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1.
BMB Rep ; 56(8): 463-468, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37156631

RESUMO

Screening for genetic defects in the cells should be examined for clinical application. The Pearson syndrome (PS) patient harbored nuclear mutations in the POLG and SSBP1 genes, which could induce systemic large-scale mitochondrial genome (mtDNA) deletion. We investigated iPSCs with mtDNA deletions in PS patient and whether deletion levels could be maintained during differentiation. The iPSC clones derived from skin fibroblasts (9% deletion) and blood mononuclear cells (24% deletion) were measured for mtDNA deletion levels. Of the 13 skin-derived iPSC clones, only 3 were found to be free of mtDNA deletions, whereas all blood-derived iPSC clones were found to be free of deletions. The iPSC clones with (27%) and without mtDNA deletion (0%) were selected and performed in vitro and in vivo differentiation, such as embryonic body (EB) and teratoma formation. After differentiation, the level of deletion was retained or increased in EBs (24%) or teratoma (45%) from deletion iPSC clone, while, the absence of deletions showed in all EBs and teratomas from deletion-free iPSC clones. These results demonstrated that non-deletion in iPSCs was maintained during in vitro and in vivo differentiation, even in the presence of nuclear mutations, suggesting that deletion-free iPSC clones could be candidates for autologous cell therapy in patients. [BMB Reports 2023; 56(8): 463-468].


Assuntos
Células-Tronco Pluripotentes Induzidas , Teratoma , Humanos , DNA Mitocondrial/genética , Diferenciação Celular/genética , Terapia Baseada em Transplante de Células e Tecidos , Teratoma/genética , Proteínas de Ligação a DNA , Proteínas Mitocondriais
2.
Cell Prolif ; 55(7): e13274, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35698260

RESUMO

OBJECTIVES: Patient-derived induced pluripotent stem cells (iPSCs) are materials that can be used for autologous stem cell therapy. We screened mtDNA mutations in iPSCs and iPSC-derived neuronal cells from patients with Alzheimer's disease (AD). Also, we investigated whether the mutations could affect mitochondrial function and deposition of ß-amyloid (Aß) in differentiated neuronal cells. MATERIALS AND METHODS: mtDNA mutations were measured and compared among iPSCs and iPSC-derived neuronal cells. The selected iPSCs carrying mtDNA mutations were subcloned, and then their growth rate and neuronal differentiation pattern were analyzed. The differentiated cells were measured for mitochondrial respiration and membrane potential, as well as deposition of Aß. RESULTS: Most iPSCs from subjects with AD harbored ≥1 mtDNA mutations, and the number of mutations was significantly higher than that from umbilical cord blood. About 35% and 40% of mutations in iPSCs were shared with isogenic iPSCs and their differentiated neuronal precursor cells, respectively, with similar or different heteroplasmy. Furthermore, the mutations in clonal iPSCs were stable during extended culture and neuronal differentiation. Finally, mtDNA mutations could induce a growth advantage with higher viability and proliferation, lower mitochondrial respiration and membrane potential, as well as increased Aß deposition. CONCLUSION: This study demonstrates that mtDNA mutations in patients with AD could lead to mitochondrial dysfunction and accelerated Aß deposition. Therefore, early screening for mtDNA mutations in iPSC lines would be essential for developing autologous cell therapy or drug screening for patients with AD.


Assuntos
Doença de Alzheimer , Genoma Mitocondrial , Células-Tronco Pluripotentes Induzidas , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Diferenciação Celular/genética , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Genoma Humano , Humanos , Mutação/genética
3.
Int J Mol Sci ; 24(1)2022 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-36614165

RESUMO

Corneal endothelial cells (CECs) do not proliferate or recover after illness or injury, resulting in decreased cell density and loss of pump/barrier function. Considering the shortage of donor cornea, it is vital to establish robust methods to generate CECs from induced pluripotent stem cells (iPSCs). We investigated the efficacy and safety of transplantation of iPSC-derived CECs into a corneal endothelial dysfunction (CED) rabbit model. iPSCs were generated from human fibroblasts. We characterized iPSCs by demonstrating the gene expression of the PSC markers OCT4, SOX2, TRA-1-60, and NANOG, teratoma formation, and differentiation into three germ layers. Differentiation of iPSCs into CECs was induced via neural crest cell (NCC) induction. CEC markers were detected using immunofluorescence and gene expression was analyzed using quantitative real-time PCR (qRT-PCR). After culturing iPSC-derived NCCs, we found the expression of zona occludens-1 (ZO-1) and Na+/K+ ATPase and a hexagonal morphology. ATP1A1, COL8A1, and AQP1 mRNA expression was higher in iPSC-derived CECs than in iPSCs and NCCs. We performed an injection of iPSC-derived CECs into the anterior chamber of a CED rabbit model and found improved levels of corneal transparency. We also found increased numbers of ZO-1- and ATP1A1-positive cells in rabbit corneas in the iPSC-derived CEC transplantation group. Usage of the coating material vitronectin (VTN) and fasudil resulted in good levels of CEC marker expression, demonstrated with Western blotting and immunocytochemistry. Combination of the VTN coating material and fasudil, instead of FNC mixture and Y27632, afforded the best results in terms of CEC differentiation's in vitro and in vivo efficacy. Successful transplantation of CEC-like cells into a CED animal model confirms the therapeutic efficacy of these cells, demonstrated by the restoration of corneal clarity. Our results suggest that iPSC-derived CECs can be a promising cellular resource for the treatment of CED.


