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1.
PLoS One ; 15(8): e0237479, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790806

RESUMO

OBJECTIVE: As native cartilage consists of different phenotypical zones, this study aims to fabricate different types of neocartilage constructs from collagen hydrogels and human mesenchymal stromal cells (MSCs) genetically modified to express different chondrogenic factors. DESIGN: Human MSCs derived from bone-marrow of osteoarthritis (OA) hips were genetically modified using adenoviral vectors encoding sex-determining region Y-type high-mobility-group-box (SOX) 9, transforming growth factor beta (TGFB) 1 or bone morphogenetic protein (BMP) 2 cDNA, placed in type I collagen hydrogels and maintained in serum-free chondrogenic media for three weeks. Control constructs contained unmodified MSCs or MSCs expressing GFP. The respective constructs were analyzed histologically, immunohistochemically, biochemically, and by qRT-PCR for chondrogenesis and hypertrophy. RESULTS: Chondrogenesis in MSCs was consistently and strongly induced in collagen I hydrogels by the transgenes SOX9, TGFB1 and BMP2 as evidenced by positive staining for proteoglycans, chondroitin-4-sulfate (CS4) and collagen (COL) type II, increased levels of glycosaminoglycan (GAG) synthesis, and expression of mRNAs associated with chondrogenesis. The control groups were entirely non-chondrogenic. The levels of hypertrophy, as judged by expression of alkaline phosphatase (ALP) and COL X on both the protein and mRNA levels revealed different stages of hypertrophy within the chondrogenic groups (BMP2>TGFB1>SOX9). CONCLUSIONS: Different types of neocartilage with varying levels of hypertrophy could be generated from human MSCs in collagen hydrogels by transfer of genes encoding the chondrogenic factors SOX9, TGFB1 and BMP2. This technology may be harnessed for regeneration of specific zones of native cartilage upon damage.


Assuntos
Proteína Morfogenética Óssea 2/genética , Hidrogéis/química , Fatores de Transcrição SOX9/genética , Fator de Crescimento Transformador beta1/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Cartilagem/citologia , Cartilagem/metabolismo , Cartilagem/patologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Condrogênese/genética , Colágeno Tipo I/química , Colágeno Tipo X/genética , Meios de Cultura Livres de Soro/química , Glicosaminoglicanos/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , RNA Mensageiro/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
2.
Gene ; 757: 144852, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32599019

RESUMO

Until now, various methods have been introduced to fabricate 3D scaffolds to provide a suitable substrate for cell growth and proliferation and subsequent use in tissue engineering to repair damaged tissues. The 3D scaffolds can simulate the natural cellular microenvironment well. Herein, the decellularized leaf spinach has been used which not only have no problems associated with artificial scaffolds, but they also do not cost significantly. Decellularized scaffolds surface properties were characterized by the investigation of scaffolds surface roughness, hydrophilicity, mechanical properties, size and shape of porosities and specific surface area. In the next step, osteogenic differentiation potential of bone marrow derived mesenchymal stem cells cultured on the scaffold and culture plate (as a control) was evaluated using alizarin staining and calcium content, alkaline phosphatase activity and bone related genes expression assays. The results indicated that the surface properties and shape of scaffold pores were effective in the stem cells binding, growth and proliferation. This higher biocompatibility due to the ideal surface hydrophilicity as well as high specific surface area due to the presence of a rough grid surface ultimately increased the efficiency of stem cell's bone differentiation. Taken together, it can be concluded that the decellularized spinach leaf scaffold, due to its easy availability, low prices and high efficiency, can be considered as a promising potential candidate for use as a proper substrate for stem cell growth and differentiation in bone tissue engineering.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteocalcina/genética , Folhas de Planta/química , Tecidos Suporte/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Biomineralização , Cálcio/metabolismo , Linhagem Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteocalcina/metabolismo , Spinacia oleracea/química
3.
Int J Nanomedicine ; 15: 3281-3290, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32440124

RESUMO

Introduction: Cells exhibit high sensitivity and a diverse response to the nanotopography of the extracellular matrix, thereby endowing materials with instructive performances formerly reserved for growth factors. This finding leads to opportunities for improvement. However, the interplay between the topographical surface and cell behaviors remains incompletely understood. Methods: In the present study, we showed nanosurfaces with various dimensions of nanopits (200-750 nm) fabricated by self-assembling polystyrene (PS) nanospheres. Human adipose-derived stem cell behaviors, such as cell morphology, adhesion, cytoskeleton contractility, proliferation, and differentiation, were investigated on the prepared PS nanopit surface. Results: The osteogenic differentiation can be enhanced by nanopits with a diameter of 300-400 nm. Discussion: The present study provided exciting new avenues to investigate cellular responses to well-defined nanoscale topographic features, which could further guide bone tissue engineering and stem cell clinical research. The capability to control developing biomaterials mimicking nanotopographic surfaces promoted functional tissue engineering, such as artificial joint replacement, bone repair, and dental applications.


