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1.
Int J Mol Sci ; 22(18)2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34576330

RESUMO

Mechanical/physical stimulations modulate tissue metabolism, and this process involves multiple cellular mechanisms, including the secretion of growth factors and the activation of mechano-physically sensitive kinases. Cells and tissue can be modulated through specific vibration-induced changes in cell activity, which depend on the vibration frequency and occur via differential gene expression. However, there are few reports about the effects of medium-magnitude (1.12 g) sonic vibration on the osteogenic differentiation of human dental pulp stem cells (HDPSCs). In this study, we investigated whether medium-magnitude (1.12 g) sonic vibration with a frequency of 30, 45, or 100 Hz could affect the osteogenic differentiation of HDPSCs. Their cell morphology changed to a cuboidal shape at 45 Hz and 100 Hz, but the cells in the other groups were elongated. FACS analysis showed decreased CD 73, CD 90, and CD 105 expression at 45 Hz and 100 Hz. Additionally, the proportions of cells in the G0/G1 phase in the control, 30 Hz, 45 Hz, and 100 Hz groups after vibration were 60.7%, 65.9%, 68.3%, and 66.7%, respectively. The mRNA levels of osteogenic-specific markers, including osteonectin, osteocalcin, BMP-2, ALP, and Runx-2, increased at 45 and 100 Hz, and the ALP and calcium content was elevated in the vibration groups compared with those in the control. Additionally, the western blotting results showed that p-ERK, BSP, osteoprotegerin, and osteonectin proteins were upregulated at 45 Hz compared with the other groups. The vibration groups showed higher ALP and calcium content than the control. Vibration, especially at 100 Hz, increased the number of calcified nodes relative to the control group, as evidenced by von Kossa staining. Immunohistochemical staining demonstrated that type I and III collagen, osteonectin, and osteopontin were upregulated at 45 Hz and 100 Hz. These results suggest that medium magnitude vibration at 45 Hz induces the G0/G1 arrest of HDPSCs through the p-ERK/Runx-2 pathway and can serve as a potent stimulator of differentiation and extracellular matrix production.


Assuntos
Polpa Dentária/citologia , Polpa Dentária/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Citometria de Fluxo , Humanos , Osteogênese/fisiologia , Osteonectina/genética , Osteonectina/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Vibração
2.
Am J Physiol Cell Physiol ; 321(3): C559-C568, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34319830

RESUMO

In organisms from flies to mammals, the initial formation of a functional tendon is completely dependent on chemical signals from muscles (myokines). However, how myokines affect the maturation, maintenance, and regeneration of tendons as a function of age is completely unstudied. Here we discuss the role of four myokines-fibroblast growth factors (FGF), myostatin, the secreted protein acidic and rich in cysteine (SPARC) miR-29-in tendon development and hypothesize a role for these factors in the progressive changes in tendon structure and function as a result of muscle wasting (disuse, aging, and disease). Because of the close relationship between mechanical loading and muscle and tendon regulation, disentangling muscle-tendon cross talk from simple mechanical loading is experimentally quite difficult. Therefore, we propose an experimental framework that hopefully will be useful in demonstrating muscle-tendon cross talk in vivo. Though understudied, the promise of a better understanding of muscle-tendon cross talk is the development of new interventions that will improve tendon development, regeneration, and function throughout the lifespan.


Assuntos
Envelhecimento/genética , Exossomos/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Tendões/metabolismo , Envelhecimento/metabolismo , Animais , Fenômenos Biomecânicos , Exossomos/genética , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Células Musculares/metabolismo , Células Musculares/patologia , Músculo Esquelético/patologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Miostatina/genética , Miostatina/metabolismo , Osteonectina/genética , Osteonectina/metabolismo , Transdução de Sinais , Tendões/patologia
3.
Sci Rep ; 11(1): 14570, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272436

RESUMO

Cleft lip and palate (CL/P) is the most prevalent craniofacial birth defect in humans. None of the surgical procedures currently used for CL/P repair lead to definitive correction of hard palate bone interruption. Advances in tissue engineering and regenerative medicine aim to develop new strategies to restore palatal bone interruption by using tissue or organ-decellularized bioscaffolds seeded with host cells. Aim of this study was to set up a new natural scaffold deriving from a decellularized porcine mucoperiosteum, engineered by an innovative micro-perforation procedure based on Quantum Molecular Resonance (QMR) and then subjected to in vitro recellularization with human bone marrow-derived mesenchymal stem cells (hBM-MSCs). Our results demonstrated the efficiency of decellularization treatment gaining a natural, non-immunogenic scaffold with preserved collagen microenvironment that displays a favorable support to hMSC engraftment, spreading and differentiation. Ultrastructural analysis showed that the micro-perforation procedure preserved the collagen mesh, increasing the osteoinductive potential for mesenchymal precursor cells. In conclusion, we developed a novel tissue engineering protocol to obtain a non-immunogenic mucoperiosteal scaffold suitable for allogenic transplantation and CL/P repair. The innovative micro-perforation procedure improving hMSC osteogenic differentiation potentially impacts for enhanced palatal bone regeneration leading to future clinical applications in humans.


