Clinical and epidemiologic studies reveal an association between vitamin D deficiency and increased risk of cardiovascular disease. Because vascular smooth muscle cell (VSMC)‐derived tissue factor ...(TF) is suggested to be critical for arterial thrombosis, we investigated whether the vitamin D molecules calcitriol and paricalcitol could reduce the expression of TF induced by the proinflammatory cytokine TNF‐α in human aortic VSMCs. We found that, compared with controls, incubation with TNF‐α increased TF expression and procoagulant activity in a NF‐κB‐dependent manner, as deduced from the increased nuclear translocation of nuclear factor κ‐light‐chain‐enhancer of activated B cells protein 65 (p65‐NF‐κB) and direct interaction of NF‐kB to the TF promoter. This was accompanied by the up‐regulation of TF signaling mediator protease‐activated receptor 2 (PAR‐2) expression and by the down‐regulation of vitamin D receptor expression in a miR‐346‐dependent way. However, addition of calcitriol or paricalcitol blunted the TNF‐α‐induced TF expression and activity (2.01 ± 0.24 and 1.32 ± 0.14 vs. 3.02 ± 0.39 pmol/mg protein, P < 0.05), which was associated with down‐regulation of NF‐kB signaling and PAR‐2 expression, as well as with restored levels of vitamin D receptor and enhanced expression of TF pathway inhibitor. Our data suggest that inflammation promotes a prothrombotic state through the up‐regulation of TF function in VSMCs and that the beneficial cardiovascular effects of vitamin D may be partially due to decreases in TF expression and its activity in VSMCs.—Martinez‐Moreno, J. M., Herencia, C., Montes de Oca, A., Muñoz‐Castañeda, J. R., Rodríguez‐Ortiz, M. E., Díaz‐Tocados, J. M., Peralbo‐Santaella, E., Camargo, A., Canalejo, A., Rodriguez, M., Velasco‐Gimena, F., Almaden, Y., Vitamin D modulates tissue factor and protease‐activated receptor 2 expression in vascular smooth muscle cells. FASEB J. 30, 1367–1376 (2016). www.fasebj.org
In chronic kidney disease patients, high phosphate (HP) levels are associated with cardiovascular disease, the major cause of morbidity and mortality. Since serum phosphate has been independently ...correlated with inflammation, the present study aimed to investigate an independent direct effect of HP as a pro-inflammatory factor in VSMCs. A possible modulatory effect of vitamin D (VitD) was also investigated. The study was performed in an
model of human aortic smooth muscle cells (HASMCs). Incubation of cells in an HP (3.3 mM) medium caused an increased expression of the pro-inflammatory mediators intercellular adhesion molecule 1 (ICAM-1), interleukins (ILs) IL-1β, IL-6, IL-8 and tumour necrosis factor α (TNF-α) (not corroborated at the protein levels for ICAM-1), as well as an increase in reactive oxygen/nitrogen species (ROS/RNS) production. This was accompanied by the activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signalling as demonstrated by the increase in the nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells protein 65 (p65-NF-κΒ) assessed by Western blotting and confocal microscopy. Since all these events were attenuated by an antioxidant pre-incubation with the radical scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), it is suggested that the inflammatory response is upstream mediated by the ROS/RNS-induced activation of NF-κΒ. Addition of paricalcitol (PC) 3·10
M to cells in HP prevented the phosphate induced ROS/RNS increase, the activation of NF-κΒ and the cytokine up-regulation. A bimodal effect was observed, however, for different calcitriol (CTR) concentrations, 10
and 10
M attenuated but 10
M stimulated this phosphate induced pro-oxidative and pro-inflammatory response. Therefore, these findings provide novel mechanisms whereby HP may directly favour vascular dysfunctions and new insights into the protective effects exerted by VitD derivatives.
Stimulation of endothelial cells (ECs) with TNF‐α causes an increase in the expression of bone morphogenetic protein‐2 (BMP‐2) and the production of endothelial microparticles (EMPs). BMP‐2 is known ...to produce osteogenic differentiation of vascular smooth muscle cells (VSMCs). It was found that EMPs from TNF‐α‐stimulated endothelial cells (HUVECs) contained a significant amount of BMP‐2 and were able to enhance VSMC osteogenesis and calcification. Calcium content was greater in VSMCs exposed to EMPs from TNF‐α‐treated HUVECs than EMPs from nontreated HUVECs (3.56 ± 0.57 vs. 1.48 ± 0.56 μg/mg protein; P < 0.05). The increase in calcification was accompanied by up‐regulation of Cbfa1 (osteogenic transcription factor) and down‐regulation of SM22α (VSMC lineage marker). Inhibition of BMP‐2 by small interfering RNA reduced the VSMC calcification induced by EMPs from TNF‐α‐treated HUVECs. Similar osteogenic capability was observed in EMPs from both patients with chronic kidney disease and senescent cells, which also presented a high level of BMP‐2 expression. Labeling of EMPs with CellTracker shows that EMPs are phagocytized by VSMCs under all conditions (with or without high phosphate, control, and EMPs from TNF‐α‐treated HUVECs). Our data suggest that EC damage results in the release of EMPs with a high content of calcium and BMP‐2 that are able to induce calcification and osteogenic differentiation of VSMCs.—Buendía, P., Montes de Oca, A., Madueño, J. A., Merino, A., Martín‐Malo, A., Aljama, P., Ramírez, R., Rodríguez, M., Carracedo, J., Endothelial microparticles mediate inflammation‐induced vascular calcification. FASEB J. 29, 173–181 (2015). www.fasebj.org
Mesenchymal stem cells (MSC) are osteoblasts progenitors and a variety of studies suggest that they may play an important role for the health in the field of bone regeneration. Magnesium ...supplementation is gaining importance as adjuvant treatment to improve osteogenesis, although the mechanisms involving this process are not well understood. The objective of this study was to investigate the effects of magnesium on MSC differentiation. Here we show that in rat bone marrow MSC, magnesium chloride increases MSC proliferation in a dose-dependent manner promoting osteogenic differentiation and mineralization. These effects are reduced by 2-APB administration, an inhibitor of magnesium channel TRPM7. Of note, magnesium supplementation did not increase the canonical Wnt/β-catenin pathway, although it promoted the activation of Notch1 signaling, which was also decreased by addition of 2-APB. Electron microscopy showed higher proliferation, organization and maturation of osteoblasts in bone decellularized scaffolds after magnesium addition. In summary, our results demonstrate that magnesium chloride enhances MSC proliferation by Notch1 signaling activation and induces osteogenic differentiation, shedding light on the understanding of the role of magnesium during bone regeneration.