Assuntos
Tecido Adiposo/citologia , Diferenciação Celular , Nanoestruturas/química , Osteogênese , Poliestirenos/farmacologia , Células-Tronco/citologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Nanoestruturas/ultraestrutura , Osteogênese/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/ultraestrutura
4.
PLoS One ; 15(5): e0234073, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470062

RESUMO

Craniosynostosis is the premature fusion of cranial bones. The goal of this study was to determine if delivery of recombinant tissue nonspecific alkaline phosphatase (TNAP) could prevent or diminish the severity of craniosynostosis in a C57BL/6 FGFR2C342Y/+ model of neonatal onset craniosynostosis or a BALB/c FGFR2C342Y/+ model of postnatal onset craniosynostosis. Mice were injected with a lentivirus encoding a mineral targeted form of TNAP immediately after birth. Cranial bone fusion as well as cranial bone volume, mineral content and density were assessed by micro CT. Craniofacial shape was measured with calipers. Alkaline phosphatase, alanine amino transferase (ALT) and aspartate amino transferase (AST) activity levels were measured in serum. Neonatal delivery of TNAP diminished craniosynostosis severity from 94% suture obliteration in vehicle treated mice to 67% suture obliteration in treated mice, p<0.02) and the incidence of malocclusion from 82.4% to 34.7% (p<0.03), with no effect on cranial bone in C57BL/6 FGFR2C342Y/+ mice. In contrast, treatment with TNAP increased cranial bone volume (p< 0.01), density (p< 0.01) and mineral content (p< 0.01) as compared to vehicle treated controls, but had no effect on craniosynostosis or malocclusion in BALB/c FGFR2C342Y/+ mice. These results indicate that postnatal recombinant TNAP enzyme therapy diminishes craniosynostosis severity in the C57BL/6 FGFR2C342Y/+ neonatal onset mouse model of Crouzon syndrome, and that effects of exogenous TNAP are genetic background dependent.


Assuntos
Fosfatase Alcalina/genética , Disostose Craniofacial/terapia , Craniossinostoses/terapia , Técnicas de Transferência de Genes , Fosfatase Alcalina/sangue , Animais , Animais Recém-Nascidos , Peso Corporal , Densidade Óssea , Suturas Cranianas/patologia , Disostose Craniofacial/diagnóstico por imagem , Craniossinostoses/diagnóstico por imagem , Modelos Animais de Doenças , Fígado/enzimologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Microtomografia por Raio-X
5.
Arterioscler Thromb Vasc Biol ; 40(7): 1680-1694, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32375544

RESUMO

OBJECTIVE: The recessive disease arterial calcification due to deficiency of CD73 (ACDC) presents with extensive nonatherosclerotic medial layer calcification in lower extremity arteries. Lack of CD73 induces a concomitant increase in TNAP (tissue nonspecific alkaline phosphatase; ALPL), a key enzyme in ectopic mineralization. Our aim was to investigate how loss of CD73 activity leads to increased ALPL expression and calcification in CD73-deficient patients and assess whether this mechanism may apply to peripheral artery disease calcification. Approach and Results: We previously developed a patient-specific disease model using ACDC primary dermal fibroblasts that recapitulates the calcification phenotype in vitro. We found that lack of CD73-mediated adenosine signaling reduced cAMP production and resulted in increased activation of AKT. The AKT/mTOR (mammalian target of rapamycin) axis blocks autophagy and inducing autophagy prevented calcification; however, we did not observe autophagy defects in ACDC cells. In silico analysis identified a putative FOXO1 (forkhead box O1 protein) binding site in the human ALPL promoter. Exogenous AMP induced FOXO1 nuclear localization in ACDC but not in control cells, and this was prevented with a cAMP analogue or activation of A2a/2b adenosine receptors. Inhibiting FOXO1 reduced ALPL expression and TNAP activity and prevented calcification. Mutating the FOXO1 binding site reduced ALPL promoter activation. Importantly, we provide evidence that non-ACDC calcified femoropopliteal arteries exhibit decreased CD73 and increased FOXO1 levels compared with control arteries. CONCLUSIONS: These data show that lack of CD73-mediated cAMP signaling promotes expression of the human ALPL gene via a FOXO1-dependent mechanism. Decreased CD73 and increased FOXO1 was also observed in more common peripheral artery disease calcification.


