RESUMO
OBJECTIVE: To investigate the effects of miR-221 and miR-222 and high glucose on human periodontal ligament (PL) cells morphology, cytoskeleton, adhesion, and migration. BACKGROUND: Chronic hyperglycemia is common in uncontrolled diabetes mellitus (DM) and plays a central role in long-term DM complications, such as impaired periodontal healing. We have previously shown that high glucose increases apoptosis of human PL cells by inhibiting miR-221 and miR-222 and consequently augmenting their target caspase-3. However, other effects of miR-221/222 downregulation on PL cells are still unknown. METHODS: Cells from young humans' premolar teeth were cultured for 7 days under 5 or 30 mM glucose. Directional and spontaneous migration on fibronectin were studied using transwell and time-lapse assays, respectively. F-actin staining was employed to study cell morphology and the actin cytoskeleton. MiR-221 and miR-222 were inhibited using antagomiRs, and their expressions were evaluated by real-time RT-PCR. RESULTS: High glucose inhibited PL cells early adhesion, spreading, and migration on fibronectin. Cells exposed to high glucose showed reduced polarization, velocity, and directionality. They formed several simultaneous unstable and short-lived protrusions, suggesting impairment of adhesion maturation. MiR-221 and miR-222 inhibition also reduced migration, decreasing cell directionality but not significantly cell velocity. After miR-221 and miR-222 downregulation cells showed morphological resemblance with cells exposed to high glucose. CONCLUSION: High glucose impairs human PL cells migration potentially through a mechanism involving reduction of microRNA-221 and microRNA-222 expression. These effects may contribute to the impairment of periodontal healing, especially after surgery and during guided regeneration therapies.
Assuntos
MicroRNAs , Humanos , MicroRNAs/metabolismo , Fibronectinas/farmacologia , Ligamento Periodontal/metabolismo , Movimento Celular , Glucose/farmacologia , Células CultivadasRESUMO
OBJECTIVES: The aim of this study was to characterize polycaprolactone-based nanofiber scaffolds (PCL) incorporated with calcium hydroxide (CH) and evaluate their bioactivity on human dental pulp cells (HDPCs) when loaded with fibronectin (FN). MATERIALS AND METHODS: CH (0.1%; 0.2%; 0.4% w/v; or 0%) was incorporated into PCL (10% w/v) scaffolds prepared by electrospinning. Morphology and composition were characterized using SEM/EDS. HDPCs were seeded on the scaffolds and evaluated for viability (alamarBlue; Live/Dead), and adhesion/spreading (F-actin). Next, scaffolds containing 0.4% CH were loaded with FN (20 µg/mL). HDPCs were evaluated for viability, adhesion/spreading, migration (Trans-well), gene expression (RT-qPCR), alkaline phosphatase activity (ALP), and mineralization nodules (Alizarin Red). Data were submitted to ANOVA and post-hoc tests (α = 5%). RESULTS: Nanofibers with larger diameter were seen as CH concentration increased, while there was no effect on interfibrillar spaces. An increase in cell viability was seen for 0.4% CH, in all periods. Incorporation of CH and FN into the scaffolds increased cellular migration, spread, and viability, all intensified when CH and FN were combined. ALPL and DSPP expression, and ALP activity were not affected by CH and FN. COL1A1 was downregulated in all groups, while DMP1 was upregulated in the presence of CH, with no differences for the groups loaded with FN. CH increased the formation of mineralized matrix, which was not influenced by FN. CONCLUSIONS: In conclusion, the incorporation of CH enhanced the odontogenic potential of HDPCs, irrespective of the presence of FN. The PCL + 0.4% CH formulation may be a useful strategy for use in dentin tissue engineering. CLINICAL RELEVANCE: A change in the form of presentation of calcium hydroxide-based materials used for direct pulp capping can increase biocompatibility and prolong the vitality of dental pulp.
