RESUMEN
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.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/enzimología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Cristalino/citología , Factor de Crecimiento Transformador beta2/farmacología , Animales , Opacificación Capsular/enzimología , Opacificación Capsular/patología , Línea Celular , Células Epiteliales/efectos de los fármacos , Fibronectinas/metabolismo , Humanos , Integrina alfa5beta1/metabolismo , Masculino , Conejos , Transducción de SeñalRESUMEN
PURPOSE: To investigate the effect of disintegrin echistatin on integrin linked kinase (ILK) and subsequent PI3-K/Akt and ERK1/2 signaling pathways in the posterior capsule opacification (PCO) model of diabetic rabbit. METHODS: 56 rabbits were injected alloxan to model diabetic. Then they accepted lens extraction surgery and randomly and intraoperatively injected distilled water (control group; n = 28) or 10.0 mg·L(-1) echistatin (echistatin-treated group; n = 28) into the anterior chamber. Each group was subdivided into ten days group (n = 14) and six weeks group (n = 14) respectively. The PCO severity was evaluated with a slit lamp microscope and light microscope for 10 days and 6 weeks postoperatively. The levels of ILK in the posterior capsule were determined by Q-PCR, Western blotting and Immunohistochemistry. Akt and ERK1/2 phosphorylation were analyzed by Western blotting. RESULTS: 10 days and 6 weeks after surgery, the grades of PCO in the echistatin-treated group were lower than the control group. The lens epithelial cells (LECs) in the posterior capsule of echistatin-treated eyes had decreased degrees of proliferation and migration than the control group. And no significant side effects appeared after treated with echistatin. Echistatin could significantly reduce the expression of ILK in terms of both mRNA and protein levels. The phosphorylation levels of Akt and ERK1/2 were decreased in the echistatin-treated group compared with the control group. CONCLUSIONS: Echistatin could inhibit postoperative PCO occurrence and development in diabetic rabbit eyes, which may be related to down-regulation the expression of ILK and inhibition the PI3-K/Akt and ERK1/2 pathways.
Asunto(s)
Opacificación Capsular/prevención & control , Diabetes Mellitus Experimental/tratamiento farmacológico , Péptidos/farmacología , Cápsula Posterior del Cristalino/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Opacificación Capsular/enzimología , Opacificación Capsular/etiología , Opacificación Capsular/genética , Opacificación Capsular/patología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/enzimología , Regulación hacia Abajo , Activación Enzimática , Femenino , Regulación Enzimológica de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Cápsula Posterior del Cristalino/enzimología , Cápsula Posterior del Cristalino/patología , Cápsula Posterior del Cristalino/cirugía , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Procedimientos Quirúrgicos Refractivos , Índice de Severidad de la Enfermedad , Factores de TiempoRESUMEN
PURPOSE: To evaluate the effect of ethylenediaminetetraacetic acid (EDTA) on posterior capsular opacification (PCO) of rabbits and to assess its effect on intraocular tissues. METHODS: Modulation of matrix metalloproteinase (MMP) activity in the aqueous following cataract surgery in rabbits and its prevention by different doses of EDTA was determined by zymography. For evaluation of PCO, lensectomized rabbits were intracamerally injected with single dose of either 5 mg EDTA or normal saline. After one month, the degree of PCO was determined by slitlamp biomicroscopy, Miyake-Apple view, and histology of the lens capsule. The effect of EDTA on intra ocular pressure (IOP), corneal endothelial cells, and the retina was evaluated by tonometry, specular microscopy and scanning electron microscopy, and electroretinography. The concentration of EDTA in the aqueous was determined by high performance liquid chromatography (HPLC) at different time points. RESULTS: The MMP activity was significantly increased in the aqueous of the operated eyes, and EDTA reduced the degree of increase in a dose-dependent manner. EDTA treatment significantly reduced the degree of PCO (p<0.05). Histopathology of the lens capsule showed a reduction in the number of proliferating and migrating cells as well as MMP2 expression in the EDTA-treated eyes. EDTA treatment did not change the IOP; density, morphology and ultrastructure of the corneal endothelial cells; and electroretinography (ERG). EDTA was detectable in the aqueous humor up to 72 h following a single intracameral injection. CONCLUSIONS: EDTA reduces the degree of PCO by suppressing the MMP activity and it is not toxic to intra ocular structures at the concentration used.
Asunto(s)
Opacificación Capsular/tratamiento farmacológico , Ácido Edético/uso terapéutico , Cápsula del Cristalino/efectos de los fármacos , Inhibidores de la Metaloproteinasa de la Matriz , Complicaciones Posoperatorias/tratamiento farmacológico , Animales , Humor Acuoso/efectos de los fármacos , Opacificación Capsular/enzimología , Opacificación Capsular/patología , Extracción de Catarata , Relación Dosis-Respuesta a Droga , Ácido Edético/administración & dosificación , Electrorretinografía , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Epitelio Corneal/citología , Epitelio Corneal/efectos de los fármacos , Inyecciones Intraoculares , Presión Intraocular/efectos de los fármacos , Cápsula del Cristalino/patología , Cápsula del Cristalino/cirugía , Metaloproteinasa 2 de la Matriz/metabolismo , Microscopía Electrónica de Rastreo , Complicaciones Posoperatorias/enzimología , Complicaciones Posoperatorias/patología , ConejosRESUMEN
Cataract is a key factor in the morbidity associated with diabetes. While the pathogenesis of diabetic cataract formation is poorly understood, previous research has identified aldose reductase (ALR2) as a key player. To elucidate a potential role for this enzyme in diabetic cataract formation, we created a series of transgenic mice designed for expression of human ALR2 (AKR1B1) in epithelial and outer cortical fiber cells of the lens. One of the founder lines, designated PAR39, developed an early onset cataract that involved formation of a plaque of cells at the anterior aspect of the lens. These cells appear to separate from the anterior epithelium and undergo a dramatic change that is reminiscent of the epithelial to mesenchymal transition (EMT). We characterized this phenotype in the PAR39 strain by examining rates of cell proliferation and by immunostaining for markers of EMT. Incorporation of the thymidine analog bromodeoxyuridine (BrdU) was used to estimate cell proliferation in two functional areas of the lens epithelium: the mitotically active germinative zone (GZ) and the less proliferative center zone (CZ). Staining cell nuclei with diamido 4',6-diamidino-2-phenylindole (DAPI) was used to establish a total cell count in the demarcated areas. Lens epithelium in PAR39 transgenic mice demonstrated a decrease in the percentage of BrdU/DAPI staining within the GZ as compared to nontransgenic littermate controls (8.1% vs. 10.9%). A similar decrease in BrdU/DAPI was observed in the CZ (0.6% compared to 3.3%). However, cell density was greater within the GZ of PAR39 mice as compared with nontransgenic controls, while it was not significantly different in the CZ among the two groups. Furthermore, cells associated with the epithelial plaque did not stain positive for BrdU, but were strongly positive for alpha-smooth muscle actin, a classical marker for EMT. These findings suggest that ALR2 over-expression is associated with an alteration in the balance between proliferation and apoptosis of epithelial cells in the mouse lens, and that cells associated with epithelial plaques in the PAR39 lens have features in common with cells undergoing EMT.
Asunto(s)
Aldehído Reductasa/metabolismo , Transición Epitelial-Mesenquimal , Cristalino/citología , Cristalino/enzimología , Aldehído Reductasa/genética , Animales , Biomarcadores/metabolismo , Opacificación Capsular/enzimología , Opacificación Capsular/patología , Proliferación Celular , Complicaciones de la Diabetes/enzimología , Complicaciones de la Diabetes/patología , Células Epiteliales/citología , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación Enzimológica de la Expresión Génica , Humanos , Cristalino/metabolismo , Cristalino/patología , Ratones , Ratones Transgénicos , FenotipoRESUMEN
A Ca(2+)-dependent TG activity, identified in the eye lens of several mammalian species, has long been implicated in cataract formation. The precise mechanism of the involvement of this enzyme in this process remains unclear. The purpose of this work was to investigate the modulatory effect of polyamines on TG activity during rabbit eye lens in vitro opacification. We observed, in an in vitro Ca(2+)-induced cataract model, a rapid decrease of the endogenous levels of SPD with the progression of opacification, paralleled by an increase of crystallin cross-linking by bis(γ-glutamyl)SPD. This pattern was reversed adding exogenous SPD to the incubation medium. Indeed, endogenous SPD levels were restored and cross-linking by bis(γ-glutamyl)SPD were drastically reduced. Surprisingly, under this experimental condition, the loss of transparency of lens was delayed. We found that exogenous SPD incubation led to a remarkable increase of mono(γ-glutamyl)SPD, likely responsible of the inhibition of cross-linking of lens crystallins and of the transparency persistence.