Assuntos
5'-Nucleotidase/deficiência , Fibroblastos/enzimologia , Proteína Forkhead Box O1/metabolismo , Doença Arterial Periférica/enzimologia , Artéria Poplítea/enzimologia , Calcificação Vascular/enzimologia , 5'-Nucleotidase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Autofagia , Estudos de Casos e Controles , Células Cultivadas , Feminino , Fibroblastos/patologia , Proteína Forkhead Box O1/genética , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Humanos , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/genética , Doença Arterial Periférica/patologia , Artéria Poplítea/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Calcificação Vascular/genética , Calcificação Vascular/patologia , Adulto Jovem
6.
Life Sci ; 248: 117475, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32119963

RESUMO

AIMS: Liver fibrosis is a crucial pathological feature which could result in cirrhosis and hepatocarcinoma. But until now, there is no favourable treatment for it. Apigenin (APG) is a flavonoid, which exhibits efficient anti-liver fibrosis activity, but its underlying mechanisms were rarely studied. So this work aims to estimate the potential therapeutic action of APG on liver fibrosis rats and to gain insight into its system-level mechanisms. MAIN METHODS: Hepatic fibrosis was induced by CCl4 in Wistar rats, and APG was given in the light of the regimen. Biochemical indexes, histopathological change and immunohistochemistry of liver were evaluated. The optimal effect group of APG was selected for further transcriptomic and proteomic analysis. KEY FINDINGS: APG ameliorated liver fibrosis via reducing the levels of AST, ALT, ALP, LDH, Hyp, TP, TB, DB, HA, LN, PCIII and IV-C, mitigating fibrosis and inflammation of liver in H&E and Masson staining. Mechanistically, APG elevated the activity of ALB, SOD and GSH-PX with reducing the level of MDA. The results of microarray and TMT revealed that 4919 genes and 4876 proteins were differentially expressed in the APG and model groups. Besides, transcriptomics and proteomics analyses unfolded 120 overlapped proteins, enriched in 111 GO terms containing apoptotic process, angiogenesis, cell migration and proliferation, etc. Meanwhile, KEGG pathway analysis showed that 26 pathways containing HIF-1/MAPK/eNOS/VEGF/PI3K/Akt signaling pathway, regulation of actin cytoskeleton and focal adhesion mostly. SIGNIFICANCE: APG can ameliorate CCl4-induced liver fibrosis via VEGF-mediated FAK phosphorylation through the MAPKs, PI3K/Akt, HIF-1, ROS, and eNOS pathways, which may hopefully become the anti-liver fibrosis activity of natural product.


Assuntos
Anti-Inflamatórios/farmacologia , Apigenina/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Cirrose Hepática/prevenção & controle , Fígado/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Alanina Transaminase/genética , Alanina Transaminase/metabolismo , Albuminas/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Aspartato Aminotransferases/genética , Aspartato Aminotransferases/metabolismo , Bilirrubina/sangue , Tetracloreto de Carbono/administração & dosagem , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Malondialdeído/antagonistas & inibidores , Malondialdeído/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Proteômica/métodos , Ratos , Ratos Wistar , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
7.
Oxid Med Cell Longev ; 2020: 7242836, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32148656

RESUMO

Bone marrow-derived mesenchymal stem cells (MSCs) have shown great promise in tissue engineering and regenerative medicine; however, the regenerative capacity of senescent MSCs is greatly reduced, thus exhibiting limited therapy potential. Previous studies uncovered that microRNA-206 (miR-206) could largely regulate cell functions, including cell proliferation, survival, and apoptosis, but whether miR-206 is involved in the senescent process of MSCs remains unknown. In this study, we mainly elucidated the effects of miR-206 on MSC senescence and the underlying mechanism. We discovered that miR-206 was upregulated in the senescent MSCs induced by H2O2, and abrogation of miR-206 could alleviate this tendency. Besides, we determined that by targeting Alpl, miR-206 could ameliorate the impaired migration and paracrine function in MSCs reduced by H2O2. In vivo study, we revealed that inhibition of miR-206 in senescent MSCs could effectively protect their potential for myocardial infarction treatment in a rat MI model. In summary, we examined that inhibition of miR-206 in MSCs can alleviate H2O2-induced senescence and dysfunction, thus protecting its therapeutic potential.


Assuntos
Fosfatase Alcalina/genética , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Animais , Senescência Celular , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Humanos , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Transfecção
8.
PLoS One ; 15(2): e0228737, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32045441

RESUMO

BACKGROUND: Higher alanine transaminase (ALT), indicating poor liver function, is positively associated with diabetes but inversely associated with body mass index (BMI) in Mendelian randomization (MR) studies, suggesting liver function affects muscle mass. To clarify, we assessed the associations of liver enzymes with muscle and fat mass observationally with two-sample MR as a validation. METHODS: In the population-representative "Children of 1997" birth cohort (n = 3,455), we used multivariable linear regression to assess the adjusted associations of ALT and alkaline phosphatase (ALP) at ~17.5 years with muscle mass and body fat percentage observationally. Genetic variants predicting ALT, ALP and gamma glutamyltransferase (GGT) were applied to fat-free and fat mass in the UK Biobank (n = ~331,000) to obtain unconfounded MR estimates. RESULTS: Observationally, ALT was positively associated with muscle mass (0.11 kg per IU/L, 95% confidence interval (CI) 0.10 to 0.12) and fat percentage (0.15% per IU/L, 95% CI 0.13 to 0.17). ALP was inversely associated with muscle mass (-0.03 kg per IU/L, 95% CI -0.04 to -0.02) and fat percentage (-0.02% per IU/L, 95% CI -0.03 to -0.01). Using MR, ALT was inversely associated with fat-free mass (-0.41 kg per 100% in concentration, 95% CI -0.64 to -0.19) and fat mass (-0.58 kg per 100% in concentration, 95% CI -0.85 to -0.30). ALP and GGT were unclearly associated with fat-free mass or fat mass. CONCLUSION: ALT reducing fat-free mass provides a possible pathway for the positive association of ALT with diabetes and suggests a potential target of intervention.


