<p>Changes in the mRNA expression of A) early markers of oste/odontogenesis and B) specific genes of mesenchymal stem cells (MSC) were determined by RT-PCR after osteo/odontogenic differentiation (OB), OB with procaine (1 μM)... more
<p>Changes in the mRNA expression of A) early markers of oste/odontogenesis and B) specific genes of mesenchymal stem cells (MSC) were determined by RT-PCR after osteo/odontogenic differentiation (OB), OB with procaine (1 μM) (OB+Proc) during 21 days. The size and intensity of amplicon was electrophoresed on agarose gel (2%). Ribosomal 18S expression was used as housekeeping. C) Runx2 transcription factor activity was determined by a commercial TransAM<sup>™</sup> assay. Only OB cells showed to be significantly positive for this transcription factor (*p<0.05 vs all groups). D) Western blot for cytoplasmatic protein smooth muscle-22 alpha after 21 days of osteo/odontogenic differentiation. Images are representative of three cultures.</p
Research Interests: Pharmacology, Biochemistry, Genetics, Biophysics, Developmental Biology, and 15 moreMolecular Biology, Biotechnology, Cancer, Cell Biology, Infectious Diseases, Medicine, Beta-Catenin, mRna expression levels, Osteo, Phosphatase Activity, LRP, Bone Resorption, Wnt, Lithium chloride, and Alveolar Bone
<p>A) Calcium content and B) alkaline phosphatase activity were significantly increased after osteo/odontogenic stimulus (OB) (* p< 0.05 vs. undifferentiated cells (UC)) at 21 days. Procaine addition (OB+Proc) significantly... more
<p>A) Calcium content and B) alkaline phosphatase activity were significantly increased after osteo/odontogenic stimulus (OB) (* p< 0.05 vs. undifferentiated cells (UC)) at 21 days. Procaine addition (OB+Proc) significantly decreased calcium content and alkaline phosphatase activity after 21 days of differentiation (• p< 0.05 vs. OB cells). C) Alizarin red staining increased after osteo/odontoblasts differentiation (OB) while procaine addition (OB+Proc) significantly decreased mineralization during the differentiation process. Images are representative of three cultures. D) MGP gene expression was increased after procaine administration, impairing the mineralization process in OB cells. The size and intensity of amplicon was analyzed on agarose gel (2%). Ribosomal 18S expression was used as housekeeping.</p
Research Interests: Pharmacology, Biochemistry, Genetics, Biophysics, Developmental Biology, and 15 moreMolecular Biology, Biotechnology, Cancer, Cell Biology, Infectious Diseases, Medicine, Beta-Catenin, mRna expression levels, Osteo, Phosphatase Activity, LRP, Bone Resorption, Wnt, Lithium chloride, and Alveolar Bone
<p>The effect of several doses of procaine (0.5, 1 and 2 μM) on cell proliferation and viability was studied onundifferentiated mesenchymal stem cells (UC) (A), MSC plus Procaine 0.5 μM (B), 1 μM (C) y 2 μM (D) for 48h, 7 and 21... more
<p>The effect of several doses of procaine (0.5, 1 and 2 μM) on cell proliferation and viability was studied onundifferentiated mesenchymal stem cells (UC) (A), MSC plus Procaine 0.5 μM (B), 1 μM (C) y 2 μM (D) for 48h, 7 and 21 days of treatment. Changes in cell proliferation and viability were also studied on MSC differentiation into osteo/odontoblasts (E) at 48h, 14d and 21d. The effects of procaine on cell proliferation and viability on these osteo/odontoblasts were studied at 0.5 μM (F), 1 μM (G) and 2 μM (H). * p<0.001 vs 48h and # p<0.001 vs 7d.</p
Research Interests: Pharmacology, Biochemistry, Genetics, Biophysics, Developmental Biology, and 15 moreMolecular Biology, Biotechnology, Cancer, Cell Biology, Infectious Diseases, Medicine, Beta-Catenin, mRna expression levels, Osteo, Phosphatase Activity, LRP, Bone Resorption, Wnt, Lithium chloride, and Alveolar Bone
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Research Interests: Complementary and Alternative Medicine, Cytokines, Ethnopharmacology, Medicinal Plants, Medicine, and 15 moreCell line, Inflammatory Bowel Disease, Nitric oxide, Female, Animals, Methanol, Glutathione, High Pressure Liquid Chromatography, Colitis, In Vivo, Edema, Lavandula, Carrageenan, Matrix Metalloproteinase, and Inflammation Mediators
