Unraveling a novel transcription factor code determining the human arterial-specific endothelial cell signature

Key Points Freshly isolated arterial/venous endothelial cells differ in their gene signature, which is only partially controlled by the Notch pathway. Eight transcription factors codetermine the arterial fingerprint in a complementary and overlapping fashion.

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Regulatory Network Analysis in Estradiol-Treated Human Endothelial Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22158193 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8193

Author(s):

Daniel Pérez-Cremades ◽

Ana B. Paes ◽

Xavier Vidal-Gómez ◽

Ana Mompeón ◽

Carlos Hermenegildo ◽

...

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Transcription Factors ◽

Regulatory Network ◽

Mirna Target ◽

Target Genes ◽

Functional Characterization ◽

Human Endothelial Cells ◽

Mrna Targets ◽

Canonical Pathways

Background/Aims: Estrogen has been reported to have beneficial effects on vascular biology through direct actions on endothelium. Together with transcription factors, miRNAs are the major drivers of gene expression and signaling networks. The objective of this study was to identify a comprehensive regulatory network (miRNA-transcription factor-downstream genes) that controls the transcriptomic changes observed in endothelial cells exposed to estradiol. Methods: miRNA/mRNA interactions were assembled using our previous microarray data of human umbilical vein endothelial cells (HUVEC) treated with 17β-estradiol (E2) (1 nmol/L, 24 h). miRNA–mRNA pairings and their associated canonical pathways were determined using Ingenuity Pathway Analysis software. Transcription factors were identified among the miRNA-regulated genes. Transcription factor downstream target genes were predicted by consensus transcription factor binding sites in the promoter region of E2-regulated genes by using JASPAR and TRANSFAC tools in Enrichr software. Results: miRNA–target pairings were filtered by using differentially expressed miRNAs and mRNAs characterized by a regulatory relationship according to miRNA target prediction databases. The analysis identified 588 miRNA–target interactions between 102 miRNAs and 588 targets. Specifically, 63 upregulated miRNAs interacted with 295 downregulated targets, while 39 downregulated miRNAs were paired with 293 upregulated mRNA targets. Functional characterization of miRNA/mRNA association analysis highlighted hypoxia signaling, integrin, ephrin receptor signaling and regulation of actin-based motility by Rho among the canonical pathways regulated by E2 in HUVEC. Transcription factors and downstream genes analysis revealed eight networks, including those mediated by JUN and REPIN1, which are associated with cadherin binding and cell adhesion molecule binding pathways. Conclusion: This study identifies regulatory networks obtained by integrative microarray analysis and provides additional insights into the way estradiol could regulate endothelial function in human endothelial cells.

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FcγRIIB on liver sinusoidal endothelial cells is essential for antibody-induced GPVI ectodomain shedding in mice

Blood ◽

10.1182/blood-2016-05-714378 ◽

2016 ◽

Vol 128 (6) ◽

pp. 862-865 ◽

Cited By ~ 5

Author(s):

David Stegner ◽

Michael Popp ◽

Viola Lorenz ◽

Jacqueline K. Wax ◽

J. Engelbert Gessner ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Sinusoidal Endothelial Cell ◽

Liver Sinusoidal Endothelial Cell ◽

Ectodomain Shedding ◽

Liver Sinusoidal Endothelial Cells ◽

Sinusoidal Endothelial Cells ◽

Key Points

Key Points Antibody-induced shedding of platelet GPVI in vivo and the associated transient thrombocytopenia depend on liver sinusoidal endothelial cell-expressed FcγRIIB.

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Genome-wide reduction in chromatin accessibility and unique transcription factor footprints in endothelial cells and fibroblasts in scleroderma skin

10.1101/2020.06.24.20138040 ◽

2020 ◽

Author(s):

