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Integrative Physiology/Experimental Medicine

Chondrocyte Rather Than Osteoblast Conversion of Vascular Cells Underlies Medial Calcification in Uremic Rats

Ellen Neven, Veerle Persy, Simonne Dauwe, Tineke De Schutter, Marc E. De Broe, Patrick C. D'Haese
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https://doi.org/10.1161/ATVBAHA.110.204834
Arteriosclerosis, Thrombosis, and Vascular Biology. 2010;30:1741-1750
Originally published August 18, 2010
Ellen Neven
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Veerle Persy
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Simonne Dauwe
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Tineke De Schutter
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Marc E. De Broe
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Patrick C. D'Haese
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Abstract

Objective— To investigate cell biological changes in calcified aortas of rats that experienced chronic renal failure.

Methods and Results— Vascular smooth muscle cells have the potential to transdifferentiate to either chondrocytes or osteoblasts, depending on the molecular pathways that are stimulated. Uremia-related medial calcification was induced by feeding rats an adenine low-protein diet for 4 weeks. Aortic calcification was evaluated biochemically and histochemically and with in vivo micro–computed tomographic scanning. Immunohistochemistry and RT-PCR were applied to analyze the time-dependent aortic expression of molecules involved in the segregation between the chondrocyte versus osteoblast differentiation pathway. After 4 weeks, 85% of the uremic rats had developed distinct aortic medial calcification, which increased to severely calcified lesions during further follow-up. The calcification process was accompanied by a significant time-dependent increase in the expression of the chondrocyte-specific markers sex determining region Y-box 9 (sox9), collagen II, and aggrecan and a nonsignificant trend toward enhanced core binding factor alpha 1 (cbfa1), and collagen I. The expression of the osteoblast marker osterix and both lipoprotein receptor–related protein 6 and β-catenin, molecules of the wingless-type MMTV integration site family member (Wnt)/β-catenin pathway induced during osteoblast differentiation, was suppressed.

Conclusion— In the aorta of uremic rats, medial smooth muscle cells acquire a chondrocyte rather than osteoblast phenotype during the calcification process.

  • chronic renal failure
  • medial calcification
  • chondrocytes
  • molecular biology
  • vascular biology

Cardiovascular disease accounts for 50% of all deaths in patients with chronic kidney disease (CKD).1 Vascular calcification is a prominent feature of vascular disease and is rapidly progressive in dialysis patients.2 In the CKD population compared with the general population, the extent of vascular calcification is significantly higher and the onset of calcium deposits in the vessel wall is detected at a much younger age.2 In addition to accelerated calcification of intimal atherosclerotic plaques, patients with CKD show characteristic calcifications of the vascular media; these are independently associated with cardiovascular mortality.3

Vascular calcification is a cell-regulated pathological process that resembles osteogenesis.4,5 When exposed to the uremic milieu, vascular cells from the tunica media retain the capacity to transdifferentiate into osteoblastlike or chondrocytelike cells, based on the expression of various bone proteins, and are able to calcify.6 In particular, under the influence of stimuli (eg, calcium, phosphate, inflammatory factors, or oxidized low-density lipoproteins), they have the potential to express bone-regulating proteins and produce matrix vesicles and an extracellular matrix prone to mineralization.7–10

Chondrocytes and osteoblasts are derived from a common mesenchymal progenitor cell. Osteochondroprogenitor cells expressing cbfa1 are bipotential and can differentiate into osteoblasts or chondrocytes. Osterix (osx) commits precursor cells to the osteoblast lineage,11 whereas sox9 induces differentiation toward chondrocytes.12 The Wnt/β-catenin signaling pathway is also involved in the segregation between osteoblasts and chondrocytes from bipotential osteochondroprogenitor cells.13 Wnt proteins are a family of secreted proteins regulating different aspects of cell growth, differentiation, and function by binding to the frizzled receptor and low-density lipoprotein receptor–related protein (LRP) 5/6. Wnt interaction with its receptors then activates a cascade of signaling molecules, resulting in hypophosphorylation of β-catenin. β-Catenin accumulates and translocates to the nucleus, where it activates the expression of several downstream genes that play a role in cell fate determination during development and tumorigenesis,14 by interaction with transcription factors.15 During skeletogenesis, β-catenin signaling is increased in differentiating osteoblasts.16 Wnt/β-catenin signaling increases bone mass through stimulation of osteoblast proliferation and differentiation and through inhibition of osteoblast and osteocyte apoptosis.17,18 Genetic ablation of β-catenin in the mouse reduced bone formation in favor of cartilage formation. Conversely, upregulation of Wnt signaling resulted in suppressed chondrocyte differentiation and enhanced ossification.16 When β-catenin was genetically inactivated in vitro in mesenchymal progenitor cells under osteogenic conditions, prominent alcian blue staining indicated that the cells formed cartilage instead of bone nodules under these conditions.16 Recently, tissue-specific overexpression of the β-catenin gene in articular chondrocytes resulted in an osteoarthritislike phenotype, characterized by the loss of articular cartilage layers and the formation of new woven bone in the subchondral bone area.19 Furthermore, the chondrocyte-specific transcription factor sox9 inhibits Wnt/β-catenin signaling by promoting β-catenin phosphorylation in the nucleus.20 Thus, the Wnt/β-catenin signaling pathway plays a critical role in the terminal differentiation of mesenchymal cells: low Wnt signaling leads to cartilage differentiation, whereas high Wnt signaling drives osteoblast differentiation.

