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1.
Am J Pathol ; 186(4): 927-37, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26968113

RESUMO

Rheumatoid arthritis is a chronic disease that results in a disabling and painful condition as it progresses to destruction of the articular cartilage and ankylosis of the joints. Although the cause of the disease is still unknown, evidence argues that autoimmunity plays an important part. There are increasing but contradictory views regarding serotonin being associated with activation of immunoinflammatory pathways and the onset of autoimmune reactions. We studied serotonin's involvement during collagen-induced arthritis in wild-type and Tph1(-/-) mice, which have markedly reduced peripheral serotonin levels. In wild-type mice, induction of arthritis triggered a robust increase in serotonin content in the paws combined with less inflammation. In Tph1(-/-) mice with arthritis, a marked increase in the clinical and pathologic arthritis scores was noticed. Specifically, in Tph1(-/-) mice with arthritis, a significant increase in osteoclast differentiation and bone resorption was observed with an increase in IL-17 levels in the paws and in Th17 lymphocytes in the draining lymph nodes, whereas T-regulatory cells were dampened. Ex vivo serotonin and agonists of the 5-HT2A and 5-HT2B receptors restored IL-17 secretion from splenocytes and Th17 cell differentiation in Tph1(-/-) mice. These findings indicate that serotonin plays a fundamental role in arthritis through the regulation of the Th17/T-regulatory cell balance and osteoclastogenesis.


Assuntos
Artrite Experimental/patologia , Doenças Autoimunes/imunologia , Reabsorção Óssea/patologia , Serotonina/metabolismo , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Artrite Experimental/imunologia , Doenças Autoimunes/patologia , Reabsorção Óssea/imunologia , Diferenciação Celular , Modelos Animais de Doenças , Camundongos Knockout , Serotonina/imunologia
2.
Purinergic Signal ; 12(2): 247-58, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26861849

RESUMO

Extracellular ATP, signalling through P2 receptors, exerts well-documented effects on bone cells, inhibiting mineral deposition by osteoblasts and stimulating the formation and resorptive activity of osteoclasts. The aims of this study were to determine the potential osteotropic effects of adenosine, the hydrolysis product of ATP, on primary bone cells in vitro. We determined the effect of exogenous adenosine on (1) the growth, alkaline phosphatase (TNAP) activity and bone-forming ability of osteoblasts derived from the calvariae of neonatal rats and mice and the marrow of juvenile rats and (2) the formation and resorptive activity of osteoclasts from juvenile mouse marrow. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed marked differences in the expression of P1 receptors in osteoblasts from different sources. Whilst mRNA for the A1 and A2B receptors was expressed by all primary osteoblasts, A2A receptor expression was limited to rat bone marrow and mouse calvarial osteoblasts and the A3 receptor to rat bone marrow osteoblasts. We found that adenosine had no detectable effects on cell growth, TNAP activity or bone formation by rodent osteoblasts in vitro. The analogue 2-chloroadenosine, which is hydrolysed more slowly than adenosine, had no effects on rat or mouse calvarial osteoblasts but increased TNAP activity and bone formation by rat bone marrow osteoblasts by 30-50 % at a concentration of 1 µM. Osteoclasts were found to express the A2A, A2B and A3 receptors; however, neither adenosine (≤100 µM) nor 2-chloroadenosine (≤10 µM) had any effect on the formation or resorptive activity of mouse osteoclasts in vitro. These results suggest that adenosine, unlike ATP, is not a major signalling molecule in the bone.


Assuntos
Adenosina/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Adenosina/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Western Blotting , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Proc Natl Acad Sci U S A ; 110(46): 18644-9, 2013 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-24167258

RESUMO

Although hyponatremia is known to be associated with osteoporosis and a high fracture risk, the mechanism through which bone loss ensues has remained unclear. As hyponatremic patients have elevated circulating arginine-vasopressin (AVP) levels, we examined whether AVP can affect the skeleton directly as yet another component of the pituitary-bone axis. Here, we report that the two Avp receptors, Avpr1α and Avpr2, coupled to Erk activation, are expressed in osteoblasts and osteoclasts. AVP injected into wild-type mice enhanced and reduced, respectively, the formation of bone-resorbing osteoclasts and bone-forming osteoblasts. Conversely, the exposure of osteoblast precursors to Avpr1α or Avpr2 antagonists, namely SR49059 or ADAM, increased osteoblastogenesis, as did the genetic deletion of Avpr1α. In contrast, osteoclast formation and bone resorption were both reduced in Avpr1α(-/-) cultures. This process increased bone formation and reduced resorption resulted in a profound enhancement of bone mass in Avpr1α(-/-) mice and in wild-type mice injected with SR49059. Collectively, the data not only establish a primary role for Avp signaling in bone mass regulation, but also call for further studies on the skeletal actions of Avpr inhibitors used commonly in hyponatremic patients.


Assuntos
Arginina Vasopressina/metabolismo , Remodelação Óssea/fisiologia , Reabsorção Óssea/etiologia , Homeostase/fisiologia , Hiponatremia/complicações , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Knockout , Receptores de Vasopressinas/genética
4.
Sci Rep ; 13(1): 6783, 2023 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-37100808

RESUMO

Idiopathic scoliosis (IS) is the deformation and/or abnormal curvature of the spine that develops progressively after birth. It is a very common condition, affecting approximately 4% of the general population, yet the genetic and mechanistic causes of IS are poorly understood. Here, we focus on PPP2R3B, which encodes a protein phosphatase 2A regulatory subunit. We found that PPP2R3B is expressed at sites of chondrogenesis within human foetuses, including the vertebrae. We also demonstrated prominent expression in myotome and muscle fibres in human foetuses, and zebrafish embryos and adolescents. As there is no rodent orthologue of PPP2R3B, we used CRIPSR/Cas9-mediated gene-editing to generate a series of frameshift mutations in zebrafish ppp2r3b. Adolescent zebrafish that were homozygous for this mutation exhibited a fully penetrant kyphoscoliosis phenotype which became progressively worse over time, mirroring IS in humans. These defects were associated with reduced mineralisation of vertebrae, resembling osteoporosis. Electron microscopy demonstrated abnormal mitochondria adjacent to muscle fibres. In summary, we report a novel zebrafish model of IS and reduced bone mineral density. In future, it will be necessary to delineate the aetiology of these defects in relation to bone, muscle, neuronal and ependymal cilia function.


Assuntos
Escoliose , Peixe-Zebra , Animais , Adolescente , Humanos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Escoliose/genética , Sistemas CRISPR-Cas , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Mutação
5.
Sci Data ; 5: 180100, 2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29809174

RESUMO

Micro-computed tomography (micro-CT) is commonly used to assess bone quality and to evaluate the outcome of experimental therapies in animal models of bone diseases. Generating large datasets is however challenging and data are rarely made publicly available through shared repositories. Here we describe a dataset of micro-CT reconstructed scans of the proximal part of 21 tibiae from wild-type mice, osteogenesis imperfecta mice (homozygous oim/oim) and oim/oim mice transplanted with human amniotic fluid stem cells. The dataset contains, for each sample, 991 8-bit Bitmap reconstructed images and a 3D reconstruction of the bone in the PLY format, available at the online repository Figshare. In line with the increasing effort to make scientific datasets open-access, our data can be downloaded and used by other researchers to compare their observations with ours and to directly test scientific questions on osteogenesis imperfecta bones without the need to generate complete datasets.


Assuntos
Osteogênese Imperfeita , Transplante de Células-Tronco , Animais , Humanos , Camundongos , Osteogênese Imperfeita/diagnóstico por imagem , Osteogênese Imperfeita/patologia , Osteogênese Imperfeita/terapia , Células-Tronco/patologia , Tíbia/diagnóstico por imagem , Microtomografia por Raio-X
6.
Sci Rep ; 8(1): 2425, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29402914

RESUMO

Human amniotic fluid contains two morphologically-distinct sub-populations of stem cells with regenerative potential, spindle-shaped (SS-hAFSCs) and round-shaped human amniotic fluid stem cells (RS-hAFSCs). However, it is unclear whether morphological differences correlate with functionality, and this lack of knowledge limits their translational applications. Here, we show that SS-hAFSCs and RS-hAFSCs differ in their neuro-protective ability, demonstrating that a single contralateral injection of SS-hAFSCs into hypoxic-ischemic P7 mice conferred a 47% reduction in hippocampal tissue loss and 43-45% reduction in TUNEL-positive cells in the hippocampus and striatum 48 hours after the insult, decreased microglial activation and TGFß1 levels, and prevented demyelination. On the other hand, RS-hAFSCs failed to show such neuro-protective effects. It is possible that SS-hAFSCs exert their neuroprotection via endoglin-dependent inhibition of TGFß1 signaling in target cells. These findings identify a sub-population of CD117+CD90+CD105+ stem cells as a promising source for the neuro-protection of the developing brain.


Assuntos
Líquido Amniótico/citologia , Isquemia Encefálica/terapia , Doenças Desmielinizantes/prevenção & controle , Hipóxia/prevenção & controle , Neuroproteção/fisiologia , Transplante de Células-Tronco , Células-Tronco/citologia , Líquido Amniótico/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptose , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Linhagem da Célula , Terapia Baseada em Transplante de Células e Tecidos/métodos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Endoglina/genética , Endoglina/metabolismo , Expressão Gênica , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Hipóxia/patologia , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/patologia , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
7.
Stem Cells Transl Med ; 7(5): 439-449, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29489062

RESUMO

Human mesenchymal stem cells (MSCs) have huge potential for regenerative medicine. In particular, the use of pluripotent stem cell-derived mesenchymal stem cells (PSC-MSCs) overcomes the hurdle of replicative senescence associated with the in vitro expansion of primary cells and has increased therapeutic benefits in comparison to the use of various adult sources of MSCs in a wide range of animal disease models. On the other hand, fetal MSCs exhibit faster growth kinetics and possess longer telomeres and a wider differentiation potential than adult MSCs. Here, for the first time, we compare the therapeutic potential of PSC-MSCs (ES-MSCs from embryonic stem cells) to fetal MSCs (AF-MSCs from the amniotic fluid), demonstrating that ES-MSCs have a superior neuroprotective potential over AF-MSCs in the mouse brain following hypoxia-ischemia. Further, we demonstrate that nuclear factor (NF)-κB-stimulated interleukin (IL)-13 production contributes to an increased in vitro anti-inflammatory potential of ES-MSC-conditioned medium (CM) over AF-MSC-CM, thus suggesting a potential mechanism for this observation. Moreover, we show that induced pluripotent stem cell-derived MSCs (iMSCs) exhibit many similarities to ES-MSCs, including enhanced NF-κB signaling and IL-13 production in comparison to AF-MSCs. Future studies should assess whether iMSCs also exhibit similar neuroprotective potential to ES-MSCs, thus presenting a potential strategy to overcome the ethical issues associated with the use of embryonic stem cells and providing a potential source of cells for autologous use against neonatal hypoxic-ischemic encephalopathy in humans. Stem Cells Translational Medicine 2018;7:439-449.


Assuntos
Encéfalo/patologia , Células-Tronco Embrionárias/citologia , Células-Tronco Fetais/citologia , Hipóxia/patologia , Células-Tronco Mesenquimais/citologia , Neuroproteção/fisiologia , Líquido Amniótico/citologia , Animais , Encéfalo/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Células-Tronco Fetais/metabolismo , Células HEK293 , Humanos , Hipóxia/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Isquemia/metabolismo , Isquemia/patologia , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Medicina Regenerativa/métodos , Transdução de Sinais/fisiologia
8.
Sci Rep ; 6: 39656, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-27995994

RESUMO

The impaired maturation of bone-forming osteoblasts results in reduced bone formation and subsequent bone weakening, which leads to a number of conditions such as osteogenesis imperfecta (OI). Transplantation of human fetal mesenchymal stem cells has been proposed as skeletal anabolic therapy to enhance bone formation, but the mechanisms underlying the contribution of the donor cells to bone health are poorly understood and require further elucidation. Here, we show that intraperitoneal injection of human amniotic mesenchymal stem cells (AFSCs) into a mouse model of OI (oim mice) reduced fracture susceptibility, increased bone strength, improved bone quality and micro-architecture, normalised bone remodelling and reduced TNFα and TGFß sigalling. Donor cells engrafted into bones and differentiated into osteoblasts but importantly, also promoted endogenous osteogenesis and the maturation of resident osteoblasts. Together, these findings identify AFSC transplantation as a countermeasure to bone fragility. These data have wider implications for bone health and fracture reduction.


Assuntos
Âmnio/citologia , Fraturas Ósseas/prevenção & controle , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Osteogênese Imperfeita/prevenção & controle , Animais , Remodelação Óssea , Osso e Ossos/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Marcadores Genéticos , Humanos , Masculino , Camundongos , Osteoblastos/metabolismo , Osteogênese , Estresse Mecânico , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Microtomografia por Raio-X
9.
Stem Cells Dev ; 25(5): 395-404, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26728561

RESUMO

Alport syndrome (AS) is a hereditary glomerulopathy caused by a mutation in type IV collagen genes, which disrupts glomerular basement membrane, leading to progressive glomerulosclerosis and end-stage renal failure. There is at present no cure for AS, and cell-based therapies offer promise to improve renal function. In this study, we found that human first trimester fetal chorionic stem cells (CSC) are able to migrate to glomeruli and differentiate down the podocyte lineage in vitro and in vivo. When transplanted into 7-week-old Alport 129Sv-Col4α3(tm1Dec)/J (-/-) mice, a single intraperitoneal injection of CSC significantly lowered blood urea and urine proteinuria levels over the ensuing 2 weeks. In addition, nearly two-thirds of transplanted -/- mice maintained their weight above the 80% welfare threshold, with both males and females weighing more than age-matched nontransplanted -/- mice. This was associated with less renal cortical fibrosis and interstitial inflammation compared to nontransplanted mice as shown by reduction in murine CD4, CD68, and CD45.2 cells. Transplanted CSC homed to glomeruli, where they expressed CR1, VEGFA, SYNAPTOPODIN, CD2AP, and PODOCIN at the RNA level and produced PODOCIN, CD2AP, and COLIVα3 proteins in nontransplanted -/- mice, indicating that CSC have adopted a podocyte phenotype. Together, these data indicate that CSC may be used to delay progression of renal pathology by a combination of anti-inflammatory effects and replacement of the defective resident podocytes.


Assuntos
Diferenciação Celular , Córion/citologia , Nefrite Hereditária/terapia , Podócitos/citologia , Células-Tronco/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Córion/transplante , Técnicas de Cocultura , Colágeno Tipo IV/farmacologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Fibrose , Humanos , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Córtex Renal/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Mutação/genética , Nefrite Hereditária/patologia , Fenótipo , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo
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