RESUMO
Vitronectin is an abundant multifunctional glycoprotein found in serum, the extracellular matrix, and bone, and is involved in diverse physiological processes. Here, we developed a new bioactive dimeric peptide (VnP-8-DN1 dimer) from a human vitronectin-derived motif (IDAAFTRINCQG; residues 206-217; VnP-8) via removal of an isoleucine residue at the N-terminus of VnP-8 and spontaneous air oxidation. The VnP-8-DN1 dimer potently enhanced cell attachment activity, and this activity was mediated by binding to cellular heparan sulfate proteoglycan receptors. Moreover, the VnP-8-DN1 dimer suppressed osteoclast differentiation by blocking the early stage of osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Furthermore, the VnP-8-DN1 dimer decreased the bone-resorbing activity of osteoclasts and increased the survival of osteoclast precursor cells by decreasing the cellular level of c-Fms and reducing RANK expression. Taken together, these results demonstrate that the VnP-8-DN1 dimer inhibits the early stages of M-CSF- and RANK-induced osteoclast differentiation by binding to c-Fms and inhibiting M-CSF signaling.
Assuntos
Reabsorção Óssea , Fator Estimulador de Colônias de Macrófagos , Reabsorção Óssea/metabolismo , Diferenciação Celular , Humanos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Glicoproteínas de Membrana/metabolismo , Osteoclastos/metabolismo , Osteogênese , Ligante RANK/metabolismo , Ligante RANK/farmacologia , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Vitronectina/metabolismo , Vitronectina/farmacologiaRESUMO
AIM: This study investigated whether a vitronectin-derived peptide (VnP-16) prevents and/or reverses alveolar bone resorption induced by ligature-induced periodontitis in rodents and identified the underlying mechanism. MATERIALS AND METHODS: We evaluated the effects of VnP-16 on osteogenic differentiation in human periodontal ligament cells (hPDLCs), lipopolysaccharide-induced inflammatory responses in gingival fibroblasts, and immune response in T lymphocytes. Ligature-induced periodontitis was induced by ligating the bilateral mandibular first molars for 14 days in rats and for 7 days in mice (n = 10/group). VnP-16 (100 µg/10 µl) was applied topically into the gingival sulcus of rats via intra-sulcular injection, whereas the peptide (50 µg/5 µl) was administered directly into the gingiva of mice via intra-gingival injection. To evaluate the preventive and therapeutic effects of VnP-16, micro-computed tomography analysis and histological staining were then performed. RESULTS: VnP-16 promoted osteogenic differentiation of periodontal ligament cells and inhibited the production of lipopolysaccharide-induced inflammatory mediators in gingival fibroblasts. Concomitantly, VnP-16 modulated the host immune response by reducing the number of receptor activator of NF-κB ligand (RANKL)-expressing lipopolysaccharide-stimulated CD4+ and CD8+ T cells, and by suppressing RANKL and interleukin (IL)-17A production. Furthermore, local administration of VnP-16 in rats and mice significantly prevented and reversed alveolar bone loss induced by ligature-induced periodontitis. VnP-16 enhanced osteoblastogenesis and simultaneously inhibited osteoclastogenesis and suppressed RANKL and IL-17A expression in vivo. CONCLUSIONS: Our findings suggest that VnP-16 acts as a potent therapeutic agent for preventing and treating periodontitis by regulating bone re-modelling and immune and inflammatory responses.
Assuntos
Perda do Osso Alveolar , Periodontite , Perda do Osso Alveolar/tratamento farmacológico , Perda do Osso Alveolar/prevenção & controle , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Humanos , Interleucina-17/uso terapêutico , Ligantes , Lipopolissacarídeos/farmacologia , Camundongos , NF-kappa B , Osteogênese , Periodontite/tratamento farmacológico , Periodontite/metabolismo , Periodontite/prevenção & controle , Ligante RANK/metabolismo , Ratos , Vitronectina/uso terapêutico , Microtomografia por Raio-XRESUMO
Gö6976 is a nonglycosidic indolocarbazole compound widely used as a specific inhibitor of PKCα/ß. In experiments probing for a role of PKCα in human laminin-2-integrin-mediated cell adhesion and spreading of PC12 cells, we observed unexpected enhancements of adhesion, spreading and stress fiber formation to 1 µM Gö6976 with concomitant increase in membrane translocation of PKCδ and autophosphorylation of focal adhesion kinase (FAK). Importantly, enhanced cellular behavior and membrane translocation of PKCδ induced by Gö6976 was retained in siRNA-transfected PC12 cells to knockdown PKCα expression. Gö6976 also induced laminin-dependent cell adhesion in NIH/3T3 and CV-1 fibroblasts, suggesting of a mechanism that may be common to multiple cell-types. A specific inhibitor of PKCδ, rottlerin, completely abrogated Gö6976-dependent increase in PC12 cell adhesion to laminin as well as the activation of small GTPases, Rac1 and Cdc42, that are downstream of PKCδ in adhesion receptor signaling. siRNA knockdown of Rac1 and Cdc42 expression inhibited cell spreading and lamellipodia formation in PC12 cells. Overall, these results suggest that Gö6976 may stimulate membrane recruitment of PKCδ through a mechanism that is independent of PKCα/ß signaling. In addition, the activation of Rac1 and Cdc42 by human laminin-2-integrin-dependent activation of PKCδ/FAK signaling mediates cell spreading and lamellipodia formation in PC12 cells.
