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1.
Cartilage ; : 1947603520903425, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32059614

RESUMO

OBJECTIVE: Circadian rhythms in cartilage homeostasis are hypothesized to temporally segregate and synchronize the activities of chondrocytes to different times of the day, and thus may provide an efficient mechanism by which articular cartilage can recover following physical activity. While the circadian clock is clearly involved in chondrocyte homeostasis in health and disease, it is unclear as to what roles it may play during early chondrogenesis. DESIGN: The purpose of this study was to determine whether the rhythmic expression of the core circadian clock was detectable at the earliest stages of chondrocyte differentiation, and if so, whether a synchronized expression pattern of chondrogenic transcription factors and developing cartilage matrix constituents was present during cartilage formation. RESULTS: Following serum shock, embryonic limb bud-derived chondrifying micromass cultures exhibited synchronized temporal expression patterns of core clock genes involved in the molecular circadian clock. We also observed that chondrogenic marker genes followed a circadian oscillatory pattern. Clock synchronization significantly enhanced cartilage matrix production and elevated SOX9, ACAN, and COL2A1 gene expression. The observed chondrogenesis-promoting effect of the serum shock was likely attributable to its synchronizing effect on the molecular clockwork, as co-application of small molecule modulators (longdaysin and KL001) abolished the stimulating effects on extracellular matrix production and chondrogenic marker gene expression. CONCLUSIONS: Results from this study suggest that a functional molecular clockwork plays a positive role in tissue homeostasis and histogenesis during early chondrogenesis.

2.
J Mol Neurosci ; 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31808034

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally secreted signaling peptide and has important regulatory roles in the differentiation of the central nervous system and its absence results in disorders in femur development. PACAP has an important function in prevention of oxidative stress or mechanical stress in chondrogenesis but little is known about its function in bone regeneration. A new callus formation model was set to investigate its role in bone remodeling. Fracturing was 5 mm distal from the proximal articular surface of the tibia and the depth was 0.5 mm. Reproducibility of callus formation was investigated with CT 3, 7, and 21 days after the operation. Absence of PACAP did not alter the alkaline phosphatase (ALP) activation in PACAP KO healing process. In developing callus, the expression of collagen type I increased in wild-type (WT) and PACAP KO mice decreased to the end of healing process. Expression of the elements of BMP signaling was disturbed in the callus formation of PACAP KO mice, as bone morphogenic protein 4 (BMP4) and 6 showed an early reduction in bone regeneration. However, elevated Smad1 expression was demonstrated in PACAP KO mice. Our results indicate that PACAP KO mice show various signs of disturbed bone healing and suggest PACAP compensatory and fine tuning effects in proper bone regeneration.

3.
Cell Commun Signal ; 17(1): 166, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842918

RESUMO

BACKGROUND: In vitro chondrogenesis depends on the concerted action of numerous signalling pathways, many of which are sensitive to the changes of intracellular Ca2+ concentration. N-methyl-D-aspartate (NMDA) glutamate receptor is a cation channel with high permeability for Ca2+. Whilst there is now accumulating evidence for the expression and function of NMDA receptors in non-neural tissues including mature cartilage and bone, the contribution of glutamate signalling to the regulation of chondrogenesis is yet to be elucidated. METHODS: We studied the role of glutamatergic signalling during the course of in vitro chondrogenesis in high density chondrifying cell cultures using single cell fluorescent calcium imaging, patch clamp, transient gene silencing, and western blotting. RESULTS: Here we show that key components of the glutamatergic signalling pathways are functional during in vitro chondrogenesis in a primary chicken chondrogenic model system. We also present the full glutamate receptor subunit mRNA and protein expression profile of these cultures. This is the first study to report that NMDA-mediated signalling may act as a key factor in embryonic limb bud-derived chondrogenic cultures as it evokes intracellular Ca2+ transients, which are abolished by the GluN2B subunit-specific inhibitor ifenprodil. The function of NMDARs is essential for chondrogenesis as their functional knock-down using either ifenprodil or GRIN1 siRNA temporarily blocks the differentiation of chondroprogenitor cells. Cartilage formation was fully restored with the re-expression of the GluN1 protein. CONCLUSIONS: We propose a key role for NMDARs during the transition of chondroprogenitor cells to cartilage matrix-producing chondroblasts.

