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1.
Kidney Int ; 95(5): 1064-1078, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30878213

RESUMO

Calcimimetics decrease parathyroid hormone (PTH) secretion in patients with secondary hyperparathyroidism. The decrease in PTH should cause a reduction in bone turnover; however, the direct effect of calcimimetics on bone cells, which express the calcium-sensing receptor (CaSR), has not been defined. In this study, we evaluated the direct bone effects of CaSR activation by a calcimimetic (AMG 641) in vitro and in vivo. To create a PTH "clamp," total parathyroidectomy was performed in rats with and without uremia induced by 5/6 nephrectomy, followed by a continuous subcutaneous infusion of PTH. Animals were then treated with either the calcimimetic or vehicle. Calcimimetic administration increased osteoblast number and osteoid volume in normal rats under a PTH clamp. In uremic rats, the elevated PTH concentration led to reduced bone volume and increased bone turnover, and calcimimetic administration decreased plasma PTH. In uremic rats exposed to PTH at 6-fold the usual replacement dose, calcimimetic administration increased osteoblast number, osteoid surface, and bone formation. A 9-fold higher dose of PTH caused an increase in bone turnover that was not altered by the administration of calcimimetic. In an osteosarcoma cell line, the calcimimetic induced Erk1/2 phosphorylation and the expression of osteoblast genes. The addition of a calcilytic resulted in the opposite effect. Moreover, the calcimimetic promoted the osteogenic differentiation and mineralization of human bone marrow mesenchymal stem cells in vitro. Thus, calcimimetic administration has a direct anabolic effect on bone that counteracts the decrease in PTH levels.


Assuntos
Compostos de Bifenilo/administração & dosagem , Remodelação Óssea/efeitos dos fármacos , Calcimiméticos/administração & dosagem , Hiperparatireoidismo Secundário/tratamento farmacológico , Falência Renal Crônica/complicações , Fenetilaminas/administração & dosagem , Animais , Modelos Animais de Doenças , Humanos , Hiperparatireoidismo Secundário/sangue , Hiperparatireoidismo Secundário/etiologia , Masculino , Osteoblastos/efeitos dos fármacos , Hormônio Paratireóideo/administração & dosagem , Hormônio Paratireóideo/sangue , Hormônio Paratireóideo/metabolismo , Ratos , Ratos Wistar , Receptores de Detecção de Cálcio/metabolismo
2.
Sci Rep ; 7(1): 7839, 2017 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-28798480

RESUMO

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/ß-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.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Cloreto de Magnésio/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Osteogênese/efeitos dos fármacos , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Osso e Ossos/citologia , Compostos de Boro/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/metabolismo , Microscopia Eletrônica , Ratos , Canais de Cátion TRPM/antagonistas & inibidores
3.
Clin Sci (Lond) ; 131(13): 1449-1463, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28442557

RESUMO

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 in vitro 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-1ß, 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) 3·10-8 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, 10-10 and 10-12 M attenuated but 10-8 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.


Assuntos
Mediadores da Inflamação/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fosfatos/farmacologia , Aorta/citologia , Aorta/metabolismo , Calcitriol/administração & dosagem , Calcitriol/farmacologia , Núcleo Celular/metabolismo , Células Cultivadas , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Ergocalciferóis/farmacologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Espécies Reativas de Nitrogênio/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição RelA/metabolismo
4.
PLoS One ; 11(6): e0156788, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27257912

RESUMO

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/ß-catenin pathway, observing a loss of nuclear ß-catenin, a decrease in Lrp5 and Frizzled 3, a significant increase of sclerostin and Gsk3ß and an increase of phosphorylated ß-catenin. The combination of osteo/odontogenic stimuli and Lithium Chloride decreased mRNA expression of Gsk3ß, recovered by Procaine. Furthermore it was proved that Procaine alone dose dependently increases the expression of Gsk3ß and ß-catenin phosphorylation. These effects of procaine were also observed on mature osteoblast. Interestingly, at this concentration of procaine no demethylant effects were observed. CONCLUSIONS: Our results demonstrated that procaine administration drastically reduced the mineralization and osteo/odontogenesis of bone marrow mesenchymal stem cells inhibiting Wnt/ß-catenin pathway through the increase of Gsk3ß expression and ß-catenin phosphorylation.


