Abstract
Current dogma dictates that, during adulthood, endothelial cells (ECs) are locked in an immutable stable homeostatic state. By contrast, herein we show that maintenance of EC fate and function are linked and active processes, which depend on the constitutive cooperativity of only two ETS transcription factors (TFs), ERG and Fli1. Although deletion of either ERG or Fli1 manifests subtle vascular dysfunction, their combined genetic deletion in adult ECs results in acute vasculopathy and multi-organ failure, due to loss of EC fate and integrity, hyperinflammation and spontaneous thrombosis, leading to death. ERG and Fli1 co-deficiency causes rapid transcriptional silencing of pan and organotypic vascular core genes, with dysregulation of inflammation and coagulation pathways. Vascular hyperinflammation leads to impaired hematopoiesis with myeloid skewing. Accordingly, enforced ERG and FLI1 expression in adult human mesenchymal stromal cells activates vascular programs and functionality, enabling in vivo engraftment of a perfusable vascular network. Genome-wide association study analysis identified vascular diseases that are associated with FLI1/ERG mutations. Constitutive expression of ERG and Fli1 upholds EC fate, physiological function and resilience in adult vasculature, whereas their functional loss can contribute to systemic human diseases.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
Data from bulk RNA-seq, ATAC-seq and scRNA-seq have been deposited in the Gene Expression Omnibus under accession number GSE210119. Additional data of this study are included in the main article, supplementary materials and associated source data.
References
Rafii, S., Butler, J. & Ding, B. Angiocrine functions of organ-specific endothelial cells. Nature 529, 316–325 (2016).
Gomez-Salinero, J. M. & Rafii, S. Endothelial cell adaptation in regeneration. Science 362, 1116–1117 (2018).
Nolan, D. et al. Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration. Dev. Cell 26, 204–219 (2013).
Sabbagh, M. et al. Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells. eLife 7, e36187 (2018).
Barry, D. et al. Molecular determinants of nephron vascular specialization in the kidney. Nat. Commun. 10, 5705 (2019).
Ginsberg, M. et al. Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGFβ suppression. Cell 151, 559–575 (2012).
Schachterle, W. et al. Sox17 drives functional engraftment of endothelium converted from non-vascular cells. Nat. Commun. 8, 13963 (2017).
Lu, T. M. et al. Pluripotent stem cell-derived epithelium misidentified as brain microvascular endothelium requires ETS factors to acquire vascular fate. Proc. Natl Acad. Sci. USA 118, e2016950118 (2021).
Birdsey, G. M. et al. Transcription factor Erg regulates angiogenesis and endothelial apoptosis through VE-cadherin. Blood 111, 3498–3506 (2008).
Sperone, A. et al. The transcription factor Erg inhibits vascular inflammation by repressing NF-κB activation and proinflammatory gene expression in endothelial cells. Arterioscler. Thromb. Vasc. Biol. 31, 142–150 (2011).
Birdsey, G. M. et al. The transcription factor Erg regulates expression of histone deacetylase 6 and multiple pathways involved in endothelial cell migration and angiogenesis. Blood 119, 894–903 (2012).
Dryden, N. H. et al. The transcription factor Erg controls endothelial cell quiescence by repressing activity of nuclear factor (NF)-κB p65. J. Biol. Chem. 287, 12331–12342 (2012).
Shah, A. V., Birdsey, G. M. & Randi, A. M. Regulation of endothelial homeostasis, vascular development and angiogenesis by the transcription factor ERG. Vascul. Pharmacol. 86, 3–13 (2016).
Nikolova-Krstevski, V. et al. ERG is required for the differentiation of embryonic stem cells along the endothelial lineage. BMC Dev. Biol. 9, 72 (2009).
Dufton, N. P. et al. Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis. Nat. Commun. 8, 895 (2017).
Shah, A. V. et al. The endothelial transcription factor ERG mediates Angiopoietin-1-dependent control of Notch signalling and vascular stability. Nat. Commun. 8, 16002 (2017).