Assuntos
Carbazóis/farmacologia , Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteína Quinase C-delta/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Células Cultivadas , Chlorocebus aethiops , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Células PC12 , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C beta , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-delta/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Regulação para Cima/efeitos dos fármacosRESUMO
Previously, we reported that various oral bacteria regulate interleukin (IL)-8 production differently in gingival epithelial cells. The aim of this study was to characterize the pattern recognition receptor(s) that mediate bacteria-induced IL-8 expression. Among ligands that mimic bacterial components, only a Toll-like receptor (TLR) 9 ligand enhanced IL-8 expression as determined by ELISA. Both normal and immortalized human gingival epithelial (HOK-16B) cells expressed TLR9 intracellularly and showed enhanced IL-8 expression in response to CpG-oligonucleotide. The ability of eight strains of four oral bacterial species to induce IL-8 expression in HOK-16B cells, and their invasion capacity were examined in the absence or presence of 2% human serum. The ability of purified bacterial DNA (bDNA) to induce IL-8 was also examined. Six out of eight strains increased IL-8 production in the absence of serum. Usage of an endosomal acidification blocker or a TLR9 antagonist inhibited the IL-8 induction by two potent strains. In the presence of serum, many strains lost the ability to induce IL-8 and presented substantially reduced invasion capacity. The IL-8-inducing ability of bacteria in the absence or presence of serum showed a strong positive correlation with their invasion index. The IL-8-inducing ability of bacteria in the absence of human serum was also correlated with the immunostimulatory activity of its bDNA. The observed immunostimulatory activity of the bDNA could not be linked to its CpG motif content. In conclusion, oral bacteria induce IL-8 in gingival epithelial cells through TLR9 and the IL-8-inducing ability depends on the invasive capacity and immunostimulating DNA.
Assuntos
Bactérias/imunologia , DNA Bacteriano/imunologia , Gengiva/imunologia , Interleucina-8/biossíntese , Boca/microbiologia , Receptor Toll-Like 9/metabolismo , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Gengiva/metabolismo , Gengiva/microbiologia , Humanos , Interleucina-8/imunologia , Receptor Toll-Like 9/antagonistas & inibidores , Receptor Toll-Like 9/imunologiaRESUMO
BACKGROUND: Bone remodeling is tightly regulated through bone resorption and bone formation; imbalances in bone remodeling can cause various pathological conditions such as osteoporosis. Antiresorptive agents commonly used for treating osteoporosis do not substantially reverse osteoporotic bone loss. METHODS: We evaluated the effects of the RVYFFKGKQYWE motif (residues 270-281; VnP-16) of human vitronectin on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and osteoclastogenesis of bone marrow-derived macrophages. The effects of VnP-16 were also assessed in a mouse model of estrogen deficiency-induced osteoporosis (ovariectomized female C57BL/6 mice). To assay whether VnP-16 can reverse ovariectomy-induced bone loss, synthetic peptides or vehicle were subcutaneously injected into ovariectomized mice once a week for 4 weeks (n = 10/group). To evaluate the bone restorative effects of VnP-16, in-vivo micro-computed tomography analysis and histological staining were performed. RESULTS: VnP-16 induced osteogenic differentiation of hMSCs and inhibited the RANKL-RANK-TRAF6 axis in the osteoclastogenesis signaling pathway. Furthermore, systemic administration of VnP-16 reversed ovariectomy-induced bone loss in the femoral neck, distal femur and lumbar spine by increasing osteoblast differentiation and promoting bone formation, and concomitantly decreasing osteoclastogenesis and inhibiting bone resorption. The bone restorative effect of VnP-16 was observed one week after subcutaneous administration, and although the timing of the effect differed according to bone location, it persisted for at least 3 weeks. CONCLUSION: Our findings suggest that VnP-16 is a potential therapeutic agent for treating osteoporosis that mediates its effects through dual regulation of bone remodeling.