Assuntos
Alanina Transaminase/metabolismo , Fosfatase Alcalina/metabolismo , Composição Corporal , Fígado/enzimologia , gama-Glutamiltransferase/metabolismo , Adolescente , Alanina Transaminase/genética , Fosfatase Alcalina/genética , Índice de Massa Corporal , Estudos de Coortes , Feminino , Força da Mão , Humanos , Modelos Lineares , Masculino , Análise da Randomização Mendeliana , Polimorfismo de Nucleotídeo Único , gama-Glutamiltransferase/genética
9.
Life Sci ; 248: 117455, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32088216

RESUMO

AIMS: Idiopathic scoliosis is a common deformity of the spine that has an especially high incidence rate in adolescents. Some studies have demonstrated a close relationship between idiopathic scoliosis and melatonin deficiency. Our team's previous research showed that melatonin can inhibit the proliferation of osteoblasts, but the mechanism remains unclear. This study aimed to determine the mechanism by which melatonin inhibits the proliferation of osteoblasts. MAIN METHODS: Cell viability experiment, DNA fragment detection and alkaline phosphatase (ALP) activity assays were performed to determine the effects of melatonin on the proliferation, apoptosis and differentiation of osteoblasts. We used immunofluorescence to detect the expression of STIM1 in melatonin-treated osteoblasts. STIM1 interference was achieved using a specific siRNA, and a TRPC inhibitor was used to block the influx of Ca2+. The mRNA expression was determined by RT-qPCR, and protein levels were measured by Western blot. KEY FINDINGS: In this study, we found that melatonin inhibited the proliferation, differentiation and apoptosis of osteoblasts in a concentration-dependent manner. Additional studies showed that melatonin elevated cytosolic calcium levels by upregulation of STIM1, leading to osteoblast apoptosis via the mitochondrial pathway. Finally, we demonstrated that the STIM1-mediated increase in cytosolic calcium levels induced apoptosis through the ERK pathway. SIGNIFICANCE: Melatonin induces mitochondrial apoptosis in osteoblasts by regulating the STIM1/cytosolic calcium elevation/ERK pathway. These basic findings provide a basis for further clinical studies on melatonin as a drug therapeutic for idiopathic scoliosis.


Assuntos
Antioxidantes/farmacologia , Cálcio/metabolismo , Sistema de Sinalização das MAP Quinases , Melatonina/farmacologia , Osteoblastos/efeitos dos fármacos , Molécula 1 de Interação Estromal/genética , Fosfatase Alcalina/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Transporte de Íons/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Molécula 1 de Interação Estromal/agonistas , Molécula 1 de Interação Estromal/antagonistas & inibidores , Molécula 1 de Interação Estromal/metabolismo , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo
10.
Life Sci ; 248: 117464, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32097667

RESUMO

AIMS: The present study was carried out to investigate the influences of Selenium/Zinc-Enriched probiotics (SeZnP) on growth performance, serum enzyme activity, antioxidant capability, inflammatory factors and gene expression associated with Wistar rats inflated under high ambient thermal-stress. MAIN METHODS: Sixty male rates with six-weeks of age were randomly allocated into five groups (12 per group) and fed basal diet (Control), basal diet supplemented with probiotics (P), Zinc-Enriched probiotics (ZnP, 100 mg/L), Selenium-Enriched Probiotics (SeP, 0.3 mg/L) and Selenium/Zinc-Enriched probiotics (SeZnP, 0.3 mg + 100 mg/L). The experiment lasted 30 days. Blood and Tissues samples were taken to investigate serum enzyme activity, antioxidants capability and inflammatory factors by using of commercial kits and antioxidant, heat shock and inflammatory related molecules expressions were determined by qRT-PCR. KEY FINDINGS: Data analysis revealed that thermal stress significantly increased the level of Aspartate-aminotransferase, Alanine-aminotransferase, Lactate-dehydrogenase, Creatine-kinase, blood urea nitrogen, Creatinine and Alkaline phosphatase compared to P, ZnP, SeP or SeZnP groups (P < 0.01). However, supplementation of ZnP, SeP, and SeZnP significantly enhanced glutathione content, glutathione-peroxidase & superoxide-dismutase activity, and decreased malondialdehyde content (P < 0.05). Moreover, the concentration of IL-2, IL-6 and IL-8 were significantly increased while IL-10 was significantly decreased (P < 0.05). Furthermore, the expression of GPx1 and SOD1 genes were significantly increased, but COX-2, iNOS, HSP70 and 90 mRNA levels were significantly decreased (P < 0.05). Finally, the highest influence of the mentioned parameters was observed in SeZnP supplemented group. SIGNIFICANCE: Our study suggests that SeZnP supplementation serves as possible and best nutritive than ZnP or SeP for Wistar rats raising under high ambient temperature.