Birdsey, G. M. et al. The endothelial transcription factor ERG promotes vascular stability and growth through Wnt/β-catenin signaling. Dev. Cell 32, 82–96 (2015).
Peghaire, C. et al. The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature. Nat. Commun. 10, 5014 (2019).
Asano, Y. et al. Endothelial Fli1 deficiency impairs vascular homeostasis: a role in scleroderma vasculopathy. Am. J. Pathol. 176, 1983–1998 (2010).
Lennard Richard, M. L. et al. Acetylation impacts Fli-1-driven regulation of granulocyte colony stimulating factor. Eur. J. Immunol. 46, 2322–2332 (2016).
Lou, N., Lennard Richard, M. L., Yu, J., Kindy, M. & Zhang, X. K. The Fli-1 transcription factor is a critical regulator for controlling the expression of chemokine C-X-C motif ligand 2 (CXCL2). Mol. Immunol. 81, 59–66 (2017).
Looney, A. et al. Synergistic role of endothelial ERG and FLI1 in mediating pulmonary vascular homeostasis. Am. J. Respir. Cell Mol. Biol. 57, 121–131 (2017).
Starck, J. et al. Inducible Fli-1 gene deletion in adult mice modifies several myeloid lineage commitment decisions and accelerates proliferation arrest and terminal erythrocytic differentiation. Blood 116, 4795–4805 (2010).
Wang, Y. et al. Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Nature 465, 483–486 (2010).
Chistiakov, D., Orekhov, A. & Bobryshev, Y. Endothelial barrier and its abnormalities in cardiovascular disease. Front. Physiol. 6, 365 (2015).
Sangwung, P. et al. KLF2 and KLF4 control endothelial identity and vascular integrity. JCI Insight 2, e91700 (2017).
Lu, Y. W. et al. MEF2 (myocyte enhancer factor 2) is essential for endothelial homeostasis and the atheroprotective gene expression program. Arterioscler Thromb Vasc Biol 41, 1105–1123 (2021).
Kalna, V. et al. The transcription factor ERG regulates super-enhancers associated with an endothelial-specific gene expression program. Circ. Res. 124, 1337–1349 (2019).
Fahmy, R. G. et al. Suppression of vascular permeability and inflammation by targeting of the transcription factor c-Jun. Nat. Biotechnol. 24, 856–863 (2006).
Liu, Y., Pelekanakis, K. & Woolkalis, M. J. Thrombin and tumor necrosis factor α synergistically stimulate tissue factor expression in human endothelial cells: regulation through c-Fos and c-Jun. J. Biol. Chem. 279, 36142–36147 (2004).
Gou, B. et al. Single-cell analysis reveals transcriptomic reprogramming in aging cardiovascular endothelial cells. Front. Cardiovasc. Med. 9, 900978 (2022).
Mazzotta, C., et al. FLI1 and ERG protein degradation is regulated via Cathepsin B lysosomal pathway in human dermal microvascular endothelial cells. Microcirculation 28, e12660 (2020).
Hogan, N. et al. Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells. eLife 6, e22536 (2017).
Itkin, T. et al. Distinct bone marrow blood vessels differentially regulate haematopoiesis. Nature 532, 323–328 (2016).
Rohde, D. et al. Bone marrow endothelial dysfunction promotes myeloid cell expansion in cardiovascular disease. Nat. Cardiovasc. Res. 1, 28–44 (2022).
Bergiers, I. et al. Single-cell transcriptomics reveals a new dynamical function of transcription factors during embryonic hematopoiesis. eLife 7, e29312 (2018).
Abedin, M. J. et al. Fli1 acts downstream of Etv2 to govern cell survival and vascular homeostasis via positive autoregulation. Circ. Res. 114, 1690–1699 (2014).
Ricard, N. et al. Endothelial ERK1/2 signaling maintains integrity of the quiescent endothelium. J. Exp. Med. 216, 1874–1890 (2019).
Fish, J. et al. Dynamic regulation of VEGF-inducible genes by an ERK/ERG/p300 transcriptional network. Development 144, 2428–2444 (2017).