Assuntos
Reabsorção Óssea , Osteoporose , Feminino , Camundongos , Humanos , Animais , Vitronectina/metabolismo , Vitronectina/farmacologia , Osteogênese , Osteoclastos , Microtomografia por Raio-X , Camundongos Endogâmicos C57BL , Ovariectomia/efeitos adversos , Remodelação Óssea , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/complicações , Reabsorção Óssea/metabolismo , Osteoporose/tratamento farmacológico , Peptídeos/farmacologia , Peptídeos/metabolismoRESUMO
PURPOSE: Spondyloarthritis (SpA) is a systemic inflammatory arthritis mediated mainly by interleukin (IL)-17. The vitronectin-derived bioactive peptide, VnP-16, exerts an anti-osteoporotic effect via ß1 and αvß3 integrin signaling. SpA is associated with an increased risk of osteoporosis, and we investigated the effect of VnP-16 in mice with SpA. METHODS: SpA was induced by curdlan in SKG ZAP-70W163C mice, which were treated with vehicle, celecoxib, VnP-16, or VnP-16+celecoxib. The clinical score, arthritis score, spondylitis score, and proinflammatory cytokine expression of the spine were evaluated by immunohistochemical staining. Type 17 helper T cell (Th17) and regulatory T cell (Treg) differentiation in the spleen was evaluated by flow cytometry and in the spine by confocal staining. Splenocyte expression of signal transducer and activator of transcription (STAT) 3 and pSTAT3 was evaluated by in vitro Western blotting. RESULTS: The clinical score was significantly reduced in the VnP16+celecoxib group. The arthritis and spondylitis scores were significantly lower in the VnP-16 and VnP16+celecoxib groups than the vehicle group. In the spine, the levels of IL-1ß, IL-6, tumor necrosis factor-α, and IL-17 expression were reduced and Th17/Treg imbalance was regulated in the VnP-16 alone and VnP-16+celecoxib groups. Flow cytometry of splenocytes showed increased polarization of Tregs in the VnP-16+celecoxib group. In vitro, VnP-16 suppressed pSTAT3. CONCLUSIONS: VnP-16 plus celecoxib prevented SpA progression in a mouse model by regulating the Th17/Treg imbalance and suppressing the expression of proinflammatory cytokines.
Assuntos
Celecoxib/administração & dosagem , Peptídeos/administração & dosagem , Espondilartrite/tratamento farmacológico , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismo , Vitronectina/química , beta-Glucanas/efeitos adversos , Animais , Celecoxib/farmacologia , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Integrina alfaVbeta3/metabolismo , Integrina beta1/metabolismo , Camundongos , Peptídeos/farmacologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Baço/imunologia , Espondilartrite/induzido quimicamente , Espondilartrite/genética , Espondilartrite/imunologiaRESUMO
PURPOSE: We aimed to introduce a new technique to reduce regional asymmetry of corneal thickness by assessing its effectiveness in four patients with myopic regression after laser refractive surgery (LRS). PATIENTS AND METHODS: Four patients (four eyes) with myopic regression after LRS were included in this study. A new technique of enhancement with laser epithelial keratomileusis-linked laser asymmetric keratectomy using semi-cylindrical ablation pattern (E-LAK-SCAP) with full integration of the Vision-Up software for analyzing the corneal thickness deviation can be used to create central symmetry by blocking laser ablation on the thin cornea. It reduces the regional asymmetry of the corneal thickness, thus improving corneal symmetry and correcting the refractive power and myopic shift due to E-LAK-SCAP. We measured refraction, visual acuity, intraocular pressure (IOP), central corneal thickness (CCT), corneal irregularities in the 3.0mm, and 5.0 zones on Orbscan maps, the sum of corneal thickness deviations in four directions (SUM), distance between the maximum posterior elevation (best-fit-sphere [BFS]) and the visual axis (DISTANCE), and angle kappa before and after LRS and E-LAK-SCAP. Blurring scores were measured before and after E-LAK-SCAP. RESULTS: The uncorrected far visual acuity (LogMAR) increased after LRS and E-LAK-SCAP. SUM (µm) increased after LRS in three cases, but decreased in all four cases after E-LAK-SCAP. DISTANCE increased after LRS, but decreased after E-LAK-SCAP. The spherical equivalent, CCT, decreased after LRS and E-LAK-SCAP. Blurring scores decreased after E-LAK-SCAP, and angle kappa was similar before and after LRS, but decreased after E-LAK-SCAP. IOP was similar before and after both LRS and E-LAK-SCAP. CONCLUSION: E-LAK-SCAP improved corneal symmetry by reducing the SUM and DISTANCE, showing good postoperative visual acuity, and blurring was reduced postoperatively. There was no myopic regression in the one-year postoperative period.
