Identification of a combination of transcription factors that synergistically increases endothelial cell barrier resistance

Abstract

Endothelial cells (ECs) display remarkable plasticity during development before becoming quiescent and functionally mature. EC maturation is directed by several known transcription factors (TFs), but the specific set of TFs responsible for promoting high-resistance barriers, such as the blood-brain barrier (BBB), have not yet been fully defined. Using expression mRNA data from published studies on ex vivo ECs from the central nervous system (CNS), we predicted TFs that induce high-resistance barrier properties of ECs as in the BBB. We used our previously established method to generate ECs from human pluripotent stem cells (hPSCs), and then we overexpressed the candidate TFs in hPSC-ECs and measured barrier resistance and integrity using electric cell-substrate impedance sensing, trans-endothelial electrical resistance and FITC-dextran permeability assays. SOX18 and TAL1 were the strongest EC barrier-inducing TFs, upregulating Wnt-related signaling and EC junctional gene expression, respectively, and downregulating EC proliferation-related genes. These TFs were combined with SOX7 and ETS1 that together effectively induced EC barrier resistance, decreased paracellular transport and increased protein expression of tight junctions and induce mRNA expression of several genes involved in the formation of EC barrier and transport. Our data shows identification of a transcriptional network that controls barrier resistance in ECs. Collectively this data may lead to novel approaches for generation of in vitro models of the BBB.Endothelial cells (ECs) from different organs display unique molecular 1 and functional 2 profiles. These organotypic profiles arise during endothelial cell development and are directed in part by signals from neighboring cells that activate TFs in ECs to activate or repress specific gene networks 3 . Organotypic differences are pronounced in ECs isolated from the central nervous system (CNS) 1,4-6 that generate the blood-brain barrier (BBB), a highly selective and semipermeable barrier. Unique properties of the BBB include suppressed transcytosis, high tight junction and specialized transporter gene expression, and low immune cell adhesion gene expression 7 . Studies suggest 8-12 that canonical Wnt, Hedgehog and retinoic acid pathways are involved in BBB development. However, other pathways are certainly involved, and the full set of TFs activated in ECs to generate the BBB has not been determined 13,14 . A more complete understanding of TF activation programs in CNS-derived ECs would greatly inform BBB biology.

Multiplexed chemogenetics in astrocytes and motoneurons restore blood–spinal cord barrier in ALS

“…Moreover, noncanonical Wnt signaling may contribute to the establishment of the BBB phenotype (Pinzón-Daza et al, 2014). Sox17 has been identified as a major target of the β-catenin pathway in endothelial cells (Corada et al, 2019), together with Lef1 and Ets1 (Roudnicky et al, 2020). These Wnt-regulated transcription factors up-regulate the transcription of several junctional proteins, including VE-cadherin and claudin-5 (Roudnicky et al, 2020), possibly involved in the repair of the BSCB.…”

“…Sox17 has been identified as a major target of the β-catenin pathway in endothelial cells ( Corada et al, 2019 ), together with Lef1 and Ets1 ( Roudnicky et al, 2020 ). These Wnt-regulated transcription factors up-regulate the transcription of several junctional proteins, including VE-cadherin and claudin-5 ( Roudnicky et al, 2020 ), possibly involved in the repair of the BSCB. Furthermore, claudin-5 is dynamically internalized and stored in intracellular vesicles upon inflammatory stimuli and trafficked back to the cell surface when the inflammatory signaling is subsided ( Stamatovic et al, 2003 , 2006 , 2009 ).…”

Human Retinal Microvasculature‐on‐a‐Chip for Drug Discovery

“…To do this, we measured macromolecular fluxes using specific dextran molecular weights chosen based on previous work investigating brain and retinal microvasculature permeability. [30][31][32][33][34] In the case of a tight endothelial barrier, tracers were retained on the luminal side of the tube, whereas tracers rapidly diffused into the matrix over 20 min in the case of a leaky barrier. Image analysis was used to quantify the leakage score (a metric that is specific to this system) and the apparent permeability (P a ; standardized metric of barrier function for comparison to other models; Figure 2a,b and Figure S2, Supporting Information).…”

Human iPSC‐Derived Blood‐Brain Barrier Models: Valuable Tools for Preclinical Drug Discovery and Development?

“…A shift from the co-differentiation protocol to direct BCECs generation was achieved by using chemically defined factors (Grifno et al, 2019;Praca et al, 2019;Qian et al, 2017). Recent advancements have been focused on the overexpression of transcription factors in upregulating BBB phenotypes (Roudnicky et al, 2020). Here, Roudnicky et al identified 17 transcription factors, including among others TAL1, SOX7, SOX18, ETS1, and LEF1, to improve the direct differentiation of vascular endothelial cells and analyzed them using gain-of-function assays (Patsch et al, 2015;Roudnicky et al, 2020).…”

“…A shift from the co‐differentiation protocol to direct BCECs generation was achieved by using chemically defined factors (Grifno et al., 2019; Praca et al., 2019; Qian et al., 2017). Recent advancements have been focused on the overexpression of transcription factors in upregulating BBB phenotypes (Roudnicky et al., 2020). Here, Roudnicky et al.…”