Endothelial Cell Fate Research Articles

Role of Glucosylceramide in Lung Endothelial Cell Fate and Emphysema.

Rationale: The loss of pulmonary endothelial cells in emphysema is associated with increased lung ceramide. Ceramide perturbations may cause adaptive alterations in other bioactive sphingolipids, with pathogenic implications. We previously reported a negative correlation between emphysema and circulating glycosphingolipids (GSLs). Glucosylceramide (GlcCer), the initial GSL synthesized from ceramide by GCS (GlcCer synthase), is required for embryonic survival, but its role in the lung is unknown.Objectives: To determine if cigarette smoke (CS) alters lung GlcCer and to elucidate the role of GCS in lung endothelial cell fate.Methods: GlcCer was measured by tandem mass spectrometry in BAL fluid of CS- or elastase-exposed mice, and GCS was detected by Western blotting in chronic obstructive pulmonary disease lungs and CS extract-exposed primary human lung microvascular endothelial cells (HLMVECs). The role of GlcCer and GCS on mTOR (mammalian target of rapamycin) signaling, autophagy, lysosomal function, and cell death were studied in HLMVECs with or without CS exposure.Measurements and Main Results: Mice exposed to chronic CS or to elastase, and patients with chronic obstructive pulmonary disease, exhibited significantly decreased lung GlcCer and GCS. In mice, lung GlcCer levels were negatively correlated with airspace size. GCS inhibition in HLMVEC increased lysosomal pH, suppressed mTOR signaling, and triggered autophagy with impaired lysosomal degradation and apoptosis, recapitulating CS effects. In turn, increasing GlcCer by GCS overexpression in HLMVEC improved autophagic flux and attenuated CS-induced apoptosis.Conclusions: Decreased GSL production in response to CS may be involved in emphysema pathogenesis, associated with autophagy with impaired lysosomal degradation and lung endothelial cell apoptosis.

Conjugated polymers optically regulate the fate of endothelial colony-forming cells.

The control of stem and progenitor cell fate is emerging as a compelling urgency for regenerative medicine. Here, we propose a innovative strategy to gain optical control of endothelial colony-forming cell fate, which represents the only known truly endothelial precursor showing robust in vitro proliferation and overwhelming vessel formation in vivo. We combine conjugated polymers, used as photo-actuators, with the advantages offered by optical stimulation over current electromechanical and chemical stimulation approaches. Light modulation provides unprecedented spatial and temporal resolution, permitting at the same time lower invasiveness and higher selectivity. We demonstrate that polymer-mediated optical excitation induces a robust enhancement of proliferation and lumen formation in vitro. We identify the underlying biophysical pathway as due to light-induced activation of TRPV1 channel. Altogether, our results represent an effective way to induce angiogenesis in vitro, which represents the proof of principle to improve the outcome of autologous cell-based therapy in vivo.

623 Changing endothelial cell fate in wound healing through modulation of Sox9 to reduce scarring

Endothelial cells that line the vascular endothelium undergo a variety of cellular transitions, with one such transition known as endothelial to mesenchymal transition (EndMT). We identified transcription factor Sox9 to be highly expressed in a progenitor population in the endothelium. We here aimed to investigate the potential molecular regulation of EndMT by the transcription factor Sox9 as well as its interaction with Notch signaling. A vascular-specific Sox9 knock-out (Sox9fl/flCdh5CreERt2Rosa-YFP (Sox9eKO)) and a vascular specific rbpj knock-out mouse model were used to assess EndMT. Wild-type mice were used for Sox9 siRNA knock-down. Loss of rbpj resulted in an increase in Sox9 expression in the vasculature. Full thickness excisional large wounds were used to assess fibrotic scar area following Sox9eKO or Sox9 siRNA knock-down. Full thickness small wounds were used to assess the changes in cellular phenotype in EndMT following Sox9eKO or Sox9 siRNA knock-down via immunofluorescence staining and flow cytometry. A significant reduction in fibrotic scar area was seen following Sox9eKO (**P<0.01) and siRNA knock-down (*P<0.05). Immunofluorescence quantification showed a significant reduction in the number of YFP labelled endothelial cells, vessels stained for endothelial (CD31) and mesenchymal/EndMT (α-SMA, FSP-1 and SLUG) markers across time-points assessed (D3 to D7), in both Sox9eKO and Sox9 siRNA knock-down. Flow cytometry analysis showed a significant reduction in the intensity of the pro-fibrotic marker CD26 in both Sox9eKO and Sox9 siRNA knock-down (***P<0.001). Loss of rbpj resulted in an increase in Sox9, EndMT and fibrosis. In conclusion, the dramatic decrease in fibrotic scar area following both Sox9eKO and Sox9 siRNA knock-down highlights Sox9s role in scar formation through EndMT. This highlights Sox9 as a novel regulator for EndMT and contributes to both an understanding into the fundamentals of cardiovascular biology and identification of a potential therapeutic target using siRNA in reducing scar area.