RESUMO
ABSTRACT: To compare and analyze the postoperative 1-year outcomes of laser refractive surgery (LRS) alone vs LRS with laser asymmetric keratectomy (LAK), in patients with myopia, for preventing and resolving LRS complications.This retrospective study compared the preoperative and 1-year postoperative outcomes between the control and comparison groups using a sum of deviations in corneal thickness in 4 directions >80âµm. The control group included 41 patients with myopia (41 eyes) who underwent LRS. The comparison group included 33 patients (33 eyes) who received LAK-linked LRS. Age, spherical equivalent (SE), sphere, cylinder, uncorrected distance visual acuity (UDVA), pupil size, kappa angle, central corneal thickness, corneal irregularity in the 3.0âmm zone on Orbscan maps (SUM), distance between the maximum posterior elevation (best-fit-sphere) and the visual axis (DISTANCE), postoperative blurring scores, frequency of postoperative myopic regression, and efficiency index were compared.Preoperative age (Pâ=â.198), SE (Pâ=â.686), sphere (Pâ=â.562), cylinder (Pâ=â.883), UDVA (Pâ=â.139), pupil size (Pâ=â.162), kappa angle (Pâ=â.807), central corneal thickness (Pâ=â.738), corneal irregularity (Pâ=â.826), SUM (Pâ=â.774), and DISTANCE (Pâ=â.716) were similar between the 2 groups. The 1-year postoperative SE (Pâ=â.024), sphere (Pâ=â.022), corneal irregularity (Pâ=â.033), SUM (Pâ=â.000), DISTANCE (Pâ=â.04), blurring scores (Pâ=â.000), and frequency of postoperative myopic regression (Pâ=â.004) were significantly decreased in the comparison group compared to the control group. UDVA (Pâ=â.014) and the efficiency index (Pâ=â.035) were higher in the comparison group.LAK with LRS improved corneal symmetry by reducing the SUM and DISTANCE. UDVA and efficiency index were also improved and blurring and myopic regression were reduced postoperatively.
Assuntos
Córnea/cirurgia , Cirurgia da Córnea a Laser/efeitos adversos , Ceratomileuse Assistida por Excimer Laser In Situ/efeitos adversos , Miopia/cirurgia , Adulto , Estudos de Casos e Controles , Terapia Combinada , Córnea/fisiopatologia , Paquimetria Corneana/estatística & dados numéricos , Cirurgia da Córnea a Laser/métodos , Feminino , Humanos , Ceratomileuse Assistida por Excimer Laser In Situ/métodos , Masculino , Miopia/diagnóstico , Miopia/fisiopatologia , Período Pós-Operatório , Refração Ocular/fisiologia , Estudos Retrospectivos , Resultado do Tratamento , Transtornos da Visão/epidemiologia , Transtornos da Visão/etiologia , Acuidade Visual/fisiologiaRESUMO
Laminin-2 promotes basement membrane assembly and peripheral myelinogenesis; however, a receptor-binding motif within laminin-2 and the downstream signaling pathways for motif-mediated cell adhesion have not been fully established. The human laminin-2 alpha2 chain cDNAs cloned from human keratinocytes and fibroblasts correspond to the laminin alpha2 chain variant sequence from the human brain. Individually expressed recombinant large globular (LG) 1 protein promotes cell adhesion and has heparin binding activities. Studies with synthetic peptides delineate the DLTIDDSYWYRI motif (Ln2-P3) within the LG1 as a major site for both heparin and cell binding. Cell adhesion to LG1 and Ln2-P3 is inhibited by treatment of heparitinase I and chondroitinase ABC. Syndecan-1 from PC12 cells binds to LG1 and Ln2-P3 and colocalizes with both molecules. Suppression of syndecan-1 with RNA interference inhibits cell adhesion to LG1 and Ln2-P3. The binding of syndecan-1 with LG1 and Ln2-P3 induces the recruitment of protein kinase Cdelta (PKCdelta) into the membrane and stimulates its tyrosine phosphorylation. A decrease in PKCdelta activity significantly reduces cell adhesion to LG1 and Ln2-P3. Taken together, these results indicate that the Ln2-P3 motif and LG1 domain, containing the motif, within the human laminin-2 alpha2 chain are major ligands for syndecan-1, which mediates cell adhesion through the PKCdelta signaling pathway.
