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Jiaoyue Hu, Zhenhao Zhang, Hui Xie, Lelei Chen, Yueping Zhou, Wensheng Chen, Zuguo Liu; Serine Protease Inhibitor A3K Protects Rabbit Corneal Endothelium From Barrier Function Disruption Induced by TNF-α. Invest. Ophthalmol. Vis. Sci. 2013;54(8):5400-5407. doi: 10.1167/iovs.12-10145.
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To determine if a serine protease inhibitor A3K (SA3K) reduces TNF-α–induced declines in rabbit corneal endothelial junctional barrier integrity.
New Zealand rabbit corneas were incubated ex vivo for 24 hours in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS with or without TNF-α, in the presence or absence of SA3K at different concentrations. Corneal endothelial barrier function permeability was determined based on measurements of FITC-dextran tissue accumulation. Apical junctional complex (AJC) integrity was evaluated of zonula occludens-1 (ZO-1), vascular endothelial (VE)-cadherin, and filamentous actin (F-actin) and associated microtubules, as well as myosin light chain (MLC) by immunofluorescent staining, Western blot analysis, and/or RT-PCR.
TNF-α (20 ng/mL) increased corneal endothelial FITC-dextran permeability by 1.8-fold compared with the untreated control. SA3K (100–200 nM) dose dependently suppressed TNF-α–induced increases in permeability. SA3K nearly completely reversed TNF-α–induced disruptions of tight junctional ZO-1 and subjacent adherens junctions VE-cadherin integrity. Interestingly, SA3K reversed TNF-α–induced disruption of AJC linkage to the cytoskeletal F-actin array by restoring F-actin double-band structures. SA3K also attenuated TNF-α–induced microtubule disassembly. Furthermore, SA3K blocked increases in MLC phosphorylation status elicited by TNF-α.
SA3K exposure markedly reduced TNF-α–induced disruption of barrier structure and function in the rabbit corneal endothelium by maintaining AJC integrity. These protective effects are due to suppression of MLC activation. SA3K may have, in vivo, a therapeutic potential to offset TNF-α–induced declines in endothelial barrier structural integrity and function.
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