Abstract 601: Radiation Therapy Induces Changes in the Endothelial Methylome Affecting Pro-angiogenic Lncrna Expression

Radiation therapy is a big part of standard of care in oncology. However, in the long- term patients treated with radiation are at higher risk of suffering cardiovascular events. It is well described that the genotoxic stress induced by Ionizing radiation in normal cardiovascular tissue triggers genetic and epigenetic changes. In addition to coding mRNAs, non-coding RNAs are also highly regulated by changes in methylation and transcription, leading to a tight regulatory response to damage. Genotoxic stress induces global hypomethylation due to decreased expression of DNA methyltransferases (DNMT). We observed that DNMT1, DNMT3A, and DNMT3b are downregulated in response to radiation treatment, in a dose-response manner in human endothelial cells. We have also observed that methylation changes produced by radiation affect a specific ncRNA cluster, DLK1-DIO3. Previous results from our lab indicate that ncRNAs from this cluster are highly responsive to different genotoxic agents, including radiation. Given the essential role of microRNAs and Long non- coding RNAs (lncRNAs) from the DLK1-DIO3 cluster in cardiovascular development and aging, we are interested in understanding how the epigenetic changes induced by radiotherapy affect to lncRNAs expression, and how they influence cardiovascular health in the long term after treatment. We have identified that a specific lncRNA from the DLK1-DIO3 cluster, MEG9, increases in ECs after exposure to ionizing radiation. Interestingly, knockdown of the individual DNMTs enzymes indicates a significant upregulation of MEG9 when DNMT3b is inhibited, more so after radiation treatment. To explore the role of this lncRNA, we performed loss-of-function studies. MEG9 inhibition not only diminished proliferation but also increased apoptosis through caspase 3/7 activation. Consistent with this phenotype, knockdown of MEG9 decreases growth factor-dependent angiogenesis in a 3D fibrin gel angiogenesis assay. Taken together, our findings illustrate how DNA methylation at particular lncRNA loci can regulate their expression and drive endothelial cell fate decisions. Our work illustrates how epigenetic changes may affect the long- term cardiovascular function of cancer patients submitted to radiation therapy.

Evolutionary Differences in the Vegf/Vegfr Code Reveal Organotypic Roles for the Endothelial Cell Receptor Kdr in Developmental Lymphangiogenesis

Lymphatic vascular development establishes embryonic and adult tissue fluid balance and is integral in disease. In diverse vertebrate organs, lymphatic vessels display organotypic function and develop in anorgan-specific manner. In all settings, developmental lymphangiogenesis is considered driven by vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3), whereas a role for VEGFR2 remains to befully explored. Here, we define the zebrafish Vegf/Vegfr code in receptor binding studies. We find that while Vegfd directs craniofacial lymphangiogenesis, it binds Kdr (a VEGFR2 homolog) but surprisingly, unlike in mammals, does not bind Flt4 (VEGFR3). Epistatic analyses and characterization of a kdr mutant confirm receptor-binding analyses, demonstrating that Kdr is indispensible for rostral craniofacial lymphangiogenesis, but not caudal trunk lymphangiogenesis, in which Flt4 is central. We further demonstrate an unexpected yet essential role for Kdr in inducing lymphatic endothelial cell fate. This work reveals evolutionary divergence in the Vegf/Vegfr code that uncovers spatially restricted mechanisms of developmental lymphangiogenesis.