Assuntos
Procaína/farmacologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Metilação de DNA/efeitos dos fármacos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Técnicas de Transferência Nuclear , Odontogênese/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
FASEB J ; 30(3): 1367-76, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26700731

RESUMO

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-κB 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-κB 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.


Assuntos
Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor PAR-2/metabolismo , Tromboplastina/metabolismo , Vitamina D/metabolismo , Calcitriol/farmacologia , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Ergocalciferóis/farmacologia , Humanos , Inflamação/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , NF-kappa B/metabolismo , Receptores de Calcitriol/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos
6.
Eur J Clin Invest ; 45(11): 1129-44, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26268950

RESUMO

BACKGROUND: Vascular calcification (VC) is highly prevalent in patients with chronic kidney disease (CKD). Low magnesium levels are associated with VC, and recent in vitro studies confirm a protective role of magnesium, which is mediated by its entry into the VSMCs through the Transient Receptor Potential Melastatin 7 (TRPM7) channel. The role of Angiotensin II (Ang II) on VC is still unclear. As Ang II is able to stimulate TRPM7 activity, we hypothesize that it might prevent VC. Thus, the aim of this study was to dissect the direct effect of Ang II on VC. MATERIALS AND METHODS: We worked with a model of high phosphate (HP)-induced calcification in human aortic smooth muscle cells, which resembles the CKD-related VC. RESULTS: Addition of Ang II to cells growing in HP decreased calcification, which was associated with the upregulation of the osteogenic factors BMP2, Runx2/Cbfa1, Osterix and ALP. A reduction of magnesium entry into the HP-calcifying cells was found. The treatment with Ang II avoided this reduction, which was reversed by the cotreatment with the TRPM7-inhibitor 2-APB. The protective effect of Ang II was related to AT1R-induced ERK1/2 MAPKinase activation. HP-induced calcification was also associated with the upregulation of the canonical Wnt/beta-catenin pathway, while its downregulation was related to attenuation of calcification by Ang II. CONCLUSION: As hypothesized, Ang II prevented phosphate-induced calcification in VSMCs, which appears mediated by the increase of magnesium influx and by the activation of the ERK1/2 and the inhibition of the canonical Wnt/beta-catenin signalling pathways.


Assuntos
Angiotensina II/farmacologia , Magnésio/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Canais de Cátion TRPM/efeitos dos fármacos , Calcificação Vascular/metabolismo , Vasoconstritores/farmacologia , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Compostos de Boro/farmacologia , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição Sp7 , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Regulação para Cima , Via de Sinalização Wnt/efeitos dos fármacos
7.
Pediatrics ; 135(6): e1518-23, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26009620

RESUMO

Tumor-induced osteomalacia/rickets is a rare paraneoplastic disorder associated with a tumor-producing fibroblast growth factor 23 (FGF23). We present a child with symptoms of rickets as the first clinical sign of a central giant cell granuloma (CGCG) with high serum levels of FGF23, a hormone associated with decreased phosphate resorption. A 3-year-old boy presented with a limp and 6 months later with painless growth of the jaw. On examination gingival hypertrophy and genu varum were observed. Investigations revealed hypophosphatemia, normal 1,25 and 25 (OH) vitamin D, and high alkaline phosphatase. An MRI showed an osteolytic lesion of the maxilla. Radiographs revealed typical rachitic findings. Incisional biopsy of the tumor revealed a CGCG with mesenchymal matrix. The CGCG was initially treated with calcitonin, but the lesions continued to grow, making it necessary to perform tracheostomy and gastrostomy. One year after onset the hyperphosphaturia worsened, necessitating increasing oral phosphate supplements up to 100 mg/kg per day of elemental phosphorus. FGF23 levels were extremely high. Total removal of the tumor was impossible, and partial reduction was achieved after percutaneous computed tomography-guided radiofrequency, local instillation of triamcinolone, and oral propranolol. Compassionate use of cinacalcet was unsuccessful in preventing phosphaturia. The tumor slowly regressed after the third year of disease; phosphaturia improved, allowing the tapering of phosphate supplements, and FGF23 levels normalized. Tumor-induced osteomalacia/rickets is uncommon in children and is challenging for physicians to diagnose. It should be suspected in patients with intractable osteomalacia or rickets. A tumor should be ruled out if FGF23 levels are high.


