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• W3014477614 abstract "•Single-cell RNA-seq reveals EC heterogeneity in choroidal neovascularization•ECs display metabolic transcriptome heterogeneity in the cell cycle and quiescence•Data integration with a genome-scale metabolic model identifies angiogenic targets•SQLE and ALDH18A1 are validated as metabolic angiogenic candidates Endothelial cell (EC) metabolism is an emerging target for anti-angiogenic therapy in tumor angiogenesis and choroidal neovascularization (CNV), but little is known about individual EC metabolic transcriptomes. By single-cell RNA sequencing 28,337 murine choroidal ECs (CECs) and sprouting CNV-ECs, we constructed a taxonomy to characterize their heterogeneity. Comparison with murine lung tumor ECs (TECs) revealed congruent marker gene expression by distinct EC phenotypes across tissues and diseases, suggesting similar angiogenic mechanisms. Trajectory inference predicted that differentiation of venous to angiogenic ECs was accompanied by metabolic transcriptome plasticity. ECs displayed metabolic transcriptome heterogeneity during cell-cycle progression and in quiescence. Hypothesizing that conserved genes are important, we used an integrated analysis, based on congruent transcriptome analysis, CEC-tailored genome-scale metabolic modeling, and gene expression meta-analysis in cross-species datasets, followed by in vitro and in vivo validation, to identify SQLE and ALDH18A1 as previously unknown metabolic angiogenic targets. Endothelial cell (EC) metabolism is an emerging target for anti-angiogenic therapy in tumor angiogenesis and choroidal neovascularization (CNV), but little is known about individual EC metabolic transcriptomes. By single-cell RNA sequencing 28,337 murine choroidal ECs (CECs) and sprouting CNV-ECs, we constructed a taxonomy to characterize their heterogeneity. Comparison with murine lung tumor ECs (TECs) revealed congruent marker gene expression by distinct EC phenotypes across tissues and diseases, suggesting similar angiogenic mechanisms. Trajectory inference predicted that differentiation of venous to angiogenic ECs was accompanied by metabolic transcriptome plasticity. ECs displayed metabolic transcriptome heterogeneity during cell-cycle progression and in quiescence. Hypothesizing that conserved genes are important, we used an integrated analysis, based on congruent transcriptome analysis, CEC-tailored genome-scale metabolic modeling, and gene expression meta-analysis in cross-species datasets, followed by in vitro and in vivo validation, to identify SQLE and ALDH18A1 as previously unknown metabolic angiogenic targets. Targeting the metabolism of endothelial cells (ECs) is a promising strategy to block pathological blood vessel growth, or angiogenesis, for the treatment of diseases like cancer. Understanding the landscape of metabolic gene expression at the single-cell level will aid in identifying novel angiogenic targets. Here, researchers in Belgium and their colleagues surveyed thousands of ECs in pre-clinical models of age-related macular degeneration and lung cancer. Their comprehensive investigation identified genes and metabolic pathways that are congruently upregulated across diseases and tissues during angiogenesis. Using an integrated analysis, the researchers generated a list of prioritized metabolic candidates and validated the importance of two candidates, SQLE and ALDH18A1, in pathological angiogenesis,supportingtheir potential as therapeutic targets. Endothelial cell (EC) metabolism regulates angiogenesis, and is an emerging target for anti-angiogenic therapy (AAT) in cancer and wet age-related macular degeneration (AMD) (Eelen et al., 2018Eelen G. de Zeeuw P. Treps L. Harjes U. Wong B.W. Carmeliet P. Endothelial cell metabolism.Physiol. Rev. 2018; 98: 3-58Crossref PubMed Scopus (55) Google Scholar). The design of new AATs by targeting EC metabolism would benefit from a better understanding of individual EC metabolism, but it remains unknown if ECs express a heterogeneous metabolic gene signature and how single ECs reprogram their metabolic transcriptome signature when forming new vessels in disease. However, metabolomics (measuring metabolite levels or metabolic fluxes) is insufficiently sensitive to determine single EC metabolism. Since we documented that changes in metabolic gene expression signatures at the bulk population level can be predictive of changes in metabolism in ECs (Bruning et al., 2018Bruning U. Morales-Rodriguez F. Kalucka J. Goveia J. Taverna F. Queiroz K.C.S. Dubois C. Cantelmo A.R. Chen R. Loroch S. et al.Impairment of angiogenesis by fatty acid synthase inhibition involves mTOR malonylation.Cell Metab. 