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Circ Res. Author manuscript; available in PMC Mar 27, 2010.
Published in final edited form as:
PMCID: PMC2716055
NIHMSID: NIHMS124860

Smooth Muscle Cells Give Rise to Osteochondrogenic Precursors and Chondrocytes in Calcifying Arteries

Abstract

Vascular calcification is a major risk factor for cardiovascular morbidity and mortality. In order 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 SM22-Cre recombinase and Rosa26-LacZ alleles to genetically trace cells derived from smooth muscle. We found that smooth muscle cells (SMCs) gave rise to osteochondrogenic precursor- and chondrocyte-like cells in calcified blood vessels of matrix Gla protein deficient (MGP−/−) mice. This lineage reprogramming of SMCs occurred prior to calcium deposition, and was associated with an early onset of Runx2/Cbfa1 expression and the down regulation of myocardin and Msx2. There was no change in the constitutive expression of Sox9 or BMP2. Osterix, Wnt3a and Wnt7a mRNAs were not detected in either calcified MGP−/− or non-calcified wild type (MGP+/+) vessels. Finally, mechanistic studies in vitro suggest that Erk signaling might be required for SMC transdifferentiation under calcifying conditions. These results provide strong support for the hypothesis that adult SMCs can transdifferentiate and that SMC transdifferentiation is an important process driving vascular calcification and the appearance of skeletal elements in calcified vascular lesions.

Keywords: Genetic fate mapping, Lineage reprogramming, Runx2/Cbfa1, Smooth muscle cells, Vascular calcification

Introduction

Vascular calcification refers to the abnormal deposition of calcium phosphate salts in blood vessels, myocardium and cardiac valves. Vascular calcification can be life-threatening, 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 Calcium phosphate salts deposit also in the media of blood vessels, known as Monckeberg’s medial sclerosis (medial calcification), and is prevalent in aging and patients with chronic kidney disease (CKD) and type II diabetes mellitus.2;4;5 Medial 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 Medial 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 Consequently, 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 CKD population.2;3;68

While previously considered a degenerative, uncontrolled process, the presence of bone-related proteins, cells of osteoblast and chondrocyte morphology, and outright bone- and 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 (SMCs) 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, SMCs retain substantial phenotypic plasticity in response to injurious stimuli in the local microenvironment. For example, SMCs 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 calcification to medial SMCs that expressed bone and cartilage marker proteins, alkaline phosphatase, bone sialoprotein, and type II collagen has been reported.4;5 Molecules regulating osteoblastic and chondrocytic differentiation, such as Runx2/Cbfa1, bone morphogenetic protein 2 (BMP2), Msx2, osterix, and Sox9, were also identified in calcified lesions of blood vessels.8;9;11;12 In addition, cultured vascular SMCs are induced to calcify by addition of supraphysiologic levels of phosphate. Concomitant with the onset of calcification, elevated phosphate levels induced cultured SMCs to undergo an osteochondrogenic phenotype change characterized by the loss of SMC markers (SM22α and SM α-actin) and gain of osteochondrogenic markers (Runx1/Cbfa1, osteopontin, osteocalcin, and alkaline phosphatase).13 Similar phenotypic changes were also triggered in vivo via adenoviral expression of transforming growth factor-β1 (TGF-β1) in arterial endothelium. Increased expression of TGF-β1 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 SMC and chondrocyte properties in human and mouse atherosclerotic lesions.9

Matrix Gla protein (MGP) 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 Since MGP requires vitamin K-dependent γ-carboxylation for activation, undercarboxylated MGP, due mainly to vitamin-K insufficiency and/or long-term warfarin treatment, accelerates the development of vascular calcification.7;17 In addition, polymorphisms of the MGP gene are associated with increased risk of myocardial infarction and cardiovascular mortality in CKD and hemodialysis patients.18 Mutation of the MGP gene causes excessive arterial calcification as seen in human autosomal recessive condition, Keutel syndrome, and the MGP mutant mouse (MGP−/−).16;19