To have a better insight in the transdifferentiation pathway of vascular smooth muscle cells toward osteochondrogenic cell types during the calcification process in the media, the time-dependent mRNA expression of molecules that are involved in the segregation of the chondrocyte versus the osteoblast differentiation pathway during the development of vascular calcification is studied in the aorta of rats with chronic renal failure (CRF).

Methods

Study Design

After 2 weeks of a high-phosphorus diet (1.03% phosphorus and 1.06% calcium) (SSNIFF Spezialdiäten, Soest, Germany), 34 male Wistar rats (Iffa Credo, Brussels, Belgium) were fed a 0.75% adenine-enriched diet (0.92% phosphorus and 1.0% calcium) with a low protein content (2.5%) for 4 weeks to induce CRF. Animals (n=4) with normal renal function (NRF) were fed a low-protein diet instead. After adenine withdrawal, all animals were maintained on a high-phosphorus diet (1.03%) until they were euthanized. Blood and urine samples were taken at regular points throughout the study after a 24-hour stay in metabolic cages. To follow both the development of vascular calcification and the expression of osteochondrogenic conversion markers in the vessel wall over time, rats with CRF were euthanized at 1, 2, 3, and 4 weeks (n=4 for each point) or at 8 weeks (n=8) after the start of CRF induction. Animals were exsanguinated through the retro-orbital plexus after anesthesia with sodium pentobarbital (Nembutal; Ceva Santé Animale, Libourne, France), 60 mg/kg, via intraperitoneal injection.

Serum and Urine Biochemistry

Serum creatinine, total calcium, and phosphorus concentrations were measured at Antwerp University Hospital using an autoanalyzer system (Vitros 5.1 Fusion; Ortho Clinical Diagnostics, Rochester, NY). The urinary calcium level was determined using flame atomic absorption spectrometry (Perkin-Elmer, Wellesley, Mass), and the urinary phosphorus level was measured with a commercially available kit (EcolineS Phosphate kit; DiaSys, Holzheim, Germany). Serum parathyroid hormone (PTH) determinations were performed with a rat PTH-immunoradiometric assay kit (Immutopics Inc, San Clemente, Calif), and serum 1,25 dihydroxyvitamin D3 levels were measured with a radioimmunoassay kit (Biosource, Nivelles, Belgium).

Evaluation of Vascular Calcification

In Vivo Micro–Computed Tomography

The development of calcification in the thoracic aorta of rats with CRF was observed over time using a desktop micro–computed tomographic (CT) scanner (Skyscan 1076; Kontich, Antwerp, Belgium) with a 10-megapixel charge-coupled device camera and a microfocal X-ray source with a 5-μm spot size.21 Therefore, a separate group of rats with CRF (n=10) was scanned at 1, 2, 3, 4, 6, and 8 weeks after the induction of CRF.

Rats were anesthetized intravenously with 35-mg/kg sodium pentobarbital. To scan the same region several times, the proximal side of the vertebrocostal articulation of the fourth rib was used as an anatomical reference point. Scans were performed with a 0.025-mm titanium filter and a voxel size of 35×35×35 μm. During an exposure time of 21 minutes, a 1.8-cm axial length of the thoracic region was scanned. A set of phantoms with known densities and a known chemical composition (hydroxyapatite; Berkeley Advanced Biomaterials Inc, Berkeley, Calif) similar to bone, was also scanned. Virtual cross sections were reconstructed using the Feldkamp cone beam algorithm. Reconstruction of a region of interest containing the aorta was performed after the scan, and aortic calcification was calculated as the number of voxels greater than a density threshold of 65 Hounsfield units and expressed as calcified volume (in cubic millimeters).

Calcium Content

The proximal abdominal aorta was weighed shortly after removal, digested in 65% nitric acid at 65°C overnight, and diluted in 0.1% lanthanum nitrate to eliminate chemical interference during measurement with flame atomic absorption spectrometry (Perkin-Elmer). Results are expressed as calcium (in milligrams) divided by wet tissue (in grams).

Microscopic Evaluation

The thoracic aorta, including the aortic arch, was fixed in neutral-buffered formalin for 90 minutes and cut into 2- to 3-mm-thick rings that were embedded upright in the same paraffin block; therefore, every paraffin section composed, on average, 21 cross sections at different sites along the vessel. Sections of 4 μm were stained using the Von Kossa method. Calcification in each arterial cross section was scored microscopically at ×100 magnification using the following semiquantitative scoring system: 0, no calcification; 1, focal calcification spots; 2, partial calcification covering 20% to 80% of the arterial circumference; and 3, circumferential calcification.

Immunohistochemistry

Immunohistochemical stainings for the chondrocyte-specific markers sox9 (sc-17340) and collagen II (sc-7764) and the osteochondrogenic transcription factor cbfa1 (sc-8566) (Santa Cruz Biotechnology, Santa Cruz, Calif) were obtained on aortic sections of rats with NRF and rats with CRF that were euthanized at different points, as previously described.22 The presence of the respective target proteins was scored semiquantitatively in 17 to 29 aortic cross sections per animal using a scoring system ranging from 0 to 3: 0, no expression; 1, focal expression; 2, partial expression taking up 20% to 80% of vessel circumference; and 3, circumferential expression (>80%). The percentage of cross sections per score was calculated for each experimental group.

To locate cells expressing cbfa1 and sox9 in relation to calcified areas, immunofluorescence double staining was performed on aortic sections of rats with CRF that were euthanized at different points. Sections were incubated with goat anti–sox9 (sc-17340) and rabbit anti–cbfa1 (sc-10758) antibodies (Santa Cruz Biotechnology). Donkey anti–goat IgG (Alexa Fluor 488) and donkey anti–rabbit IgG (Alexa Fluor 555) (Invitrogen, Carlsbad, Calif) were used as secondary antibodies, and all sections were counterstained with Hoechst to visualize cell nuclei.