Assuntos
Células-Tronco Pluripotentes Induzidas , Animais , Humanos , Coelhos , Endotélio Corneano , Células Endoteliais/metabolismo , Córnea , Diferenciação Celular , Células Cultivadas
4.
Int J Ophthalmol ; 14(12): 1805-1812, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34926192

RESUMO

AIM: To characterize the anti-inflammatory and anti-apoptotic effects of N-acetylcysteine (NAC) in streptozotocin (STZ)-induced diabetic rat corneal epithelium and human corneal epithelial cells (HCECs) exposed to a high-glucose environment. METHODS: HCECs were incubated in 0, 5, 50 mmol/L glucose medium, or 50 mmol/L glucose medium with NAC for 24h. Diabetes was induced in rats by intraperitoneal injection of 65 mg/kg STZ and some of these rats were topically administered NAC to corneas with 3 mice per group. We characterized receptor for advanced glycation end-products (RAGE) expression using immunofluorescence, and interleukin (IL)-1ß and cleaved caspase-3 (CCAP-3) expression using immunohistochemistry. Circulating tumor necrosis factor (TNF)-α concentration was measured by ELISA and cleaved poly-ADP ribose polymerase (PARP) concentration was quantified by Western blotting. Apoptotic cells were detected using TUNEL assay and annexin V and propidium iodide staining. RESULTS: Diabetic rats had higher expression of RAGE (2.46±0.13 fold), IL-1ß, and CCAP-3 in apoptotic cells of their corneas than control rats. The expression of RAGE (1.83±0.11 fold), IL-1ß, and CCAP-3, and the number of apoptotic cells, were reduced by topical NAC treatment. HCECs incubated in 50 mmol/L glucose medium showed high concentrations of TNF-α (310±2.00 pg/mL) and cleaved PARP (7.43±0.56 fold), and more extensive apoptosis than cells in 50 mmol/L glucose medium. However, the addition of NAC reduced the concentrations of TNF-α (153.67±2.31 pg/mL) and cleaved PARP (5.55±0.31 fold) and the number of apoptotic cells. CONCLUSION: NAC inhibits inflammation and apoptosis in the corneas of diabetic rats and HCECs maintained in a high-glucose environment.

5.
J Ophthalmol ; 2021: 4668056, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35154818

RESUMO

This study aimed to evaluate and compare the effectiveness of a newly developed epithelial removal brush with conventional methods in a rabbit model of corneal epithelial defects. The corneal epithelia of thirty-seven rabbits were removed by three different methods including blades (blade group), newly developed epithelial brushes (Ocu group), and conventional rotating brushes (Amo group). The defect area was measured with light microscopy immediately and at 4, 18, 24, and 50 hours after removal. Corneas were obtained immediately and at 24 and 50 hours and subjected to hematoxylin and eosin (H&E) and immunofluorescence staining using proliferating cell nuclear antigen (PCNA) and phosphorylated heat shock protein 27 (pHSP27) antibodies. The residual stromal surface was observed by scanning electron microscopy (SEM). In the Ocu group, epithelia were significantly recovered at 18, 24, and 50 hours compared with immediately after removal, and in the blade and Amo groups, epithelia were significantly recovered only at 50 hours after epithelial removal. The expression levels of PCNA and pHSP27 did not differ among three groups. There was significantly more inflammatory cell infiltration in the blade group than in the other groups. SEM showed a more regular and uniform residual stromal surface in the Ocu group than in the other groups. The newly developed epithelial brush showed better polishing ability and led to earlier significant epithelial recovery and a more regular and uniform stromal surface than conventional methods in this rabbit model of epithelial defects. Accumulation of clinical data is expected to expand the scope of application of new brushes for laser surface ablation.

6.
Exp Eye Res ; 202: 108319, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33080303

RESUMO

The purpose of this study was to investigate whether and how topical nerve growth factor (NGF) attenuates streptozotocin (STZ)-induced diabetic cataracts in vivo. Rats were randomly divided into three groups, including the normal control rat group, STZ-induced diabetic cataract rat group (DM group), and STZ-induced diabetic cataract rat group treated with 200 µg/mL recombinant rat ß-NGF (DM + NGF group). Cataract formation was evaluated by portable slit lamp biomicroscopy following pupil dilation at 8 weeks. The expression levels of NGF, aldose reductase (AR), and Na+/K+-ATPase in the lens epithelial cells (LECs) of the three groups were measured in the presence or absence of topical NGF. TUNEL-positive LECs were quantified to determine if hyperglycemia caused LEC apoptosis. At 8 weeks, the mean cataract score in the control group was significantly lower than that in DM and DM + NGF groups, and the score in the DM + NGF group was significantly lower than that in the DM group. At the equatorial zone and anterior central zone of lens, NGF and Na+/K+-ATPase expression levels were significantly decreased in the DM group; however, they were partially restored in the DM + NGF group. At the equatorial zone and anterior central zone of lens, AR expression and TUNEL-positive apoptotic LECs were significantly increased in the DM group compared with the control group, however, they were significantly decreased in the DM + NGF group. In conclusion, topical NGF could delay the progression of diabetic cataracts by attenuating polyol pathway activation and increasing Na+/K+-ATPase protein levels.