Assuntos
Neoplasias Ósseas/complicações , Granuloma de Células Gigantes/complicações , Neoplasias de Tecido Conjuntivo/etiologia , Raquitismo/etiologia , Pré-Escolar , Fator de Crescimento de Fibroblastos 23 , Humanos , Masculino , Osteomalacia , Síndromes Paraneoplásicas
8.
Regen Med ; 10(1): 25-38, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25562350

RESUMO

AIM: It is unknown if the beneficial effects of mesenchymal stromal cells (MSC) transplantation into the liver are dependent on their anchorage and differentiation into hepatocytes or rather the result of the release of stem cell intracellular content with hepatoprotector properties. MATERIALS & METHODS: The effects of intact MSC transplantation were compared with the infusion of MSC lysates in an experimental rat model of acute liver failure. RESULTS: A more powerful hepatoprotective and antiapoptotic effect was obtained after infusion of MSC lysates than intact MSC. Changes in IL-6 levels and miRNAs might explain the beneficial effects of MSC lysates. CONCLUSION: Infusion of MSC lysates show a better hepatoprotective effect than the transplantation of intact MSC.


Assuntos
Extratos Celulares/farmacologia , Extratos Celulares/uso terapêutico , Fígado/fisiologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Feminino , Humanos , Imunofenotipagem , Fígado/efeitos dos fármacos , Hepatopatias/patologia , Hepatopatias/terapia , MicroRNAs/metabolismo , Células-Tronco Pluripotentes/citologia , Veia Porta/fisiologia , Ratos Wistar , Tioacetamida/administração & dosagem , Antígenos Thy-1/metabolismo
9.
J Surg Res ; 193(1): 119-25, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25085703

RESUMO

BACKGROUND: Cardiotrophin-1 (CT1) has been used to prevent cell death in different models of liver injury in rats. D-galactosamine induces cell death in culture rat and human hepatocytes. The present study evaluated the cytoprotective effects of CT1 in an experimental model of apoptosis induced by D-galactosamine in hepatocytes. METHODS: DNA fragmentation, calpain activity and Western blots of caspase-3, calpastatin and Stat3, and Akt phosphorylation were measured. Stat3 and Akt inhibitors were used to analyze the mechanisms of action of CT1. RESULTS: CT1 caused an increase in Stat3 and Akt phosphorylation and a decrease of DNA fragmentation, calpain activity, and caspase-3 induced by D-galactosamine. The reduction of calpain activity by CT1 was associated with an increase of calpastatin (its endogenous inhibitor). The effects of CT1 were also dependent on the activation of Sta3 or Akt. CONCLUSIONS: CT1 decreases cell death through a mechanism related to Stat3 and Akt phosphorylation and activation of calpastatin in D-galactosamine-treated hepatocytes.


Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/metabolismo , Citoproteção/efeitos dos fármacos , Galactosamina/farmacologia , Hepatócitos/efeitos dos fármacos , Animais , Calpaína/metabolismo , Caspase 3/metabolismo , Citocinas/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Hepatócitos/citologia , Masculino , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Suínos
10.
PLoS One ; 9(2): e89179, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24586576

RESUMO

BACKGROUND: Transforming growth factor-ß (TGF-ß) 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. RESULTS: Our results showed that TGF-ß 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/ß-catenin in addition to an increase in BMP-2 expression, calcium deposition and alkaline phosphatase activity. The administration of TGF-ß to MSC treated with high phosphate abolished all these effects by inhibiting canonical Wnt, BMP and TGF-ß 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/ß-catenin activators CHIR98014 and lithium chloride enhanced the effect of high phosphate on BMP-2, calcium deposition and alkaline phosphatase activity. CONCLUSIONS: Full VSMC differentiation induced by TGF-ß may not be achieved when extracellular phosphate levels are high. Moreover, TGF-ß prevents high phosphate-induced osteogenesis by decreasing the nuclear translocation of Smad 1/5/8 and avoiding the activation of Wnt/ß-catenin pathway.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Perfilação da Expressão Gênica , Imunofenotipagem , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fosfatos/metabolismo , Transporte Proteico , Ratos , Proteína Smad3/metabolismo
11.
PLoS One ; 9(2): e89525, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24586847

RESUMO

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/ß-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/ß-catenin pathway as demonstrated by the translocation of ß-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/ß-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/ß-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/ß-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/ß-catenin by magnesium is one potential intracellular mechanism by which this anti-calcifying effect is achieved.


Assuntos
Magnésio/farmacologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Calcificação Vascular/tratamento farmacológico , Proteínas Wnt/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Compostos de Boro/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Humanos , Músculo Liso Vascular/metabolismo , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Proteínas Serina-Treonina Quinases , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Calcificação Vascular/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Proteína de Matriz Gla
12.
PLoS One ; 7(4): e34656, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22506042

RESUMO

Wnt/ß-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/ß-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, ß-catenin nuclear translocation, up-regulation of genes related to the Wnt/ß-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/ß-catenin inactivation. Hepatocytes with nuclear translocation of ß-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 ß-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 ß-chain were down-regulated in this protocol. In conclusion, our results suggest that activation of the Wnt/ß-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype.


Assuntos
Núcleo Celular/metabolismo , Hepatócitos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , beta Catenina/metabolismo , Biomarcadores/metabolismo , Carcinoma Hepatocelular/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Regulação para Baixo/fisiologia , Perfilação da Expressão Gênica/métodos , Humanos , Imunofenotipagem/métodos , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Neoplásicas/metabolismo , Transporte Proteico , Esferoides Celulares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/fisiologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia
13.
J Bone Miner Res ; 25(9): 1996-2005, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20499380

RESUMO

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.


Assuntos
Calcinose/complicações , Metilação de DNA , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso Vascular/metabolismo , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Primers do DNA , Humanos , Ratos
14.
Am J Physiol Renal Physiol ; 296(6): F1376-85, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19321594

RESUMO

The purpose of the present study was to test the hypothesis that extraskeletal calcification regresses in uremic rats after reduction in phosphorus intake and treatment with calcimimetics. Extraosseous calcification was induced in five to six nephrectomized rats fed a high-phosphorus (1.2%) diet who received calcitriol (80 ng/kg ip) every other day for a period of 14 days. Next, dietary phosphorus was reduced to 0.6%, and rats were treated with vehicle (n = 20), calcitriol [80 ng/kg ip/48 h (n = 20)], or the calcimimetic AMG 641 [1.5 mg/kg sc/48 h (n = 20)]. Aortic and soft-tissue calcium and phosphorus content was evaluated after 14 and 28 days. At 28 days, reduction of phosphorus intake resulted in a significant decrease in tissue mineral content in vehicle- and AMG 641-treated rats but not in rats receiving calcitriol. Aortic calcium and phosphorus was lower in rats treated with AMG 641 (96.7 +/- 26.4 mg/g) than in rats receiving vehicle (178.3 +/- 38.6 mg/g). An infiltrate of phagocytic cells expressing the calcium-sensing receptor was identified in areas surrounding foci of calcification. Additional studies in parathyroidectomized rats demonstrated that AMG 641 increased the urinary excretion of calcium (6.2 +/- 0.6 vs. 3.1 +/- 0.5 mg/day, vehicle) (P < 0.001). In conclusion, experimentally induced extraosseous calcification in uremic rats can be partially resolved by reducing phosphorus intake; the addition of calcimimetics may accelerate the regression process through mechanisms potentially involving a direct stimulatory effect on mineral phagocytic cells plus an increase in urinary calcium excretion.