RT-PCR Analysis

Total mRNA of the distal part of the abdominal aorta was extracted using a commercially available kit (Rneasy Fibrous Tissue mini kit; Qiagen, Hilden, Germany) and reverse transcribed to cDNA by another kit (High Capacity cDNA archive kit; Applied Biosystems, Foster City, Calif). RT-PCR with an available system (ABI Prism 7000 Sequence Detection System; Applied Biosystems), based on the Taqman fluorescence method, was used for mRNA quantification. Taqman probe and primers were purchased as Taqman gene expression assays on demand (Applied Biosystems) for GAPDH (Rn99999916_s1), sox9 (Rn01751069_mH), collagen II (Rn00491926_g1), Indian hedgehog (Rn03810376_m1), aggrecan (Rn006729206_g1), cbfa1 (Rn01512296_m1), collagen I (Rn00801649_g1), osx (Rn01761789_m1), phosphate transporter (Pit)-1 (Rn00579811_m1), Wnt4 (Rn0058477_m1), LRP6 (Rn01492711_m1), and β-catenin (Rn01246634_m1). The expression of each tested gene was analyzed in triplicate for each sample. The expression of each gene was normalized to the expression of the housekeeping gene GAPDH. Gene expression was calculated with the comparative CT method using aortas of rats with NRF as calibrator samples.

Statistical Analysis

Data are expressed as mean±SD unless otherwise noted. Statistical analyses were performed with SPSS 16.0. Differences between multiple points for each study group were determined by the Friedman test, followed by a Wilcoxon signed-rank test with Bonferroni correction. Comparisons between the study groups for each point were assessed using a Kruskal-Wallis test, followed by a Mann-Whitney U test in combination with Bonferroni correction. P<0.05 was considered significant.

Results

Biochemical Analyses

The findings of serum and urine biochemistry analyses are summarized in the Table.

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Table. Serum and Urine Biochemistry Data

During adenine administration, serum creatinine concentrations significantly increased over time compared with rats with NRF eating a low-protein diet (0.48±0.05 mg/dL) and reached maximum values at week 4 (4.05±1.15 mg/dL), reflecting the installation of severe CRF. After adenine withdrawal, serum creatinine levels decreased to 2.03±0.62 mg/dL.

A substantial increase in serum phosphate concentrations in rats with CRF was observed up to a maximum value of 23.8±3.1 mg/dL at week 4. Two weeks after adenine withdrawal, serum phosphate levels returned to baseline values; however, at the end of the study, significantly higher serum phosphate concentrations were noted compared with animals with NRF. Because of renal impairment, phosphate excretion significantly decreased from 2 weeks after the start of adenine feeding onward compared with baseline values. Serum calcium concentrations time dependently decreased from 1 week after the induction of CRF onward. During the entire study period, urinary calcium excretion was significantly lower in rats with CRF. The serum calcium-by-phosphate product was remarkably elevated in rats with CRF during adenine loading.

A time-dependent increase in serum PTH concentrations was observed in rats with CRF, reflecting severe secondary hyperparathyroidism. Renal dysfunction caused a significant decrease in serum 1,25-dihydroxyvitamin D3 concentrations.

Vascular Calcification

After 4 weeks of an adenine low-protein diet, 85% of the rats had developed aortic calcification, as presented by total calcium content (Figure 1) and the percentage of cross sections with moderate-to-severe medial calcification (ie, scores of 2 and 3 on Von Kossa–stained sections) (Figure 2). Eight weeks after the induction of CRF, all rats had extensive calcifications in the aorta (Figures 1 and 2⇓). No arterial calcification was observed in rats with NRF at the end of the study.

Figure1
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Figure 1. Calcium content in the aorta in rats with NRF and CRF. Gray diamonds represent individual data of rats with CRF that died before the planned euthanasia at week 8. *P<0.05 vs rats with NRF.

Figure2
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Figure 2. Evaluation of vascular calcification by semiquantitative scoring of Von Kossa–stained sections in the aorta in rats with NRF and CRF.

In vivo micro-CT scanning could detect aortic calcification in half of the rats with CRF 4 weeks after CRF induction; at the end of the study and in agreement with total calcium measurement and Von Kossa staining, calcification could be demonstrated with this technique in all rats receiving an adenine low-protein diet. Figure 3 presents micro-CT data, including the calcified volume of the scanned aortic part, on the evolution of the development of calcification in the aorta of adenine-induced rats with CRF eating a low-protein diet. After 4 weeks of CRF, mild calcification was detected in the aorta; this dramatically increased to distinct and dense calcification after 6 and 8 weeks of CRF.

Figure3
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Figure 3. Onset and progression of calcification in the thoracic aorta (arrows) of a rat with CRF. In vivo micro-CT cross sections of the middle thoracic spine (between T4 and T7) demonstrate that treatment with an adenine-rich diet combined with low-protein content induced discernable aortic calcification after 4 weeks of CRF; this calcification progressed during further follow-up. The calcified volume of the scanned region is displayed in each panel and shows the degree of calcification at each point.