<p>Osteo/odontoblast stimulus (OB) increased the mRNA expression of A) Lrp5/6 and B) Frizzled 1 and decreased the mRNA expression of C) Gsk3β (***p<0.05 vs. undifferentiated cells, UC. Treatments with procaine (OB+Proc) decreased... more
<p>Osteo/odontoblast stimulus (OB) increased the mRNA expression of A) Lrp5/6 and B) Frizzled 1 and decreased the mRNA expression of C) Gsk3β (***p<0.05 vs. undifferentiated cells, UC. Treatments with procaine (OB+Proc) decreased expression of Lrp5/6 and Frizzled 1 and increased expression of Gsk3β (### p<0.001 vs OB cells and ## p<0.01 vs OB cells). D) Rat mesenchymal stem cells treated with osteo/odontoblasts stimulus (OB) or osteo/odontoblasts stimulus plus procaine (OB+Proc) were stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue) to determine β-catenin subcellular localization. Merged images of β-catenin immunofluorescence and DAPI staining are shown. Original magnification: 40x. Images were representatives of three independent experiments. E) phospho-β-catenin analysis by western blot showed that osteo/odontogenesis of MSC leads to a decrease of this protein while the presence of Procaine recovered the cytoplasmic levels of phospho β-catenin after 21 days of culture. Images are representative of three cultures. F) mRNA expression of sclerostin was decreased by osteo/odontoblast stimulus (OB) (p<0.05 vs UC) while procaine administration significantly increased the expression of this inhibitor (p<0.05 vs OB). Ribosomal 18S expression was used as housekeeping.</p
Research Interests: Pharmacology, Biochemistry, Genetics, Biophysics, Developmental Biology, and 15 moreMolecular Biology, Biotechnology, Cancer, Cell Biology, Infectious Diseases, Medicine, Beta-Catenin, mRna expression levels, Osteo, Phosphatase Activity, LRP, Bone Resorption, Wnt, Lithium chloride, and Alveolar Bone
<p>mRNA expression of mature markers of oste/odontoblast such as A) DMP1 and B) RANKL were measured by RT-PCR after 21 days in culture. The expression in mesenchymal stem cell differentiated into osteo/odontoblasts (OB) was... more
<p>mRNA expression of mature markers of oste/odontoblast such as A) DMP1 and B) RANKL were measured by RT-PCR after 21 days in culture. The expression in mesenchymal stem cell differentiated into osteo/odontoblasts (OB) was significantly higher than in undifferentiated cells (UC). This expression was reduced by the addition of Procaine (OB+Proc) during the differentiation process. Ribosomal 18S expression was used as housekeeping (* p<0.05 vs all groups).</p
Research Interests: Pharmacology, Biochemistry, Genetics, Biophysics, Developmental Biology, and 15 moreMolecular Biology, Biotechnology, Cancer, Cell Biology, Infectious Diseases, Medicine, Beta-Catenin, mRna expression levels, Osteo, Phosphatase Activity, LRP, Bone Resorption, Wnt, Lithium chloride, and Alveolar Bone
En los pacientes con enfermedad renal crónica (ERC), los niveles elevados del factor de crecimiento de fibroblastos 23 (FGF23) se asocian con la enfermedad cardiovascular y la mortalidad. Las células de músculo liso vascular (CMLV)... more
En los pacientes con enfermedad renal crónica (ERC), los niveles elevados del factor de crecimiento de fibroblastos 23 (FGF23) se asocian con la enfermedad cardiovascular y la mortalidad. Las células de músculo liso vascular (CMLV) presentan dos fenotipos funcionales diferenciados, contráctil y sintético. La abundancia del fenotipo sintético se asocia con la disfunción vascular, y se desconoce si el FGF23 puede promover el cambio fenotípico ocasionando complicaciones fisiopatológicas vasculares, como por ejemplo la rigidez vascular. El presente estudio evaluó si el FGF23 afecta al fenotipo de las CMLV y en consecuencia a la rigidez arterial. Además, en este se exploró un posible biomarcador clínico relacionado con la ingesta de P y su excreción. Dicho marcador permite predecir la existencia de altos niveles de FGF23 a nivel clínico y por tanto identificar qué pacientes tienen mayor riesgo de disfunción vascular. Nuestros resultados indicaron que altos niveles de FGF23 promovieron la...