Assuntos
Fenda Labial/terapia , Fissura Palatina/terapia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Engenharia Tecidual/métodos , Tecidos Suporte , Transplante de Tecidos/métodos , Animais , Regeneração Óssea , Diferenciação Celular , Microambiente Celular , Colágeno Tipo I/metabolismo , Humanos , Osteogênese , Osteonectina/metabolismo , Medicina Regenerativa , Fatores de Transcrição SOXB1/metabolismo , Suínos
4.
Theranostics ; 11(13): 6173-6192, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995652

RESUMO

Rationale: Alternative therapeutic strategies based on tumor-specific molecular targets are urgently needed for triple-negative breast cancer (TNBC). The protease cathepsin D (cath-D) is a marker of poor prognosis in TNBC and a tumor-specific extracellular target for antibody-based therapy. The identification of cath-D substrates is crucial for the mechanistic understanding of its role in the TNBC microenvironment and future therapeutic developments. Methods: The cath-D substrate repertoire was investigated by N-Terminal Amine Isotopic Labeling of Substrates (TAILS)-based degradome analysis in a co-culture assay of TNBC cells and breast fibroblasts. Substrates were validated by amino-terminal oriented mass spectrometry of substrates (ATOMS). Cath-D and SPARC expression in TNBC was examined using an online transcriptomic survival analysis, tissue micro-arrays, TNBC cell lines, patient-derived xenografts (PDX), human TNBC samples, and mammary tumors from MMTV-PyMT Ctsd-/- knock-out mice. The biological role of SPARC and its fragments in TNBC were studied using immunohistochemistry and immunofluorescence analysis, gene expression knockdown, co-culture assays, western blot analysis, RT-quantitative PCR, adhesion assays, Transwell motility, trans-endothelial migration and invasion assays. Results: TAILS analysis showed that the matricellular protein SPARC is a substrate of extracellular cath-D. In vitro, cath-D induced limited proteolysis of SPARC C-terminal extracellular Ca2+ binding domain at acidic pH, leading to the production of SPARC fragments (34-, 27-, 16-, 9-, and 6-kDa). Similarly, cath-D secreted by TNBC cells cleaved fibroblast- and cancer cell-derived SPARC at the tumor pericellular acidic pH. SPARC cleavage also occurred in TNBC tumors. Among these fragments, only the 9-kDa SPARC fragment inhibited TNBC cell adhesion and spreading on fibronectin, and stimulated their migration, endothelial transmigration, and invasion. Conclusions: Our study establishes a novel crosstalk between proteases and matricellular proteins in the tumor microenvironment through limited SPARC proteolysis, revealing a novel targetable 9-kDa bioactive SPARC fragment for new TNBC treatments. Our study will pave the way for the development of strategies for targeting bioactive fragments from matricellular proteins in TNBC.


Assuntos
Catepsina D/metabolismo , Matriz Extracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Osteonectina/metabolismo , Fragmentos de Peptídeos/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catepsina D/deficiência , Catepsina D/genética , Adesão Celular , Feminino , Fibroblastos , Regulação Neoplásica da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Neoplasias Mamárias Experimentais/enzimologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Peso Molecular , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Osteonectina/genética , Fragmentos de Peptídeos/metabolismo , Domínios Proteicos , Proteólise , Especificidade por Substrato , Migração Transendotelial e Transepitelial , Neoplasias de Mama Triplo Negativas/enzimologia
5.
Neuroscience ; 467: 91-109, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34033869

RESUMO

Hevin is a matricellular glycoprotein that plays important roles in neural developmental processes such as neuronal migration, synaptogenesis and synaptic plasticity. In contrast to other matricellular proteins whose expression decreases when development is complete, hevin remains highly expressed, suggesting its involvement in adult brain function. In vitro studies have shown that hevin can have different post-translational modifications. However, the glycosylation pattern of hevin in the human brain remains unknown, as well as its relative distribution and localization. The present study provides the first thorough characterization of hevin protein expression by Western blot in postmortem adult human brain. Our results demonstrated two major specific immunoreactive bands for hevin: an intense band migrating around 130 kDa, and a band migrating around 100 kDa. Biochemical assays revealed that both hevin bands have a different glycosylation pattern. Subcellular fractionation showed greater expression in membrane-enriched fraction than in cytosolic preparation, and a higher expression in prefrontal cortex (PFC) compared to hippocampus (HIP), caudate nucleus (CAU) and cerebellum (CB). We confirmed that a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) and matrixmetalloproteinase 3 (MMP-3) proteases digestion led to an intense double band with similar molecular weight to that described as SPARC-like fragment (SLF). Finally, hevin immunoreactivity was also detected in human astrocytoma, meningioma, cerebrospinal fluid and serum samples, but was absent from any blood cell type.