To provide definitive evidence that SMCs contribute to the development of skeletal elements seen in calcified vasculature, we undertook a genetic fate mapping approach in MGP−/− mice. MGP−/− 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. Calcification in these mice is associated with profound changes in cell differentiation as arterial SMCs are replaced by chondrocyte-like cells undergoing progressive mineralization. There are no fatty streaks or atherosclerotic plaques in the affected arteries of MGP−/− mice. MGP 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 SM tissues of these mice.13;16;20 Thus, using MGP−/− mice as an arterial medial calcification model in this report, we genetically labeled SMCs with the SM22-Cre recombinase (SM22-Cre)21 and the Cre reporter Rosa26-LacZ (R26R–LacZ)22 alleles during embryonic development of the mice. This lineage tracing approach permits a direct test of whether vascular SMCs can undergo lineage reprogramming, and contribute to the development of skeletal elements in calcified blood vessels.

Materials and Methods

MGP mutant mice generated in the C57BL/6J background were kind gifts from Dr. Karsenty.16 To generate MGP mutant mice in which cells of SM origin were genetically marked by LacZ transgenes, MGP heterozygotes (MGP+/−) were bred respectively to SM22α-Cre recombinase transgenic mice (gift from Dr. Herz, UT) and Rosa26 Cre reporter transgenic mice (gift from Dr. Soriano, FHCRC) to produce SM22α-Cre+/0:MGP+/− and R26R–LacZ+/0:MGP+/−. The F1 offspring were inbred to produce male SM22α-Cre+/+:MGP+/− and female R26R–LacZ+/+:MGP+/− mice which were then used as breeders to produce SM22α-Cre+/0:R26R–LacZ+/0:MGP−/− experimental mice and SM22α-Cre+/0:R26R–LacZ+/0:MGP+/+ controls. Mice were maintained in a specific pathogen-free environment, and genotypes were determined.16;21;22 One- to eight-week-old mice were sacrificed by lethal intraperitoneal injection of nembutol (0.3 mg/g) for necropsy. A total of 52 mice were examined for these studies. All protocols were approved by the Institutional Animal Care and Use Committee, University of Washington.

Tissues dissected from SM22α-Cre+/0:R26R–LacZ+/0:MGP−/− and SM22α-Cre+/0:R26R–LacZ+/0:MGP+/+ were stained with X-gal prior to processing and embedding in paraffin. Five-micrometer sections were used for histochemical and immunohistochemical analyses. Other materials and methods used for these studies are available in the online data supplement section.

Results

Characterization of MGP Mutant Mice Carrying SM22-Cre and R26R–LacZ Transgenes

As described in Figure 1A, mice carrying both SM22-Cre and R26R–LacZ transgenes delete the floxed stuffer sequence exclusively in SM22α-positive cells during embryonic development, generating intracellular β-galactosidase activity. Because Cre recombination occurs at the level of genomic DNA and is irreversible, β-galactosidase expression persists in the SM22α-expressing cells irrespective of subsequent down-regulation of SMC lineage proteins including SM22α. As shown in Figure 1C, MGP+/+ mice hemizygous for R26R–LacZ and SM22-Cre transgenes had blue SMCs in the arterial media. Outside the vasculature, with rare exceptions (e.g., occasional β-galactosidase positive cells in the outer fibrous layer of the epiphysial perichondrium), β-galactosidase expression was confined to SM-rich tissue (Figure 1D – 1H) and to a lesser extent, cardiomyocytes that transiently express SM22α early in development (data not shown). No β-galactosidase-positive cells were found in the bone marrow (Figure 1H). In addition, calcification of blood vessels in MGP−/− mice did not affect β-galactosidase expression in cells of the vascular media (Figure 1I and 1J). The homogeneity of X-gal staining indicated excellent Cre excision efficiency (Figure 1C – 1J), identical to the findings in floxed tgfbr2 mice.23 Finally, tissues from mice carrying only the R26R–LacZ transgene did not stain with X-gal (Figure 1B and 1K).