4.
Geroscience ; 41(6): 775-793, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31655957

RESUMO

Pituitary adenylate cyclase activating polypeptide (PACAP) is an evolutionarly conserved neuropeptide which is produced by various neuronal and non-neuronal cells, including cartilage and bone cells. PACAP has trophic functions in tissue development, and it also plays a role in cellular and tissue aging. PACAP takes part in the regulation of chondrogenesis, which prevents insufficient cartilage formation caused by oxidative and mechanical stress. PACAP knockout (KO) mice have been shown to display early aging signs affecting several organs. In the present work, we investigated articular cartilage of knee joints in young and aged wild-type (WT) and PACAP KO mice. A significant increase in the thickness of articular cartilage was detected in aged PACAP gene-deficient mice. Amongst PACAP receptors, dominantly PAC1 receptor was expressed in WT knee joints and a remarkable decrease was found in aged PACAP KO mice. Expression of PKA-regulated transcription factors, Sox5, Sox9 and CREB, decreased both in young and aged gene deficient mice, while Sox6, collagen type II and aggrecan expressions were elevated in young but were reduced in aged PACAP KO animals. Increased expression of hyaluronan (HA) synthases and HA-binding proteins was detected parallel with an elevated presence of HA in aged PACAP KO mice. Expression of bone related collagens (I and X) was augmented in young and aged animals. These results suggest that loss of PACAP signaling results in dysregulation of cartilage matrix composition and may transform articular cartilage in a way that it becomes more prone to degenerate.

5.
Differentiation ; 107: 24-34, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31152959

RESUMO

Bone graft substitutes and bone void fillers are predominantly used to treat bone defects and bone fusion in orthopaedic surgery. Some aragonite-based scaffolds of coralline exoskeleton origin exhibit osteoconductive properties and are described as useful bone repair scaffolds. The purpose of this study was to evaluate the in vitro osteogenic potential of the bone phase of a novel aragonite-based bi-phasic osteochondral scaffold (Agili-C™, CartiHeal Ltd.) using adult human bone marrow-derived mesenchymal stem cells (MSCs). Analyses were performed at several time intervals: 3, 7, 14, 21, 28 and 42 days post-seeding. Osteogenic differentiation was assessed by morphological characterisation using light microscopy after Alizarin red and von Kossa staining, and scanning electron microscopy. The transcript levels of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), bone gamma-carboxyglutamate (BGLAP), osteonectin (SPARC) and osteopontin (SPP1) were determined by quantitative PCR. Proliferation was assessed by a thymidine incorporation assay and proliferating cell nuclear antigen (PCNA) immunocytochemistry. Our results demonstrate that the bone phase of the bi-phasic aragonite-based scaffold supports osteogenic differentiation and enhanced proliferation of bone marrow-derived MSCs at both the molecular and histological levels. The scaffold was colonized by differentiating MSCs, suggesting its suitability for incorporation into bone voids to accelerate bone healing, remodelling and regeneration. The mechanism of osteogenic differentiation involves scaffold surface modification with de novo production of calcium phosphate deposits, as revealed by energy dispersive spectroscopy (EDS) analyses. This novel coral-based scaffold may promote the rapid formation of high quality bone during the repair of osteochondral lesions.


Assuntos
Carbonato de Cálcio , Células-Tronco Mesenquimais/citologia , Osteogênese , Tecidos Suporte , Substitutos Ósseos/química , Carbonato de Cálcio/química , Fosfatos de Cálcio/metabolismo , Células Cultivadas , Humanos , Engenharia Tecidual
6.
Int J Mol Sci ; 20(1)2019 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-30621194

RESUMO

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide also secreted by non-neural cells, including chondrocytes. PACAP signaling is involved in the regulation of chondrogenesis, but little is known about its connection to matrix turnover during cartilage formation and under cellular stress in developing cartilage. We found that the expression and activity of hyaluronidases (Hyals), matrix metalloproteinases (MMP), and aggrecanase were permanent during the course of chondrogenesis in primary chicken micromass cell cultures, although protein levels changed daily, along with moderate and relatively constant enzymatic activity. Next, we investigated whether PACAP influences matrix destructing enzyme activity during oxidative and mechanical stress in chondrogenic cells. Exogenous PACAP lowered Hyals and aggrecanase expression and activity during cellular stress. Expression and activation of the majority of cartilage matrix specific MMPs such as MMP1, MMP7, MMP8, and MMP13, were also decreased by PACAP addition upon oxidative and mechanical stress, while the activity of MMP9 seemed not to be influenced by the neuropeptide. These results suggest that application of PACAP can help to preserve the integrity of the newly synthetized cartilage matrix via signaling mechanisms, which ultimately inhibit the activity of matrix destroying enzymes under cellular stress. It implies the prospect that application of PACAP can ameliorate articular cartilage destruction in joint diseases.