Assuntos
Nanofibras , Engenharia Tecidual , Hidróxido de Cálcio/farmacologia , Diferenciação Celular , Células Cultivadas , Polpa Dentária , Dentina , Fibronectinas/farmacologia , Humanos , Alicerces TeciduaisRESUMO
Mammalian ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, collectively called capacitation, in order to fertilize the oocyte. We reported that fibronectin (Fn), a glycoprotein from the extracellular matrix, and anandamide (AEA), one of the major members of the endocannabinoid family, are present in the bovine oviductal fluid and regulate bull sperm function. Also, AEA induces bovine sperm capacitation, through CB1 and TRPV1 receptors. In this work, we investigated if Fn induces bovine sperm capacitation thought the activation of the endocannabinoid system in this process. We incubated sperm with Fn (100 µg/ml) and/or capsazepine, a TRPV1 antagonist (0.1 µM) and some events related to sperm capacitation such as LPC-induced acrosome reaction, sperm-release from the oviduct, induction of PKA phosphorylated substrates (pPKAs) and protein tyrosine phosphorylation (pY) and nitric oxide (NO) production were assessed. Also, we studied the activity of fatty acid amide hydrolase (FAAH), the enzyme that degrades AEA. We found that Fn, via α5ß1 integrin, induced capacitation-associated events. Also, Fn stimulated signaling pathways associated to capacitation as cAMP/PKA and NO/NO synthase. Moreover, Fn decreased the FAAH activity and this correlated with sperm capacitation. Capsazepine reversed fibronectin-induced capacitation, and pPKAs and NO levels. The incubation of spermatozoa with R-methanandamide (1.4 nM), a stable analogue of AEA, increased cAMP and pPKAs levels. The presence of H89 (50 µM) or KT5720 (100 nM) (PKA inhibitors) prevented AEA-induced capacitation. In addition, R-methanandamide and capsaicin (0.01 µM), a TRPV1 agonist, increased NO production via the PKA pathway. These results indicate that Fn, through α5ß1, supports capacitation in bovine spermatozoa. This effect is dependent on the activation of TRPV1 through cAMP/PKA and NO signaling pathways. We propose that Fn could be considered as a new agent that promotes sperm capacitation in bull sperm. Our findings contribute to better understand the significance of Fn signaling in the capacitating events that lead to successful fertilization and embryo development in mammals including humans.
Assuntos
Bovinos , Endocanabinoides/metabolismo , Fibronectinas/farmacologia , Preservação do Sêmen/veterinária , Capacitação Espermática/efeitos dos fármacos , Animais , Criopreservação/veterinária , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocanabinoides/genética , Integrina alfa5beta1/genética , Integrina alfa5beta1/metabolismo , Masculino , Óxido Nítrico , Motilidade dos EspermatozoidesRESUMO
Early steps of tissue invasion by Entamoeba histolytica are mediated by adhesion and migration through matrix components such as fibronectin with the participation of the actin cytoskeleton. Striking differences in their produced structures, movement, and migration were found. These observations suggest differential changes in their ability to organize the actin cytoskeleton and, therefore, to modify its morphology after adhesion to fibronectin. To understand these observations, we explore deeper the cytoskeleton pathway of E. histolytica compared to Entamoeba dispar, analyzing the activation and involvement of actin cytoskeleton regulatory proteins such as small GTPases (Rho, Rac1 and Cdc42), myosin IB, paxillin, alpha-actinin, and ARP2/3 during interaction with fibronectin. Results showed a higher activation of Rac1 in E. histolytica compared to E. dispar, while Cdc42 and RhoA were equally activated in both amebae; besides, variations in the amount of myosin IB, paxillin, and ARP2/3 were detected among these species, coinciding and reflected in formation of lamellipodia in E. histolytica and filopodia in E. dispar. These could partially explain the higher invasive capacity of E. histolytica compared to E. dispar, due to its pleomorphic ability, high motility, migration, activation, and abundance of proteins involved in the cytoskeleton arrangement.
