VE-cadherin Transcription Research Articles

Long noncoding RNA Hotair facilitates retinal endothelial cell dysfunction in diabetic retinopathy.

Retinal endothelial cell (REC) dysfunction induced by diabetes mellitus (DM) is an important pathological step of diabetic retinopathy (DR). Long noncoding RNAs (lncRNAs) have emerged as novel modulators in DR. The present study aimed to investigate the role and mechanism of lncRNA Hotair in regulating DM-induced REC dysfunction. The retinal vascular preparations and immunohistochemical staining assays were conducted to assess the role of Hotair in retinal vessel impairment in vivo. The EdU, transwell, cell permeability, CHIP, luciferase activity, RIP, RNA pull-down, and Co-IP assays were employed to investigate the underlying mechanism of Hotair-mediated REC dysfunction in vitro. Hotair expression was significantly increased in diabetic retinas and high glucose (HG)-stimulated REC. Hotair knockdown inhibited the proliferation, invasion, migration, and permeability of HG-stimulated REC in vitro and reduced the retinal acellular capillaries and vascular leakage in vivo. Mechanistically, Hotair bound to LSD1 to inhibit VE-cadherin transcription by reducing the H3K4me3 level on its promoter and to facilitate transcription factor HIF1α-mediated transcriptional activation of VEGFA. Furthermore, LSD1 mediated the effects of Hotair on REC function under HG condition. The Hotair exerts its role in DR by binding to LSD1, decreasing VE-cadherin transcription, and increasing VEGFA transcription, leading to REC dysfunction. These findings revealed that Hotair is a potential therapeutic target of DR.

IL-1β suppression of VE-cadherin transcription underlies sepsis-induced inflammatory lung injury.

Unchecked inflammation is a hallmark of inflammatory tissue injury in diseases such as acute respiratory distress syndrome (ARDS). Yet the mechanisms of inflammatory lung injury remain largely unknown. Here we showed that bacterial endotoxin lipopolysaccharide (LPS) and cecal ligation and puncture-induced (CLP-induced) polymicrobial sepsis decreased the expression of transcription factor cAMP response element binding (CREB) in lung endothelial cells. We demonstrated that endothelial CREB was crucial for VE-cadherin transcription and the formation of the normal restrictive endothelial adherens junctions. The inflammatory cytokine IL-1β reduced cAMP generation and CREB-mediated transcription of VE-cadherin. Furthermore, endothelial cell-specific deletion of CREB induced lung vascular injury whereas ectopic expression of CREB in the endothelium prevented the injury. We also observed that rolipram, which inhibits type 4 cyclic nucleotide phosphodiesterase-mediated (PDE4-mediated) hydrolysis of cAMP, prevented endotoxemia-induced lung vascular injury since it preserved CREB-mediated VE-cadherin expression. These data demonstrate the fundamental role of the endothelial cAMP-CREB axis in promoting lung vascular integrity and suppressing inflammatory injury. Therefore, strategies aimed at enhancing endothelial CREB-mediated VE-cadherin transcription are potentially useful in preventing sepsis-induced lung vascular injury in ARDS.

Long Noncoding RNA Hotair Facilitates Retinal Endothelial Cell Dysfunction in Diabetic Retinopathy

Background: Retinal endothelial cell (REC) dysfunction induced by diabetes mellitus (DM) is an important pathological step of diabetic retinopathy (DR). Long noncoding RNAs (lncRNAs) have emerged as novel modulators in DR. This study aimed to investigate the role and mechanism of lncRNA Hotair in regulating DM-induced REC dysfunction. Methods: The retinal vascular preparations and immunohistochemical staining assays were conducted to assess the role of Hotair in retinal vessel impairment in vivo. The EdU, transwell, cell permeability, CHIP, luciferase activity, RIP, RNA pull-down, and Co-IP assays were employed to investigate the underlying mechanism of Hotair-mediated REC dysfunction in vitro. Results: Hotair expression was significantly increased in diabetic retinas and high glucose (HG)-stimulated REC. Hotair knockdown inhibited the proliferation, migration, and permeability of HG-stimulated REC in vitro and reduced the retinal acellular capillaries and vascular leakage in vivo. Mechanistically, Hotair bound to LSD1 to inhibit VE-cadherin transcription by reducing the H3K4me3 level on its promoter or to facilitate transcription factor HIF1α-mediated transcriptional activation of VEGFA. Furthermore, LSD1 mediated the effects of Hotair on REC function under HG condition. Conclusion: The Hotair exerts its role in DR by binding to LSD1, decreasing VE-cadherin transcription and increasing VEGFA transcription, leading to REC dysfunction. These findings revealed that Hotair is a potential therapeutic target of DR. Funding Statement: This study was supported by the funding of the National science foundation for youth (81700729). Declaration of Interests: All authors declare that they have no conflicts of interest in this work. Ethics Approval Statement: All animal experiments were approved by the Ethics Committees of the First Affiliated Hospital of Zhengzhou University.

