RESUMO
With the aim to increase type II collagen content in the scaffold-free cartilage-like cell sheet using human bone marrow mesenchymal stem cells, we examined the effect of epigallocatechin-3-gallate (EGCG) addition to the chondrogenic medium for the cell sheet culture. The addition of EGCG (10 µM) increased the content of type II collagen 2-fold, while the addition did not markedly change the expression level of the genes encoding type II collagen and Sox 9. The reactive oxygen species level in the cells in cell sheets was thought to be too low to suppress the accumulation of type II collagen. On the other hand, the addition of EGCG markedly decreased both the matrix metalloproteinase-13 concentration in the supernatant of cell sheet culture and the type II collagen degradation activity in that supernatant. Taken together, EGCG may enhance the accumulation of type II collagen by suppressing type II collagen degradation.
Assuntos
Cartilagem/efeitos dos fármacos , Catequina/análogos & derivados , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Colágeno Tipo II/agonistas , Células-Tronco Mesenquimais/efeitos dos fármacos , Idoso , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Cartilagem/citologia , Cartilagem/metabolismo , Catequina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Meios de Cultura/química , Meios de Cultura/farmacologia , Regulação da Expressão Gênica , Humanos , Masculino , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Cultura Primária de Células , Proteólise/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Engenharia TecidualRESUMO
Hepatic stellate cells (HSCs) activation represents an essential event during alcoholic liver fibrosis (ALF). Previous studies have demonstrated that the rat HSCs could be significantly activated after exposure to 200 µmol/L acetaldehyde for 48 h, and the cAMP/PKA signaling pathways were also dramatically upregulated in activated HSCs isolated from alcoholic fibrotic rat liver. Exchange protein activated by cAMP (EPAC) is a family of guanine nucleotide exchange factors (GEFs) for the small Ras-like GTPases Rap, and is being considered as a vital mediator of cAMP signaling in parallel with the principal cAMP target protein kinase A (PKA). Our data showed that both cAMP/PKA and cAMP/EPAC signaling pathways were involved in acetaldehyde-induced HSCs. Acetaldehyde could reduce the expression of EPAC1 while enhancing the expression of EPAC2. The cAMP analog Me-cAMP, which stimulates the EPAC/Rap1 pathway, could significantly decrease the proliferation and collagen synthesis of acetaldehyde-induced HSCs. Furthermore, depletion of EPAC2, but not EPAC1, prevented the activation of HSC measured as the production of α-SMA and collagen type I and III, indicating that EPAC1 appears to have protective effects on acetaldehyde-induced HSCs. Curiously, activation of PKA or EPAC perhaps has opposite effects on the synthesis of collagen and α-SMA: EPAC activation by Me-cAMP increased the levels of GTP-bound (activated) Rap1 while PKA activation by Phe-cAMP had no significant effects on such binding. These results suggested that EPAC activation could inhibit the activation and proliferation of acetaldehyde-induced HSCs via Rap1.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/agonistas , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática Alcoólica/metabolismo , Proteínas rap1 de Ligação ao GTP/agonistas , Acetaldeído/antagonistas & inibidores , Acetaldeído/toxicidade , Actinas/agonistas , Actinas/antagonistas & inibidores , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/agonistas , Colágeno Tipo I/antagonistas & inibidores , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/agonistas , Colágeno Tipo II/antagonistas & inibidores , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , AMP Cíclico/agonistas , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática Alcoólica/patologia , Cirrose Hepática Alcoólica/prevenção & controle , Interferência de RNA , Ratos , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Proteínas rap1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
Transforming growth factor (TGF)ß regulates the anabolic metabolism of articular cartilage and prevents cartilage degradation. TGFß1 influences cellular proliferation, differentiation and the extracellular matrix through activation of the extracellular signalregulated kinase (ERK)1/2 and Smad2/3 signaling pathways. However, it has remained to be fully elucidated precisely how the ERK1/2 and Smad2/3 signaling pathways mediate anabolic processes of articular cartilage. The present study investigated how ERK1/2 and Smad2/3 signaling mediate TGFß1stimulated type II collagen and aggrecan expression in rat chondrocytes. The results confirmed that TGFß1 stimulates type II collagen and aggrecan expression in rat chondrocytes, and furthermore, that the ERK1/2 and Smad2/3 signaling pathways were activated by TGFß1. Conversely, the TGFß receptor I (ALK5) kinase inhibitor SB525334 significantly impaired TGFß1induced type II collagen and aggrecan expression, coinciding with a reduction of ERK1/2 and Smad3 phosphorylation. In addition, TGFß1induced type II collagen and aggrecan expression were significantly suppressed by ERK1/2 inhibitor PD98059. Similarly, TGFß1stimulated type II collagen and aggrecan expression were decreased in the presence of a Smad3 phosphorylation inhibitor SIS3. Therefore, the present study demonstrated that the ERK1/2 and Smad2/3 signaling pathways regulate type II collagen and aggrecan expression in rat chondrocytes.
Assuntos
Agrecanas/genética , Colágeno Tipo II/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta1/farmacologia , Agrecanas/agonistas , Agrecanas/metabolismo , Animais , Cartilagem Articular/citologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno Tipo II/agonistas , Colágeno Tipo II/metabolismo , Ativação Enzimática , Feminino , Flavonoides/farmacologia , Regulação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Isoquinolinas/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , Pirróis/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Proteína Smad2/agonistas , Proteína Smad2/metabolismo , Proteína Smad3/agonistas , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
PURPOSE: Previously, we demonstrated that scleral stem/progenitor cells (SSPCs) from mice have a chondrogenic differentiation potential, which is stimulated by transforming growth factor-ß (TGF-ß). In the present study, we hypothesized that chondrogenesis in the sclera could be a possible mechanism in myopia development. Therefore, we investigated the association of form-deprivation myopia (FDM) with expressions in mice sclera representing the chondrogenic phenotype: collagen type II (Col2) and α-smooth muscle actin (α-SMA). METHODS: The mRNA levels of α-SMA and Col2 in cultured murine SSPCs during chondrogenesis stimulated by TGF-ß2 were determined by real-time quantitative RT-PCR (qRT-PCR). The expression patterns of α-SMA and Col2 were assessed by immunohistochemistry in a three dimensional pellet culture. In an FDM mouse model, a western blot analysis and immunofluorescence study were used to detect the changes in the α-SMA and Col2 protein expressions in the sclera. In the RPE-choroid complex, qRT-PCR was used to detect any changes in the TGF-ß mRNA expression. RESULTS: The treatment of SSPCs in vitro with TGF-ß2 for 24 h at 1 or 10 ng/ml led to increased levels of both the α-SMA and Col2 expressions. In addition, we observed the formation of cartilage-like pellets from TGF-ß2-treated SSPCs. Both α-SMA and Col2 were expressed in the pellet. In an in-vivo study, the α-SMA and Col2 protein expressions were significantly increased in the sclera of FDM eyes in comparison to contralateral control eyes. Similarly, the levels of TGF-ß in the RPE-choroid complex of an FDM eye were also significantly elevated. CONCLUSION: Based on the concept of stem cells possessing multipotent differentiation potentials, scleral chondrogenesis induced by SSPCs may play a role in myopia development. The increased expressions of the cartilage-associated proteins Col2 and α-SMA during scleral chondrogenesis may be potential markers for myopia development. In addition, the increased levels of TGF-ß mRNA in the RPE-choroid complex might induce the chondrogenic change in the sclera during myopia development.
Assuntos
Condrogênese/genética , Corioide/patologia , Miopia/patologia , Epitélio Pigmentado da Retina/patologia , Esclera/patologia , Células-Tronco/patologia , Actinas/agonistas , Actinas/genética , Actinas/metabolismo , Animais , Células Cultivadas , Corioide/metabolismo , Colágeno Tipo II/agonistas , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miopia/genética , Miopia/metabolismo , RNA Mensageiro/agonistas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Esclera/efeitos dos fármacos , Esclera/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/agonistas , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta2/farmacologiaRESUMO
AIMS: We previously reported that Lactobacillus casei (L. casei) has beneficial effects in experimental rheumatoid arthritis (RA) by suppressing inflammatory immune responses. The major purpose of this study was to evaluate therapeutic effects of L. casei on pathological responses in experimental rodent model of osteoarthritis (OA). MAIN METHODS: Experimental OA was induced by intra-articular injection of monosodium iodoacetate (MIA) in Wistar rats. L. casei alone or together with type II collagen (CII) and glucosamine (Gln) was orally administered into OA rats. The pathophysiological aspects of OA were investigated by analyzing mechanical hyperalgesia and histology of articular tissues. Expression of inflammatory molecules was analyzed in CD4(+) T cells, synovial fibroblasts, and chondrocytes by quantitative real-time PCR. KEY FINDINGS: Oral administration of L. casei together with CII and Gln more effectively reduced pain, cartilage destruction, and lymphocyte infiltration than the treatment of Gln or L. casei alone. This co-administration also decreased expression of various pro-inflammatory cytokines (interleukin-1ß (IL-1ß), IL-2, IL-6, IL-12, IL-17, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)) and matrix metalloproteinases (MMP1, MMP3, and MMP13), while up-regulating anti-inflammatory cytokines (IL-4 and IL-10). These results are concomitant with reduced translocation of NF-κB into the nucleus and increased expression of the tissue inhibitor of MMP1 (TIMP1) and CII in chondrocytes. SIGNIFICANCE: Our study provides evidence that L. casei could act as a potent nutraceutical modulator for OA treatment by reducing pain, inflammatory responses, and articular cartilage degradation.
Assuntos
Colágeno Tipo II/agonistas , Glucosamina/agonistas , Inflamação/terapia , Lacticaseibacillus casei/fisiologia , Osteoartrite/terapia , Probióticos/uso terapêutico , Animais , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/fisiologia , Condrócitos/química , Condrócitos/efeitos dos fármacos , Condrócitos/fisiologia , Colágeno Tipo II/fisiologia , Citocinas/análise , Feminino , Glucosamina/fisiologia , Inflamação/imunologia , NF-kappa B/análise , Osteoartrite/microbiologia , Medição da Dor , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Líquido Sinovial/citologia , Líquido Sinovial/efeitos dos fármacos , Líquido Sinovial/fisiologiaRESUMO
The regeneration of diseased hyaline cartilage continues to be a great challenge, mainly because degeneration--caused either by major injury or by age-related processes--can overextend the tissue's self-renewal capacity. We show that repair tissue from human articular cartilage during the late stages of osteoarthritis harbors a unique progenitor cell population, termed chondrogenic progenitor cells (CPCs). These exhibit stem cell characteristics such as clonogenicity, multipotency, and migratory activity. The isolated CPCs, which exhibit a high chondrogenic potential, were shown to populate diseased tissue ex vivo. Moreover, downregulation of the osteogenic transcription factor runx-2 enhanced the expression of the chondrogenic transcription factor sox-9. This, in turn, increased the matrix synthesis potential of the CPCs without altering their migratory capacity. Our results offer new insights into the biology of progenitor cells in the context of diseased cartilage tissue. Our work may be relevant in the development of novel therapeutics for the later stages of osteoarthritis.