Assuntos
Entamoeba/fisiologia , Fibronectinas/farmacologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas dos Microfilamentos/metabolismo , Entamoeba/efeitos dos fármacos , Entamoeba/ultraestrutura , Entamoeba histolytica/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Microscopia Confocal , Proteínas de Protozoários/metabolismoRESUMO
Fibronectin (Fn) enhances human sperm capacitation via the cAMP/PKA pathway, and the endocannabinoid system participates in this process. Moreover, Fn has been linked to endocannabinoid system components in different cellular models, even though no evidence of such interactions in human sperm is available. Normal semen samples were evaluated over a 4-year period. Our findings suggest that (a) the capacitating effects of Fn were reversed by preincubating the sperm with a cannabinoid receptor 1 (CB1) or transient receptor potential cation channel subfamily V member 1 (TRPV1) antagonist ( p < 0.001 and p < 0.05, respectively); (b) cooperation between CB1 and TRPV1 may exist ( p < 0.01); (c) the activity of specific fatty acid amide hydroxylase (FAAH) decreased after 1 min ( p < 0.01) and increased after 60 min ( p < 0.01) of capacitation in the presence of Fn; (d) the effects of Fn on FAAH activity were prevented by preincubating spermatozoa with a protein kinase A (PKA) inhibitor ( p < 0.01); (e) Fn modulated both the cyclic adenosine monophosphate concentration and PKA activity ( p < 0.05) during early capacitation; and (f) FAAH was a PKA substrate modulated by phosphorylation. These findings indicate that Fn stimulates human sperm capacitation via the cAMP/PKA pathway through modulation of the endocannabinoid system. Understanding the functional competence of human spermatozoa is essential for facilitating clinical advances in infertility treatment and for developing novel contraceptive strategies.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Endocanabinoides/farmacologia , Fibronectinas/farmacologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/metabolismo , Humanos , Masculino , Espermatozoides/citologiaRESUMO
The aim of this study was to determine the proliferation and osteogenic activity of fibroblasts induced with fibronectin and their possible dose-dependent relationship. The fibroblasts obtained by tissue explants adherent method were induced with fibronectin at different concentrations of 0, 10, 20, 40, 60, and 80 µg/mL for 14 days. The 3H-thymidine and 3H-proline incorporation test was used to evaluate the synthesis of DNA and collagen by fibroblasts, respectively. The mineralized nodules and osteocalcin secretion, as vital osteogenic indicators, were detected with tetracycline labeling and 125I-labeled competitive immunoassay, respectively. Fibronectin significantly increased the synthesis of DNA and collagen by fibroblasts, especially at the concentration of 40 µg/mL (P<0.05). The increased secretion of osteocalcin in the supernatant was also statistically significant at the concentration of 40 µg/mL (P<0.05). The mineralized nodules with trabecula-like structure derived from induced fibroblasts were positive for tetracycline labeling. The granulation tissue-derived fibroblasts induced with fibronectin exhibited increased proliferative, functional and osteogenic potential. Fibroblasts are considered a possible in situ stem cell in tissue engineering.
Assuntos
Proliferação de Células/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibronectinas/farmacologia , Osteogênese/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Fibroblastos/fisiologia , Humanos , CoelhosRESUMO
ABSTRACT Objective Patients infected with the human immunodeficiency virus (HIV) have an increased risk of metabolic disorders and alterations on irisin levels. Therefore, the purpose of the current investigation was to quantify the circulating irisin concentration in HIV-infected subjects under highly active antiretroviral therapy and to determine possible correlations between irisin levels with fat mass, fat-free mass, body mass index (BMI), and muscle strength. Subjects and methods Cross-sectional study of 10 men (36.7 ± 11.3 years) and 10 women (42.5 ± 10.3 years) infected with HIV, recruited from the Specialized Service Center