Intelly Lee, Claudia Garcia, Justin De Villar, Jia Jan Fu, David Beebe. Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO.

Secondary cataracts form as the result of an injury response in lens epithelial cells of the lens capsule following introduction of the artificial lens in cataract surgery. This injury response prompts lens epithelial cells to differentiate into myofibroblasts that express large amounts of proteins such collagen and actin, a process known as fibrosis. In this project, the effect of peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist drugs has been studied for potential use in inhibiting myofibroblast differentiation in mouse lens epithelia grown in vitro, with the aim of developing more widely available treatments for secondary cataracts. PPAR-γ agonists are of particular interest not only due to previous research demonstrating their antifibrotic properties in other human systems but also their widespread commercial availability. The explantation of epithelial cells in vitro induces the same injury response observed in the formation of secondary cataracts, resulting in the differentiation of epithelial cells into myofibroblasts that express α-smooth muscle actin (α-SMA). The effects of PPAR-γ agonists in inhibiting fibrosis can thus be measured by comparing α-SMA protein expression in epithelial cells exposed to different drug treatments for equal time periods in growth culture. Lower α-SMA protein concentrations in treated explants would indicate effective anti-fibrotic capabilities.

Lens epithelial cells were extracted from BalbC mice and either analyzed immediately as time zero (T0) controls or grown for four days (D4) at 37C in the presence or absence of drugs in DMEM growth solution with 0.1% BSA and antibiotics. The effects of both BMP-7, a member of the TGF-β superfamily of ligands shown to exhibit antifibrotic capabilities, and troglitazone, a true PPAR-γ agonist, were examined on mouse lens epithelial cells. At the end of the growth period, cells were removed from culture and placed in lysis buffer, followed by DNA concentration assaying. Protein samples were normalized and isolated through SDS-PAGE in a 4-20% gradient gel run at 110 V for 2 hours, and Western blotting at 300 milliamps for another 2 hours transferred protein samples onto nitrocellulose paper; α-SMA protein was then stained using appropriate antibodies. Pierce ECL reagents were added as substrates for HRP and chemiluminescence of samples was recorded on film. Relative intensity measurements of α-SMA bands were determined with Kodak ID Imaging software.

Preliminary results indicate that BMP-7 is not a very effective inhibitor of fibrosis, with treated explants exhibiting 67% as much α-SMA expression as similar controls explants grown without drug treatment.  Troglitazone, in contrast, shows significantly more promise as an anti-fibrotic agent in inhibiting myofibroblast differentiation, reducing α-SMA expression in D4-treated explants to comparable levels found in T0-control explants that experienced no myofibroblast differentiation growth period. However, Western blotting and antibody-staining from these preliminary results are of poor quality and dubious accuracy, and further trials should be conducted. Future directions for this research include immunostaining for other proteins associated with myofibroblasts such as CTGF, SM22 and Type I collagen, analysis of chemically similar PPAR-γ agonists such as pioglitazone and rosiglitazone, and plotting of dose response curves to different drug treatments