Assuntos
Adesão Celular , Membrana Celular/metabolismo , Laminina/metabolismo , Proteína Quinase C-delta/metabolismo , Sindecana-1/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Células Cultivadas , Chlorocebus aethiops , Dicroísmo Circular , Células Epidérmicas , Epiderme/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Queratinócitos/metabolismo , Dados de Sequência Molecular , Células PC12 , Fosforilação , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Sindecana-1/genéticaRESUMO
We reported previously that Treponema denticola, one of the periodontal pathogens, suppresses the expression of human beta-defensins (HBDs) in human gingival epithelial cells. To identify the mechanisms involved in this suppression, immortalized and normal human gingival epithelial cells were infected with live or heat-killed T. denticola for 24 h, and then the expression of HBDs was examined by real-time RT-PCR. Live T. denticola suppressed the expression of HBD-3 substantially and also suppressed the expression of HBD-1 and HBD-2. However, heat-killed bacteria did not produce a suppressive effect but instead slightly upregulated the levels of HBD-2 and HBD-3. In contrast to live T. denticola, which reduced the activation of mitogen-activated protein kinase (MAPK) and NF-kappaB within an hour of infection, heat-killed bacteria did not show any inhibitory effect on the MAPK and NF-kappaB signaling pathways. Knockdown of Toll-like receptor 2 (TLR2) via RNA interference abolished the suppressive effect of T. denticola on the expression of HBD-3. Heat-killed T. denticola but not live bacteria could activate TLR2 in CHO/CD14/TLR2 reporter cells, suggesting that T. denticola contains a heat-labile inhibitor(s) of TLR2 in addition to ligands recognized by TLR2. Indeed, live T. denticola was able to inhibit TLR2 activation by Pam(3)CSK. In conclusion, T. denticola suppressed the expression of HBD-3 by inhibiting the TLR2 axis in gingival epithelial cells. These results may provide new insight into the pathogenesis of periodontitis caused by T. denticola.
Assuntos
Células Epiteliais/microbiologia , Gengiva/microbiologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologia , Infecções por Treponema/imunologia , Separação Celular , Células Cultivadas , Células Epiteliais/imunologia , Citometria de Fluxo , Gengiva/imunologia , Humanos , Periodontite/imunologia , Periodontite/microbiologia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Treponema denticola/imunologia , beta-Defensinas/biossíntese , beta-Defensinas/imunologiaRESUMO
Laminin-5 and alpha3beta1 integrin promote keratinocyte survival; however, the downstream signaling pathways for laminin-5/alpha3beta1 integrin-mediated cell survival had not been fully established. We report the unexpected finding of multiple interactions between 14-3-3 isoforms and proapoptotic proteins in the survival signaling pathway. Ln5-P4 motif within human laminin-5 alpha3 chain promotes cell survival and anti-apoptosis by inactivating Bad and YAP. This effect is achieved through the formation of 14-3-3zeta/p-Bad and 14-3-3sigma/p-YAP complexes, which is initiated by alpha3beta1 integrin and FAK/PI3K/Akt signaling. These complexes result in cytoplasmic sequestration of Bad and YAP and their subsequent inactivation. An increase in Akt1 activity in cells induces 14-3-3zeta and sigma, p-Bad, and p-YAP, promoting cell survival, whereas decreasing Akt activity suppresses the same proteins and inhibits cell survival. Suppression of 14-3-3zeta with RNA-interference inhibits cell viability and promotes apoptosis. These results reveal a new mechanism of cell survival whereby the formation of 14-3-3zeta/p-Bad and 14-3-3sigma/p-YAP complexes is initiated by laminin-5 stimulation via the alpha3beta1 integrin and FAK/PI3K/Akt signaling pathways, thereby resulting in cell survival and anti-apoptosis.
Assuntos
Proteínas 14-3-3/metabolismo , Integrina alfa3beta1/metabolismo , Queratinócitos/metabolismo , Cicatrização , Proteínas 14-3-3/agonistas , Proteínas 14-3-3/genética , Motivos de Aminoácidos/fisiologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Moléculas de Adesão Celular/farmacologia , Proteínas de Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Pré-Escolar , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Quinase 1 de Adesão Focal/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Lactente , Integrina alfa3beta1/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Morfolinas/farmacologia , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Proteína de Morte Celular Associada a bcl/efeitos dos fármacos , Proteína de Morte Celular Associada a bcl/metabolismo , CalininaRESUMO
This study compared one-year postoperative outcomes of laser refractive surgery combined with laser asymmetric keratectomy (LAK) and laser in situ keratomileusis (LASIK)for myopia correction in middle-aged patients (aged 40-49 years) with a total corneal thickness deviation (summed across four directions) ≥ 80 microns. The control group (n = 26; 52 eyes) underwent LASIK; the comparison group (n = 26; 52 eyes) underwent combined laser refractive surgery and LAK. Age, spherical equivalence, uncorrected visual acuity (near and far), corneal irregularity on the Orbscan map, sum of corneal thickness deviations in four directions, corneal thickness distribution, distance between the maximum posterior elevation (best-fit sphere; BFS) and visual axis, and postoperative blurring scores were analysed retrospectively between the groups. Both groups had similar preoperative findings. Postoperatively, the sum of corneal thickness deviations in four directions (p = 0.000), distance between maximum posterior elevation (BFS) and visual axis (p = 0.003),blurring score (p = 0.001), and corneal irregularity in the 3.0 and 5.0 mm zones on the Orbscan map (p = 0.033 and p < 0.0001, respectively) were significantly lower in the comparison group (p = 0.000). LAK reduced total corneal thickness deviation, improved corneal symmetry, and reduced blurring scores significantly, one-year postoperatively. LAK could resolve shortcomings of LASIK, producing better surgical outcomes.
