Abstract
Purpose: :
Previous work has shown that neither Smad4 signalling or induction of the transdifferentiation marker, smooth muscle actin, are critical for TGFβ induced matrix contraction to occur. We therefore tested the novel hypothesis that myosin activity is a key regulator in TGFβ mediated contraction by human lens cells.
Methods: :
The human lens line FHL 124 and the human capsular bag model were employed. Contraction by FHL 124 cells was assessed using a patch assay, whereby the area covered by cells, was measured using imaging techniques. Following a 24hr period of serum starvation, cells were maintained in the following conditions: Control medium ± 15µM Myosin light chain kinase inhibitor (ML-7); 10ng/ml TGFß2 ± ML-7; Control medium ± 50nM myosin phosphatase inhibitor (tautomycin); 10ng/ml TGFß2 ± tautomycin. Western blots were performed to determine levels of phosphorylated myosin light chain (pMLC). The human capsular bag model was also used to study TGFβ induced matrix contraction.
Results: :
Addition of 10ng/ml TGFß2 to cell patches caused a significant contractile event to take place following a 3 day culture period. Maintenance of cultures in the presence of 15µM ML-7 when added to control medium had no significant effect on patch area; however, when added in the presence of TGFß2, contraction was inhibited. In contrast, application of 50nM tautomycin promoted the rate of TGFβ mediated contraction. TGFβ also significantly increased the level of pMLC relative to controls. This induction was inhibited by 15µM ML-7, 10µM U0126 (MEK inhibitor) and 10µM Y-27632 (Rho kinase inhibitor); the latter two agents also suppress TGFβ induced contraction. Application of tautomycin increased the level of pMLC ± TGFβ. In capsular bags, TGFβ induced marked contraction (wrinkling) of the posterior capsule. Application of 15µM ML-7 to TGFβ treated capsular bags inhibited wrinkling.
Conclusions: :
Myosin appears to be a key component in the contractile apparatus driving TGFβ mediated matrix contraction. This finding provides a novel target for the treatment of posterior capsule opacification and other fibrotic conditions within the eye.
Keywords: posterior capsular opacification (PCO) • growth factors/growth factor receptors • wound healing