Abstract
The corneal endothelium is the interior of the cornea, comparable to the window through which our body looks at the outside. Proper function of this corneal endothelium is essential to obtain a crystal- clear cornea. Current consensus holds that endothelial cells do not display any significant regenerative capacity. Damaged or non-functional cells may consequently lead to corneal blindness. In this regard, stimulation of the regenerative capacity of the corneal endothelium is of exceptional importance in the search for new therapeutic possibilities. On the one hand, these alternative strategies will spare transplantable corneal endothelial donor tissue which will be beneficial for visually impaired patients in terms of shorter donor waiting lists. Furthermore, a regenerative compound could be used as a supplementary treatment in corneal endothelial surgery or in the development of novel cell therapies. On the other hand, for in vitro biomedical research the culture of primary corneal endothelium is an extremely time-intensive process without full guarantee of cell expansion. Stimulation of the regenerative capacity of primary tissue would therefore increase the amount of available starting material for in vitro research, thereby drastically improve the translatability of basic ophthalmological research.
In this project we intend to provide a solution for these issues by pharmacological stimulation of the regenerative capacity of corneal endothelial cells through ROCK inhibitors (ROCKi), given their growth potential within tissue regeneration. However, there is no golden standard ROCKi available yet to boost corneal endothelial regeneration that is rationally described and characterized for this purpose, which leaves space for improved compound characterization. In this project, customized ROCK inhibitor libraries, containing inhibitors with different IC50 values and variable selectivity to its key target proteins will be screened. As such we will characterize the regenerative capacity of those compounds in terms of proliferation and migration processes. During this project all the ROCKi will be subject to a two-fold screening method, creating an innovative drug repurposing pipeline. This screening setup is based on the synergy of high throughput live cell imaging and genomic analysis (i.e., RNA sequencing and kinomics) to connect the cellular to the subcellular level. Live cell imaging applied on the different ROCKi will generate kinetic drug profiles and will indicate the most efficient timepoint for downstream genomic analysis. In its turn, these genomic sequencing techniques provide tremendous insights for unravelling activated pathways and signaling molecules. The combination of cellular and subcellular compound characterization will result in the selection of the most promising ROCKi for hit-to-lead optimization for corneal endothelial regeneration purposes.
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