Assuntos
Antioxidantes/administração & dosagem , Suplementos Nutricionais , Regulação da Expressão Gênica/efeitos dos fármacos , Resposta ao Choque Térmico/efeitos dos fármacos , Probióticos/administração & dosagem , Selênio/administração & dosagem , Zinco/administração & dosagem , Alanina Transaminase/genética , Alanina Transaminase/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Aspartato Aminotransferases/genética , Aspartato Aminotransferases/metabolismo , Nitrogênio da Ureia Sanguínea , Creatina Quinase/genética , Creatina Quinase/metabolismo , Creatinina/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Glutationa/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Resposta ao Choque Térmico/genética , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Masculino , Malondialdeído/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Wistar , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
11.
Nat Genet ; 52(2): 167-176, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959995

RESUMO

The kidneys integrate information from continuous systemic processes related to the absorption, distribution, metabolism and excretion (ADME) of metabolites. To identify underlying molecular mechanisms, we performed genome-wide association studies of the urinary concentrations of 1,172 metabolites among 1,627 patients with reduced kidney function. The 240 unique metabolite-locus associations (metabolite quantitative trait loci, mQTLs) that were identified and replicated highlight novel candidate substrates for transport proteins. The identified genes are enriched in ADME-relevant tissues and cell types, and they reveal novel candidates for biotransformation and detoxification reactions. Fine mapping of mQTLs and integration with single-cell gene expression permitted the prioritization of causal genes, functional variants and target cell types. The combination of mQTLs with genetic and health information from 450,000 UK Biobank participants illuminated metabolic mediators, and hence, novel urinary biomarkers of disease risk. This comprehensive resource of genetic targets and their substrates is informative for ADME processes in humans and is relevant to basic science, clinical medicine and pharmaceutical research.


Assuntos
Biotransformação/genética , Rim/metabolismo , Locos de Características Quantitativas , Insuficiência Renal Crônica/urina , Acetiltransferases/genética , Acetiltransferases/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Biomarcadores/urina , Estudos de Coortes , Citocromo P-450 CYP2D6/genética , Estudo de Associação Genômica Ampla , Humanos , Inativação Metabólica , Rim/citologia , Metoprolol/farmacocinética , Polimorfismo de Nucleotídeo Único , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo , Urina/fisiologia , Xenobióticos/farmacocinética , Xenobióticos/urina
12.
Am J Physiol Lung Cell Mol Physiol ; 318(2): L356-L365, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31800264

RESUMO

Airway surface dehydration is a pathological feature of cystic fibrosis (CF) lung disease. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated Cl- channel controlled in part by the adenosine A2B receptor. An alternative CFTR-independent mechanism of fluid secretion is regulated by ATP via the P2Y2 receptor (P2Y2R) that activates Ca2+-regulated Cl- channels (CaCC/TMEM16) and inhibits Na+ absorption. However, due to rapid ATP hydrolysis, steady-state ATP levels in CF airway surface liquid (ASL) are inadequate to maintain P2Y2R-mediated fluid secretion. Therefore, inhibiting airway epithelial ecto-ATPases to increase ASL ATP levels constitutes a strategy to restore airway surface hydration in CF. Using [γ32P]ATP as radiotracer, we assessed the effect of a series of ATPase inhibitory compounds on the stability of physiologically occurring ATP concentrations. We identified the polyoxometalate [Co4(H2O)2(PW9O34)2]10- (POM-5) as the most potent and effective ecto-ATPase inhibitor in CF airway epithelial cells. POM-5 caused long-lasting inhibition of ATP hydrolysis in airway epithelia, which was reversible upon removal of the inhibitor. Importantly, POM-5 markedly enhanced steady-state levels of released ATP, promoting increased ASL volume in CF cell surfaces. These results provide proof of concept for ecto-ATPase inhibitors as therapeutic agents to restore hydration of CF airway surfaces. As a test of this notion, cell-free sputum supernatants from CF subjects were studied and found to have abnormally elevated ATPase activity, which was markedly inhibited by POM-5.


Assuntos
Trifosfato de Adenosina/metabolismo , Fibrose Cística/metabolismo , Mucosa Respiratória/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Brônquios/patologia , Fibrose Cística/patologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Hidrólise , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/patologia , Escarro/enzimologia , Compostos de Tungstênio/farmacologia
13.
Gene ; 726: 144193, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31669647