Expression of Osteochondrogenic Markers in the Aorta

Immunohistochemical analysis of aortic sections showed that the expression of the chondrocyte-specific proteins sox9 and collagen II and the osteochondrogenic transcription factor cbfa1 increased with the duration of CRF (Figure 4). After 4 weeks of CRF, when aortic calcification was observed in nearly all rats, almost all moderately to severely calcified (scores, 2 and 3, respectively) aortic cross sections abundantly expressed sox9. At the end of the study, when extensive calcification was observed in all animals, sox9, collagen II, and cbfa1 were highly expressed in more than 80% of the aortic cross sections. No or only limited expression of cbfa1 and collagen II was found in the aorta of animals with NRF. Unexpectedly, sox9 was expressed in the aorta of rats with NRF. However, the degree of protein expression in this study was based on the spatial extent (ie, tissue surface presenting positive staining) of the signal in the vessel wall without considering the differences in signal intensity. As shown in Figure 4, the expression of sox9 in rats with NRF was weak compared with the intensity of the signal in rats after 8 weeks of CRF. Fluorescent double staining for sox9 and cbfa1 revealed that cells expressing cbfa1 were located close to mineral deposits (Figure 5). These cbfa1-positive cells also frequently expressed sox9 (Figure 5, right panels). At a certain distance from the calcification, and often strikingly at the luminal side of it, cells abundantly expressed sox9.

Figure4
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Figure 4. Expression of cbfa1, sox9, and collagen II in the aortic tunica media of rats with CRF. The graphs show the percentage of aortic cross sections with a respective score reflecting the degree of protein expression for each chondrocyte/osteogenic marker. Abundant aortic expression of cbfa1, sox9, and collagen II after 4 and 8 weeks of CRF is present.

Figure5
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Figure 5. Fluorescent double staining for sox9 and cbfa1 expression in calcified aortas of rats after 4 weeks of CRF. Vascular cells in the media expressing cbfa1 are located close to the mineral, whereas sox9-positive cells are also found at regions farther from the calcified area (left panels, original magnification ×200). Coexpression of cbfa1 and sox9 in medial cells was also detected (right panels, original magnification ×640). L indicates lumen.

The mRNA expression of the chondrocyte-specific transcription factor sox9 in the aorta of rats with CRF was significantly increased after 4 and 8 weeks of CRF compared with rats with NRF (Figure 6A). In animals with CRF, a dramatic induction of the mRNA expression of the factor’s downstream target proteins collagen II and aggrecan was seen (Figure 6B and C). However, CRF did not significantly change aortic Indian hedgehog expression (Figure 6D). The expression of the osteochondrogenic transcription factor cbfa1 and its downstream target protein collagen I in the aorta of rats with CRF increased time dependently; however, no significant differences were observed between rats with CRF and rats with NRF (Figure 7A and B). In contrast, the osteoblast transcription factor osx was significantly downregulated after 3 and 4 weeks of CRF compared with NRF (Figure 7C).

Figure6
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Figure 6. A through D, mRNA expression of sox9 (A), collagen II (B), aggrecan (C), and Indian hedgehog (Ihh) (D) relative to GAPDH in the aorta of rats with CRF vs NRF. *P<0.05 vs rats with NRF.

Figure7
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Figure 7. A through G, mRNA expression of cbfa1 (A), collagen I (B), osx (C), Pit1 (D), Wnt4 (E), LRP6 (F), and β-catenin (G) relative to GAPDH in the aorta of rats with CRF vs NRF. *P<0.05 vs rats with NRF.

The expression of the sodium/inorganic phosphate cotransporter Pit1 mRNA in the aortic wall of rats with CRF rapidly and steadily increased with the duration of renal function impairment, reaching significance after 8 weeks of CRF compared with control aortas (Figure 7D). In the aorta of rats with CRF, no difference was observed in Wnt4 mRNA expression compared with rats with NRF (Figure 7E). However, the induction of CRF resulted in significantly reduced LRP6 and β-catenin mRNA expression in the aorta after 2, 3, and 4 weeks of CRF and after 3 and 4 weeks of CRF, respectively (Figure 7F and G).

Discussion

A previous study22 reported the presence of chondrocytelike cells in the aortas of uremic rats, based on immunohistochemical localization of sox9 and collagen II in the vicinity of medial calcification. These findings led us to the hypothesis that medial smooth muscle cells more likely transdifferentiate into chondrocytes than toward osteoblasts during medial calcification. To test this hypothesis, the mRNA and protein expressions of various segregation markers for both differentiation pathways were investigated in the aorta of rats with uremia-related medial calcifications. The abundant expression of chondrocyte-specific markers and the suppression of molecules involved in the osteoblast differentiation pathway in the aorta of rats with CRF during the initial phase of the calcification process confirm that chondrogenic transdifferentiation occurs in the vessel wall. Speer et al23 provided direct evidence that the osteochondrogenic cells found in calcifying arteries are derived from medial smooth muscle cells that undergo phenotypic reprogramming.

