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M. Shivanna, M. Satpathy, S. P. Srinivas; Role of MMPs in TNF--Induced Loss of Barrier Integrity of Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1815.
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TNF- is a pleiotropic cytokine implicated in corneal endothelial failure during allograft rejection. In this study, we have investigated the role of matrix metalloproteinases (MMPs) in the TNF--induced loss of barrier integrity in cultured bovine corneal endothelial cells (BCEC).
Changes in the barrier integrity were assessed in terms of trans-endothelial electrical resistance (TER) obtained from electrical cell-substrate impedance sensing (ECISTM, Applied Biophysics, Inc, NY). Apical junctional assembly was visualized by immunolocalization of cadherins (with a pan-cadherin a/b) and ZO-1. MMP activity in the conditioned media from cells treated with TNF- was visualized by gelatin zymography.
Exposure to TNF- (20 ng/ml; 6 hr) induced dispersion of ZO-1 and cadherins and also led to loss of peri-junctional actomyosin ring. TNF- also caused a decline in TER, which sustained for more than 20 hr. These effects were opposed by pre-treatment with GM-6001 (a broad spectrum MMP inhibitor; 50 µM; 1 hr) and minocycline (50 µg/ml; 1 hr), a selective MMP-2 and MMP-9 inhibitor. Pronounced inhibition of the loss in TER was also noticed upon pretreatment with a selective p38 MAPK inhibitor, SB-203580. An increase in MMP-9 activity in the conditioned media was observed upon exposure to TNF- for 20 hr.
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