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

Methods

Results

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.

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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.

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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.

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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.

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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.

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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).

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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.

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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 studies^21,22 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 suppressed^15,44 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.