Assuntos
Proteínas da Matriz Extracelular , Osteonectina , Adulto , Western Blotting , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio , Cerebelo/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Humanos , Neurogênese , Osteonectina/metabolismo
6.
Elife ; 102021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34036938

RESUMO

Phenotypic plasticity represents the most relevant hallmark of the carcinoma cell as it bestows it with the capacity of transiently altering its morphological and functional features while en route to the metastatic site. However, the study of phenotypic plasticity is hindered by the rarity of these events within primary lesions and by the lack of experimental models. Here, we identified a subpopulation of phenotypic plastic colon cancer cells: EpCAMlo cells are motile, invasive, chemo-resistant, and highly metastatic. EpCAMlo bulk and single-cell RNAseq analysis indicated (1) enhanced Wnt/ß-catenin signaling, (2) a broad spectrum of degrees of epithelial to mesenchymal transition (EMT) activation including hybrid E/M states (partial EMT) with highly plastic features, and (3) high correlation with the CMS4 subtype, accounting for colon cancer cases with poor prognosis and a pronounced stromal component. Of note, a signature of genes specifically expressed in EpCAMlo cancer cells is highly predictive of overall survival in tumors other than CMS4, thus highlighting the relevance of quasi-mesenchymal tumor cells across the spectrum of colon cancers. Enhanced Wnt and the downstream EMT activation represent key events in eliciting phenotypic plasticity along the invasive front of primary colon carcinomas. Distinct sets of epithelial and mesenchymal genes define transcriptional trajectories through which state transitions arise. pEMT cells, often earmarked by the extracellular matrix glycoprotein SPARC together with nuclear ZEB1 and ß-catenin along the invasive front of primary colon carcinomas, are predicted to represent the origin of these (de)differentiation routes through biologically distinct cellular states and to underlie the phenotypic plasticity of colon cancer cells.


Assuntos
Movimento Celular , Plasticidade Celular , Neoplasias do Colo/patologia , Animais , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/mortalidade , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Células HCT116 , Humanos , Masculino , Camundongos Endogâmicos NOD , Invasividade Neoplásica , Metástase Neoplásica , Osteonectina/genética , Osteonectina/metabolismo , Fenótipo , Via de Sinalização Wnt , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
7.
Elife ; 102021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33904394

RESUMO

Cortical interneurons establish inhibitory microcircuits throughout the neocortex and their dysfunction has been implicated in epilepsy and neuropsychiatric diseases. Developmentally, interneurons migrate from a distal progenitor domain in order to populate the neocortex - a process that occurs at a slower rate in humans than in mice. In this study, we sought to identify factors that regulate the rate of interneuron maturation across the two species. Using embryonic mouse development as a model system, we found that the process of initiating interneuron migration is regulated by blood vessels of the medial ganglionic eminence (MGE), an interneuron progenitor domain. We identified two endothelial cell-derived paracrine factors, SPARC and SerpinE1, that enhance interneuron migration in mouse MGE explants and organotypic cultures. Moreover, pre-treatment of human stem cell-derived interneurons (hSC-interneurons) with SPARC and SerpinE1 prior to transplantation into neonatal mouse cortex enhanced their migration and morphological elaboration in the host cortex. Further, SPARC and SerpinE1-treated hSC-interneurons also exhibited more mature electrophysiological characteristics compared to controls. Overall, our studies suggest a critical role for CNS vasculature in regulating interneuron developmental maturation in both mice and humans.


Assuntos
Movimento Celular/efeitos dos fármacos , Córtex Cerebral/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Eminência Mediana/irrigação sanguínea , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Osteonectina/farmacologia , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Potenciais de Ação , Animais , Córtex Cerebral/embriologia , Córtex Cerebral/cirurgia , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Interneurônios/metabolismo , Interneurônios/transplante , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Eminência Mediana/embriologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Neovascularização Fisiológica , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/transplante , Osteonectina/metabolismo , Comunicação Parácrina , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Transdução de Sinais
8.
Biochem Biophys Res Commun ; 558: 134-140, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33910127

