An in vitro model of posterior capsular opacity: SPARC and TGF-beta2 minimize epithelial-to-mesenchymal transition in lens epithelium.
OBJECTIVE
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-beta2 and 20 mug/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-beta2 inhibited the migration and proliferation of both WT and SP-null LECs in the presence of rhSP. TGF-beta2 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-beta2 on PCO in vitro. The action of TGF-beta2 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-beta2 and SPARC in the formation of PCO.
Gotoh
Norihito
N
Department of Biological Structure, University of Washington School of Medicine, Seattle, Washington 98195-7420, USA.
Perdue
Nikole R
NR
Matsushima
Hiroyuki
H
Sage
E Helene
EH
Yan
Qi
Q
Clark
John I
JI
D000199:Actins / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002386:Cataract / Q000378:metabolism* / Q000473:pathology; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004195:Disease Models, Animal; D004847:Epithelial Cells / Q000187:drug effects*; D005109:Extracellular Matrix / Q000187:drug effects*; D005353:Fibronectins / Q000378:metabolism; D007150:Immunohistochemistry; D007903:Lens Capsule, Crystalline / Q000378:metabolism* / Q000473:pathology; D008648:Mesoderm / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D015676:Osteonectin / Q000494:pharmacology / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology*
D000199:Actins; D015415:Biomarkers; D024042:Collagen Type I; D005353:Fibronectins; D015676:Osteonectin; D011994:Recombinant Proteins; D053781:Transforming Growth Factor beta2; D004247:DNA