2018; 28: 866-880.e15Abstract Full Text Full Text PDF PubMed Google Scholar, Cantelmo et al., 2016Cantelmo A.R. Conradi L.C. Brajic A. Goveia J. Kalucka J. Pircher A. Chaturvedi P. Hol J. Thienpont B. Teuwen L.A. et al.Inhibition of the glycolytic activator PFKFB3 in endothelium induces tumor vessel normalization, impairs metastasis, and improves chemotherapy.Cancer Cell. 2016; 30: 968-985Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, Kalucka et al., 2018Kalucka J. Bierhansl L. Conchinha N.V. Missiaen R. Elia I. Brüning U. Scheinok S. Treps L. Cantelmo A.R. Dubois C. et al.Quiescent endothelial cells upregulate fatty acid β-oxidation for vasculoprotection via redox homeostasis.Cell Metab. 2018; 28: 881-894.e13Abstract Full Text Full Text PDF PubMed Google Scholar, Vandekeere et al., 2018Vandekeere S. Dubois C. Kalucka J. Sullivan M.R. García-Caballero M. Goveia J. Chen R. Diehl F.F. Bar-Lev L. Souffreau J. et al.Serine synthesis via PHGDH is essential for heme production in endothelial cells.Cell Metab. 2018; 28: 573-587.e13Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), we analyzed the metabolic transcriptome of ECs at the single-cell level. During vessel sprouting, a navigating tip EC leads the way, while proliferating stalk cells elongate the vessel sprout (Potente et al., 2011Potente M. Gerhardt H. Carmeliet P. Basic and therapeutic aspects of angiogenesis.Cell. 2011; 146: 873-887Abstract Full Text Full Text PDF PubMed Scopus (1306) Google Scholar); once newly formed vessels become perfused, ECs adopt a quiescent phalanx phenotype (Welti et al., 2013Welti J. Loges S. Dimmeler S. Carmeliet P. Recent molecular discoveries in angiogenesis and antiangiogenic therapies in cancer.J. Clin. Invest. 2013; 123: 3190-3200Crossref PubMed Scopus (348) Google Scholar). ECs rely on metabolic reprogramming when switching from quiescence to vessel sprouting (Eelen et al., 2018Eelen G. de Zeeuw P. Treps L. Harjes U. Wong B.W. Carmeliet P. Endothelial cell metabolism.Physiol. Rev. 2018; 98: 3-58Crossref PubMed Scopus (55) Google Scholar, Li et al., 2019Li X. Kumar A. Carmeliet P. Metabolic pathways fueling the endothelial cell drive.Annu. Rev. Physiol. 2019; 81: 483-503Crossref PubMed Scopus (3) Google Scholar, Sawada and Arany, 2017Sawada N. Arany Z. Metabolic regulation of angiogenesis in diabetes and aging.Physiology (Bethesda). 2017; 32: 290-307PubMed Google Scholar, Yu et al., 2018Yu P. Alves T.C. Kibbey R.G. Simons M. Metabolic analysis of lymphatic endothelial cells.Methods Mol. Biol. 2018; 1846: 325-334Crossref PubMed Scopus (0) Google Scholar). In tumors, bulk metabolic gene expression profiling identified metabolic targets in tumor ECs (Cantelmo et al., 2016Cantelmo A.R. Conradi L.C. Brajic A. Goveia J. Kalucka J. Pircher A. Chaturvedi P. Hol J. Thienpont B. Teuwen L.A. et al.Inhibition of the glycolytic activator PFKFB3 in endothelium induces tumor vessel normalization, impairs metastasis, and improves chemotherapy.Cancer Cell. 2016; 30: 968-985Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). AMD is a common blinding disease of elderly people, characterized by ocular neovascularization. Laser-induced choroid neovascularization (CNV) is a preclinical model of AMD (Ambati and Fowler, 2012Ambati J. Fowler B.J. Mechanisms of age-related macular degeneration.Neuron. 2012; 75: 26-39Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). Since angiogenic ECs in AMD/CNV have not been studied at the single-cell level, we used single-cell RNA sequencing (scRNA-seq) to profile their (metabolic) transcriptome heterogeneity. Anti-VEGF drugs are used for the treatment of cancer and AMD, but resistance limits their efficacy (Jain, 2014Jain R.K. Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia.Cancer Cell. 2014; 26: 605-622Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar, Yang et al., 2016Yang S. Zhao J. Sun X. Resistance to anti-VEGF therapy in neovascular age-related macular degeneration: a comprehensive review.Drug Des. Devel. Ther. 2016; 10: 1857-1867Crossref PubMed Scopus (9) Google Scholar). Hence, there is an unmet clinical need to identify novel angiogenic targets. scRNA-seq is a powerful technology to identify such candidates, but an outstanding challenge is to prioritize targets for further clinical translation. Here, we present a strategy, starting from scRNA-seq and complemented with orthogonal techniques, to prioritize metabolic targets that control angiogenesis. To model CNV in mice, we laser-induced 10 lesions per eye and microdissected choroids 7 days later. We pooled choroids from 6 mice and repeated this procedure 3 times, using choroids from healthy mice as controls (6 mice per sample, in triplicate) (Figures 1A and 1B ). For comparative analysis, we generated a pooled sample of two choroids from one healthy human donor (see below). Single-cell suspensions were MACS-enriched for CD45−/CD31+ ECs (Cantelmo et al., 2016Cantelmo A.R. Conradi L.C. Brajic A. Goveia J. Kalucka J. Pircher A. Chaturvedi P. Hol J. Thienpont B. Teuwen L.A. et al.Inhibition of the glycolytic activator PFKFB3 in endothelium induces tumor vessel normalization, impairs metastasis, and improves chemotherapy.Cancer Cell. 2016; 30: 968-985Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) and subjected to scRNA-seq. After quality filtering (Table S1), batch correction, and in silico EC selection, graph-based clustering was performed to group a total of 28,337 ECs according to their gene expression profile. Clusters were annotated based on marker genes (Tables S2 and S3) and results were visualized using t-distributed stochastic neighbor embedding (t-SNE) (Figures 1C, 1D, S1A, and S1B). CECs from control mice were indistinguishable from healthy peripheral CECs from lasered mice and clustered together (Figures 1C–1F). We detected a new separate population in lasered mice, not present in healthy CECs, representing CNV-ECs (Figure 1D). Compared to healthy CECs, CNV-ECs expressed activation markers associated with response to injury such as Sparc (Bradshaw and Sage, 2001Bradshaw A.D. Sage E.H. SPARC, a matricellular protein that functions in cellular differentiation and tissue response to injury.J. Clin. Invest. 2001; 107: 1049-1054Crossref PubMed Google Scholar) and Col18a1, a source of the angiostatic endostatin, previously used for CNV treatment (Marneros et al., 2007Marneros A.G. She H. Zambarakji H. Hashizume H. Connolly E.J. Kim I. Gragoudas E.S. Miller J.W. Olsen B.R. Endogenous endostatin inhibits choroidal neovascularization.FASEB J. 2007; 21: 3809-3818Crossref PubMed Scopus (0) Google Scholar), a finding confirmed at the protein level by quantitative mass cytometry (CyTOF) (Figures 1G, S1C, and S1D; Table S2). In CECs, we identified previously unknown sublineages of the classical arterial, capillary, and venous EC phenotypes (Figures 1E–1G; for more complete description of marker genes and putative inferred biological activity, see Table S3). For instance, we identified large supply arteries (P1 on Figure 1D), smaller ramifying arterioles (P2) and arterial ECs expressing the shear stress marker (Pi16) (tentatively coined shear-stress induced arterial ECs [P3]), and a laser-induced arterial subpopulation (activated arterial CEC) that upregulated activation markers and matricellular proteins (P4) (Figure 1H). Activated arterial CECs clustered together with other arterial phenotypes (Figure 1D), suggesting a relatively normal transcriptome. Capillary CEC phenotypes expressed signatures of the outer (P5) and inner choriocapillaries (P6), characterized by the differential expression of genes involved in fenestration and VEGF signaling (Blaauwgeers et al., 1999Blaauwgeers H.G. Holtkamp G.M. Rutten H. Witmer A.N. Koolwijk P. Partanen T.A. Alitalo K. Kroon M.E. Kijlstra A. van Hinsbergh V.W. Schlingemann R.O. Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris. Evidence for a trophic paracrine relation.Am. J. Pathol. 