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M. Shivanna, S. P. Srinivas; Role of Microtubules in TNF- -Induced Loss of Barrier Integrity of Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3948.
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TNF-α is a pro-inflammatory cytokine implicated in corneal endothelial failure during graft rejection. It is known to induce a loss in barrier integrity of the corneal endothelial cells but the underlying molecular mechanisms are not well understood. In this study, we have investigated the role of microtubules in TNF-α-induced loss of barrier integrity in cultured bovine corneal endothelial cells.
Changes in the organization of actin cytoskeleton and microtubules were examined by staining for actin and α-tubulin, respectively. Localization of ZO-1, a marker of TJ assembly, was visualized by immunostaining. Changes in the barrier function were monitored continuously by transendothelial electrical resistance (TER) using ECIS (Applied Biophysics, Inc, NY).
Exposure to TNF-α (20 ng/ml; 6hr) induced disruption of microtubules along with thinning of PAMR (peri-junctional actomyosin ring) and dispersion of ZO-1. Similar exposure to TNF-α also led to a continued decline in TER, which sustained for more than 15 hr. These effects of TNF-α were significantly opposed by pretreatment with paclitaxel ( 10 µM; 1 hr), a microtubule stabilizing agent and SB 203580 (20 µM; 1 hr), a p38 MAPK (mitogen activated protein kinase) inhibitor.
TNF-α may induce loss of barrier integrity through microtubule disassembly and thinning of PAMR. These events appear to be downstream of activation of p38 MAPK.
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