Vascular calcification refers to the abnormal deposition of

Transcription

1 olecular edicine Smooth uscle ells Give Rise to Osteochondrogenic Precursors and hondrocytes in alcifying rteries ei Y. Speer, Hsueh-Ying Yang, Thea Brabb, Elizabeth eaf, my ook, Wei-ing in, ndrew Frutkin, David Dichek, ecilia. Giachelli bstract Vascular calcification is a major risk factor for cardiovascular morbidity and mortality. To develop appropriate prevention and/or therapeutic strategies for vascular calcification, it is important to understand the origins of the cells that participate in this process. In this report, we used the S22-re recombinase and Rosa26-acZ alleles to genetically trace cells derived from smooth muscle. We found that smooth muscle cells (Ss) gave rise to osteochondrogenic precursor- and chondrocyte-like cells in calcified blood vessels of matrix Gla protein deficient (GP / ) mice. This lineage reprogramming of Ss occurred before calcium deposition and was associated with an early onset of Runx2/bfa1 expression and the downregulation of myocardin and sx2. There was no change in the constitutive expression of Sox9 or bone morphogenetic protein 2. Osterix, Wnt3a, and Wnt7a mrns were not detected in either calcified GP / or noncalcified wild-type (GP / ) vessels. Finally, mechanistic studies in vitro suggest that Erk signaling might be required for S transdifferentiation under calcifying conditions. These results provide strong support for the hypothesis that adult Ss can transdifferentiate and that S transdifferentiation is an important process driving vascular calcification and the appearance of skeletal elements in calcified vascular lesions. (irc Res. 2009;104: ) Key Words: genetic fate mapping lineage reprogramming Runx2/bfa1 smooth muscle cells vascular calcification Vascular calcification refers to the abnormal deposition of calcium phosphate salts in blood vessels, myocardium, and cardiac valves. Vascular calcification can be lifethreatening, as in the case of generalized infantile arterial calcification, calcific uremic arteriolopathy, and calcific valve disease. 1,2 In atherosclerotic lesions, calcification is mainly found in the intima of blood vessels as dispersed punctate or patchy crystals associated with the necrotic core of atheromas (intimal calcification) and has been shown to positively correlate to the atherosclerotic plaque burden and the increased risk of myocardial infarction. 3 alcium phosphate salts deposit also in the media of blood vessels, known as onckeberg s medial sclerosis (medial calcification), and is prevalent in aging and patients with chronic kidney disease and type 2 diabetes mellitus. 2,4,5 edial calcification in these patients can occur independently of intimal calcification and/or atherosclerotic lesions and features linear calcium phosphate deposits along the elastic lamina and, when advanced, circumferential mineral deposits throughout the media. 4,5 edial calcification results in increased vessel wall stiffness and decreased vessel compliance and therefore leads to increased arterial pulse wave velocity and pulse pressure that eventually affects coronary artery perfusion and heart function. 6,7 onsequently, medial calcification-associated loss of arterial compliance may at least partially underlie increased coronary ischemic syndromes including myocardial infarction and left ventricular hypertrophy in diabetes and chronic kidney disease population. 2,3,6 8 lthough previously considered a degenerative, uncontrolled process, the presence of bone-related proteins, cells of osteoblast and chondrocyte morphology, and outright boneand cartilage-like tissue in calcified lesions has underscored the active, cell-mediated nature of vascular calcification. 4,5,8,9 These findings have also led to the important questions of what cell type(s) give rise to the skeletal elements found in vascular calcification and what mechanisms regulate vascular calcification. Smooth muscle cells (Ss) are the predominant cell type found in the arterial wall and are essential for the structural and functional integrity of the vessel. Unlike most cell types that undergo terminal differentiation, Ss retain substantial phenotypic plasticity in response to injurious stimuli in the local microenvironment. For example, Ss convert from a quiescent, contractile phenotype to a proliferative, synthetic phenotype following arterial injury and in atherosclerotic disease. 10 In calcified blood vessels, direct apposition of Original received arch 28, 2008; resubmission received July 14, 2008; revised resubmission received January 24, 2009; accepted January 26, From the Departments of Bioengineering (.Y.S., H.-Y.Y., E..,.., W.-..,..G.), omparative edicine (T.B.), and edicine (.F., D.D.), University of Washington, Seattle. orrespondence to ecilia. Giachelli, Professor, Department of Bioengineering, University of Washington, Box , 1705 NE Pacific St Foege N330, Seattle, W eci@u.washington.edu 2009 merican Heart ssociation, Inc. irculation Research is available at DOI: /IRRESH