Figure 1
Genetic tracing and characterization of cells that transcribe β-galactosidase transgene

SMCs Give Rise to Osteochondrogenic Precursors and Chondrocytes in Calcifying Arteries of MGP–/– Mice

MGP−/− mice develop arterial medial calcification that has features similar to human calcified vessels.13;16;20 As shown in Online Figure 1A – 1D, SMCs of non-calcified one-week-old MGP−/− vessels showed expression of SMMHC, SM22α, and SM α-actin genes, demonstrating a normal SMC differentiation in this mutant strain. In calcified blood vessels of 4-wk-old mice (Figure 1I, 1J, Figure 2E, dark brown and black), medial cells did not express SMC lineage proteins, SMMHC (Figure 2F, vs brown in 2B) and SM22α (Figure 2G, vs brown in 2C), 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 SM22α at an earlier time point) and because they were present at the precise locations that were occupied by X-gal positive, SMC-marker-expressing cells in non-calcified arteries, they appeared to have undergone a dramatic phenotypic change consistent with transdifferentiation from SMCs to osteochondrogenic progenitor cells. Moreover, in older mice, blue-staining type II collagen-expressing chondrocyte-like cells were often observed in the calcified media (Figure 3A and3B, arrows). Of eleven calcified aortas, nine 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 β-galactosidase (Figure 3D, blue), suggesting strongly that they differentiated in situ from SMCs. 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 SMCs. Of 617 type II collagen positive cells counted in calcified aortic media, 599 cells were stained blue by X-gal (97%), supporting that osteochondrogenic precursor- and chondrocyte-like cells observed in the calcified MGP−/− vessels derive from SMCs that transdifferentiate in situ.

Figure 2
Transdifferentiation of SMCs in calcified arteries of MGP−/− mice
Figure 3
SMCs gave rise to chondrocyte-like cells in calcifying MGP−/− vessels

To understand whether there was an increased turnover of medial cells in the MGP−/− arteries, which would support circulating and/or residential multipotent mesenchymal progenitors as possible sources of the observed osteochondrogenic precursor- and chondrocyte-like cells, we stained the arteries for active caspase-3 (apoptotic cells) and proliferating cell nuclear antigen (PCNA; proliferating cells). All sixteen stained MGP−/− aortas (1-week- to 8-week-old) showed only rare active caspase-3 positive cells in either adventitia or outer layer of the media (Online Figure 2B – 2D). Very few PCNA positive cells were occasionally seen in the neointima and adventitia of the MGP−/− vessels (Online Figure 2G and 2H).

To further determine whether BM-derived progenitors make a significant contribution to the osteochondrogenic precursor- and chondrocyte-like cells that appear in arteries of MGP−/− mice, we attempted to engraft GFP-expressing BM cells into MGP−/− neonates. Because MGP−/− mice start to develop vascular calcification at ~2 weeks old and do not survive lethal irradiation, we used a non-ablative neonatal BM transplantation strategy. The engraftment rate of the MGP−/− chimeras was low (~0.5% in peripheral blood vs ~10% by Soper et al24), although GFP-positive cells were easily detected in thymus and spleen of recipients (Online Figure 3A and 3B). We also found GFP-positive cells in recipients’ aortae but these cells were rare (two in the 2-week-old aorta and one in the 5-week-old aorta) and were all positive for CD45, identifying them as inflammatory cells (Online Figure 3D – 3F). Interpretation of this study is limited by a low engraftment rate (due most likely to use of a nonablative approach and non-congenic BM donors) and the possibility that only certain subpopulations of BM progenitor cells successfully engrafted. Therefore, the BM transplant study does not alone exclude a role for BM-derived cells as a source of osteochondrogenic precursor- and chondrocyte-like cells that appear in calcifying arteries of MGP−/− mice. However, combined with the genetic fate mapping study and the apoptosis and proliferation studies of the MGP−/− vessels that showed very low turnover rate of artery wall cells, the results of the BM transplantation study support our conclusion that BM-derived progenitors do not make a significant contribution to osteochondrogenesis in calcified MGP−/− vessels.