1999; 155: 421-428Abstract Full Text Full Text PDF PubMed Google Scholar, McLeod et al., 1995McLeod D.S. Lefer D.J. Merges C. Lutty G.A. Enhanced expression of intracellular adhesion molecule-1 and P-selectin in the diabetic human retina and choroid.Am. J. Pathol. 1995; 147: 642-653PubMed Google Scholar) (Table S2). Venous subclusters included cells expressing markers of large caliber vessels (P7), venules (P8), shear stress (P9), post-capillary venules (pcvs) (P10) that upregulated a previously identified CEC signature of resident endothelial stem cells (ESCs) (Naito et al., 2012Naito H. Kidoya H. Sakimoto S. Wakabayashi T. Takakura N. Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels.EMBO J. 2012; 31: 842-855Crossref PubMed Scopus (88) Google Scholar, Wakabayashi et al., 2013Wakabayashi T. Naito H. Takara K. Kidoya H. Sakimoto S. Oshima Y. Nishida K. Takakura N. Identification of vascular endothelial side population cells in the choroidal vessels and their potential role in age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 2013; 54: 6686-6693Crossref PubMed Scopus (9) Google Scholar), and an activated pcv CEC phenotype (p11) (Figures 1I and S1E). We observed two putative lymphatic EC phenotypes (LEC [P12] and LEC-like [P13]) that differentially expressed Lyve-1 (Figure 1D; Table S2). The existence of lymphatics in the choroid remains debated (Heindl et al., 2015Heindl L.M. Kaser-Eichberger A. Schlereth S.L. Bock F. Regenfuss B. Reitsamer H.A. McMenamin P. Lutty G.A. Maruyama K. Chen L. et al.Sufficient evidence for lymphatics in the developing and adult human choroid?.Invest. Ophthalmol. Vis. Sci. 2015; 56: 6709-6710Crossref PubMed Scopus (8) Google Scholar, Koina et al., 2015Koina M.E. Baxter L. Adamson S.J. Arfuso F. Hu P. Madigan M.C. Chan-Ling T. Evidence for lymphatics in the developing and adult human choroid.Invest. Ophthalmol. Vis. Sci. 2015; 56: 1310-1327Crossref PubMed Scopus (31) Google Scholar). Angiogenic CNV-ECs were distinct from normal CECs and included proliferating ECs (C1 in Figure 1D) and tip ECs (C2), but also 3 previously unknown phenotypes that expressed signatures associated with transitioning from pcv to angiogenic EC phenotypes (transitioning CNV-ECs [C3]), and immature (immature [C4]) and maturing (neophalanx [C5]) neovasculature (Figure 1J). Tip cells upregulated transcripts of the disease-restricted angiogenic factor Pgf (encoding placental growth factor, Plgf) (Figures 1J and S1F). Immature ECs were characterized by the lack of specific marker gene expression, but expressed activation markers and upregulated ribosomal gene expression consistent with an activated intermediate phenotype. Neophalanx ECs expressed markers of mature capillaries and arteries, and were characterized by upregulation of a Notch signaling gene signature (Figure S1G). Interestingly, transcription factor activity analysis using single-cell regulatory network inference and clustering (SCENIC) (Aibar et al., 2017Aibar S. González-Blas C.B. Moerman T. Huynh-Thu V.A. Imrichova H. Hulselmans G. Rambow F. Marine J.C. Geurts P. Aerts J. et al.SCENIC: single-cell regulatory network inference and clustering.Nat. Methods. 2017; 14: 1083-1086Crossref PubMed Scopus (168) Google Scholar) indicated differential transcription factor activity in EC subtypes (Figure 2A). Consistent with previous reports, Nr2f2 expression was induced in activated pcvs and transitioning ECs (Jeong et al., 2017Jeong H.W. Hernández-Rodríguez B. Kim J. Kim K.P. Enriquez-Gasca R. Yoon J. Adams S. Schöler H.R. Vaquerizas J.M. Adams R.H. Transcriptional regulation of endothelial cell behavior during sprouting angiogenesis.Nat. Commun. 2017; 8: 726Crossref PubMed Scopus (20) Google Scholar), while Sox17 expression was highest in arterial ECs (Corada et al., 2013Corada M. Orsenigo F. Morini M.F. Pitulescu M.E. Bhat G. Nyqvist D. Breviario F. Conti V. Briot A. Iruela-Arispe M.L. et al.Sox17 is indispensable for acquisition and maintenance of arterial identity.Nat. Commun. 2013; 4: 2609Crossref PubMed Scopus (111) Google Scholar, You et al., 2005You L.R. Lin F.J. Lee C.T. DeMayo F.J. Tsai M.J. Tsai S.Y. Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity.Nature. 