2 734 irculation Research arch 27, 2009 calcification to medial Ss that expressed bone and cartilage marker proteins, alkaline phosphatase, bone sialoprotein, and type II collagen has been reported. 4,5 olecules regulating osteoblastic and chondrocytic differentiation, such as Runx2/bfa1, bone morphogenetic protein (BP)2, sx2, osterix, and Sox9, were also identified in calcified lesions of blood vessels. 8,9,11,12 In addition, cultured vascular Ss are induced to calcify by addition of supraphysiological levels of phosphate. oncomitant with the onset of calcification, elevated phosphate levels induced cultured Ss to undergo an osteochondrogenic phenotype change characterized by the loss of S markers (S22 and S -actin) and gain of osteochondrogenic markers (Runx1/bfa1, osteopontin, osteocalcin, and alkaline phosphatase). 13 Similar phenotypic changes were also triggered in vivo via adenoviral expression of transforming growth factor (TGF)- 1 in arterial endothelium. Increased expression of TGF- 1 in arterial endothelium caused cartilaginous metaplasia in the underlying media of rats. 14 Finally, electron microscopic and immunochemical studies identified putative transitional cells, termed myochondrocytes, that showed hybrid S and chondrocyte properties in human and mouse atherosclerotic lesions. 9 atrix Gla protein (GP) is a calcification inhibitor that accumulates at the border of calcified areas and normal media of blood vessels, and appears to act locally to limit calcium phosphate deposition in the vessel wall. 4,8,15,16 Because GP requires vitamin K-dependent -carboxylation for activation, undercarboxylated GP, attributable mainly to vitamin K insufficiency and/or long-term warfarin treatment, accelerates the development of vascular calcification. 7,17 In addition, polymorphisms of the GP gene are associated with increased risk of myocardial infarction and cardiovascular mortality in chronic kidney disease and hemodialysis patients. 18 utation of the GP gene causes excessive arterial calcification as seen in human autosomal recessive condition, Keutel syndrome, and the GP mutant mouse (GP / ). 16,19 To provide definitive evidence that Ss contribute to the development of skeletal elements seen in calcified vasculature, we undertook a genetic fate mapping approach in GP / mice. GP / mice develop calcification of the arterial media with predominance of the elastic lamellae in elastic and muscular arteries, such as aortas, carotids, and coronary arteries. alcification in these mice is associated with profound changes in cell differentiation as arterial Ss are replaced by chondrocyte-like cells undergoing progressive mineralization. There are no fatty streaks or atherosclerotic plaques in the affected arteries of GP / mice. GP deficiency also causes aortic valve calcification, peripheral pulmonary artery stenosis, and skeletal defects including abnormal cartilage and bone calcification and nasal hypoplasia, but no ectopic calcification was found in the myocardium and other S tissues of these mice. 13,16,20 Thus, using GP / mice as an arterial medial calcification model in this report, we genetically labeled Ss with the S22-re recombinase (S22-re) 21 and the re reporter Rosa26- acz (R26R-acZ) 22 alleles during embryonic development of the mice. This lineage tracing approach permits a direct test of whether vascular Ss can undergo lineage reprogramming and contribute to the development of skeletal elements in calcified blood vessels. aterials and ethods GP mutant mice generated in the 57B/6J background were kind gifts from Dr Karsenty. 16 To generate GP mutant mice in which cells of S origin were genetically marked by acz transgenes, GP heterozygotes (GP / ) were bred respectively to S22 -re recombinase transgenic mice (gift from Dr Herz, University of Texas Southwestern edical enter, Dallas) and Rosa26 re reporter transgenic mice (gift from Dr Soriano, Fred Hutchinson ancer Research enter, Seattle, Wash) to produce S22 -re /0 :GP / and R26R- acz /0 :GP /. The F1 offspring were inbred to produce male S22 -re / :GP / and female R26R-acZ / :GP / mice which were then used as breeders to produce S22 -re /0 :R26R- acz /0 :GP / experimental mice and S22 -re /0 :R26R- acz /0 :GP / controls. ice were maintained in a specific pathogen-free environment, and genotypes were determined. 16,21,22 One- to 8-week-old mice were euthanized by lethal intraperitoneal injection of Nembutal (0.3 mg/g) for necropsy. total of 52 mice were examined for these studies. ll protocols were approved by the Institutional nimal are and Use ommittee of the University of Washington. Tissues dissected from S22 -re /0 :R26R-acZ /0 :GP / and S22 -re /0 :R26R-acZ /0 :GP / were stained with X-gal before processing and embedding in paraffin. Five-micron sections were used for histochemical and immunohistochemical analyses. n expanded aterials and ethods is available in the online data supplement at Results haracterization of GP utant ice arrying S22-re and R26R-acZ Transgenes s described in Figure 1, mice carrying both S22-re and R26R-acZ transgenes delete the floxed stuffer sequence exclusively in S22 -positive cells during embryonic development, generating intracellular -galactosidase activity. Because re recombination occurs at the level of genomic DN and is irreversible, -galactosidase expression persists in the S22 -expressing cells irrespective of subsequent downregulation of S lineage proteins including S22. s shown in Figure 1, GP / mice hemizygous for R26R- acz and S22-re transgenes had blue Ss in the arterial media. Outside the vasculature, with rare exceptions (eg, occasional -galactosidase positive cells in the outer fibrous layer of the epiphysial