To study cell biological changes during the calcification process, a variant on the adenine-induced CRF model, as described by Price et al,24 was used. Previous studies21,22 in our laboratory demonstrated that the induction of CRF with an adenine-rich diet containing a normal protein content results in calcification of the aortic tunica media in approximately 50% of the animals after 4 to 6 weeks. However, lowering the protein content from 19% to 2.5% during the 4-week adenine treatment period resulted in a remarkably increased prevalence of severe medial calcifications up to 100% in rats with CRF. In the present study, 12 of 14 animals eating an adenine low-protein diet and euthanized at week 4 already had developed moderate-to-severe calcifications in the aortic media. Eight weeks after the induction of CRF, all rats eating a low-protein diet exhibited extensive medial calcifications in the aorta. Remarkably, the combination of an adenine-enriched diet with a low protein content induced more pronounced hyperphosphatemia than the standard adenine diet (23.8±3.1 mg/dL versus 9.2±1.8 mg/dL22 after 4 weeks of CRF). To keep the total phosphorus concentration in the adenine low-protein diet equal to that of the standard adenine diet (0.92% phosphorus), phosphorus under the form of anorganic phosphate, such as monocalcium phosphate, was added to the animal food. The bioavailability of this phosphorus source is much higher compared with that of protein-bound phosphorus, which explains the higher serum phosphate concentrations during adenine feeding in rats receiving a low-protein diet compared with those receiving a standard protein adenine diet. Moreover, casein is used as a phosphorus protein source in the synthetic low-protein diet, whereas the standard protein diet is a grain-based diet in which approximately half of the phosphorus is present under the form of phytic acid, a poorly bioavailable source of phosphorus.25 As recently reported by Moe et al,26 rats fed a casein-based diet showed higher serum and urinary phosphate concentrations than those fed a grain-based diet, with equivalent total phosphorus content. The prominent hyperphosphatemia most probably explains the consistent development of severe medial calcification in rats receiving an adenine low-protein diet. However, the possibility of the lack of certain calcification inhibitors in the low-protein diet cannot be excluded.

The expression of the type III sodium/inorganic phosphate cotransporter Pit1 mRNA increased with the duration of CRF and was significantly elevated after 8 weeks compared with expression in rats with NRF. Mizobuchi et al27 also found elevated Pit1 mRNA in the calcified aorta of uremic rats. Pit1 is expressed in a wide variety of tissues and cell types, such as osteoblasts, chondrocytes, and vascular smooth muscle cells.28–30 The incubation of cultured arterial smooth muscle cells with high phosphate concentrations induced mineralization and transdifferentiation toward an osteochondrogenic phenotype with expression of cbfa1, osteocalcin, and osteopontin.9 Blocking phosphate uptake in Pit1 knockdown cells inhibited the induction of cbfa1 and osteopontin and decreased calcification of vascular smooth muscle cells exposed to high phosphate concentrations,31 indicating that cellular uptake of phosphate through Pit1 is involved in osteochondrogenic transdifferentiation of medial smooth muscle cells and the subsequent mineralization process. In our experimental study, the high circulating phosphate levels are probably responsible for the distinct upregulation of Pit1 in the aortic vessel wall. High intracellular phosphate accumulation will then contribute to the phenotypic change of arterial smooth muscle cells preceding the calcification process.32

A time-dependent increased expression of the chondrocyte-specific transcription factor sox9 and its downstream matrix protein collagen II was found in immunohistochemically stained aortic sections of rats from the beginning of CRF induction onward, thus before the initiation of the calcification process in the media. Slight basal sox9 expression was also noted in the aortas of rats with NRF, probably reflecting the presence of mesenchymal progenitor cells with multilineage potential, as reported by Tintut and colleagues.33 During limb bud development, multipotent mesenchymal cells that give rise to both chondrocytes and osteoblasts express sox9 before mesenchymal condensation occurs.34 In addition to immunohistochemical observations, RT-PCR analysis of the abdominal aorta of rats with CRF revealed that sox9 and aggrecan mRNA expression increased with the duration of CRF and that collagen II mRNA was also highly induced after 8 weeks of CRF compared with rats with NRF. Similar trends in mRNA and protein expression of the chondrocyte transcription factor sox9 and strong induction of collagen II and aggrecan, important structural components of cartilage tissue, confirm the presence of chondrocytelike cells and cartilage metaplasia, providing further evidence for the involvement of endochondral mineralization processes during medial calcification. In contrast, Indian hedgehog expression did not change in the aortas of rats with CRF. This protein is secreted by prehypertrophic chondrocytes; together with parathyroid hormone–related peptide, it coordinates the chondrocyte proliferation and differentiation rate in the growth plate.35 Although chondrocyte differentiation and cartilage formation occur during media calcification, it is clear that this pathological process is similar, but not quite identical, to the physiological process occurring in the growth plate during endochondral bone formation.

CRF time dependently and markedly induced cbfa1 protein expression; however, no significant changes in the aortic expression of cbfa1 mRNA were found compared with rats with NRF. This is in agreement with the findings of Lomashvili et al,36 who did not find significant upregulation of cbfa1 mRNA in cultured rat aortas under calcifying conditions. As previously reported,37 cbfa1 expression is predominantly regulated at the translational level by a ubiquitin- or proteosome-mediated protein degradation mechanism controlled by E3 ligases, such as Smurf1,38 C-terminus of Hsc70-interacting protein (CHIP),39 and WW domain containing E3 ubiquitin protein ligase (WWP)-1/Schnurri-3,40 which likely explains the incongruity between cbfa1 transcripts and protein levels observed immunohistochemically and suggests that the degradation of cbfa1 transcripts is inhibited in the aortas of rats with CRF. Cbfa1 mRNA is constitutively expressed in osteoblasts and in osteoblast precursors and fibroblasts, whereas cbfa1 protein could only be detected in differentiated osteoblasts.37,38 The aortic expression of cbfa1 protein also went along with the early stages of calcification in uremic rats receiving high PTH infusion41 and in uremic mice receiving a high phosphorus diet.42 Furthermore, the expression of osteochondrogenic transcription factor cbfa1 is not restricted to osteoblasts. Indeed, cbfa1 is also expressed in hypertrophic chondrocytes and in the common osteochondroprogenitor cells that still can differentiate to either osteoblasts or chondrocytes.43 Concerning the spatial relationship of sox9 and cbfa1 with the mineral, cbfa1 expression was mainly located close to the mineral. These cells were also often, although not consistently, positive for sox9. The expression of sox9 was more widespread and more extended to a certain distance from the mineral, preferentially toward the luminal side, compared with cbfa1 expression. These results, together with the early upregulation of sox9 preceding cbfa1 expression, suggest that chondrocyte differentiation is the first step in the cellular mechanism initiating medial calcification. In a further differentiation stage, vascular cells start to express cbfa1, which is probably the cellular signal to activate the mineralization process.