2005; 435: 98-104Crossref PubMed Scopus (387) Google Scholar). SCENIC analysis of CNV-ECs also identified transcription factors not previously implicated in EC specification, such as in tip (Tgif1), immature (Smad1 and Sox4), and proliferating (Trp53) CNV-ECs (Figure 2A). We validated the taxonomy using orthogonal in situ localization techniques. Quantitative RNAscope to count transcript numbers, combined with staining for the EC marker CD105, confirmed that arterial (Gja4) and venous (Nr2f2) marker transcripts did not colocalize in the same CNV-ECs (Figure S1H). We confirmed by immunostaining of healthy choroids the expression of the following EC markers: (1) artery ECs (ELN) and arteriole ECs (CXCL12) (Figures S2A–S2C), (2) capillary ECs (VEGFR2) (Figure S2D), and (3) venous ECs (VWF and SELP) (Figures S2E and S2F). Immunostaining of CNV lesions confirmed the expression of a marker of immature ECs (APLNR), tip ECs (PlGF, LXN, and CXCR4), and pcv ECs (SPARCL1) (Figures S2G–S2J). We explored if ECs underwent a differentiation trajectory during vessel sprouting and if EC differentiation was associated with metabolic transcriptome changes. Trajectory inference analysis predicted that the hierarchy of angiogenic phenotypes resulted from differentiation of activated pcv CECs to transitioning CNV-ECs, then to immature CNV-ECs, which thereafter differentiated to tip cells and finally to more mature neophalanx CNV-ECs (Figure 2B). This prediction extends previous morphological evidence that neovessels may originate from pcvs (Folkman, 1982Folkman J. Angiogenesis: initiation and control.Ann. N Y Acad. Sci. 1982; 401: 212-227Crossref PubMed Google Scholar). Since pcv CECs expressed a previously validated signature of resident ESCs, our analysis provides further suggestion that ESCs might contribute to new vessel sprouting, as previously established by lineage tracing (Corey et al., 2016Corey D.M. Rinkevich Y. Weissman I.L. Dynamic patterns of clonal evolution in tumor vasculature underlie alterations in lymphocyte-endothelial recognition to foster tumor immune escape.Cancer Res. 2016; 76: 1348-1353Crossref PubMed Scopus (11) Google Scholar, Manavski et al., 2018Manavski Y. Lucas T. Glaser S.F. Dorsheimer L. Günther S. Braun T. Rieger M.A. Zeiher A.M. Boon R.A. Dimmeler S. Clonal expansion of endothelial cells contributes to ischemia-induced neovascularization.Circ. Res. 2018; 122: 670-677Crossref PubMed Scopus (21) Google Scholar, McDonald et al., 2018McDonald A.I. Shirali A.S. Aragón R. Ma F. Hernandez G. Vaughn D.A. Mack J.J. Lim T.Y. Sunshine H. Zhao P. et al.Endothelial regeneration of large vessels is a biphasic process driven by local cells with distinct proliferative capacities.Cell Stem Cell. 2018; 23: 210-225.e6Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, Mondor et al., 2016Mondor I. Jorquera A. Sene C. Adriouch S. Adams R.H. Zhou B. Wienert S. Klauschen F. Bajénoff M. Clonal proliferation and stochastic pruning orchestrate lymph node vasculature remodeling.Immunity. 2016; 45: 877-888Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, Red-Horse et al., 2010Red-Horse K. Ueno H. Weissman I.L. Krasnow M.A. Coronary arteries form by developmental reprogramming of venous cells.Nature. 2010; 464: 549-553Crossref PubMed Scopus (314) Google Scholar, Wakabayashi et al., 2013Wakabayashi T. Naito H. Takara K. Kidoya H. Sakimoto S. Oshima Y. Nishida K. Takakura N. Identification of vascular endothelial side population cells in the choroidal vessels and their potential role in age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 2013; 54: 6686-6693Crossref PubMed Scopus (9) Google Scholar, Wakabayashi et al., 2018Wakabayashi T. Naito H. Suehiro J.I. Lin Y. Kawaji H. Iba T. Kouno T. Ishikawa-Kato S. Furuno M. Takara K. et al.CD157 marks tissue-resident endothelial stem cells with homeostatic and regenerative properties.Cell Stem Cell. 2018; 22: 384-397.e6Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Interestingly, when focusing on metabolic genes and pathways, we noted that membrane transport, ATP synthase, and glycolysis gene signatures were dynamically regulated during differentiation from quiescent vein to angiogenic ECs (Figure 2B). Maximal differences in metabolic gene expression of central carbon metabolism were observed in the most angiogenic EC phenotypes (immature and tip ECs), possibly suggesting that these ECs had higher metabolic demands to