perichondrium), -galactosidase expression was confined to S-rich tissue (Figure 1D through 1H) and to a lesser extent, cardiomyocytes that transiently express S22 early in development (data not shown). No -galactosidase positive cells were found in the B (Figure 1H). In addition, calcification of blood vessels in GP / mice did not affect -galactosidase expression in cells of the vascular media (Figure 1I and 1J). The homogeneity of X-gal staining indicated excellent re excision efficiency (Figure 1 through 1J), identical to the findings in floxed tgfbr2 mice. 23 Finally, tissues from mice carrying only the R26R- acz transgene did not stain with X-gal (Figure 1B and 1K). Ss Give Rise to Osteochondrogenic Precursors and hondrocytes in alcifying rteries of GP / ice GP / mice develop arterial medial calcification that has features similar to human calcified vessels. 13,16,20 s shown in

3 Speer et al Transdifferentiation of Smooth uscle ells 735 Rosa 26 promoter Stuffer sequence acz R26R-acZ (Inactivated acz gene) Rosa 26 promoter acz S22α-specific re recombination (ctivated acz gene) B D E F G H Figure I ( through D in the online data supplement), Ss of noncalcified 1-week-old GP / vessels showed expression of SH, S22, and S -actin genes, demonstrating a normal S differentiation in this mutant strain. In calcified blood vessels of 4-week-old mice (Figures 1I, 1J, and 2E, dark brown and black), medial cells did not express S lineage proteins, SH (Figure 2F versus brown in Figure 2B) and S22 (Figure 2G versus brown in Figure 2) but gained expression of the osteochondrogenic protein osteopontin (Figure 2H and 2D, brown). Because these medial cells stained blue with X-gal (marking them as cells that expressed S22 at an earlier time point) and because they were present at the precise locations that were occupied by X-gal positive, S marker expressing cells in noncalcified arteries, they appeared to have undergone a dramatic phenotypic change consistent with transdifferentiation from Ss to osteochondrogenic progenitor cells. oreover, in older mice, blue-staining type II collagen expressing I 50 µm J Figure 1. Genetic tracing and characterization of cells that transcribe -galactosidase transgene., Schematic description of re recombination that leads to acz gene activation in cells expressing S22. B through K, X-gal stained tissues dissected from GP / (B through H) and GP / (I through K) mice that carry R26R-acZ and S22-re transgenes ( through J) or R26R-acZ transgene only (B, K). B,, and I through K, aorta. D, Spleen. E, iver. F, Trachea. G, Growth plate of a femur. H, marrow of a femur. Sections were counterstained by nuclear fast red (B through H and K) or hematoxylin (J). GP / aorta was stained for mineral by von Kossa method (I). indicates lumen;, media;, adventitia. 50 µm K chondrocyte-like cells were often observed in the calcified media (Figure 3 and 3B, arrows). Of 11 calcified aortas, 9 contained chondrocyte-like cells in the calcified aortic media (82%), as identified by morphology and type II collagen staining (Figure 3D, brown). Importantly, nearly all of these cells also expressed -galactosidase (Figure 3D, blue), suggesting strongly that they differentiated in situ from Ss. Finally, X-gal, type II collagen antibody, and nuclear fast red triple-stained sections were used to quantify the proportion of chondrocyte-like cells that were derived from Ss. Of 617 type II collagen positive cells counted in calcified aortic media, 599 cells were stained blue by X-gal (97%), supporting the notion that osteochondrogenic precursor- and chondrocyte-like cells observed in the calcified GP / vessels derive from Ss that transdifferentiate in situ. To understand whether there was an increased turnover of medial cells in the GP / arteries, which would support circulating and/or residential multipotent mesenchymal pro-

4 736 irculation Research arch 27, 2009 B D E F G H Figure 2. Transdifferentiation of Ss in calcified arteries of GP / mice. ortas were dissected from 4-week-old S22 -re /0 :R26R /0 : GP / ( through D) and S22 -re /0 :R26R /0 :GP / (E through H) mice. ells of S origin were stained by X-gal before embedding. jacent sections were stained for mineral by von Kossa method ( and E) and for various cell markers by immunohistochemistry: SH (B and F), S22 ( and G), and osteopontin (D and H). Inset, Higher-power magnification of the boxed region shows colocalization of -galactosidase (blue) and osteochondrogenic marker, osteopontin (brown). indicates lumen;, media;, adventitia. B Figure 3. Ss gave rise to chondrocyte-like cells in calcifying GP / vessels. ortas were dissected from 6-week-old S22 -re /0 :R26R /0 :GP / mice. ells of S origin were stained by X-gal before embedding. jacent sections were stained by hematoxylin/eosin (), lizarin red S (B), osteopontin (), and type II collagen (D). rrows designate chondrocytes. Inset, Higher-power magnification of the boxed region shows colocalization of -galactosidase (blue) and osteochondrogenic marker, osteopontin (brown) and chondrocyte marker, type II collagen (brown). indicates lumen;, media;, adventitia. D genitors as possible sources of the observed osteochondrogenic precursor- and chondrocyte-like cells, we stained the arteries for active caspase-3 (apoptotic cells) and PN (proliferating-cell nuclear antigen) (proliferating cells). ll sixteen stained