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Edema stemming from leaky blood vessels is common in eye diseases such as age-related macular degeneration and diabetic retinopathy. Whereas therapies targeting vascular endothelial growth factor A (VEGFA) can suppress leakage, side-effects include vascular rarefaction and geographic atrophy. By challenging mouse models representing different steps in VEGFA/VEGF receptor 2 (VEGFR2)-induced vascular permeability, we show that targeting signaling downstream of VEGFR2 pY949 limits vascular permeability in retinopathy induced by high oxygen or by laser-wounding. Although suppressed permeability is accompanied by reduced pathological neoangiogenesis in oxygen-induced retinopathy, similarly sized lesions leak less in mutant mice, separating regulation of permeability from angiogenesis. Strikingly, vascular endothelial (VE)-cadherin phosphorylation at the Y685, but not Y658, residue is reduced when VEGFR2 pY949 signaling is impaired. These findings support a mechanism whereby VE-cadherin Y685 phosphorylation is selectively associated with excessive vascular leakage. Therapeutically, targeting VEGFR2-regulated VE-cadherin phosphorylation could suppress edema while leaving other VEGFR2-dependent functions intact., Competing Interests: RS, TN, EG, HA, ED, DV, AK, LC No competing interests declared, (© 2020, Smith et al.)

Objective: Activation of endothelial β-catenin signaling by neural cell-derived Norrin or Wnt ligands is vital for the vascularization of the retina and brain. Mutations in members of the Norrin/β-catenin pathway contribute to inherited blinding disorders because of defective vascular development and dysfunctional blood-retina barrier. Despite a vital role for endothelial β-catenin signaling in central nervous system health and disease, its contribution to central nervous system angiogenesis and its interactions with downstream signaling cascades remains incompletely understood. Approach and Results: Here, using genetically modified mouse models, we show that impaired endothelial β-catenin signaling caused hypovascularization of the postnatal retina and brain because of deficient endothelial cell proliferation and sprouting. Mosaic genetic analysis demonstrated that endothelial β-catenin promotes but is not required for tip cell formation. In addition, pharmacological treatment revealed that angiogenesis under conditions of inhibited Notch signaling depends upon endothelial β-catenin. Importantly, impaired endothelial β-catenin signaling abrogated the expression of the VEGFR (vascular endothelial growth factor receptor)-2 and VEGFR3 in brain microvessels but not in the lung endothelium., Conclusions: Our study identifies molecular crosstalk between the Wnt/β-catenin and the Notch and VEGF-A signaling pathways and strongly suggest that endothelial β-catenin signaling supports central nervous system angiogenesis by promoting endothelial cell sprouting, tip cell formation, and VEGF-A/VEGFR2 signaling.

Rationale: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability., Objective: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization., Methods and Results: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5 , vascular endothelial-protein tyrosine phosphatase ( VE-PTP ), and von Willebrand factor ( vWf ). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5 , VE-PTP , and vWf . VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5 , VE-PTP , and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP., Conclusions: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system., (© 2017 The Authors.)

T-cell infiltration of solid tumors is associated with improved prognosis and favorable responses to immunotherapy. Mechanisms that enable tumor infiltration of CD8 + T cells have not been defined, nor have drugs that assist this process been discovered. Here we address these issues with a focus on VE-cadherin, a major endothelial cell-specific junctional protein that controls vascular integrity. A decrease in VE-cadherin expression is associated with tumor pathology. We developed an oligonucleotide-based inhibitor (CD5-2), which disrupted the interaction of VE-cadherin with its regulator miR-27a, resulting in increased VE-cadherin expression. Administration of CD5-2 in tumor-bearing mice enhanced expression of VE-cadherin in tumor endothelium, activating TIE-2 and tight junction pathways and normalizing vessel structure and function. CD5-2 administration also enhanced tumor-specific T-cell infiltration and spatially redistributed CD8 + T cells within the tumor parenchyma. Finally, CD5-2 treatment enhanced the efficacy of anti-PD-1 blocking antibody. Our work establishes a role for VE-cadherin in T-cell infiltration in tumors and offers a preclinical proof of concept for CD5-2 as a therapeutic modifier of cancer immunotherapy via effects on the tumor vasculature. Cancer Res; 77(16); 4434-47. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Fluid shear stress due to blood flow on the vascular endothelium regulates blood vessel development, remodeling, physiology, and pathology [1, 2]. A complex consisting of PECAM-1, VE-cadherin, and vascular endothelial growth factor receptors (VEGFRs) that resides at endothelial cell-cell junctions transduces signals important for flow-dependent vasodilation, blood vessel remodeling, and atherosclerosis. PECAM-1 transduces forces to activate src family kinases (SFKs), which phosphorylate and transactivate VEGFRs [3-5]. By contrast, VE-cadherin functions as an adaptor that interacts with VEGFRs through their respective cytoplasmic domains and promotes VEGFR activation in flow [6]. Indeed, shear stress triggers rapid increases in force across PECAM-1 but decreases the force across VE-cadherin, in close association with downstream signaling [5]. Interestingly, VE-cadherin cytoplasmic tyrosine Y658 can be phosphorylated by SFKs [7], which is maximally induced by low shear stress in vitro and in vivo [8]. These considerations prompted us to address the involvement of VE-cadherin cytoplasmic tyrosines in flow sensing. We found that phosphorylation of a small pool of VE-cadherin on Y658 is essential for flow sensing through the junctional complex. Y658 phosphorylation induces dissociation of p120ctn, which allows binding of the polarity protein LGN. LGN is then required for multiple flow responses in vitro and in vivo, including activation of inflammatory signaling at regions of disturbed flow, and flow-dependent vascular remodeling. Thus, endothelial flow mechanotransduction through the junctional complex is mediated by a specific pool of VE-cadherin that is phosphorylated on Y658 and bound to LGN., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: VE-cadherin is the chief constituent of endothelial adherens junctions. However, the role of VE-cadherin in the pathogenesis of cerebrovascular diseases including brain ischemia has not yet been investigated., Methods: VE-cadherin heterozygous (VEC(+/-)) mice and wildtype controls were subjected to transient brain ischemia by 30 min filamentous middle cerebral artery occlusion (MCAo)/reperfusion., Results: Acute lesion sizes as assessed by MR-imaging on day 3 did not differ between genotypes. Unexpectedly, however, partial loss of VE-cadherin resulted in long-term stroke protection measured histologically on day 28. Equally surprisingly, VEC(+/-) mice displayed no differences in post-stroke angiogenesis compared to littermate controls, but showed increased absolute regional cerebral blood flow in ischemic striatum at four weeks. The early induction of VE-cadherin mRNA transcription after stroke was reduced in VEC(+/-) mice. By contrast, N-cadherin and β-catenin mRNA expression showed a delayed, but sustained, upregulation up to 28 days after MCAo, which was increased in VEC(+/-) mice. Furthermore, partial loss of VE-cadherin resulted in a pattern of elevated ischemia-triggered mRNA transcription of pericyte-related molecules α-smooth muscle actin (α-SMA), aminopeptidase N (CD13), and platelet-derived growth factor receptor β (PDGFR-β)., Conclusions: Partial loss of VE-cadherin results in long term stroke protection. On the cellular and molecular level, this effect appears to be mediated by improved endothelial/pericyte interactions and the resultant increase in cerebral blood flow. Our study reinforces accumulating evidence that long-term stroke outcome depends critically on vascular mechanisms.