Assuntos
Cirurgia da Córnea a Laser/métodos , Ceratomileuse Assistida por Excimer Laser In Situ/métodos , Adulto , Córnea/patologia , Córnea/cirurgia , Cirurgia da Córnea a Laser/efeitos adversos , Feminino , Humanos , Ceratomileuse Assistida por Excimer Laser In Situ/efeitos adversos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Transtornos da Visão/etiologiaRESUMO
We previously reported that the PPFEGCIWN motif (Ln2-LG3-P2-DN3), residues 2678-2686 of the human laminin α2 chain, promotes cell attachment of normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts (NHDFs); however, its in vivo effects on cutaneous wound healing have not yet been examined. In this study, we sought to determine whether Ln2-LG3-P2-DN3 could promote full-thickness cutaneous wound healing by accelerating wound reepithelialization and wound closure in vivo. Ln2-LG3-P2-DN3 had significantly higher cell attachment and spreading activities than vehicle or scrambled peptide control in both NHEKs and NHDFs in vitro. The wound area was significantly smaller in rats treated with Ln2-LG3-P2-DN3 than in those treated with vehicle or scrambled peptide in the early phase of wound healing. Furthermore, Ln2-LG3-P2-DN3 significantly accelerated wound reepithelialization relative to vehicle or scrambled peptide and promoted FAK-Tyr397 phosphorylation and Rac1 activation. Collectively, our findings suggest that the PPFEGCIWN motif has potential as a therapeutic agent for cutaneous regeneration via the acceleration of wound reepithelization and wound closure.
Assuntos
Quinase 1 de Adesão Focal/metabolismo , Guanosina Trifosfato/metabolismo , Laminina/química , Peptídeos , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões , Proteínas rac1 de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Animais , Masculino , Peptídeos/química , Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/terapiaRESUMO
Early implant loading is very important for reducing the duration of missing teeth in human patients. The laminin-derived peptide, DLTIDDSYWYRI motif (Ln2-P3), accelerates bone healing. Therefore, to investigate the hypothesis that Ln2-P3 increases the bone response to sandblasted, large-grit, acid-etched (SLA) titanium implants, the effect of the Ln2-P3 peptide on the osseointegration of SLA titanium implants was evaluated in vitro and in vivo. Human osteoblast-like cells were cultured on untreated, scrambled peptide (SP)-treated, and Ln2-P3-treated SLA titanium discs, and the cellular responses of these cells were evaluated. The Ln2-P3 treatment augmented osteoblast attachment and spreading, alkaline phosphatase activity, and the expression of osteogenic marker genes. Furthermore, the untreated and Ln2-P3-treated SLA titanium implants were inserted into the tibiae of rabbits for 9 and 11 days. Compared with the untreated implants, the Ln2-P3-treated implants showed a significantly higher bone-to-implant contact ratio at Day 9 after implantation and an increased bone area. The Ln2-P3 treatment of the SLA titanium implant surface augmented osteoblastic activity and accelerated peri-implant bone formation at the bone-implant interface. Overall, these results indicated that compared with the SLA titanium surface alone, the Ln2-P3 peptide-treated SLA titanium surface enhances initial osseointegration, thereby facilitating earlier implant loading.