GP / aortas (1-week- to 8-week-old) showed only rare active caspase-3 positive cells in either adventitia or outer layer of the media (supplemental Figure II, B through D). Very few PN-positive cells were occasionally seen in the neointima and adventitia of the GP / vessels (supplemental Figure II, G and H). To further determine whether bone marrow (B)-derived progenitors make a significant contribution to the osteochondrogenic precursor- and chondrocyte-like cells that appear in arteries of GP / mice, we attempted to engraft green fluorescent protein (GFP)-expressing B cells into GP / neonates. Because GP / mice start to develop vascular calcification at 2 weeks old and do not survive lethal irradiation, we used a nonablative neonatal B transplantation strategy. The engraftment rate of the GP / chimeras was low ( 0.5% in peripheral blood versus 10% by Soper et al 24 ), although GFP-positive cells were easily detected in thymus and spleen of recipients (supplemental Figure III, and B). We also found GFP-positive cells in the aortae of recipients but these cells were rare (2 in the 2-week-old aorta and 1 in the 5-week-old aorta), and were all positive for D45, identifying them as inflammatory cells (supplemental Figure III, D through F). Interpretation of this study is limited by a low engraftment rate (most likely attributable to use of a nonablative approach and noncongenic B donors) and the possibility that only certain subpopulations of B progenitor cells successfully engrafted. Therefore, the B transplant study does not alone exclude a role for B-derived cells as a source of osteochondrogenic precursor- and chondrocyte-like cells that appear in calcifying arteries of GP / mice. However, combined with the genetic fate mapping study and the apoptosis and proliferation studies of the GP / vessels that showed very low turnover rate of artery wall cells, the results of the B transplantation study support our conclusion that B-derived progenitors do not make a significant contribution to osteochondrogenesis in calcified GP / vessels. Runx2/bfa1 Is an Early arker of S Transdifferentiation, and Its Upregulation Precedes atrix alcification To identify potential regulators of S transdifferentiation in vivo, we extracted RN from mildly calcified carotids of 2-week-old GP / mice and measured expression of genes associated with differentiation of Ss, osteoblasts, and

5 Speer et al Transdifferentiation of Smooth uscle ells 737 S-H S22α yocardin Runx2/ cbfa1 BP2 Sox 9 sx2 Osterix Wnt 3a Wnt 7a GPDH WT KO +trl Figure 4. Expression of genes associated with differentiation of Ss, osteoblasts, and chondrocytes in mouse arteries. Total RN was extracted from 2 to 4 carotids of 2-week-old GP / or GP / mice. Total RN (1 g) of these pooled samples was reverse transcribed to cdn. Various gene expression levels were determined by RT-PR using specific primers as listed in supplemental Table I. Total RN (1 g) extracted from cementoblasts was used as positive controls for osteochondrogenic genes. Expression levels of a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GPDH), were determined to assure equal loading. Data shown represent 1 of the 2 preparations; similar results were achieved in another independent preparation. WT indicates wild type; KO, knockout; trl, positive control of osteochondrogenic genes. chondrocytes. s shown in Figure 4, the S master transcription coactivator, myocardin, and its target genes, SH and S22, were downregulated in GP / arteries compared to wild-type counterparts. In contrast, the osteochondrogenic transcription factor, Runx2/bfa1 was highly upregulated in calcified arteries. BP2, a potent inducer of ectopic calcification, 25 and Sox9, a transcription factor required for chondrocyte differentiation, 26 were present in equal amounts in GP / (calcified) and GP / (noncalcified) arteries. On the other hand, expression of sx2, an inhibitor of chondrocytic differentiation, 26 was decreased in GP / compared to GP / arteries. No detectable expression of osteoblast differentiation factors, osterix, Wnt3a, or Wnt7a was observed in either GP / or GP / vessels. To further investigate the time course of S transdifferentiation in relation to arterial calcification, immunohistochemistry for Runx2/bfa1 was performed in arteries of 1- to 8-week-old GP / mice. s shown in Figure 5, Runx2/ bfa1 was selectively localized to the nucleus of the majority of arterial medial cells in all GP / mice examined by 2 weeks of age (Figure 5, brown). Staining of adjacent sections with an antibody recognizing -galactosidase confirmed the S lineage of these cells (Figure 5B, brown). small number of Runx2/bfa1 positive cells were sometimes observed in the adventitia (Figure 5, brown), but these cells were not -galactosidase positive (Figure 5B), indicating that they were not of S origin. Of particular interest, many of the Runx2/bfa1/ -galactosidase positive cells coexpressed S22 at this time point (Figure 5, brown), suggesting that they were transitional cells in an early stage of transdifferentiation to osteochondrogenic progenitors. Finally, the process of S transdifferentiation appeared to start before mineral deposition, because no arterial medial calcification could be detected in adjacent sections by von Kossa (Figure 5D) or lizarin red S (data not shown) staining. In contrast to the findings in 2-week-old