Background and Purpose: Cerebral cavernous malformation (CCM) is characterized by multiple lumen vascular malformations in the central nervous system that can cause neurological symptoms and brain hemorrhages. About 20% of CCM patients have an inherited form of the disease with ubiquitous loss-of-function mutation in any one of 3 genes CCM1, CCM2, and CCM3. The rest of patients develop sporadic vascular lesions histologically similar to those of the inherited form and likely mediated by a biallelic acquired mutation of CCM genes in the brain vasculature. However, the molecular phenotypic features of endothelial cells in CCM lesions in sporadic patients are still poorly described. This information is crucial for a targeted therapy., Methods: We used immunofluorescence microscopy and immunohistochemistry to analyze the expression of endothelial-to-mesenchymal transition markers in the cavernoma of sporadic CCM patients in parallel with human familial cavernoma as a reference control., Results: We report here that endothelial cells, a cell type critically involved in CCM development, undergo endothelial-to-mesenchymal transition in the lesions of sporadic patients. This switch in endothelial phenotype has been described only in genetic CCM patients and in murine models of the disease. In addition, TGF-β/p-Smad- and β-catenin-dependent signaling pathways seem activated in sporadic cavernomas as in familial ones., Conclusions: Our findings support the use of common therapeutic strategies for both sporadic and genetic CCM malformations., (© 2016 American Heart Association, Inc.)

Vascular endothelial (VE)-cadherin transfers intracellular signals contributing to vascular hemostasis. Signaling through VE-cadherin requires association and activity of different intracellular partners. Yes-associated protein (YAP)/TAZ transcriptional cofactors are important regulators of cell growth and organ size. We show that EPS8, a signaling adapter regulating actin dynamics, is a novel partner of VE-cadherin and is able to modulate YAP activity. By biochemical and imaging approaches, we demonstrate that EPS8 associates with the VE-cadherin complex of remodeling junctions promoting YAP translocation to the nucleus and transcriptional activation. Conversely, in stabilized junctions, 14-3-3-YAP associates with the VE-cadherin complex, whereas Eps8 is excluded. Junctional association of YAP inhibits nuclear translocation and inactivates its transcriptional activity both in vitro and in vivo in Eps8-null mice. The absence of Eps8 also increases vascular permeability in vivo, but did not induce other major vascular defects. Collectively, we identified novel components of the adherens junction complex, and we introduce a novel molecular mechanism through which the VE-cadherin complex controls YAP transcriptional activity., (© 2015 Giampietro et al.)

Vascular lumen formation is a fundamental step during angiogenesis; yet, the molecular mechanisms underlying this process are poorly understood. Recent studies have shown that neural and vascular systems share common anatomical, functional and molecular similarities. Here we show that the organization of endothelial lumen is controlled at the post-transcriptional level by the alternative splicing (AS) regulator Nova2, which was previously considered to be neural cell-specific. Nova2 is expressed during angiogenesis and its depletion disrupts vascular lumen formation in vivo. Similarly, Nova2 depletion in cultured endothelial cells (ECs) impairs the apical distribution and the downstream signalling of the Par polarity complex, resulting in altered EC polarity, a process required for vascular lumen formation. These defects are linked to AS changes of Nova2 target exons affecting the Par complex and its regulators. Collectively, our results reveal that Nova2 functions as an AS regulator in angiogenesis and is a novel member of the 'angioneurins' family.

Purpose of Review: The control of the endothelial barrier function is essential for vascular homeostasis and is mainly mediated by cell-to-cell junctions that tightly regulate permeability to plasma solutes and circulating cells such as leukocytes and tumor cells. While in some circumstances the transient dismantling of endothelial cell junctions might be beneficial, in pathological conditions, such as cancer, severe alterations of endothelial junction composition and function are detrimental, causing massive edema and increased interstitial pressure. Here, we aim to discuss the newly and most recently identified molecular mechanisms that cooperate in the control of vascular permeability., Recent Findings: Although the involvement of vascular endothelial-cadherin in the regulation of vascular leakage is well known, recent findings shed light on additional molecules involved in the control of vascular endothelial-cadherin phosphorylation in physiological and pathological conditions, and identified new unknown regulators of the endothelial barrier function., Summary: In the past years, several studies explored the contribution of various signaling pathways in the regulation of vascular leakage. Despite encouraging results, a more comprehensive understanding of the molecular mechanisms involved in this process will define druggable targets for new therapeutic interventions to limit endothelial barrier dysfunctions.

Blood vessel stability is essential for embryonic development; in the adult, many diseases are associated with loss of vascular integrity. The ETS transcription factor ERG drives expression of VE-cadherin and controls junctional integrity. We show that constitutive endothelial deletion of ERG (Erg(cEC-KO)) in mice causes embryonic lethality with vascular defects. Inducible endothelial deletion of ERG (Erg(iEC-KO)) results in defective physiological and pathological angiogenesis in the postnatal retina and tumors, with decreased vascular stability. ERG controls the Wnt/β-catenin pathway by promoting β-catenin stability, through signals mediated by VE-cadherin and the Wnt receptor Frizzled-4. Wnt signaling is decreased in ERG-deficient endothelial cells; activation of Wnt signaling with lithium chloride, which stabilizes β-catenin levels, corrects vascular defects in Erg(cEC-KO) embryos. Finally, overexpression of ERG in vivo reduces permeability and increases stability of VEGF-induced blood vessels. These data demonstrate that ERG is an essential regulator of angiogenesis and vascular stability through Wnt signaling., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The establishment of arterial and venous identity of endothelial cells is critical for the proper anatomic configuration and function of the vascular tree. Arterial and venous specification of endothelial cells is determined by genetic factors, although surrounding cells and hemodynamic forces may also contribute to vascular remodeling. This review provides an overview of the signaling pathways and related transcription factors implicated in differentiation of endothelial cells. We will discuss, in particular, the role of upstream and downstream effectors of Wnt, Sox, and Notch pathways. The understanding of the molecular mechanisms that orchestrate endothelial differentiation may have therapeutic relevance for diseases such as atherosclerosis, arteriovenous malformations, aneurysms, and others., (© 2014 American Heart Association, Inc.)