execute their biological functions (Figure 2B). We explored whether metabolic transcriptome reprogramming was specific to CNV-ECs or a more general hallmark of the angiogenic switch in pathological angiogenesis (such as in tumors), as this would address a fundamental question in vascular biology of whether vessels in different tissues and diseases form via similar or different mechanisms. We therefore explored to which extent CNV and tumors contained similar EC phenotypes, and whether they expressed congruent genes. We analyzed a publicly available, previously in-house generated dataset of murine lung tumor ECs (TECs) (Goveia et al., 2020Goveia J. Rohlenova K. Taverna F. Treps L. Conradi L.C. Pircher A. Geldhof V. de Rooij L.P.M.H. Kalucka J. Sokol L. et al.An integrated gene expression landscape profiling approach to identify lung tumor endothelial cell heterogeneity and angiogenic candidates.Cancer Cell. 2020; 37: 21-36.e13Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar), which comprised largely similar EC phenotypes as CNV-ECs. However, in addition, murine lung TECs contained breach and pre-breach ECs (that expressed both tip cell and podosome rosette markers, presumably involved in vessel sprouting initiation), and interferon ECs (displaying a transcriptome response to interferon, possibly involved in immune surveillance) (Goveia et al., 2020Goveia J. Rohlenova K. Taverna F. Treps L. Conradi L.C. Pircher A. Geldhof V. de Rooij L.P.M.H. Kalucka J. Sokol L. et al.An integrated gene expression landscape profiling approach to identify lung tumor endothelial cell heterogeneity and angiogenic candidates.Cancer Cell. 2020; 37: 21-36.e13Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). Focusing on all detected genes, we explored whether similar EC phenotypes could be detected in these diseases, and whether they expressed congruent genes. We performed differential gene expression and gene set enrichment analysis to determine which processes were upregulated in CNV-ECs and TECs versus CECs and NECs, respectively (Figures 2C and 2D). Gene sets associated with proliferation, hypoxia signaling, and extracellular matrix formation were commonly upregulated (Figure 2C). Consistently, many of the 175 commonly upregulated genes were involved in extracellular matrix remodeling, cytoskeleton, glycolysis, EC activation, and others. Interestingly, Aplnr, an angiogenic and vasculoprotective gene that regulates EC metabolism (Apostolidis et al., 2018Apostolidis S.A. Stifano G. Tabib T. Rice L.M. Morse C.M. Kahaleh B. Lafyatis R. Single cell RNA sequencing identifies HSPG2 and APLNR as markers of endothelial cell injury in systemic sclerosis skin.Front. Immunol. 2018; 9: 2191Crossref PubMed Scopus (1) Google Scholar, Hwangbo et al., 2017Hwangbo C. Wu J. Papangeli I. Adachi T. Sharma B. Park S. Zhao L. Ju H. Go G.W. Cui G. et al.Endothelial APLNR regulates tissue fatty acid uptake and is essential for apelin’s glucose-lowering effects.Sci. Transl. Med. 2017; 9https://doi.org/10.1126/scitranslmed.aad4000Crossref PubMed Scopus (15) Google Scholar), was identified as a congruent marker of CNV-ECs and TECs (Figure 2D). Since differential analysis of pooled populations may not adequately discover genes with restricted expression in small EC subpopulations such as tip and proliferating cells, we determined whether the same EC subpopulations were present in CNV-ECs and TECs. We used the Jaccard similarity index to score the similarity of marker gene sets of all EC subpopulations, and observed that marker gene sets across CNV-ECs and TECs were relatively similar for several EC subpopulations (Figures 2E and S3A). Further, TECs and CNV-ECs of the same phenotype expressed congruent marker genes (Figure 2F). Similar to CNV-ECs, trajectory inference analysis predicted that the hierarchy of TEC phenotypes originated in veins that expressed resident ESC markers (Goveia et al., 2020Goveia J. Rohlenova K. Taverna F. Treps L. Conradi L.C. Pircher A. Geldhof V. de Rooij L.P.M.H. Kalucka J. Sokol L. et al.An integrated gene expression landscape profiling approach to identify lung tumor endothelial cell heterogeneity and angiogenic candidates.Cancer Cell. 2020; 37: 21-36.e13Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, Wakabayashi et al., 2018Wakabayashi T. Naito H. Suehiro J.I. Lin Y. Kawaji H. Iba T. Kouno T. Ishikawa-Kato S. Furuno M. Takara K. et al.CD157 marks tissue-resident endothelial stem cells with homeostatic and regenerative properties.Cell Stem Cell. 2018; 22: 384-397.e6Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) differentiating to postcapillary veins and further to an immature TEC phenotype, tip cells, neophalanx TECs, and activated arteries (Figure S3B). Focusing on metabolic genes, we observed that proliferating ECs in both disease models upregulated the expression of metabolic genes involved in one-carbon metabolism, nucleotide synthesis, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS) (Figure 3A). In contrast, glycolytic gene expression was upregulated in proliferating, tip, and immature ECs in tumors, and was elevated in CNV in proliferating ECs, but less in tip and immature ECs (Figure 3B). These observations might suggest that the metabolic demands of proliferating ECs are disease- or tissue-type independent, while metabolic adaptations of other subtypes may be more plastic. The metabolic gene expression signatures between the different TEC phenotypes were more outspoken, possibly reflecting the harsh nutrient-deprived micro-environment in tumors and the fact that TECs grow in an uncontrolled, non-resolving manner. Indeed, heatmap analysis revealed that most TECs exhibited a different metabolic transcriptome signature (Figure S3C). Subsequent analysis at the gene level showed that capillary TECs upregulated the expression of genes controlling lipid uptake (Figures 3C and S3C), raising the question of whether they need lipids for internal use when switching to quiescence (Kalucka et al., 2018Kalucka J. Bierhansl L. Conchinha N.V. Missiaen R. Elia I. Brüning U. Scheinok S. Treps L. Cantelmo A.R. Dubois C. et al.Quiescent endothelial cells upregulate fatty acid β-oxidation for vasculoprotection via redox homeostasis.Cell Metab. 2018; 28: 881-894.e13Abstract Full Text Full Text PDF PubMed Google Scholar) and/or for trans-EC transport to cancer cells for energy production or lipogenesis (Santos and Schulze, 2012Santos C.R. Schulze A. Lipid metabolism in cancer.FEBS J. 2012; 279: 2610-2623Crossref PubMed Scopus (571) Google Scholar). Venous TECs upregulated transcripts of genes involved in prostaglandin metabolism (Figures 3C and S3C), suggesting a role in vasoregulation, sprouting, or vascular inflammation (Félétou et al., 2011Félétou M. Huang Y. Vanhoutte P.M. Endothelium-mediated control of vascular tone: COX-1 and COX-2 products.Br. J. Pharmacol. 2011; 164: 894-912Crossref PubMed Scopus (0) Google Scholar, Iñiguez et al., 2003Iñiguez M.A. Rodríguez A. Volpert O.V. Fresno M. Redondo J.M. Cyclooxygenase-2: a therapeutic target in angiogenesis.Trends Mol. Med. 2003; 9: 73-78Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Interferon (IFN)-activated TECs upregulated genes involved in nucleotide catabolism to salvage/lower nucleotide content (Figure 3C) (Barankiewicz et al., 1986Barankiewicz J. Kaplinsky C. Cohen A. Modification of ribonucleotide and deoxyribonucleotide metabolism in interferon-treated human B-lymphoblastoid cells.J. Interferon Res. 1986; 6: 717-727Crossref PubMed Scopus (0) Google Scholar). In turn, breach TECs upregulated genes involved in extracellular matrix production the most, in line with their presumed role in vessel sprouting initiation (Goveia et al., 2020Goveia J. Rohlenova K. Taverna F. Treps L. Conradi L.C. Pircher A. Geldhof V. de Rooij L.P.M.H. Kalucka J. Sokol L. et al.An integrated gene expression landscape profiling approach to identify lung tumor endothelial cell heterogeneity and angiogenic candidates.Cancer Cell. 2020; 37: 21-36.e13Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar) (Figures 3C and S3C). Consistent with literature reports (Kanda et al., 2009Kanda T. Brown J.D. Orasanu G. Vog" @default.
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• W3014477614 date "2020-04-01" @default.
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• W3014477614 title "Single-Cell RNA Sequencing Maps Endothelial Metabolic Plasticity in Pathological Angiogenesis" @default.
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