mice, GP / arteries of 4- to 5-week-old mice were substantially calcified (Figure 5E, dark brown and black) and had much less Runx2/bfa1 expression. Only 2 of 5 mice showed a few Runx2/bfa1-positive cells in the calcified area (Figure 5F, brown) at this age. Runx2/bfa1 staining was exclusively in -galactosidase positive cells (smooth muscle [S] origin) of the vessel (Figure 5F, blue and brown). By 6 to 8 weeks of age, GP / arteries no longer expressed Runx2/bfa1 either in the media or in the adventitia (Figure 5G), despite high levels of calcification, increased expression of the Runx2/bfa1 downstream target gene osteopontin (Figures 2H and 3), and the presence of chondrocyte-like cells (Figure 3B and 3D). No Runx2/bfa1 staining was detected in 1-week-old GP / arteries (Figure 5H) or in GP / arteries at all ages examined (data not shown). Thus, temporal expression of Runx2/bfa1 correlated with early stages of S transdifferentiation, and preceded matrix calcification. Phosphorylation of Erk1/2 Is Required for S Transdifferentiation To study the potential mechanisms of S transdifferentiation, we isolated aortic medial cells from wild-type mice carrying S22-re and R26R-acZ transgenes. ultures were induced to undergo calcification (Figure 6) with elevated phosphate as previously described. 13 Under these conditions, Ss downregulated the expression of S lineage markers, S22 and S -actin (Figure 6B), and upregulated the expression of osteochondrogenic markers, osteopontin, Runx2/bfa1, and alkaline phosphatase (Figure 6), a phenomenon that was also observed in clonal populations of mouse Ss (data not shown). Thus, these calcifying cell culture experiments, performed with wild-type S, seemed to reproduce the S transdifferentiation observed in calcified GP / arteries. Because extracellular signal-regulated kinases (Erks) have been implicated in the regulation of S and osteoblast differentiation, we focused on the early stages of calcification and used this in vitro calcification model to examine the role of Erks in S transdifferentiation. Phosphorylation

6 738 irculation Research arch 27, 2009 B D E F G 50 µm 50 µm H 20 µm 20 µm 50 µm 50 µm Figure 5. Runx2/bfa1 expression in GP / arteries of various ages. ortas were dissected from 1-week-old (H), 2-week-old ( through D), 4-week-old (E and F), and 6-week-old (G) S22 -re /0 :R26R /0 :GP / mice. jacent sections were stained immunohistochemically for Runx2/bfa1 ( and F through H), -galactosidase (B), and S22 (), and for mineral by von Kossa (D and E). B and were counterstained with nuclear fast red, D and E were counterstained with ethyl green, and F and G were prestained with X-gal. rrows designate Runx2/bfa1-positive cells that originate from S lineage ( -galactosidase positive). Inset, Higher-power magnification of the boxed region shows colocalization of -galactosidase (blue) and Runx2/bfa1 (brown). indicates lumen;, media;, adventitia. of Erk1/2 was augmented in calcifying Ss that were treated with high phosphate for 1 to 7 days. The increase of phosphorylated Erk1/2 levels occurred before a decrease in the levels of S lineage marker (Figure 7). Furthermore, inhibition of Erk phosphorylation by the EK inhibitor U0126 prevented the down regulation of S lineage markers (Figure 7B) and the S-specific transcription coactivator myocardin (Figure 7) in calcifying Ss. oreover, Erk1/2 activation was accompanied by an increase of Runx2/ bfa1. Upregulation of Runx2/bfa1 mrn levels in calci- B alcium (µg/mg cellular protein) m Pi 1 S α-actin 3.0 m Pi Osteopontin Runx2/bfa1 80 µm S22α P 1.0 m Pi 3.0 m Pi 1.0 m Pi 3.0 m Pi Figure 6. S calcification and transdifferentiation in culture on exposure to elevated inorganic phosphate. Ss were cultured in DE culture medium containing basal (1.0 mmol/) or high (3.0 mmol/) inorganic phosphate levels. t day 5, calcium content of the cultures was determined as described in aterials and ethods. Data shown are means SD (n 3) (). Ss treated as in were double stained by X-gal and antibodies specific for S lineage proteins (B) and bone tissue associated proteins (). Similar results were achieved in another independent experiment. P indicates alkaline phosphatase.

7 Speer et al Transdifferentiation of Smooth uscle ells 739 Day 1 Day 2 Day 4 Day 7 Day 10 Day 15 High Pi P-Erk1/2 Total Erk1/2 S22α B P-Erk1/2 Total Erk1/2 S22α S α-actin yocardin Expression (myocardin/18s) P < D bfa1 Expression (bfa1/18s) P < High Pi U-0126 (µ) ontrol 3.0 m Pi 3.0 m Pi 2 µ U ontrol 3.0 m Pi 3.0 m Pi 2 µ U0126 Figure 7. Erk signaling in S transdifferentiation to osteochondrogenic precursors. Ss were cultured in the presence ( ) or absence ( ) of 3.0 mmol/ inorganic phosphate for various days () or in the presence of 3.0 mmol/ inorganic phosphate with or without EK inhibitor U-0126 for 4 days (B through D). ell lysate was collected for Western blot analysis ( and B), and total RN was extracted for real-time RT-PR ( and D). Similar results were obtained in another independent experiment. fying Ss was also inhibited by U0126 (Figure 7D). Therefore, the early molecular events that likely initiate the process of S calcification are inhibited by U1026. Because prolonged treatment with U1026 was toxic to S, we were unable to determine whether prolonged blockade of EK in this model would prevent S calcification. Discussion The cellular origins and mechanisms