Histone acetyltransferase p300 promotes MRTF-A-mediates transactivation of VE-cadherin gene in human umbilical vein endothelial cells

Vascular endothelial cadherin (VE-cadherin) is the major determinant of endothelial cell contact integrity and is required in vascular development and angiogenesis. Serum response factor (SRF) plays essential roles in postnatal retinal angiogenesis and adult neovascularization. It is unclear whether transcription of VE-cadherin is mediated by a SRF co-activator, myocardin-related transcription factor-A (MRTF-A). Here we have demonstrated that MRTF-A is a key regulatory factor to activate the transcription of VE-cadherin in human umbilical vein endothelial cells (HUVECs). siRNA-mediated knockdown of MRTF-A decreased the level of VE-cadherin in HUVECs. Vascular endothelial growth factor (VEGF) induced MRTF-A binding to the SRF-binding site (CArG box) within VE-cadherin promoter. Histone acetyltransferase p300 and MRTF-A could synergistically augment the expression of VE-cadherin by enhancing acetylation of histone3K9 (H3K9Ac), histone3K14 (H3K14Ac) and histone4 at the SRF-binding site within VE-cadherin promoter. Taken together, these data identified a detailed regulatory mechanism of VE-cadherin gene expression.

The C-terminus region of β-arrestin1 modulates VE-cadherin expression and endothelial cell permeability

BackgroundThe endothelial specific cell-cell adhesion molecule, VE-cadherin, modulates barrier function and vascular homeostasis. In this context, we have previously characterized that VEGF (vascular endothelial growth factor) leads to VE-cadherin phosphorylation, β-arrestin2 recruitment and VE-cadherin internalization in mouse endothelial cells. However, exactly how this VE-cadherin/β-arrestin complex contributes to VEGF-mediated permeability in human endothelial cells remains unclear. In this study, we investigated in-depth the VE-cadherin/β-arrestin interactions in human endothelial cells exposed to VEGF.FindingsFirst, we demonstrated that VEGF induces VE-cadherin internalization in a clathrin-dependent manner in human umbilical vein endothelial cells (HUVEC). In addition to the classical components of endocytic vesicles, β-arrestin1 was recruited and bound to phosphorylated VE-cadherin. Molecular mapping of this interaction uncovered that the C-terminus tail of β-arrestin1, that comprises amino acids 375 to 418, was sufficient to directly interact with the phosphorylated form of VE-cadherin. Interestingly, the expression of the C-terminus tail of β-arrestin1 induced loss of surface exposed-VE-cadherin, promoted monolayer disorganization and enhanced permeability. Finally, this effect relied on decreased VE-cadherin expression at the transcriptional level, through inhibition of its promoter activity.ConclusionsAltogether, our results demonstrate that β-arrestin1 might play multiple functions collectively contributing to endothelial barrier properties. Indeed, in addition to a direct implication in VE-cadherin endocytosis, β-arrestin1 could also control VE-cadherin transcription and expression. Ultimately, understanding the molecular mechanisms involved in VE-cadherin function might provide therapeutic tools for many human diseases where the vascular barrier is compromised.

Suicide Gene Reveals the Myocardial Neovascularization Role of Mesenchymal Stem Cells Overexpressing CXCR4 (MSCCXCR4)