Runx2/Cbfa1 is an early marker of SMC transdifferentiation and its upregulation precedes matrix calcification

To identify potential regulators of SMC transdifferentiation in vivo, we extracted RNA from mildly calcified carotids of 2-week-old MGP−/− mice and measured expression of genes associated with differentiation of SMCs, osteoblasts, and chondrocytes. As shown in Figure 4, the SMC master transcription co-activator, myocardin, and its target genes, SMMHC and SM22α, were down-regulated in MGP−/− arteries compared to wild type counterparts. In contrast, the osteochondrogenic transcription factor, Runx2/Cbfa1 was highly up-regulated in calcified arteries. BMP2, a potent inducer of ectopic calcification,25 and Sox9, a transcription factor required for chondrocyte differentiation,26 were present in equal amounts in MGP−/− (calcified) and MGP+/+ (non-calcified) arteries. On the other hand, expression of Msx2, an inhibitor of chondrocytic differentiation,26 was decreased in MGP−/− compared to MGP+/+ arteries. No detectable expression of osteoblast differentiation factors, osterix, Wnt3a, or Wnt7a was observed in either MGP−/− or MGP+/+ vessels.

Figure 4
Expression of genes associated with differentiation of SMCs, osteoblasts, and chondrocytes in mouse arteries

To further investigate the time course of SMC transdifferentiation in relation to arterial calcification, immunohistochemistry for Runx2/Cbfa1 was performed in arteries of 1- to 8-week-old MGP−/− mice. As shown in Figure 5, Runx2/Cbfa1 was selectively localized to the nucleus of the majority of arterial medial cells in all MGP−/− mice examined by 2 weeks of age (5A, brown). Staining of adjacent sections with an antibody recognizing β-galactosidase confirmed the SM lineage of these cells (5B, brown). A small number of Runx2/Cbfa1 positive cells were sometimes observed in the adventitia (5A, brown), but these cells were not β-galactosidase positive (5B), indicating that they were not of SM origin. Of particular interest, many of the Runx2/Cbfa1/β-galactosidase positive cells co-expressed SM22α at this time point (5C, brown), suggesting that they were transitional cells in an early stage of transdifferentiation to osteochondrogenic progenitors. Finally, the process of SMC transdifferentiation appeared to start prior to mineral deposition, since no arterial medial calcification could be detected in adjacent sections by von Kossa (5D) or Alizarin red S (data not shown) staining.

Figure 5
Runx2/Cbfa1 expression in MGP−/− arteries of various ages

In contrast to the findings in 2-week-old mice, MGP−/− arteries of 4- to 5-week-old mice were substantially calcified (5E, dark brown and black) and had much less Runx2/Cbfa1 expression. Only two out of five mice showed a few Runx2/Cbfa1 positive cells in the calcified area (5F, brown) at this age. Runx2/Cbfa1 staining was exclusively in β-galactosidase positive cells (SM origin) of the vessel (5F, blue and brown). By 6 to 8 weeks of age, MGP−/− arteries no longer expressed Runx2/Cbfa1 either in the media or in the adventitia (5G), despite high levels of calcification, increased expression of the Runx2/Cbfa1 downstream target gene osteopontin (Figure 2H and Figure 3C), and the presence of chondrocyte-like cells (Figure 3B and 3D). No Runx2/Cbfa1 staining was detected in 1-week-old MGP−/− arteries (5H) or in MGP+/+ arteries at all ages examined (data not shown). Thus, temporal expression of Runx2/Cbfa1 correlated with early stages of SMC transdifferentiation, and preceded matrix calcification.

Phosphorylation of Erk1/2 Is Required for SMC Transdifferentiation

To study the potential mechanisms of SMC transdifferentiation, we isolated aortic medial cells from wild type mice carrying SM22-Cre and R26R–LacZ transgenes. Cultures were induced to undergo calcification (Figure 6A) with elevated phosphate as previously described.13 Under these conditions, SMCs down-regulated the expression of SM lineage markers, SM22α and SM α-actin (Figure 6B), and up-regulated the expression of osteochondrogenic markers, osteopontin, Runx2/Cbfa1, and alkaline phosphatase (Figure 6C), a phenomenon that was also observed in clonal populations of mouse SMCs (data not shown). Thus, these calcifying cell culture experiments, performed with wild-type SMC, seemed to reproduce the SMC transdifferentiation observed in calcified MGP−/− arteries.