Liu, T. et al. A screen for Fli-1 transcriptional modulators identifies PKC agonists that induce erythroid to megakaryocytic differentiation and suppress leukemogenesis. Oncotarget 8, 16728–16743 (2017).
Falkenberg, K. D., Rohlenova, K., Luo, Y. & Carmeliet, P. The metabolic engine of endothelial cells. Nat. Metab. 1, 937–946 (2019).
Xiong, J. et al. A metabolic basis for endothelial-to-mesenchymal transition. Mol. Cell 69, 689–698 (2018).
De Val, S. et al. Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors. Cell 135, 1053–1064 (2008).
Nagai, N. et al. Downregulation of ERG and FLI1 expression in endothelial cells triggers endothelial-to-mesenchymal transition. PLoS Genet. 14, e1007826 (2018).
Poulos, M. et al. Endothelial transplantation rejuvenates aged hematopoietic stem cell function. J. Clin. Invest. 127, 4163–4178 (2017).
Donner, D., Kiriazis, H., Du, X., Marwick, T. & McMullen, J. Improving the quality of preclinical research echocardiography: observations, training, and guidelines for measurement. Am. J. Physiol. Heart. Circ. Physiol. 315, 58–70 (2018).
Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat. Methods 14, 959–962 (2017).
Ewels, P. A. et al. The nf-core framework for community-curated bioinformatics pipelines. Nat. Biotechnol. 38, 276–278 (2020).
Ramírez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).
Bentsen, M. et al. ATAC-seq footprinting unravels kinetics of transcription factor binding during zygotic genome activation. Nat. Commun. 11, 4267 (2020).
Fornes, O. et al. JASPAR 2020: update of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 48, D87–D92 (2020).
Ziemann, M., Kaspi, A. & El-Osta, A. Digital expression explorer 2: a repository of uniformly processed RNA sequencing data. Gigascience 8, giz022 (2019).
Jiang, H., Lei, R., Ding, S. & Zhu, S. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics 15, 182 (2014).
Davis, M., van Dongen, S., Abreu-Goodger, C., Bartonicek, N. & Enright, A. Kraken: a set of tools for quality control and analysis of high-throughput sequence data. Methods 62, 41–49 (2013).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
Mootha, V. K. et al. PGC-1α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat. Genet. 34, 267–273 (2003).
Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).
Kolde, R. Pheatmap: pretty heatmaps. R package version 1. https://scholar.google.com/citations?view_op=view_citation&hl=en&user=IYhbHFMAAAAJ&citation_for_view=IYhbHFMAAAAJ:M3NEmzRMIkIC (2012).
Walter, W., Sánchez-Cabo, F. & Ricote, M. GOplot: an R package for visually combining expression data with functional analysis. Bioinformatics 31, 2912–2914 (2015).
Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411–420 (2018).
Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902 (2019).
Acknowledgements
We would like to thank the Microscopy and Image Analysis Core Facility at Weill Cornell Medicine for taking the TEM images. J.M.G.-S. was a New York Stem Cell Foundation Druckenmiller Fellow and is currently supported by the Weill Cornell Medicine Fund for the Future program and the JumpStart career development program. The New York Stem Cell Foundation, New York State Stem Cell Science and the British Heart Foundation supported this research. S.R. is funded by the Hartman Institute for Therapeutic Organ Regeneration; the Ansary Stem Cell Institute; grants from the National Institutes of Health (R35 HL150809, RC2 DK114777 and U01AI138329); the Empire State Stem Cell Board (C030160); the Daedalus Fund for Innovation; the Selma and Lawrence Ruben Science to Industry Bridge Fund from Weill Cornell Medicine; and the Starr Foundation stem cell core project and initiatives (TRI-SCI 2019-029). A.M.R., G.M.B., N.D. and C.P. were funded by the British Heart Foundation (grant nos. RG/11/17/29256 and RG/17/4/32662). V.K. was funded by the British Heart Foundation PhD Studentship (FS/15/65/32036).
