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1.
Dev Growth Differ ; 61(7-8): 410-418, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31608440

RESUMO

Macular fibrosis is a vital obstacle of vision acuity improvement of age-related macular degeneration patients. This study was to investigate the effects of interleukin 2 (IL-2) on epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) synthesis and transforming growth factor ß2 (TGF-ß2) expression in retinal pigment epithelial (RPE) cells. 10 µg/L IL-2 was used to induce fibrosis in RPE cells for various times. Western blot was used to detect the EMT marker α-smooth muscle actin (α-SMA), ECM markers fibronectin (Fn) and type 1 collagen (COL-1), TGF-ß2, and the activation of the JAK/STAT3 and NF-κB signaling pathway. Furthermore, JAK/STAT3 and NF-κB signaling pathways were specifically blocked by WP1066 or BAY11-7082, respectively, and the expression of α-SMA, COL-1, Fn and TGF-ß2 protein were detected. Wound healing and Transwell assays were used to measure cell migration ability of IL-2 with or without WP1066 or BAY11-7082. After induction of IL-2, the expressions of Fn, COL-1, TGF-ß2 protein were significantly increased, and this effect was correlated with IL-2 treatment duration, while α-SMA protein expression did not change significantly. Both WP1066 and BAY11-7082 could effectively downregulate the expression of Fn, COL-1 and TGF-ß2 induced by IL-2. What's more, both NF-κB and JAK/STAT3 inhibitors could suppress the activation of the other signaling pathway. Additionally, JAK/STAT3 inhibitor WP1066 and NF-κB inhibitor BAY 11-7082 could obviously decrease RPE cells migration capability induced by IL-2. IL-2 promotes cell migration, ECM synthesis and TGF-ß2 expression in RPE cells via JAK/STAT3 and NF-κB signaling pathways, which may play an important role in proliferative vitreoretinopathy.

2.
Cell Cycle ; 18(15): 1714-1726, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31234714

RESUMO

Objective: The present study was conducted to determine the role of gremlin during the development of posterior capsular opacification (PCO) via in vitro and in vivo experiments. Methods: The activation, roles and relationships of the BMPs/Smad1/5, MAPK, FAK and AKT signaling pathways in human lens epithelial cells (HLECs) after gremlin induction were detected by western blotting and real-time PCR. Wound-healing, transwell, capsular bag models and rat PCO models assays were used to test the effects of gremlin on HLECs' migration, proliferation, EMT-specific protein α-smooth muscle actin(α-SMA)and development of PCO in rats. Results: Our data showed that knockdown of the gremlin inhibited the development of PCO and reduced expression of α-SMA in rats. While gremlin did not alter the migration of HLECs, it increased the expression of p-ERK and p-AKT. Knockout of Smad2 or Smad3 inhibited the expression of p-ERK and p-AKT proteins induced by gremlin. Gremlin also reduced BMP4-induced expression of the p-Smad1/5 protein. Finally, knockout of Smad1/5 increased gremlin-induced expression of α-SMA, fibronectin and type I collagen (COL-1) in HLECs. Conclusion: These results suggested that gremlin contributed to the development of PCO by promoting LEC proliferation, activation of TGF-ß/Smad, ERK and AKT signaling and inhibition of BMPs/Smad1/5 signaling. Furthermore, inhibiting gremlin effectively impaired both PCO development in rats and EMT in the lens capsule. Thus, our data suggest that gremlin might be a potential target for PCO.

3.
Int J Mol Med ; 42(6): 3591-3601, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30280182

RESUMO

The migration of lens epithelial cells towards the posterior capsule is a key event in the development of posterior capsule opacification (PCO). Accumulating evidence has described crosstalk between growth factors and adhesive signaling pathways in wound healing and cell migration. The aim of the present study was to elucidate an aberrant transforming growth factor (TGF)­ß2 signaling pathway that regulated the migration of lens epithelial cells in the pathological context of PCO. The expression of fibronectin, focal adhesion kinase (FAK) and phosphorylated (p)­FAK in HLE­B3 cells following TGF­ß2 treatment was determined by western blot analysis and the expression of integrin α5ß1 was detected by flow cytometry. Cell migration capacity was measured by wound healing and Transwell assays in the presence of 1,2,4,5­tetraaminobenzene tetrahydrochloride, a selective FAK inhibitor, fibronectin small interfering RNA interference, arginylglycylaspartic acid peptides or α5ß1­integrin neutralizing antibodies. The 1,2,4,5­tetraaminobenzene tetrahydrochloride was administered daily to 16 rabbits following cataract surgery. Fibronectin and TGF­ß expression were increased in the PCO group, demonstrated by immunofluorescence assays. PCO grading was conducted by slit­lamp biomicroscopy and evaluation of posterior capsule opacification software. It was observed that TGF­ß2 promoted HLE­B3 cell migration and upregulated fibronectin expression, which was followed by an increased phosphorylation of FAK. In addition, TGF­ß2 treatment and fibronectin surface coating significantly increased cell migration and FAK activation, which was inhibited by disrupting fibronectin­integrin α5ß1 interaction with the arginylglycylaspartic acid peptide, α5ß1­integrin neutralizing antibody or fibronectin depletion. Finally, suppression of FAK signaling by its inhibitor significantly decreased cell migration in vitro and attenuated PCO development in vivo. In summary, TGF­ß2 was indicated to promote the migration of lens epithelial cells through the TGF­ß2/fibronectin/integrin/FAK axis. Inhibition of FAK activity decreased TGF­ß2­mediated cell migration in vitro and improved the symptoms of PCO in a rabbit model.