Assuntos
Calcinose/tratamento farmacológico , Agonistas dos Canais de Cálcio/uso terapêutico , Uremia/complicações , Animais , Aorta/patologia , Calcinose/complicações , Calcinose/patologia , Calcitriol/farmacologia , Calcitriol/uso terapêutico , Cálcio/sangue , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Relação Dose-Resposta a Droga , Esquema de Medicação , Pulmão/patologia , Masculino , Fósforo/sangue , Fósforo/metabolismo , Ratos , Ratos Wistar , Estômago/patologia
15.
J Bone Miner Metab ; 24(2): 164-8, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16502126

RESUMO

This review summarizes the factors involved in the development of hyperparathyroidism secondary (2nd-HPTH) to chronic kidney disease (CKD). Calcium and calcitriol act on their respective specific parathyroid cell receptors to inhibit parathyroid function. As well as the well-known effect of calcium and calcitriol on parathyroid cell function, there is experimental work that demonstrates that phosphate, changes in pH, PTHrP, estrogens, and some cytokines also have an effect on PTH secretion. These factors are relevant in patients with chronic kidney disease. However, low calcium, vitamin D deficiency, and an accumulation of phosphate due to the decrease in renal function are the main pathogenic factors involved in the pathogenesis of 2nd-HPTH in CKD patients.


Assuntos
Regulação da Expressão Gênica , Falência Renal Crônica/metabolismo , Falência Renal Crônica/patologia , Glândulas Paratireoides/fisiologia , Acidose , Actinas/química , Calcitriol/metabolismo , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Estrogênios/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hiperparatireoidismo/metabolismo , Glândulas Paratireoides/metabolismo , Hormônio Paratireóideo/análogos & derivados , Hormônio Paratireóideo/metabolismo , Fosfatos/metabolismo , RNA Mensageiro/metabolismo , Receptores de Calcitriol/metabolismo , Regulação para Cima , Vitamina D/metabolismo , Deficiência de Vitamina D
16.
J Am Soc Nephrol ; 17(3): 795-804, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16467452

RESUMO

Calcimimetics decrease parathyroid hormone (PTH) levels in uremic patients with secondary hyperparathyroidism without increasing serum calcium (Ca). The aim of this study was to evaluate the effect of calcimimetic R-568 alone or in combination with calcitriol on vascular and other soft tissue calcifications in uremic rats with secondary hyperparathyroidism. Sham-operated and 5/6 nephrectomized Wistar rats were studied. 5/6 Nephrectomized rats were treated with vehicle, calcitriol (80 ng/kg every other day), R-568 (1.5 and 3 mg/kg per d), and both calcitriol and R-568 1.5 mg/kg, as above. Rats were killed after 14 or 56 d of treatment. Blood was drawn for biochemical measurements. Aortic, heart, kidney, lung, and stomach tissue samples were processed for histopathology and measurement of tissue Ca and phosphorus content. PTH concentrations were significantly reduced by all treatments. Treatment with calcitriol induced significant vascular calcification (aortic Ca increased to 4.2+/-1.2 mg/g at day 14 and to 11.4+/-0.7 mg/g at day 56; P<0.05 versus vehicle). Treatment with R-568 did not induce vascular calcification. Concurrent administration of R-568 with calcitriol reduced the aortic Ca (1.9+/-0.2 mg/g at day 14 and 7.5+/-1.4 mg/g at day 56) in relation to calcitriol alone. Soft tissue calcifications mirrored aortic mineralizations. Survival was significantly (P<0.001) reduced in calcitriol-treated rats, and mortality was attenuated (P=0.01) by concurrent treatment with R-568. In uremic rats, R-568 reduces elevated PTH levels without inducing vascular calcification, prevents calcitriol-induced vascular calcification, and decreases mortality.


