Combined effects of ascorbic acid and phosphate on rat VSMC osteoblastic differentiation

Ciceri, Paola; Volpi, Elisa; Brenna, Irene; Arnaboldi, Lorenzo; Neri, Luca; Brancaccio, Diego; Cozzolino, Mario

doi:10.1093/ndt/gfr284

Cited by 46 publications

(34 citation statements)

References 24 publications

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“…Previous studies have shown that VSMCs cultured in high concentrations of phosphate undergo osteogenic transformation and calcification [27,28], and the role of BMPs in the regulation of osteoblastic differentiation of VSMCs and VC has been studied extensively [2,29]. In the present study, we showed that exposure of VSMCs to high phosphate increased the expression of BMP-2 and calcium deposition, and induced VSMC apoptosis in parallel with the differentiation into an osteoblastic phenotype.…”

Section: Discussionsupporting

confidence: 68%

Vascular Calcification in Chronic Kidney Disease is Induced by Bone Morphogenetic Protein-2 via a Mechanism Involving the Wnt/�-Catenin Pathway

Shu

Zhao

Jin

et al. 2014

Cell Physiol Biochem

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Background: Vascular calcification (VC), in which vascular smooth muscle cells (VSMCs) undergo a phenotypic transformation into osteoblast-like cells, is one of the emergent risk factors for the accelerated atherosclerosis process characteristic of chronic kidney disease (CKD). Phosphate is an important regulator of VC. Methods: The expression of different smooth muscle cell or osteogenesis markers in response to high concentrations of phosphate or exogenous bone morphogenetic protein 2 (BMP-2) was examined by qRT-PCR and western blotting in rat VSMCs. Osteocalcin secretion was measured by radioimmunoassay. Differentiation and calcification of VSMCs were examined by alkaline phosphatase (ALP) activity assay and Alizarin staining. Short hairpin RNA-mediated silencing of β-catenin was performed to examine the involvement of Wnt/β-catenin signaling in VSMC calcification and osteoblastic differentiation induced by high phosphate or BMP-2. Apoptosis was determined by TUNEL assay and immunofluorescence imaging. Results: BMP-2 serum levels were significantly higher in CKD patients than in controls. High phosphate concentrations and BMP-2 induced VSMC apoptosis and upregulated the expression of β-catenin, Msx2, Runx2 and the phosphate cotransporter Pit1, whereas a BMP-2 neutralization antibody reversed these effects. Knockdown of β-catenin abolished the effect of high phosphate and BMP-2 on VSMC apoptosis and calcification. Conclusions: BMP-2 plays a crucial role in calcium deposition in VSMCs and VC in CKD patients via a mechanism involving the Wnt/β-catenin pathway.

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Section: Discussionsupporting

confidence: 68%

Vascular Calcification in Chronic Kidney Disease is Induced by Bone Morphogenetic Protein-2 via a Mechanism Involving the Wnt/�-Catenin Pathway

Shu

Zhao

Jin

et al. 2014

Cell Physiol Biochem

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“…VSMCs were isolated from the descending thoracic aortas of rats by enzymatic digestion, as described elsewhere [13]. Cells were grown in monolayers at 37°C in a humidified atmosphere of CO 2 in Dulbecco's modiﬁed Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 0.1 mg/ml streptomycin, 20 m M Tricine buffer, and 1% non-essential amino acid solution.…”

Section: Methodsmentioning

confidence: 99%

High Phosphorus Level Leads to Aortic Calcification via β-Catenin in Chronic Kidney Disease

Sun

et al. 2015

Am J Nephrol

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Aims: Vascular calcification is a risk factor for causing cardiovascular events and has a high prevalence among chronic kidney disease (CKD) patients. However, the molecular mechanism underlying this pathogenic process is still obscure. Methods: Vascular smooth muscle cells (VSMCs) were induced by a concentration of phosphorus (Pi) of 2.5 mM, and were subjected to cell calcification analyses. The effect of high Pi on the Wnt/β-catenin pathway was measured using a TOP/FOP-Flash reporter assay. The transcriptional regulation of β-catenin on PIT1 (a type III sodium-dependent phosphate cotransporter) was confirmed by promoter reporter and chromatin immunoprecipitation assays. The 5/6 nephrectomized rat was used as an in vivo model and was fed a high Pi diet to induce aortic calcification. Serum levels of phosphate, calcium, creatine, and blood urea nitrogen were measured, and abdominal aortic calcification was examined. Results: High Pi induced VSMC calcification, downregulated expression levels of VSMC markers, and upregulated levels of osteogenic markers. High Pi activated the Wnt/β-catenin pathway and β-catenin activity. β-Catenin was involved in the process of high Pi-induced VSMC calcification. Further investigation revealed that β-catenin transcriptionally regulated Pit1, a necessary player in VSMC osteogenic phenotype change and calcification. The in vivo study showed that β-catenin was involved in rat abdominal aortic calcification induced by high Pi. When knockdown expression of β-catenin in the rat model was investigated, we found that aortic calcification was reduced. Conclusion: These results suggest that β-catenin is an important player in high phosphorus level-induced aortic calcification in CKD.

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“…Rat VSMCs were obtained by enzymatic digestion as previously described [6], grown in monolayers at 37°C in humidified atmosphere of CO 2 , and routinely subcultured in growth medium (DMEM containing 10% FBS supplemented with 100 U·ml -1 penicillin, 0.1 mg·ml -1 streptomycin). At 80% confluence, the cells were switched to calcification medium (DMEM containing 12% FBS, 10 m M sodium pyruvate and supplemented with 5 m M Pi and 50 μg/ml AA, 100 U·ml -1 penicillin and 0.1 mg·ml -1 streptomycin) for up to 8 days.…”

Section: Methodsmentioning

confidence: 99%

“…It has been well elucidated that vascular smooth muscle cells (VSMCs) are a reproducible in vitro model to investigate the cellular and molecular mechanisms involved in the physiopathology of VC [6]. In fact, high Pi levels in culture media not only cause calcium deposition in VSMCs, but also induce a phenotypical change towards osteoblastic-like cells [7].…”

Section: Introductionmentioning

confidence: 99%

The Calcimimetic Calindol Prevents High Phosphate-Induced Vascular Calcification by Upregulating Matrix GLA Protein

Ciceri

Elli

Brenna

et al. 2013

Nephron Exp Nephrol

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Background: High serum phosphate (Pi) levels represent a major issue in dialysis patients, because associate with secondary hyperparathyroidism, vascular calcification (VC), and cardiovascular outcomes. In this population, calcimimetics are used to control secondary hyperparathyroidism, hyperphosphatemia, and, more recently, to delay the progression of VC. The aim of this in vitro study was to investigate the direct effects of the calcimimetic calindol on the progression of high Pi-induced VC. Methods: Rat vascular smooth muscle cells (VSMCs) were incubated with high Pi concentrations, and the effects of calindol were investigated on vascular calcium deposition and VSMC osteoblastic differentiation. Results: Calindol inhibited calcium deposition concentration-dependently with a maximal inhibition of 64.0 ± 5.2% achieved at 100 nM. Furthermore, calindol was able to partially prevent the high Pi-induced bone morphogenic protein 2 (BMP-2) expression upregulation (32.4 ± 4.6% of inhibition; p < 0.01). Interestingly, the pretreatment with calindol enhanced the matrix Gla protein (MGP) gene expression significantly, compared to high Pi-treated cells (40.2 ± 6.6% of increase, p < 0.01). Conclusions: In conclusion, we demonstrated that the calcimimetic calindol prevents high Pi-induced VC by affecting osteoblastic differentiation in vitro. In particular, the inhibitory effect of calindol on VC is probably due to its stimulatory role on the calcium-sensing receptor, leading to an increase in the synthesis of MGP by VSMCs.

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Combined effects of ascorbic acid and phosphate on rat VSMC osteoblastic differentiation

Cited by 46 publications

References 24 publications

Vascular Calcification in Chronic Kidney Disease is Induced by Bone Morphogenetic Protein-2 via a Mechanism Involving the Wnt/�-Catenin Pathway

Vascular Calcification in Chronic Kidney Disease is Induced by Bone Morphogenetic Protein-2 via a Mechanism Involving the Wnt/�-Catenin Pathway

High Phosphorus Level Leads to Aortic Calcification via β-Catenin in Chronic Kidney Disease

The Calcimimetic Calindol Prevents High Phosphate-Induced Vascular Calcification by Upregulating Matrix GLA Protein

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