The cornea is the transparent, outermost part of the eye which protects the fragile intraocular contents from mechanical and pathogenic perturbations. It consists of three distinct layers: a tear-bathed, stratified layer of epithelial cells; a thick stroma of collagen fibrils embedded with quiescent fibroblasts; and an inner epithelial layer, called the endothelium. Poor cell growth in any one of these layers can result in a loss of corneal transparency and thus blindness. The endothelium in particular lacks a robust proliferative response to cell loss, and this can hinder recovery after eye surgery and in diseases such as keratoconus, which affects between 1 in 500 and 1 in 2000 people in the U.S. <issue of endothelial to mesenchymal (fibroblast?) transfer>.
Our goal is to determine whether we can enhance the proliferation of human corneal endothelial cells (HCECs) while maintaining their phenotype. We will use ROCK inhibitor and upregulation of NGF to increase the proliferation of HCECs and study whether the presence of these proteins allow the cells to maintain their phenotype in culture.
Pipparelli, Aurélien, Yvan Arsenijevic, et al. "ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells." PLoS One. 8.4 (2013): n. page. Web. 5 May. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633902/>.
--> ROCK inhibitor Y-27632 promotes adhesion and cell proliferation in monkey CECs in vitro. The addition of ROCK inhibitor to HCECs was hoped to increase anti-apoptotic activity, and thereby increase endothelial cell density in the cornea. Unfortunately, it was not found to enhance cell proliferation in HCECs ex vivo. ROCK inhibitor does, however, promote cell adhesion and wound healing in HCECs.
Hong, J., Qian, T., NGF promotes cell cycle progression by regulating D-type cyclins via PI3K/Akt and MAPK/Erk activation in human corneal epithelial cells, Molecular Vision, Vol 18, Mar 30, 2012, Pages 758-764, <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324366/>.
--> Nerve growth factor is a protein that promotes cell survival through the MAPKcascade. Without NGF the expression of pro-apoptotic proteins are increased, leading to cell death. NGF positively regulates HCEC cell cycle progression which can enhance cell proliferation. We would like to up-regulate NGF expression in order to allow the HCECs to proliferate during wound healing in vitro.
Okumura, Naoki, EunDuck Kay, et al. "Inhibition of TGF-β Signaling Enables Human Corneal Endothelial Cell Expansion In Vitro for Use in Regenerative Medicine." PLoS One. 8.2 (2013): n. page. Web. 5 May. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581499/>.
-->This paper demonstrated that the use of SB431542, a selective inhibitor of the TGF-β receptor, in culture media counteracted the development of fibroblastic phenotypes in HCECs. This suggests that SB431542 would be a useful additive to our HCEC growth media because it may allow the cells to maintain their phenotype as they proliferate.