BackgroundOur previous studies indicated that MSCCXCR4 improved cardiac function after myocardial infarction (MI). This study was aimed to investigate the specific role of MSCCXCR4 in neovascularization of infarcted myocardium using a suicide gene approach.MethodsMSCs were transduced with either lentivirus-null vector/GFP (MSCNull as control) or vector encoding for overexpressing CXCR4/GFP. The MSC derived-endothelial cell (EC) differentiation was assessed by a tube formation assay, Dil-ac-LDL uptake, EC marker expression, and VE-cadherin promoter activity assay. Gene expression was analyzed by quantitative RT-PCR or Western blot. The suicide gene approach was under the control of VE-cadherin promoter. In vivo studies: Cell patches containing MSCNull or MSCCXCR4 were transduced with suicide gene and implanted into the myocardium of MI rat. Rats received either ganciclovir (GCV) or vehicle after cell implantation. After one month, the cardiac functional changes and neovascularization were assessed by echocardiography, histological analysis, and micro-CT imaging.ResultsThe expression of VEGF-A and HIF-1α was significantly higher in MSCCXCR4 as compared to MSCNull under hypoxia. Additionally, MSCCXCR4 enhanced new vessel formation and EC differentiation, as well as STAT3 phosphorylation under hypoxia. STAT3 participated in the transcription of VE-cadherin in MSCCXCR4 under hypoxia, which was inhibited by WP1066 (a STAT3 inhibitor). In addition, GCV specifically induced death of ECs with suicide gene activation. In vivo studies: MSCCXCR4 implantation promoted cardiac functional restoration, reduced infarct size, improved cardiac remodeling, and enhanced neovascularization in ischemic heart tissue. New vessels derived from MSCCXCR4 were observed at the injured heart margins and communicated with native coronary arteries. However, the derived vessel networks were reduced by GCV, reversing improvement of cardiac function.ConclusionThe transplanted MSCCXCR4 enhanced neovascularization after MI by boosting release of angiogenic factors and increasing the potential of endothelial differentiation.

Hypoxia-induced vasculogenic mimicry formation via VE-cadherin regulation by Bcl-2

Vasculogenic mimicry (VM) refers to the unique ability of highly aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. Hypoxia plays a pivotal role in the formation of VM. Hypoxia-induced Bcl-2 overexpression is observed in many types of tumors including melanoma, in which it is associated with tumorigenicity and angiogenesis. VE-cadherin, the major endothelial adhesion molecule controlling cellular junctions and blood vessel formation, is also overexpressed in melanoma. Despite these connections, whether hypoxia induces VM formation via VE-cadherin regulation by Bcl-2 is not confirmed. We used human melanoma cells to upregulate or knockdown the expression of Bcl-2 to investigate the possible molecular mechanism of VM formation under hypoxia. Bcl-2 overexpression increased VE-cadherin expression and VM formation under normoxia, whereas Bcl-2 siRNA significantly decreased VE-cadherin expression and VM formation under hypoxia. We then demonstrated that Bcl-2 regulated VE-cadherin transcription activity by Western blot, three-dimensional cultures, reporter gene assay, and clinical analysis. Therefore, Bcl-2-dependent VE-cadherin overexpression may be an important mechanism by which hypoxia induces VM.

TNFα Accelerates Monocyte to Endothelial Transdifferentiation in Tumors by the Induction of Integrin α5 Expression and Adhesion to Fibronectin

Tumor-associated myeloid cells are believed to promote tumor development by stimulating tumor growth, angiogenesis, invasion, and metastasis. Tumor-associated myeloid cells that coexpress endothelial and myeloid markers represent a proangiogenic subpopulation known as vascular leukocytes. Recently, we and others had shown that tumor-derived TNFα promotes local tumor growth and vascularity. Our data suggested that tumor growth is in part due to TNFα-mediated increased numbers of tumor-associated vascular leukocytes (i.e., myeloid-endothelial biphenotypic cells). The work detailed herein explored the mechanism by which TNFα mediates endothelial differentiation of myeloid cells. Our studies showed that fibronectin is a robust facilitator of endothelial differentiation of blood mononuclear cells in vitro. We have found that TNFα treatment of monocytes significantly increased expression of α(5)β(1) integrin, a major fibronectin receptor enriched on endothelial cells, leading to a consequent fourfold increase in fibronectin adhesion. Furthermore, TNFα-treated monocytes upregulated expression of endothelial markers, flk-1(VEGFR2/KDR) and VE-cadherin. Integrin α(5) subunit inhibitory antibodies blocked adhesion to fibronectin as well as consequent upregulation of flk-1 and VE-cadherin transcripts, implying a role for outside-in signaling by the α(5)β(1) integrin after binding fibronectin. Finally, treatment of mouse tumors with anti-α(5) antibodies reduced accumulation of tumor vascular leukocytes in vivo. Our studies suggest that tumor cell-derived TNFα constitutes a tumor microenvironment signal that promotes differentiation of tumor-associated monocytes toward a proangiogenic/provasculogenic myeloid-endothelial phenotype via upregulation of the fibronectin receptor α(5)β(1).