RESUMO

OBJECTIVE: miR-199a-5p was increased during osteoblast differentiation, which may target and regulate TET2, a gene attracted a lot of attention in the osteoblast differentiation in the past few years. However, the role of miR-199a-5p in osteoblast differentiation by targeting TET2 is not established. METHODS: The correlation between miR-199a-5p and TET2 was verified through dual luciferase reporter assay, and their expressions in human bone marrow stromal cells (hBMSCs) during the osteoblast differentiation were detected. hBMSCs were transfected with TET2 siRNA, miR-199a-5p mimic or/and TET2 CRISPR activation plasmid., and then prepared for the induction of osteoblast differentiation, followed by alkaline phosphatase (ALP) and alizarin red staining, qRT-PCR and Western blotting. In vivo, ovariectomized (OVX) mice were injected with agomir-miR-199a-5p, antagomiR-199a-5p or/and TET2 siRNA to calculate the BMD and BV/TV ratio of mice, as well as to measure the expressions of osteogenesis-related genes in bone tissues. RESULTS: A gradual increase of miR-199a-5p was observed in hBMSCs during the induction of osteoblast differentiation, while TET2 expression was decreased. Besides, miR-199a-5p was reduced in the bone tissue of OVX mice, while TET2 was up-regulated. In addition, overexpression of miR-199a-5p and inhibition of TET2 augmented ALP activity in hBMSCs, with the enhanced calcification and the up-regulated expressions of Runx2, OSX and OCN, which also increased the quality of bone in OVX mice accompanying the enhancement BV/TV ratio, BMD and osteogenesis-related genes. CONCLUSION: MiR-199a-5p may promote the osteoblast differentiation and prevent OVX-induced osteoporosis by targeting TET2.


Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , MicroRNAs/genética , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas/genética , Fosfatase Alcalina/genética , Animais , Células da Medula Óssea/metabolismo , Osso e Ossos/metabolismo , Calcificação Fisiológica/genética , Células Cultivadas , Feminino , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteogênese/genética , Regulação para Cima/genética
14.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140291, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678193

RESUMO

The biocatalytic activity of a so far underexploited alkaline phosphatase, PhoK from Sphingomonas sp. BSAR-1, was extensively studied in transphosphorylation and hydrolysis reactions. The use of high-energy phosphate donors and oligophosphates as suitable phosphate donors was evaluated, as well as the hydrolytic activity on a variety of phosphate monoesters. While substrates bearing free hydroxy group displayed only moderate reactivity as acceptors for transphosphorylation by PhoK, strong hydrolytic activity on a broad variety of phosphate monoesters under alkaline conditions was observed. Site-directed mutagenesis of selected amino acid residues in the active site provided valuable insights on their involvement in enzyme catalysis. The key residue Thr89 so far postulated to engage in enzyme phosphorylation was confirmed to be crucial for catalysis and could be replaced by serine, albeit with much lower catalytic efficiency.


Assuntos
Fosfatase Alcalina/química , Proteínas de Bactérias/química , Ésteres/química , Fosfatos/química , Sphingomonas/enzimologia , Fosfatase Alcalina/genética , Proteínas de Bactérias/genética , Biocatálise , Hidrólise , Fosforilação , Treonina/química
15.
Arch Oral Biol ; 109: 104582, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31605918

RESUMO

OBJECTIVE: The aim of this study was to evaluate the proliferation and odontogenic differentiation of human dental pulp cells (hDPCs) and human umbilical cord mesenchymal stem cells (hUCMSCs) in three-dimensional co-culture system which was established with the help of bone morphogenetic protein-2 (BMP-2) and hydrogel. METHODS: hDPCs and hUCMSCs were cultured in different concentrations of hydrogel to explore the more suitable concentrations for subsequent experiments. hUCMSCs and hDPCs induced by BMP-2 were co-cultured in the hydrogel. MTT assay was used to measure the cell viability. The differentiation into odontoblast-like cells were measured by the mRNA expression of dentin salivary phosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), alkaline phosphatase and osteocalcin. Alizarin red staining was performed for the formation of mineralized nodules. RESULTS: hUCMSCs and hDPCs could grow and proliferate in hydrogel scaffold. The growth rate of cells in lower concentrations hydrogels were higher than that of high concentrations hydrogels (P < 0.05). The study showed that 0.25% hydrogel scaffold was more suitable for subsequent experiments than other groups. Compared with hUCMSCs-monoculture and hDPCs-monoculture, the co-culture groups exhibited more proliferative potential, alkaline phosphatase activity and mineralization nodule formation (P < 0.05). The mRNA expression in co-culture groups were higher than that of hUCMSCs-monoculture, closed to or even higher than that of hDPCs-monoculture. CONCLUSION: 0.25% hydrogel was the suitable concentration in co-culture system for subsequent experiments. The co-culture groups had stronger abilities of odontoblastic differentiation and mineralization than cells-monoculture groups, indicated that the co-culture conditions could regulate cell proliferation and differentiation within a certain range.


Assuntos
Diferenciação Celular , Polpa Dentária/citologia , Hidrogéis , Células-Tronco Mesenquimais/citologia , Fosfatase Alcalina/genética , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Proteínas da Matriz Extracelular/genética , Humanos , Odontoblastos/citologia , Osteocalcina/genética , Fosfoproteínas/genética , Sialoglicoproteínas/genética , Cordão Umbilical/citologia
16.
Ann Anat ; 227: 151420, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31563571

RESUMO

BACKGROUND: Cholesterol esters (CEs) accumulate in the cells of the adrenal cortex and are used for the synthesis of steroid hormones. The full molecular pathways involved in mediating the accumulation of CEs within the adrenal cortex are yet to be elucidated. Tissue non-specific alkaline phosphatase (TNAP) is needed for intracellular lipid accumulation of triglycerides in adipocytes and is also expressed in the cortical cells of the adrenal gland. Therefore we aimed to determine if TNAP is needed for the accumulation of CEs within the murine Y1 adrenal cortex cell line. METHODS: Y1 cells were induced to accumulate lipids. Lipid accumulation and TNAP activity and expression were determined throughout intracellular lipid accumulation. The location of TNAP within the cell was determined through immunohistochemical analysis. Lipid accumulation in the cells was associated with a rise in TNAP activity and TNAP was localised to lipid droplets within the Y1 cells. Inhibition of TNAP with a specific inhibitor (levamisole) resulted in the cessation of CE accumulation. DISCUSSION AND CONCLUSIONS: These data demonstrate that TNAP plays a role in the control of lipid accumulation in this adrenal cortex cell line. Therefore, in both triglyceride and CE storing cell types TNAP would seem to be essential for intra-cellular lipid storage.


Assuntos
Córtex Suprarrenal/metabolismo , Fosfatase Alcalina/metabolismo , Ésteres do Colesterol/metabolismo , Córtex Suprarrenal/citologia , Córtex Suprarrenal/efeitos dos fármacos , Fosfatase Alcalina/análise , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/genética , Animais , Compostos Azo , Linhagem Celular , Corantes , Corantes Fluorescentes , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Levamisol/farmacologia , Lipídeos/química , Camundongos , Oxazinas , Regulação para Cima
17.
J Pathol ; 250(1): 30-41, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31509234

RESUMO

Medial arterial calcification (MAC) is a major complication of chronic kidney disease (CKD) and an indicator of poor prognosis. Aortic overexpression of tissue-nonspecific alkaline phosphatase (TNAP) accelerates MAC formation. The present study aimed to assess whether a TNAP inhibitor, SBI-425, protects against MAC and improves survival probability in a CKD-mineral and bone disorder (MBD) mouse model. CKD-MBD mice were divided in three groups: vehicle, SBI-10, and SBI-30. They were fed a 0.2% adenine and 0.8% phosphorus diet from 14 to 20 weeks of age to induce CKD, followed by a high-phosphorus (0.2% adenine and 1.8% phosphorus) diet for another 6 weeks. At 14-20 weeks of age, mice in the SBI-10 and SBI-30 groups were given 10 and 30 mg/kg SBI-425 by gavage once a day, respectively, while vehicle-group mice were given distilled water as vehicle. Control mice were fed a standard chow (0.8% phosphorus) between the ages of 8 and 20 weeks. Computed tomography imaging, histology, and aortic tissue calcium content revealed that, compared to vehicle animals, SBI-425 nearly halted the formation of MAC. Mice in the control, SBI-10 and SBI-30 groups exhibited 100% survival, which was significantly better than vehicle-treated mice (57.1%). Aortic mRNA expression of Alpl, encoding TNAP, as well as plasma and aortic tissue TNAP activity, were suppressed by SBI-425 administration, whereas plasma pyrophosphate increased. We conclude that a TNAP inhibitor successfully protected the vasculature from MAC and improved survival rate in a mouse CKD-MBD model, without causing any adverse effects on normal skeletal formation and residual renal function. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Fosfatase Alcalina/antagonistas & inibidores , Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Distúrbio Mineral e Ósseo na Doença Renal Crônica/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Niacinamida/análogos & derivados , Sulfonamidas/farmacologia , Calcificação Vascular/prevenção & controle , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Aorta/enzimologia , Aorta/patologia , Doenças da Aorta/enzimologia , Doenças da Aorta/etiologia , Doenças da Aorta/patologia , Distúrbio Mineral e Ósseo na Doença Renal Crônica/complicações , Distúrbio Mineral e Ósseo na Doença Renal Crônica/enzimologia , Distúrbio Mineral e Ósseo na Doença Renal Crônica/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Niacinamida/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Osteoblastos/patologia , Fatores de Tempo , Calcificação Vascular/enzimologia , Calcificação Vascular/etiologia , Calcificação Vascular/patologia
18.
Ecotoxicol Environ Saf ; 188: 109904, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31704326

RESUMO

Tetrabromobisphenol A (TBBPA) is the largest brominated flame retardant which can be released to environment and cause long-term hazard. In this work, we developed a rapid and highly sensitive fluorescence enzyme-linked immunosorbent assay (FELISA) for monitoring of TBBPA in soil samples. TBBPA specific nanobody derived from camelid was fused with alkaline phosphatase to obtain the bi-functional fusion protein, which enable the specific binding of TBBPA and the generation of detection signal simultaneously. The assay showed an IC50 of 0.23 ng g-1, limit detection of 0.05 ng g-1 and linear range from 0.1 to 0.55 ng g-1 for TBBPA in soil samples. Due to the high resistance to organic solvents of the fusion protein, a simple pre-treatment by using 40% dimethyl sulfoxide (DMSO) as extract solvent can eliminate matrix effect and obtain good recoveries (ranging from 93.4% to 112.4%) for spiked soil samples. Good relationship between the results of the proposed FELISA and that of liquid chromatography tandem mass spectrometry (LC-MS/MS) was obtained, which indicated it could be a powerful analytical tool for determination of TBBPA to monitor human and environmental exposure.


Assuntos
Monitoramento Ambiental/métodos , Ensaio de Imunoadsorção Enzimática , Retardadores de Chama/análise , Bifenil Polibromatos/análise , Poluentes do Solo/análise , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Camelídeos Americanos , Limite de Detecção , Bifenil Polibromatos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo
19.
J Hum Genet ; 65(3): 337-343, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31857675

RESUMO

Although alkaline phosphatase (ALP) activity is relatively low in carriers of recessive type hypophosphatasia (HPP), most are asymptomatic and therefore do not undergo medical evaluations. We analyzed the association of ALP-encoding ALPL variants with serum ALP and bone traits in the general Japanese population. Study participants (n = 9671) were from the Nagahama Study, which was a longitudinal cohort study of an apparently healthy general Japanese population. ALPL variants were analyzed by whole-genome sequencing or TaqMan probe assays using DNA extracted from peripheral blood samples. The speed of sound in calcaneal bone was assessed by quantitative ultrasound (QUS) and used as surrogate measures of bone mineral density. We identified 13 ALPL variants. Minor allele frequencies of three variants were higher than expected. Variant c.529G > A has been reported as a possible pathogenic variant for adult type HPP. Variants c.979C > T and c.1559delT are reported as pathogenic variants for perinatal severe HPP or infantile HPP. The allele frequencies of c.529G > A, c.979C > T, and c.1559delT were 0.0107, 0.0040, and 0.0014, respectively. Serum ALP activity was significantly lower and differed among the three variants (P < 0.001), as well as between individuals with and without any of the three variants (P < 0.001). Serum ALP activity was inversely associated with QUS values, although no direct association was observed between the ALPL variants and QUS values. An association between serum ALP activity and QUS was confirmed; however, we failed to detect an association between ALPL variants and bone traits in the general Japanese population.


Assuntos
Fosfatase Alcalina/genética , Densidade Óssea/genética , Desenvolvimento Ósseo/genética , Predisposição Genética para Doença , Adulto , Fosfatase Alcalina/sangue , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Análise Mutacional de DNA , Feminino , Humanos , Hipofosfatasia/sangue , Hipofosfatasia/epidemiologia , Hipofosfatasia/genética , Japão/epidemiologia , Estudos Longitudinais , Masculino , Fenótipo , Gravidez , Sequenciamento Completo do Genoma
20.
Cell Mol Biol Lett ; 24: 64, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31827540

RESUMO

Background: Hypoxia in the vicinity of bone defects triggers the osteogenic differentiation of precursor cells and promotes healing. The activation of STAT3 signaling in mesenchymal stem cells (MSCs) has similarly been reported to mediate bone regeneration. However, the interaction between hypoxia and STAT3 signaling in the osteogenic differentiation of precursor cells during bone defect healing is still unknown. Methods: In this study, we assessed the impact of different durations of CoCl2-induced cellular hypoxia on the osteogenic differentiation of MSCs. Role of STAT3 signaling on hypoxia induced osteogenic differentiation was analyzed both in vitro and in vivo. The interaction between cellular hypoxia and STAT3 signaling in vivo was investigated in a mouse femoral bone defect model. Results: The peak osteogenic differentiation and expression of vascular endothelial growth factor (VEGF) occurred after 3 days of hypoxia. Inhibiting STAT3 reversed this effect. Hypoxia enhanced the expression of hypoxia-inducible factor 1-alpha (HIF-1α) and STAT3 phosphorylation in MSCs. Histology and µ-CT results showed that CoCl2 treatment enhanced bone defect healing. Inhibiting STAT3 reduced this effect. Immunohistochemistry results showed that CoCl2 treatment enhanced Hif-1α, ALP and pSTAT3 expression in cells present in the bone defect area and that inhibiting STAT3 reduced this effect. Conclusions: The in vitro study revealed that the duration of hypoxia is crucial for osteogenic differentiation of precursor cells. The results from both the in vitro and in vivo studies show the role of STAT3 signaling in hypoxia-induced osteogenic differentiation of precursor cells and bone defect healing.


Assuntos
Regeneração Óssea/genética , Células-Tronco Mesenquimais/metabolismo , Fator de Transcrição STAT3/genética , Transdução de Sinais , Cicatrização/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular , Hipóxia Celular/genética , Cobalto/farmacologia , Fêmur/citologia , Fêmur/metabolismo , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/genética , Fosforilação , Cultura Primária de Células , Fator de Transcrição STAT3/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
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