Abstract
Purpose :
Vascular deficits are associated with vision loss, and are characteristic of multiple retinal diseases including diabetic retinopathy (DR) and wet AMD (wAMD). DR and wAMD patients can experience excessive vascular permeability and leakage that negatively impacts neuronal survival. Therapeutically stabilizing diseased vessels can significantly improve patient outcomes. We developed an endothelial cell line that fluoresces when high barrier resistance is achieved. Using this tool, we can identify novel compounds that can impact blood vessel integrity that might ultimately be used to treat features of human diabetic retinopathy and/or wet AMD.
Methods :
Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) were developed and validated. The hPSC-ECs were maintained in culture until high resistances were achieved. Gene-profiling was performed to identify novel biomarkers of high endothelial cell barrier resistance, and of the genes examined, Claudin 5 was the most significantly upregulated. We next generated Claudin 5-GFP reporter endothelial cells through gene-editing, and screened the cells with a library of factors to identify candidates that potentiate barrier resistance. Lead candidates were further validated in vitro, and efforts to validate their impacts on developing and diseased blood vessels in vivo are preliminary, but encouraging.
Results :
Claudin 5-GFP cells faithfully express GFP in patterns correlating with enhanced barrier resistance. From the screen to identify novel factors that promote endothelial cell resistance, we determined that Repsox, a commercially available tyrosine kinase inhibitor, potently induced GFP fluorescence and enhanced barrier resistance in Claudin 5-GFP cells.
Conclusions :
Claudin 5-GFP cells are useful for identifying novel factors that enhance endothelial cell barrier resistance. Repsox was identified as a potent modulator of endothelial cell resistance, and work is underway to characterize its effects in vivo. This may be important since the use of tyrosine kinase inhibitors is being explored to prevent vision loss in various neovascular diseases.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.