Pei-Suen Tsou ◽

Pamela J. Palisoc ◽

Mustafa Ali ◽

Dinesh Khanna ◽

Amr H Sawalha

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Transcription Factors ◽

Target Genes ◽

Critical Role ◽

Vascular Complications ◽

Chromatin Accessibility ◽

Unknown Etiology ◽

Healthy Controls ◽

Genome Wide

AbstractSystemic sclerosis (SSc) is a rare autoimmune disease of unknown etiology characterized by widespread fibrosis and vascular complications. We utilized an assay for genome-wide chromatin accessibility to examine the chromatin landscape and transcription factor footprints in both endothelial cells (ECs) and fibroblasts isolated from healthy controls and patients with diffuse cutaneous (dc) SSc. In both cell types, chromatin accessibility was significantly reduced in SSc patients compared to healthy controls. Genes annotated from differentially accessible chromatin regions were enriched in pathways and gene ontologies involved in the nervous system. In addition, our data revealed that chromatin binding of transcription factors SNAI2, ETV2, and ELF1 was significantly increased in dcSSc ECs, while recruitment of RUNX1 and RUNX2 was enriched in dcSSc fibroblasts. Significant elevation of SNAI2 and ETV2 levels in dcSSc ECs, and RUNX2 levels in dcSSc fibroblasts were confirmed. Further analysis of publicly available ETV2-target genes suggests that ETV2 may play a critical role in EC dysfunction in dcSSc. Our data, for the first time, uncovered the chromatin blueprint of dcSSc ECs and fibroblasts, and suggested that neural-related characteristics of SSc ECs and fibroblasts could be a culprit for dysregulated angiogenesis and enhanced fibrosis. Targeting these pathways and the key transcription factors identified might present novel therapeutic approaches for this disease.

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ETS1, ELK1, and ETV4 Transcription Factors Regulate Angiopoietin-1 Signaling and the Angiogenic Response in Endothelial Cells

Frontiers in Physiology ◽

10.3389/fphys.2021.683651 ◽

2021 ◽

Vol 12 ◽

Author(s):

Sharon Harel ◽

Veronica Sanchez ◽

Alaa Moamer ◽

Javier E. Sanchez-Galan ◽

Mohammad N. Abid Hussein ◽

...

Keyword(s):

Endothelial Cells ◽

Transcription Factors ◽

Endothelial Cell ◽

Binding Sites ◽

Umbilical Vein ◽

Intracellular Localization ◽

Mrna Levels ◽

Mobility Shift ◽

Angiogenic Response ◽

Angiopoietin 1

BackgroundAngiopoietin-1 (Ang-1) is the main ligand of Tie-2 receptors. It promotes endothelial cell (EC) survival, migration, and differentiation. Little is known about the transcription factors (TFs) in ECs that are downstream from Tie-2 receptors.ObjectiveThe main objective of this study is to identify the roles of the ETS family of TFs in Ang-1 signaling and the angiogenic response.MethodsIn silico enrichment analyses that were designed to predict TF binding sites of the promotors of eighty-six Ang-1-upregulated genes showed significant enrichment of ETS1, ELK1, and ETV4 binding sites in ECs. Human umbilical vein endothelial cells (HUVECs) were exposed for different time periods to recombinant Ang-1 protein and mRNA levels of ETS1, ELK1, and ETV4 were measured with qPCR and intracellular localization of these transcription factors was assessed with immunofluorescence. Electrophoretic mobility shift assays and reporter assays were used to assess activation of ETS1, ELK1, and ETV4 in response to Ang-1 exposure. The functional roles of these TFs in Ang-1-induced endothelial cell survival, migration, differentiation, and gene regulation were evaluated by using a loss-of-function approach (transfection with siRNA oligos).ResultsAng-1 exposure increased ETS1 mRNA levels but had no effect on ELK1 or ETV4 levels. Immunostaining revealed that in control ECs, ETS1 has nuclear localization whereas ELK1 and ETV4 are localized to the nucleus and the cytosol. Ang-1 exposure increased nuclear intensity of ETS1 protein and enhanced nuclear mobilization of ELK1 and ETV4. Selective siRNA knockdown of ETS1, ELK1, and ETV4 showed that these TFs are required for Ang-1-induced EC survival and differentiation of cells, while ETS1 and ETV4 are required for Ang-1-induced EC migration. Moreover, ETS1, ELK1, and ETV4 knockdown inhibited Ang-1-induced upregulation of thirteen, eight, and nine pro-angiogenesis genes, respectively.ConclusionWe conclude that ETS1, ELK1, and ETV4 transcription factors play significant angiogenic roles in Ang-1 signaling in ECs.

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Uncovering a Distinct Gene Signature in Endothelial Cells Associated With Contrast Enhancement in Glioblastoma

Frontiers in Oncology ◽

10.3389/fonc.2021.683367 ◽

2021 ◽

Vol 11 ◽

Author(s):

Fan Yang ◽

Yuan Xie ◽

Jiefu Tang ◽

Boxuan Liu ◽

Yuancheng Luo ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Vascular Permeability ◽

Contrast Enhancement ◽

Vascular Endothelial Cells ◽

Gene Signature ◽

Special Focus ◽

Vascular Endothelial ◽

Treatment Optimization ◽

Mri Features

PurposeGlioblastoma (GBM) is the most aggressive and lethal type of brain tumors. Magnetic resonance imaging (MRI) has been commonly used for GBM diagnosis. Contrast enhancement (CE) on T1-weighted sequences are presented in nearly all GBM as a result of high vascular permeability in glioblastomas. Although several radiomics studies indicated that CE is associated with distinct molecular signatures in tumors, the effects of vascular endothelial cells, the key component of blood brain barrier (BBB) controlling vascular permeability, on CE have not been thoroughly analyzed.MethodsEndothelial cell enriched genes have been identified using transcriptome data from 128 patients by a systematic method based on correlation analysis. Distinct endothelial cell enriched genes associated with CE were identified by analyzing difference of correlation score between CE-high and CE–low GBM cases. Immunohistochemical staining was performed on in-house patient cohort to validate the selected genes associated with CE. Moreover, a survival analysis was conducted to uncover the relation between CE and patient survival.ResultsWe illustrated that CE is associated with distinct vascular molecular imprints characterized by up-regulation of pro-inflammatory genes and deregulation of BBB related genes. Among them, PLVAP is up-regulated, whereas TJP1 and ABCG2 are down-regulated in the vasculature of GBM with high CE. In addition, we found that the high CE is associated with poor prognosis and GBM mesenchymal subtype.ConclusionWe provide an additional insight to reveal the molecular trait for CE in MRI images with special focus on vascular endothelial cells, linking CE with BBB disruption in the molecular level. This study provides a potential new direction that may be applied for the treatment optimization based on MRI features.

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Hemato-vascular specification requires arnt1 and arnt2 genes in zebrafish embryos

10.1101/2022.01.04.474920 ◽

2022 ◽

Author(s):

Hailey E Edwards ◽

Jaclyn Paige Souder ◽

Daniel A Gorelick

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Transcription Factors ◽

Progenitor Cells ◽

Bhlh Transcription Factor ◽

Zebrafish Embryos ◽

Pas Domain ◽

Hematopoietic Stem ◽

Vascular Progenitor Cells ◽

Bhlh Transcription Factors

During embryonic development, a subset of cells in the mesoderm germ layer are specified as hemato-vascular progenitor cells, which then differentiate into endothelial cells and hematopoietic stem and progenitor cells. In zebrafish, the transcription factor npas4l, also known as cloche, is required for the specification of hemato-vascular progenitor cells. However, it is unclear if npas4l is the sole factor at the top of the hemato-vascular specification cascade. Here we show that arnt1 and arnt2 genes are required for hemato-vascular specification. We found that arnt1;arnt2 double homozygous mutant zebrafish embryos (herein called arnt1/2 mutants), but not arnt1 or arnt2 single mutants, lack blood cells and most vascular endothelial cells. arnt1/2 mutants have reduced or absent expression of etv2 and tal1, the earliest known endothelial and hematopoietic transcription factor genes. npas4l and arnt genes are PAS domain-containing bHLH transcription factors that function as dimers. We found that Npas4l binds both Arnt1 and Arnt2 proteins in vitro, consistent with the idea that PAS domain-containing bHLH transcription factors act in a multimeric complex to regulate gene expression. Our results demonstrate that npas4l, arnt1 and arnt2 act together as master regulators of endothelial and hematopoietic cell fate. Our results also demonstrate that arnt1 and arnt2 act redundantly in a transcriptional complex containing npas4l, but do not act redundantly when interacting with another PAS domain-containing bHLH transcription factor, the aryl hydrocarbon receptor. Altogether, our data enhance our understanding of hemato-vascular specification and the function of PAS domain-containing bHLH transcription factors.

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Abstract 611: AP-1 and ETS Family Transcription Factors Co-localize at Enhancers in Human Aortic Endothelial Cells

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.611 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Nicholas T Hogan ◽

Casey E Romanoski ◽

Michael T Lam ◽

Christopher K Glass

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Transcription Factors ◽

De Novo ◽

Regulatory Elements ◽

Specific Gene ◽

Motif Analysis ◽

Aortic Endothelial Cells ◽

Human Aortic Endothelial Cells ◽

Aortic Endothelial

Introduction: Sequence-specific transcription factors bind DNA regulatory elements and play a key role in establishing cellular identity. Studies comparing macrophages to B cells have revealed that small numbers of such collaborative or lineage-determining transcription factors (LDTF) establish distinct enhancers in each cell type. These factors also allow for the binding of signal dependent transcription factors. Here we present data which suggest members of the AP-1, ETS, and STAT transcription factor families serve as collaborative transcriptional regulators in human aortic endothelial cells (HAEC). Hypothesis: We hypothesize that a set of AP-1 and ETS transcription factors collaborate to establish key endothelial cell enhancers. Methods: Working in HAEC, we measured poised and active enhancers using ChIP-seq for the epigenetic histone modifications H3K4me2 and H3K27Ac, performed motif analysis, and measured transcription factor binding for candidate factors. Knockdowns of JUN, ERG, and STAT3 followed by RNA-seq were used to evaluate altered enhancer function and gene targets of candidate factors. Results: Our de novo motif analysis revealed that motifs for ETS and AP-1 transcription factors are highly enriched at HAEC enhancers. ChIP-seq experiments for JUN, JUNB, ERG, and STAT3 showed between 8,000 and 55,000 intergenic peaks for each factor. Together these peaks bind 50% of poised enhancers, with a subset co-localizing at these sites. Gene ontology analysis showed that gene targets of these enhancers are involved in endothelial-specific functions. Further, knockdown of JUN, ERG, and STAT3 resulted in a twofold or greater change in expression of hundreds of HAEC transcripts. Conclusion: The genome-wide pattern of JUN, JUNB, ERG, and STAT3 co-localization at enhancers in HAEC suggests these factors serve as key regulators that collaboratively modulate endothelial-specific gene expression. Further investigation of candidate lineage-determining transcription factors using pro-atherogenic signals could reveal regulatory mechanisms of disease-relevant endothelial transcriptional programs.

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Abstract 547: TGFß Response Coupled to Stress Signaling by TGRL Lipolysis in Vascular Endothelial Cells

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a547 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Hnin Aung ◽

Larissa Eiselein ◽

Michael W Lamé ◽

Kit Ng ◽

Dennis W Wilson ◽

...

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Endothelial Cell ◽

Map Kinases ◽

Gene Array ◽

Receptor Activation ◽

Type I ◽

Tgfβ Signaling ◽

Tgfβ Receptor ◽

Activating Transcription Factor

Elevation of circulating triglyceride-rich lipoproteins (TGRL) in the postprandial state is associated with increased endothelial cell inflammation and dysfunction potentially contributing to atherosclerosis. Previous studies from our laboratory showed that lipolysis products of TGRL induced endothelial cell barrier permeability, up-regulation of cytokines and adhesion molecules, and apoptosis. Gene array studies implicated the Stress Associated Protein Kinase and c-Jun N-terminal kinase (SAPK/JNK) pathway and identified the transcription factor activating transcription factor 3 (ATF3) as highly induced in this process. Additional studies demonstrated activation of second messengers for transforming growth factor beta (TGFβ) signaling (SMAD2/SMAD4) in response to lipolysis product treatment. In the present study, we asked whether TGFβ signaling was implicated in the induction of apoptosis by lipolysis products and whether there was a linkage between TGFβ receptor activation and pro-inflammatory responses. siRNA knockdown of ATF3 transcription abrogated both IL-8 and E-selectin up-regulation in lipolysis product-treated human aortic endothelial cells (HAEC). In addition, ATF3 knockdown prevented activation of caspase 3/7 and induction of p53, markers of apoptosis. Inhibition of the TGFβ Type I activin receptor-like kinase (ALK) receptors (ALKs 4, 5 and 7) by SB 431542 (10 μM) also inhibited inflammatory up-regulation, ATF3 induction and apoptosis. Both ATF3 knockdown and TGFβ receptor blockade prevented c-Jun phosphorylation, a co-activating transcription factor with ATF3 of AP-1 transcription sites. Our findings implicate TGFβ signaling in the inflammatory and apoptotic responses to TGRL lipolysis and suggest a complex interaction between TGFβ receptors and the SAPK subset of MAP Kinases.