controlling development of ectopic cartilage and bone in diseased blood vessels are largely unknown. Decades of studies have raised 2 possibilities: transdifferentiation from mature Ss or differentiation from immature multipotent mesenchymal progenitors that reside within the vessel wall or migrate from the circulation. In this report, we used a genetic fate mapping strategy to identify the origin of the cells that give rise to osteochondrogenic precursor- and chondrocyte-like cells in the calcifying blood vessels of GP / mice. Ss of the vascular media are labeled with -galactosidase during embryonic development. oexistence within a single vascular medial cell of -galactosidase activity and osteochondrogenic or chondrocytic markers along with simultaneous loss of S lineage markers provides strong evidence supporting lineage reprogramming of Ss to osteochondrogenic precursors and chondrocytes. ccording to this reasoning, our experiments reveal that the majority of the osteochondrogenic precursor- and chondrocyte-like cells observed in the calcified arterial media of GP / mice were derived from Ss. This conclusion is supported by localization of Runx2/bfa1, osteopontin, and type II collagen expression within -galactosidase positive cells. Previous studies have implicated multipotent mesenchymal progenitors as possible sources of skeletal elements observed in vascular calcification. Demer and colleagues have identified a clonal population of bovine arterial medial cells, termed calcifying vascular cells (Vs). The Vs lack S marker proteins and display pericyte-like properties early in culture. With time in culture these V spontaneously form calcifying nodules and develop osteoblastic features. 11 ore recently, the Vs have been shown to undergo additional developmental fates, including chondrogenesis, leiomyogenesis, and stromagenesis, depending on the culture conditions. 30 Thus, the Vs behave like pericytes, a cell type that has long been postulated as a reservoir of multipotent stem cells in adult vasculature and can be induced to differentiate into multiple lineages, including osteoblasts. In our studies of GP / medial calcification, osteochondrogenic cells seen in the calcified arterial media were unlikely to be derived from pericytes or multipotent mesenchymal progenitors because no -galactosidase activity was found in the B (Figure 1H) or in the vascular adventitia of the S22 -re:r26r-acz mice at any age examined (see also 23 ). In addition, no cells expressing the mesenchymal/ hematopoietic stem cell markers Sca-1 and D34 were observed in the calcifying GP / vessels (data not shown). oreover, a hypothesis that attributed chondrogenesis to intramural migration of extramural precursor cells would need to account for the simultaneous near-total disappearance of resident Ss from vascular media. This seems highly unlikely as supported by the rare occurrence of apoptotic cells and the low number of both proliferating cells in calcified GP / arteries (supplemental Figure II) and engrafted GFP-expressing cells in the GP / neonatal chimeric arteries (supplemental Figure III). Nevertheless, our studies cannot exclude rare events and although they overwhelmingly support a major role for S lineage reprogramming in arterial medial calcification of GP / mice, they do not completely exclude limited contribution of non-s

8 740 irculation Research arch 27, 2009 GP BP2 Wnt/β-catenin sx2 Osterix Osteoblasts cell types or circulating B-derived precursors to the population of osteochondrogenic cells and chondrocytes that appear in GP / arteries. oreover, we cannot assume that the lineage reprogramming shown in this model would also explain observations in other vascular calcification models. 9,12,14,30,31 GP is a 10-kDa protein containing 5 -carboxyglutamic acid residues. It is normally expressed at high levels in cartilage and S, and serves as a calcification inhibitor in cartilage and vasculature. 25 Part of this inhibitory effect has been attributed to its capacity to bind and inhibit BP2, a major regulator of Runx2/bfa1 expression and potent osteoinductive factor. GP also abolishes BP2 receptor binding and phosphorylation of Smad1, acting as an inhibitor of BP2-dependent activation of Smads, critical cofactors that are involved in Runx2/bfa1-dependent osteochondrogenic differentiation. 25 Because Ss of 1-week-old GP / arteries showed normal S differentiation as evidenced by expression of the S lineage genes, SH, S22, and S -actin, and because 1-week-old GP / arteries showed no expression of osteochondrogenic genes such as Runx2/bfa1, transdifferentiation of Ss toward osteochondrogenic precursor- and chondrocyte-like cells in the calcifying GP / vessels is unlikely to be due simply to a lack of functional GP. It is proposed that lack of GP gene expression in Ss of GP / mice leaves BP2 activity unopposed, resulting in Runx2/bfa1 expression and inhibited myocardin expression, and thus transdifferentiation of Ss to osteochondrogenic precursors (Figure 8). Our data suggest that differentiation of these precursors preferentially down the chondrocyte path is favored by low levels of sx2, Pi Ss Erk1/2 phosphorylation Osteochondrogenic precursors Runx2/cbfa1 S lineage genes (myocardin, S22α, S α-actin) +Sox 9 sx2 hondrocytes Figure 8. Proposed mechanisms of S transdifferentiation in calcified arteries. oss of GP with subsequent release from inhibition of BP2, or exposure to elevated phosphate, leads to increased BP2 and phosphorylation of Erk1/2 in Ss. Erk1/2 activation increases Runx2/bfa1 and decreases myocardin and S lineage markers to generate the osteochondrogenic precursor state. In the presence of high Sox9 and the absence of sx2, Wnts, and osterix, osteochondrogenic precursors preferentially differentiate toward a chondrocytic lineage. an inhibitor of chondrogenesis, 26 expression of Sox9, a chondrocyte differentiation factor, 26 and lack of expression of osterix and Wnts, factors that are required for osteoblastic differentiation and prevention of osteoblast differentiation to chondrocytes respectively. 