RESUMO

Previous studies have shown that secreted protein acidic and rich in cysteine (SPARC) proteins can inhibit the development of cancer cells in various ways, such as by inhibiting angiogenesis and inhibiting cell proliferation. In fact, SPARC proteins may have an effect on the chemoresistance of gastric cancer cells to 5-Fluorouracil (5-FU), which needs further research in the future. Therefore, the purpose of this study was to explore the relationship between SPARC proteins and the chemosensitivity of gastric cancer cells to 5-FU. In vitro, after SPARC protein levels were regulated by plasmid, siRNA and human recombinant SPARC protein transfection in MGC-803, SGC-7901 and BGC-823 cells, we detected epithelial-mesenchymal transition (EMT), apoptosis markers and cell viability after 5-FU treatment. In vivo, we implanted BGC-823 cells with stable SPARC overexpression into nude mice. Tumour size was measured to assess the effect of SPARC protein on tumour formation and 5-FU chemosensitivity. In SGC-7901 and BGC-823 cells, both endogenous and exogenous upregulation of SPARC protein levels decreased cell viability, destroyed cytoskeletal F-actin, inhibited cell migration, and downregulated a series of transcription factors to inhibit cell EMT; it also upregulated cell apoptosis-related proteins to promote cell apoptosis. However, we obtained opposite results in SPARC knockdown MGC-803 cells. In vivo, compared with the control group, the group engrafted with BGC-823 cells stably overexpressing SPARC had a significant smaller tumour size. After 5-FU treatment, the new tumour gradually decreased in size. Our results show that the SPARC protein could enhance 5-FU chemosensitivity in gastric cancer cell lines by inhibiting EMT and promoting cell apoptosis.


Assuntos
Fluoruracila/farmacologia , Osteonectina/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Actinas/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/fisiologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Osteonectina/antagonistas & inibidores , Osteonectina/genética , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/genética , Neoplasias Gástricas/patologia , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Oncol Rep ; 45(6)2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33907853

RESUMO

Tumor­stroma interactions serve a crucial role in the development of colorectal cancer (CRC), in which secreted protein acidic and rich in cysteine (SPARC) has been implicated. Due to interactions between cancer and stromal cells [mesenchymal stem cells (MSCs)], SPARC gene expression is markedly upregulated in CRC cells. The present study investigated the role of SPARC in CRC development and its potential as a biomarker. Specifically, the present study examined the association between SPARC expression and clinicopathological characteristics in 42 cases of CRC. SPARC expression in cancer cells was associated with T grade, N grade (TNM classification), stage and poor prognosis. Furthermore, the area of fibroblast­activating protein­positive staining around the cancer cells was increased in SPARC­positive compared with SPARC­negative cases. Proliferation and wound healing assays in SPARC­silenced KM12SM cells [short hairpin RNA SPARC (shSPARC)], the reduced SPARC expression of which was demonstrated by reverse transcription­quantitative PCR, revealed that the proliferative and migratory capacity of shSPARC cells did not differ from that of wild­type (WT) cells. However, it was markedly reduced when co­cultured with MSCs. Furthermore, in vivo, immunohistological analysis and RNA sequencing were conducted in an orthotopic implanted mouse model. Tumor growth and lymph node metastasis were markedly suppressed in shSPARC­transplanted tumors compared with WT­transplanted tumors, with a more marked suppression observed following shSPARC co­transplantation with MSCs. Immunohistological examination further revealed that the stromal reaction and epithelial­mesenchymal transition (EMT) were markedly suppressed in tumors co­transplanted with shSPARC and MSCs, and these results were consistent with RNA sequencing using RNA extracted from orthotopic tumors. Overall, these results suggested that SPARC expression in CRC cells is dependent on the interaction between cancer cells and stromal cells to induce EMT and promote stromal formation in the tumor microenvironment, suggesting its suitability as a novel target molecule for CRC treatment.


Assuntos
Neoplasias Colorretais/patologia , Células-Tronco Mesenquimais/patologia , Osteonectina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Técnicas de Cocultura , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/cirurgia , Transição Epitelial-Mesenquimal , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Osteonectina/genética , Cultura Primária de Células , Microambiente Tumoral , Adulto Jovem
10.
Med Sci Monit ; 27: e929558, 2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33758160

RESUMO

BACKGROUND Gastric cancer is the most common gastrointestinal tumor, and the rates of recurrence and metastasis are high. Research results on molecular biomarkers used for prognosis of gastric cancer remain inconclusive. This study aimed to explore the gene expression module of gastric cancer and to determine potential prognostic biomarkers. MATERIAL AND METHODS Three microarray datasets (GSE13911, GSE79973, and GSE29272) from Gene Expression Omnibus (GEO), including 206 pairs of gastric tumors and adjacent normal samples, were used for analysis of differentially expressed genes (DEGs). The 3 microarray datasets yielded 144 genes associated with the progression and prognosis of gastric cancer. After this, a risk score model was developed for result validation using an independent dataset from The Cancer Genome Atlas. RESULTS The validation of the independent dataset showed significantly increased NID2, SPARC, and MFAP2 expression in gastric tumor tissues, which were associated with poor outcomes in gastric cancer patients. Moreover, the high risk score obtained was associated with poor overall survival (HR: 1.787; 1.069-2.986; P=0.027). Subgroup analyses revealed that these significant prognostic values were detected in patients aged <65.0 years, tumors in the antrum/distal colon, grade 3 tumors, or TNM-M0 stages of cancer. CONCLUSIONS The findings of this study show that NID2, SPARC, and MFAP2 are upregulated in gastric tumor tissues and are significantly associated with poor overall survival. Therefore, the predictive values of the risk score model employed for the prognosis of gastric cancer could be improved by using these 3 upregulated DEGs.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Moléculas de Adesão Celular/genética , Osteonectina/genética , Fatores de Processamento de RNA/genética , Neoplasias Gástricas/genética , Biomarcadores Tumorais/genética , Proteínas de Ligação ao Cálcio/metabolismo , Moléculas de Adesão Celular/metabolismo , Bases de Dados Genéticas , Progressão da Doença , Mucosa Gástrica/metabolismo , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes , Humanos , Análise em Microsséries , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Osteonectina/metabolismo , Prognóstico , Fatores de Processamento de RNA/metabolismo , Neoplasias Gástricas/mortalidade , Análise de Sobrevida
11.
Iran Biomed J ; 25(3): 180-92, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33639637

RESUMO

Background: Bioactive glasses 58S, are silicate-based materials containing calcium and phosphate, which dissolved in body fluid and bond to the bone tissue. This type of bioactive glass is highly biocompatible and has a wide range of clinical applications. Methods: The 58S glass powders were synthesized via sol-gel methods, using tetraethyl orthosilicate, triethyl phosphate, and calcium nitrate, as precursors. Upon the analyses of phase and chemical structures of bioactive glass in different gelation times (12, 48, and 100 h), the appropriate heat treatment (at 525, 575, and 625 °C) was performed to eliminate nitrate compounds and stabilize the glass powder samples. The in vitro assay in SBF solution revealed the bioactivity of the synthesized 58S glass through the morphological (SEM), chemical structure (FTIR), release of calcium, phosphorous and silicon elements, pH variations, and weight loss measurements. The behavior of MSCs in the presence of bioactive glass powders was studied by MTT cytotoxicity, cell staining, ALP activity and biomineralization tests, as well as by the evaluation of ALP, osteocalcin, osteonectin, collagen I, and RUNX2 gene expression. Results: The results confirmed a gelation time of 100 h and a calcination temperature of 575 °C at optimal conditions for the synthesis of nitrate-free bioactive glass powders. Conclusion: The glass spherical nanoparticles in the range of 20-30 nm possess the improved bioactivity and osteogenic properties as demanded for bone tissue engineering.


Assuntos
Diferenciação Celular , Vidro/química , Células-Tronco Mesenquimais/citologia , Transição de Fase , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Cálcio/análise , Proliferação de Células , 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 , Regulação da Expressão Gênica , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Osteocalcina/genética , Osteocalcina/metabolismo , Osteonectina/genética , Osteonectina/metabolismo , Fósforo/análise , Pós , Silício/análise , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
12.
Endocrinology ; 162(4)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33539507

RESUMO

CONTEXT: Basal-ganglia calcification (BGC) is common (70%) in patients with chronic hypoparathyroidism. Interestingly, cortical gray matter is spared from calcification. The mechanism of BGC, role of hyperphosphatemia, and modulation of osteogenic molecules by parathyroid hormone (PTH) in its pathogenesis is not clear. OBJECTIVE: We assessed the expression of a large repertoire of molecules with proosteogenic or antiosteogenic effects, including neuroprogenitor cells in caudate, dentate, and cortical gray matter from normal autopsy tissues. The effect of high phosphate and PTH was assessed in an ex vivo model of BGC using striatum tissue culture of the Sprague-Dawley rat. METHODS: The messenger RNA and protein expression of 39 molecules involved in multiple osteogenic pathways were assessed in 25 autopsy tissues using reverse-transcriptase polymerase chain reaction, Western blot, and immunofluorescence. The striatal culture was maintained in a hypoparathyroid milieu for 24 days with and without (a) high phosphate (10-mm ß-glycerophosphate) and (b) PTH(1-34) (50 ng/mL Dulbecco's modified Eagle's medium-F12 media) for their effect on striatal calcification and osteogenic molecules. RESULTS: Procalcification molecules (osteonectin, ß-catenin, klotho, FZD4, NT5E, LRP5, WNT3A, collagen-1α, and SOX2-positive neuroprogenitor stem cells) had significantly higher expression in the caudate than gray matter. Caudate nuclei also had higher expression of antiosteogenic molecules (osteopontin, carbonic anhydrase-II [CA-II], MGP, sclerostin, ISG15, ENPP1, and USP18). In an ex vivo model, striatum culture showed an increased propensity for calcified nodules with mineral deposition similar to that of bone tissue on Fourier-transformed infrared spectroscopy, alizarin, and von Kossa stain. Mineralization in striatal culture was enhanced by high phosphate and decreased by exogenous PTH through increased expression of CA-II. CONCLUSION: This study provides a conceptual advance on the molecular mechanisms of BGC and the possibility of PTH therapy to prevent this complication in a hypoparathyroid milieu.


Assuntos
Gânglios da Base/fisiopatologia , Hipoparatireoidismo/fisiopatologia , Osteogênese , Animais , Gânglios da Base/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Calcinose , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Núcleo Caudado/metabolismo , Marcadores Genéticos/genética , Substância Cinzenta/metabolismo , Humanos , Hipoparatireoidismo/genética , Hipoparatireoidismo/metabolismo , Técnicas In Vitro , Masculino , Osteonectina/genética , Osteonectina/metabolismo , Hormônio Paratireóideo/metabolismo , Fosfatos/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/genética , Pirofosfatases/metabolismo , Ratos , Ratos Sprague-Dawley
13.
Liver Int ; 41(7): 1677-1693, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33641248

RESUMO

BACKGROUND AND AIMS: Non-alcoholic fatty liver (NAFLD) and its more serious form non-alcoholic steatohepatitis increase risk of hepatocellular carcinoma (HCC). Lipid metabolic alterations and its role in HCC development remain unclear. SPARC (Secreted Protein, Acidic and Rich in Cysteine) is involved in lipid metabolism, NAFLD and diabetes, but the effects on hepatic lipid metabolism and HCC development is unknown. The aim of this study was to evaluate the role of SPARC in HCC development in the context of NAFLD. METHODS: Primary hepatocyte cultures from knockout (SPARC-/- ) or wild-type (SPARC+/+ ) mice, and HepG2 cells were used to assess the effects of free fatty acids on lipid accumulation, expression of lipogenic genes and de novo triglyceride (TG) synthesis. A NAFLD-HCC model was stabilized on SPARC-/- or SPARC+/+ mice. Correlations among SPARC, lipid metabolism-related gene expression patterns and clinical prognosis were studied using HCC gene expression dataset. RESULTS: SPARC-/- mice increases hepatic lipid deposits over time. Hepatocytes from SPARC-/- mice or inhibition of SPARC by an antisense adenovirus in HepG2 cells resulted in increased TG deposit, expression of lipid-related genes and nuclear translocation of SREBP1c. Human HCC database analysis revealed that SPARC negatively correlated with genes involved in lipid metabolism, and with poor survival. In NAFLD-HCC murine model, the absence of SPARC accelerates HCC development. RNA-seq study revealed that pathways related to lipid metabolism, cellular detoxification and proliferation were upregulated in SPARC-/- tumour-bearing mice. CONCLUSIONS: The absence of SPARC is associated with an altered hepatic lipid metabolism, and an accelerated NAFLD-related HCC development.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Animais , Carcinoma Hepatocelular/metabolismo , Metabolismo dos Lipídeos , Lipídeos , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Osteonectina/genética , Osteonectina/metabolismo
14.
Mol Med Rep ; 23(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33576452

RESUMO

Colorectal cancer (CRC), one of the most common cancer types, causes a large number of cancer­related mortalities annually worldwide. Dysregulated microRNAs (miRNAs/miR) are closely associated with the malignant progression of CRC. Therefore, the present study aimed to investigate the expression and regulatory role of miR­592 in CRC. It was found that miR­592 expression was significantly elevated in CRC tissues and cell lines, and was associated with the prognosis of patients. Cellular phenotype assays demonstrated that miR­592 could promote CRC cell proliferation, migration and invasion. Bioinformatics analysis demonstrated that miR­592 mainly participated in the positive regulation of transcription, as well as the regulation of cell motility. Moreover, miR­592 targets were enriched in several signaling pathways, such as the 'mTOR' and 'FoxO' signaling pathways. In addition, secreted protein acidic and rich in cysteine (SPARC) was identified as a target of miR­592 in CRC. The present results suggested that miR­592 acts as an oncogene in CRC, in part, by directly inhibiting SPARC expression. Collectively, the present study provides a novel potential therapeutic strategy for CRC.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Osteonectina/genética , Adulto , Idoso , Linhagem Celular Tumoral , Células Cultivadas , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Perfilação da Expressão Gênica/métodos , Células HCT116 , Células HT29 , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Osteonectina/metabolismo , Prognóstico , Transdução de Sinais/genética
15.
Blood ; 137(12): 1641-1651, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33529332

RESUMO

Secreted modular calcium-binding protein 1 (SMOC1) is an osteonectin/SPARC-related matricellular protein, whose expression is regulated by microRNA-223 (miR-223). Given that platelets are rich in miR-223, this study investigated the expression of SMOC1 and its contribution to platelet function. Human and murine platelets expressed SMOC1, whereas platelets from SMOC1+/- mice did not present detectable mature SMOC1 protein. Platelets from SMOC1+/- mice demonstrated attenuated responsiveness to thrombin (platelet neutrophil aggregate formation, aggregation, clot formation, Ca2+ increase, and ß3 integrin phosphorylation), whereas responses to other platelet agonists were unaffected. SMOC1 has been implicated in transforming growth factor-ß signaling, but no link to this pathway was detected in platelets. Rather, the SMOC1 Kazal domain directly bound thrombin to potentiate its activity in vitro, as well as its actions on isolated platelets. The latter effects were prevented by monoclonal antibodies against SMOC1. Platelets from miR-223-deficient mice expressed high levels of SMOC1 and exhibited hyperreactivity to thrombin that was also reversed by preincubation with monoclonal antibodies against SMOC1. Similarly, SMOC1 levels were markedly upregulated in platelets from individuals with type 2 diabetes, and the SMOC1 antibody abrogated platelet hyperresponsiveness to thrombin. Taken together, we have identified SMOC1 as a novel thrombin-activating protein that makes a significant contribution to the pathophysiological changes in platelet function associated with type 2 diabetes. Thus, strategies that target SMOC1 or its interaction with thrombin may be attractive therapeutic approaches to normalize platelet function in diabetes.


Assuntos
Plaquetas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Osteonectina/metabolismo , Trombina/metabolismo , Adulto , Animais , Plaquetas/citologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Ativação Plaquetária , Agregação Plaquetária
16.
PLoS One ; 16(2): e0245653, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33534863

RESUMO

Collagen deposition contributes to both high mammographic density and breast cancer progression. Low stromal PTEN expression has been observed in as many as half of breast tumors and is associated with increases in collagen deposition, however the mechanism connecting PTEN loss to increased collagen deposition remains unclear. Here, we demonstrate that Pten knockout in fibroblasts using an Fsp-Cre;PtenloxP/loxP mouse model increases collagen fiber number and fiber size within the mammary gland. Pten knockout additionally upregulated Sparc transcription in fibroblasts and promoted collagen shuttling out of the cell. Interestingly, SPARC mRNA expression was observed to be significantly elevated in the tumor stroma as compared to the normal breast in several patient cohorts. While SPARC knockdown via shRNA did not affect collagen shuttling, it notably decreased assembly of exogenous collagen. In addition, SPARC knockdown decreased fibronectin assembly and alignment of the extracellular matrix in an in vitro fibroblast-derived matrix model. Overall, these data indicate upregulation of SPARC is a mechanism by which PTEN regulates collagen deposition in the mammary gland stroma.


Assuntos
Colágeno/metabolismo , Glândulas Mamárias Humanas/metabolismo , Osteonectina/metabolismo , PTEN Fosfo-Hidrolase/fisiologia , Animais , Linhagem Celular , Matriz Extracelular/metabolismo , Fibroblastos , Humanos , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/patologia , Camundongos , Camundongos Knockout
17.
BMC Cancer ; 21(1): 156, 2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33579227

RESUMO

BACKGROUND: Matricellular glycoprotein, SPARC is a secreted molecule, that mediates the interaction between cells and extracellular matrix. SPARC functions as a regulator of matrix organization and modulates cell behavior. In various kinds of cancer, strong SPARC expression was observed in stromal tissues as well as in cancer epithelial cells. The function of SPARC in cancer cells is somewhat controversial and its impact on peritumoral stromal cells remains to be resolved. METHODS: We investigated the effects of SPARC expression in endometrial cancer cells on the surrounding stromal fibroblasts using in vitro co-culture system. Changes in characteristics of fibroblasts were examined by analysis of fibroblast-specific markers and in vitro contraction assay. RESULTS: SPARC induced AKT phosphorylation and epithelial-to-mesenchymal transition, consistent with previous reports. Cancer-associated fibroblasts of endometrial cancer expressed higher levels of mesenchymal- and fibroblast-associated factors and had a stronger contraction ability. Unexpectedly, cancer-associated fibroblasts expressed comparable levels of SPARC compared with fibroblasts from normal endometrium. However, co-culture of normal fibroblasts with SPARC-expressing Ishikawa cells resulted in activation of the fibroblasts. Immunodepletion of SPARC did not affect the activation of fibroblasts. CONCLUSIONS: Our data indicated that SPARC activated fibroblasts only in the presence of fibronectin, which was abundantly secreted from SPARC-expressing endometrial cancer cells. These results suggested that a SPARC-fibronectin-mediated activation of fibroblasts might be involved in enhanced mobility and invasion of cancer cells.


Assuntos
Movimento Celular , Neoplasias do Endométrio/patologia , Transição Epitelial-Mesenquimal , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Osteonectina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Neoplasias do Endométrio/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Osteonectina/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética
18.
Sci Rep ; 11(1): 1470, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446775

RESUMO

Small interfering RNA (siRNA) therapy is a promising epigenetic silencing strategy. However, its widespread adoption has been severely impeded by its ineffective delivery into the cellular environment. Here, a biocompatible injectable gelatin-based hydrogel with positive-charge tuned surface charge is presented as an effective platform for siRNA protection and delivery. We demonstrate a two-step synthesis of a gelatin-tyramine (Gtn-Tyr) hydrogel with simultaneous charge tunability and crosslinking ability. We discuss how different physiochemical properties of the hydrogel interact with siSPARC (siRNA for secreted protein, acidic and rich in cysteine), and study the positive-charge tuned gelatin hydrogel as an effective delivery platform for siSPARC in anti-fibrotic treatment. Through in vitro studies using mouse tenon fibroblasts, the positive-charge tuned Gtn-Tyr hydrogel shows sustained siSPARC cellular internalization and effective SPARC silencing with excellent biocompatibility. Similarly, the same hydrogel platform delivering siSPARC in an in vivo assessment employing a rabbit model shows an effective reduction in subconjunctival scarring in post glaucoma filtration surgery, and is non-cytotoxic compared to a commonly used anti-scarring agent, mitomycin-C. Overall, the current siRNA delivery strategy involving the positive-charge tuned gelatin hydrogel shows effective delivery of gene silencing siSPARC for anti-fibrotic treatment. The current charge tunable hydrogel delivery system is simple to fabricate and highly scalable. We believe this delivery platform has strong translational potential for effective siRNA delivery and epigenetic silencing therapy.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Gelatina/química , Hidrogéis/química , Animais , Células Cultivadas , Cicatriz/genética , Cicatriz/terapia , Doenças da Túnica Conjuntiva/genética , Feminino , Fibroblastos/metabolismo , Fibrose , Inativação Gênica/fisiologia , Glaucoma/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteonectina/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Coelhos
19.
J Invest Dermatol ; 141(7): 1792-1801.e5, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33484701

RESUMO

Immunoregulatory effects of IL-4 and IL-13 and alterations of keratinocyte (KC) differentiation are important factors in the pathogenesis of atopic dermatitis. This study investigated the role of IL-4 and IL-13 in KC responses to changes in extracellular calcium (Ca2+) and analyzed differentiation signals elicited via a Ca2+ sensor, SMOC1. Real-time dynamics of transmembrane Ca2+ influx were assessed in live KCs by flow cytometry and microscopy. Exposure of KCs to a high Ca2+ environment (1.3 mM) triggered a rapid intracellular Ca2+ influx, whereas IL-4- and IL-13-treated cells exhibited a significant decrease in the peak amplitude of Ca2+ influx (P < 0.01). IL-17A and IL-22 did not elicit such responses. Evaluation of intracellular Ca2+ dynamics by microscopy confirmed these observations and revealed heterogeneity of individual KC responses. IL-4 and IL-13 significantly inhibited the expression of Ca2+-binding protein SMOC1 (P < 0.001). Inhibition of epidermal differentiation markers were also observed in SMOC1 small interfering RNA-transfected KCs. Concurrently, the deletion of SMOC1 increased the amplitude of Ca2+ peak response (P < 0.05). In conclusion, our results provide innovative data that IL-4 and IL-13 regulate KC sensitivity to microenvironmental Ca2+ changes and inhibit Ca2+-induced KC differentiation signals. SMOC1 inhibition by IL-4 and IL-13 alters Ca2+ transport in KCs and inhibits differentiation, suggesting a new target for treatment of atopic dermatitis.


Assuntos
Cálcio/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Queratinócitos/metabolismo , Osteonectina/metabolismo , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Dermatite Atópica/imunologia , Dermatite Atópica/patologia , Perfilação da Expressão Gênica , Humanos , Microscopia Intravital , Queratinócitos/imunologia , Osteonectina/genética , Cultura Primária de Células , Análise de Célula Única
20.
Blood ; 137(11): 1517-1526, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32932520

RESUMO

The cells and mechanisms involved in blood clot resorption are only partially known. We show that regulatory T cells (Tregs) accumulate in venous blood clots and regulate thrombolysis by controlling the recruitment, differentiation and matrix metalloproteinase (MMP) activity of monocytes. We describe a clot Treg population that forms the matricellular acid- and cysteine-rich protein SPARC (secreted protein acidic and rich in cysteine) and show that SPARC enhances monocyte MMP activity and that SPARC+ Tregs are crucial for blood clot resorption. By comparing different treatment times, we define a therapeutic window of Treg expansion that accelerates clot resorption.


Assuntos
Osteonectina/metabolismo , Linfócitos T Reguladores/metabolismo , Trombose Venosa/metabolismo , Animais , Fibrinólise , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Monócitos/patologia , Linfócitos T Reguladores/patologia , Trombose Venosa/sangue , Trombose Venosa/patologia
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