Assuntos
Movimento Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Cristalino/citologia , Fator de Crescimento Transformador beta2/farmacologia , Animais , Opacificação da Cápsula/enzimologia , Opacificação da Cápsula/patologia , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Fibronectinas/metabolismo , Humanos , Integrina alfa5beta1/metabolismo , Masculino , Coelhos , Transdução de Sinais
4.
Int J Biol Sci ; 14(4): 437-448, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29725265

RESUMO

Connective tissue growth factor (CTGF) is a crucial factor that plays a major role in the process of posterior capsule opacification (PCO). However, the effects of CTGF on the proliferation and migration of lens epithelial cells (LECs) and on the mechanism of the epithelial mesenchymal transition (EMT) and extracellular matrix (ECM) in human lens epithelial cells (HLECs) as well as the effects of shRNA-mediated CTGF knockdown on the development of PCO in rats remain unclear. In the present study, we found that CTGF promoted EMT, proliferation, migration and the expression of p-ERK1/2 protein in HLECs but exerted little effect on the expression of p-p38 and p-JNK1/2 proteins. MEK inhibitor U0126 effectively restrained the CTGF-induced expression of α-smooth muscle actin (α-SMA), fibronectin (Fn) and type I collagen (COL-1) in HLECs. CTGF knockdown effectively postponed the onset of PCO in the rats and significantly reduced the expression of α-SMA in the capsule. In conclusion, CTGF contributed to the development of PCO presumably by promoting proliferation, migration of LECs, EMT specific protein expression and ECM synthesis in HLECs, which is dependent on ERK signalling. Furthermore, blocking CTGF effectively inhibited PCO in the rats and the EMT specific protein expression in the lens capsule.


Assuntos
Fator de Crescimento do Tecido Conjuntivo/fisiologia , Cápsula Posterior do Cristalino/patologia , Animais , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Células Epiteliais , Técnicas de Silenciamento de Genes , Humanos , Cristalino/citologia , Sistema de Sinalização das MAP Quinases/genética , Interferência de RNA , Ratos , Suínos
5.
Exp Eye Res ; 172: 94-103, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29617629

RESUMO

The purpose of this work was to determine the effects of interleukin-6 (IL-6) on the development of posterior capsular opacification (PCO) in vitro and in vivo. Western blot and real-time PCR were used to test the IL-6-induced epithelial-mesenchymal transition (EMT) marker α-smooth muscle actin (α-SMA), the extracellular matrix (ECM) markers fibronectin (Fn) and type I collagen (COL-1), transforming growth factor ß2 (TGF-ß2), and the activation and role of the JAK/STAT3 signaling pathway in human lens epithelial cells (HLECs). Immunocytofluorescence staining was performed to detect gp130 and IL-6Rα expression in HLECs. Rat PCO models were then established to examine the impact of STAT3 knockdown by shRNA adeno-associated virus on PCO development, and immunohistochemical staining was performed to detect the expression of Fn in the anterior and posterior capsule in vivo. We found that IL-6 promotes the expression of Fn, COL-1, TGF-ß2, p-JAK2 and p-STAT3 in HLECs but exerts little effect on α-SMA. The JAK/STAT3 inhibitor WP1066 effectively suppressed the IL-6-induced expression of Fn and COL-1 in lens epithelial cells. STAT3 knockdown effectively inhibited the development of PCO in rats and significantly reduced the expression of Fn in the anterior and posterior capsule. These data suggest that IL-6 contributes to the development of PCO by promoting TGF-ß2 activation and ECM synthesis through a JAK/STAT3 signaling-dependent mechanism. Furthermore, inhibiting JAK/STAT3 signaling effectively impairs both PCO development in rats and ECM synthesis in the lens capsule.


Assuntos
Opacificação da Cápsula/etiologia , Células Epiteliais/efeitos dos fármacos , Interleucina-6/farmacologia , Cristalino/efeitos dos fármacos , Cápsula Posterior do Cristalino/efeitos dos fármacos , Actinas/metabolismo , Animais , Western Blotting , Opacificação da Cápsula/metabolismo , Colágeno Tipo I/metabolismo , Receptor gp130 de Citocina/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/fisiologia , Fibronectinas/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-6/metabolismo , Janus Quinases/metabolismo , Cristalino/metabolismo , Cápsula Posterior do Cristalino/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição STAT3/metabolismo , Fator de Crescimento Transformador beta2/metabolismo
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