Energy-dense diets increase FGF23, lead to phosphorus retention and promote vascular calcifications in rats

Rats with normal renal function (Experiment 1, n = 12) and uninephrectomized (1/2Nx) rats (Experiment 2, n = 12) were fed diets with normal P (NP) and either normal (NF) or high fat (HF). Rats with intact renal function (Experiment 3, n = 12) were also fed NF or HF diets with high P (HP). Additionally, uremic (5/6Nx) rats (n = 16) were fed HP diets with NF or HF. Feeding the HF diets resulted in significant elevation of plasma FGF23 vs rats fed NF diets: Experiment 1, 593 ± 126 vs 157 ± 28 pg/ ml (p < 0.01); Experiment 2, 538 ± 105 vs 250 ± 18 pg/ml (p < 0.05); Experiment 3, 971 ± 118 vs 534 ± 40 pg/ml (p < 0.01). Rats fed HF diets showed P retention and decreased renal klotho (ratio klotho/actin) vs rats fed NF diets: Experiment 1, 0.75 ± 0.06 vs 0.97 ± 0.02 (p < 0.01); Experiment 2, 0.69 ± 0.07 vs 1.12 ± 0.08 (p < 0.01); Experiment 3, 0.57 ± 0.19 vs 1.16 ± 0.15 (p < 0.05). Uremic rats fed HF diet showed more severe vascular calcification (VC) than rats fed NF diet (aortic Ca = 6.3 ± 1.4 vs 1.4 ± 0.1 mg/g tissue, p < 0.001). In conclusion, energy-rich diets increased plasma levels of FGF23, a known risk factor of cardiovascular morbidity and mortality. Even though FGF23 has major phosphaturic actions, feeding HF diets resulted in P retention, likely secondary to decreased renal klotho, and aggravated uremic V

Nuclear Translocation of b-Catenin during Mesenchymal Stem Cells Differentiation into Hepatocytes Is Associated with a Tumoral Phenotype

Wnt/b-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/b-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, b-catenin nuclear translocation, up-regulation of genes related to the Wnt/b-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/b-catenin inactivation. Hepatocytes with nuclear translocation of b-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase b-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin b-chain were downregulated in this protocol. In conclusion, our results suggest that activation of the Wnt/b-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotyp

Procaine Inhibits Osteo/Odontogenesis through Wnt/β-Catenin Inactivation

Introduction 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. Methods 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. Results 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. PLO

In vascular smooth muscle cells paricalcitol prevents phosphate-induced Wnt/β-catenin activation

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 10−8M (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

Hypertriglyceridemia Influences the Degree of Postprandial Lipemic Response in Patients with Metabolic Syndrome and Coronary Artery Disease: From the Cordioprev Study

Objective To determine whether metabolic syndrome traits influence the postprandial lipemia response of coronary patients, and whether this influence depends on the number of MetS criteria. Materials and Methods 1002 coronary artery disease patients from the CORDIOPREV study were submitted to an oral fat load test meal with 0.7 g fat/kg body weight (12% saturated fatty acids, 10% polyunsaturated fatty acids, 43% monounsaturated fatty acids), 10% protein and 25% carbohydrates. Serial blood test analyzing lipid fractions were drawn at 0, 1, 2, 3 and 4 hours during the postprandial state. Total and incremental area under the curves of the different postprandial parameters were calculated following the trapezoid rule to assess the magnitude of change during the postprandial state Results Postprandial lipemia response was directly related to the presence of metabolic syndrome. We found a positive association between the number of metabolic syndrome criteria and the response of postprandial plasma triglycerides (p<0.001), area under the curve of triglycerides (p<0.001) and incremental area under the curve of triglycerides (p<0.001). However, the influence of them on postprandial triglycerides remained statistically significant only in those patients without basal hypertriglyceridemia. Interestingly, in stepwise multiple linear regression analysis with the AUC of triglycerides as the dependent variable, only fasting triglycerides, fasting glucose and waist circumference appeared as significant independent (P<0.05) contributors. The multiple lineal regression (R) was 0.77, and fasting triglycerides showed the greatest effect on AUC of triglycerides with a standardized coefficient of 0.75. Conclusions Fasting triglycerides are the major contributors to the postprandial triglycerides levels. MetS influences the postprandial response of lipids in patients with coronary heart disease, particularly in non-hypertriglyceridemic patients

Magnesium Inhibits Wnt/β-Catenin Activity and Reverses the Osteogenic Transformation of Vascular Smooth Muscle Cells

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

Dietary Mg Supplementation Decreases Oxidative Stress, Inflammation, and Vascular Dysfunction in an Experimental Model of Metabolic Syndrome with Renal Failure

whether a dietary Mg supplementation might attenuate vascular dysfunction through the modulation of oxidative stress and inflammation in concurrent MetS and CKD. Methods: A rat model of MetS (Zucker strain) with CKD (5/6 nephrectomy, Nx) was used. Nephrectomized animals were fed a normal 0.1%Mg (MetS+Nx+Mg0.1%) or a supplemented 0.6%Mg (MetS+Nx+Mg0.6%) diet; Sham-operated rats with MetS receiving 0.1%Mg were used as controls. Results: As compared to controls, the MetS+Nx-Mg0.1% group showed a significant increase in oxidative stress and inflammation biomarkers (lipid peroxidation and aortic interleukin-1b and -6 expression) and Endothelin-1 levels, a decrease in nitric oxide and a worsening in uremia and MetS associated pathology as hypertension, and abnormal glucose and lipid profile. Moreover, proteomic evaluation revealed changes mainly related to lipid metabolism and CVD markers. By contrast, in the MetS+Nx+Mg0.6% group, these parameters remained largely similar to controls. Conclusion: In concurrent MetS and CKD, dietary Mg supplementation reduced inflammation and oxidative stress and improved vascular function

The direct effect of fibroblast growth factor 23 on vascular smooth muscle cell phenotype and function

[Background] In chronic kidney disease (CKD) patients, increased levels of fibroblast growth factor 23 (FGF23) are associated with cardiovascular mortality. The relationship between FGF23 and heart hypertrophy has been documented, however, it is not known whether FGF23 has an effect on vasculature. Vascular smooth muscle cells VSMCs may exhibit different phenotypes; our hypothesis is that FGF23 favours a switch from a contractile to synthetic phenotype that may cause vascular dysfunction. Our objective was to determine whether FGF23 may directly control a change in VSMC phenotype.[Methods] This study includes in vitro, in vivo and ex vivo experiments and evaluation of patients with CKD stages 2–3 studying a relationship between FGF23 and vascular dysfunction.[Results] In vitro studies show that high levels of FGF23, by acting on its specific receptor FGFR1 and Erk1/2, causes a change in the phenotype of VSMCs from contractile to synthetic. This change is mediated by a downregulation of miR-221/222, which augments the expression of MAP3K2 and PAK1. miR-221/222 transfections recovered the contractile phenotype of VSMCs. Infusion of recombinant FGF23 to rats increased vascular wall thickness, with VSMCs showing a synthetic phenotype with a reduction of miR-221 expression. Ex-vivo studies on aortic rings demonstrate also that high FGF23 increases arterial stiffening. In CKD 2–3 patients, elevation of FGF23 was associated with increased pulse wave velocity and reduced plasma levels of miR-221/222.[Conclusion] In VSMCs, high levels of FGF23, through the downregulation of miR-221/222, causes a change to a synthetic phenotype. This change in VSMCs increases arterial stiffening and impairs vascular function, which might ultimately worsen cardiovascular disease.This work was supported by a Spanish government grant from the Programa Nacional I+D+I 2013–2016 and Instituto de Salud Carlos III (ISCIII) grants PI18/0138 and PI21/0654 co-financing from European Funds (FEDER), Consejería de Salud (grants PI-0136 and PI-0169-2020) from the Junta de Andalucía, Framework Programme 7 Syskid UE grant FP7-241544, and EUTOX and REDinREN from the ISCIII. N.V. and J.M.D.-T. were supported by Consejería de Economía, Innovación, Ciencia y Empleo (grant CVI-7925) from the Junta de Andalucía. Y.A. and J.R.M.-C. are senior researchers supported by the Nicolás Monardes Programme, Consejería de Salud-Servicio Andaluz de Salud (Junta de Andalucía).Peer reviewe

Supplemental Material The direct effect of fibroblast growth factor 23 on vascular smooth muscle cell phenotype and function

3 pages. -- Figure S1. Supplemental Material. Effects of anti-miR-221 and miR-222. -- Figure S1. Supplemental Material: A) Anti-miR-221 and B) anti-miR-222 transfection for 48 h decreased not significantly the expression of miR-221 and miR-222 in VSMC. -- Figure S2. Supplemental Material. Recombinant Klotho administration did not modify the expression of contractile markers of VSMC. -- Figure S3. Histological quantifications in thoracic aortas of rats of synthetic markers of VSMC.Peer reviewe

Nuclear Translocation of β-Catenin during Mesenchymal Stem Cells Differentiation into Hepatocytes Is Associated with a Tumoral Phenotype

Wnt/β-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/β-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, β-catenin nuclear translocation, up-regulation of genes related to the Wnt/β-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/β-catenin inactivation. Hepatocytes with nuclear translocation of β-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase β-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin β-chain were down-regulated in this protocol. In conclusion, our results suggest that activation of the Wnt/β-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype