Magnesium reduces vascular smooth muscle cell (VSMC) calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a) whether inhibition of magnesium transport into the cell influences VSMC calcification, b) whether Wnt/β-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c) whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM) and moderately elevated magnesium (1.4 mM) significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP) and osteoprotegerin (OPG). The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]). High phosphate induced activation of Wnt/β-catenin pathway as demonstrated by the translocation of β-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/β-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/β-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/β-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro. Inhibition of Wnt/β-catenin by magnesium is one potential intracellular mechanism by which this anti-calcifying effect is achieved.

Hyperphosphatemia is closely related to vascular calcification in patients with chronic kidney disease. Vascular smooth muscle cells (VSMCs) exposed to high phosphate concentrations in vitro undergo phenotypic transition to osteoblast‐like cells. Mechanisms underlying this transdifferentiation are not clear. In this study we used two in vitro models, human aortic smooth muscle cells and rat aortic rings, to investigate the phenotypic transition of VSMCs induced by high phosphate. We found that high phosphate concentration (3.3 mmol/L) in the medium was associated with increased DNA methyltransferase activity and methylation of the promoter region of SM22α. This was accompanied by loss of the smooth muscle cell–specific protein SM22α, gain of the osteoblast transcription factor Cbfa1, and increased alkaline phosphatase activity with the subsequent in vitro calcification. The addition of a demethylating agent (procaine) to the high‐phosphate medium reduced DNA methyltransferase activity and prevented methylation of the SM22α promoter, which was accompanied by an increase in SM22α expression and less calcification. Additionally, downregulation of SM22α, either by siRNA or by a methyl group donor (S‐adenosyl methionine), resulted in overexpression of Cbfa1. In conclusion, we demonstrate that methylation of SM22α promoter is an important event in vascular smooth muscle cell calcification and that high phosphate induces this epigenetic modification. These findings uncover a new insight into mechanisms by which high phosphate concentration promotes vascular calcification. © 2010 American Society for Bone and Mineral Research

Cardiovascular disease is a major cause of mortality in chronic kidney disease patients. Indoxyl sulfate (IS) is a typical protein-bound uremic toxin that cannot be effectively cleared by conventional dialysis. Increased IS is associated with the progression of chronic kidney disease and development of cardiovascular disease. After endothelial activation by IS, cells release endothelial microvesicles (EMV) that can induce endothelial dysfunction. We developed an in vitro model of endothelial damage mediated by IS to evaluate the functional effect of EMV on the endothelial repair process developed by endothelial progenitor cells (EPCs). EMV derived from IS-treated endothelial cells were isolated by ultracentrifugation and characterized for miRNAs content. The effects of EMV on healthy EPCs in culture were studied. We observed that IS activates endothelial cells and the generated microvesicles (IsEMV) can modulate the classic endothelial roles of progenitor cells as colony forming units and form new vessels in vitro. Moreover, 23 miRNAs were contained in IsEMV including four (miR-181a-5p, miR-4454, miR-150-5p, and hsa-let-7i-5p) that were upregulated in IsEMV compared with control endothelial microvesicles. Other authors have found that miR-181a-5p, miR-4454, and miR-150-5p are involved in promoting inflammation, apoptosis, and cellular senescence. Interestingly, we observed an increase in NFκB and p53, and a decrease in IκBα in EPCs treated with IsEMV. Our data suggest that IS is capable of inducing endothelial vesiculation with different membrane characteristics, miRNAs and other molecules, which makes maintaining of vascular homeostasis of EPCs not fully functional. These specific characteristics of EMV could be used as novel biomarkers for diagnosis and prognosis of vascular disease.

Transforming growth factor-β (TGF-β) is a key cytokine during differentiation of mesenchymal stem cells (MSC) into vascular smooth muscle cells (VSMC). High phosphate induces a phenotypic transformation of vascular smooth muscle cells (VSMC) into osteogenic-like cells. This study was aimed to evaluate signaling pathways involved during VSMC differentiation of MSC in presence or not of high phosphate. Our results showed that TGF-β induced nuclear translocation of Smad3 as well as the expression of vascular smooth muscle markers, such as smooth muscle alpha actin, SM22α, myocardin, and smooth muscle-myosin heavy chain. The addition of high phosphate to MSC promoted nuclear translocation of Smad1/5/8 and the activation of canonical Wnt/β-catenin in addition to an increase in BMP-2 expression, calcium deposition and alkaline phosphatase activity. The administration of TGF-β to MSC treated with high phosphate abolished all these effects by inhibiting canonical Wnt, BMP and TGF-β pathways. A similar outcome was observed in high phosphate-treated cells after the inhibition of canonical Wnt signaling with Dkk-1. Conversely, addition of both Wnt/β-catenin activators CHIR98014 and lithium chloride enhanced the effect of high phosphate on BMP-2, calcium deposition and alkaline phosphatase activity. Full VSMC differentiation induced by TGF-β may not be achieved when extracellular phosphate levels are high. Moreover, TGF-β prevents high phosphate-induced osteogenesis by decreasing the nuclear translocation of Smad 1/5/8 and avoiding the activation of Wnt/β-catenin pathway.