Abstract 3480: TNFα accelerates tumor vascular leukocyte formation through the induction of integrin α5 expression and adhesion to fibronectin

Abstract Tumor associated myeloid cells are believed to promote tumor development by stimulating tumor growth, angiogenesis, invasion and metastasis. Tumor associated myeloid cells that co-express endothelial and myeloid markers represent a pro-angiogenic subpopulation known as vascular leukocytes. Recently, we and others have demonstrated that tumor-derived TNFα promotes local tumor growth and vascularity. Our data suggest that tumor growth is in part due to TNFα-mediated increased numbers of tumor-associated vascular leukocytes (ie myeloid-endothelial biphenotypic cells). The work detailed herein explored the mechanism by which TNFα mediates endothelial differentiation of myeloid cells. Our studies showed that fibronectin is a robust facilitator of endothelial differentiation of blood mononuclear cells in vitro. We have found that TNFα treatment of monocytes significantly increased expression of α5β1 integrin, a major fibronectin receptor enriched on endothelial cells, leading to a consequent 4-fold increase in fibronectin adhesion. Furthermore, TNFα treated monocytes upregulated expression of endothelial markers, flk-1(VEGFR2/KDR) and VE-cadherin. Integrin α5 subunit inhibitory antibodies blocked adhesion to fibronectin as well as consequent upregulation of flk-1 and VE-cadherin transcripts, implying a role for outside-in signaling by the α5β1 integrin after binding fibronectin. Finally, treatment of mouse tumors with anti-α5 antibodies reduced accumulation of tumor vascular leukocytes in vivo. Our studies suggest that tumor-cell derived TNFα constitutes a tumor microenvironment signal that promotes differentiation of tumor-associated monocytes towards a proangiogenic/provasculogenic myeloid-endothelial phenotype via upregulation of the fibronectin receptor α5β1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3480. doi:10.1158/1538-7445.AM2011-3480

Wilms’ tumour protein Wt1 stimulates transcription of the gene encoding vascular endothelial cadherin

The Wilms' tumour gene, Wt1, encodes a zinc finger protein, which is mutated in a subset of paediatric renal carcinomas known as Wilms' tumours (nephroblastomas). Recent findings indicate that Wt1, beside its role in genitourinary development, is also necessary for normal vascularisation of the embryonic heart, and may even be involved in tumour angiogenesis. The original purpose of this study was to decipher potential downstream signalling pathways of Wt1 for blood vessel formation. We found that the Wt1(-KTS) protein, which functions as a transcription factor, stimulated the expression of cadherin 5 (CDH5, vascular endothelial (VE) cadherin) and other vascular genes, i.e. those encoding vascular endothelial growth factor receptors 1 and 2, and angiopoietin-2. Furthermore, an enhancer element was identified in the first intron of the CDH5 gene, which bound to the Wt1(-KTS) protein and was necessary for reporter gene activation by Wt1(-KTS) in transiently transfected cell lines. Wt1 and VE-cadherin proteins could be co-localised by double immunofluorescence staining in maturating glomeruli of embryonic murine kidneys. VE-cadherin transcripts were reduced in some but not all tissues of Wt1-deficient mouse embryos. These results indicate that Wt1 can stimulate vascular gene transcription. By demonstrating that Wt1(-KTS) protein trans-activates an enhancer element in the first intron we identified CDH5 as a novel target gene of Wt1. It is suggested that transcriptional activation of CDH5 by Wt1 fulfils regulatory functions during vascular development and kidney formation.

The Wilms’ tumor transcription factor WT1 regulates expression of the VE‐cadherin gene

The Wilms’ tumor transcription factor, WT1, is an important regulator of embryonic development. A predominant feature of Wt1‐deficient mouse embryos is their lack of kidneys and gonads in addition to a failure of normal formation of the vasculature in the heart. Identification of downstream target genes is necessary to understand the function of WT1 during normal embryogenesis.Here we demonstrate, that WT1 stimulates expression of the vascular endothelial (VE) cadherin gene. VE‐cadherin transcripts were increased more then 15‐fold in a cell line with inducible WT1 expression. WT1 enhanced VE‐cadherin expression also at the protein level in these cells. With the combinatorial use of luciferase reporter gene assays and electrophoretic mobility shift experiments we identified cis‐regulatory elements in the promoter and the first intron of the VE‐cadherin gene. Activation of the reporter constructs by WT1 was abolished upon introducing mutations into the identified binding sites.Immunohistochemical staining revealed co‐expression of WT1 and VE‐cadherin in the developing glomeruli of normal kidneys. VE‐cadherin expression was significantly reduced in the hearts and livers of Wt1‐deficient mouse embryos. These findings indicate that WT1 stimulates transcription of the VE‐cadherin gene, which, in turn, may promote vascular endothelial cell survival through stabilization of cell‐cell contacts.

The Basic Helix-Loop-Helix TAL1/SCL and Its Partners E47 and LMO2 Act Upstream of the VE-Cadherin Gene in Endothelial Cells.

The basic helix-loop-helix protein TAL-1/SCL, essential for the formation of the hematopoietic system, is also required for vascular development and more particularly for embryonic angiogenesis. We previously reported that TAL-1 acts as a positive factor for post-natal angiogenesis by stimulating endothelial morphogenesis. To understand how TAL-1 modulates angiogenesis, we investigated the functional consequences of TAL-1 silencing, mediated by small-interfering RNAs, in human primary endothelial cells (ECs). We found that TAL-1 knockdown impaired in vitro EC tubulomorphogenesis (in 2-D on Matrigel or 3-D in collagen I gel), with the notable absence of cell-cell contacts, a prerequisite for morphogenesis initiation. This cellular deficiency was associated with a dramatic reduction in the vascular-endothelial (VE)-cadherin at intercellular junctions, the major component of endothelial adherens junctions. In contrast, PECAM (or CD31) was present at cell-cell junctions at the same levels as control cells. Importantly, silencing of two known TAL-1-partners in hematopoietic cells, E47 or LMO2, produce the same effects as TAL-1. Accordingly, silencing of TAL-1, as well as E47 and LMO2, provoked down-regulation of VE-cadherin at both the mRNA and protein levels. Transient transfection experiments in HUVECs showed that TAL-1 and E47 regulate the VE-cadherin promoter through a specialized E-box element. Finally, endogenous VE-cadherin transcription could be directly activated in non-endothelial HEK-293 cells that neither express TAL-1 or LMO2, by the sole concomitant ectopic expression of TAL-1, E47 and LMO2. Overall, our data demonstrate that a multiprotein complex containing at least TAL-1, LMO2 and E47 act upstream of the VE-cadherin gene. We are currently performing chromatin immunoprecipitation (ChIP) to investigate whether the TAL-1-containing complex binds in vivo the VE-cadherin promoter. This study identifies VE-cadherin as an upstream TAL-1-target gene in the endothelial lineage, and provides a first clue in TAL-1 function in the control of angiogenesis.

Vascular Endothelial Cadherin-Expressing Cells Involved in Both Developmental and Adult Neovascularization.

Vessel formation, including vasculogenesis and angiogenesis, is thought to be directly mediated by adhesive molecules between vascular endothelial cells and adjacent cells. Vascular endothelial cadherin (VE-cadherin, CD144) is a adhesive molecule exclusively expressed in adherens junctions of cultured endothelial cells. VE-cadherin transcripts have been detected at first in mesodermal cells. However, no work has focused on which cell type expresses VE-cadherin during vessel formation and after vessel stabilization. To monitor VE-cadherin in vivo, we generated transgenic mice expressing a VE-cadherin promoter-driven Cre (VE-cad-Cre). By crossing VE-cad-Cre with loxP-based EGFP reporter mice (CAG-CAT-EGFP) and LacZ reporter mice (cAct-XstopX-LacZ), we obtained two double transgenic mice; one expressing EGFP (VE/EG) another expressing LacZ (VE/Z) dependently upon Cre driven by VE-cadherin promoter. Both reporter expression was increased in developing vasculature during embryogenesis, while it was decreased in postnatal vessel maturation. Intriguingly, LacZ reporter was found in the budding cells and lining cells of the dorsal aorta in the aorta-gonad-mesonephros region, suggesting that VE-cadherin-expressing cells belongs to hemangioblasts. Consistently, although VE-cadherin and its mRNA was detected in vasculature by immunohistochemistry and in situ hybridization, respectively, during embryogenesis, it was not detected in maturated vessels. These results indicated that VE-cadherin promoter activity reflects the endogeneous VE-cadherin expression and that VE-cadherin promoter is activated during developmental vasculogenesis and angiogenesis. To test whether the VE-cadherin promoter is re-activated in postnatal neovascularization, we used two models; a VEGF-induced cornea angiogenesis model and a myocardial ischemia model. VEGF implanted in the VE/EG eyes promoted the neovascularization visualized by EGFP expression. Myocardial ischemia in which the coronary artery was banded triggered the VE-cadherin promoter, thereby inducing reporter expression in vascular cells (endothelial cells and mural cells) of undamaged region and in infiltrated cells of damaged region. Furthermore, by flow cytometric analyses, we confirmed that myocardial ischemia re-activated the VE-cadherin promoter of both bone marrow cells and peripheral blood cells, as indicated by the remarkable increase of EGFP-positive cells. These data show that the VE-cadherin promoter is re-activated in pre-existing vascular cells and infiltrated cells during adult neovascularization. Collectively, VE-cadherin-expressing cells participates in both developmental and postnatal vasulogenesis and angiogenesis.

Targeted null-mutation in the vascular endothelial-cadherin gene impairs the organization of vascular-like structures in embryoid bodies.

Vascular endothelial-cadherin (VE-cadherin) is exclusively expressed in endothelial cells and is strictly located at cell-to-cell junctions. As the other members of the cadherin family, VE-cadherin is able to mediate a homotypic type of cellular interaction in a Ca2+-dependent manner. In the mouse embryo, VE-cadherin transcripts are detected at the earliest stages of vascular development. To ascertain if VE-cadherin expression is required for the assembly of endothelial cells into vascular structures, we generated VE-cadherin-negative mouse embryonic stem cells (VE-cadherin-/- ES cells) by gene targeting and examined the consequences on vascular development of ES-derived embryoid bodies (EBs). In contrast to wild-type EBs, we observed that endothelial cells remained dispersed and failed to organize a vessel-like pattern in VE-cadherin-/- ES-derived EBs. However, dispersed VE-cadherin-/- ES-derived endothelial cells expressed a large range of other endothelial markers. Moreover, the targeted null-mutation in the VE-cadherin locus did not interfere with the hematopoietic differentiation potential of ES cells. These in vitro experiments are consistent with a pivotal role of VE-cadherin in vascular structure assembly.

Molecular Cloning and Expression of Murine Vascular Endothelial-Cadherin in Early Stage Development of Cardiovascular System

An early step in the formation of the extraembryonic and intraembryonic vasculature is endothelial cell differentiation and organization in blood islands and vascular structures. This involves the expression and function of specific adhesive molecules at cell-to-cell junctions. Previous work showed that endothelial cells express a cell-specific cadherin (vascular endothelial [VE]-cadherin, or 7B4/cadherin-5) that is organized at cell-to-cell contacts in cultured cells and is able to promote intercellular adhesion. In this study, we investigated whether VE-cadherin could be involved in early cardiovascular development in the mouse embryo. We first cloned and sequenced the mouse VE-cadherin cDNA. At the protein level, murine VE-cadherin presented 75% identity (90%, considering conservative amino acid substitutions) with the human homologue. Transfection of murine VE-cadherin cDNA in L cells induced Ca(++)-dependent cell-to-cell aggregation and reduced cell detachment from monolayers. In situ hybridization of adult tissues showed that the murine molecule is specifically expressed by endothelial cells. In mouse embryos, VE-cadherin transcripts were detected at the very earliest stages of vascular development (E7.5) in mesodermal cells of the yolk sac mesenchyme. At E9.5, expression of VE-cadherin was restricted to the peripheral cell layer of blood islands that gives rise to endothelial cells. Hematopoietic cells in the center of blood islands were not labeled. At later embryonic stages, VE-cadherin transcripts were detected in vascular structures of all organs examined, eg, in the ventricle of the heart, the inner cell lining of the atrium and the dorsal aorta, in intersomitic vessels, and in the capillaries of the developing brain. A comparison with flk-1 expression during brain angiogenesis revealed that brain capillaries expressed relatively low amounts of VE-cadherin. In the adult brain, the level of VE-cadherin transcript was further reduced. By immunohistochemistry, murine VE-cadherin protein was detected at cell-to-cell junctions of endothelial cells. Overall, these data demonstrate that VE-cadherin is an early, constitutive, and specific marker of endothelial cells. This distinguishes this molecule from other cadherins and suggests that its expression is associated with the early assembly of vascular structures.