Journal Article

Inducers of the endothelial cell barrier identified through chemogenomic screening in genome-edited hPSC-endothelial cells

Modrusan, Zora, Kim, Bo Kyoung, Pandya, Nikhil J., Meyer, Claas A., Banfai, Balazs, Westenskow, Peter D., Ragelle, Heloise, Graf, Martin, Roudnicky, Filip, Sturm, Gregor, Goldstein, Leonard D., Uhles, Sabine, Kueppers, Verena, Tzouros, Manuel, Burcin, Mark, Christensen, Klaus, Strassburger, Pamela, Lazendic, Mirjana, Lan, Yanjun, Gruener, Sabine, Revelant, Franco, Cowan, Chad A., Patsch, Christoph, Sach-Peltason, Lisa, Eidam, Oliv

2020

Overview

The blood–retina barrier and blood–brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-β pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in themouse retina during developmentwhen delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-β, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibitingmultiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.

• Publisher: National Academy of Sciences
• Subject: Animals / Applied Biological Sciences / Biological Sciences / Blood-brain barrier / Blood-Brain Barrier - drug effects / Blood-Brain Barrier - metabolism / Blood-Retinal Barrier - drug effects / Blood-Retinal Barrier - metabolism / Cell Differentiation / Cell Line / Cell Proliferation - drug effects / Central nervous system / Claudin-5 - genetics / Claudin-5 - metabolism / Development strategies / Dextran / Dextrans / Drug Evaluation, Preclinical / Electrical junctions / Electrical resistivity / Endothelial cells / Endothelial Cells - cytology / Endothelial Cells - drug effects / Endothelial Cells - metabolism / Flow cytometry / Fluorescein / Fluorescein isothiocyanate / Gene Editing / Gene expression / Genome / Genome editing / Genomes / Growth factors / Humans / Inflammation / Mice / Mice, Knockout / Permeability / Pluripotency / Pluripotent Stem Cells - cytology / Pluripotent Stem Cells - drug effects / Pluripotent Stem Cells - metabolism / Proteomes / Pyrazoles - pharmacology / Pyridines - pharmacology / Regulators / Retina / Screening / Small Molecule Libraries - pharmacology / Stem cell transplantation / Stem cells / Tight junctions / Tight Junctions - metabolism / Vascular endothelial growth factor / Vascular Endothelial Growth Factor A - metabolism / Wnt protein