While tumor blood vessels share many characteristics with normal vasculature, they also exhibit morphological and functional aberrancies. For example, the neural adhesion molecule L1, which mediates neurite outgrowth, fasciculation, and pathfinding, is expressed on tumor vasculature. Here, using an orthotopic mouse model of pancreatic carcinoma, we evaluated L1 functionality in cancer vessels. Tumor-bearing mice specifically lacking L1 in endothelial cells or treated with anti-L1 antibodies exhibited decreased angiogenesis and improved vascular stabilization, leading to reduced tumor growth and metastasis. In line with these dramatic effects of L1 on tumor vasculature, the ectopic expression of L1 in cultured endothelial cells (ECs) promoted phenotypical and functional alterations, including proliferation, migration, tubulogenesis, enhanced vascular permeability, and endothelial-to-mesenchymal transition. L1 induced global changes in the EC transcriptome, altering several regulatory networks that underlie endothelial pathophysiology, including JAK/STAT-mediated pathways. In particular, L1 induced IL-6-mediated STAT3 phosphorylation, and inhibition of the IL-6/JAK/STAT signaling axis prevented L1-induced EC proliferation and migration. Evaluation of patient samples revealed that, compared with that in noncancerous tissue, L1 expression is specifically enhanced in blood vessels of human pancreatic carcinomas and in vessels of other tumor types. Together, these data indicate that endothelial L1 orchestrates multiple cancer vessel functions and represents a potential target for tumor vascular-specific therapies.

Vascular permeability is frequently associated with inflammation and is triggered by a cohort of secreted permeability factors such as vascular endothelial growth factor (VEGF). Here, we show that the physiological vascular permeability that precedes implantation is directly controlled by progesterone receptor (PR) and is independent of VEGF. Global or endothelial-specific deletion of PR blocks physiological vascular permeability in the uterus, whereas misexpression of PR in the endothelium of other organs results in ectopic vascular leakage. Integration of an endothelial genome-wide transcriptional profile with chromatin immunoprecipitation sequencing revealed that PR induces an NR4A1 (Nur77/TR3)-dependent transcriptional program that broadly regulates vascular permeability in response to progesterone. Silencing of NR4A1 blocks PR-mediated permeability responses, indicating a direct link between PR and NR4A1. This program triggers concurrent suppression of several junctional proteins and leads to an effective, timely, and venous-specific regulation of vascular barrier function that is critical for embryo implantation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Adherens junctions have an important role in the control of vascular permeability. These structures are located at cell-to-cell contacts, mediate cell adhesion and transfer intracellular signals. Adhesion is mediated by cadherins, which interact homophilically in trans and form lateral interactions in cis. VE-cadherin (also known as CDH5 and CD144) is the major component of endothelial adherens junctions and is specific to endothelial cells. Endothelial cells from different types of vessels, such as lymphatic vessels, arteries and veins, show differences in junction composition and organization. Vascular permeability is increased by modifications in the expression and function of adherens junction components. In some cases these defects might be cause of pathology. In this Cell Science at a Glance article, we present the example of the so-called cerebral cavernous malformation (CCM), where adherens junctions are dismantled in the vessels contributing to brain microcirculation. This causes the loss of endothelial cell apical-basal polarity and the formation of cavernomas, which are fragile and hemorrhagic. Other diseases are accompanied by persistent alterations of vascular morphology and permeability, such as seen in tumors. It will be important to achieve a better understanding of the relationship between vascular fragility, malformations and junctional integrity in order to develop more effective therapies.

Understanding and accelerating the mechanisms of endothelial wound healing is of fundamental interest for biotechnology and of significant medical utility in repairing pathologic changes to the vasculature induced by invasive medical interventions. We report the fundamental mechanisms that determine the influence of substrate topography and flow on the efficiency of endothelial regeneration. We exposed endothelial monolayers, grown on topographically engineered substrates (gratings), to controlled levels of flow-induced shear stress. The wound healing dynamics were recorded and analyzed in various configurations, defined by the relative orientation of an inflicted wound, the topography and the flow direction. Under flow perpendicular to the wound, the speed of endothelial regeneration was significantly increased on substrates with gratings oriented in the direction of the flow when compared to flat substrates. This behavior is linked to the dynamic state of cell-to-cell adhesions in the monolayer. In particular, interactions with the substrate topography counteract Vascular Endothelial Cadherin phosphorylation induced by the flow and the wounding. This effect contributes to modulating the mechanical connection between migrating cells to an optimal level, increasing their coordination and resulting in coherent cell motility and preservation of the monolayer integrity, thus accelerating wound healing. We further demonstrate that the reduction of vascular endothelial cadherin phosphorylation, through specific inhibition of Src activity, enhances endothelial wound healing in flows over flat substrates., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Vascular endothelial growth factor (VEGF) guides the path of new vessel sprouts by inducing VEGF receptor-2 activity in the sprout tip. In the stalk cells of the sprout, VEGF receptor-2 activity is downregulated. Here, we show that VEGF receptor-2 in stalk cells is dephosphorylated by the endothelium-specific vascular endothelial-phosphotyrosine phosphatase (VE-PTP). VE-PTP acts on VEGF receptor-2 located in endothelial junctions indirectly, via the Angiopoietin-1 receptor Tie2. VE-PTP inactivation in mouse embryoid bodies leads to excess VEGF receptor-2 activity in stalk cells, increased tyrosine phosphorylation of VE-cadherin and loss of cell polarity and lumen formation. Vessels in ve-ptp(-/-) teratomas also show increased VEGF receptor-2 activity and loss of endothelial polarization. Moreover, the zebrafish VE-PTP orthologue ptp-rb is essential for polarization and lumen formation in intersomitic vessels. We conclude that the role of Tie2 in maintenance of vascular quiescence involves VE-PTP-dependent dephosphorylation of VEGF receptor-2, and that VEGF receptor-2 activity regulates VE-cadherin tyrosine phosphorylation, endothelial cell polarity and lumen formation.

Purpose of Review: Vascular integrity is characterized by a tight control of permeability to cells and solutes and by resistance to blood flow. In several pathologies including tumor angiogenesis, vascular malformations, hemorrhagic stroke and others, there is the need to stabilize the vessels and prevent undesired bleeding or edema. Here, we discuss the current knowledge on the role of endothelial cell-to-cell adherens junctions in maintaining vascular integrity., Recent Findings: The identification of several components of adherens junctions in endothelial cells helped understanding of the complex role of these structures not only in maintaining cell-to-cell adhesion but also in transferring intracellular signals. Vascular endothelial (VE)-cadherin, an endothelial-specific adhesion protein at adherens junctions, was found to interact with several signaling partners that induce contact inhibition of growth, decrease in permeability, tight junction organization and others. Changes in VE-cadherin levels in vivo may significantly affect vascular permeability, and induce uncontrolled growth and vascular fragility., Summary: In the past years, the research on angiogenesis was mostly directed to the definition of the mechanisms able to modulate vascular growth. We now understand that in many pathological conditions we do not simply need to increase or inhibit vascularization but we also need to develop tools able to stabilize organ perfusion and to avoid hemorrhages or edema.