Author information
Authors and Affiliations
Contributions
J.M.G.-S., T.I., A.M.R., C.B. and S.R. designed and initiated the ERG and Fli1 dKO studies. J.M.G.-S., T.I., A.M.R. and S.R. designed and completed experimental studies described in all figures. J.M.G.-S., T.I. and C.B. performed the initial characterization, electron microscopy of the phenotype and blood abnormalities in dKO mice. J.M.G.-S., C.B., T.I. and T.M.L. collected data for in vivo RNA-seq analysis. J.M.G.-S. and T.I. performed data collection for in vitro RNA-seq analysis. J.M.G.-S., Y.L. and G.L. carried out functional characterization of the Fli1, ERG and VE-cadherin CREERT2 mice for EC barrier functions and immunofluorescence analysis. T.I., along with J.M.G.-S. and Y.L., executed the breeding, crossing and phenotypic characterization and analysis of Td-Tomato Fli1, ERG and VE-cadherin CREERT2. S.H., D.R., J.M.G.-S. and T.I. performed bioinformatic analysis of the samples for the bulk RNA-seq analysis and scRNA-seq analysis. J.M.G.-S., along with T.I. and Y.L., performed EC isolation and in vitro characterization and analysis of ECs. T.I. carried out human in vitro and in vivo ERG and FLI1 MSC-EC conversion and murine hematopoiesis relevant studies. G.M.B., V.K., N.D. and C.R.P. assisted with in vitro design and experiments and bioinformatic analysis. M.Y. and M.W. performed and interpreted the echocardiography analysis. J.Z.X. helped in the processing and analyzing of all RNA-seq bulk and single-cell samples. Y.-M.S.H. provided samples and technical support for the in vitro human conversion studies. R.S. carried out acquisition and analysis of time-lapse imaging and provided technical support for the immunofluorescence experiments. J.M.G.-S., T.I., A.M.R., G.M.B. and S.R. wrote, edited, compiled and revised the manuscript. All authors read and approved the final manuscript.
Corresponding authors
Ethics declarations
Competing interests
S.R. is the co-founder and a non-paid consultant to Angiocrine Bioscience.
Peer review
Peer review information
Nature Cardiovascular Research thanks Philip Wenzel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 Endothelial in vivo deletion of ERG is achieved at a multiorgan level.
a-h, Immunofluorescence analysis of ERG (green) expression at day 12 after tamoxifen administration in the endothelium counterstained with VE-cadherin (magenta), Endomucin (Emcn, cyan) and Dapi (white). a-b, brain, c-d, heart, e-f, kidney, g-h, liver. ERG∆ECFli1∆EC dKO mice are represented as dKO. Control WT mice are represented as Ctl. Representative image of n = 5 mice stained in each organ. Bar size = 50 μm.
Extended Data Fig. 2 Endothelial in vivo deletion of Fli1 is achieved at a multiorgan level.
a-h, Immunofluorescence analysis of Fli1 (green) expression at day 12 after tamoxifen administration in the endothelium counterstained with VE-cadherin (magenta), Endomucin (Emcn, cyan) and Dapi (white). a-b, brain, c-d, heart, e-f, kidney, g-h, liver. ERG∆ECFli1∆EC dKO mice are represented as dKO. Control WT mice are represented as Ctl. Representative image of n = 5 mice stained in each organ. Bar size = 50μm.
Extended Data Fig. 3 Endothelial deletion of ERG and Fli1 leads to multiorgan alterations.
a-e, Blood parameters were analyzed in Control and dKO mice 12 days post tamoxifen administration. Hematocrit – HCT (a), Red blood cells – RBC (b), Hemoglobin – HGB (c), Mean corpuscular volume – MCV (d), mean corpuscular hemoglobin concentration – MCHC (e). Student t-test analysis was performed comparing Ctl and dKO mice ±SEM from n=11 Ctl and n=10 dKO mice. f-q, Plasma parameters were measured in Control and dKO mice 12 days post tamoxifen administration. Alanine aminotransferase – ALT (f), Aspartate aminotransferase – AST (g), Alkaline phosphatase – ALP (h), Albumin – ALB (i), Globin – GLOB (j), Albumin / Globin ratio – A/G ratio (k), Blood urea nitrogen – BUN (l), Creatinine – CREA (m), Blood urea nitrogen/ Creatinine ratio – BUN/CREA ratio (n), Lactic acid dehydrogenase – LDH (o), Cholesterol – CHOL (p), Glucose – GLU (q). Student t-test analysis was performed comparing Ctl and dKO mice ±SEM from n=10 mice per group, and n=9 mice in Ctl group of CHOL. r, Brain tissue of Control and dKO mice 12 days post tamoxifen administration injected IV with Evans Blue Dye (EBD). Representative image of n = 5.
Extended Data Fig. 4 Expression of ERG and Fli1 in endothelium is essential for proper cardiac and circulatory function.
a, b, Left ventricular internal dimension in diastole (LVIDd) or systole (LVIDs). c, d, Left ventricular cardiac volume in diastole (LV Vol d) or systole (LV Vol s). e, Ejection fraction, measured as the percentage of ejected blood (EF). Student t-test analysis was performed comparing Ctl and dKO mice ±SEM from n = 6 Ctl mice and 8 dKO mice.
Extended Data Fig. 5 Endothelial deletion of ERG and Fli1 disrupts the vascular program in favor of a maladapted inflammatory phenotype.
a, Endothelial cells from Control (Ctl) and dKO mice were collected at days 7, 10 and 12 following tamoxifen administration as indicated before. Cells were sorted based on CD45neg, CD31+, and VE-cadherin+ markers followed by RNA-seq analysis. b-n, Representation of genes involved in EC signaling pathways based on their expression pattern in dKO versus Ctl ECs. All genes with an FDR < 0.05 were colored based on their Fold induction <±2 or >±2, as indicated in the color legend at the top right. b, Cell membrane and cell junction genes. c, Gap junction genes. d, Vascular permeability genes. e, Tip cell markers. f, Coagulation genes. g, Integrin genes. h, Matrix genes classified in the subfamilies of MMPs, Collagens, Adam family and Crosslinkers. i, Inflammation. j, BMP/TGFβ, k, Lamins. l, Glycolysis. m, Notch. n, Igfbp genes. o, Shear stress. p, Graphical representation of the physiological model based on the alterations observed in dKO versus Ctl ECs.
Extended Data Fig. 6 Deletion of ERG and Fli1 alters chromatin accessibility landscape.
a, Principal component analysis of ATAC-seq analysis of control (Ctl) and dKO mice at day 10 after been treated with tamoxifen as indicated before. b, ATAC-seq distance matrix showing association between subclusters.
Extended Data Fig. 7 In vivo deletion of ERG and Fli1 induces early hematopoietic mobilization and multiorgan myeloid infiltration.
a, BM H&E images of Control and dKO mice 7 days post tamoxifen administration. Representative image of n = 5 mice. Enlarged areas display perivascular megakaryocytes. Scale bar = 100 μm. b, c, Number of PB white blood cells (WBC)(b) and platelets (c) at day 7, as determined by HESKA veterinary hematology system. n = 5 mice. Student t-test analysis was performed comparing Ctl and dKO mice ±SEM. d, Immunofluorescence of VE-cadherin (magenta) and CD45 (cyan) in control dKO mice in liver, heart and kidney. Representative images of n = 5 mice, bar represents 100μm. e-g, Flow cytometry quantification for the frequency of total CD45+ cells (hematopoietic), myeloid (CD45+Gr-1+CD11b+), B-cells (CD45+B220+) or T-cells (CD45+CD3+) present in the heart (e), kidneys (f) and liver (g) of control and dKO mice. n = 5 mice per group. Student t-test analysis was performed comparing Ctl and dKO mice ± SEM.
Extended Data Fig. 8 ERG and FLI1 are associated with diverse cardiovascular disorders in GWAS.
a, b, Analysis of the FinnGen and Uk Biobank GWAS associated pathologies common to both studies with vascular and vascular associated pathologies are labeled. Identification of associated GWAS was performed by expanding ±100 kb of ERG and FLI1 genes and significantly enriched pathologies within the FinnGen were included and compared to the Uk biobank.
Extended Data Fig. 9 ERG and FLI1 cooperatively induce a vascular EC program in non-vascular human MSCs.
a, RNA-seq analysis was performed after 3 weeks in culture for isolated Ctl and ERG and FLI1 MSCs and for cultured HUVECs. Heatmaps of normalized raw counts expression levels of coagulation pathway related genes. n = 4 donors per each MSC group and n = 3 HUVEC donors. b, Schema of experimental design for panels c, d, After 3 weeks of culture control (Ctl) and VE-cadherin+ ERG and FLI1 MSCs were isolated, plated in transwells (8 µM pore size for transmigration assays (c) and 0.4 µM pore size for permeability assays (d)) and in regular culture plate wells, and incubated in EC media until confluency was observed in all culture plate wells. Media in lower well and upper transwell was exchanged for O/N with fresh EC media (control) or with fresh EC media supplemented with both IL1β (10 ng ml−1) and TNFα (10 ng ml−1). c, Following incubation, for migration assays, lower well media was replaced with fresh serum free media supplemented with CXCL12(SDF1) (125 ng ml−1), and upper transwell media was exchanged with serum free media containing isolated CD34+ HSPCs from mobilized peripheral blood donors. n = 3 BM MSC donors and n = 2 HSPC donors. HSPCs were allowed to migrate for 4 hours and then collected for flow analysis to determine frequency of migration in lower well relatively to loaded HSPCs in the upper transwell. Two independent experiments were performed each time with a different HSPC donor. Each mark represents an average of n = 3 technical repeats. Two-way ANOVA statistical analysis was performed comparing all groups. d, For permeability assays, lower well media was replaced with fresh serum free media, and upper transwell media was exchanged with serum free media containing FITC-dextran 70 kDa (1 ng ml−1). n = 3 BM MSC donors. FITC-dextran was allowed to diffuse for 4 hours and then collected for analysis by fluorescent plate reader to determine FITC-dextran concentration in lower well. Two independent experiments were performed. Each mark represents an average of n = 3 technical repeats. Two-way ANOVA statistical analysis was performed comparing all groups. e, Representative stromal monolayer images from n = 3 MSC donors displaying each experimental condition at the time point of beginning of either transmigration or permeability assays, after achieving full confluency. Bars indicate 1000 µm. f, Representative images from n = 4 donors of tube network formation assay 24 h post seeding on top of Matrigel. Ctl and ERG and FLI1 MSCs were isolated after 7 days culture in EC medium. Bars indicate 1000 µm.
Extended Data Fig. 10 Cooperative ERG and Fli1 co-expression is essential for sustaining and induction of endothelial genetic core programs and physiological vascular functions.
a, In vivo deletion of ERG and Fli1 (dKO) induces acute systemic coagulopathy and death in adult mice, associated with a decrease in vascular junctional proteins, EC detachment, increase in vascular leakiness, aberrant expression of cytokines/chemokines and abnormal inflammatory response. b, ERG and Fli1 (in vitro) sustain the expression of the EC core program and ERG and Fli1 combined genetic deletion leads to acquisition of a non-endothelial cell fate which exhibits inadequate response to inflammatory stimulation. c, Overexpression of ERG and FLI1 is sufficient for the induction of a transcriptional vascular EC core programs in human adult non-vascular mesenchymal stromal cells, conferring them with functional vascular tubulogenic network capacity in vitro and in vivo, with the acquisition of an anti-coagulation gene signature, and with the capacity to mount an immune response to facilitate transmigration of hematopoietic cells following inflammatory stimulation.
Supplementary information
Supplementary Tables 1–3
Supplementary Table 1. Endothelial vascular cell enriched list of genes. List of genes enriched in the ECs from the heart, lung, kidney, lung and fat with a fold change of 4.5 and FDR < 0.001. Supplementary Table 2. Comparison of ERG Fli1 deletion days 7, 10 and 12. List of genes enriched with an FDR < 0.05 between control samples and dKO at days 7, 10 and 12. Supplementary Table 3. Comparison of control (Ctl) MSCs to ERG and FLI1-transduced CD31+VE-cadherin+ MSCs.
Supplementary Video 1
ERG and Fli1 are essential for in vitro maintenance of EC morphology. Ctl and ERG∆ECFli1∆EC dKO cells were cultured with tamoxifen for 48 hours to induce Cre activation. Time-lapse analysis shows morphological changes observed in Fig. 7a from day 0 to day 6. At the end, an immunofluorescence analysis of CD31 and DAPI of the same area is presented.
Supplementary Video 2
ERG and FLI1-converted MSCs form tubular structures in fibrin gel. ERG and FLI1-transfected MSCs and HUVECs (n = 3 donors each) were embedded in fibrin gel and introduced into a microfluidics chip device for 3 days. Formed tubes were perfused with anti-human-VE-cadherin antibody (clone BV9, blue color) and imaged. Representative videos display 3D reconstitution of HUVEC-formed blood vessels (left) and ERG and FLI1 MSC-formed tubes (right)
Supplementary Video 3
ERG and FLI1-converted MSCs form tubular structures in fibrin gel with the capacity of blood transport. ERG and FLI1-transfected MSCs (n = 3 donors each) were embedded in fibrin gel and introduced into a microfluidics chip device for 3 days. Formed tubes were perfused with heparinized diluted blood and imaged. Representative video display ×40 magnification of tubes perfused with ×50 diluted blood (Video 3).
Supplementary Video 4
ERG and FLI1-converted MSCs form tubular structures in fibrin gel with the capacity of blood transport. ERG and FLI1-transfected MSCs (n = 3 donors each) were embedded in fibrin gel and introduced into a microfluidics chip device for 3 days. Formed tubes were perfused with heparinized diluted blood and imaged. Representative video display ×5 magnification of tubes perfused with ×5,000 diluted blood.
Supplementary Video 5
ERG and FLI1-converted MSCs establish functional anastomosed vascular blood vessels in vivo after transplantation. Control (Ctl) and ERG and FLI1-transfected cells (n = 4 donors) were lentivirally labeled with mCherry (red color) and cultured for 3 weeks, harvested, embedded in Matrigel and transplanted subcutaneously as plugs into NSG mice. Mice were intravenously injected with anti-human-VE-cadherin antibody (clone BV9, blue color) before sacrifice and plug harvesting. Matrigel plugs were fixed, permeabilized and stained with anti-human-CD31 antibody (green color). Video exhibits 3D reconstitution of plugs embedded with either Ctl (left) or ERG and FLI1-transfected (right) cells.
Supplementary Dataset 1
This file contains the individual genes from each organ used to generate the list of enriched genes in Supplementary Table 1. Each list represents genes that are EC-specific of each organ.
Source data
Source Data Fig. 1
Platelet numbers and EBD data
Source Data Fig. 5
Circulating Td-Tomato+ cells and percentages of ECs in tissues
Source Data Fig. 6
Analysis of BM leakiness and cell populations
Source Data Fig. 7
Flow cytometry analysis of expression of CD31 levels after ERG and Fli1 deletion in mouse ECs in culture
Source Data Fig. 8
Flow cytometry analysis of percentage of CD31 and VE-cadherin+ cells after ERG and FLI1 overexpression
Source Data Extended Data Fig. 3
PB cell parameters and serum analysis
Source Data Extended Data Fig. 4
Echocardiography parameters
Source Data Extended Data Fig. 7
PB data and analysis of hematopoietic infiltrated cells in peripheral tissues by flow cytometry
Source Data Extended Data Fig. 9
Analysis of MSCs and ERG and FLI1 cells response to inflammatory stimulation and leakiness
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Gomez-Salinero, J.M., Itkin, T., Houghton, S. et al. Cooperative ETS transcription factors enforce adult endothelial cell fate and cardiovascular homeostasis. Nat Cardiovasc Res 1, 882–899 (2022). https://doi.org/10.1038/s44161-022-00128-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s44161-022-00128-3
This article is cited by
-
A Notch between vascular morphogenesis and transcriptional identity
Nature Cardiovascular Research (2023)