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In chronic kidney disease (CKD) patients, it would be desirable to reduce the intake of inorganic phosphate (P) rather than limit the intake of P contained in proteins. Urinary excretion of P should reflect intestinal absorption of... more
In chronic kidney disease (CKD) patients, it would be desirable to reduce the intake of inorganic phosphate (P) rather than limit the intake of P contained in proteins. Urinary excretion of P should reflect intestinal absorption of P(inorganic plus protein-derived). The aim of the present study is to determine whether the ratio of urinary P to urinary urea nitrogen (P/UUN ratio) helps identify patients with a high intake of inorganic P.A cross-sectional study was performed in 71 patients affected by metabolic syndrome with CKD (stages 2–3) with normal serum P concentration. A 3-day dietary survey was performed to estimate the average daily amount and the source of P ingested. The daily intake of P was 1086.5 ± 361.3 mg/day; 64% contained in animal proteins, 22% in vegetable proteins, and 14% as inorganic P. The total amount of P ingested did not correlate with daily phosphaturia, but it did correlate with the P/UUN ratio (p < 0.018). Patients with the highest tertile of the P/UUN...
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Background and Aims In patients with chronic kidney disease (CKD), high levels of c-terminal fibroblast growth factor 23 (FGF23) are associated with cardiovascular disease and mortality. Vascular smooth muscle cells (VSMC) may present two... more
Background and Aims In patients with chronic kidney disease (CKD), high levels of c-terminal fibroblast growth factor 23 (FGF23) are associated with cardiovascular disease and mortality. Vascular smooth muscle cells (VSMC) may present two clearly differentiated functional phenotypes, contractile and synthetic. The abundance of synthetic phenotype is associated with vascular dysfunction and it is unknown whether in FGF23 may promote transition from a contractile to a synthetic phenotype in VSMC causing vascular stiffness. The present study was conducted to evaluate whether FGF23 affects VSMC phenotype and arterial stiffness. Method and Results High levels of FGF23 promoted VSMC transition from a contractile to a synthetic phenotype. These effects were mediated through FGFR1 and Ras/MAPK signaling activation. Inhibition of both pathways enhanced contractile phenotype of VSMC. The pro-contractile microRNAs, miR-221 and miR-222 were reduced by FGF23 and miR-221 transfection recovered th...
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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... more
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...
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Although magnesium has been shown to prevent vascular calcification in vitro, controlled in vivo studies in uremic animal models are limited. To determine whether dietary magnesium supplementation protects against the development of... more
Although magnesium has been shown to prevent vascular calcification in vitro, controlled in vivo studies in uremic animal models are limited. To determine whether dietary magnesium supplementation protects against the development of vascular calcification, 5/6 nephrectomized Wistar rats were fed diets with different magnesium content increasing from 0.1 to 1.1%. In one study we analyzed bone specimens from rats fed 0.1%, 0.3%, and 0.6% magnesium diets, and in another study we evaluated the effect of intraperitoneal magnesium on vascular calcification in 5/6 nephrectomized rats. The effects of magnesium on established vascular calcification were also evaluated in uremic rats fed on diets with either normal (0.1%) or moderately increased magnesium (0.6%) content. The increase in dietary magnesium resulted in a marked reduction in vascular calcification, together with improved mineral metabolism and renal function. Moderately elevated dietary magnesium (0.3%), but not high dietary magn...