Cerebral cavernous malformations (CCM) are vascular malformations of the central nervous system (CNS) that lead to cerebral hemorrhages. Familial CCM occurs as an autosomal dominant condition caused by loss-of-function mutations in one of the three CCM genes. Constitutive or tissue-specific ablation of any of the Ccm genes in mice previously established the crucial role of Ccm gene expression in endothelial cells for proper angiogenesis. However, embryonic lethality precluded the development of relevant CCM mouse models. Here, we show that endothelial-specific Ccm2 deletion at postnatal day 1 (P1) in mice results in vascular lesions mimicking human CCM lesions. Consistent with CCM1/3 involvement in the same human disease, deletion of Ccm1/3 at P1 in mice results in similar CCM lesions. The lesions are located in the cerebellum and the retina, two organs undergoing intense postnatal angiogenesis. Despite a pan-endothelial Ccm2 deletion, CCM lesions are restricted to the venous bed. Notably, the consequences of Ccm2 loss depend on the developmental timing of Ccm2 ablation. This work provides a highly penetrant and relevant CCM mouse model., (© 2011 Boulday et al.)

Endothelial cells (ECs) differ in morphology and functional responses in the different regions of the vascular tree. During embryo development they acquire organ specific characteristics to respond to the needs of the perfused organs. The brain microvasculature is a striking example of this process. This particular vasculature develops unique properties to assure a tight control of permeability between blood and the underlying nervous system. To this end, these cells present well developed cell to cell junctions, tight basement membrane and express a series of transporters which support the passage of nutrients and toxic agents inside or outside the brain, respectively. This highly differentiated EC phenotype is induced and maintained by the cross-talk with the surrounding cells such as pericytes and astrocytes. Recent evidence highlights the molecular basis of this cross-talk (constituting the neurovascular unit) and opens new perspectives in the development of drugs which modulate blood-brain-barrier (BBB) permeability properties. In this review we describe the specific features of the BBB and we discuss recent data on the role of Wnt as a mediator of brain angiogenesis and BBB differentiation., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Early stages of vascular development include endothelial cell differentiation in a network of arteries, veins, and lymphatics. Subsequently, to respond to the specific needs of the organs, endothelial cells acquire specialized properties such as permeability control, expression of specific transcellular transport systems, membrane adhesive molecules, and others. Endothelial cell differentiation depends on communication between the surrounding tissues, which is mediated by growth and differentiation factors able to activate specific gene expression programs. Recent reports underline the important role of the Wnt system in vascular morphogenesis in the embryo and in organ-specific endothelial differentiation. Wnt signaling regulates fundamental aspects of development, including cell fate specification, proliferation, and survival, and may use different receptors and signaling pathways. Both loss- and gain-of-function experiments of members of the Wnt signaling pathway were found to cause marked alterations of vascular development and endothelial cell specification. Furthermore, altered Wnt signaling in the endothelium may contribute to pathological conditions such as retinopathies, pulmonary arterial hypertension, stroke, and others. Continued progress in this field holds the potential to identify novel therapeutics for the treatment of these diseases.

In vertebrates, endothelial cells (ECs) form blood vessels in every tissue. Here, we investigated vascular lumen formation in the developing aorta, the first and largest arterial blood vessel in all vertebrates. Comprehensive imaging, pharmacological manipulation, and genetic approaches reveal that, in mouse embryos, the aortic lumen develops extracellularly between adjacent ECs. We show that ECs adhere to each other, and that CD34-sialomucins, Moesin, F-actin, and non-muscle Myosin II localize at the endothelial cell-cell contact to define the luminal cell surface. Resultant changes in EC shape lead to lumen formation. Importantly, VE-Cadherin and VEGF-A act at different steps. VE-Cadherin is required for localizing CD34-sialomucins to the endothelial cell-cell contact, a prerequisite to Moesin and F-actin recruitment. In contrast, VEGF-A is required for F-actin-nm-Myosin II interactions and EC shape change. Based on these data, we propose a molecular mechanism of in vivo vascular lumen formation in developing blood vessels.

Leukocyte transmigration is mediated by endothelial cell (EC) junctional molecules, but the associated mechanisms remain unclear. Here we investigate how intercellular adhesion molecule-2 (ICAM-2), junctional adhesion molecule-A (JAM-A), and platelet endothelial cell adhesion molecule (PECAM-1) mediate neutrophil transmigration in a stimulus-dependent manner (eg, as induced by interleukin-1beta [IL-1beta] but not tumor necrosis factor-alpha [TNF-alpha]), and demonstrate their ability to act in sequence. Using a cell-transfer technique, transmigration responses of wild-type and TNF-alpha p55/p75 receptor-deficient leukocytes (TNFR(-/-)) through mouse cremasteric venules were quantified by fluorescence intravital microscopy. Whereas wild-type leukocytes showed a normal transmigration response to TNF-alpha in ICAM-2(-/-), JAM-A(-/-), and PECAM-1(-/-) recipient mice, TNFR(-/-) leukocytes exhibited a reduced transmigration response. Hence, when the ability of TNF-alpha to directly stimulate neutrophils is blocked, TNF-alpha-induced neutrophil transmigration is rendered dependent on ICAM-2, JAM-A, and PECAM-1, suggesting that the stimulus-dependent role of these molecules is governed by the target cell being activated. Furthermore, analysis of the site of arrest of neutrophils in inflamed tissues from ICAM-2(-/-), JAM-A(-/-), and PECAM-1(-/-) mice demonstrated that these molecules act sequentially to mediate transmigration. Collectively, the findings provide novel insights into the mechanisms of action of key molecules implicated in leukocyte transmigration.

Human pathologies such as vascular malformations, hemorrhagic stroke, and edema have been associated with defects in the organization of endothelial cell junctions. Understanding the molecular basis of these diseases requires different integrated approaches which include basic cell biology, clinical studies, and studies in animal models such as mice and zebrafish. In this review we discuss recent findings derived from these approaches and their possible integration in a common picture.

Endothelial cells control the passage of plasma constituents and circulating cells from blood to the underlying tissues. This specialized function is lost or impaired in several pathological conditions - including inflammation, sepsis, ischemia and diabetes - which leads to severe, and sometimes fatal, organ dysfunction. Endothelial permeability is regulated in part by the dynamic opening and closure of cell-cell adherens junctions (AJs). In endothelial cells, AJs are largely composed of vascular endothelial cadherin (VE-cadherin), an endothelium-specific member of the cadherin family of adhesion proteins that binds, via its cytoplasmic domain, to several protein partners, including p120, beta-catenin and plakoglobin. Endogenous pathways that increase vascular permeability affect the function and organization of VE-cadherin and other proteins at AJs in diverse ways. For instance, several factors, including vascular endothelial growth factor (VEGF), induce the tyrosine phosphorylation of VE-cadherin, which accompanies an increase in vascular permeability and leukocyte diapedesis; in addition, the internalization and cleavage of VE-cadherin can cause AJs to be dismantled. From the knowledge of how AJ organization can be modulated, it is possible to formulate several pharmacological strategies to control the barrier function of the endothelium. We discuss the possible use of inhibitors of SRC and other kinases, of agents that increase cAMP levels, and of inhibitors of lytic enzymes as pharmacological tools for decreasing endothelial permeability.

Lymphocytes emigrate from the circulation to target tissues through the microvascular endothelial cell (EC) barrier. During paracellular transmigration cell-cell junctions have been proposed to disengage and provide homophilic and heterophilic interaction surfaces in a zip-like process. However, it is not known whether ECs modulate junction proteins during this process. Here we show that tyrosine phosphorylation of adherens junction vascular endothelial cadherin (VEC) is required for successful transendothelial lymphocyte migration. We found that adhesion of lymphocytes or activation of the endothelial intercellular adhesion molecule 1 (ICAM1) led to tyrosine phosphorylation of VEC. Substitution of tyrosine for phenylalanine in VEC at positions 645, 731 or 733 produced ECs that were significantly less permissive to lymphocyte migration. We also found that these same tyrosine residues are involved in ICAM1-dependent changes of VEC phosphorylation. ICAM1 activation enhanced transendothelial permeability, suggesting the occurrence of junction disassembly. In agreement, the expression of VEC mutated at Y645F, Y731F or Y733F predominantly affected lymphocyte transmigration in paracellular areas. Taken together, these results demonstrate that phosphorylation of adherens junctions constitutes a molecular endpoint of lymphocyte-induced vascular EC signaling and may be exploited as a new target of anti-inflammatory therapies.

During tumour growth the surrounding vasculature forms new vessels which penetrate into the stroma and bring oxygen and nutrients to the proliferating cancer cells. The ability to control and reduce this phenomenon may have important therapeutic implications. Angiogenesis is a complex event which requires endothelial cell sprouting, lumen formation, tubulogenesis and is regulated by the coordinated action of different transcription factors. Studies on promoters of endothelial cell-specific genes or gene inactivation experiments reveal the extreme complexity of the system. Many transcription factors are implicated in vascular development and the majority are not endothelial-specific. Their interaction leads to endothelial cell differentiation and acquisition of arterial, venous and lymphatic properties. Two large families of transcription factors, Foxs and Ets, play a major role in these events. They participate in both embryonic and adult angiogenesis. The FoxO subgroup regulates the correct organization of the vascular system, controlling excessive endothelial growth and inducing apoptosis both in embryos and adult mice. Ets factors participate in early endothelial differentiation and angiogenesis. Many members of this family are expressed very early in the developing vasculature and Ets consensus binding domains are present in essentially all endothelial cell-specific gene promoters. In this review we discuss the overall transcriptional regulation of vascular development with a particular focus on some specific members of these two families considered important in the formation and maintenance of the vascular network.

Junctional adhesion molecules (JAMs) of the immunoglobulin superfamily are important in the control of vascular permeability and leukocyte transmigration across endothelial-cell surfaces, by engaging in homophilic, heterophilic and lateral interactions. Through their localization on the endothelial-cell surface and expression by platelets, JAMs contribute to adhesive interactions with circulating leukocytes and platelets. Antibody-blocking studies and studies using genetically modified mice have implicated these functions of JAMs in the regulation of leukocyte recruitment to sites of inflammation and ischaemia-reperfusion injury, in growth-factor-mediated angiogenesis, atherogenesis and neointima formation. The comparison of different JAM-family members and animal models, however, shows that the picture remains rather complex. This Review summarizes recent progress and future directions in understanding the role of JAMs as 'gate keepers' in inflammation and vascular pathology.

An effective immune response depends not only on the proper activation, regulation, and function of immune cells, but also on their distribution and retention in diverse tissue microenvironments where they encounter a number of stimuli and other cell types. These activities are mediated by endothelial cells, which form specialized microcirculatory networks used by immune cells under both physiological and pathological circumstances. Endothelial cells represent a highly heterogeneous population of cells with the ability to interact with and modulate the function of immune cells. This review is focused on the role of microvascular endothelial cells in innate and adaptive immunity, inflammation, coagulation, angiogenesis, and the therapeutic implications of targeting endothelial cells in selected autoimmune and chronic inflammatory disorders.

Unlabelled: Liver neo-angiogenesis plays a fundamental role in physiological and pathological processes such as regeneration, cirrhosis, autoimmune hepatitis, and alcoholic liver disease. How liver parenchymal cells influence angiogenesis is largely unknown. We studied the influence of soluble factors released by hepatocytes on hematopoietic and endothelial cell differentiation. Human CD34+ cells cultured for several weeks in a hepatocyte-conditioned medium gradually decrease the expression of CD34 and CD133 markers (i.e. after 4 weeks from 85% and 69%, respectively, to 6% and 3%, respectively), whereas expression of CD144 and CD14 cell markers increased (from 2% and 8%, respectively, to 54% and 55%, respectively). The cells' capacity to form hematopoietic colonies in methylcellulose declined with time, whereas they acquired endothelial morphology, expressed endothelial markers, and incorporated into newly forming vascular structures both in vitro and in vivo. Cultured single CD34+ cells formed colonies expressing both hematopoietic (CD45+) and endothelial (CD144+) markers, suggesting they constitute a bona fide hemangioblast population., Conclusion: This system allowed subsequent stages of differentiation of hematopoietic cells to endothelial cells to be defined, underlining the strict interrelationship between endothelial and hematopoietic cells in a hepatocyte environment.

Adherens junctions and more specifically cadherins play an important role in endothelial cell integrity and growth and, in general, in vascular morphogenesis. Besides their adhesive properties, cadherins may act by transferring intracellular signals through interaction with a complex network of cytoskeletal and signaling molecules. Cadherins may signal in different ways: through direct activation of signaling pathways, through interaction with cell-specific growth factor receptors or by controlling beta-catenin and/or other transcription factors' translocation to the nucleus. Endothelial cells present different cadherins which may transfer specific signals and exert distinct functional roles. VE-cadherin is endothelial-specific and the major constituent of adherens junctions. This protein is able to protect endothelial cells from apoptosis and contributes to contact inhibition of endothelial cell growth. N-cadherin is also abundantly expressed in the endothelium and may be important in modulating VE-cadherin expression. T cadherin, R-cadherin and VE-cadherin 2 were found in specific regions of the vascular tree but their role in vascular development or angiogenesis is still unclear.

Endoglin is an accessory receptor for transforming growth factor beta (TGFbeta) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFbeta type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFbeta pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFbeta, Eng-/- cells displayed a potent anti-proliferative response. TGFbeta-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFbeta treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFbeta concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFbeta type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFbeta receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFbeta signaling in endothelial cells by regulating surface TGFbeta receptors and suppressing Smad1 activation. Thus an altered balance in TGFbeta receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.

Junctional adhesion molecule-A (JAM-A) regulates key inflammatory responses, such as edema formation and leukocyte transmigration. Although it has been reported that the inflammatory cytokine tumor necrosis factor (TNF) causes the disassembly of JAM-A from the intercellular junctions, the mechanism has not been elucidated fully. Here, we report that TNF enhances the solubility of JAM-A in Triton X-100 and increases the amount of Triton-soluble JAM-A dimers at the cell surface but does not change the total levels of cellular JAM-A. Thus we hypothesized that TNF causes the redistribution of JAM-A from the junctions to the cell surface and that junction disassembly is sufficient to account for JAM-A redistribution. Intriguingly, however, even after complete disassembly of the junctions (with EDTA and trypsin), higher levels of JAM-A are detectable at the cell surface (by FACS analysis) in cells that had been previously incubated in the presence of TNF than in its absence. Thus we propose that TNF causes not only the disassembly of JAM-A from the junctions and its subsequent redistribution to the cell surface but also its dispersal in such a way that JAM-A becomes more easily accessible to the antibodies used for FACS analysis. Finally, we evaluated whether soluble fibronectin might attenuate the effects of TNF on JAM-A, as some inflammatory conditions are associated with the depletion of plasma fibronectin. We found that fibronectin reduces the effect of TNF on the disassembly of JAM-A, but not on its dispersal, thus further stressing that disassembly and dispersal can be functionally dissociated.

Objective: To test the potential of mesoangioblasts (Mabs) in reducing postischemic injury in comparison with bone marrow progenitor cells (BMPCs), fibroblasts (Fbs), and embryonic stem cell-derived endothelial cells (ECs), and to identify putative cellular protective mechanisms., Methods and Results: Cells were injected percutaneously in the left ventricular (LV) chamber of C57BL/6 mice, 3 to 6 hours after coronary ligation, and detected in the hearts 2 days and 6 weeks later. Echocardiographic examinations were performed at 6 weeks. LV dilation was reduced and LV shortening fraction was improved with Mabs and BMPCs but not with ECs and Fbs. Donor cell colonization of the host myocardium was modest and predominantly in the smooth muscle layer of vessels. Capillary density was higher in the peripheral infarct area and apoptotic cardiomyocytes were fewer with Mabs and BMPCs. Mabs and BMPCs, but not Fbs or ECs, promoted survival of cultured cardiocytes under low-oxygen in culture. This activity was present in Mab-conditioned medium and could be replaced by a combination of basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-1, and hepatocyte growth factor (HGF), all of which are produced by these cells. Conditioned medium from Mabs, but not from Fbs, stimulated proliferation of smooth muscle cells in vitro., Conclusions: Mabs appear as effective as BMPCs in reducing postinfarction LV dysfunction, likely through production of antiapoptotic and angiogenic factors.

Intercellular junctions mediate adhesion and communication between adjoining endothelial and epithelial cells. In the endothelium, junctional complexes comprise tight junctions, adherens junctions, and gap junctions. The expression and organization of these complexes depend on the type of vessels and the permeability requirements of perfused organs. Gap junctions are communication structures, which allow the passage of small molecular weight solutes between neighboring cells. Tight junctions serve the major functional purpose of providing a "barrier" and a "fence" within the membrane, by regulating paracellular permeability and maintaining cell polarity. Adherens junctions play an important role in contact inhibition of endothelial cell growth, paracellular permeability to circulating leukocytes and solutes. In addition, they are required for a correct organization of new vessels in angiogenesis. Extensive research in the past decade has identified several molecular components of the tight and adherens junctions, including integral membrane and intracellular proteins. These proteins interact both among themselves and with other molecules. Here, we review the individual molecules of junctions and their complex network of interactions. We also emphasize how the molecular architectures and interactions may represent a mechanistic basis for the function and regulation of junctions, focusing on junction assembly and permeability regulation. Finally, we analyze in vivo studies and highlight information that specifically relates to the role of junctions in vascular endothelial cells.

Survivin is strongly expressed in embryonic organs and in tumor cells but is low or absent in differentiated normal tissues. Resting endothelium expresses low levels of survivin but can up-regulate its synthesis on activation to proliferate. The mechanisms responsible for survivin down-regulation in resting conditions are still unknown. We report here that confluence and vascular endothelial-cadherin (VE-cadherin) expression induce contact inhibition of cell growth and survivin down-regulation in the endothelium. Using beta-catenin null and positive isogenic endothelial cell lines we found that the effect requires beta-catenin expression and its association to VE-cadherin cytoplasmic tail. Furthermore, in allantois organ cultures, survivin expression is up-regulated in areas of growing vessels where VE-cadherin is partially dismantled from junctions or in VE-cadherin -/- specimens. Overall, these data indicate that VE-cadherin and beta-catenin may negatively regulate survivin synthesis in endothelial cells. Consistently, in epidermal and pancreatic cell lines or ovarian tumors, epithelial-cadherin (E-cadherin) and survivin expression is inversely related, suggesting a non-cell-specific role of cadherins in reducing survivin synthesis.

The junctional membrane protein vascular endothelial (VE)-cadherin mediates contact inhibition of growth and inhibits apoptosis of endothelial cells. In this article we show that VE-cadherin induces expression of growth arrest-specific 1 (Gas1), an integral membrane protein up-regulated in nonproliferating cells. By comparing syngenic endothelial cell lines, we found that Gas1 mRNA was increased by 3-fold in VE-cadherin-positive cells in comparison to VE-cadherin-null cells. Ectopic expression of Gas1 in endothelial or 293 cells strongly reduced apoptosis without affecting cell growth. Addition of vascular endothelial growth factor (VEGF) also up-regulated Gas1 and this effect was augmented more so in confluent nonproliferating cells than in sparse cultures. VE-cadherin-blocking antibody partially inhibited VEGF-induced Gas1, suggesting that VE-cadherin clustering is required for an optimal response to this stimulus. Inhibition of phosphoinositole-3-OH kinase (PI3-kinase) pathway by Wortmannin prevented Gas1 synthesis and the antiapoptotic effect of VEGF, but, in cells ectopically expressing Gas1, Wortmannin was ineffective. Furthermore, inhibition of Gas1 expression by short interfering RNA (siRNA) both in vitro and in allantois organ cultures made endothelial cells refractory to the antiapoptotic effect of VEGF. Overall these data indicate that Gas1 induction by VE-cadherin and VEGF in endothelial cells requires activation of PI3-kinase. Gas1 expression positively correlates with inhibition of endothelial cell apoptosis and may contribute to the integrity of resting endothelium.

We previously showed that clonable skeletal myogenic cells can be derived from the embryonic aorta but become very rare in the more mature and structured fetal aorta. The aim of this study was to investigate whether, during fetal and postnatal development, these myogenic progenitors progressively disappear or may rather associate with the microvascular district, being thus distributed to virtually all tissues. To test this hypothesis, we used F1 embryos (or mice) from a transgenic line expressing a striated muscle-specific reporter gene (LacZ) crossed with a transgenic line expressing a different endothelial-specific reporter genes (GFP). Endothelial cells were isolated from yolk sac (at E11) and lung (at E11, E17, P1, P10, and P60), two organs embryologically unrelated to paraxial mesoderm, rich in vessels, and devoid of skeletal muscle. Endothelial cells, purified by magnetic bead selection (CD31/PECAM-1(+)) or cell sorting (Tie2-GFP(+)) were then challenged for their skeletal myogenic potential in vitro and in vivo. The results demonstrated that both yolk sac and lung contain progenitor cells, which express endothelial markers and are endowed with a skeletal myogenic potential that they reveal when in the presence of differentiating myoblasts, in vitro, and regenerating muscle, in vivo. The number (or potency to generate skeletal muscle) of these vessels associated cells decreases rapidly with age and is very low in mature animals, possibly correlating with reduced regenerative capacity of adult mammalian tissues.

Using the Cre/loxP system we conditionally inactivated beta-catenin in endothelial cells. We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered. In addition, in many regions, the vascular lumen was irregular with the formation of lacunae at bifurcations, vessels were frequently hemorrhagic, and fluid extravasation in the pericardial cavity was observed. Cultured beta-catenin -/- endothelial cells showed a different organization of intercellular junctions with a decrease in alpha-catenin in favor of desmoplakin and marked changes in actin cytoskeleton. These changes paralleled a decrease in cell-cell adhesion strength and an increase in paracellular permeability. We conclude that in vivo, the absence of beta-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts. This may become more marked when the vessels are exposed to high or turbulent flow, such as at bifurcations or in the beating heart, leading to fluid leakage or hemorrhages.

Several platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) family members display C-terminal protein motifs that confer retention of the secreted factors within the pericellular space. To address the role of PDGF-B retention in vivo, we deleted the retention motif by gene targeting in mice. This resulted in defective investment of pericytes in the microvessel wall and delayed formation of the renal glomerulus mesangium. Long-term effects of lack of PDGF-B retention included severe retinal deterioration, glomerulosclerosis, and proteinuria. We conclude that retention of PDGF-B in microvessels is essential for proper recruitment and organization of pericytes and for renal and retinal function in adult mice.

Vascular endothelial cadherin (VE-cadherin) is an endothelial-specific, trans-membrane protein that promotes homophilic cell adhesion. Inhibition of VE-cadherin by the blocking monoclonal antibody (mAb) BV13 inhibited angiogenesis and tumor growth in vivo. However, this effect was accompanied by a marked increase in lung and heart permeability. In the present paper, we characterize a different VE-cadherin mAb (BV14) that is able to inhibit angiogenesis without affecting vascular permeability. In vitro studies show that BV14, in contrast to BV13, did not increase paracellular permeability of endothelial monolayers and did not disrupt VE-cadherin clusters at junctions. However, both antibodies could inhibit formation of vascularlike structures in collagen gels and increase migration of endothelial cells into wounded areas. In vivo, BV14 and BV13 were equally active in inhibiting angiogenesis in the mouse cornea and in reducing the growth of hemangioma and C6 glioma. In contrast to BV13, BV14 did not change vascular permeability in all the organs tested and at any dose used. BV14 and BV13 bind to VE-cadherin extracellular repeats EC4 and EC1, respectively. We propose that, in resting vessels, where junctions are stable and well-structured, antibody binding to EC1 but not EC4 disrupts their organization and increases permeability. In contrast, in growing vessels, where endothelial cells are migrating and junctions are weaker, antibody binding to EC4 may be sufficient to disrupt cell-to-cell adhesion and inhibit assembly of new vascular structures.

Blood-flow interactions with the vascular endothelium represents a specialized example of mechanical regulation of cell function that has important physiological and pathophysiological cardiovascular consequences. Yet, the mechanisms of mechanostransduction are not understood fully. This study shows that shear stress induces a rapid induction as well as nuclear translocation of the vascular endothelial growth factor (VEGF) receptor 2 and promotes the binding of the VEGF receptor 2 and the adherens junction molecules, VE-cadherin and beta-catenin, to the endothelial cytoskeleton. These changes are accompanied by the formation of a complex containing the VEGF receptor 2-VE-cadherin-beta-catenin. In endothelial cells lacking VE-cadherin, shear stress did not augment nuclear translocation of the VEGF receptor 2 and phosphorylation of Akt1 and P38 as well as transcriptional induction of a reporter gene regulated by a shear stress-responsive promoter. These results suggest that VEGF receptor 2 and the adherens junction act as shear-stress cotransducers, mediating the transduction of shear-stress signals into vascular endothelial cells.

Vascular endothelial-cadherin (VE-cadherin) is an endothelial cell-specific adhesion molecule that is localized exclusively at cell-cell contacts referred to as adherens junctions. VE-cadherin-mediated adhesion is crucial for proper assembly of vascular structures during angiogenesis as well as for maintenance of a normal vascular integrity. We have shown previously that a monoclonal antibody (BV13) to VE-cadherin not only inhibits the formation of vascular tubes during tumor angiogenesis but also disrupts adherens junctions of normal vasculature with a concomitant increase in vascular permeability. The goal of the current studies was to block VE-cadherin function during angiogenesis without disrupting existing junctions on normal endothelium. Using in vitro screening assays to test for functional blocking of adherens junction formation and in vivo assays to detect antibody effects on vascular permeability in normal tissues, we have identified a novel blocking antibody (E4G10) that inhibits VE-cadherin function during angiogenesis but does not disrupt existing adherens junctions on normal vasculature. E4G10 inhibited formation of vascular tubes in vivo in the Matrigel plug and corneal micropocket assays. E4G10 also inhibited tumor growth in three models of mouse and human tumors via an antiangiogenic mechanism. Examination of normal mouse and tumor tissues showed that E4G10 bound to endothelial cells in a subset of tumor vasculature but not to normal vasculature. Bromodeoxyuridine labeling experiments showed that E4G10 specifically targeted a subset of tumor endothelium that is undergoing active cell proliferation, which likely reflects the activated, angiogenic endothelium. These findings indicate that VE-cadherin can be selectively targeted during states of pathological angiogenesis, despite its ubiquitous distribution throughout the entire vasculature. Our data also suggest that antibody E4G10 recognizes VE-cadherin epitopes that are only accessible on endothelial cells forming new adherens junctions, such as in angiogenic tumor vasculature.

Previously published reports support the concept that, besides promoting homotypic intercellular adhesion, cadherins may transfer intracellular signals. However, the signaling pathways triggered by cadherin clustering and their biological significance are still poorly understood. We report herein that transfection of VE-cadherin (VEC) cDNA in VEC null endothelial cells induces actin rearrangement and increases the number of vinculin positive adhesion plaques. VEC expression augments the level of active Rac but decreases active Rho. Microinjection of a dominant negative Rac mutant altered stress fiber organization, whereas inhibition of Rho was ineffective. VEC expression increased protein and mRNA levels of the Rac-specific guanosine exchange factor Tiam-1 and induced its localization at intercellular junctions. In addition, in the presence of VEC, the amounts of Tiam, Rac, and the Rac effector PAK as well as the level of PAK phosphorylation were found increased in the membrane/cytoskeletal fraction. These observations are consistent with a role of VEC in localizing Rac and its signaling partners in the same membrane compartment, facilitating their reciprocal interaction. Through this mechanism VEC may influence the constitutive organization of the actin cytoskeleton.

The concept of tissue-restricted differentiation of postnatal stem cells has been challenged by recent evidence showing pluripotency for hematopoietic, mesenchymal, and neural stem cells. Furthermore, rare but well documented examples exist of already differentiated cells in developing mammals that change fate and trans-differentiate into another cell type. Here, we report that endothelial cells, either freshly isolated from embryonic vessels or established as homogeneous cells in culture, differentiate into beating cardiomyocytes and express cardiac markers when cocultured with neonatal rat cardiomyocytes or when injected into postischemic adult mouse heart. Human umbilical vein endothelial cells also differentiate into cardiomyocytes under similar experimental conditions and transiently coexpress von Willebrand factor and sarcomeric myosin. In contrast, neural stem cells, which efficiently differentiate into skeletal muscle, differentiate into cardiomyocytes at a low rate. Fibroblast growth factor 2 and bone morphogenetic protein 4, which activate cardiac differentiation in embryonic cells, do not activate cardiogenesis in endothelial cells or stimulate trans-differentiation in coculture, suggesting that different signaling molecules are responsible for cardiac induction during embryogenesis and in successive periods of development. The fact that endothelial cells can generate cardiomyocytes sheds additional light on the plasticity of endothelial cells during development and opens perspectives for cell autologous replacement therapies.

Endothelial cell-cell junctions play an important role in vascular hemostasis. The two junctional proteins VE-cadherin and JAM-1 are localized at adherens and tight junctions, respectively. VE-cadherin is only expressed by endothelial cells, suggesting that it can exert cell specific function. Absence of VE-cadherin or blocking of its adhesive activity prevents a normal organization of new vascular structures, suggesting that VE-cadherin may be a molecular target of antiangiogenic therapy. In addition, the ability of permeability-increasing agents and adherent leukocytes to modify VE-cadherin/catenin organization may be related to a role in the control of vascular permeability and leukocyte infiltration. JAM-1 is an integral membrane protein expressed in endothelial and epithelial cells. Its extracellular domain can dimerize and bind homophilically. The intracellular domain (and in particular a PDZ-binding motif) enables JAM-1 to interact with structural and signaling proteins. Study of the molecular interactions of JAM-1 may help explain mechanisms of JAM-mediated function, such as control of paracellular permeability and leukocyte transmigration.

Exchange of solutes and ions between the luminal and abluminal compartments of the circulation is critically dependent on the barrier properties of the vascular endothelium. Transport of solutes and fluids occurs along the transcellular and paracellular pathways that are mediated by intracellular vesicles and intercellular junctions, respectively. Although the ability of endothelial cells to dynamically regulate permeability has long been recognized, the precise mechanism and the signaling pathways involved have not been fully elucidated. Finally, current definition of the complex molecular composition of intercellular junctions is expected to explain the difference in permeability between diverse segments of the circulation and possibly to highlight the existence of specific junctional channels. The properties of junctional adhesion molecule-1 (JAM-1) and vascular endothelial cadherin (VE-cadherin), two transmembrane components of interendothelial junctions, are described in detail.

Vascular endothelial cadherin (VE-cadherin) is an endothelial cell-specific cadherin that plays an important role in the control of vascular organization. Blocking VE-cadherin antibodies strongly inhibit angiogenesis, and inactivation of VE-cadherin gene causes embryonic lethality due to a lack of correct organization and remodeling of the vasculature. Hence, inhibitors of VE-cadherin adhesive properties may constitute a tool to prevent tumor neovascularization. In this paper, we tested different monoclonal antibodies (mAbs) directed to human VE-cadherin ectodomain for their functional activity. Three mAbs (Cad 5, BV6, BV9) were able to increase paracellular permeability, inhibit VE-cadherin reorganization, and block angiogenesis in vitro. These mAbs could also induce endothelial cell apoptosis in vitro. Two additional mAbs, TEA 1.31 and Hec 1.2, had an intermediate or undetectable activity, respectively, in these assays. Epitope mapping studies show that BV6, BV9, TEA 1.31, and Hec 1.2 bound to a recombinant fragment spanning the extracellular juxtamembrane domains EC3 through EC4. In contrast, Cad 5 bound to the aminoterminal domain EC1. By peptide scanning analysis and competition experiments, we defined the sequences TIDLRY located on EC3 and KVFRVDAETGDVFAI on EC1 as the binding domain of BV6 and Cad 5, respectively. Overall, these results support the concept that VE-cadherin plays a relevant role on human endothelial cell properties. Antibodies directed to the extracellular domains EC1 but also EC3-EC4 affect VE-cadherin adhesion and clustering and alter endothelial cell permeability, apoptosis, and vascular structure formation.

Murine vascular endothelial cadherin-2 (VE-cad-2) is a cellular adhesion molecule that is distinct from vascular endothelial cadherin 1 (VE-cad-1) in that it does not interact with catenins and does not appear to affect cell migration or growth. In this study, we have cloned a full-length cDNA of the human homolog of VE-cad-2 and used it to map the chromosomal locus of the VE-cad-2 gene. Human VE-cad-2 maps to Chromosome (Chr) 5q31. The cDNA of human VE-cad-2 is highly homologous to mouse VE-cad-2, except for a C-terminal tail. The genomic structure of VE-cad-2 is strikingly similar to that reported for a large family of neuronal protocadherin genes mapped to Chr 5q, yet the amino acid sequences between VE-cad-2 and the protocadherins are substantially divergent. The promoter of human VE-cad-2 contains two TATA boxes and transcription initiates from a single site 3' to these elements. Similar to mouse VE-cad-2, the human gene is expressed primarily in highly vascularized tissues.