Assuntos
Proteínas Reguladoras de Apoptose/farmacologia , Condrócitos/efeitos dos fármacos , Estresse Oxidativo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Estresse Mecânico , Animais , Proteínas Reguladoras de Apoptose/administração & dosagem , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Técnicas de Cultura de Células , Embrião de Galinha , Condrócitos/metabolismo , Endopeptidases/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Hialuronoglucosaminidase/metabolismo , Peróxido de Hidrogênio/farmacologia , Metaloproteinases da Matriz/metabolismo , Oxidantes/farmacologia
7.
J Neuroinflammation ; 15(1): 335, 2018 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-30509328

RESUMO

OBJECTIVE: The regulatory role of capsaicin-sensitive peptidergic sensory nerves has been shown in acute inflammation, but little is known about their involvement in T/B-cell driven autoimmune arthritis. This study integratively characterized the function of these nerve endings in the proteoglycan-induced chronic arthritis (PGIA), a translational model of rheumatoid arthritis. METHODS: Peptidergic afferents were defunctionalized by resiniferatoxin (RTX) pretreatment in BALB/c mice, PGIA was induced by repeated antigen challenges. Hind paw volume, arthritis severity, grasping ability and the mechanonociceptive threshold were monitored during the 17-week experiment. Myeloperoxidase activity, vascular leakage and bone turnover were evaluated by in vivo optical imaging. Bone morphology was assessed using micro-CT, the intertarsal small joints were processed for histopathological analysis. RESULTS: Following desensitization of the capsaicin-sensitive afferents, ankle edema, arthritis severity and mechanical hyperalgesia were markedly diminished. Myeloperoxidase activity was lower in the early, but increased in the late phase, whilst plasma leakage and bone turnover were not altered. Desensitized mice displayed similar bone spurs and erosions, but increased trabecular thickness of the tibia and bony ankylosis of the spine. Intertarsal cartilage thickness was not altered in the model, but desensitization increased this parameter in both the non-arthritic and arthritic groups. CONCLUSION: This is the first integrative in vivo functional and morphological characterization of the PGIA mouse model, wherein peptidergic afferents have an important regulatory function. Their overall effect is proinflammatory by increasing acute inflammation, immune cell activity and pain. Meanwhile, their activation decreases spinal ankylosis, arthritis-induced altered trabecularity, and cartilage thickness in small joints.


Assuntos
Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/patologia , Capsaicina/farmacologia , Proteoglicanas/toxicidade , Fármacos do Sistema Sensorial/farmacologia , Limiar Sensorial/efeitos dos fármacos , Animais , Tornozelo/diagnóstico por imagem , Cartilagem/patologia , Modelos Animais de Doenças , Diterpenos/farmacologia , Feminino , Membro Posterior/efeitos dos fármacos , Membro Posterior/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Neurotoxinas/farmacologia , Peptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Índice de Gravidade de Doença , Coluna Vertebral/diagnóstico por imagem
8.
Int J Mol Sci ; 19(9)2018 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-30150589

RESUMO

: Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with diverse developmental roles, including differentiation of skeletal elements. It is a positive regulatory factor of chondrogenesis and osteogenic differentiation in vitro, but little is known about its in vivo role in bone formation. In our experiments, diaphyses of long bones from hind limbs of PACAP gene-deficient mice showed changes in thickness and increased staining intensity. Our main goal was to perform a detailed morphological and molecular biological analysis of femurs from PACAP knockout (KO) and wild type (WT) mice. Transverse diameter and anterior cortical bone thickness of KO femurs showed significant alterations with disturbed Ca2+ accumulation and collagen type I expression. Higher expression and activity of alkaline phosphatase were also observed, accompanied by increased fragility PACAP KO femurs. Increased expression of the elements of bone morphogenic protein (BMP) and hedgehog signalling was also observed, and are possibly responsible for the compensation mechanism accounting for the slight morphological changes. In summary, our results show that lack of PACAP influences molecular and biomechanical properties of bone matrix, activating various signalling cascade changes in a compensatory fashion. The increased fragility of PACAP KO femur further supports the role of endogenous PACAP in in vivo bone formation.


Assuntos
Condrogênese/genética , Osteogênese/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Transdução de Sinais/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cálcio/metabolismo , Diferenciação Celular/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fêmur/diagnóstico por imagem , Fêmur/metabolismo , Expressão Gênica , Camundongos Knockout , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/deficiência , Microtomografia por Raio-X
9.
Int J Mol Sci ; 19(7)2018 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-29966365

RESUMO

Heterotetrameric N-methyl-d-aspartate type glutamate receptors (NMDAR) are cationic channels primarily permeable for Ca2+. NR1 and NR3 subunits bind glycine, while NR2 subunits bind glutamate for full activation. As NR1 may contain a nuclear localization signal (NLS) that is recognized by importin-α, our aim was to investigate if NMDARs are expressed in the nuclei of melanocytes and melanoma cells. A detailed NMDAR subunit expression pattern was examined by RT-PCRs (reverse transcription followed by polymerase chain reaction), fractionated western blots and immunocytochemistry in human epidermal melanocytes and in human melanoma cell lines A2058, HT199, HT168M1, MEL35/0 and WM35. All kind of NMDAR subunits are expressed as mRNAs in melanocytes, as well as in melanoma cells, while NR2B protein remained undetectable in any cell type. Western blots proved the exclusive presence of NR1 and NR3B in nuclear fractions and immunocytochemistry confirmed NR1-NR3B colocalization inside the nuclei of all melanoma cells. The same phenomenon was not observed in melanocytes. Moreover, protein database analysis revealed a putative NLS in NR3B subunit. Our results support that unusual, NR1-NR3B composed NMDAR complexes are present in the nuclei of melanoma cells. This may indicate a new malignancy-related histopathological feature of melanoma cells and raises the possibility of a glycine-driven, NMDA-related nuclear Ca2+-signalling in these cells.


Assuntos
Melanoma/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Humanos , Melanócitos/metabolismo , Melanoma/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
10.
Sci Rep ; 7: 39863, 2017 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-28067251

RESUMO

Semicarbazide-sensitive amine oxidase (SSAO) catalyses oxidative deamination of primary amines. Since there is no data about its function in pain and arthritis mechanisms, we investigated the effects of our novel SSAO inhibitor SzV-1287 in chronic mouse models of joint inflammation. Effects of SzV-1287 (20 mg/kg i.p./day) were investigated in the K/BxN serum-transfer and complete Freund's adjuvant (CFA)-evoked active immunization models compared to the reference SSAO inhibitor LJP-1207. Mechanonociception was assessed by aesthesiometry, oedema by plethysmometry, clinical severity by scoring, joint function by grid test, myeloperoxidase activity by luminescence, vascular leakage by fluorescence in vivo imaging, histopathological changes by semiquantitative evaluation, and cytokines by Luminex assay. SzV-1287 significantly inhibited hyperalgesia and oedema in both models. Plasma leakage and keratinocyte chemoattractant production in the tibiotarsal joint, but not myeloperoxidase activity was significantly reduced by SzV-1287 in K/BxN-arthritis. SzV-1287 did not influence vascular and cellular mechanisms in CFA-arthritis, but significantly decreased histopathological alterations. There was no difference in the anti-hyperalgesic and anti-inflammatory actions of SzV-1287 and LJP-1207, but only SzV-1287 decreased CFA-induced tissue damage. Unlike SzV-1287, LJP-1207 induced cartilage destruction, which was confirmed in vitro. SzV-1287 exerts potent analgesic and anti-inflammatory actions in chronic arthritis models of distinct mechanisms, without inducing cartilage damage.


Assuntos
Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Analgésicos não Entorpecentes/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Artrite/tratamento farmacológico , Hidrazinas/uso terapêutico , Articulações/patologia , Oxazóis/uso terapêutico , Oximas/uso terapêutico , Animais , Células Cultivadas , Doença Crônica , Modelos Animais de Doenças , Progressão da Doença , Adjuvante de Freund/imunologia , Humanos , Hidrazinas/farmacologia , Articulações/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Oxazóis/farmacologia , Oximas/farmacologia
11.
Int J Oral Sci ; 8(1): 24-31, 2016 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-27025262

RESUMO

Although several heat shock proteins have been investigated in relation to tooth development, no available information is available about the spatial and temporal expression pattern of heat shock protein 60 (Hsp 60). To characterize Hsp 60 expression in the structures of the developing tooth germ, we used Western blotting, immunohistochemistry and in situ hybridization. Hsp 60 was present in high amounts in the inner and outer enamel epithelia, enamel knot (EK) and stratum intermedium (SI). Hsp 60 also appeared in odontoblasts beginning in the bell stage. To obtain data on the possible effect of Hsp 60 on isolated lower incisors from mice, we performed in vitro culturing. To investigate the effect of exogenous Hsp 60 on the cell cycle during culturing, we used the 5-bromo-2-deoxyuridine (BrdU) incorporation test on dental cells. Exogenously administered Hsp 60 caused bluntness at the apical part of the 16.5-day-old tooth germs, but it did not influence the proliferation rate of dental cells. We identified the expression of Hsp 60 in the developing tooth germ, which was present in high concentrations in the inner and outer enamel epithelia, EK, SI and odontoblasts. High concentration of exogenous Hsp 60 can cause abnormal morphology of the tooth germ, but it did not influence the proliferation rate of the dental cells. Our results suggest that increased levels of Hsp 60 may cause abnormalities in the morphological development of the tooth germ and support the data on the significance of Hsp during the developmental processes.


Assuntos
Chaperonina 60/farmacologia , Odontogênese/efeitos dos fármacos , Animais , Western Blotting , Imuno-Histoquímica , Hibridização In Situ , Incisivo , Camundongos , Germe de Dente/efeitos dos fármacos
12.
Cell Calcium ; 59(2-3): 108-16, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26925979

RESUMO

It is now widely recognised that changes of the intracellular calcium concentration have deep impact on the differentiation of various non-excitable cells including the elements of the vertebrate skeleton. It has become evident that purinergic signalling is one of the most ancient cellular mechanisms that can cause such alterations in the intracellular Ca(2+)-homeostasis, which are precisely set either spatially or temporally. Purinergic signalling is believed to regulate intracellular Ca(2+)-concentration of developing cartilage and skeletal muscle cells and suggested to play roles in the modulation of various cellular functions. This idea is supported by the fact that pluripotent mesenchymal cells, chondroprogenitors or muscle precursors, as well as mature chondrocytes all are capable of releasing ectonucleotides, and express various types of purinoreceptors and ectonucleotidases. The presence of the basic components of purinergic signalling proves that cells of the chondrogenic lineage can utilise this mechanism for modulating their intracellular Ca(2+) concentration independently from the surrounding skeletal muscle and bone tissues, which are well known to release ectopurines during development and mechanical stress. In this review, we summarize accumulating experimental evidence supporting the importance of purinergic signalling in the regulation of chondrogenesis and during skeletal muscle formation.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Condrogênese , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Humanos , Músculo Esquelético/citologia , Células-Tronco Pluripotentes/citologia
13.
Int J Oncol ; 48(3): 983-97, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26717964

RESUMO

Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Ácido Hialurônico/biossíntese , Melanoma/metabolismo , Proteína Fosfatase 2/metabolismo , Calcineurina/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ciclosporina/química , Flavonoides/química , Glucuronosiltransferase/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases , Sistema de Sinalização das MAP Quinases , Fosfoproteínas/metabolismo , Fosforilação
14.
Front Genet ; 7: 213, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28066501