Canonical Wnt Signaling Promotes Neovascularization Through Determination of Endothelial Progenitor Cell Fate via Metabolic Profile Regulation.

Endothelial progenitor cells (EPCs) contribute to blood vessel formation. Canonical Wnt signaling plays an important role in physiological and pathological angiogenesis and EPC fate regulation. However, the mechanism for Wnt signaling to regulate EPC fate in neovascularization (NV) has not been clearly defined. Here, we showed that very low‐density lipoprotein receptor knockout (Vldlr −/−) mice, a model of ocular NV induced by Wnt signaling overactivation, have increased EPC numbers in the bone marrow, blood, and retina, as well as an elevated mitochondrial membrane potential indicating higher mitochondrial function of EPCs in the circulation. Isolated EPCs from Vldlr −/− mice showed overactivated Wnt signaling, correlating with increased mitochondrial function, mass, and DNA copy numbers, compared with WT EPCs. Our results also demonstrated that Wnt signaling upregulated mitochondrial biogenesis and function, while inhibiting glycolysis in EPCs, which further decreased EPC stemness and promoted EPCs to a more active state toward differentiation, which may contribute to pathologic vascular formation. Fenofibric acid, an active metabolite of fenofibrate, inhibited Wnt signaling and mitochondrial function in EPCs and decreased EPC numbers in Vldlr −/−mice. It also decreased mitochondrial biogenesis and reactive oxygen species production in Vldlr −/− EPCs, which may be responsible for its therapeutic effect on diabetic retinopathy. These findings demonstrated that Wnt signaling regulates EPC fate through metabolism, suggesting potential application of the EPC metabolic profile as predictor and therapeutic target for neovascular diseases. stem cells 2019;37:1331–1343

Substrate stiffness directs diverging vascular fates

Embryonic stem cells (ESC) are excellent cell culture systems for elucidating developmental signals that may be part of the stem cell niche. Although stem cells are traditionally induced using predominately soluble signals, the mechanical environment of the niche can also play a role in directing cells towards differential cell lineages. Interested in diverging vascular fates, we set out to examine to what extent mechanical signaling played a role in endothelial cell and/or smooth muscle fate. Using chemically-defined staged vascular differentiation methods, vascular progenitor cells (VPC) fate was examined on single stiffness polyacrylamide hydrogels of 10 kPa, 40 kPa and >0.1 GPa. Emergence of vascular cell populations aligned with corresponding hydrogel stiffness: EC-lineages favoring the softer material and SMC lineages favoring the stiffest material. Statistical significance was observed on both cell lines on almost all days. Transcriptome analysis indicated that the populations on the varying stiffness emerge in distinct categories. Lastly, blocking studies show that αvβ1, and not αvβ6, activation mediates stiffness-directed vascular differentiation. Overall, these studies indicate that softer materials direct VPCs into a more EC-like fate compared to stiffer materials. Statement of SignificanceAlthough stem cells are traditionally induced using predominately soluble signals, the mechanical environment of the niche also plays a role in directing cell fate. Several studies have examined the stiffness-induced cell fate from mesenchymal stem cells (MSCs) and undifferentiated embryonic stem cells (ESCs). This is the first study that rigorously examines the role of matrix stiffness in diverging vascular fates from a purified population of vascular progenitor cells (VPCs). We show that the emergence of endothelial cell (EC) versus smooth muscle cell (SMC) populations corresponds with hydrogel stiffness: EC-lineages favoring the softness material and SMC lineages favoring the stiffest material, and that αvβ1 activation mediates this stiffness-directed vascular differentiation.

Sox7 Regulates Lineage Decisions in Cardiovascular Progenitor Cells.

