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S.R. Gordon; A Corneal Endothelial Surface Protein That Binds Soybean Agglutinin Is Involved in Cell Reorganization During Wound Repair . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4515.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To determine the function(s) of a corneal endothelial cell surface soybean–agglutinin (SBA) binding protein expressed during explantation and injury. Methods: Transcorneal freezing was used to produce a circular wound area on the endothelium. Noninjured and injured corneas were organ cultured in order to express the SBA–binding protein. To determine the function(s) of this protein, noninjured and injured tissues were treated with either SBA, N–acetylgalactosamine or N–acetylgalactosaminidase. Synthesis of the SBA–binding protein was investigated using puromycin, actinomycin D and alpha–amanitin. SBA– binding protein distribution was visualized using fluorochrome–conjugated SBA and the actin cytoskeleton was observed with rhodamine–conjugated phalloidin. Flat mounts of the tissues were observed by light and fluorescence microscopy using standard protocols, as well as by electron microscopy. Results:Exposure of noninjured, organ cultured endothelia to SBA results in the disruption of cell–cell and cell/matrix contacts causing a loss of monolayer integrity. Rhodamine–phalloidin staining reveals that this also results in the loss of the actin cytoskeleton. This effect is reversed if tissues are transferred into medium without SBA, and minimized when tissues are pretreated prior to SBA exposure with phalloidin to stabilize their microfilaments. The expression of the SBA–binding protein is sensitive to puromycin but does not appear to be effected by incubation in either actinomycin D or alpha–amanitin, suggesting it may be post–transcriptionally regulated.When injured endothelia are treated with N–acetylgalactosaminidase to remove the terminal SBA–binding sugar there is no effect on the actin cytoskeleton, cell migration or mitosis. If injured tissues are incubated in the presence of SBA, migrating cells fail to exhibit extended processes typical of translocating cells and appear blunt–shaped. In contrast, migrating cells exposed to N–acetylgalactosamine maintain normal appearing processes. In each case, cells repopulate the wound area by 72 h post–injury. However, in both instances, cells within the wound area only exhibit minimal cell–cell interaction with their neighbors compared to their control counterparts. Conclusions:Experimental evidence indicates that the SBA–binding protein, together with the actin cytoskeleton, plays a role in maintaining endothelial monolayer integrity under conditions such as organ culture. Wound repair studies also suggest that this protein is involved in reestablishing cell–cell contacts during reorganization of the injured monolayer.
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