Somewhat unexpectedly, the induction of CRF resulted in significantly diminished expression of the osteoblast-specific transcription factor osx in the aorta after 3 and 4 weeks. Interestingly, a similar reduced expression pattern was simultaneously noted in the β-catenin signaling pathway. The mRNA expression of both LRP6, the receptor on which Wnt proteins bind, and β-catenin was significantly decreased in the aorta at 3 and 4 weeks after CRF induction compared with aortic expression in rats with NRF, pointing toward an inhibition of the Wnt/β-catenin signaling pathway. The reduced expression of osx, LRP6, and β-catenin in the aorta of rats with uremia-related medial calcifications suggests that the transdifferentiation pathway of arterial smooth muscle cells toward osteoblastlike cells is suppressed15,44 and further supports our hypothesis that medial smooth muscle cells acquire a chondrocyte rather than an osteoblast phenotype during the calcification process. Chondrogenic differentiation of aortic smooth muscle cells was also detected in mice lacking matrix γ-carboxyglutamic acid (Gla) protein, which typically develop medial calcifications, as indicated by the induction of collagen II expression45 and the lack of expression of Msh homebox (msx)-2, osx, Wnt3a, and Wnt7a,23 factors promoting osteoblast differentiation. Furthermore, our results are in line with the recent results of Roman-Garcia et al,46 who found a significant induction of secreted frizzled related proteins 1, 2, and 4, inhibitors of the Wnt/β-catenin signaling pathway, in the calcified aorta of 7/8 nephrectomized rats. Our observation of chondrocyte-specific proteins induced in arteries from transplant donors with medial calcification22 and the finding of cartilage metaplasia in lower-extremity arteries with extensive intima and medial calcifications of 2 diabetic patients with renal failure47 suggest that chondrocyte differentiation is a more general mechanism underlying medial calcification, which also occurs in humans. However, in diabetic low-density lipoprotein receptor knockout mice, high circulating concentrations of tumor necrosis factor α induced the expression of the osteoblast differentiation markers msx2, Wnt3a, and Wnt7a in aortas with atherosclerotic and medial calcification.48 These findings suggest that the segregation between osteoblast and chondrocyte differentiation from vascular smooth muscle cells depends on local/circulating stimuli. To find out whether the conversion of vascular cells toward osteochondrogenic cells can be stopped and, with regard to the outcome in patients with CKD and other populations at risk, whether inhibition of this process modulates the degree of calcification, it would be of particular interest to continue research on the molecular pathways and their triggers underlying medial calcification.

In summary, the induction of chondrocyte-specific markers sox9, aggrecan, and collagen II and the simultaneous downregulation of osx and β-catenin, molecules involved in the osteoblast differentiation pathway, in the aorta of uremic rats indicate that a transdifferentiation of vascular smooth muscle cells toward chondrocytes rather than osteoblasts underlies the calcification process in the vessel wall. This study contributes to a better understanding of the cell biological mechanism initiating mineralization in the vascular media. Unraveling step by step the molecular and cellular processes in this pathology may lead to new predictive markers of the calcification process and to novel targets for intervention.

Acknowledgments

We especially thank N. De Clerck, PhD, and A. Postnov, PhD, for the analysis of the micro-CT data; Caroline Verstraete, MS, Antwerp University Hospital, for the biochemical analysis; Geert Dams, BS, Hilde Geryl, BS, and Rita Marynissen, BS, for their excellent technical assistance; and Dirk De Weerdt, MS, for his help with the graphics.

Sources of Funding

This study was supported by a doctoral grant from the Institute for the Promotion of Innovation through Science and Technology, Flanders (Dr Neven). Dr Persy was a Postdoctoral Fellow of the Fund for Scientific Research–Flanders.

Disclosures

None.

Footnotes

  • Received on: February 15, 2010; final version accepted on: May 18, 2010.