Assuntos
Compostos de Anilina/farmacologia , Calcinose/patologia , Calcinose/prevenção & controle , Calcitriol/farmacologia , Uremia/tratamento farmacológico , Animais , Biópsia por Agulha , Análise Química do Sangue , Modelos Animais de Doenças , Quimioterapia Combinada , Imuno-Histoquímica , Masculino , Nefrectomia , Fenetilaminas , Probabilidade , Propilaminas , RNA Mensageiro/análise , Ratos , Ratos Wistar , Sensibilidade e Especificidade , Taxa de Sobrevida , Uremia/mortalidade , Uremia/fisiopatologia
17.
Kidney Int ; 64(1): 63-70, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12787396

RESUMO

BACKGROUND: The secretion of parathyroid hormone (PTH) from the parathyroid glands might be regulated by autocrine/paracrine factors. We have previously shown that N-terminal parathyroid hormone-related protein (PTHrP) enhanced the secretory PTH response to low calcium in vivo and in vitro in rat parathyroid glands. N-terminal PTHrP fragments are equipotent to N-terminal PTH as ligands for the PTH/PTHrP receptor that is demonstrated in parathyroid tissue. This supports the possibility that the parathyroid cells respond to PTH/PTHrP receptor ligands and as such are target for an autoregulatory action of PTH and PTHrP. Our aim was to search for the PTH/PTHrP receptor in rat parathyroid glands and to examine the effects of PTHrP 1-40 on PTH secretion in in vivo models of secondary hyperparathyroidism (HPT) in uremic rats. METHODS: PTH secretion was examined during ethyleneglycol tetraacetic acid (EGTA)-induced hypocalcemia both with and without PTHrP. Five groups, each of six normal rats, received a bolus of increasing doses of 0.1, 1.0, 10, and 100 microg of PTHrP 1-40, or vehicle only. Chronic renal failure (CRF) was induced by 5/6 nephrectomy. One group of 12 CRF rats received a standard diet, while another CRF group of 18 rats received a high phosphorus diet to induce more severe HPT. After 8 weeks of uremia, the rats were infused with EGTA and PTHrP 1-40 or vehicle. The presence of the PTH/PTHrP receptor in the rat parathyroid glands was examined by reverse transcription-polymerase chain reaction (RT-PCR) technique. PTH was measured by a rat PTH assay that does not cross-react with PTHrP. RESULTS: In a dose-related manner, PTHrP enhanced the PTH response to hypocalcemia in normal rats. A similar rate of decrease of plasma Ca++ was induced by EGTA in all experimental groups. In CRF rats, plasma creatinine (0.99 +/- 0.10 mmol/L vs. 0.33 +/- 0.01 mmol/L, P < 0.001) and plasma PTH (226 +/- 32 pg/mL vs. 69 +/- 16 pg/mL, P < 0.001) levels were significantly increased. The CRF rats on high phosphorus diet had significant hypocalcemia (Ca++, 1.04 +/- 0.02 mmol/L vs. 1.28 +/- 0.03 mmol/L, P < 0.001), hyperphosphatemia (3.48 +/- 0.3 mmol/L vs. 2.25 +/- 0.1 mmol/L, P < 0.001) and severe secondary HPT, PTH (984 +/- 52 pg/mL vs. 226 +/- 32 pg/mL, P < 0.001) compared to CRF rats on a standard phosphorus diet. The maximal PTH response to hypocalcemia was enhanced in CRF rats (maximum PTH 382 +/- 58 pg/mL vs. 196 +/- 29 pg/mL in normal rats, P < 0.01) and further enhanced by PTHrP 1-40 to 826 +/- 184 pg/mL (P < 0.01). The secretory capacity of the parathyroid glands in response to low Ca++ was severely diminished in uremia. In CRF rats given a high phosphorus diet, the basal PTH levels were at the upper part of the calcium/PTH curve, and the induction of more marked hypocalcemia did not stimulate PTH secretion further (maximum PTH 1475 +/- 208 pg/mL vs. basal 1097 +/- 69 pg/mL, NS). PTHrP, however, further enhanced the maximal PTH levels significantly (maximum PTH 3142 +/- 296 pg/mL, P < 0.01). The presence of the PTH/PTHrP receptor in the rat parathyroid glands was confirmed by RT-PCR technique. CONCLUSION: PTHrP enhanced significantly, in a dose-related manner, the low Ca++-stimulated PTH secretion in normal rats. The PTH/PTHrP receptor is present in rat parathyroid glands. The impaired secretory capacity of the parathyroid glands in uremic rats is significantly enhanced by PTHrP. An autocrine/paracrine role in the parathyroid glands of the PTH/PTHrP receptor targeting peptides, PTHrP and PTH, is suggested. Thus, it is hypothesized that PTH during hypocalcemia might have a positive auto-feedback regulatory role on its own secretion.