Figure 6
SMC calcification and transdifferentiation in culture upon exposure to elevated inorganic phosphate

Because extracellular signal-regulated kinases (Erks) have been implicated in the regulation of SMC and osteoblast differentiation,2729 we focused on the early stages of calcification and used this in vitro calcification model to examine the role of Erks in SMC transdifferentiation. Phosphorylation of Erk1/2 was augmented in calcifying SMCs that were treated with high phosphate for 1 to 7 days. The increase of phosphorylated Erk1/2 levels occurred prior to a decrease in the levels of SMC lineage marker (Figure 7A). Furthermore, inhibition of Erk phosphorylation by the MEK inhibitor, U0126, prevented the down regulation of SMC lineage markers (Figure 7B) and the SMC-specific transcription co-activator myocardin (Figure 7C) in calcifying SMCs. Moreover, Erk1/2 activation was accompanied by an increase of Runx2/Cbfa1. Upregulation of Runx2/Cbfa1 mRNA levels in calcifying SMCs was also inhibited by U0126 (Figure 7D). Therefore, the early molecular events that likely initiate the process of SMC calcification are inhibited by U1026. Because prolonged treatment with U1026 was toxic to SMC, we were unable to determine whether prolonged blockade of MEK in this model would prevent SMC calcification.

Figure 7
Erk signaling in SMC transdifferentiation to osteochondrogenic precursors

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 two possibilities: transdifferentiation from mature SMCs 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 MGP−/− mice. SMCs of the vascular media are labeled with β-galactosidase during embryonic development. Coexistence within a single vascular medial cell of β-galactosidase activity and osteochondrogenic or chondrocytic markers along with simultaneous loss of SM lineage markers provides strong evidence supporting lineage reprogramming of SMCs to osteochondrogenic precursors and chondrocytes. According to this reasoning, our experiments reveal that the vast majority of the osteochondrogenic precursor- and chondrocyte-like cells observed in the calcified arterial media of MGP−/− mice were derived from SMCs. This conclusion is supported by localization of Runx2/Cbfa1, osteopontin, and type II collagen expression within β-galactosidase positive cells.

Previous studies have implicated multipotent mesenchymal progenitors as possible sources of skeletal elements observed in vascular calcification. Demer and co-workers have identified a clonal population of bovine arterial medial cells, termed calcifying vascular cells (CVCs). The CVCs lack SMC marker proteins and display pericyte-like properties early in culture. With time in culture these CVC spontaneously form calcifying nodules and develop osteoblastic features.11 More recently, the CVCs have been shown to undergo additional developmental fates, including chondrogenesis, leiomyogenesis, and stromogenesis, depending on the culture conditions.30 Thus, the CVCs 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 MGP−/− medial calcification, osteochondrogenic cells seen in the calcified arterial media were unlikely to be derived from pericytes or multipotent mesenchymal progenitors because no β-galactosidase activity was found in the bone marrow (Figure 1H) or in the vascular adventitia of the SMC22α-Cre:R26R–LacZ mice at any age examined (see also 23). In addition, no cells expressing the mesenchymal/hematopoietic stem cell markers Sca-1 and CD34 were observed in the calcifying MGP−/− vessels (data not shown). Moreover, a hypothesis that attributed chondrogenesis to intramural migration of extramural precursor cells would need to account for the simultaneous near-total disappearance of resident SMCs 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 MGP−/− arteries (Online Figure 2) and engrafted GFP-expressing cells in the MGP−/− neonatal chimeric arteries (Online Figure 3). Nevertheless, our studies cannot exclude rare events and although they overwhelmingly support a major role for SMC lineage reprogramming in arterial medial calcification of MGP−/− mice, they do not completely exclude limited contribution of non-SM cell types or circulating BM-derived precursors to the population of osteochondrogenic cells and chondrocytes that appear in MGP−/− arteries. Moreover, 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