Assuntos
Substitutos Ósseos/farmacologia , Laminina/farmacologia , Osseointegração/efeitos dos fármacos , Peptídeos/farmacologia , Titânio/farmacologia , Animais , Prótese Ancorada no Osso , Linhagem Celular , Feminino , Humanos , Osteoblastos/citologia , Osteogênese/efeitos dos fármacos , Coelhos , Tíbia/efeitos dos fármacos , Tíbia/lesões , Tíbia/fisiologia , Tíbia/cirurgiaRESUMO
We previously reported that infection by Fusobacterium nucleatum strongly induced the expression of both human beta-defensin 2 (HBD-2) and HBD-3 by gingival epithelial cells. The aim of this study was to characterize the pattern recognition receptors (PRRs) and regulatory mechanisms involved in the induction of HBD-2 and -3 expression by F. nucleatum in gingival epithelial cells. Immortalized human gingival epithelial HOK-16B cells were infected with live or heat-killed F. nucleatum, and the expression of HBDs and interleukin-8 (IL-8) was examined by real-time reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively. Live, but not heat-killed, F. nucleatum invaded HOK-16B cells, as seen by confocal microscopy and flow cytometry. Live F. nucleatum induced both HBD-2 and -3 efficiently, whereas heat-killed bacteria induced only HBD-3 at a reduced level. Knockdown of NACHT-LRR- and pyrin domain-containing protein 2 (NALP2), the most abundant intracellular PRR in HOK-16B cells, by RNA interference (RNAi) significantly reduced the induction of HBD-3 but not HBD-2 and IL-8. In addition, knockdown of Toll-like receptor 2 (TLR2) by RNAi reduced the upregulation of HBD-2 and -3 but not IL-8. Heat-killed F. nucleatum was hindered in its ability to activate TLR2 and JNK signaling pathways. Theses data show that TLR2 and NALP2 mediate the induction of HBDs by F. nucleatum in gingival epithelial cells, but thresholds for the two HBD genes are different.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Epiteliais/metabolismo , Infecções por Fusobacterium/metabolismo , Gengiva/metabolismo , Receptor 2 Toll-Like/metabolismo , beta-Defensinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Adulto , Proteínas Reguladoras de Apoptose , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Citometria de Fluxo , Infecções por Fusobacterium/imunologia , Fusobacterium nucleatum/imunologia , Expressão Gênica , Gengiva/imunologia , Gengiva/microbiologia , Humanos , Interleucina-8/metabolismo , Microscopia Confocal , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Receptor 2 Toll-Like/imunologia , beta-Defensinas/imunologiaRESUMO
PURPOSE: This study aimed to investigate the in vitro and in vivo bone-forming potential of a sandblasted, large-grit, acid-etched (SLA) titanium (Ti) surface treated with a laminin-derived functional peptide, PPFEGCIWN (DN3). MATERIALS AND METHODS: Human osteoblast-like MG63 cells were cultured with SLA Ti discs untreated or treated with DN3 or a control scrambled peptide (SP). Cell adhesion, spreading, and viability on the discs were tested. Alkaline phosphatase gene expression and enzyme activity were also evaluated. Four DN3-coated SLA Ti implants and four untreated implants were placed into the tibiae of two rabbits (two implants/tibia). Ten days later, the bone-implant interfaces were subjected to histomorphometry to measure the bone response. The surface properties of the discs and implants were determined using scanning electron, widefield confocal, and confocal laser microscopy and x-ray photoelectron spectroscopy. RESULTS: The peptide-treated and untreated discs and implants were similar in terms of physical surface properties, but the peptide-treated surfaces had significantly higher nitrogen levels (P < .05). The DN3 peptide promoted cell adhesion, spreading, and alkaline phosphatase expression and enzyme activity (P < .05). Histomorphometry of the harvested implants showed rapid bone formation and affinity of the motif. CONCLUSION: This study suggests that treatment with the cell adhesion peptide DN3 promotes bone healing at the SLA Ti surface.
Assuntos
Implantes Dentários , Titânio , Animais , Linhagem Celular , Humanos , Laminina , Microscopia Eletrônica de Varredura , Osteoblastos , Osteogênese , Peptídeos , Coelhos , Propriedades de SuperfícieRESUMO
In this study, we evaluated early bone responses to a vitronectin-derived, minimal core bioactive peptide, RVYFFKGKQYWE motif (VnP-16), both in vitro and in vivo, when the peptide was treated on sandblasted, large-grit, acid-etched (SLA) titanium surfaces. Four surface types of titanium discs and of titanium screw-shaped implants were prepared: control, SLA, scrambled peptide-treated, and VnP-16-treated surfaces. Cellular responses, such as attachment, spreading, migration, and viability of human osteoblast-like HOS and MG63 cells were evaluated in vitro on the titanium discs. Using the rabbit tibia model with the split plot design, the implants were inserted into the tibiae of four New Zealand white rabbits. After two weeks of implant insertion, the rabbits were sacrificed, the undecalcified specimens were prepared for light microscopy, and the histomorphometric data were measured. Analysis of variance tests were used for the quantitative evaluations in this study. VnP-16 was non-cytotoxic and promoted attachment and spreading of the human osteoblast-like cells. The VnP-16-treated SLA implants showed no antigenic activities at the interfaces between the bones and the implants and indicated excellent bone-to-implant contact ratios, the means of which were significantly higher than those in the SP-treated implants. VnP-16 reinforces the osteogenic potential of the SLA titanium dental implant.