Specification of the mesodermal lineages requires a complex set of morphogenetic events orchestrated by interconnected signaling pathways and gene regulatory networks. The transcription factor Sox7 has critical functions in differentiation of multiple mesodermal lineages, including cardiac, endothelial, and hematopoietic. Using a doxycycline-inducible mouse embryonic stem cell line, we have previously shown that expression of Sox7 in cardiovascular progenitor cells promotes expansion of endothelial progenitor cells (EPCs). In this study, we show that the ability of Sox7 to promote endothelial cell fate occurs at the expense of the cardiac lineage. Using ChIP-Seq coupled with ATAC-Seq we identify downstream target genes of Sox7 in cardiovascular progenitor cells and by integrating these data with transcriptomic analyses, we define Sox7-dependent gene programs specific to cardiac and EPCs. Furthermore, we demonstrate a protein-protein interaction between SOX7 and GATA4 and provide evidence that SOX7 interferes with the transcriptional activity of GATA4 on cardiac genes. In addition, we show that Sox7 modulates WNT and BMP signaling during cardiovascular differentiation. Our data represent the first genome-wide analysis of Sox7 function and reveal a critical role for Sox7 in regulating signaling pathways that affect cardiovascular progenitor cell differentiation.

SOX18 mutations associated with human HLTRS syndrome reveal modification of protein‐protein interactions

Key transcription factors (TFs) including SRY‐related HMG box‐containing protein 18 (SOX18), COUP‐TFII, and prospero‐related homeobox 1 (PROX1) are responsible for the molecular control of the lymphatic endothelial cell fate. Mutations or truncations in the coding sequence of these TFs can cause a variety of human diseases. In particular, mutations in the transcription factor SOX18 cause the Hypotricosis‐Lymphedema‐Telangiectasia syndrome (HTLS), a congenital condition characterized for presenting swelling of tissue due to malformation or malfunction of lymphatics (Lymphedema), furthermore the people who present the disease also present alopecia (Hypotricosis) and the presence of small dilated vessels near the surface of the skin (Telangiectasia). Some patients also present with renal defect and hypertension, in a new variant of the disease called HLTRS (Hypotricosis‐Lymphedema‐Telangiectasia renal defect syndrome). So far two types of SOX18 variants have been described to be associated with HTLS: missense mutations that lack the transactivation domain and nonsense mutations, in the DNA binding domain. We aimed at identifying how the protein‐protein interactions (PPI) network is affected by these mutations and correlate interactions losses with phenotypic outcomes.We first established a SOX18 interactome, validating interaction with 31 SOX18 potential co‐factors. Then we characterized the stability of the mutated proteins by single‐molecule fluorescence and measured physical interactions using different in vitro techniques. Our data show that the SOX18 mutants are thermally stable and still able to bind DNA. However they are losing a significant number of protein partners, the extent and profile of these interaction losses was found to depend on the mutations. This preliminary data set is slowly providing a molecular view of the disease and is opening the door for potential pharmaceutical treatment.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genome-wide strategies reveal target genes of Npas4l associated with vascular development in zebrafish.

The development of a vascular network is essential to nourish tissues and sustain organ function throughout life. Endothelial cells (ECs) are the building blocks of blood vessels, yet our understanding of EC specification remains incomplete. Zebrafish cloche/npas4l mutants have been used broadly as an avascular model, but little is known about the molecular mechanisms of action of the Npas4l transcription factor. Here, to identify its direct and indirect target genes, we have combined complementary genome-wide approaches, including transcriptome analyses and chromatin immunoprecipitation. The cross-analysis of these datasets indicates that Npas4l functions as a master regulator by directly inducing a group of transcription factor genes that are crucial for hematoendothelial specification, such as etv2, tal1 and lmo2 We also identified new targets of Npas4l and investigated the function of a subset of them using the CRISPR/Cas9 technology. Phenotypic characterization of tspan18b mutants reveals a novel player in developmental angiogenesis, confirming the reliability of the datasets generated. Collectively, these data represent a useful resource for future studies aimed to better understand EC fate determination and vascular development.

Bone Morphogenetic Protein 9 Regulates Early Lymphatic-Specified Endothelial Cell Expansion during Mouse Embryonic Stem Cell Differentiation.

