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A. Banh, P. A. Deschamps, M. M. Vijayan, J. A. West-Mays, J. G. Sivak; The Role of Molecular Chaperones in TGF-ß Induced Epithelial-Mesenchymal Transition in Rat Lens Epithelial Explants. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3188.
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The treatment with TGF-ß in cultured rat lens epithelial explants has shown to induced epithelial-mesenchymal transition (EMT) in lens epithelial cells (LECs). It has been suggested that Hsp70 and α-crystallins are upregulated in the lens under stressed condition and play a protective role in the LECs. The purpose of this study is to investigate the effects of heat shock treatment and the role of molecular chaperones on TGF-ß2 induced EMT in rat lens epithelial explants.
Rat lens epithelial explants from 7-10 days old Wistar rats were dissected and incubated with M199 with antibiotics at 37 o C with 4% CO2 for 24 hours before treatment. The explants were divided into eight treatment groups: control (culture medium), 10 ng/ml FGF-2, 8 ng/ml TGF-ß2 and 8 ng/ml TGF-ß2+10ng/ml FGF-2 under normal culture conditions and heat shocked control (culture medium), 10 ng/ml FGF-2, 8 ng/ml TGF-ß2, and 8 ng/ml TGF-ß2+10ng/ml FGF-2. The heat shocked explants were heat shocked at 45°C for 1 hour and stabilized 37 °C with 4.0% CO2 for 3 hours prior to treatment with the respective media as mentioned above. H&E staining was performed on whole-mount epithelial explants from each group. Immunofluorescence staining for α-smooth muscle (α-SMA) actin, F-actin and E-cadherin was also used to determine EMT in the lens epithelial explants. A Zeiss confocal microscope was use to visualize the immunoreactivity in the whole-mount epithelial explants. Western blot analysis of α-SMA, E-cadherin, Hsp70 and Hsp90, and αA-and αB-crystallin were also performed.
TGF-ß2 induced EMT and plaque formation in the lens epithelial explants. The treatment of epithelial explants with TGF-ß2+FGF-2 induced the most significant morphological changes and EMT. Heat shock treatment of lens epithelial explants show significant reduction of α-SMA expression and greater E-cadherin expression when compared to the non-heat shocked TGF-ß2 treated explants. There was also a significant reduction of Hsp70, Hsp90 and αA-crystallin expression in the heat shocked groups at day 4 of treatment.
TGF-ß2 induced EMT is significantly reduced in the heat shocked TGF-ß2 lens epithelial explants. The results also show that there is a reduction in expression of Hsps in the heat-shocked groups indicating that the LECs are under less stressful condition than the non-heat shocked groups. In conclusion, molecular chaperones can play a protective role against TGF-ß2 induced EMT.
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