Assuntos
Homeostase , Glândulas Paratireoides/fisiopatologia , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Uremia/fisiopatologia , Animais , Relação Dose-Resposta a Droga , Hipocalcemia/metabolismo , Ligantes , Masculino , Glândulas Paratireoides/metabolismo , Hormônio Paratireóideo/sangue , Hormônio Paratireóideo/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Fósforo na Dieta/administração & dosagem , Ratos , Ratos Endogâmicos
18.
J Am Soc Nephrol ; 11(10): 1865-1872, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11004217

RESUMO

Calcitriol treatment is used to reduce parathyroid hormone levels in azotemic patients with secondary hyperparathyroidism (HPT). Whether long-term calcitriol administration reduces parathyroid gland size in patients with severe secondary hyperparathyroidism is not clear. The aim of the study was to evaluate in vitro the effect of calcitriol on parathyroid cell proliferation and apoptosis in normal parathyroid glands and in adenomatous and hyperplastic human parathyroid glands. Freshly harvested parathyroid glands from normal dogs and hyperplastic and adenomatous glands from patients with secondary (2 degrees) and primary (1 degree) HPT undergoing parathyroidectomy were studied. Flow cytometry was used to quantify the cell cycle and apoptosis of parathyroid cells. Apoptosis was also evaluated by DNA electrophoresis and light and electron microscopy. In normal dog parathyroid glands, culture with calcitriol (10(-10) to 10(-7) M) for 24 h produced a dose-dependent inhibitory effect on the progression of cells into the cell cycle and into apoptosis. When glands from patients with 2 degrees HPT were cultured for 24 h, only high calcitriol concentrations (10(-7) M) inhibited the progression through the cell cycle and the induction of apoptosis. In parathyroid adenomas (1 degrees HPT), even a high concentration of calcitriol (10(-7) M) had no significant effect on the cell cycle or apoptosis. The present study shows that in vitro, calcitriol inhibits in a dose-dependent manner in normal parathyroid glands both parathyroid cell proliferation and apoptosis. However, in secondary hyperplasia, only high concentrations of calcitriol inhibited cell proliferation and apoptosis. In 1 degree HPT, even high concentrations of calcitriol had no effect. Because calcitriol simultaneously inhibits both cell proliferation and apoptosis, a reduction in the parathyroid gland mass may not occur as a direct effect of calcitriol treatment.


Assuntos
Apoptose/efeitos dos fármacos , Calcitriol/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Glândulas Paratireoides/citologia , Glândulas Paratireoides/fisiologia , Adenoma/patologia , Animais , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Técnicas de Cultura , Cães , Humanos , Hiperplasia/patologia , Glândulas Paratireoides/patologia , Neoplasias das Paratireoides/patologia , Valores de Referência
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