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Abstract 267: Opposing Functions of Two Splice Variants of the Human Transcription Factor Grainyhead-like 3 in Endothelial Cells And in vivo

Circulation Research ◽

10.1161/res.113.suppl_1.a267 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Joachim Altschmied ◽

Nicole Büchner ◽

Sascha Jakob ◽

Sabrina Farrokh ◽

Christine Goy ◽

...

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Transcription Factors ◽

Target Genes ◽

Splice Variants ◽

Luciferase Reporter ◽

Dependent Manner ◽

Active Transcription ◽

Human Transcription Factor

Grainyhead-like 3 (GRHL3) is a member of the evolutionary conserved Grainyhead family of transcription factors. In humans, three isoforms are derived from differential first exon usage and alternative splicing, which differ only in their N-terminus. Isoform 2, the only variant also present in mouse, is required for endothelial cell (EC) migration and protects against apoptosis. The functions of the human specific isoforms 1 and 3, which are derived from an alternatively spliced pre-mRNA, have not yet been investigated, although all three isoforms are expressed in EC. Therefore, we have assessed their effects on EC migration and apoptosis. Overexpression of the two proteins had opposite effects on EC migration, with isoform 1 acting pro-migratory. This protein also protected EC against apoptosis in an eNOS-dependent manner, whereas isoform 3 had no effect. These opposing outcomes with respect to apoptosis EC were corroborated by isoform-specific knockdowns. With reporter assays using a GRHL3-specific luciferase reporter we demonstrated that both are active transcription factors. Microarray analyses revealed that they induce divergent target gene sets in EC. Two validated targets, Akt2 and Mxi1, which are upregulated by isoform1, are regulators of Akt1-, and thus eNOS-phosphorylation and apoptosis, which could explain the effects of this protein on these processes. In vivo, overexpression of isoform 3 in zebrafish embryos resulted in increased lethality and severe deformations, while isoform 1 had no deleterious effect. In conclusion, our data demonstrate that the splice variant derived isoforms 1 and 3 of the human transcription factor GRHL3 induce opposing effects in primary human endothelial cells and in a whole animal model, most likely through the induction of different target genes.

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Regulation of Tissue Factor by NF-kB Transcription Factor p50 Is Essential for the Pathogeneses of Deep Vein Thrombosis and Arterial Restenosis.

Blood ◽

10.1182/blood.v108.11.1458.1458 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1458-1458

Author(s):

Jian-Guo Geng ◽

Jian-Guo Wang ◽

Nigel Mackman ◽

Arne Slungaard ◽

Yuqing Huo ◽

...

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Transcription Factors ◽

Deep Vein Thrombosis ◽

Venous Thrombosis ◽

Tissue Factor ◽

Coagulation Cascade ◽

Vein Thrombosis ◽

Arterial Restenosis ◽

Deep Vein

Abstract NF-kB transcription factors regulate the expression of tissue factor (TF), a principal initiator for the coagulation cascade. Dominant among the five cellular members of NF-kB transcription factors is the p50/p65 heterodimer. Here we report that Andrographolide (Andro; a 350-dalton antagonist that targets reduced cysteine62 of p50 for inhibition of NF-kB activation) and genetic deletion of p50 potently attenuated TF activity in stimulated endothelial cells and monocytes/macrophages. The direct binding of p50/p65 heterodimer to the TF-kB site in human TF promoter was demonstrated by p50 and p65 antibody ‘supershift’ using electrophoretic mobility shift assay and immunoprecipitation of the promoter of the human TF gene from chromatins of TNF-a-stimulated human umbilical vein endothelial cells. Andro-treated and p50 null mice both exhibited suppressed TF expression, blunted fibrin deposition, reduced venous thrombosis, and decreased neointimal hyperplasia. Blockade of TF activity by an anti-murine TF antibody also attenuated venous thrombosis and neointimal proliferation in vivo. Our findings thus indicate that NF-kB transcription factor p50 critically regulates TF activity in the pathogeneses of deep vein thrombosis and arterial restenosis, and suggest that specific inhibitors of p50, such as Andro, have the potential to be therapeutically valuable for preventing and perhaps treating arterial and venous thrombosis.

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