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Inducers of the endothelial cell barrier identified through chemogenomic screening in genome-edited hPSC-endothelial cells.
Proceedings of the National Academy of Sciences of the United States of America ( IF 9.4 ) Pub Date : 2020-08-18 , DOI: 10.1073/pnas.1911532117
Filip Roudnicky 1 , Jitao David Zhang 2 , Bo Kyoung Kim 3 , Nikhil J Pandya 4 , Yanjun Lan 1 , Lisa Sach-Peltason 5 , Heloise Ragelle 3 , Pamela Strassburger 3 , Sabine Gruener 3 , Mirjana Lazendic 3 , Sabine Uhles 3 , Franco Revelant 3 , Oliv Eidam 5 , Gregor Sturm 4 , Verena Kueppers 3 , Klaus Christensen 1 , Leonard D Goldstein 6 , Manuel Tzouros 4 , Balazs Banfai 4 , Zora Modrusan 6 , Martin Graf 1 , Christoph Patsch 1 , Mark Burcin 1 , Claas A Meyer 1 , Peter D Westenskow 7 , Chad A Cowan 8, 9, 10
Affiliation  

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 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-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 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.



中文翻译:

通过化学基因组筛选在基因组编辑的hPSC-内皮细胞中鉴定出的内皮细胞屏障诱导物。

血视网膜屏障和血脑屏障(BRB / BBB)具有选择性和半渗透性,对支持和保护驻留中枢神经系统(CNS)的细胞至关重要。BRB / BBB中的内皮细胞(ECs)紧密耦合,表达高水平的Claudin-5(CLDN5),这是一种稳定ECs的连接蛋白,对于正常的神经元功能很重要。为了鉴定新型的CLDN5调节剂(以及最终的EC稳定剂),我们从人多能干细胞(hPSC)生成了CLDN5-P2A-GFP稳定细胞系,将其分化为EC(CLDN5-GFP hPSC-EC),并进行了流式细胞术的化学基因组文库筛选,以测量GFP表达,作为屏障完整性的替代报道基因。使用这种方法,我们确定了激活CLDN5-GFP的62种独特化合物。其中包括TGF-β途径抑制剂,包括RepSox。当应用于hPSC-EC,原发性脑EC和视网膜EC时,RepSox可以显着提高屏障抵抗力(跨内皮电阻),降低细胞旁通透性(异硫氰酸荧光素-右旋糖酐),并防止血管内皮生长因子A(VEGFA)诱导的屏障破坏体外。当外源递送时,RepSox还可以在发育过程中改变小鼠视网膜的血管模式。为了确定RepSox的作用机理,我们进行了激酶组,转录组和蛋白质组图谱分析,发现RepSox抑制了TGF-β,VEGFA和炎症基因网络。此外,RepSox不仅激活了血管稳定和建立障碍的Notch和Wnt通路,而且还诱导了重要的紧密连接和转运蛋白的表达。在一起

更新日期:2020-08-19
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