IOVS Human Reproduction
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(Investigative Ophthalmology and Visual Science. 2007;48:4679-4687.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-0091
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An In Vitro Model of Posterior Capsular Opacity: SPARC and TGF-ß2 Minimize Epithelial-to-Mesenchymal Transition in Lens Epithelium

Norihito Gotoh,1,2 Nikole R. Perdue,3 Hiroyuki Matsushima,2 E. Helene Sage,1,4 Qi Yan,3,4 and John I. Clark3

1From the Departments of Biological Structure and 3Ophthalmology, University of Washington School of Medicine, Seattle, Washington; the 2Department of Ophthalmology, Dokkyo University School of Medicine, Tochigi, Japan; and the 4Hope Heart Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington.

PURPOSE. This report presents a novel model for studies of extracellular matrix (ECM) in posterior capsular opacification (PCO) in vitro. Lens epithelial cells (LEC) were cultured with an intraocular lens (IOL) on a surface of type IV collagen in an evaluation of the importance of the ECM–cell interaction in formation of PCO. Abnormal migration, proliferation, and expression of proteins associated with the epithelial-to-mesenchymal transition (EMT) that characterizes PCO were observed in the presence and absence of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine), which regulates matrix–cell interactions.

METHODS. The model for PCO in vitro consisted of an IOL placed on a membrane coated with collagen IV, a major constituent of the lens capsule. LECs from the lenses of wild-type (WT) and SPARC-null (SP-null) mice were cultured in the presence or absence of 10 ng/mL TGF-ß2 and 20 µg/mL recombinant human SPARC (rhSP) for up to 6 days. The migration of LECs was quantified. Labeling with BrdU and the measurement of DNA synthesis were assays for cell proliferation. Expression of the EMT markers, collagen type I, fibronectin, and {alpha}-smooth muscle actin were assessed using immunocytochemistry or Western immunoblots.

RESULTS. LEC migration, proliferation, and the synthesis of EMT markers were enhanced in SP-null compared with WT LECs. TGF-ß2 inhibited the migration and proliferation of both WT and SP-null LECs in the presence of rhSP. TGF-ß2 increased the production of collagen type I, fibronectin, and {alpha}-SMA. The responses of SP-null LECs were rescued by the addition of recombinant human (rh)SP.

CONCLUSIONS. A simple IOL culture system was useful for the evaluation of the effects of SPARC and TGF-ß2 on PCO in vitro. The action of TGF-ß2 on LEC migration and proliferation is influenced by SPARC, a regulator of matrix–cell interactions. The results indicate a functional intersection between pathways activated by TGF-ß2 and SPARC in the formation of PCO.






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