The present study investigates the differential effect of two vitamin D receptor agonists, calcitriol and paricalcitol, on human aortic smooth muscle cells calcification in vitro. Human vascular ...smooth muscle cells were incubated in a high phosphate (HP) medium alone or supplemented with either calcitriol 10(-8)M (HP + CTR) or paricalcitol 3·10(-8) M (HP + PC). HP medium induced calcification, which was associated with the upregulation of mRNA expression of osteogenic factors such as bone morphogenetic protein 2 (BMP2), Runx2/Cbfa1, Msx2, and osteocalcin. In these cells, activation of Wnt/β-catenin signaling was evidenced by the translocation of β-catenin into the nucleus and the increase in the expression of direct target genes as cyclin D1, axin 2, and VCAN/versican. Addition of calcitriol to HP medium (HP + CTR) further increased calcification and also enhanced the expression of osteogenic factors together with a significant elevation of nuclear β-catenin levels and the expression of cyclin D1, axin 2, and VCAN. By contrast, the addition of paricalcitol (HP + PC) not only reduced calcification but also downregulated the expression of BMP2 and other osteoblastic phenotype markers as well as the levels of nuclear β-catenin and the expression of its target genes. The role of Wnt/β-catenin on phosphate- and calcitriol-induced calcification was further demonstrated by the inhibition of calcification after addition of Dickkopf-related protein 1 (DKK-1), a specific natural antagonist of the Wnt/β-catenin signaling pathway. In conclusion, the differential effect of calcitriol and paricalcitol on vascular calcification appears to be mediated by a distinct regulation of the BMP and Wnt/β-catenin signaling pathways.
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.
Periodontitis is a complex pathology characterized by the loss of alveolar bone. The causes and the mechanisms that promote this bone resorption still remain unknown. The knowledge of the critical ...regulators involved in the alteration of alveolar bone homeostasis is of great importance for developing molecular therapies. Procaine is an anesthetic drug with demethylant properties, mainly used by dentists in oral surgeries. The inhibitor role of Wnt signaling of procaine was described in vitro in colon cancer cells.
In this work we evaluated the role of procaine (1 uM) in osteo/odontogenesis of rat bone marrow mesenchymal stem cells. Similarly, the mechanisms whereby procaine achieves these effects were also studied.
Procaine administration led to a drastic decrease of calcium content, alkaline phosphatase activity, alizarin red staining and an increase in the expression of Matrix Gla Protein. With respect to osteo/odontogenic markers, procaine decreased early and mature osteo/odontogenic markers. In parallel, procaine inhibited canonical Wnt/β-catenin pathway, observing a loss of nuclear β-catenin, a decrease in Lrp5 and Frizzled 3, a significant increase of sclerostin and Gsk3β and an increase of phosphorylated β-catenin. The combination of osteo/odontogenic stimuli and Lithium Chloride decreased mRNA expression of Gsk3β, recovered by Procaine. Furthermore it was proved that Procaine alone dose dependently increases the expression of Gsk3β and β-catenin phosphorylation. These effects of procaine were also observed on mature osteoblast. Interestingly, at this concentration of procaine no demethylant effects were observed.
Our results demonstrated that procaine administration drastically reduced the mineralization and osteo/odontogenesis of bone marrow mesenchymal stem cells inhibiting Wnt/β-catenin pathway through the increase of Gsk3β expression and β-catenin phosphorylation.
Vitamin D sterols may modulate vascular response to inflammation and vascular calcification (VC).
Rat aortic rings (RARs) and human vascular smooth muscle cells (HVSMCs) were treated in vitro with ...phosphate (P), tumour necrosis factor alpha (TNF-α), calcitriol (CTR) and paricalcitol (PCT). Rats having undergone subtotal nephrectomy (Nx) (n = 66) on a high-phosphorus diet were treated with Escherichia coli lipopolysacharide (LPS) (40-400 μg/kg/day) or LPS plus CTR (80 ng/kg/48 h) or LPS plus PCT (240 ng/kg/48 h) for 14 days.
In vitro, the addition of TNF-α to the medium increased the mineral content of RAR and HVSMC. Treatment with both vitamin D analogues decreased bone morphogenetic protein 2 but did not modify Runx-2. Calcification was prevented only by PCT. In vivo, treatment with LPS increased plasma levels of TNF-α, monocyte chemotactic protein-1 and interleukin-1alfa and induced calcification. The concomitant administration of LPS with either CTR or PCT led to a significant decrease in cytokine plasma levels and the decrease was more accentuated after treatment with PCT than with CTR. Rats treated with CTR showed an elevation in aortic Ca and marked Von Kossa staining; however, rats treated with PCT did not increase aortic Ca and did not show Von Kossa staining.
Treatment with PCT resulted in more marked anti-inflammatory effect than treatment with CTR and, as opposed to CTR, PCT prevented VC.
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.
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