SummaryExogenous cues involved in the regulation of the initial steps of lymphatic endothelial development remain largely unknown. We have used an in vitro model based on the co-culture of vascular precursors derived from mouse embryonic stem cell (ESC) differentiation and OP9 stromal cells to examine the first steps of lymphatic specification and expansion. We found that bone morphogenetic protein 9 (BMP9) induced a dose-dependent biphasic effect on ESC-derived vascular precursors. At low concentrations, below 1 ng/mL, BMP9 expands the LYVE-1-positive lymphatic progeny and activates the calcineurin phosphatase/NFATc1 signaling pathway. In contrast, higher BMP9 concentrations preferentially enhance the formation of LYVE-1-negative endothelial cells. This effect results from an OP9 stromal cell-mediated VEGF-A secretion. RNA-silencing experiments indicate specific involvement of ALK1 and ALK2 receptors in these different BMP9 responses. BMP9 at low concentrations may be a useful tool to generate lymphatic endothelial cells from stem cells for cell-replacement strategies.

Fibroblast growth factor-transforming growth factor beta dialogues, endothelial cell to mesenchymal transition, and atherosclerosis.

Despite much effort, atherosclerosis remains an important public health problem, leading to substantial morbidity and mortality worldwide. The purpose of this review is to provide an understanding of the role of endothelial cell fate change in atherosclerosis process. Recent studies indicate that a process known as endothelial-to-mesenchymal transition (EndMT) may play an important role in atherosclerosis development. Transforming growth factor beta (TGFβ) has been shown to be an important driver of the endothelial cell phenotype transition. The current review deals with the current state of knowledge regarding EndMT's role in atherosclerosis and its regulation by fibroblast growth factor (FGF)-TGFβ cross-talk. A better understanding of FGF-TGFβ signaling in the regulation of endothelial cell phenotypes is key to the development of novel therapeutic agents.

The Warburg Effect in Endothelial Cells and its Potential as an Anti-angiogenic Target in Cancer.

Endothelial cells (ECs) make up the lining of our blood vessels and they ensure optimal nutrient and oxygen delivery to the parenchymal tissue. In response to oxygen and/or nutrient deprivation, ECs become activated and sprout into hypo-vascularized tissues forming new vascular networks in a process termed angiogenesis. New sprouts are led by migratory tip cells and extended through the proliferation of trailing stalk cells. Activated ECs rewire their metabolism to cope with the increased energetic and biosynthetic demands associated with migration and proliferation. Moreover, metabolic signaling pathways interact and integrate with angiogenic signaling events. These metabolic adaptations play essential roles in determining EC fate and function, and are perturbed during pathological angiogenesis, as occurs in cancer. The angiogenic switch, or the growth of new blood vessels into an expanding tumor, increases tumor growth and malignancy. Limiting tumor angiogenesis has therefore long been a goal for anticancer therapy but the traditional growth factor targeted anti-angiogenic treatments have met with limited success. In recent years however, it has become increasingly recognized that focusing on altered tumor EC metabolism provides an attractive alternative anti-angiogenic strategy. In this review, we will describe the EC metabolic signature and how changes in EC metabolism affect EC fate during physiological sprouting, as well as in the cancer setting. Then, we will discuss the potential of targeting EC metabolism as a promising approach to develop new anti-cancer therapies.

Abstract 006: Role of Endothelial YingYang1 in Controlling Angiogenetic Sprouting and Maturation

