Abstract
Purpose: :
TGFβ/Smad signalling is proposed to regulate transdifferentiation of lens epithelial cells and matrix contraction; both clinical features of posterior capsule opacification (PCO) following cataract surgery. FGF is reported to exacerbate TGFβ induced cataract formation (Cerra et al, 2003) The objective of the current study was to determine the interplay between TGFβ and FGF in relation to transdifferentiation and matrix contraction.
Methods: :
Matrix contraction was assessed using a patch assay; following a 24hr period of serum starvation, cells were maintained in experimental conditions for 1-3 days. All areas covered by cells were measured using imaging techniques following fixation in 4% formaldehyde and cell staining with Coomassie Blue. Total protein content was determined by dye extractions and used to give an estimate of cell population. Gene and protein expression of the transdifferentiation marker alpha smooth muscle actin (αSMA) was determined using Real-time PCR and western blotting respectively. To observe Smad2/3 distribution patterns immunocytochemistry was used and nuclear translocation quantified by Cellomics analysis.
Results: :
Patch assays cultured for 3 days in the presence of 10ng/ml TGFβ2 showed significant contraction, with no decrease in cell population. However, patches cultured for 1 day in TGFβ2 alone did not demonstrate any significant difference in either area or population relative to untreated controls i.e. no contraction. However, cultures maintained in 10ng/ml TGFβ2 and 10ng/ml basic FGF for 1 day showed a marked difference in detectable area i.e. contraction; basic FGF alone did not statistically differ from controls. Real time PCR and western blots showed a significant induction of αSMA expression in response to 10ng/ml TGFβ2; 10ng/ml basic FGF alone did not cause a significant increase. Co-addition of 10ng/ml TGFβ2 and 10ng/ml basic FGF resulted in a significant reduction of αSMA levels relative to 10ng/ml TGFβ2 alone. Smad2/3 translocation to the nucleus was significantly increased following 2 hr exposure to 10ng/ml TGFβ2; this was unaffected by the presence of 10ng/ml basic FGF.
Conclusions: :
Basic FGF enhances TGFβ induced matrix contraction, but suppresses expression of the transdifferentiation marker αSMA. Interplay between TGFβ and FGF is likely to contribute to light scattering changes in PCO formation; transdifferentiation does not appear critical for TGFβ induced matrix contraction to occur.
Keywords: posterior capsular opacification (PCO) • growth factors/growth factor receptors • EMT (epithelial mesenchymal transition)