RESUMEN
PURPOSE: Transforming growth factor-ß2 (TGF-ß2) is a potent inducer of posterior capsular opacification (PCO), a critical Smad-dependent event. This study was conducted to investigate the contributions of Smad2 and Smad3 to PCO development based on selective over-expression of either Smad2 or Smad3. METHODS: We selectively activated the TGF-ß/Smad pathway in cell lines transfected with expression plasmids containing Smad2 or Smad3. These cell lines were then analyzed to determine the individual contributions of Smad2 and Smad3 to TGF-ß2 treatment response in an in vitro culture of HLE B-3 cells. The effects of Smad2 and Smad3 on cell viability were assessed by MTT and flow cytometry assay. A transwell assay was used to observe the role of Smad2 and Smad3 in the migration of HLE B-3 cells. Western blotting, real-time PCR, and immunocytofluorescence staining were performed to detect the accumulation of ECM proteins and EMT in response to selective Smad2 or Smad3 activation. The presence of soluble collagen I, and fibronectin in the culture medium supernatant were detected by ELISA. RESULTS: Selective Smad3 activation via gene transfection enhanced TGF-ß2-responsive growth inhibition and apoptosis. Transwell assay results showed that TGF-ß2-induced cell migration was Smad2 dependent and Smad3 independent. Analysis by Western blot, RT-PCR and ELISA demonstrated that the determinant factor in ECM secretion was Smad3 signaling rather than Smad2 signaling. Western blot and RT-PCR showed that the loss of E-Cadherin and acquisition of α-SMA, the hallmark of epithelial-mesenchymal transition (EMT), were both reliant on Smad2 signaling. Immunocytofluorescence staining confirmed the role of Smad2 in the accumulation of α-SMA. CONCLUSIONS: Smad2 and Smad3 are both necessary for the formation of PCO. The discovery of additional TGF-ß2/Smad signaling mechanisms may provide potential therapeutic targets to help combat PCO.
Asunto(s)
Opacificación Capsular/metabolismo , Células Epiteliales/efectos de los fármacos , Cápsula Posterior del Cristalino/efectos de los fármacos , Proteína Smad2/fisiología , Proteína smad3/fisiología , Factor de Crecimiento Transformador beta2/farmacología , Western Blotting , Opacificación Capsular/inducido químicamente , Opacificación Capsular/patología , Línea Celular , Movimiento Celular/efectos de los fármacos , Supervivencia Celular , Colágeno Tipo I/metabolismo , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Fibronectinas/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Células HEK293 , Humanos , Cristalino/citología , Plásmidos/genética , Cápsula Posterior del Cristalino/metabolismo , Cápsula Posterior del Cristalino/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , TransfecciónRESUMEN
PURPOSE: Because hyaluronic acid (HA) is found in many surgical viscoelastic agents, this study aimed to determine (1) if HA receptors are present in the canine lens, (2) if the rate of lens epithelial cell (LEC) migration is altered following treatment with HA, and (3) if introduction of exogenous HA into the lens capsule promotes lenticular migration, thus contributing to posterior capsule opacification (PCO). METHODS: Normal and cataractous canine LECs were evaluated for expression of the HA receptor CD44 and the receptor for HA mediated motility (RHAMM) using immunohistochemistry, immunoblotting, and real-time PCR. Canine LEC were treated with various concentrations of HA, and induction of migration was monitored over time. Commercially available surgical viscoelastics were utilized ex vivo, and rates of PCO formation were analyzed. RESULTS: Basal protein and mRNA expression of both CD44 and RHAMM was noted. Cataractous canine LEC demonstrated significantly (P < 0.01) higher expression of CD44 but not RHAMM. Treatment with higher concentrations of HA resulted in a significant (P < 0.01) increase in CD44 mRNA and increased LEC migration in vitro. Use of CD44-neutralizing antibodies confirmed the role of CD44 in HA-induced lenticular migration. Viscoelastic material containing higher concentrations of HA led to increased rates of ex vivo PCO. CONCLUSIONS: Exogenous HA can induce lenticular migration and CD44 expression. Use of surgical viscoelastics that contained HA resulted in increased rates of ex vivo PCO suggesting that judicious selection and use of viscoelastic material during cataract surgery is warranted.
Asunto(s)
Opacificación Capsular/inducido químicamente , Ácido Hialurónico/toxicidad , Cápsula del Cristalino/efectos de los fármacos , Animales , Western Blotting , Opacificación Capsular/metabolismo , Opacificación Capsular/patología , Movimiento Celular , Células Cultivadas , Modelos Animales de Enfermedad , Perros , Regulación de la Expresión Génica/efectos de los fármacos , Receptores de Hialuranos/biosíntesis , Receptores de Hialuranos/genética , Ácido Hialurónico/administración & dosificación , Inmunohistoquímica , Cápsula del Cristalino/metabolismo , Cápsula del Cristalino/patología , Soluciones Oftálmicas , Pronóstico , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Viscosuplementos/administración & dosificación , Viscosuplementos/toxicidadRESUMEN
PURPOSE: To evaluate the level of matrix metalloproteinase (MMP)-2 and MMP-9 activities in patients with steroid induced posterior subcapsular cataract (PSC). METHODS: This prospective, observational study comprised of 156 patients having either steroid induced PSC (n=50) or non-steroidal PSC (n=106) were performed to evaluate the level of MMP-2 and MMP-9 activities in the lens epithelial cells (LECs) and the serum. Anterior lens capsules harboring LECs were obtained during phacoemulsification and peripheral blood was collected from patients before administration of anesthesia. Serum was separated by centrifugation at 10,000× g for 15 min at 4 °C. The LECs and serum samples were processed to analyze MMP-2 and MMP-9 activities using succinylated gelatin assay. Quantitative real time-PCR (qRT-PCR) was performed to determine the mRNA levels of MMP-2 and MMP-9 in LECs. The mRNA levels were expressed as a ratio, using the delta-delta method for comparing the relative expression results between cases with steroid induced PSC and cases with non-steroidal PSC. MMP-2 and MMP-9 levels were also compared in the two groups using immunolocalization. RESULTS: The level of MMP-2 and MMP-9 activity was found to be high in LECs and serum of cases with steroid induced PSC. Further in all steroid induced cases, a 1.4 fold increase was observed in MMP-2 activity in LECs and a 1.4 fold increase in MMP-9 activity in the serum. Both qRT-PCR and immunolocalization showed increased expression of MMP-2 and MMP-9 activity. CONCLUSIONS: MMP-2 and MMP-9 activity in both LECs and serum was significantly higher in cases with steroid induced PSC. The possible use of MMP-9 as a non-invasive biomarker in ascertaining the presence of steroid induced PSC should be evaluated using a larger sample size.
Asunto(s)
Opacificación Capsular/sangre , Células Epiteliales/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Cápsula Posterior del Cristalino/efectos de los fármacos , Adolescente , Adulto , Anciano , Biomarcadores/sangre , Opacificación Capsular/inducido químicamente , Niño , Ciclosporina/efectos adversos , Dexametasona/efectos adversos , Células Epiteliales/patología , Femenino , Expresión Génica , Glucocorticoides/efectos adversos , Humanos , Inmunosupresores/efectos adversos , Masculino , Metaloproteinasa 2 de la Matriz/sangre , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/sangre , Metaloproteinasa 9 de la Matriz/genética , Persona de Mediana Edad , Cápsula Posterior del Cristalino/patología , Prednisolona/efectos adversos , Estudios Prospectivos , ARN Mensajero/sangreRESUMEN
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.