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In renal failure, hyperphosphatemia occurs despite a marked elevation in serum fibroblast growth factor (FGF)-23. Abnormal regulation of the FGFR1-Klotho receptor complex may cause a resistance to the phosphaturic action of FGF23. The... more
In renal failure, hyperphosphatemia occurs despite a marked elevation in serum fibroblast growth factor (FGF)-23. Abnormal regulation of the FGFR1-Klotho receptor complex may cause a resistance to the phosphaturic action of FGF23. The purpose of the present study was to investigate the regulation of renal Klotho and FGF receptor (FEFR)-1 in healthy and uremic rats induced by 5/6 nephrectomy. In normal rats, the infusion of rat recombinant FGF23 enhanced phosphaturia and increased renal FGFR1 expression; however, Klotho expression was reduced. Uremic rats on a high-phosphate (HP) diet presented hyperphosphatemia with marked elevation of FGF23 and an increased fractional excretion of phosphate (P) that was associated with a marked reduction of Klotho expression and an increase in FGFR1. After neutralization of FGF23 by anti-FGF23 administration, phosphaturia was still abundant, Klotho expression remained low, and the FGFR1 level was reduced. These results suggest that the expression o...
Research Interests: Endocrinology, Physiology, Medicine, Humans, Internal Medicine, and 15 moreAnimals, Male, Glucuronidase, Medical Physiology, Fibroblast Growth Factor, Calcitriol, Rats, Phosphates, Recombinant Proteins, Biochemistry and cell biology, Gene Expression Regulation, hyperphosphatemia, KLOTHO, renal insufficiency, and Fibroblast growth factors
In chronic kidney disease patients, high phosphate (HP) levels are associated to cardiovascular disease, the major cause of morbidity and mortality. Since serum phosphate has been independently correlated with inflammation, the present... more
In chronic kidney disease patients, high phosphate (HP) levels are associated to 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 was also investigated. The study was performed in an in vitro model of human aortic smooth muscle cells (HASMCs). Incubation of cells in a HP (3.3 mM) medium caused an increased expression of the pro-inflammatory mediators ICAM-1, IL-1β, IL-6, IL-8 and 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 NF-kB signalling as demonstrated by the increase in the nuclear translocation of NF-kB -p65 assessed by western blotting and confocal microscopy. Since all these events were attenuate...
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Research Interests:
Research Interests: Chemistry, Calcium, Transcription Factors, Medicine, Multidisciplinary, and 15 moreCell Differentiation, Mesenchymal stem cells, Animals, Male, DNA methylation, PLoS one, Rats, Odontogenesis, Wistar Rats, Osteogenesis, Cell Proliferation, Osteoblasts, Mesenchymal Stromal Cells, Alveolar Bone, and reverse transcriptase polymerase chain reaction
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,... more
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 ...
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Nitric-oxide synthase, the enzyme responsible for mammalian nitric oxide generation, and cytochrome P450, the major enzymes involved in drug metabolism, share striking similarities. Therefore, it makes sense that cytochrome P450 drug... more
Nitric-oxide synthase, the enzyme responsible for mammalian nitric oxide generation, and cytochrome P450, the major enzymes involved in drug metabolism, share striking similarities. Therefore, it makes sense that cytochrome P450 drug mediated biotransformations might play an important role in the pharmacological modulation of nitric oxide synthase. In this work, we have undertaken an integrated in vitro assessment of the hepatic metabolism and nitric oxide modulation of previously described dual inhibitors (imidazoles and macrolides) of these enzymes in order assess the implication of CYP450 activities over production of nitric oxide. In vitro systems based in human liver microsomes and activated mouse macrophages were developed for these purposes. Additionally in vitro production the hepatic metabolites of dual inhibitor, roxithromycin, was investigated achieving the identification and isolation of main hepatic biotransformation products. Our results suggested that for some macroli...