32 Our finding of endochondral differentiation in calcifying GP / vessels differs from the osteoblastic differentiation described by Towler and colleagues 31 in arteries of diabetic, atherosclerotic, D receptor knockout (Dr / ) mice. This is likely attributable to differences in signaling pathways present in calcifying arteries of GP / versus Dr / mice. In Dr / mice, hyperlipidemia induced upregulation of aortic BP2 expression and promoted adventitial sx2- Wnt signaling, which triggered mural Vs and other resident osteoprogenitors to proceed down the osteoblast differentiation path via tumor necrosis factor- dependent signals. 31 In agreement with this model, hyperlipidemic V-sx2 transgenic Dr / mice exhibited marked cardiovascular calcification. Intraperitoneal administration of BP2 enhanced aortic sx2 expression and canonical Wnt signaling in the tunica media of the blood vessels of TOPG mice (Wnt signaling reporters). 31 In contrast, calcifying GP / vessels were characterized by Sox9 expression, downregulation of sx2 expression, and no detectable expression of osterix, Wnt3a, or Wnt7a. Thus, chondrocytic versus osteoblastic differentiation is likely to depend on the local signaling milieu, which can differ substantially depending on the underlying disease and/or deficiency state. Our in vitro studies suggest that transdifferentiation of Ss is initiated by activation of the Erk1/2 signaling pathway, suppression of the S master transcription coactivator myocardin, and induction of the osteochondrogenic transcription factor Runx2/bfa1 (Figure 8). Because BP2 provokes phosphate uptake and S phenotypic transition toward osteochondroprogenitors 33 and is known to induce Erk1/2 signaling, 27 BP2 and elevated phosphate appear to share a common downstream signaling pathway for induction of S transdifferentiation. In support of these possibilities, Olson and coworkers reported that platelet-derived growth factor-bb mediated inhibition of S-specific gene expression was attributable to the Erk1/2-dependent phosphorylation of the transcriptional repressor Elk1. Phosphorylation of Elk1 promoted its binding to SRF and prevented the association of SRF with myocardin and thus inhibited the expression of S-specific genes. 28 onsistent with these findings, Erk phosphorylation was associated with injury- or fibronectininduced phenotypic modulation of vascular Ss. 34,35 Finally, Erk1/2 is important in osteoblast differentiation, as identified by its essential role in expression of osteogenic genes including Runx2/bfa1, osteopontin, osteocalcin, and bone sialoprotein, as well as its function in Runx2/bfa1-dependent skeletal development. 27,29 Our findings suggest a crucial role for Ss in mediating the onset and development of vascular medial calcification especially under conditions of GP deficiency. The osteochondrogenic state of Ss may be exquisitely designed to repair and/or adapt to a calcifying microenvironment, with enhanced expression of a number of calcification regulatory molecules. Understanding the mechanisms that control S

9 Speer et al Transdifferentiation of Smooth uscle ells 741 transdifferentiation to osteochondroprogenitors and subsequent vascular calcification may help developing novel strategies that prevent or reverse vascular calcification. Sources of Funding This work was supported by NIH grants R01 H (to..g.), R01 H62329 (to..g.), and K01 DK (to.y.s.) and NIH training grant H (to.y.s.). None. Disclosures References 1. Rutsch F, Ruf N, Vaingankar S, Toliat R, Suk, Hohne W, Schauer G, ehmann, Roscioli T, Schnabel D, Epplen JT, Knisely, Superti-Furga, cgill J, Filippone, Sinaiko R, Vallance H, Hinrichs B, Smith W, Ferre, Terkeltaub R, Nurnberg P. utations in ENPP1 are associated with idiopathic infantile arterial calcification. Nat Genet. 2003;34: Block G. ontrol of serum phosphorus: implications for coronary artery calcification and calcific uremic arteriolopathy (calciphylaxis). urr Opin Nephrol Hypertens. 2001;10: Taylor J, Burke P, O alley PG, Farb, alcom GT, Smialek J, Virmani R. comparison of the Framingham risk index, coronary artery calcification, and culprit plaque morphology in sudden cardiac death. irculation. 2000;101: Shanahan, ary NR, Salisbury JR, Proudfoot D, Weissberg P, Edmonds E. edial localization of mineralization-regulating proteins in association with onckeberg s sclerosis: evidence for smooth muscle cell-mediated vascular calcification. irculation. 1999;100: oe S, O Neill KD, Duan D, hmed S, hen NX, eapman SB, Fineberg N, Kopecky K. edial artery calcification in ESRD patients is associated with deposition of bone matrix proteins. Kidney Int. 2002;61: Guerin P, Blacher J, Pannier B, archais SJ, Safar E, ondon G. Impact of aortic stiffness attenuation on survival of patients in end-stage renal failure. irculation. 2001;103: Essalihi R, Dao HH, Yamaguchi N, oreau P. new model of isolated systolic hypertension induced by chronic warfarin and vitamin K1 treatment. m J Hypertens. 2003;16: Tyson K, Reynolds J, cnair R, Zhang Q, Weissberg P, Shanahan. Osteo/chondrocytic transcription factors and their target genes exhibit distinct patterns of expression in human arterial calcification. rterioscler Thromb Vasc Biol. 2003;23: Bobryshev YV. Transdifferentiation of smooth muscle cells into chondrocytes in atherosclerotic arteries in situ: implications for diffuse intimal calcification. J Pathol. 2005;205: Ross R. therosclerosis an inflammatory disease. N Engl J ed. 1999; 340: Bostrom K, Watson KE, Horn S, Wortham, Herman I, Demer. Bone morphogenetic protein expression in human atherosclerotic lesions. J lin Invest. 1993;91: Towler D, Bidder, atifi T, oleman T, Semenkovich F. Dietinduced diabetes activates an osteogenic gene regulatory program in the aortas of low density lipoprotein receptor-deficient mice. J Biol hem. 1998;273: Steitz S, Speer Y, uringa G, Yang HY, Haynes P, ebersold R, Schinke T, Karsenty G, Giachelli. Smooth muscle cell phenotypic transition associated with calcification - upregulation of bfa1 and downregulation of smooth muscle lineage markers. irc Res. 2001;89: Schulick H, Taylor J, Zuo W, Qiu B, Dong G, Woodward RN, gah R, Roberts B, Virmani R, Dichek D. Overexpression of transforming growth factor beta 1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia. Proc Natl cad Sci U S. 1998;95: Spronk H, Soute B, Schurgers J, leutjens JP, Thijssen HH, De ey JG, Vermeer. atrix Gla protein accumulates at the border of regions of calcification and normal tissue in the media of the arterial vessel wall. Biochem Biophys Res ommun. 2001;289: uo G, Ducy P, ckee D, Pinero GJ, oyer E, Behringer RR, Karsenty G. Spontaneous calcification of arteries and cartilage in mice lacking matrix G protein. Nature. 1997;386: Schori TR, Stungis GE. ong-term warfarin treatment may induce arterial calcification in humans: case report. lin Invest ed. 2004;27: Brancaccio D, Biondi, Gallieni, Turri O, Galassi, ecchini F, Russo D, ndreucci V, ozzolino. atrix G protein gene polymorphisms: clinical correlates and cardiovascular mortality in chronic kidney disease patients. m J Nephrol. 2005;25: unroe PB, Olgunturk RO, Fryns J-P, aldergem V, Ziereisen F, Yuksel B, Gardiner R, hung E. utations in the gene encoding the human matrix Gla protein cause Keutel syndrome. Nat Genet. 1999;21: El-aadawy S, Kaartinen T, Schinke T, urshed, Karsenty G, ckee D. artilage formation and calcification in arteries of mice lacking matrix Gla protein. onnect Tissue Res. 2003;44(suppl 1): Boucher P, Gotthardt, i WP, nderson RG, Herz J. RP: role in vascular wall integrity and protection from atherosclerosis. Science. 2003; 300: Soriano P. Generalized lacz expression with the ROS26 re reporter strain. Nat Genet. 1999;21: Frutkin D, Shi H, Otsuka G, eveen P, Karlsson S, Dichek D. critical developmental role for tgfbr2 in myogenic cell lineages is revealed in mice expressing S22-re, not SH-re. J ol ell ardiol. 2006;41: Soper BW, essard D, Vogler, evy B, Beamer WG, Sly WS, Barker JE. Nonablative neonatal marrow transplantation attenuates functional and physical defects of beta-glucuronidase deficiency. Blood. 2001; 97: Zebboudj F, Imura, Bostrom K. atrix G protein, a regulatory protein for bone morphogenetic protein-2. J Biol hem. 2002;277: Semba I, Nonaka K, Takahashi I, Takahashi K, Dashner R, Shum, Nuckolls GH, Slavkin H. Positionally-dependent chondrogenesis induced by BP4 is co-regulated by Sox9 and sx2. Dev Dyn. 2000; 217: Gallea S, allemand F, tfi, Rawadi G, Ramez V, Spinella-Jaegle S, Kawai S, Faucheu, Huet, Baron R, Roman-Roman S. ctivation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent 212 cells. Bone. 2001;28: Wang Z, Wang DZ, Hockemeyer D, cnally J, Nordheim, Olson EN. yocardin and ternary complex factors compete for SRF to control smooth muscle gene expression. Nature. 2004;428: Xiao G, Jiang D, Thomas P, Benson D, Guan K, Karsenty G, Franceschi RT. PK pathways activate and phosphorylate the osteoblast-specific transcription factor, bfa1. J Biol hem. 2000;275: Tintut Y, lfonso Z, Saini T, Radcliff K, Watson K, Bostrom K, Demer. ultilineage potential of cells from the artery wall. irculation. 2003;108: l ly Z, Shao JS, ai F, Huang E, ai J, Behrmann, heng S, Towler D. ortic sx2-wnt calcification cascade is regulated by TNF- {alpha} dependent signals In diabetic dlr / mice. rterioscler Thromb Vasc Biol. 2007;27: Hill TP, Spater D, Taketo, Birchmeier W, Hartmann. anonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev ell. 2005;8: i X, Yang HY, Giachelli. BP-2 promotes phosphate uptake, phenotypic modulation, and calcification of human vascular smooth muscle cells. therosclerosis. 2008;199: ovdahl, Thyberg J, Hultgardh-Nilsson. The synthetic metalloproteinase inhibitor batimastat suppresses injury-induced phosphorylation of P kinase ERK1/ERK2 and phenotypic modification of arterial smooth muscle cells in vitro. J Vasc Res. 2000;37: Ding HT, Wang G, Zhang T, Wang K. Fibronectin enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular smooth muscle cells via ERK pathway. J ell Biochem. 2006; 99: