; 36: 1603–1611.
Clarke E. Culture of human and mouse mesenchymal cells. Methods Mol Biol. 2004; 290: 173–186.
Haas AR, Tuan RS. Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: II. Stimulation by bone morphogenetic protein-2 requires modulation of N-cadherin expression and function. Differentiation. 1999; 64: 77–89.
Salminen H, Vuorio E, Saamanen AM. Expression of Sox9 and type IIA procollagen during attempted repair of articular cartilage damage in a transgenic mouse model of osteoarthritis. Arthritis Rheum. 2001; 44: 947–955.
Johnson K, Jung A, Murphy A, Andreyev A, Dykens J, Terkeltaub R. Mitochondrial oxidative phosphorylation is a downstream regulator of nitric oxide effects on chondrocyte matrix synthesis and mineralization. Arthritis Rheum. 2000; 43: 1560–1570.
Arakawa E, Hasegawa K, Yanai N, Obinata M, Matsuda Y. A mouse bone marrow stromal cell line, TBR-B, shows inducible expression of smooth muscle-specific genes. FEBS Lett. 2000; 481: 193–196.
Mimura Y, Kobayashi S, Notoya K, Okabe M, Kimura I, Horikoshi I, Kimura M. Activation by alpha 1-adrenergic agonists of the progression phase in the proliferation of primary cultures of smooth muscle cells in mouse and rat aorta. Biol Pharm Bull. 1995; 18: 1373–1376.
Yamazaki M, Moriya H, Goto S, Saitoh Y, Arai K, Nagai Y. Increased type XI collagen expression in the spinal hyperostotic mouse (TWY/TWY). Calcif Tiss Int. 1991; 48: 182–189.
Akiyama H, Lyons JP, Mori-Akiyama Y, Yang X, Zhang R, Zhang Z, Deng JM, Taketo MM, Nakamura T, Behringer RR, McCrea PD, de Crombrugghe B. Interactions between Sox9 and beta-catenin control chondrocyte differentiation. Genes Dev. 2004; 18: 1072–1087.
Goding JW, Grobben B, Slegers H. Physiological and pathophysiological functions of the ecto-nucleotide pyrophosphatase/phosphodiesterase family. Biochim Biophys Acta. 2003; 1638: 1–19.
Tyson KL, Reynolds JL, McNair R, Zhang Q, Weissberg PL, Shanahan CM. Osteo/chondrocytic transcription factors and their target genes exhibit distinct patterns of expression in human arterial calcification. Arterioscler Thromb Vasc Biol. 2003; 23: 489–494.
Wang D, Canaff L, Davidson D, Corluka A, Liu H, Hendy GN, Henderson JE. Alterations in the sensing and transport of phosphate and calcium by differentiating chondrocytes. J Biol Chem. 2001; 276: 33995–34005.
Cecil DL, Rose DM, Terkeltaub R, Liu-Bryan R. IL-8 induces chondrocyte expression of Pit-1 and CXCR1-mediated Pi uptake. Arthritis Rheum. In press.
Jono S, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, Morii H, Giachelli CM. Phosphate regulation of vascular smooth muscle cell calcification. Circ Res. 2000; 87: E10–E17.
Lomashvili KA, Cobbs S, Hennigar RA, Hardcastle KI, O’Neill WC. Phosphate-induced vascular calcification: role of pyrophosphate and osteopontin. J Am Soc Nephrol. 2004; 15: 1392–1401.
Beck GR Jr. Inorganic phosphate as a signaling molecule in osteoblast differentiation. J Cell Biochem. 2003; 90: 234–243.
Johnson K, Terkeltaub R. Upregulated ANK expression in osteoarthritis can promote both chondrocyte MMP-13 expression and calcification via chondrocyte extracellular PPi excess. Osteoarthritis Cartilage. 2004; 12: 321–335.
Matsui Y, Rittling SR, Okamoto H, Inobe M, Jia N, Shimizu T, Akino M, Sugawara T, Morimoto J, Kimura C, Kon S, Denhardt D, Kitabatake A, Uede T. Osteopontin deficiency attenuates atherosclerosis in female apolipoprotein e-deficient mice. Arterioscler Thromb Vasc Biol. 2003; 23: 1029–1034.
Sartore S, Chiavegato A, Faggin E, Franch R, Puato M, Ausoni S, Pauletto P. Contribution of adventitial fibroblasts to neointima formation and vascular remodeling: from innocent bystander to active participant. Circ Res. 2001; 89: 1111–1121.(Kristen Johnson; Monika P)
Correspondence to Robert Terkeltaub, 111K, Veterans Affairs Medical Center, 3350 La Jolla Village Dr, San Diego, CA 92161. E-mail rterkeltaub@ucsd.edu
Abstract
Objective— We recently linked human arterial media calcification of infancy to heritable PC-1/nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) deficiency. NPP1 hydrolyzes ATP to generate PPi, a physicochemical inhibitor of hydroxyapatite crystal growth. But pathologic calcification in NPP1 deficiency states is tissue-restricted and in perispinal ligaments is endochondral differentiation–mediated rather than simply a dystrophic process. Because ectopic chondro-osseous differentiation promotes artery calcification in atherosclerosis and other disorders, we tested the hypothesis that NPP1 and PPi deficiencies regulate cell phenotype plasticity to promote artery calcification.
Methods and Results— Using cultured multipotential NPP1–/– mouse bone marrow stromal cells, we demonstrated spontaneous chondrogenesis inhibitable by treatment with exogenous PPi. We also demonstrated cartilage-specific gene expression, upregulated alkaline phosphatase, decreased expression of the physiological calcification inhibitor osteopontin, and increased calcification in NPP1–/– aortic smooth muscle cells (SMCs). Similar changes were demonstrated in aortic SMCs from ank/ank mice, which are extracellular PPi–depleted because of defective ANK transmembrane PPi transport activity. Moreover, NPP1–/– and ank/ank mice demonstrated aortic media calcification by von Kossa staining, and intra-aortic cartilage-specific collagen gene expression was demonstrated in situ in NPP1–/– mice.
Conclusions— NPP1 and PPi deficiencies modulate phenotype plasticity in artery SMCs and chondrogenesis in mesenchymal precursors, thereby stimulating artery calcification by modulating cell differentiation.
Human "idiopathic" infantile arterial media calcification is linked to deficient PPi-generating PC-1/nucleotide pyrophosphatase phosphodiesterase 1 (NPP1). We demonstrate that NPP1 and extracellular PPi deficiencies promote chondrogenic differentiation in mesenchymal precursors and vascular smooth muscle cells. Therefore, NPP1 and PPi deficiencies promote active rather than simply dystrophic artery calcification, mediated partly by primary alterations in cell differentiation.
Key Words: nucleotide pyrophosphatase phosphodiesterase 1 ? osteopontin ? vascular smooth muscle cells ? ank/ank
Introduction
Arterial calcification frequently develops in association with atherosclerosis, aging, end-stage renal failure, and diabetes mellitus1,2 and is associated with an increased risk of ischemic cardiovascular events.3,4 Arterial calcification promotes decreased arterial compliance, and induction of cytokines and matrix metalloproteinases by deposited hydroxyapatite in atherosclerotic plaques could affect plaque stability.3,4
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Arterial calcification in atherosclerosis, which is primarily intimal, is driven by inflammation, stimulated by low-density lipoprotein oxidation, and suppressed by high-density lipoprotein.1 In contrast, artery calcification in aging, diabetes mellitus, and renal failure (M?nckeberg’s sclerosis) is predominantly limited to tunica media, concentrated mainly at the internal elastic lamina and not directly localized with atherosclerotic lesions.1,2 Wide distribution of media calcification in the arterial tree likely reflects systemic alterations of regulators of calcification, exemplified by hyperparathyroidism and hyperphosphatemia in renal failure.1,2 But both artery intima and media calcification appear to be mediated by ectopic cartilage and bone formation, a consequence of the well-characterized transdifferentiation potential of vascular smooth muscle cells (VSMCs) and the multilineage potential of pericytes.5–7 Intra-arterial differentiation to bone-forming chondrocytic or osteoblastic or cells associated with artery calcification is illustrated by expression of tissue-nonspecific alkaline phosphatase (AP) and certain matrix proteins (eg, osteocalcin, osteopontin [OPN]) characteristic of mature osteoblasts and of terminally differentiated chondrocytes.1,2
Analogous to bone remodeling, arterial calcification is subject to regulation by several physiological inhibitors whose identification has been expedited by analyses of genetically deficient mice.1 For example, fetuin appears to act primarily by limiting hydroxyapatite crystal growth,8 an effect shared by OPN which also promotes hydroxyapatite dissolution.9,10 Requisite physiological inhibition of artery calcification by osteoprotegerin (OPG) may be mediated by effects on macrophage lineage cell differentiation that influence mineral resorption.11 Certain inhibitors of differentiation to bone-forming cells also physiologically suppress artery calcification.1,2 Specifically, targeted gene inactivation of murine matrix Gla protein (MGP) causes spontaneous intra-arterial chondroid metaplasia9 reflecting loss of physiological MGP inhibition of bone morphogenic protein (BMP)-2–induced chondrogenesis.12 Intra-arterial chondroid metaplasia and artery calcification resembling endochondral bone formation also occur in mice with targeted gene inactivation of the BMP signal transduction inhibitor Smad6.13
Recently, we discovered that recessive (or compound heterozygotic) inheritance of mutations that inhibit catalytic activity of PC-1/nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), which generates the potent physiological hydroxyapatite crystal deposition inhibitor PPi,14 cause the majority of cases of human "idiopathic" infantile media calcification, a spontaneous and frequently lethal form of widely disseminated artery media calcification.15,16 Significantly, idiopathic infantile arterial calcification linked to human NPP1 deficiency is associated with concurrent periarticular and aortic calcifications,17 similar to phenotypic features described in NPP1-deficient ttw/ttw mice.18,19
The shared NPP1–/– and ttw/ttw mouse skeletal phenotype of spontaneous bone fusion of periarticular and perispinal soft tissues in early life is remarkably similar to that of the ank/ank mouse.20 The ank/ank mouse is homozygous for a C-terminal cytosolic domain truncation mutation of the multiple-pass transmembrane protein ANK that disables PPi channeling.20 Furthermore, the marked depletion of extracellular PPi and the rapid extensive calcification by both NPP1–/– and ank/ank osteoblasts in culture are corrected by soluble NPP1, which suggests a common pathogenic role of PPi deficiency.21 Shared features of pathologic bony fusion in NPP1-deficient and ank/ank mice include de novo chondrogenesis and endochondral bone formation involving periarticular and perispinal ligaments.18,19,22 Hence, we studied NPP1–/– and ank/ank mice to test the hypothesis that transdifferentiation of VSMCs and intra-arterial chondrogenic differentiation mediated by PPi depletion promotes spontaneous aortic calcification.
Methods
Reagents
All chemical reagents were obtained from Sigma, unless otherwise indicated.
Source and Breeding of Mice
NPP1–/– mice were on a C57BL/6 background21 and the ank/ank mice on a BALB/c X C3H and C57BL/6 hybrid background.22 NPP1 and ANK genotypes were analyzed by polymerase chain reaction, and heterozygote breeders were used to generate distinct litters (NPP1–/– and ank/ank mice and respective littermates).21
Isolation, Culture, and Analyses of Mouse Bone Marrow Stromal Cells
To isolate primary plastic-adherent bone marrow stromal cells, mouse femurs were flushed with 1% FCS-containing DMEM low glucose medium, with hematopoietic cells removed using 1.44 g/L Ficoll density gradient centrifugation. Stromal cells were cultured for 14 days in basal mesenchymal stem cell media (Cambrex, Walkersville, Md) and at confluence transferred to high-density culture conditions in complete serum-free medium (Mediatech, Herndon, Va) for chondrogenesis assays. To assay for multipotential bone marrow stromal precursor cells (colony-forming unit–fibroblastoid cells [CFU-F]), viable washed stromal cells (assessed by trypan blue staining) were resuspended (2.25x105 cells per ml) and plated in 2-cm2 dishes in 0.4 mL DMEM containing 10% FCS and 3.7 g/L HEPES, pH 7.3 (medium B, which was replaced on days 3 and 8). CFU-F were counted on day 13 after fixation and Giemsa staining, with a colony defined as constituting a minimum of 5 cells per group.23
To quantify chondrogenesis by measuring 35S incorporation into sulfated proteoglycans,24 plastic-adherent bone marrow stromal cells in high-density culture were labeled with 1μCi/ml of 35S sulfate and 3H proline for 24 hours. Cellular proteoglycans were extracted with 8 mol/L guanidine HCl, 0.01 mol/L sodium acetate, 0.02 mol/L EDTA, 0.2 mol/L 6-aminocaproic acid, 5 mmol/L benzamidine HCl, 10 mmol/L N-ethylmaleimide, and 0.5 mmol/L phenylmethylsulphonyl fluoride for 24 hours at 4°C for scintillation counting.
RT-PCR Analyses
Total RNA was isolated using TriZOL (Invitrogen), reverse-transcribed, and amplified for 40 cycles as described.21 Primers were as previously described for Sox9,25 type II collagen,26 SM22,27 osteocalcin, MGP, OPG, cbfa1, and the ribosomal housekeeping gene L30.21 Type I collagen primers were forward 5'-CGA GAA AGG ATC CCC TGG TGC-3 and reverse 5'-TTT ACC GGT CTC ACC ACG GTG A-3'.
Isolation and Analyses of Cultured Murine Aortic VSMCs
Enriched preparations of aortic SMCs from 2-week-old mice were obtained by collagenase and elastase digestion as described.28 After digestion, aortic cells (1x104 cells per mouse aorta) were cultured in DMEM low glucose medium supplemented with 100 U/mL Penicillin, 50 μg/mL streptomycin, 1% glutamine, and 1x nonessential amino acids (medium A) containing 10% FCS.28 After 2 days, in which an approximate doubling of SMC numbers had occurred, cells from animals of the same genotype were pooled in medium A supplemented with 1% FCS. In each case, aortic VSMC preparations were confirmed to have typical VSMC morphology and to be >95% positive for SMC-specific -actin staining (using a monoclonal antibody from Chemicon, Temecula, Calif) and <1% positive for von Willebrand factor staining (using polyclonal antibody from Chemicon). To quantify VSMC matrix calcification, we used a previously described Alizarin red S binding assay21 and analyzed aliquots of 5x103 VSMCs in medium A supplemented with 1% FCS and 1 mmol/L NaPi. BMP-2 was measured in conditioned media by ELISA (R&D Systems) according to manufacturer’s instructions.
PPi concentrations were determined radiometrically and equalized for DNA concentration.21 AP-specific activity was determined colorimetrically.21 To measure OPN by ELISA,21 we used plates coated with monoclonal antibody to native OPN (Chemicon, Temecula, Calif). Recombinant murine OPN was the standard. Washed wells were incubated for 1 hour at 37°C with rabbit anti-OPN (1:1000; Chemicon, Temecula, Calif) followed by detection as described.21
von Kossa Staining and Immunohistochemistry
Aortic specimens were embedded in optimal cutting temperature compound (Tissue Tek). Using a cryostat, 20-μm-thick sagittal frozen sections were stained for calcium phosphate by the von Kossa reaction.9 For immunohistochemical analyses, frozen sections were fixed with cold acetone for 10 minutes and stained using polyclonal antibodies specific for type X collagen (Calbiochem) or types IX/XI collagen (Chemicon) using the HistoMouse staining kit (Zymed).
Statistics
Where indicated, error bars represent SD. Statistical analyses used the Student t test (paired 2-sample testing for means).
Results
Spontaneous Chondrogenesis in NPP1–/– Multipotential Bone Marrow Stromal Cells
We placed NPP1–/– plastic-adherent monocyte-depleted primary bone marrow stromal cells in high-density culture. By avoiding standard addition of BMP-2 or transforming growth factor ? to stimulate chondrogenesis,24 we tested for emergence of "spontaneous" chondrogenesis from a mixed cell population enriched in multipotential cells. We used noncalcifying conditions to avoid secondary effects due to matrix calcification. We observed increased 35S incorporation into sulfated proteoglycans in NPP1–/– bone marrow stromal cells relative to littermate NPP1+/+ cells (Figure 1A). NPP1–/– bone marrow stromal cells demonstrated upregulated expression of the chondrogenic master transcription factor Sox9 and of cartilage-selective type II collagen, which were inhibited by regular media supplementation with 5 pmol of exogenous PPi (Figure 1B). Significantly, upregulated chondrogenesis was not caused by altered multipotential bone marrow stromal precursor cell (CFU-F) pool size (Figure 1C).
Figure 1. Spontaneous emergence of chondrogenesis in NPP1–/–mouse plastic-adherent bone marrow stromal cells. A, 35S incorporation into sulfated proteoglycans. Plastic-adherent bone marrow stromal cells from euthanized 3-month-old NPP1–/– and littermate NPP1+/+ mice were studied in high-density culture in complete serum-free medium as described in Methods. Cells were labeled with 1μCi/ml of 35S sulfate and 3H proline for 24 hours before collection. Washed cell extracts were collected on the days indicated for analysis of incorporation of 35S into sulfated proteoglycans. Data were pooled from 3 mice of each genotype, each studied in 3 separate experiments. *P<0.05. B, RT-PCR analyses for chondrogenic gene expression in NPP1–/– bone marrow stromal cells. Sox9 and collagen II expression were determined by RT-PCR of bone marrow stromal cells in high-density cultures on the days indicated as described in Methods. Where indicated, NPP1–/– cells were treated with 5 pmol sodium PPi at time 0 and every 72 hours thereafter to raise the extracellular concentration of PPi by 2.5 nM. Results shown are from cultures pooled from 5 mice of each genotype. C, Lack of significant change in multipotential bone marrow stromal precursor cells (CFU-F) from NPP1–/– mice. Bone marrow stromal cell preparations were harvested as described above from 4-month-old mice and aliquots of viable cells studied during 13 days in culture as described in Methods. CFU-F were counted on day 13 after fixation and Giemsa staining, with a colony defined as constituting a minimum of 5 cells per group. Results shown are from cultures pooled from the marrow of 5 mice of each genotype.
Altered Differentiation and Calcification in Cultured Aortic NPP1–/– VSMCs
Type II collagen expression was consistent with chondrogenic differentiation in preparations of enriched aortic VSMCs of NPP1–/– mice but not littermate NPP1+/+ mice (Figure 2). Under these conditions, both NPP1–/– and NPP1+/+ VSMC preparations demonstrated expression of type I collagen and the SMC-specific marker SM22 as well as MGP and OPG (Figure 2). Parallel experiments revealed similarly upregulated collagen II expression in aortic ank/ank VSMCs (not shown). Cultured aortic SMC preparations demonstrated significantly decreased extracellular PPi and OPN and increased AP activity and calcification in NPP1–/– and ank/ank mice cells relative to normal littermates (Figure 3).
Figure 2. RT-PCR analyses for chondro-osseous gene expression in cultured aortic SMC preparations from NPP1–/– mice. Aortic SMCs from euthanized 2-week-old NPP1–/– mice and littermate control NPP1+/+ mice were harvested by elastase and collagenase digestion as described in Methods. We studied expression of the indicated mRNAs by RT-PCR in aliquots of 5x104 primary cells cultured for 9 days as described in Methods. Representative of RNA isolated from 4 individual animals of each genotype are shown.
Figure 3. Comparable changes in extracellular PPi and OPN in cellular AP-specific activity and in calcification in primary mouse VSMCs from NPP1–/– and ank/ank mice. Primary VSMCs from NPP1 –/– and ank/ank mice and their respective congenic wild-type littermates at 2 weeks of age were harvested as described above and aliquots of 5x104 cells cultured in medium supplemented with 1 mmol/L sodium phosphate. At the times indicated, conditioned media and cell lysates were collected for determination of the extracellular PPi levels (A), cell AP-specific activity (B), and extracellular OPN (C) as described in Methods. To quantify matrix calcification (D), aliquots of 5x103 cells per well of primary VSMCs from NPP1–/–, ank/ank, and respective wild-type littermates were analyzed by Alizarin red S staining as described in Methods. Each experiment used VSMCs pooled from 8 animals from each genotype, run in triplicate. Data were pooled from 3 experiments. *P<0.05.
Conditioned media BMP-2 levels of the VSMCs of 2-week-old mice at 7 days in culture did not significantly differ between NPP1+/+ and NPP1–/– states (13.44±5.5 pg/mL versus 10.43±4.5 pg/mL, respectively, analyzing cells from 5 mice of each genotype, run in replicates of 3). Aortic cell preparations contained not only typical VSMCs but also cells with irregular borders and ruffled edges that assembled in nodular clusters in the postconfluent state (not shown). But the pattern of matrix calcification was diffuse in these cultured VSMCs preparations, unlike the typical nodular pattern of in vitro calcification by pericytes,21,22 suggesting calcification driven by transdifferentiated PPi-depleted VSMCs.
Aortic Media Calcification and Chondrogenic Differentiation In Situ in NPP1–/– Mice
von Kossa staining demonstrated aortic tunica media calcifications at 21 to 22 weeks of age in NPP1–/– and to a lesser degree in ank/ank mice (Figure 4). Assessment of whole aortic tissue by RT-PCR demonstrated in situ expression of collagen II in NPP1–/– and ank/ank mice (Figure 5). In addition, NPP1–/– and ank/ank mouse aortas demonstrated expression of the chondrocyte hypertrophy and late osteoblast differentiation marker osteocalcin and of cbfa1, a transcription factor centrally involved in both chondrocyte maturation to hypertrophy and osteoblastic differentiation.5 In contrast, expression of SM22, type I collagen, MGP, and OPG were detected in aortas from normal and NPP1–/– and ank/ank mice (Figure 5).
Figure 4. Pathological calcification involving the tunica media of the aorta in NPP1–/– and ank/ank mice. Thoracic aortas were removed from NPP1–/–, ank/ank, and respective wild-type littermates at 21 to 22 weeks of age. We performed von Kossa staining on 20 μmol/L frozen sagittal sections as described in Methods. As shown, black/brown staining calcium phosphate mineral deposition foci were seen in the media of NPP1–/– and ank/ank but not wild-type mice.
Figure 5. Aortic chondro-osseous mRNA expression in situ in both NPP1–/– and ank/ank mice. We extracted total RNA from aortas from 3-month-old mice and pooled RNA from 5 animals of each genotype indicated. After reverse transcription, 40 cycles of polymerase chain reaction amplification were performed for each mRNA analyzed.
Last, we immunohistochemically assessed for ectopic cartilage-specific collagen expression in NPP1–/– mouse aortas in comparison with NPP1+/+ controls. We studied types IX/XI collagen, whose ectopic expression at sites of perispinal ligament calcification was described in NPP1-deficient mice.29 Because chondrocyte maturation to hypertrophy is intimately linked with matrix calcification,30 we also tested for the stereotypic chondrocyte hypertrophy marker type X collagen. We detected aortic foci of expression of types IX/XI collagen in the media and adventitia, and of type X collagen in the media in 21-week-old NPP1–/– mice but not normal littermates (Figure 6).
Figure 6. Immunohistochemical detection of chondrocyte-specific type X and types IX/XI collagens in NPP1–/– aortas. Aortic specimens from 21-week-old NPP1–/– mice and NPP1+/+ littermate controls were immunohistochemically analyzed for chondrocyte-specific type X and types IX/XI collagen expression as described in Methods, using 20-μm thick sagittal frozen sections and using nonimmune rabbit IgG as a control. Brown staining foci of expression of types IX/XI collagen in the media and adventitia and of type X collagen in the media were detected in aortas of NPP1–/– mice but not the normal age-matched littermates. Representative of 4 animals of each genotype are shown. All images are displayed at x160 magnification.
Discussion
Human aortic SMCs constitutively express the physiological calcification inhibitor NPP1.17 Moreover, IIAC is linked to NPP1 mutations that decrease catalytic activity for PPi generation.15–17 Here we demonstrated that NPP1–/– mice, previously characterized to have marked extracellular PPi depletion,21 develop aortic media calcification. Chondro-osseous differentiation of cells in the artery wall mediates both atherosclerotic intima and nonatherosclerotic media forms of artery calcification.1,2 Here we discovered that NPP1 deficiency promoted the spontaneous emergence of chondrogenesis from bone marrow stromal cells. Our arterial studies were limited by small yields of VSMC-enriched mixed cell populations from mouse aortas. Nevertheless, it was significant that cultured NPP1–/– aortic SMC preparations and NPP1–/– aortic cells in situ expressed cbfa1, osteocalcin, and chondrocyte-specific collagens. Procalcifying AP specific activity was markedly upregulated in cultured NPP1–/– VSMCs. In contrast, there was no gross alteration in expression of the physiological artery calcification inhibitors MGP and OPG in NPP1–/– mouse arterial cells, consistent with previous results for NPP1–/– versus NPP1+/+ osteoblasts21
NPP1 not only generates PPi but also scavenges ATP and regulates purinergic receptor signaling, N-glycosylation, secretion of glycoproteins, proteoglycans sulfation, and insulin receptor signaling.31 But the capacity of exogenous PPi to correct spontaneous chondrogenesis in NPP1–/– bone marrow stromal cells under noncalcifying conditions suggested that extracellular PPi deficiency directly promoted chondrogenesis. Critical evidence supporting this conclusion was provided by our studies of ank/ank mice, which we observed to develop aortic media calcification. Cultured ank/ank mouse VSMCs demonstrated comparable changes to those in NPP1–/– VSMCs for extracellular PPi levels, OPN expression, AP-specific activity, and matrix calcification. Moreover, collagen II, cbfa1, and osteocalcin were expressed in situ in the ank/ank mouse aorta.
The extent of aortic media calcification was substantially less in ank/ank than in NPP1–/– mice between 4 to 5 months of age. Similar to NPP1–/– mice, we have observed that ank/ank mice also develop perispinal ligament calcification at 2 months of age (our unpublished observations, 2004). It will be of interest to assess whether the lesser extent of aortic media calcification in ank/ank mice relative to NPP1–/– mice persists beyond 21 to 22 weeks of age.
Chondrogenesis is a complex process mediated by precursor cell recruitment, condensation, and transcriptional pathways modulating chondrocyte-specific gene expression, including direct transcriptional regulatory effects of Sox9, effects of Wnt-frizzled receptor signaling, and the interplay between these factors exerted partly at the level of Sox9 interaction with ?-catenin.24,30 In this study, we observed that Sox9 expression was upregulated in NPP1–/– bone marrow stromal cells, an effect reversed by exogenous PPi. Definition of the primary mechanism(s) by which NPP1 and extracellular PPi regulate Sox9 expression and chondrogenesis will be of interest. We did not observe altered expression of BMP-2 by NPP1–/– VSMCs, but this finding did not rule out deficiency of 1 or more BMP inhibitors or changes in BMP signaling in NPP1–/– cells. It will also be of interest to assess whether NPP1 deficiency selectively promotes chondrogenesis in both mice and human arteries or whether NPP1 deficiency also impacts on Msx2-mediated vascular ossification programs that circumvent chondrogenesis.32
Cell surface receptors or active cellular or subcellular uptake mechanisms for PPi have yet to be identified in eukaryotic cells.14 But extracellular inorganic phosphate (Pi) generated partly through tissue-nonspecific AP pyrophosphohydrolysis of PPi is taken up by cells through defined sodium-dependent cotransporters.33 Though Pi uptake can promote chondrogenic differentiation, chondrocyte and osteoblastic maturation, and calcification,33–35 it also appears likely that extracellular PPi by itself can directly regulate differentiation of arterial cells. First, PPi inhibits calcification triggered by experimental artery injury, an effect that is removed by AP.36 Second, nonhydrolyzable bisphosphonate PPi analogues, which suppress artery calcification in states including IIAC,17 clearly regulate cellular signal transduction, gene expression, and viability.14 Third, intracellular PPi is elevated in ank/ank cells but decreased in NPP1–/– skeletal cells,21 pointing to shared critical effects of extracellular PPi depletion on VSMC function and chondrogenic differentiation. Fourth, analogous to effects of Pi,37 we have observed direct effects of extracellular PPi on expression of genes that regulate skeletal remodeling, such as matrix metalloproteinase-13 and OPN.21,38 In this context, the observation of decreased OPN expression by NPP1–/– and ank/ank VSMCs was compelling because OPN promotes atherogenesis.39 Hence, diminished OPN expression by NPP1–/– VSMCs may help explain why inflammation is not observed at IIAC artery lesions.
We did not quantitatively examine for SMC phenotype suppression5 in NPP1–/– aortic cells in this study, but transdifferentiation of VSMCs to calcifying chondro-osseous cells5 appeared central to the observed artery media calcification in NPP1–/– mice. Pericytes6,7 and other arterial cells also likely contributed. For example, we observed ectopic expression of types IX/XI collagen in the NPP1–/– mouse aortic adventitia, a potential source of multipotential cells (stenmark) and migratory fibroblasts capable of transdifferentiation to myofibroblasts, which drive vascular remodeling and fibrosis.40 Interestingly, a marked myointimal fibroproliferative response is a cardinal feature of IIAC associated with NPP1 "loss-of-function" mutations17 but, for undefined reasons, was not seen in NPP1–/– or ank/ank mouse aortas.
In conclusion, NPP1 and PPi physiologically function to prevent IIAC by suppressing artery calcification at the level of cell differentiation and not simply at the level of mineral formation and resorption in the extracellular matrix. Extracellular PPi levels are regulated by both genetic and acquired factors14 such as oxidative stress, which can depress PPi and promote calcification in vitro.26 In addition, cell stimulation by the atherogenesis mediators interleukin-1 or insulin-like growth factor-I depress extracellular PPi, partly through suppression of NPP1 and ANK expression.14 Therefore, acquired decrements in lesion NPP1 and ANK expression and extracellular PPi could contribute to intra-arterial chondro-osseous metaplasia and calcification in atherosclerosis and other conditions.
Acknowledgments
This work was supported by the Department of Veterans Affairs and National Institutes of Health (NIH) grants HL077360, P01AGO7996, and AR049366.
References
Demer LL, Tintut Y. Mineral Exploration: search for the Mechanism of Vascular Calcification and Beyond. The 2003 Jeffrey M. Hoeg Award Lecture. Arterioscler Thromb Vasc Biol. 2003; 23: 1739–1743.
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.
Wayhs R, Zelinger A, Raggi P. High coronary artery calcium scores pose an extremely elevated risk for heart events. J Am Coll Cardiol. 2002; 39: 225–230.
Abedin M, Tintut Y, Demer LL. Vascular calcification: mechanisms and clinical ramifications. Arterioscler Thromb Vasc Biol. 2004; 24: 1161–1170.
Steitz SA, Speer MY, Curinga G, Yang HY, Haynes P, Aebersold R, Schinke T, Karsenty G, Giachelli CM. Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 and downregulation of smooth muscle lineage markers. Circ Res. 2001; 89: 1147–1154.
Tintut Y, Alfonso Z, Saini T, Radcliff K, Watson K, Bostrom K, Demer LL. Multilineage potential of cells from the artery wall. Circulation. 2003; 108: 2505–2510.
Canfield AE, Doherty MJ, Wood AC, Farrington C, Ashton B, Begum N, Harvey B, Poole A, Grant ME, Boot-Handford RP. Role of pericytes in vascular calcification: a review. Z Kardiol. 2000; 89 (suppl 2): 20–27.
Ketteler M, Wanner C, Metzger T, Bongartz P, Westenfeld R, Gladziwa U, Schurgers LJ, Vermeer C, Jahnen-Dechent W, Floege J. Deficiencies of calcium-regulatory proteins in dialysis patients: a novel concept of cardiovascular calcification in uremia. Kidney Int Suppl. 2003; S84–S87.
Speer MY, McKee MD, Guldberg RE, Liaw L, Yang HY, Tung E, Karsenty G, Giachelli CM. Inactivation of the osteopontin gene enhances vascular calcification of matrix Gla protein-deficient mice: evidence for osteopontin as an inducible inhibitor of vascular calcification in vivo. J Exp Med. 2002; 196: 1047–1055.
Steitz SA, Speer MY, McKee MD, Liaw L, Almeida M, Yang H, Giachelli CM. Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification. Am J Pathol. 2002; 161: 2035–2046.
Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu W, Lacey DL, Boyle WJ, Simonet WS. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 1998; 12: 1260–1268.
B?strom K, Tsao D, Shen S, Wang Y, Demer LL. Matrix GLA protein modulates differentiation induced by bone morphogenetic protein-2 in C3H10T1/2 cells. J Biol Chem. 2001; 276: 14044–14052.
Galvin KM, Donovan MJ, Lynch CA, Meyer RI, Paul RJ, Lorenz JN, Fairchild-Huntress V, Dixon KL, Dunmore JH, Gimbrone MA Jr, Falb D, Huszar D. A role for smad6 in development and homeostasis of the cardiovascular system. Nat Genet. 2000; 24: 171–174.
Terkeltaub RA. Inorganic pyrophosphate generation and disposition in pathophysiology. Am J Physiol Cell Physiol. 2001; 281: C1–C11.
Rutsch F, Ruf N, Vaingankar S, Toliat MR, Suk A, H?hne W, Schauer G, Schnabel D, Epplen JT, Knisely A, Superti-Furga A, McGill J, Roscioli T, Filippone M, Vallance H, Hinrichs B, Smith W, Ferre M, Terkeltaub R, Nürnberg P. Mutations in ENPP1 are associated with "idiopathic infantile arterial" calcification. Nat Genet. 2003; 34: 379–381.
Ruf N, Uhlenberg B, Terkeltaub R, Nürnberg P, Rutsch F. The mutational spectrum of ENPP1 as arising after the analysis of 23 unrelated patients with generalized arterial calcification of infancy (GACI). Hum Mutat. 2005; 25: 98.
Rutsch F, Vaingankar S, Johnson K, Goldfine I, Maddux B, Schauerte P, Kalhoff H, Boisvert WA, Superti-Furga A, Terkeltaub R. PC-1 nucleoside triphosphate pyrophosphohydrolase deficiency in idiopathic infantile arterial calcification. Am J Pathol. 2001; 158: 543–554.
Hosoda Y, Yoshimura Y, Higaki S. A new breed of mouse showing multiple osteochondral lesions: twy mouse. Ryumachi. 1981; 21 (suppl 1): 157–164.
Okawa A, Nakamura I, Goto S, Moriya H, Nakamura Y, Ikegawa S. Mutation in Npps in a mouse model of ossification of the posterior longitudinal ligament of the spine. Nat Genet. 1998; 19: 271–273.
Ho AM, Johnson MD, Kingsley DM. Role of the mouse ank gene in control of tissue calcification and arthritis. Science. 2000; 289: 265–270.
Johnson K, Goding J, van Etten D, Sali A, Hu SI, Farley D, Krug H, Hessle L, Millan JL, Terkeltaub R. Linked deficiencies in extracellular inorganic pyrophosphate and osteopontin expression mediate pathologic calcification in PC-1 null mice. Am J Bone Min Res. 2003; 18: 994–1004.
Krug HE, Mahowald ML, Halverson PB, Sallis JD, Cheung HS. Phosphocitrate prevents disease progression in murine progressive ankylosis. Arthritis Rheum. 1993; 36: 1603–1611.
Clarke E. Culture of human and mouse mesenchymal cells. Methods Mol Biol. 2004; 290: 173–186.
Haas AR, Tuan RS. Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: II. Stimulation by bone morphogenetic protein-2 requires modulation of N-cadherin expression and function. Differentiation. 1999; 64: 77–89.
Salminen H, Vuorio E, Saamanen AM. Expression of Sox9 and type IIA procollagen during attempted repair of articular cartilage damage in a transgenic mouse model of osteoarthritis. Arthritis Rheum. 2001; 44: 947–955.
Johnson K, Jung A, Murphy A, Andreyev A, Dykens J, Terkeltaub R. Mitochondrial oxidative phosphorylation is a downstream regulator of nitric oxide effects on chondrocyte matrix synthesis and mineralization. Arthritis Rheum. 2000; 43: 1560–1570.
Arakawa E, Hasegawa K, Yanai N, Obinata M, Matsuda Y. A mouse bone marrow stromal cell line, TBR-B, shows inducible expression of smooth muscle-specific genes. FEBS Lett. 2000; 481: 193–196.
Mimura Y, Kobayashi S, Notoya K, Okabe M, Kimura I, Horikoshi I, Kimura M. Activation by alpha 1-adrenergic agonists of the progression phase in the proliferation of primary cultures of smooth muscle cells in mouse and rat aorta. Biol Pharm Bull. 1995; 18: 1373–1376.
Yamazaki M, Moriya H, Goto S, Saitoh Y, Arai K, Nagai Y. Increased type XI collagen expression in the spinal hyperostotic mouse (TWY/TWY). Calcif Tiss Int. 1991; 48: 182–189.
Akiyama H, Lyons JP, Mori-Akiyama Y, Yang X, Zhang R, Zhang Z, Deng JM, Taketo MM, Nakamura T, Behringer RR, McCrea PD, de Crombrugghe B. Interactions between Sox9 and beta-catenin control chondrocyte differentiation. Genes Dev. 2004; 18: 1072–1087.
Goding JW, Grobben B, Slegers H. Physiological and pathophysiological functions of the ecto-nucleotide pyrophosphatase/phosphodiesterase family. Biochim Biophys Acta. 2003; 1638: 1–19.
Tyson KL, Reynolds JL, McNair R, Zhang Q, Weissberg PL, Shanahan CM. Osteo/chondrocytic transcription factors and their target genes exhibit distinct patterns of expression in human arterial calcification. Arterioscler Thromb Vasc Biol. 2003; 23: 489–494.
Wang D, Canaff L, Davidson D, Corluka A, Liu H, Hendy GN, Henderson JE. Alterations in the sensing and transport of phosphate and calcium by differentiating chondrocytes. J Biol Chem. 2001; 276: 33995–34005.
Cecil DL, Rose DM, Terkeltaub R, Liu-Bryan R. IL-8 induces chondrocyte expression of Pit-1 and CXCR1-mediated Pi uptake. Arthritis Rheum. In press.
Jono S, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, Morii H, Giachelli CM. Phosphate regulation of vascular smooth muscle cell calcification. Circ Res. 2000; 87: E10–E17.
Lomashvili KA, Cobbs S, Hennigar RA, Hardcastle KI, O’Neill WC. Phosphate-induced vascular calcification: role of pyrophosphate and osteopontin. J Am Soc Nephrol. 2004; 15: 1392–1401.
Beck GR Jr. Inorganic phosphate as a signaling molecule in osteoblast differentiation. J Cell Biochem. 2003; 90: 234–243.
Johnson K, Terkeltaub R. Upregulated ANK expression in osteoarthritis can promote both chondrocyte MMP-13 expression and calcification via chondrocyte extracellular PPi excess. Osteoarthritis Cartilage. 2004; 12: 321–335.
Matsui Y, Rittling SR, Okamoto H, Inobe M, Jia N, Shimizu T, Akino M, Sugawara T, Morimoto J, Kimura C, Kon S, Denhardt D, Kitabatake A, Uede T. Osteopontin deficiency attenuates atherosclerosis in female apolipoprotein e-deficient mice. Arterioscler Thromb Vasc Biol. 2003; 23: 1029–1034.
Sartore S, Chiavegato A, Faggin E, Franch R, Puato M, Ausoni S, Pauletto P. Contribution of adventitial fibroblasts to neointima formation and vascular remodeling: from innocent bystander to active participant. Circ Res. 2001; 89: 1111–1121.(Kristen Johnson; Monika P)