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KH Svoboda, P Moessner, P Moessner, J Acevedo; Collagen, Fibronectin and Lysophosphatidic Acid decreased Caspase-3 activity while inhibiting ROCK (Y27632) increased Caspase-3 activity in Whole Epithelial Sheets . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3217.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Whole embryonic corneal epithelial sheets can be isolated without the basal lamina. These sheets of cells respond to bombesin, lysophosphatidic acid (LPA) and extracellular matrix (ECM) molecules including fibronectin (FN), laminin and collagen (COL), by reorganizing the actin cortical mat (ACM). As other epithelial and endothelial tissues are attachment dependent, we asked: are these sheets of epithelia susceptible to apoptosis? Caspase-3 activity is associated with the apoptosis pathway. Caspase-3 activity can be assessed using synthetic substrates with a consensus cleavage site (DEVD) attached to reporter molecules (colorimetric or fluorescent). Methods: Epithelia were isolated without the basal lamina and incubated overnight with either control media or media containing COL I (50, 100 µg/ml), 50 µg/ml FN, 10 µM LPA in the presence or absence of 10 µM Y27632 and inhibitor specific for p160 Rho kinase. All treatment groups were also cultured with or without pretreating with ZVAD, a caspase-3 inhibitor. Epithelia (25/group) were homogenized, and assayed for active caspase-3 with the caspACETM (Promega) system. To visualize caspase-3 activity, PhiPhiLux was added to the media of the live tissues for 1hr. The tissues were rinsed, fixed and stained the Texas Red phalloidin. Whole tissues were morphologically analyzed using a Leica TSP SPII microscope for actin and FITC-PhiPhiLux. Results: Control tissues incubated without ECM had moderate levels of caspase-3 activity that was decreased to background with the ZVAD caspase-3 inhibitor. FN, COL and LPA decreased caspase-3 activity 0.2 to 0.5 fold, whereas, Y27632 increased caspase-3 activity 1.4 fold in the presence of COL and 2 fold without COL. Increasing doses of Y27632 also caused increasing amounts of FITC-PhiPhiLux, indicating that the tissues were positive for caspase-3. Conclusion: Corneal epithelial cells increase the apoptosis marker, caspase-3 activity in the absence of extracellular matrix. In addition, blocking the reorganization of the actin cortical mat by inhibiting Rho kinase increased the level of caspase-3 activity.
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