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

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-ß 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 the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-ß, 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 inhibiting multiple 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.

N-Terminomics identifies HtrA1 cleavage of thrombospondin-1 with generation of a proangiogenic fragment in the polarized retinal pigment epithelial cell model of age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population. Variants in the HTRA1-ARMS2 locus have been linked to increased AMD risk. In the present study we investigated the impact of elevated HtrA1 levels on the retina pigment epithelial (RPE) secretome using a polarized culture system. Upregulation of HtrA1 alters the abundance of key proteins involved in angiogenesis and extracellular matrix remodeling. Thrombospondin-1, an angiogenesis modulator, was identified as a substrate for HtrA1 using terminal amine isotope labeling of substrates in conjunction with HtrA1 specificity profiling. HtrA1 cleavage of thrombospondin-1 was further corroborated by in vitro cleavage assays and targeted proteomics together with small molecule inhibition of HtrA1. While thrombospondin-1 is anti-angiogenic, the proteolytically released N-terminal fragment promotes the formation of tube-like structure by endothelial cells. Taken together, our findings suggest a mechanism by which increased levels of HtrA1 may contribute to AMD pathogenesis. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier. For quantitative secretome analysis, project accession: PXD007691, username: reviewer45093@ebi.ac.uk, password: 1FUpS6Yq. For TAILS analysis, project accession: PXD007139, username: reviewer76731@ebi.ac.uk, password: sNbMp7xK.