Angiogenesis is essential for growth and repair, and irregularity of angiogenesis contributes to numerous malignant, inflammatory and ischemic disorders. Transcription factors that regulate angiogenic gene expression are critical for proper vascular development and homeostasis. Here we show that YingYang1( YY1) is essential for vascular development both in embryonic and postnatal angiogenesis. We first found that endothelium-specific deletion of YY1 leads to embryonic lethality due to vascular defects. We further found inducible endothelial YY1 knockout (YY1 iΔEC ) mice exhibited abnormality of sprouting angiogenesis with a blunted-end, aneurysm-like structure with fewer and dysmorphic filopodia in EC tip cells compared with those of wild-type mice. Similar results are also identified in vitro by using cultured HUVEC cells. Moreover, we found that endothelial deletion of YY1 also leads to severe impairments in retinal vessel remodeling and maturation. Mechanistically, we show that YY1 is a robust endogenous inhibitor of Notch1 signaling that controls explicitly endothelial cell fate. YY1 deficiency remarkably enhances Hey1 expression, resulting in impaired retinal sprouting angiogenesis, which can be rescued by the treatment of the Notch inhibitor. Furthermore, YY1 regulates Hey1 expression by binding to RBP-J via a highly conserved ankyrin (ANK) repeat domain, then inhibits the NICD-MAML1- RBPJ complex formation by competing with MAML1 binding to RBP-J, which attenuates expression of downstream target genes Hey1. Lastly, we found that tumor growth and angiogenesis is reduced significantly in YY1 iΔEC mice, suggesting that endothelial YY1 is required for pathological angiogenesis in mouse tumor models. Altogether, our study reveals a new role for YY1 as a central mediator of Notch signaling and an essential regulator in both physiologic and pathologic angiogenesis.

Spatial patterning of the Notch ligand Dll4 controls endothelial sprouting in vitro

Angiogenesis, the formation of new blood vessels, is a vital process for tissue growth and development. The Notch cell-cell signalling pathway plays an important role in endothelial cell specification during angiogenesis. Dll4 - Notch1 signalling directs endothelial cells into migrating tip or proliferating stalk cells. We used the directing properties of Dll4 to spatially control endothelial cell fate and the direction of endothelial sprouts. We created linear arrays of immobilized Dll4 using micro contact printing. HUVECs were seeded perpendicular to these Dll4 patterns using removable microfluidic channels. The Notch activating properties of surface immobilized Dll4 were confirmed by qPCR. After induction of sprouting, microscopic images of fluorescently labelled endothelial sprouts were analysed to determine the direction and the efficiency of controlled sprouting (Ecs). Directionality analysis of the sprouts showed the Dll4 pattern changes sprout direction from random to unidirectional. This was confirmed by the increase of Ecs from 54.5 ± 3.1% for the control, to an average of 84.7 ± 1.86% on the Dll4 patterned surfaces. Our data demonstrates a surface-based method to spatially pattern Dll4 to gain control over endothelial sprout location and direction. This suggests that spatial ligand patterning can be used to provide control over (neo) vascularization.

Arteriovenous specification: BMPER and TWSG1 determine endothelial cell fate via activation of synergistic BMP and Notch signaling.

Two extracellular BMP modulators, BMPER and TWSG1, act in a pro-BMP fashion to activate endothelial-specific members of the TGF-β/BMP receptor family. Through cross-talk with the Notch signaling pathways, they are key regulators of downstream Notch targets, including ephrin B2. This adds to our understanding of BMP and Notch signaling, how these pathways converge, and thereby control arteriovenous specification.

PTEN suppresses epigenetic modulation of ERG transcription factor to maintain endothelial lineage and vascular integrity

Homeostatic replenishment of endothelial cells (ECs) after injury is required for the efficient formation of a stable endothelial lineage and to prevent long‐lasting lung vascular injury such as protein‐rich edema in the interstitial tissue, a hallmark of tissue inflammation. However, mechanisms maintaining EC lineage remains unclear. Phosphatase and tensin homologue (PTEN), a tumor suppressor, is well‐known to function in cell membrane to negatively regulate PIP3 levels required for angiogenesis and endothelial cell proliferation and thereby may have role in regulating EC fate. Thus, we induced the conditional deletion of PTEN in ECs of adult mice using tamoxifen to address the hypothesis that PTEN regulates EC lineage. Intriguingly, we found that EC‐specific loss of PTEN led to intractable vascular injury basally due to marked reduction in EC number in the lung. FACS analysis showed that these ECs acquired fibroblast‐like phenotype. RNA seq analysis of PTEN depleted ECs showed PTEN was required for maintaining ECs genes including VE‐cadherin and PECAM. PTEN maintained ECs genes by activating ETS (E26 Transformation Specific) family member transcription factor, ERG. We show that this function of PTEN occur in nucleus since loss of PTEN increased the activity of epigenetic modulating gene, HDAC1 leading to upregulation of non‐EC genes and thereby converting these ECs into fibroblasts like cells. Inhibition of HDAC1 activity by velproic acid and trichostatin rescued ERG expression and transcription of VE‐cadherin in PTEN depleted cells. Thus, we identify a novel role of PTEN in suppressing HDAC1 activity enabling thereby ERG transcription of EC genes leading to EC generation and lung‐vascular homeostasis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Single-cell transcriptomics reveals a new dynamical function of transcription factors during embryonic hematopoiesis.

Recent advances in single-cell transcriptomics techniques have opened the door to the study of gene regulatory networks (GRNs) at the single-cell level. Here, we studied the GRNs controlling the emergence of hematopoietic stem and progenitor cells from mouse embryonic endothelium using a combination of single-cell transcriptome assays. We found that a heptad of transcription factors (Runx1, Gata2, Tal1, Fli1, Lyl1, Erg and Lmo2) is specifically co-expressed in an intermediate population expressing both endothelial and hematopoietic markers. Within the heptad, we identified two sets of factors of opposing functions: one (Erg/Fli1) promoting the endothelial cell fate, the other (Runx1/Gata2) promoting the hematopoietic fate. Surprisingly, our data suggest that even though Fli1 initially supports the endothelial cell fate, it acquires a pro-hematopoietic role when co-expressed with Runx1. This work demonstrates the power of single-cell RNA-sequencing for characterizing complex transcription factor dynamics.

Identification, molecular characterization and analysis of the expression pattern of $${\varvec{SoxF}}$$ SoxF subgroup genes the Yellow River carp, $${\varvec{Cyprinus} \varvec{carpio}}$$ Cyprinus carpio

Sox7, Sox17 and Sox18 are the members of the Sry-related high-mobility group box family (SoxF) of transcription factors. SoxF factors regulate endothelial cell fate as well as development and differentiation of blood cells and lymphatic vessels. There is very less information about the functions of these genes in fish. We obtained the full-length cDNA sequence of SoxF genes including Sox7, Sox17 and Sox18 in Cyprinus carpio, where Sox7 and Sox18 had two copies. The construction of a phylogenetic tree showed that these genes were homologous to the genes in other species. Chromosome synteny analysis indicated that the gene order of Sox7 and Sox18 was highly conserved in fish. However, immense change in genomic sequences around Sox17 had taken place. Numerous putative transcription factor binding sites were identified in the 5_ flanking regions of SoxF genes which may be involved in the regulation of the nervous system, vascular epidermal differentiation and embryonic development. The expression levels of SoxF genes were highest in gastrula, and was abundantly expressed in the adult brain.We investigated the expression levels of SoxF genes in five specific parts of the brain. The expression levels of Sox7 and Sox18 were highest in the mesencephalon, while the expression level of Sox17 was highest in the epencephalon. In carp, the expression patterns of SoxF genes indicated a potential function of these genes in neurogenesis and in vascular development. These results provide new information for further studies on the potential functions of SoxF genes in carp.

A Metabolic Basis for Endothelial-to-Mesenchymal Transition

Endothelial-to-mesenchymal transition (EndoMT) isa cellular process often initiated by the transforming growth factor β (TGF-β) family of ligands. Although required for normal heart valve development, deregulated EndoMT is linked to a wide range of pathological conditions. Here, we demonstrate that endothelial fatty acid oxidation (FAO) is a critical invitro and invivo regulator of EndoMT. We further show that this FAO-dependent metabolic regulation of EndoMT occurs through alterations in intracellular acetyl-CoA levels. Disruption of FAO via conditional deletion of endothelial carnitine palmitoyltransferaseII (Cpt2E-KO) augments the magnitude of embryonic EndoMT, resulting in thickening of cardiac valves. Consistent with the known pathological effects ofEndoMT, adult Cpt2E-KO mice demonstrate increased permeability in multiple vascular beds. Taken together, these results demonstrate that endothelial FAO is required to maintain endothelial cell fate and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions.