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M. Wormstone, L.J. Dawes, J.A. Eldred, J.R. Reddan, I.K. Anderson, M. Sleeman, G. Duncan; TGFß Induced Matrix Contraction Is Not Promoted by Fibronectin/Fibronectin Receptor Interaction or Alpha Smooth Muscle Actin Expression . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4741.
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© ARVO (1962-2015); The Authors (2016-present)
Matrix contraction causes light scatter and contributes to posterior capsule opacification (PCO) following cataract surgery. TGFß can induce matrix contraction and increase expression of transdifferentiation markers, alpha smooth muscle actin (αSMA) and fibronectin. We therefore aimed to determine the relationship between transdifferentiation of human lens cells and matrix contraction.
The human lens line FHL 124 was routinely cultured and cells seeded onto tissue culture dishes, such that distinct patches form. Contraction was assessed using a patch growth assay, whereby all areas covered by cells were measured using imaging techniques following fixation and cell staining with Coomassie Blue. In addition, total protein content, determined by dye extractions was used to give an estimate of total cell population. Following a 24hr period of serum starvation, cells were maintained in the following conditions for 1–3 days: Control medium +/– 100µM RGDS (Fn/Fn receptor inhibitor); 10ng/ml TGFß2 +/– RGDS; Control medium +/– siRNA–αSMA; 10ng/ml TGFß2 +/– siRNA–αSMA. SiRNA efficiency was tested at message and protein levels using Real–time PCR and western blots.
Real–time PCR analysis showed 10ng/ml TGFß2 significantly increased expression of αSMA (466 ± 85%), fibronectin (568 ± 192%) plus α5 (190 ± 29%) and ß1 (149 ± 19%) integrin (Fibronectin receptor components). Patch assays cultured for 3 days in the presence of TGFß showed significant contraction, with no decrease in cell population. Patches cultured for 1 day in TGFß alone did not demonstrate any difference in either area or population relative to unstimulated controls i.e. no contraction. Cultures maintained in TGFß and RGDS for 1 day showed a marked difference in detectable area without any reduction in total protein i.e. contraction. RGDS alone did not statistically differ from controls. Real time PCR and western blots showed reduced levels of αSMA message and protein when transfected with siRNA. αSMA knockdown in the presence of TGFß was associated with an enhanced reduction in patch area after 1 day. αSMA knockdown alone did not statistically differ from controls.
Disruption of transdifferentiation markers actively promotes TGFß induced contraction; therefore it appears that transdifferentiation is not a pre–requisite for contraction.
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