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K.J. Mandell, G.P. Holley, C.A. Parkos, H.F. Edelhauser; Expression and Function of Junctional Adhesion Molecule–A (JAM–A) in Tight Junctions of the Rabbit Corneal Endothelium . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4387.
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Junctional adhesion molecule–A (JAM–A) is an immunoglobulin superfamily (IgSF) adhesion protein implicated in tight junction assembly and barrier function. In the corneal endothelium, tight junctions play a central role in regulating diffusion of salt, water, and nutrients into and out of the corneal stroma. Although corneal endothelial tight junctions have been studied extensively at the ultrastructural level, little is known about their molecular composition. In this study, we have investigated the role of JAM–A in rabbit corneal endothelium.
A novel monoclonal antibody (mAb) 7G2C9 was raised in mice against JAM–A. Immunoreactivity of mAb 7G2C9 with JAM–A was confirmed by Western blot. Corneas were isolated from New Zealand white rabbits, and the cellular localization of JAM–A in the corneal endothelium was assessed by immunofluorescence confocal microscopy. For functional experiments, whole rabbit corneas were mounted in a perfusion specular microscope. Corneal swelling was induced by transient exposure of the endothelium to calcium–free medium. Corneas were then perfused with either calcium–enriched medium containing mAb 7G2C9 or vehicle alone. Changes in corneal thickness were measured over time using the specular microscope.
Western blots revealed that mAb 7G2C9 specifically reacted with a single 37 kDa protein band corresponding to JAM–A. Immunofluorescence confocal microscopy demonstrated expression of JAM–A at cell–cell junctions in rabbit corneal endothelium with a localization pattern identical to that of ZO–1, a known marker of the tight junction. In functional experiments, treatment of the rabbit corneal endothelium with mAb 7G2C9 after transient calcium depletion increased corneal swelling by 63% compared to controls.
Our results provide evidence that JAM–A is expressed in tight junctions of the corneal endothelium. The observed effect of the anti–JAM–A antibody on corneal swelling suggests that JAM–A may play a functional role in regulation of the corneal endothelial barrier.
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