MGP is a 10-kDa protein containing 5 γ-carboxyglutamic acid residues. It is normally expressed at high levels in cartilage and smooth muscle, 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 BMP2, a major regulator of Runx2/Cbfa1 expression and potent osteoinductive factor. MGP also abolishes BMP2 receptor binding and phosphorylation of Smad1, acting as an inhibitor of BMP2-dependent activation of Smads, critical co-factors that are involved in Runx2/Cbfa1-dependent osteochondrogenic differentiation.25 Because SMCs of 1-week-old MGP−/− arteries showed normal SMC differentiation as evidenced by expression of the SM lineage genes, SMMHC, SM22α, and SM α-actin, and because 1-week-old MGP−/− arteries showed no expression of osteochondrogenic genes such as Runx2/Cbfa1, transdifferentiation of SMCs toward osteochondrogenic precursor- and chondrocyte-like cells in the calcifying MGP−/− vessels is unlikely to be due simply to a lack of functional MGP. It is proposed that lack of MGP gene expression in SMCs of MGP−/− mice leaves BMP2 activity unopposed, resulting in Runx2/Cbfa1 expression and inhibited myocardin expression, and thus transdifferentiation of SMCs to osteochondrogenic precursors (Figure 8). Our data suggest that differentiation of these precursors preferentially down the chondrocyte path is favored by low levels of Msx2, 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

Figure 8
Proposed mechanisms of SMC transdifferentiation in calcified arteries

Our finding of endochondral differentiation in calcifying MGP−/− vessels differs from the osteoblastic differentiation described by Towler and co-workers in arteries of diabetic, atherosclerotic, LDL receptor knockout (LDLr−/−) mice. This is likely due to differences in signaling pathways present in calcifying arteries of MGP−/− versus LDLr−/− mice. In LDLr–/– mice, hyperlipidemia induced upregulation of aortic BMP2 expression and promoted adventitial Msx2-Wnt signaling, which triggered mural CVCs and other resident osteoprogenitors to proceed down the osteoblast differentiation path via TNF-α-dependent signals.31 In agreement with this model, hyperlipidemic CMV-Msx2 transgenic LDLr−/− mice exhibited marked cardiovascular calcification. Intraperitoneal administration of BMP2 enhanced aortic Msx2 expression and canonical Wnt signaling in the tunica media of the blood vessels of TOPGAL mice (Wnt signaling reporters).31 In contrast, calcifying MGP−/− vessels were characterized by Sox9 expression, downregulation of Msx2 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 SMCs is initiated by activation of the Erk1/2 signaling pathway, suppression of the SMC master transcription co-activator myocardin and induction of the osteochondrogenic transcription factor Runx2/Cbfa1 (Figure 8). Because BMP2 provokes phosphate uptake and SMC phenotypic transition toward osteochondroprogenitors33 and is known to induce Erk1/2 signaling,27 BMP2 and elevated phosphate appear to share a common downstream signaling pathway for induction of SMC transdifferentiation. In support of these possibilities, Olson and co-workers reported that PDGF-BB-mediated inhibition of SMC-specific gene expression was due 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 SM-specific genes.28 Consistent with these findings, Erk phosphorylation was associated with injury- or fibronectin-induced phenotypic modulation of vascular SMCs.34;35 Finally, Erk1/2 is important in osteoblast differentiation, as identified by its essential role in expression of osteogenic genes including Runx2/Cbfa1, osteopontin, osteocalcin, and bone sialoprotein as well as its function in Runx2/Cbfa1-dependent skeletal development.27;29

Our findings suggest a crucial role for SMCs in mediating the onset and development of vascular medial calcification especially under conditions of MGP deficiency. The osteochondrogenic state of SMCs 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 SMC transdifferentiation to osteochondroprogenitors and subsequent vascular calcification may help developing novel strategies that prevent or reverse vascular calcification.

Supplementary Material

Suppl

Acknowledgements

This work was supported by NIH grants R01 HL081785 (CMG), R01 HL62329 (CMG), K01 DK075665 (MYS), and NIH training grant HL07828-06 (MYS).

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