RESUMO

Current cell-based repair strategies have proven unsuccessful for treating cartilage defects and osteoarthritic lesions, consequently advances in innovative therapeutics are required and mesenchymal stem cell-based (MSC) therapies are an expanding area of investigation. MSCs are capable of differentiating into multiple cell lineages and exerting paracrine effects. Due to their easy isolation, expansion, and low immunogenicity, MSCs are an attractive option for regenerative medicine for joint repair. Recent studies have identified several MSC tissue reservoirs including in adipose tissue, bone marrow, cartilage, periosteum, and muscle. MSCs isolated from these discrete tissue niches exhibit distinct biological activities, and have enhanced regenerative potentials for different tissue types. Each MSC type has advantages and disadvantages for cartilage repair and their use in a clinical setting is a balance between expediency and effectiveness. In this review we explore the challenges associated with cartilage repair and regeneration using MSC-based cell therapies and provide an overview of phenotype, biological activities, and functional properties for each MSC population. This paper also specifically explores the therapeutic potential of each type of MSC, particularly focusing on which cells are capable of producing stratified hyaline-like articular cartilage regeneration. Finally we highlight areas for future investigation. Given that patients present with a variety of problems it is unlikely that cartilage regeneration will be a simple "one size fits all," but more likely an array of solutions that need to be applied systematically to achieve regeneration of a biomechanically competent repair tissue.

15.
Int J Mol Sci ; 16(8): 18412-38, 2015 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-26262612

RESUMO

Mature and developing chondrocytes exist in a microenvironment where mechanical load, changes of temperature, osmolarity and acidic pH may influence cellular metabolism. Polymodal Transient Receptor Potential Vanilloid (TRPV) receptors are environmental sensors mediating responses through activation of linked intracellular signalling pathways. In chondrogenic high density cultures established from limb buds of chicken and mouse embryos, we identified TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6 mRNA expression with RT-PCR. In both cultures, a switch in the expression pattern of TRPVs was observed during cartilage formation. The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation. Incubating cell cultures at 41 °C elevated the expression of TRPV1, and increased cartilage matrix production. When chondrogenic cells were exposed to mechanical load at the time of their differentiation into matrix producing chondrocytes, we detected increased mRNA levels of TRPV3. Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation. As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells.


Assuntos
Condrócitos/metabolismo , Condrogênese , Canais de Cátion TRPV/metabolismo , Animais , Cartilagem/citologia , Cartilagem/fisiologia , Técnicas de Cultura de Células , Células Cultivadas , Embrião de Galinha , Condrócitos/citologia , Condrogênese/efeitos dos fármacos , Temperatura Alta , Camundongos , RNA Mensageiro/genética , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Transcriptoma , Suporte de Carga
16.
Int J Mol Sci ; 16(8): 17344-67, 2015 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-26230691

RESUMO

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load.


Assuntos
Condrócitos/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas Hedgehog/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Estresse Mecânico , Animais , Células Cultivadas , Embrião de Galinha , Proteínas Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Proteína GLI1 em Dedos de Zinco
17.
Curr Rheumatol Rep ; 17(7): 43, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25980668

RESUMO

Chondrocytes, the single cell type in adult articular cartilage, have conventionally been considered to be non-excitable cells. However, recent evidence suggests that their resting membrane potential (RMP) is less negative than that of excitable cells, and they are fully equipped with channels that control ion, water and osmolyte movement across the chondrocyte membrane. Amongst calcium-specific ion channels, members of the voltage-dependent calcium channel (VDCC) family are expressed in chondrocytes where they are functionally active. L-type VDCC inhibitors such as nifedipine and verapamil have contributed to our understanding of the roles of these ion channels in chondrogenesis, chondrocyte signalling and mechanotransduction. In this narrative review, we discuss published data indicating that VDCC function is vital for chondrocyte health, especially in regulating proliferation and maturation. We also highlight the fact that activation of VDCC function appears to accompany various inflammatory aspects of osteoarthritis (OA) and, based on in vitro data, the application of nifedipine and/or verapamil may be a promising approach for ameliorating OA severity. However, very few studies on clinical outcomes are available regarding the influence of calcium antagonists, which are used primarily for treating cardiovascular conditions in OA patients. This review is intended to stimulate further research on the chondrocyte 'channelome', contribute to the development of novel therapeutic strategies and facilitate the retargeting and repositioning of existing pharmacological agents currently used for other comorbidities for the treatment of OA.


Assuntos
Canais de Cálcio/fisiologia , Condrócitos/fisiologia , Osteoartrite/fisiopatologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Cartilagem/fisiologia , Condrócitos/efeitos dos fármacos , Humanos , Inflamação , Osteoartrite/tratamento farmacológico
18.
PLoS One ; 10(4): e0123583, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25893964

RESUMO

The sarcoplasmic/endoplasmic reticulum Ca2+ATPases (SERCAs) are the main Ca2+ pumps which decrease the intracellular Ca2+ level by reaccumulating Ca2+ into the sarcoplasmic reticulum. The neonatal SERCA1b is the major Ca2+ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca2+-transients were evoked by applications of 120 mM KCl. The significantly higher [Ca2+]i measured at the 20th and 40th seconds after the beginning of KCl application (112±3 and 110±3 nM vs. 150±7 and 135±5 nM, in control and in cloneC1 cells, respectively) indicated a decreased Ca2+-uptake capability which was quantified by extracting the maximal pump rate (454±41 µM/s vs. 144±24 µM/s, in control and in cloneC1 cells). Furthermore, the rate of calcium release from the SR (610±60 vs. 377±64 µM/s) and the amount of calcium released (843±75 µM vs. 576±80 µM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca2+]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation.


Assuntos
Cálcio/metabolismo , Diferenciação Celular , Inativação Gênica , Homeostase , Células Musculares/metabolismo , Músculo Esquelético/citologia , Animais , Sinalização do Cálcio , Linhagem Celular , Proliferação de Células , Regulação para Baixo , Camundongos , Modelos Biológicos , Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , RNA Interferente Pequeno/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Transfecção
19.
Maturitas ; 80(3): 237-44, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25637957

RESUMO

Aging and inflammation are major contributing factors to the development and progression of arthritic and musculoskeletal diseases. "Inflammaging" refers to low-grade inflammation that occurs during physiological aging. In this paper we review the published literature on cartilage aging and propose the term "chondrosenescence" to define the age-dependent deterioration of chondrocyte function and how it undermines cartilage function in osteoarthritis. We propose the concept that a small number of senescent chondrocytes may be able to take advantage of the inflammatory tissue microenvironment and the inflammaging and immunosenescence that is concurrently occurring in the arthritic joint, further contributing to the age-related degradation of articular cartilage, subchondral bone, synovium and other tissues. In this new framework "chondrosenescence" is intimately linked with inflammaging and the disturbed interplay between autophagy and inflammasomes, thus contributing to the age-related increase in the prevalence of osteoarthritis and a decrease in the efficacy of articular cartilage repair. A better understanding of the basic mechanisms underlying chondrosenescence and its modification by drugs, weight loss, improved nutrition and physical exercise could lead to the development of new therapeutic and preventive strategies for osteoarthritis and a range of other age-related inflammatory joint diseases. Aging is inevitable but age-related diseases may be modifiable.


Assuntos
Cartilagem Articular/patologia , Condrócitos/patologia , Osteoartrite/patologia , Idoso , Senescência Celular , Progressão da Doença , Humanos , Osteoartrite/etiologia
20.
Peptides ; 66: 51-7, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25701761

RESUMO

Skeletal development is a complex process regulated by multifactorial signaling cascades that govern proper tissue specific cell differentiation and matrix production. The influence of certain regulatory peptides on cartilage or bone development can be predicted but are not widely studied. In this review, we aimed to assemble and overview those signaling pathways which are modulated by PACAP and VIP neuropeptides and are involved in cartilage and bone formation. We discuss recent experimental data suggesting broad spectrum functions of these neuropeptides in osteogenic and chondrogenic differentiation, including the canonical downstream targets of PACAP and VIP receptors, PKA or MAPK pathways, which are key regulators of chondro- and osteogenesis. Recent experimental data support the hypothesis that PACAP is a positive regulator of chondrogenesis, while VIP has been reported playing an important role in the inflammatory reactions of surrounding joint tissues. Regulatory function of PACAP and VIP in bone development has also been proved, although the source of the peptides is not obvious. Crosstalk and collateral connections of the discussed signaling mechanisms make the system complicated and may obscure the pure effects of VIP and PACAP. Chondro-protective properties of PACAP during oxidative stress observed in our experiments indicate a possible therapeutic application of this neuropeptide.


Assuntos
Condrogênese/fisiologia , Osteogênese/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Cartilagem/metabolismo , Condrogênese/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Modelos Biológicos , Osteogênese/genética , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Peptídeo Intestinal Vasoativo/genética
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