The corneal endothelium plays a pivotal role in maintaining corneal transparency. Unlike other species, the human corneal endothelium is notorious for its limited proliferative capacity in vivo after injury, aging, and surgery. Persistet corneal endothelial dysfunction leads to sight- threatening bullous keratopathy. Presently, the only solution to restore vision in eyes inflicted with bullous keratopathy relies upon transplantation of a cadaver donor cornea containing a healthy corneal endothelium. Because of a severe global shortage of donor corneas in conjunction with an increasing trend toward transplanting only the corneal endothelium in procedures collectively termed "endothelial keratoplasty", it is timely and paramount to develop a tissue engineering strategy to produce surgical grafts containing human corneal endothelial cells (HCEC). Using our reported in vitro model system, in which the mitotic block is mediated by contact inhibition when cell junctions mature, our preliminary studies showed that such mitotic block unlocked by the conventional engineering method based on EDTA/bFGF activates ?-catenin/Wnt signaling and runs the risk of losing the normal phenotype to fibrous metaplasia because of endothelial-mesenchymal transition (EMT). We have further discovered such mitotic block can also uniquely be unlocked by knockdown of p120-catenin to selectively activate p120- catenin/Kaiso but not ?-catenin/Wnt signaling. Consequently, our novel tissue engineering technology has successfully produced HCEC monolayers with a hexagonal shape and high cell density and an average size of 3.7 ? 0.7 mm2 (2.1 ? 0.4 mm in diameter) from stripped Descemet membrane of 1/8 of the corneoscleral rim normally discarded after conventional corneal transplantation. Thus, in this Phase I application, we would like to prove the concept that the size of HCEC monolayers can further be enlarged by optimizing the regimen of p120- catenin siRNA knockdown followed by additional Kaiso siRNA knockdown (Aim 1), and by addition of nocodazole to enhance p120-catenin nuclear translocation (Aim 2). Completion of these two Aims will allow us further fabricate expanded HCEC monolayers on epithelially- denuded amniotic membrane to ultimately produce 8 HCEC grafts from one donor rim and to conduct pre-clinical animal experiments in Phase II. We envision that this novel tissue engineering technology based on siRNA can also be applied to switch on and off proliferation both in vivo and ex vivo without risking the loss of normal function to EMT in other contact- inhibited tissues. Further exploration of how contact inhibition is controlled by p120- catenin/Kaiso signaling may unravel other therapeutic potentials in burgeoning regenerative medicine for treating a number of diseases characterized by the lack of regeneration due to aging, surgery, or degeneration.
This Phase I application proposes to develop a novel strategy of engineering human corneal endothelium based on selective activation of p120ctn/Kaiso signaling using interference RNA technology targeted at p120-catenin. Using our reported in vitro model system, we have provided strong preliminary data supporting the plausibility of further expanding human corneal endothelial monolayers by additional Kaiso siRNA knockdown with or without nocodazole. By switching on and off p120/Kaiso signaling, our novel engineering strategy may control cellular proliferation without disrupting their intercellular junctions, hence avoiding both the use of enzymatically dissociated single cells and the risk of losing the normal cell phenotype to fibrous metaplasia. Such engineered grafts may one day be used to improve the surgical procedure of endothelial keratoplasty for restoring sight in patients suffering from bullous keratopathy due to dysfunctional human corneal endothelium. Furthermore, the said technology may also be applied to engineer other similar tissues, such as the retinal pigment epithelium, in the future.
|Zhu, Ying-Ting; Han, Bo; Li, Fu et al. (2014) Knockdown of both p120 catenin and Kaiso promotes expansion of human corneal endothelial monolayers via RhoA-ROCK-noncanonical BMP-NF?B pathway. Invest Ophthalmol Vis Sci 55:1509-18|
|Zhu, Ying-Ting; Li, Fu; Han, Bo et al. (2014) Activation of RhoA-ROCK-BMP signaling reprograms adult human corneal endothelial cells. J Cell Biol 206:799-811|