RESUMO
Electrospinning of collagen (COL)/silk fibroin (SF) blend solutions in 1,1,1,3,3,3-hexafluoro-2-propanol was investigated for fabrication of a biocompatible and biomimetic nanostructured scaffold for tissue engineering. The morphology of the electrospun COL/SF blend nanofibers was observed by scanning electron microscopy. The average diameters of COL/SF blend fibers ranged from 320 to 360 nm, irrespective of SF content in the blends. Both COL and SF components in the as-spun COL/SF blend matrices were stabilized by glutaraldehyde and water vapor, respectively, under the saturated glutaraldehyde aqueous solution at 25 degrees C. The glutaraldehyde vapor chemically stabilized the COL component via cross-linking, whereas the water vapor physically stabilized the SF component via crystallization to the beta-sheet structure. These structural changes of after-treated COL/SF blend matrices were examined using ATR-IR and CP/MAS (13)C NMR spectroscopy. To assay the cytocompatibility and cellular behavior of the COL/SF blend nanofibrous scaffolds, cell attachment and the spreading of normal human epidermal keratinocytes (NHEK) and fibroblasts (NHEF) seeded on the scaffolds were studied. In addition, both morphological changes and cellular responses of COL/SF blend nanofibrous matrices were also compared with COL/SF hybrid nanofibrous matrices. Generally similar levels of cell attachment and spreading of NHEF were shown in the COL/SF blend nanofibrous matrix compared with those of the pure COL and pure SF matrices; the cellular responses of NHEK were, however, markedly decreased in the COL/SF blend nanofibrous matrix as compared to the pure matrices. In contrast, cell attachment and spreading of NHEK on the COL/SF hybrid nanofibrous matrix were significantly higher than that of the COL/SF blend nanofibrous matrix. Our results indicate that a COL/SF hybrid nanofibrous matrix may be a better candidate than a COL/SF blend nanofibrous matrix for biomedical applications such as wound dressing and scaffolds for tissue engineering.
Assuntos
Biomimética , Colágeno/química , Fibroínas/química , Queratinócitos/metabolismo , Nanoestruturas/ultraestrutura , Seda/química , Engenharia Tecidual/métodos , Animais , Bovinos , Adesão Celular , Células Cultivadas , Criança , Cristalização , Células Epidérmicas , Epiderme/metabolismo , Humanos , Queratinócitos/citologia , Espectroscopia de Ressonância Magnética , Nanoestruturas/química , Pele/citologia , Pele/metabolismoRESUMO
Embryonic stem cells (ESCs) are established from blastocysts and give rise to various types of cells and tissues. In the present study, we assessed the osteogenic potential of ESCs using in vitro culture conditions and in vivo differentiation in tooth sockets. An ESC-derived embryoid body (EB) was formed and subsequently induced to an osteogenic lineage. The differentiated EB cells exhibited increased expression of various osteogenic markers as determined by real-time PCR analysis. Likewise, the differentiated EB-derived cells had enhanced alkaline phosphatase activity and calcium accumulation, as determined by cytochemical methods. For in vivo transplantation, mixtures of ESCs and hydroxyapatite/ tricalcium phosphate particles or EBs alone were transplanted into female rat tooth sockets. After 12 weeks, we observed formation of osteogenic structure in the tooth sockets without evidence of teratomas. These data suggest that pluripotent ESCs can serve as an alternative source for the reconstruction of craniofacial structures, as well as for further applications.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Osteogênese/fisiologia , Células-Tronco Pluripotentes/fisiologia , Transplante de Células-Tronco , Alvéolo Dental/citologia , Animais , Biomarcadores/metabolismo , Fosfatos de Cálcio/metabolismo , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Feminino , Hidroxiapatitas/metabolismo , Células-Tronco Pluripotentes/citologia , Ratos , Ratos Sprague-DawleyRESUMO
To fabricate a biomimetic nanostructured bicomponent scaffolds, two types of chitin/silk fibroin (SF) nanofibrous scaffolds (blend scaffolds and hybrid scaffolds) were prepared by electrospinning or simultaneous electrospinning of chitin/SF solutions. The chitin/SF bicomponent scaffolds were after-treated with water vapor, and their nanofibrous structures were almost maintained. From the cytocompatibility and cell behavior on the chitin/SF blend or hybrid nanofibrous scaffolds, the hybrid matrix with 25% chitin and 75% SF as well as the chitin/SF blend nanofibers could be a potential candidate for tissue engineering scaffolds.