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Audrey E. K. Hutcheon, Xiaoqing Q Guo, James D Zieske; Corneal Extracellular Vesicles Studied in 3D Construct and Ex Vivo Model. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2279.
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© ARVO (1962-2015); The Authors (2016-present)
Cell-cell communication has always been an important component of most functions in the cornea—fibrosis, regeneration, and homeostasis. Recently, a new player has come to the forefront of this research, extracellular vesicles (EVs). These small nanovesicles carry “cargo” consisting of proteins, mRNA, miRNA, and DNA that allow cells to communicate with one another and thus, influence the functions of recipient cells. Previous studies have found that both corneal epithelium and fibroblasts release EVs, and we, along with Dr. Azar’s group, showed that epithelial EVs stimulate keratocytes to express α-smooth muscle actin (SMA), thus indicating that EVs are involved in stimulating fibrosis. In this study, we examined our 3D fibroblast constructs and co-cultures (3D fibroblast construct with either epithelial or endothelial cells), as well as rabbit ex vivo corneas for the presence of EVs.
Human corneal fibroblasts (HCF) were grown in our 3D culture model producing a construct consisting of HCF and self-assembled extracellular matrix. Some of these constructs then were cultured with human corneal epithelial (HCE) or endothelial (HCEndo) cells producing co-cultures. In addition, rabbit corneas were obtained. Constructs, co-cultures, and rabbit corneas were then processed for TEM, examined for evidence of EVs, and imaged.
Interestingly, in the 3D cultures, a large number of vesicles were observed between cells that were adjacent to but not touching each other, and a group of EVs were observed near a deep pit that may either be a method of EV release or engulfment by the cell. Numerous EVs also were present in the ex vivo rabbit tissue, and one intriguing observation was that EVs appeared to be able to move through the Descemet’s membrane.
In all samples examined, numerous EVs were present and they appeared to move considerable distances between cells, which supports the use of our 3D culture system and ex vivo corneas in the examination of corneal EVs.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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