Assuntos
Catarata/prevenção & controle , Diabetes Mellitus Experimental/prevenção & controle , Fator de Crescimento Neural/uso terapêutico , ATPase Trocadora de Sódio-Potássio/metabolismo , Administração Oftálmica , Animais , Apoptose , Catarata/induzido quimicamente , Catarata/enzimologia , Catarata/patologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Hiperglicemia/metabolismo , Marcação In Situ das Extremidades Cortadas , Masculino , Soluções Oftálmicas , Polímeros , Ratos , Ratos Sprague-Dawley , Estreptozocina/toxicidade , Regulação para Cima
7.
Cornea ; 39(4): 508-513, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31895086

RESUMO

PURPOSE: To compare the protective effects of cyclosporine A emulsion (Restasis: 0.05% cyclosporine A) (CsAE) and cyclosporine A cationic emulsion (Ikervis: 0.1% cyclosporine A) (CsACE) on cellular inflammation, apoptosis, proliferation, and survival in an in vitro dry eye model. METHODS: The concentration of CsA in CsAE and CsACE was verified using a liquid chromatography tandem mass spectrometry system. Human corneal epithelial cells were subjected to desiccation stress. Human corneal epithelial cells were incubated with or without 3 groups of cyclosporine A medium (CsAE 1:50, CsACE 1:50, and CsACE 1:100). p-NF-κB p65, p-IκBα, Bax, Bcl-xL, p-Erk1/2, and p-Akt levels were determined using Western blots, and TNF-α levels were quantified using an enzyme-linked immunosorbent assay. RESULTS: The CsA concentration of CsACE 1:100 was nearly the same as that of CsAE 1:50. Compared with CsAE 1:50 (0.78 ± 0.19 fold), the p-NF-κB p65 level was further reduced in CsACE 1:50 (0.38 ± 1.20 fold) and 1:100 (0.29 ± 0.11 fold) as well as p-IκBα. Levels of TNF-α were also lower in CsACE 1:50 and 1:100 than in CsAE 1:50. Induction of the apoptotic protein Bax was significantly decreased in CsACE 1:50 and 1:100 compared with CsAE 1:50, whereas that of the antiapoptotic protein Bcl-xL was increased in CsACE 1:50 and 1:100. p-ERK1/2 and p-Akt levels were higher in CsACE 1:50 and 1:100 than in CsAE 1:50. CONCLUSIONS: CsACE had more potent anti-inflammatory and antiapoptotic effects than CsAE in a transwell desiccation stress model. CsACE also enhanced proliferation and survival factors under desiccation stress compared with CsAE in this in vitro dry eye model.


Assuntos
Apoptose/efeitos dos fármacos , Ciclosporina/uso terapêutico , Dessecação/métodos , Síndromes do Olho Seco/tratamento farmacológico , Epitélio Corneano/patologia , Estresse Mecânico , Lágrimas/metabolismo , Linhagem Celular , Síndromes do Olho Seco/patologia , Emulsões , Epitélio Corneano/efeitos dos fármacos , Humanos , Imunossupressores/uso terapêutico
8.
Invest Ophthalmol Vis Sci ; 59(12): 5108-5115, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30372737

RESUMO

Purpose: To evaluate the effect of diquafosol on corneal epithelium in a dry eye model using Transwell culture and a scopolamine-induced dry eye rat model. Methods: Desiccation stress induced in an in vitro dry eye model using human corneal epithelial cells was used, and the cells were incubated with or without diquafosol media diluted at 1:100. Reactive oxygen species (ROS) generation was measured using 2',7'-dichlorofluorescein diacetate (DCFH-DA). Apoptosis was analyzed, and levels of phosphorylated Erk1/2, phosphorylated p90RSK, phosphorylated Akt, IκB-α, and NF-κB-p65 were determined. Levels of IL-1ß, TNF-α, IL-6, IL-8, and GM-CSF were quantified. To investigate the in vivo effects of diquafosol, we induced dry eye in Wistar rats using scopolamine hydrobromide. The rats were divided into three groups: control, dry eye, and dry eye diquafosol; topical DIQUAS was applied four times daily for 28 days. We used immunohistochemistry to detect the levels of phosphorylated Erk1/2, phosphorylated p90RSK, and IL-1ß, and used the TUNEL assay in corneal tissue. Results: The distribution of highly fluorescent dichlorofluorescein and the proportion of annexin V- and PI-positive cells decreased in the diquafosol medium. Diquafosol increased the levels of phospho-Erk1/2, phospho-90RSK, phospho-Akt, and IκB-α, whereas it significantly decreased the levels of NF-κB-p65, IL-1ß, and TNF-α. In vivo, apoptosis was enhanced in dry eye group. This response was markedly reduced and the level of phosphorylated p90RSK and phosphorylated ERK1/2 were upregulated and IL-1ß was downregulated by DIQUAS. Conclusions: Diquafosol treatment reduced intracellular ROS levels, apoptosis, and inflammation, all of which were increased in the dry eye model through desiccation.


Assuntos
Apoptose/efeitos dos fármacos , Síndromes do Olho Seco/tratamento farmacológico , Ceratite/prevenção & controle , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Polifosfatos/uso terapêutico , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Nucleotídeos de Uracila/uso terapêutico , Animais , Western Blotting , Citocinas/metabolismo , Modelos Animais de Doenças , Síndromes do Olho Seco/metabolismo , Epitélio Corneano/efeitos dos fármacos , Feminino , Inflamação/metabolismo , Inflamação/prevenção & controle , Ceratite/metabolismo , Soluções Oftálmicas/uso terapêutico , Fosforilação , Agonistas do Receptor Purinérgico P2Y/uso terapêutico , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo
9.
Invest Ophthalmol Vis Sci ; 57(15): 6767-6775, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27978558

RESUMO

Purpose: To examine the effects of nerve growth factor (NGF) on apoptosis and inflammation in the diabetic cornea. Methods: To investigate the effects of NGF on glucose-induced apoptosis, we stained human corneal epithelial cells (HCECs) for annexin-V and propidium iodide (PI), and measured expression of cleaved caspase-3 and the Bcl-2-associated X protein (BAX). Moreover, to examine the effects of NGF on inflammation, we quantified the expression of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) using multiplex cytokine analysis, and analyzed nuclear factor-κB (NF-κB) activation and NF-κ-B inhibitor α (IκBα) degradation using Western blot analysis. To investigate the effects in vivo, we induced diabetes in male Sprague-Dawley rats using streptozotocin. The rats were divided into three groups: control, diabetic control, and diabetic NGF; topical NGF was applied three times daily for 3 weeks. We used the TUNEL assay to detect apoptosis in corneal tissue, and immunohistochemistry to identify cleaved caspase-3 and IL-1ß. Results: In HCECs, high glucose concentration (≥25 mM) led to reactive oxygen species (ROS) generation, apoptosis, and the release of inflammatory cytokines. Nerve growth factor markedly reduced ROS activation, annexin-PI-positive cells, and levels of cleaved caspase-3, BAX, IL-1ß, and TNF-α. In the diabetic rat cornea, apoptosis and inflammation were enhanced, as were levels of cleaved caspase-3 and IL-1ß. These responses were markedly reduced by NGF. Conclusions: Apoptosis and inflammation are enhanced in the diabetic cornea; NGF attenuates these responses-both in vivo and in vitro. Therefore, NGF therapy may represent a novel approach for the management of diabetic keratopathy.


Assuntos
Apoptose , Diabetes Mellitus Experimental , Epitélio Corneano/metabolismo , Regulação da Expressão Gênica , Ceratite/metabolismo , Fator de Crescimento Neural/genética , RNA Mensageiro/genética , Animais , Western Blotting , Linhagem Celular , Citocinas/metabolismo , Epitélio Corneano/patologia , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Ceratite/etiologia , Ceratite/genética , Masculino , Fator de Crescimento Neural/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
J Vis Exp ; (115)2016 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-27768052

RESUMO

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.


Assuntos
Córnea/fisiologia , Proteínas de Choque Térmico HSP27/fisiologia , Cicatrização/fisiologia , Western Blotting , Linhagem Celular , Células Cultivadas , Córnea/citologia , Córnea/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Citometria de Fluxo , Técnicas de Silenciamento de Genes/métodos , Proteínas de Choque Térmico HSP27/deficiência , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Fosforilação , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transfecção/métodos
11.
Curr Eye Res ; 41(5): 676-82, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26237412

RESUMO

PURPOSE: To evaluate the effects of prostaglandin analogs (PGAs) on cell viability and apoptosis in cultured astrocytes obtained from the lamina cribrosa (LC) of the human optic nerve head (ONH). METHODS: Astrocytes were cultured from LC samples obtained from human donor ONH and treated with three kinds of acid form of PGAs: latanoprost (LAT-A), tafluprost (TAF-A), and bimatoprost (BIM-A) (0.1, 1, 10, 50 and 100 ug/mL). Cell viability was assessed using the WST-1 assay. Cell apoptosis was measured using the deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay. Apoptotic protein expression was evaluated using western blot analysis. RESULTS: ONH astrocytes expressed FP receptor in western blot analysis. In the presence of 0.1 ug/mL of LAT-A, BIM-A, and TAF-A, the cell viability was 85%, 85% and 82%, respectively. WST-1 assay revealed about 50% of cell viability following treatment with 50 ug/mL of all PGAs. After exposing astrocytes to 10 ug/mL of each PGA for 24 hours, apoptotic cells were stained in TUNEL assay. Western blot analysis revealed that the PGAs up-regulated Bax (pro-apoptotic protein) and down-regulated Bcl-xL (anti-apoptotic protein) in the astrocytes. CONCLUSIONS: PGAs affected cell viability in cultured astrocytes obtained from human ONH LC. PGA treatment may induce apoptosis in ONH astrocytes.


Assuntos
Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Disco Óptico/citologia , Prostaglandinas Sintéticas/farmacologia , Receptores de Prostaglandina/biossíntese , Regulação para Cima , Adulto , Astrócitos/citologia , Astrócitos/metabolismo , Western Blotting , Sobrevivência Celular , Células Cultivadas , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Marcação In Situ das Extremidades Cortadas , Pessoa de Meia-Idade , Disco Óptico/metabolismo
12.
Invest Ophthalmol Vis Sci ; 56(9): 5614-21, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26305534

RESUMO

PURPOSE: We evaluated the effects of N-acetylcysteine (NAC), which is known to inhibit reactive oxygen species (ROS)-dependent apoptosis, on high glucose-induced ROS, apoptosis, inflammation, and delayed-wounding closure in primary cultured human conjunctival epithelial cells (pHCECs), and the regulatory effects of cleaved caspase-3, BAX, nuclear factor κB (NF-κB), IL-6, and TNF-α on these processes. METHODS: High glucose-induced ROS generation was measured using 2',7'-dichlorofluorescein diacetate (DCFH-DA). The effects of NAC on high glucose-induced apoptosis were investigated in pHCECs using Annexin-V and PI staining, and cleaved caspase-3 and BAX expression levels using immunoblotting. To evaluate the inflammatory response, IL-6 and TNF-α expression levels were quantified by multiplex cytokine analysis, and NF-κB activation and IkB-α degradation were assessed by Western blot analysis. The effects of NAC on high glucose-delayed conjunctival epithelial wound healing were assessed by a scratch-induced directional wounding assay. RESULTS: Compared to the untreated control and normal glucose (5 mM), high glucose at 25 mM stimulated ROS generation, apoptosis, and release of inflammatory cytokines, and delayed wound healing in pHCECs. The addition of NAC markedly reduced the high glucose-induced ROS activation, Annexin-PI-positive cells, and levels of cleaved caspase-3, BAX, IL-6, and TNF-α. N-acetylcysteine also prevented high glucose-delayed wound healing. CONCLUSIONS: High glucose levels promote apoptosis by affecting mitochondria-dependent caspase activity through elevation of ROS production, a process that can be reversed by the antioxidant NAC. These findings demonstrate that NAC has a beneficial effect on conjunctival epithelial cell wound healing, antiapoptosis, and anti-inflammation in the conjunctival epithelial cell.


Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Túnica Conjuntiva/patologia , Células Epiteliais/metabolismo , Glucose/farmacologia , Hiperglicemia/patologia , Western Blotting , Células Cultivadas , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Citometria de Fluxo , Humanos , Hiperglicemia/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Biochem Biophys Res Commun ; 450(1): 105-9, 2014 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-24866235

RESUMO

Secretory clusterin (sCLU)/apolipoprotein J is a multifunctional glycoprotein that is ubiquitously expressed in various tissues. Reduced sCLU in the joints of patients with bone erosive disease is associated with disease activity; however, its exact role has yet to be elucidated. Here, we report that CLU is expressed and secreted during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) that are treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CLU-deficient BMMs obtained from CLU(-/-) mice exhibited no significant alterations in OC differentiation in comparison with BMMs obtained from wild-type mice. In contrast, exogenous sCLU treatment significantly inhibited OC formation in both BMMs and OC precursor cultures. The inhibitory effect of sCLU was more prominent in BMMs than OC precursor cultures. Interestingly, treating BMMs with sCLU decreased the proliferative effects elicited by M-CSF and suppressed M-CSF-induced ERK activation of OC precursor cells without causing apoptotic cell death. This study provides the first evidence that sCLU reduces OC formation by inhibiting the actions of M-CSF, thereby suggesting its protective role in bone erosion.


Assuntos
Clusterina/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/fisiologia , Osteoclastos/citologia , Osteoclastos/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Ativação de Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL
14.
Invest Ophthalmol Vis Sci ; 55(1): 535-41, 2014 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-24327613

RESUMO

PURPOSE: To evaluate the effects of nerve growth factor (NGF), which is activated during inflammatory episodes of ocular diseases, on the apoptotic response in cultured human primary conjunctival epithelial cells (pHCECs). METHODS: Levels of NGF transcripts and NGF protein in pHCEC grown in medium with normal osmolarity (307 mOsm/L) or hyperosmolar medium (350, 400, and 450 mOsm/L) were determined using RT-PCR or ELISA, respectively. To assess apoptosis, pHCEC were cultured in normal or 400 mOsm/L hyperosmolar medium with neutralizing anti-NGF antibody or recombinant human NGF for 6 hours before analysis by flow cytometry. Levels of Bcl-xL, Bax, phosphorylated JNK, and cleaved caspase-3 were detected using Western blotting. Levels of the inflammatory cytokine IL-6 was analyzed using ELISA. RESULTS: Hyperosmolar conditions increased NGF levels in cultured pHCECs. Hyperosmolarity and exposure to neutralizing anti-NGF antibody significantly increased the number of apoptotic cells. Addition of recombinant human NGF to 400 mOsm/L medium decreased the number of apoptotic cells, reduced the expression of phosphorylated JNK, Bax, and cleaved caspase-3 and increased the expression of Bcl-xL. Levels of IL-6 were increased by hyperosmotic stress but decreased by exposure to recombinant human NGF. CONCLUSIONS: Hyperosmolarity induces apoptosis of pHCECs by activating JNK signaling. Increased levels of NGF under hyperosmolar conditions may contribute, at least in part, to the reduced apoptosis of pHCECs and may be beneficial in recovering conjunctival damage due to chronic hyperosmolar stress.


Assuntos
Apoptose/efeitos dos fármacos , Túnica Conjuntiva/patologia , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Fator de Crescimento Neural/genética , RNA/genética , Western Blotting , Linhagem Celular , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Citometria de Fluxo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Concentração Osmolar , Fosforilação , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
15.
Exp Eye Res ; 118: 36-41, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24239511

RESUMO

We reported the expression of phosphorylated HSP27 during epithelial wound healing in murine corneas (Jain et al., 2012) in July of 2012. This in vivo investigation demonstrated that the expression levels of phosphorylated HSP27 were greater in wounded corneal epithelial cells than in unwounded controls and that the localization of phosphorylated HSP27 was in the basal and superficial epithelia three days following corneal epithelial wounding. We suggested that phosphorylated HSP27 had a role in the early phase of corneal epithelial wound healing. The purpose of this study was to investigate the exact role of heat shock protein 27 (HSP27) phosphorylation for the wound healing of cultured human corneal epithelial cells (HCECs). HSP27-specific siRNAs and control-siRNAs, with no known homologous targets in HCECs, were created. The cultured HCECs were divided into two groups: Scrambled control-siRNA-transfected group vs. HSP27-specific siRNA-transfected group. The scratch-induced directional wounding assay, Western blotting, using antibodies against non-phosphorylated and phosphorylated HSP27, non-phosphorylated and phosphorylated Akt, and Bcl-2-associated X protein (Bax), immunofluorescence staining to determine the filament actin, flow cytometry to measure apoptosis, and proliferation assay were performed to determine the role of HSP27. Western blot assay showed that the expression of phosphorylated HSP27 significantly increased at 5, 10, and 30 min after scratch wounding, compared with those in unwounded HCECs (all p < 0.05). Western blot assay also showed HSP27-specific siRNAs effectively blocked the expression of non-phosphorylated HSP27. The HSP27-specific siRNA-transfected group had more Bax expression, less phosphorylated Akt expression, and less non-phosphorylated and phosphorylated HSP27 expression (all p < 0.05). The scratch-induced directional wounding assay showed the HSP27-specific siRNA-transfected group with a less migrating cell number than the control-siRNA-transfected group (p < 0.05). Immunofluorescence staining showed that reorganization of actin cytoskeleton prominently decreased in the HSP27-specific siRNA-transfected group, compared with the control siRNA-tranfected group. Flow cytometry revealed that the HSP27-specific siRNA-transfected group had more HCEC apoptosis. Proliferation assay showed no difference between the two groups. In conclusion, the role of HSP27 in corneal epithelial wound healing can be epithelial cell apoptosis, as well as epithelial migration. HSP27 is involved in HCEC migration by the reorganization of actin cytoskeleton.


Assuntos
Apoptose , Epitélio Corneano/metabolismo , Traumatismos Oculares/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP27/genética , RNA Interferente Pequeno/genética , Cicatrização/fisiologia , Western Blotting , Linhagem Celular , Movimento Celular , Proliferação de Células , Epitélio Corneano/lesões , Epitélio Corneano/patologia , Traumatismos Oculares/genética , Traumatismos Oculares/patologia , Citometria de Fluxo , Proteínas de Choque Térmico HSP27/biossíntese , Humanos , Fosforilação , RNA Interferente Pequeno/metabolismo
16.
Graefes Arch Clin Exp Ophthalmol ; 251(8): 1945-53, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23677487

RESUMO

BACKGROUND: To analyze the effects of supplemental epidermal growth factor (EGF) and the roles of inflammatory cytokines (interleukin [IL]-6) in an ex vivo dry-eye model under hyperosmotic stress using a multilayered culture of human conjunctival epithelial cells (HCECs). METHODS: Multilayered cultures of HCECs were exposed to hyperosmotic stress (400 mOsm/L) for 24 h in addition to 0.5 ng/mL EGF (low-EGF group) or 25 ng/mL EGF (high-EGF group). Apoptosis was analyzed using the TUNEL assay. Cell proliferation was measured using the [3H]-thymidine incorporation assay. The expression of IL-6, EGF, EGF receptor (EGFR), and phosphorylated extracellular signal-regulated kinase (p-ERK) was measured by western blot analysis. The secretion of IL-6 was measured using ELISA. Western blot analysis was also performed using antibodies against cleaved caspase-3. RESULTS: The percentage of apoptotic cells was lower in the high-EGF group (6.7%) than in the low-EGF group (10.3%). The high-EGF group demonstrated increased proliferation (323.7 counts/min in the low-EGF group vs 649.1 counts/min in the high-EGF group). EGF induced higher phosphor-EGFR expression and upregulated p-ERK in HCECs. In addition, EGF significantly decreased the secretion of IL-6 and cleaved caspase-3 in HCECs. CONCLUSIONS: The level of IL-6 was increased in the ex vivo HCEC dry-eye model that was under hyperosmotic stress. Supplemental EGF reduces the level of IL-6, decreases apoptosis, and increases proliferation. These findings indicate that EGF has potential as a therapeutic agent for the treatment of dry eyes.


Assuntos
Túnica Conjuntiva/efeitos dos fármacos , Síndromes do Olho Seco/prevenção & controle , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/efeitos dos fármacos , Modelos Biológicos , Pressão Osmótica , Apoptose , Western Blotting , Caspase 3/metabolismo , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/patologia , Síndromes do Olho Seco/genética , Síndromes do Olho Seco/patologia , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Interleucina-6/genética , Interleucina-6/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Doadores de Tecidos
17.
Arthritis Res Ther ; 14(1): R14, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-22264405

RESUMO

INTRODUCTION: Interleukin-34 (IL-34) is a recently defined cytokine, showing a functional overlap with macrophage colony stimulating factor (M-CSF). This study was undertaken to address the expression of IL-34 in rheumatoid arthritis (RA) patients and to investigate its regulation and pathogenic role in RA. METHODS: IL-34 levels were determined in the RA synovium, synovial fluid (SF) and fibroblast-like synovial cells (FLS) by immunohistochemistry, real-time PCR, enzyme-linked immunosorbent assay and immunoblotting. RA activity was assessed using Disease Activity Score 28 (DAS28) activity in the plasma collected at baseline and one year after treatment. Conditioned media (CM) were prepared from RA FLS culture with tumor necrosis factor alpha (TNFα) for 24 hours and used for functional assay. RESULTS: IL-34 was expressed in the synovium, SF, and FLS from RA patients. The production of IL-34 in FLS was up-regulated by TNFα in RA samples compared with osteoarthritis (OA) patients. Importantly, the preferential induction of IL-34 rather than M-CSF by TNFα in RAFLS was mediated by the transcription factor nuclear factor kappa B (NF-κB) and activation of c-Jun N-terminal kinase (JNK). IL-34 elevation in plasma from RA patients was decreased after the administration of disease-modifying anti-rheumatic drugs (DMARDs) in accordance with a decrease in DAS28. CM from RAFLS cultured with TNFα promoted chemotactic migration of human peripheral blood mononuclear cells (PBMCs) and subsequent osteoclast (OC) formation, effects that were attenuated by an anti-IL-34 antibody. CONCLUSIONS: These data provide novel information about the production of IL-34 in RA FLS and indicate that IL-34 is an additional osteoclastogenic factor regulated by TNFα in RA, suggesting a discrete role of IL-34 in inflammatory RA diseases.


Assuntos
Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Membrana Sinovial/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adulto , Idoso , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Immunoblotting , Imuno-Histoquímica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Líquido Sinovial/metabolismo , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/farmacologia
18.
Int J Biochem Cell Biol ; 43(11): 1612-20, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21810480

RESUMO

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is an adaptor protein which involves in the activation of the transcription factor, nuclear factor kappaB (NF-κB), in the tumor necrosis factor (TNF) receptor pathway. This signaling is modulated by proteins that interact with tumor necrosis factor receptor-associated factor 2. In this study, we identified the zinc-finger protein AWP1 as a tumor necrosis factor receptor-associated factor 2-interacting protein through yeast two-hybrid screening. We found that AWP1 directly interacted with the C-terminal tumor necrosis factor receptor-associated factor (TRAF) domain of tumor necrosis factor receptor-associated factor 2. Knockdown of AWP1 using small hairpin RNA significantly decreased nuclear factor kappaB activity but increased tumor necrosis factor alpha (TNFα)-induced apoptosis, presumably by decreasing the induction of nuclear factor kappaB-responsive anti-apoptotic molecules, including FICE-like inhibitory protein (FLIP), X-linked inhibitor of apoptosis protein (XIAP), Bcl-2, and Bcl-xL. In contrast, overexpression of wild-type AWP1 inhibited nuclear factor kappaB activation. Detailed domain mapping experiments showed that the AN1 domain of AWP1 mediated the functional interaction with tumor necrosis factor receptor-associated factor 2, and the A20 domain was responsible for the negative regulation of nuclear factor kappaB activation. Importantly, ectopic AWP1 overexpression led to an A20 domain-dependent increase in K-48 ubiquitination of tumor necrosis factor receptor-associated factor 2. These data indicate that AWP1 binds to tumor necrosis factor receptor-associated factor 2 and that regulates tumor necrosis factor alpha-induced nuclear factor kappaB activation through two distinct domains.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Transdução de Sinais/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sítios de Ligação , Inativação Gênica , Células HEK293 , Células HeLa , Humanos , NF-kappa B , Plasmídeos , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Fator 1 Associado a Receptor de TNF/genética , Fator 1 Associado a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Transfecção , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
19.
Cardiovasc Res ; 84(3): 378-86, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19596672

RESUMO

AIMS: We investigated the mechanism by which cannabinoid receptors-1 (CB1) and -2 (CB2) modulate inflammatory activities of macrophages. METHODS AND RESULTS: Real-time polymerase chain reaction showed the predominant CB2 expression in freshly isolated human monocytes. PMA, a potent inducer of differentiation, upregulated CB1 and increased CB1:CB2 transcript ratio from 1:17.5 to 1:3 in 5 days of culture. Immunohistochemistry showed that CB1 protein was colocalized in CD68- and CD36-positive macrophages in human atheroma. Through selective expression of CB1 or CB2 to thioglycollate-elicited peritoneal macrophages, we proved that CB1 and CB2 mediate opposing influences on the production of reactive oxygen species (ROS). Flow cytometry showed that cannabinoid-induced ROS production by macrophages was CB1-dependent. Immunoblotting assays confirmed that macrophage CB1, not CB2, induced phosphorylation of p38-mitogen-activated protein kinase, which modulated ROS production and the subsequent synthesis of tumour necrosis factor-alpha and monocyte chemoattractant protein-1. Pull-down assays showed that the Ras family small G protein, Rap1 was activated by CB2. Dominant-negative Rap1 profoundly enhanced CB1-dependent ROS production by macrophages, suggesting CB2 Rap1-dependently inhibits CB1-stimulated ROS production. CONCLUSION: CB1 promotes pro-inflammatory responses of macrophages through ROS production, which is negatively regulated by CB2 through Rap1 activation. Blocking CB1 together with selective activation of CB2 may suppress pro-inflammatory responses of macrophages.


Assuntos
Macrófagos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Quimiocina CCL2/metabolismo , Citocinas/metabolismo , Humanos , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Receptor CB1 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/agonistas , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo
20.
J Immunol ; 177(8): 5727-35, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-17015762

RESUMO

Rheumatoid arthritis (RA) synoviocytes are resistant to apoptosis and exhibit a transformed phenotype, which might be caused by chronic exposure to genotoxic stimuli including reactive oxygen species and growth factors. In this study, we investigated the role of vascular endothelial growth factor165 (VEGF165), a potent angiogenic factor, and its receptor in the apoptosis of synoviocytes. We demonstrated here that neuropilin-1, rather than fms-like tyrosine kinase-1 and kinase insert domain-containing receptor, is the major VEGF165 receptor in the fibroblast-like synoviocytes. Neuropilin-1 was highly expressed in the lining layer, infiltrating leukocytes, and endothelial cells of rheumatoid synovium. The production of VEGF165, a ligand for neuropilin, was significantly higher in the RA synoviocytes than in the osteoarthritis synoviocytes. The ligation of recombinant VEGF165 to its receptor prevented the apoptosis of synoviocytes induced by serum starvation or sodium nitroprusside (SNP). VEGF165 rapidly triggered phospho-Akt and phospho-ERK activity and then induced Bcl-2 expression in the rheumatoid synoviocytes. The Akt or ERK inhibitor cancelled the protective effect of VEGF165 on SNP-induced synoviocyte apoptosis. Moreover, VEGF165 blocks SNP-induced Bcl-2 down-regulation as well as SNP-induced Bax translocation from the cytosol to the mitochondria. The down-regulation of the neuropilin-1 transcripts by short interfering RNA caused spontaneous synoviocyte apoptosis, which was associated with both the decrease in Bcl-2 expression and the increase in Bax translocation to mitochondria. Collectively, our data suggest that the interaction of VEGF165 with neuropilin-1 is crucial to the survival of rheumatoid synoviocytes and provide important implications for the abnormal growth of synoviocytes and therapeutic intervention in RA.


Assuntos
Apoptose/efeitos dos fármacos , Artrite Reumatoide/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Neuropilina-1/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Membrana Sinovial/patologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteína X Associada a bcl-2/metabolismo , Humanos , Neuropilina-1/metabolismo , Nitroprussiato/farmacologia , Osteoartrite/patologia , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
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