in the State Center of Reference for High and Medium Complexity. Blood samples were collected to determine plasma irisin levels, glucose, HDL, total cholesterol, triglycerides, and LDL. Body composition (fat mass, fat-free mass) and anthropometrics (body mass index; BMI) were measured by bioelectrical impedance. Muscle strength was assessed using a mechanic hand dynamometer and one maximum repetition tests. Results Irisin levels correlated positively with fat mass (r = 0.67; p = 0.001) and BMI (r = 0.48; p = 0.036). In contrast, there was an inverse correlation between irisin levels and fat-free mass (r = -0.41; p = 0.008) and five strength parameters: right hand grip (r = -0.46; p = 0.044); left hand grip (r = -0.50; p = 0.027), relative hand grip (r = -0.79; p = 0.001), bench press (r = -0.58; p = 0.009), leg press (r = -0.40; p = 0.085), and biceps curl (r = -0.059; p = 0.009). Conclusion Irisin levels correlated positively with body fat and negatively with fat-free mass and strength parameters in HIV-infected patients. Female patients infected with HIV receiving highly active antiretroviral therapy have higher levels of irisin compared with men in a similar circumstance.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Infecções por HIV/sangue , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/patologia , Fibronectinas/sangue , Composição Corporal/efeitos dos fármacos , Infecções por HIV/metabolismo , Infecções por HIV/tratamento farmacológico , Fatores Sexuais , Estudos Transversais , Fibronectinas/metabolismo , Fibronectinas/farmacologia , Força da Mão , Terapia Antirretroviral de Alta Atividade , Antirretrovirais/uso terapêutico , Força Muscular/efeitos dos fármacosRESUMO
OBJECTIVE: To assess the effect of fibronectin (Fn) and porcine type I collagen (PCOL) on odontoblast-like cells in vitro. MATERIAL AND METHODS: Rat odontoblast-like cells (MDPC-23 cells) were inoculated and cultured on Fn-coated or type I collagen-coated substrates. Proliferation assay, alkaline phosphatase activity (ALP activity), mRNA expression of hard tissue-forming markers, and Alizarin red staining were investigated over a period of 10 days. RESULTS: Cells maintained a high proliferation activity on Fn and PCOL even at a low seeding concentration (0.5×104/mL) as demonstrated by CCK-8 assay. The proliferation activity of cells on Fn increases in a concentration-dependent manner while it reached a plateau after 10 µg/mL. Cells adopted long, thin and spindle shape on Fn(10-50) and PCOL. Parallel actin filaments were observed in MDPC-23 cells cultured on Fn and PCOL. ALP activity was markedly up-regulated on Fn and PCOL-coated surfaces. Importantly, gene expression of BSP (Fn10: 2.44±0.32; Fn20: 3.05±0.01; Fn30: 2.90±0.21; Fn40: 2.74±0.30; Fn50: 2.64±0.12; PCOL: 2.20±0.03) and OCN (Fn10: 2.52±0.23; Fn20: 2.28±0.24; Fn30: 2.34±0.21; Fn40: 2.34±0.25; Fn50: 2.20±0.22; PCOL: 1.56±0.16) was significantly enhanced on Fn and PCOL substrates as compared with control; moreover, expression of integrin beta 1 (ITGB1), an ubiquitous cell surface receptor was augmented in Fn(10-50) and PCOL groups simultaneously. In accordance with the ALP activity and gene expression data, calcific deposition in cells grown on Fn(10-50) and PCOL was observed as well. CONCLUSION: Despite the limitation of this study, the findings indicate that a surface coating of Fn enhances the proliferation, differentiation and mineralization of odontoblast-like cells by activation of integrin beta 1 (ITG B1). The promoting effects of Fn on MDPC-23 cells were achieved at a comparatively lower coating concentration than type I collagen (300 µg/mL). Specifically, it is suggested that the optimum coating concentration of Fn to be 10 µg/mL.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fibronectinas/farmacologia , Odontoblastos/efeitos dos fármacos , Fosfatase Alcalina/análise , Animais , Antraquinonas , Células Cultivadas , Colágeno Tipo I/farmacologia , Imunofluorescência , Expressão Gênica , Humanos , Integrina beta1/farmacologia , Ratos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
OBJECTIVE: Patients infected with the human immunodeficiency virus (HIV) have an increased risk of metabolic disorders and alterations on irisin levels. Therefore, the purpose of the current investigation was to quantify the circulating irisin concentration in HIV-infected subjects under highly active antiretroviral therapy and to determine possible correlations between irisin levels with fat mass, fat-free mass, body mass index (BMI), and muscle strength. SUBJECTS AND METHODS: Cross-sectional study of 10 men (36.7 ± 11.3 years) and 10 women (42.5 ± 10.3 years) infected with HIV, recruited from the Specialized Service Center in the State Center of Reference for High and Medium Complexity. Blood samples were collected to determine plasma irisin levels, glucose, HDL, total cholesterol, triglycerides, and LDL. Body composition (fat mass, fat-free mass) and anthropometrics (body mass index; BMI) were measured by bioelectrical impedance. Muscle strength was assessed using a mechanic hand dynamometer and one maximum repetition tests. RESULTS: Irisin levels correlated positively with fat mass (r = 0.67; p = 0.001) and BMI (r = 0.48; p = 0.036). In contrast, there was an inverse correlation between irisin levels and fat-free mass (r = -0.41; p = 0.008) and five strength parameters: right hand grip (r = -0.46; p = 0.044); left hand grip (r = -0.50; p = 0.027), relative hand grip (r = -0.79; p = 0.001), bench press (r = -0.58; p = 0.009), leg press (r = -0.40; p = 0.085), and biceps curl (r = -0.059; p = 0.009). CONCLUSION: Irisin levels correlated positively with body fat and negatively with fat-free mass and strength parameters in HIV-infected patients. Female patients infected with HIV receiving highly active antiretroviral therapy have higher levels of irisin compared with men in a similar circumstance.
Assuntos
Tecido Adiposo , Fibronectinas/sangue , Infecções por HIV/sangue , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/patologia , Adulto , Antirretrovirais/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Composição Corporal/efeitos dos fármacos , Estudos Transversais , Feminino , Fibronectinas/metabolismo , Fibronectinas/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Força da Mão , Humanos , Masculino , Pessoa de Meia-Idade , Força Muscular/efeitos dos fármacos , Fatores SexuaisRESUMO
Abstract Objective To assess the effect of fibronectin (Fn) and porcine type I collagen (PCOL) on odontoblast-like cells in vitro. Material and Methods Rat odontoblast-like cells (MDPC-23 cells) were inoculated and cultured on Fn-coated or type I collagen-coated substrates. Proliferation assay, alkaline phosphatase activity (ALP activity), mRNA expression of hard tissue-forming markers, and Alizarin red staining were investigated over a period of 10 days. Results Cells maintained a high proliferation activity on Fn and PCOL even at a low seeding concentration (0.5×104/mL) as demonstrated by CCK-8 assay. The proliferation activity of cells on Fn increases in a concentration-dependent manner while it reached a plateau after 10 µg/mL. Cells adopted long, thin and spindle shape on Fn(10-50) and PCOL. Parallel actin filaments were observed in MDPC-23 cells cultured on Fn and PCOL. ALP activity was markedly up-regulated on Fn and PCOL-coated surfaces. Importantly, gene expression of BSP (Fn10: 2.44±0.32; Fn20: 3.05±0.01; Fn30: 2.90±0.21; Fn40: 2.74±0.30; Fn50: 2.64±0.12; PCOL: 2.20±0.03) and OCN (Fn10: 2.52±0.23; Fn20: 2.28±0.24; Fn30: 2.34±0.21; Fn40: 2.34±0.25; Fn50: 2.20±0.22; PCOL: 1.56±0.16) was significantly enhanced on Fn and PCOL substrates as compared with control; moreover, expression of integrin beta 1 (ITGB1), an ubiquitous cell surface receptor was augmented in Fn(10-50) and PCOL groups simultaneously. In accordance with the ALP activity and gene expression data, calcific deposition in cells grown on Fn(10-50) and PCOL was observed as well. Conclusion Despite the limitation of this study, the findings indicate that a surface coating of Fn enhances the proliferation, differentiation and mineralization of odontoblast-like cells by activation of integrin beta 1 (ITG B1). The promoting effects of Fn on MDPC-23 cells were achieved at a comparatively lower coating concentration than type I collagen (300 µg/mL). Specifically, it is suggested that the optimum coating concentration of Fn to be 10 µg/mL.