References

  1. ↵
    US Renal Data System: USRDS Annual Data Report. Bethesda, MD: National Institutes of Health National Institute of Diabetes, Digestive, Kidney Diseases; 1998.
  2. ↵
    Goodman WG, Goldin J, Kuizon BD, Yoon C, Gales B, Sider D, Wang Y, Chung J, Emerick A, Greaser L, Elashoff RM, Salusky IB. Coronary-artery calcification in young adults with end-stage renal disease who are undergoing dialysis. N Engl J Med. 2000; 342: 1478–1483.
    OpenUrlCrossRefPubMed
  3. ↵
    London GM, Guerin AP, Marchais SJ, Metivier F, Pannier B, Adda H. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant. 2003; 18: 1731–1740.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Deneke T, Langner K, Grewe PH, Harrer E, Muller KM. Ossification in atherosclerotic carotid arteries. Z Kardiol. 2001; 90 (suppl 3): 106–115.
    OpenUrlPubMed
  5. ↵
    Bostrom K. Insights into the mechanism of vascular calcification. Am J Cardiol. 2001; 88: 20E–22E.
    OpenUrlPubMed
  6. ↵
    Chen NX, Duan D, O'Neill KD, Wolisi GO, Koczman JJ, Laclair R, Moe SM. The mechanisms of uremic serum-induced expression of bone matrix proteins in bovine vascular smooth muscle cells. Kidney Int. 2006; 70: 1046–1053.
    OpenUrlCrossRefPubMed
  7. ↵
    Bear M, Butcher M, Shaughnessy SG. Oxidized low-density lipoprotein acts synergistically with beta-glycerophosphate to induce osteoblast differentiation in primary cultures of vascular smooth muscle cells. J Cell Biochem. 2008; 105: 185–193.
    OpenUrlCrossRefPubMed
  8. ↵
    Chen NX, O'Neill KD, Duan D, Moe SM. Phosphorus and uremic serum up-regulate osteopontin expression in vascular smooth muscle cells. Kidney Int. 2002; 62: 1724–1731.
    OpenUrlCrossRefPubMed
  9. ↵
    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.
    OpenUrlCrossRefPubMed
  10. ↵
    Tintut Y, Patel J, Parhami F, Demer LL. Tumor necrosis factor-alpha promotes in vitro calcification of vascular cells via the cAMP pathway. Circulation. 2000; 102: 2636–2642.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell. 2002; 108: 17–29.
    OpenUrlCrossRefPubMed
  12. ↵
    Akiyama H, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev. 2002; 16: 2813–2828.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Hill TP, Spater D, Taketo MM, Birchmeier W, Hartmann C. Canonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell. 2005; 8: 727–738.
    OpenUrlCrossRefPubMed
  14. ↵
    Lustig B, Behrens J. The Wnt signaling pathway and its role in tumor development. J Cancer Res Clin Oncol. 2003; 129: 199–221.
    OpenUrlPubMed
  15. ↵
    Day TF, Yang Y. Wnt and hedgehog signaling pathways in bone development. J Bone Joint Surg Am. 2008; 90 (suppl 1): 19–24.
    OpenUrlCrossRefPubMed
  16. ↵
    Day TF, Guo X, Garrett-Beal L, Yang Y. Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev Cell. 2005; 8: 739–750.
    OpenUrlCrossRefPubMed
  17. ↵
    Kato M, Patel MS, Levasseur R, Lobov I, Chang BH, Glass DA, Hartmann C, Li L, Hwang TH, Brayton CF, Lang RA, Karsenty G, Chan L. Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. J Cell Biol. 2002; 157: 303–314.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Bodine PV, Zhao W, Kharode YP, Bex FJ, Lambert AJ, Goad MB, Gaur T, Stein GS, Lian JB, Komm BS. The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice. Mol Endocrinol. 2004; 18: 1222–1237.
    OpenUrlCrossRefPubMed
  19. ↵
    Zhu M, Tang D, Wu Q, Hao S, Chen M, Xie C, Rosier RN, O'Keefe RJ, Zuscik M, Chen D. Activation of beta-catenin signaling in articular chondrocytes leads to osteoarthritis-like phenotype in adult beta-catenin conditional activation mice. J Bone Miner Res. 2009; 24: 12–21.
    OpenUrlCrossRefPubMed
  20. ↵
    Topol L, Chen W, Song H, Day TF, Yang Y. Sox9 inhibits Wnt signaling by promoting beta-catenin phosphorylation in the nucleus. J Biol Chem. 2009; 284: 3323–3333.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Persy V, Postnov A, Neven E, Dams G, De BM, D'Haese P, De Clerck N. High-resolution X-ray microtomography is a sensitive method to detect vascular calcification in living rats with chronic renal failure. Arterioscler Thromb Vasc Biol. 2006; 26: 2110–2116.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Neven E, Dauwe S, De Broe ME, D'Haese PC, Persy V. Endochondral bone formation is involved in media calcification in rats and in men. Kidney Int. 2007; 72: 574–581.
    OpenUrlCrossRefPubMed
  23. ↵
    Speer MY, Yang HY, Brabb T, Leaf E, Look A, Lin WL, Frutkin A, Dichek D, Giachelli CM. Smooth muscle cells give rise to osteochondrogenic precursors and chondrocytes in calcifying arteries. Circ Res. 2009; 104: 733–741.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Price PA, Roublick AM, Williamson MK. Artery calcification in uremic rats is increased by a low protein diet and prevented by treatment with ibandronate. Kidney Int. 2006; 70: 1577–1583.
    OpenUrlCrossRefPubMed
  25. ↵
    Pallauf J, Rimbach G. Nutritional significance of phytic acid and phytase. Arch Tierernahr. 1997; 50: 301–319.
    OpenUrlCrossRefPubMed
  26. ↵
    Moe SM, Chen NX, Seifert MF, Sinders RM, Duan D, Chen X, Liang Y, Radcliff JS, White KE, Gattone VH. A rat model of chronic kidney disease-mineral bone disorder. Kidney Int. 2009; 75: 176–184.
    OpenUrlCrossRefPubMed
  27. ↵
    Mizobuchi M, Ogata H, Hatamura I, Koiwa F, Saji F, Shiizaki K, Negi S, Kinugasa E, Ooshima A, Koshikawa S, Akizawa T. Up-regulation of Cbfa1 and Pit-1 in calcified artery of uraemic rats with severe hyperphosphataemia and secondary hyperparathyroidism. Nephrol Dial Transplant. 2006; 21: 911–916.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Boyer CJ, Baines AD, Beaulieu E, Beliveau R. Immunodetection of a type III sodium-dependent phosphate cotransporter in tissues and OK cells. Biochim Biophys Acta. 1998; 1368: 73–83.
    OpenUrlPubMed
  29. ↵
    Palmer G, Guicheux J, Bonjour JP, Caverzasio J. Transforming growth factor-beta stimulates inorganic phosphate transport and expression of the type III phosphate transporter Glvr-1 in chondrogenic ATDC5 cells. Endocrinology. 2000; 141: 2236–2243.
    OpenUrlCrossRefPubMed
  30. ↵
    Kakita A, Suzuki A, Nishiwaki K, Ono Y, Kotake M, Ariyoshi Y, Miura Y, Ltoh M, Oiso Y. Stimulation of Na-dependent phosphate transport by platelet-derived growth factor in rat aortic smooth muscle cells. Atherosclerosis. 2004; 174: 17–24.
    OpenUrlCrossRefPubMed
  31. ↵
    Li X, Yang HY, Giachelli CM. Role of the sodium-dependent phosphate cotransporter, Pit-1, in vascular smooth muscle cell calcification. Circ Res. 2006; 98: 905–912.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Giachelli CM. Vascular calcification: in vitro evidence for the role of inorganic phosphate. J Am Soc Nephrol. 2003; 14: S300–S304.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    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.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Akiyama H, Kim JE, Nakashima K, Balmes G, Iwai N, Deng JM, Zhang Z, Martin JF, Behringer RR, Nakamura T, de Crombrugghe B. Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors. Proc Natl Acad Sci U S A. 2005; 102: 14665–14670.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science. 1996; 273: 613–622.
    OpenUrlAbstract
  36. ↵
    Lomashvili KA, Monier-Faugere MC, Wang X, Malluche HH, O'Neill WC. Effect of bisphosphonates on vascular calcification and bone metabolism in experimental renal failure. Kidney Int. 2009; 75: 617–625.
    OpenUrlCrossRefPubMed
  37. ↵
    Sudhakar S, Li Y, Katz MS, Elango N. Translational regulation is a control point in RUNX2/Cbfa1 gene expression. Biochem Biophys Res Commun. 2001; 289: 616–622.
    OpenUrlCrossRefPubMed
  38. ↵
    Zhao M, Qiao M, Oyajobi BO, Mundy GR, Chen D. E3 ubiquitin ligase Smurf1 mediates core-binding factor alpha1/Runx2 degradation and plays a specific role in osteoblast differentiation. J Biol Chem. 2003; 278: 27939–27944.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Li X, Huang M, Zheng H, Wang Y, Ren F, Shang Y, Zhai Y, Irwin DM, Shi Y, Chen D, Chang Z. CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation. J Cell Biol. 2008; 181: 959–972.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Jones DC, Wein MN, Glimcher LH. Schnurri-3 is an essential regulator of osteoblast function and adult bone mass. Ann Rheum Dis. 2007; 66 (suppl 3): iii49–iii51.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Graciolli FG, Neves KR, dos Reis LM, Graciolli RG, Noronha IL, Moyses RM, Jorgetti V. Phosphorus overload and PTH induce aortic expression of Runx2 in experimental uraemia. Nephrol Dial Transplant. 2009; 24: 1416–1421.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    El-Abbadi MM, Pai AS, Leaf EM, Yang HY, Bartley BA, Quan KK, Ingalls CM, Liao HW, Giachelli CM. Phosphate feeding induces arterial medial calcification in uremic mice: role of serum phosphorus, fibroblast growth factor-23, and osteopontin. Kidney Int. 2009; 75: 1297–1307.
    OpenUrlCrossRefPubMed
  43. ↵
    Tchetina E, Mwale F, Poole AR. Distinct phases of coordinated early and late gene expression in growth plate chondrocytes in relationship to cell proliferation, matrix assembly, remodeling, and cell differentiation. J Bone Miner Res. 2003; 18: 844–851.
    OpenUrlCrossRefPubMed
  44. ↵
    Nakashima K, de Crombrugghe B. Transcriptional mechanisms in osteoblast differentiation and bone formation. Trends Genet. 2003; 19: 458–466.
    OpenUrlCrossRefPubMed
  45. ↵
    Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer RR, Karsenty G. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature. 1997; 386: 78–81.
    OpenUrlCrossRefPubMed
  46. ↵
    Roman-Garcia P, Carrillo-Lopez N, Fernandez-Martin JL, Naves-Diaz M, Ruiz-Torres MP, Cannata-Andia JB. High phosphorus diet induces vascular calcification, a related decrease in bone mass and changes in the aortic gene expression. Bone. 2010; 46: 121–128.
    OpenUrlCrossRefPubMed
  47. ↵
    Qiao JH, Mertens RB, Fishbein MC, Geller SA. Cartilaginous metaplasia in calcified diabetic peripheral vascular disease: morphologic evidence of enchondral ossification. Hum Pathol. 2003; 34: 402–407.
    OpenUrlCrossRefPubMed
  48. ↵
    Al-Aly Z, Shao JS, Lai CF, Huang E, Cai J, Behrmann A, Cheng SL, Towler DA. Aortic Msx2-Wnt calcification cascade is regulated by TNF-alpha-dependent signals in diabetic Ldlr-/- mice. Arterioscler Thromb Vasc Biol. 2007; 27: 2589–2596.
    OpenUrlAbstract/FREE Full Text
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    Chondrocyte Rather Than Osteoblast Conversion of Vascular Cells Underlies Medial Calcification in Uremic Rats
    Ellen Neven, Veerle Persy, Simonne Dauwe, Tineke De Schutter, Marc E. De Broe and Patrick C. D'Haese
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2010;30:1741-1750, originally published August 18, 2010
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    Ellen Neven, Veerle Persy, Simonne Dauwe, Tineke De Schutter, Marc E. De Broe and Patrick C. D'Haese
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2010;30:1741-1750, originally published August 18, 2010
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