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Background: While endothelial dysfunction is suggested to contribute to heart failure with preserved ejection fraction pathophysiology, understanding the importance of the endothelium alone, in the pathogenesis of diastolic abnormalities has not yet been fully elucidated. Here, we investigated the consequences of specific endothelial dysfunction on cardiac function, independently of any comorbidity or risk factor (diabetes or obesity) and their potential effect on cardiomyocyte. Methods: The ubiquitine ligase Pdzrn3 , expressed in endothelial cells (ECs), was shown to destabilize tight junction. A genetic mouse model in which Pdzrn3 is overexpressed in EC (iEC-Pdzrn3) in adults was developed. Results: EC-specific Pdzrn3 expression increased cardiac leakage of IgG and fibrinogen blood-born molecules. The induced edema demonstrated features of diastolic dysfunction, with increased end-diastolic pressure, alteration of dP/dt min, increased natriuretic peptides, in addition to limited exercise capacity, without major signs of cardiac fibrosis and inflammation. Electron microscopic images showed edema with disrupted EC-cardiomyocyte interactions. RNA sequencing analysis of gene expression in cardiac EC demonstrated a decrease in genes coding for endothelial extracellular matrix proteins, which could be related to the fragile blood vessel phenotype. Irregularly shaped capillaries with hemorrhages were found in heart sections of iEC- Pdzrn3 mice. We also found that a high-fat diet was not sufficient to provoke diastolic dysfunction; high-fat diet aggravated cardiac inflammation, associated with an altered cardiac metabolic signature in EC- Pdzrn3 mice, reminiscent of heart failure with preserved ejection fraction features. Conclusions: An increase of endothelial permeability is responsible for mediating diastolic dysfunction pathophysiology and for aggravating detrimental effects of a high-fat diet on cardiac inflammation and metabolism.

BACKGROUND: Endothelial cells (ECs) are sensitive to physical forces created by blood flow, especially to laminar shear stress. Among the cell responses to laminar flow, EC polarization against the flow direction emerges as a key event, particularly during the development and remodeling of the vascular network. EC adopt an elongated planar cell shape with an asymmetrical distribution of intracellular organelles along the axis of blood flow. This study aimed to investigate the involvement of planar cell polarity via the receptor ROR2 (receptor tyrosine kinase-like orphan receptor 2) in endothelial responses to laminar shear stress. METHODS: We generated a genetic mouse model with EC-specific deletion of Ror2 , in combination with in vitro approaches involving loss- and gain-of-function experiments. RESULTS: During the first 2 weeks of life, the endothelium of the mouse aorta undergoes a rapid remodeling associated with a loss of EC polarization against the flow direction. Notably, we found a correlation between ROR2 expression and endothelial polarization levels. Our findings demonstrate that deletion of Ror2 in murine ECs impaired their polarization during the postnatal development of the aorta. in vitro experiments further validated the essential role of ROR2 in both EC collective polarization and directed migration under laminar flow conditions. Exposure to laminar shear stress triggered the relocalization of ROR2 to cell-cell junctions where it formed a complex with VE-Cadherin and β-catenin, thereby regulating adherens junctions remodeling at the rear and front poles of ECs. Finally, we showed that adherens junctions remodeling and cell polarity induced by ROR2 were dependent on the activation of the small GTPase Cdc42. CONCLUSIONS: This study identified ROR2/planar cell polarity pathway as a new mechanism controlling and coordinating collective polarity patterns of EC during shear stress response.

Background: Genome-wide association studies have revealed robust associations of common genetic polymorphisms in an intron of the PHACTR-1 (phosphatase and actin regulator 1) gene (chr6p24), with cervical artery dissection, spontaneous coronary artery dissection, and fibromuscular dysplasia. The aim was to assess its role in the pathogenesis of cervical artery dissection or fibromuscular dysplasia. Methods: Using various tissue-specific Cre-driver mouse lines, Phactr1 was deleted either in endothelial cells using 2 tissue-specific Cre-driver (PDGFB [platelet-derived growth factor B]-Cre ERT2 mice and Tie2 [tyrosine kinase with immunoglobulin and EGF homology domains]-Cre) and smooth muscle cells (smooth muscle actin-Cre ERT2 ) with a third tissue-specific Cre-driver. Results: To test the efficacy of the Phactr1 deletion after cre-induction, we confirmed first, a decrease in Phactr1 transcription and Phactr1 expression in endothelial cell and smooth muscle cell isolated from Phactr1 iPDGFB and Phactr1 iSMA mice. Irrespective to the tissue or the duration of the deletion, mice did not spontaneously display pathological phenotype or vascular impairment: mouse survival, growth, blood pressure, large vessel morphology, or actin organization were not different in knockout mice than their comparatives littermates. Challenging vascular function and repair either by angiotensin II–induced hypertension or limb ischemia did not lead to vascular morphology or function impairment in Phactr1-deleted mice. Similarly, there were no more consequences of Phactr1 deletion during embryogenesis in endothelial cells. Conclusions: Loss of PHACTR-1 function in the cells involved in vascular physiology does not appear to induce a pathological vascular phenotype. The in vivo effect of the intronic variation described in genome-wide association studies is unlikely to involve downregulation in PHACTR-1 expression.

résumé de communication dans un congrès; International audience; Blood brain barrier (BBB) disruption is a critical component of the pathophysiology of cognitive impairment of vascular etiology (VCI) and associated with Alzheimer's disease (AD). The Wnt pathway plays a crucial role in BBB maintenance, but there is limited data on its role in cognitive pathologies. The E3 ubiquitin ligase PDZRN3 is a regulator of the Wnt pathway. In a murine model of VCI, overexpressing Pdzrn3 in endothelial cell (EC) exacerbated BBB hyperpermeability and accelerated cognitive decline. We extended these observations, in both VCI and AD models, showing that EC-specific depletion of Pdzrn3, reinforced the BBB, with a decrease in vascular permeability and a subsequent spare in cognitive decline. We found that in cerebral vessels, Pdzrn3 depletion protects against AD-induced Wnt target gene alterations and enhances endothelial tight junctional proteins. Our results provide evidence that Wnt signaling could be a molecular link regulating BBB integrity and cognitive decline under VCI and AD pathologies.

Introduction Genome-wide association studies have revealed robust associations of common genetic polymorphisms in the Phactr1 locus (6p24) (encoding Phosphatase and Actin Regulator 1) with cervico-cerebral artery dissection (CCeAD), spontaneous coronary artery dissection (SCAD) and fibromuscular dysplasia (FMD). These common elements of genetic predisposition provide a proof of concept for important shared mechanisms at the molecular level between CCeAD, SCAD and FMD vascular diseases. Objective Deciphering genetic, molecular and physiological importance of PHACTR1. Method Using various tissue-specific Cre-driver mouse lines, Phactr1 was deleted either in endothelial cells (EC) using 2 tissue-specific Cre-driver (one of them was embryonic) and Smooth Muscle Cells (SMC) with a third tissue-specific Cre-driver to assess its role in the pathogenesis of CCeAD, SCAD or FMD. Results To test the efficacy of the Phactr1 deletion after cre-induction, we confirmed firstly, a decrease in Phactr1 transcription and Phactr1 expression in EC and SMC isolated from Phactr1iPDGFB and Phactr1iSMA mice. Irrespective to the tissue or the duration of the deletion, mice did not spontaneously display pathological phenotype or vascular impairment: mouse survival, growth, blood pressure, large vessel morphology or actin organization were not different in KO mice than their comparatives littermates. Challenging vascular function and repair either by angiotensin-II-induced hypertension or limb ischemia did not lead to vascular morphology or function impairment in Phactr1-deleted mice. Similarly, there was no more consequences of Phactr1 deletion during embryogenesis in ECs. Conclusion Loss of function of PHACTR-1 in the cell types mostly involved in vascular physiology does not appear to induce a pathological vascular phenotype. Funding Acknowledgement Type of funding sources: None.

Sense organs acquire their distinctive shapes concomitantly with the differentiation of sensory cells and neurons necessary for their function. Although our understanding of the mechanisms controlling morphogenesis and neurogenesis in these structures has grown, how these processes are coordinated remains largely unexplored. Neurogenesis in the zebrafish olfactory epithelium requires the bHLH proneural transcription factor Neurogenin 1 (Neurog1). To address whether Neurog1 also controls morphogenesis, we analysed the migratory behaviour of early olfactory neural progenitors in neurog1 mutant embryos. Our results indicate that the oriented movements of these progenitors are disrupted in this context. Morphogenesis is similarly affected by mutations in the chemokine receptor gene, cxcr4b, suggesting it is a potential Neurog1 target gene. We find that Neurog1 directly regulates cxcr4b through an E-box cluster located just upstream of the cxcr4b transcription start site. Our results suggest that proneural transcription factors, such as Neurog1, directly couple distinct aspects of nervous system development.

Heart failure is the final common stage of most cardiopathies. Cardiomyocytes connect with others via their extremities by intercalated disk protein complexes. This planar and directional organization of myocytes is crucial for mechanical coupling and anisotropic conduction of the electric signal in the heart. One of the hallmarks of heart failure is alterations in the contact sites between cardiomyocytes. Yet no factor on its own is known to coordinate cardiomyocyte polarized organization. We report enhanced levels of an ubiquitine ligasePdzrn3in diseased hypertrophic human and mouse myocardium, which correlates with a loss of cardiomyocyte polarized elongation. We provide evidence thatPdzrn3has a causative role in heart failure. We found that cardiacPdzrn3deficiency protected against heart failure while over expression ofPdzrn3in mouse cardiomyocytes during the first weeks of life, impaired postnatal cardiomyocyte maturation leading to premature death. Our results reveal a novel signaling pathway that controls a genetic program essential for heart maturation and maintenance of overall geometry, as well as the contractile function of cardiomyocytes, and implicates PDZRN3 as a potential therapeutic target for the prevention of human heart failure.

International audience; The Wnt/frizzled signaling pathway is one of the major regulators of endothelial biology, controlling key cellular activities. Many secreted Wnt ligands have been identified and can initiate diverse signaling via binding to a complex set of Frizzled (Fzd) transmembrane receptors and coreceptors. Roughly, Wnt signaling is subdivided into two pathways: the canonical Wnt/β-catenin signaling pathway whose main downstream effector is the transcriptional coactivator β-catenin, and the noncanonical Wnt signaling pathway, which is subdivided into the Wnt/Ca2+ pathway and the planar cell polarity pathway. Here, we will focus on its cross talk with other angiogenic pathways and on its role in blood-retinal- and blood-brain-barrier formation and its maintenance in a differentiated state. We will unravel how retinal vascular pathologies and neurovascular degenerative diseases result from disruption of the Wnt pathway related to vascular instability, and highlight current research into therapeutic options.

Retinopathies remain major causes of visual impairment in diabetic patients and premature infants. Introduction of anti-angiogenic drugs targeting vascular endothelial growth factor (VEGF) has transformed therapy for these proliferative retinopathies. However, limitations associated with anti-VEGF medications require to unravel new pathways of vessel growth to identify potential drug targets. Here, we investigated the role of Wnt/Frizzled-7 (Fzd7) pathway in a mouse model of oxygen-induced retinopathy (OIR). Using transgenic mice, which enabled endothelium-specific and time-specific Fzd7 deletion, we demonstrated that Fzd7 controls both vaso-obliteration and neovascular phases (NV). Deletion of Fzd7 at P12, after the ischemic phase of OIR, prevented formation of aberrant neovessels into the vitreous by suppressing proliferation of endothelial cells (EC) in tufts. Next we validated in vitro two Frd7 blocking strategies: a monoclonal antibody (mAbFzd7) against Fzd7 and a soluble Fzd7 receptor (CRD). In vivo a single intravitreal microinjection of mAbFzd7 or CRD significantly attenuated retinal neovascularization (NV) in mice with OIR. Molecular analysis revealed that Fzd7 may act through the activation of Wnt/β-catenin and Jagged1 expression to control EC proliferation in extra-retinal neovessels. We identified Fzd7/β-catenin signaling as new regulator of pathological retinal NV. Fzd7 appears to be a potent pharmacological target to prevent or treat aberrant angiogenesis of ischemic retinopathies.

Sense organs acquire their distinctive shapes concomitantly with the differentiation of sensory cells and neurons necessary for their function. While our understanding of the mechanisms controlling morphogenesis and neurogenesis in these structures has grown, how these processes are coordinated remains largely unexplored. Neurogenesis in the zebrafish olfactory epithelium requires the bHLH proneural transcription factor Neurogenin1 (Neurog1). To address whether Neurog1 also controls morphogenesis in this system, we analysed the morphogenetic behaviour of early olfactory neural progenitors inneurog1mutant embryos. Our results indicate that the oriented movements of these progenitors are disrupted in this context. Morphogenesis is similarly affected by mutations in the chemokine receptor gene,cxcr4b, suggesting it is a potential Neurog1 target gene. We find that Neurog1 directly regulatescxcr4bthrough an E-boxes cluster located just upstream of thecxcr4btranscription start site. Our results suggest that proneural transcription factors, such as Neurog1, directly couple distinct aspects of nervous system development.One Sentence SummaryNeurog1 controls olfactory organ morphogenesis viacxcr4b

Pitx2c, a homeodomain transcription factor, is classically known for its left-right patterning role. However, an early wave of pitx2 expression occurs at the onset of gastrulation in several species, indicating a possible earlier role that remains relatively unexplored. Here we show that in zebrafish, maternal-zygotic (MZ) pitx2c mutants exhibit a shortened body axis indicative of convergence and extension (CE) defects. Live imaging reveals that MZpitx2c mutants display less persistent mesendodermal migration during late stages of gastrulation. Transplant data indicate that Pitx2c functions cell non-autonomously to regulate this cell behavior by modulating cell shape and protrusive activity. Using transcriptomic analyses and candidate gene approaches, we identify transcriptional changes in components of the chemokine-ECM-integrin dependent mesendodermal migration network. Together, our results define pathways downstream of Pitx2c that are required during early embryogenesis and reveal novel functions for Pitx2c as a regulator of morphogenesis.

Vertebrate olfactory placodes consists of a variety of neuronal populations, which are thought to have distinct embryonic origins. In the zebrafish, while ciliated sensory neurons arise from preplacodal ectoderm (PPE), previous lineage tracing studies suggest that both Gonadotropin releasing hormone 3 (Gnrh3) and microvillous sensory neurons derive from cranial neural crest (CNC). We find that the expression of Islet1/2 is restricted to Gnrh3 neurons associated with the olfactory placode. Unexpectedly, however, we find no change in Islet1/2+ cell numbers insox10mutant embryos, calling into question their CNC origin. Lineage reconstruction based on backtracking in time-lapse confocal datasets, and confirmed by photoconversion experiments, reveals that Gnrh3 neurons derive from the anterior/medial PPE. Similarly, all of the microvillous sensory neurons we have traced arise from preplacodal progenitors. Our results suggest that rather than originating from separate ectodermal populations, cell-type heterogeneity is generated from overlapping pools of progenitors within the preplacodal ectoderm.

Introduction Endothelial cells (ECs) are sensitive to physical forces created by blood flow, especially to shear stress (SS). ECs transduce this mechanical forces into intracellular signals leading to gene expression, cytoskeleton reorganization, cell elongation and polarization against blood flow direction. Defects in ECs adaption to blood flow are highly correlated with cardiovascular diseases including atherosclerosis. Recently, it has been shown that non-canonical Wnt signaling modulates the endothelial shear stress flow sensor in vascular remodeling. Objective ROR2, a receptor tyrosine kinase, is known to regulate non canonical Wnt/PCP signaling. This study investigates the role of ROR2 in ECs response to flow. Method Different types of ECs were exposed to low orbital shear stress (OSS) (3,6 dynes/cm2). After 24 h or 72 h of OSS, ROR2 and Klf2, a flow-induced gene, expression were assessed by qRT-PCR and/or Western Blot. ROR2 gain- (lentivirus) and loss-of-function (siRNA) were used to decipher its role in ECs flow response. ECs polarization under laminar shear stress (LSS) were studied using laminar flow chamber slide. After 18 h of LSS (5 dynes/cm2), ECs were immunostained for VE-cadherin, WGA, ROR2 and pericentrin. Results As expected, OSS induced Klf2 expression as compared to static condition. Interestingly, ROR2 expression was induced at the protein and gene level by OSS. To analyse whether ROR2 could be upstream or downstream Klf2 induction, ECs were treated with ROR2 or control siRNA and subjected to OSS for 24 h. siRNA depletion of ROR2 decreased flow-induced Klf2 expression. In LSS conditions, ECs was mostly polarized against flow direction (pericentrin, Hoechst localization) and we observed ROR2 relocalization at cell-cell junctions, as compared to static conditions. Conclusion This study shows that ROR2 is involved in ECs responses to flow. Study of ROR2 signaling is under investigation to understand how ROR2 could regulate ECs properties.

Endothelial cells serve as a barrier between blood and tissues. Maintenance of the endothelial cell barrier depends on the integrity of intercellular junctions, which is regulated by a polarity complex that includes the ζ isoform of atypical protein kinase C (PKCζ) and partitioning defective 3 (PAR3). We revealed that the E3 ubiquitin ligase PDZ domain-containing ring finger 3 (PDZRN3) regulated endothelial intercellular junction integrity. Endothelial cell-specific overexpression of Pdzrn3 led to early embryonic lethality with severe hemorrhaging and altered organization of endothelial intercellular junctions. Conversely, endothelial-specific loss of Pdzrn3 prevented vascular leakage in a mouse model of transient ischemic stroke, an effect that was mimicked by pharmacological inhibition of PKCζ. PDZRN3 regulated Wnt signaling and associated with a complex containing PAR3, PKCζ, and the multi-PDZ domain protein MUPP1 (Discs Lost-multi-PDZ domain protein 1) and targeted MUPP1 for proteasomal degradation in transfected cells. Transient ischemic stroke increased the ubiquitination of MUPP1, and deficiency of MUPP1 in endothelial cells was associated with decreased localization of PKCζ and PAR3 at intercellular junctions. In endothelial cells, Pdzrn3 overexpression increased permeability through a PKCζ-dependent pathway. In contrast, Pdzrn3 depletion enhanced PKCζ accumulation at cell-cell contacts and reinforced the cortical actin cytoskeleton under stress conditions. These findings reveal how PDZRN3 regulates vascular permeability through a PKCζ-containing complex.

Objective— Vessel formation requires precise orchestration of a series of morphometric and molecular events controlled by a multitude of angiogenic factors and morphogens. Wnt/frizzled signaling is required for proper vascular formation. In this study, we investigated the role of the Fzd7 (frizzled-7) receptor in retinal vascular development and its relationship with the Wnt/β-catenin canonical pathway and Notch signaling. Approach and Results— Using transgenic mice, we demonstrated that Fzd7 is required for postnatal vascular formation. Endothelial cell (EC) deletion of fzd7 ( fzd7 ECKO ) delayed retinal plexus formation because of an impairment in tip cell phenotype and a decrease in stalk cell proliferation. Dvl (dishevelled) proteins are a main component of Wnt signaling and play a functionally redundant role. We found that Dvl3 depletion in dvl1 −/− mice mimicked the fzd7 ECKO vascular phenotype and demonstrated that Fzd7 acted via β-catenin activation by showing that LiCl treatment rescued impairment in tip and stalk cell phenotypes induced in fzd7 mutants. Deletion of fzd7 or Dvl1/3 induced a strong decrease in Wnt canonical genes and Notch partners’ expression. Genetic and pharmacological rescue strategies demonstrated that Fzd7 acted via β-catenin activation, upstream of Notch signaling to control Dll4 and Jagged1 EC expression. Conclusions— Fzd7 expressed by EC drives postnatal angiogenesis via activation of Dvl/β-catenin signaling and can control the integrative interaction of Wnt and Notch signaling during postnatal angiogenesis.

Rationale: A growing body of evidence supports the hypothesis that the Wnt/planar cell polarity (PCP) pathway regulates endothelial cell proliferation and angiogenesis, but the components that mediate this regulation remain elusive. Objective: We investigated the involvement of one of the receptors, Frizzled4 (Fzd4), in this process because its role has been implicated in retinal vascular development. Methods and Results: We found that loss of fzd4 function in mice results in a striking reduction and impairment of the distal small artery network in the heart and kidney. We report that loss of fzd4 decreases vascular cell proliferation and migration and decreases the ability of the endothelial cells to form tubes. We show that fzd4 deletion induces defects in the expression level of stable acetylated tubulin and in Golgi organization during migration. Deletion of fzd4 favors Wnt noncanonical AP1-dependent signaling, indicating that Fzd4 plays a pivotal role favoring PCP signaling. Our data further demonstrate that Fzd4 is predominantly localized on the top of the plasma membrane, where it preferentially induces Dvl3 relocalization to promote its activation and α-tubulin recruitment during migration. In a pathological mouse angiogenic model, deletion of fzd4 impairs the angiogenic response and leads to the formation of a disorganized arterial network. Conclusions: These results suggest that Fzd4 is a major receptor involved in arterial formation and organization through a Wnt/PCP pathway.

International audience; Mesenchymal stem cells (MSC) are multipotent postnatal stem cells, involved in the treatment of ischemic vascular diseases. We investigate the ability of MSC, exposed to short-term hypoxic conditions, to participate in vascular and tissue regeneration in an in vivo model of hindlimb ischemia. Transplantation of hypoxic preconditioned murine MSC (HypMSC) enhanced skeletal muscle regeneration at day 7, improved blood flow and vascular formation compared to injected nonpreconditioned MSC (NormMSC). These observed effects were correlated with an increase in HypMSC engraftment and a putative role in necrotic skeletal muscle fiber clearance. Moreover, HypMSC transplantation resulted in a large increase in Wnt4 (wingless-related MMTV integration site 4) expression and we demonstrate its functional significance on MSC proliferation and migration, endothelial cell (EC) migration, as well as myoblast differentiation. Furthermore, suppression of Wnt4 expression in HypMSC, abrogated the hypoxia-induced vascular regenerative properties of these cells in the mouse hindlimb ischemia model. Our data suggest that hypoxic preconditioning plays a critical role in the functional capabilities of MSC, shifting MSC location in situ to enhance ischemic tissue recovery, facilitating vascular cell mobilization, and skeletal muscle fiber regeneration via a paracrine Wnt-dependent mechanism.

Introduction Vessel formation requires a precise orchestration of morphometric and molecular events controlled by a multitude of angiogenic factors and morphogens. Growing evidence has shown that Wnt/PCP pathway is important for vessel formation. We previously demonstrated that ubiquitin ligase E3 PDZRN3 drives non-canonical Wnt/PCP signaling to control post-natal angiogenesis in the retina. Endothelial cell polarity is essential to control vascular properties and endothelial cell polarization. Objectifs Orphan tyrosine receptor kinase ROR2 has been described to regulate Wnt/PCP signaling. The goal of this study was to investigate the role of ROR2 during vascular formation and its role in EC polarity. Methods and results For this purpose we first analyzed the expression of ROR2 during the kinetic of vascular formation in the retina. After in toto immunofluorescence staining, ROR2 is observed in different cells in the retina and in CD31+ cells. By Western blot and qRT-PCR, we showed that ROR2 is strongly expressed at P4-P7 and decreased after P8. To analyze the role of ROR2 in the endothelium, transgenic mice deleted for ROR2 specifically in the endothelium (CDH5-iCre;ROR2F/F and Pdgfb-iCre; ROR2F/F mice) were generated. The retinas of pups sacrificed 5, 7 and 11 days after birth were immunostained (CD31, isolectin B4, NG2, SMA, ColIV, KI67) to study the kinetics of vascular plexus formation. Deletion of ROR2 gene induced a significant decrease in vessel plexus formation at P5, P7 and P11 in Pdgfb-ROR2iECKO and CDH5-ROR2iECKO mice compared to littermates. Conclusion This study shows that ROR2 is required for the formation of primary but also ternary vascular network. Investigation of the ROR2 signaling and molecular mechanisms are under investigation to understand whether and how ROR2/PCP pathway could regulate EC properties.

The translocator protein (18 kDa) (TSPO), also known as peripheral-type benzodiazepine receptor, is directly or indirectly associated with many biological processes. Although extensively characterized, the specific function of TSPO during development remains unclear. It has been reported that TSPO is involved in a variety of mechanisms, including cell proliferation, apoptosis, regulation of mitochondrial functions, cholesterol transport and steroidogenesis, and porphyrin transport and heme synthesis. Although the literature has reported a murine knockout model, the experiment did not generate information because of early lethality. We then used the zebrafish model to address the function of tspo during development. Information about spatiotemporal expression showed that tspo has a maternal and a zygotic contribution which, during somatogenesis, seems to be erythroid restricted to the intermediate cell mass. Genetic and pharmacological approaches used to invalidate Tspo function resulted in embryos with specific erythropoietic cell depletion. Although unexpected, this lack of blood cells is independent of the Tspo cholesterol binding site and reveals a new in vivo key role for Tspo during erythropoiesis.

Consistent with findings of Wnt pathway members involved in vascular cells, a role for Wnt/Frizzled signaling has recently emerged in vascular cell development. Among the few Wnt family members implicated in vessel formation in adult, Wnt7b and Frizzled 4 have been shown as involved in vessel formation in the lung and in the retina, respectively. Our previous work has shown a role for secreted Frizzled-related protein-1 (sFRP-1), a proposed Wnt signaling inhibitor, in neovascularization after an ischemic event and demonstrated its role as a potent angiogenic factor. However the mechanisms involved have not been investigated. Here, we show that sFRP-1 treatment increases endothelial cell spreading on extracellular matrix as revealed by actin stress fiber reorganization in an integrin-dependent manner. We demonstrate that sFRP-1 can interact with Wnt receptors Frizzled 4 and 7 on endothelial cells to transduce downstream to cellular machineries requiring Rac-1 activity in cooperation with GSK-3β. sFRP-1 overexpression in endothelium specifically reversed the inactivation of GSK-3β and increased neovascularization in ischemia-induced angiogenesis in mouse hindlimb. This study illustrates a regulated pathway by sFRP-1 involving GSK-3β and Rac-1 in endothelial cell cytoskeletal reorganization and in neovessel formation.

An atypical kinesin, Kif26b, is identified as a signaling effector of the Wnt PCP under Daam1 signaling. Molecular evidence shows that it interacts with Dvl3/Daam1. Kif26b is involved in endothelial microtubule network stabilization and has a crucial role together with the Dvl3/Daam1 complex in establishing endothelial cell polarity., Angiogenesis involves the coordinated growth and migration of endothelial cells (ECs) toward a proangiogenic signal. The Wnt planar cell polarity (PCP) pathway, through the recruitment of Dishevelled (Dvl) and Dvl-associated activator of morphogenesis (Daam1), has been proposed to regulate cell actin cytoskeleton and microtubule (MT) reorganization for oriented cell migration. Here we report that Kif26b—a kinesin—and Daam1 cooperatively regulate initiation of EC sprouting and directional migration via MT reorganization. First, we find that Kif26b is recruited within the Dvl3/Daam1 complex. Using a three-dimensional in vitro angiogenesis assay, we show that Kif26b and Daam1 depletion impairs tip cell polarization and destabilizes extended vascular processes. Kif26b depletion specifically alters EC directional migration and mislocalized MT organizing center (MTOC)/Golgi and myosin IIB cell rear enrichment. Therefore the cell fails to establish a proper front–rear polarity. Of interest, Kif26b ectopic expression rescues the siDaam1 polarization defect phenotype. Finally, we show that Kif26b functions in MT stabilization, which is indispensable for asymmetrical cell structure reorganization. These data demonstrate that Kif26b, together with Dvl3/Daam1, initiates cell polarity through the control of PCP signaling pathway–dependent activation.

Although the left and right hemispheres of our brains develop with a high degree of symmetry at both the anatomical and functional levels, it has become clear that subtle structural differences exist between the two sides and that each is dominant in processing specific cognitive tasks. As the result of evolutionary conservation or convergence, lateralization of the brain is found in both vertebrates and invertebrates, suggesting that it provides significant fitness for animal life. This widespread feature of hemispheric specialization has allowed the emergence of model systems to study its development and, in some cases, to link anatomical asymmetries to brain function and behavior. Here, we present some of what is known about brain asymmetry in humans and model organisms as well as what is known about the impact of environmental and genetic factors on brain asymmetry development. We specifically highlight the progress made in understanding the development of epithalamic asymmetries in zebrafish and how this model provides an exciting opportunity to address brain asymmetry at different levels of complexity.

Phosphorylation and subsequent inactivation of glycogen synthase kinase (GSK)-3beta via the Akt/PI3-Kinase pathway during ischemic preconditioning (PC) has been shown to be cardioprotective. As FrzA/sFRP-1, a secreted antagonist of the Wnt/Frizzled pathway, is expressed in the heart and is able to decrease the phosphorylation of GSK-3beta in vitro on vascular cells, we examined its effect during PC using transgenic mouse overexpressing FrzA in cardiomyocytes (alpha-MHC promoter) under a conditional transgene expression approach (tet-off system). Overexpression of FrzA inhibited the increase in GSK-3beta phosphorylation as well as protein kinase C (PKC) epsilon activation in transgenic mice after PC as compared with littermates. Phospho-Akt (P-Akt), phospho-JNK, or the cytoplasmic beta-catenin levels were not modified, phospho-p38 (P-p38) was slightly increased in transgenic mice after PC as compared with littermates. FrzA transgenic mice displayed a larger infarct size and a greater worsening of cardiac function compared with littermates. All these differences were reversed by the addition of doxycycline. This study demonstrates for the first time that disruption of a beta-catenin independent Wnt/Frizzled pathway induces the activation of GSK-3beta and reverses the benefit of preconditioning.

The incidence of peripheral arterial disease is rising and despite advances in clinical management, many problems remain unsolved. Better knowledge of the mechanisms and consequences associated with chronic muscle ischemia has opened the way for development of new treatment strategies, including therapeutic angiogenesis. Therapeutic angiogenesis is a promising technique based on experimental studies showing that growth factors or genes able to increase capillary density can be used to reduce the impact of muscle ischemia and increase blood flow to ischemic tissue. Enthusiasm for this technique has prompted numerous clinical trials with encouraging results, but data are still inconclusive. Optimal indications for gene therapy must be defined and further experimental progress is needed to respond to ethical issues. Therapeutic angiogenesis should be viewed as an adjunct to rather than as a competitor of current surgical revascularization techniques.

Background Myocardial infarction results in irreversible myocyte loss. In a murine model, we tested the feasibility of a novel repair technique combining bone marrow cell (BMC) transplantation and cardiomyoplasty. Methods Myocardial infarction was induced cryogenically in backcrossed ROSA 26 transgenic × C57BL/6J mice (n = 75). Thirty days later, surviving mice (n = 69) were randomized to sham treatment (rethoracotomy only; n=11), patch only treatment (n = 29), or patch + BMC treatment (n = 29). Abdominal muscle patches were harvested from donor littermates not expressing the β-galactosidase reporter gene and sutured on the epicardium directly above the infarct zone. Patch only–treated mice received uncoated patches. Patch + BMC–treated mice received patches coated with 5 × 10 6 β-galactosidase-expressing BMCs embedded in a collagen-rich three-dimensional matrix. Results Mortality rate was 52% after muscle patch implantation. Bone marrow cells were able to migrate from muscle patch into the infarct zone, as demonstrated by β-galactosidase immunostaining, and ultimately constituted 8% of all cells in scar tissue (mean ± standard deviation, 219 ± 111/mm 2 ). Angiogenesis and cell survival in the scar were improved by patch + BMC treatment. Left ventricular geometry and cardiac function were improved by patch treatment, with or without BMC, although the effects were stronger after patch + BMC treatment. Conclusions Epicardial deposition of a BMC-coated muscle patch is a promising approach to restoring cardiac function after myocardial infarction.

Objective: FrzA, a member of the group of secreted frizzled related proteins (sFRP) that is expressed in the cardiovascular system, has been shown to antagonize the Wnt/frizzled signaling pathway. We have recently demonstrated its role in vascular cell growth control in vitro. In this study, we aimed to examine the mechanisms by which FrzA exerts its antiproliferative effect on vascular cells in vitro and its potential effect in vivo. Methods and results: On synchronized, growth-arrested endothelial cells (EC) and smooth muscle cells (SMC) treated with the recombinant purified FrzA protein, flow cytometry analysis showed that the recombinant FrzA protein delayed G1 phase and entry into S-phase. Western blot experiments demonstrated that the treatment of EC or SMC with FrzA was associated with a decrease in the level of the cyclins and cyclin-dependent kinases and an increase in cytosolic phospho-β-catenin levels. The FrzA-induced cell cycle delay was resolved by 24 h. C57BL/6J mice underwent surgery to produce unilateral hindlimb ischemia and empty adenoviruses (AdE) or adenoviruses coding for FrzA (AdFrzA) were injected at the time of the surgery. In AdFrzA-treated mice in the 7 days following surgery, we showed a decrease in cell proliferation, capillary density, and blood flow recovery and a reduced expression of cyclin and cdk activity in the ischemic muscle compared to that in the AdE-treated ischemic muscle. To gain insight into the pathway activated by FrzA overexpression, we showed an increase in the level of cytosolic phospho-β-catenin, a marker of β-catenin degradation, in AdFrzA-treated ischemic muscle compared to that in control AdE-treated ischemic muscle. Conclusion: We provided the first evidence that an impairment of the Wnt-Frizzled pathway, via FrzA overexpression, controlled proliferation and neovascularization after muscle ischemia.

Background— The secreted frizzled related proteins (sFRP) are soluble proteins thought to interfere with the Wnt signaling. Our group previously demonstrated that one of these members, sFRP-1/FrzA, is strongly expressed during early phases of the vascularization process in embryonic vasculature and in the endothelium of arteries and capillaries in adults and modulated vascular cell proliferation. Methods and Results— Analysis of the expression of sFRP-1 during cyclic ovarian angiogenesis revealed that sFRP-1 is expressed during the formation of neovessels and becomes undetectable when the vasculature is fully maturated. We then studied the role of FrzA in several distinct angiogenic models. FrzA induced angiogenesis in a chick chorioallantoic membrane model. Moreover, gene transfer of AdFrzA in grafted mesenchymal and glioma cells increased vessel density and tumor growth. FrzA induced formation of vessels, which were enlarged, longer, and appeared to be more mature compared with vessels formed under control treatments. In vitro, FrzA increased migration and tube formation of endothelial cells and seemed to protect them from apoptosis. FrzA-angiogenic effect in vitro was independent of vascular endothelial growth factor, fibroblast growth factor-2, or angiopiotin-1 induction and Akt activation. In contrast, FrzA decreased glycogen synthase kinase-3 phosphorylation. Conclusions— These results showed that FrzA has proangiogenic effects and suggest that Wnt signaling may be involved in normal differentiation as well as in the pathological development of vasculature.

Development and stabilization of a vascular plexus requires the coordination of multiple signalling processes. Wnt planar cell polarity (PCP) signalling is critical in vertebrates for diverse morphogenesis events, which coordinate cell orientation within a tissue-specific plane. However, its functional role in vascular morphogenesis is not well understood. Here we identify PDZRN3, an ubiquitin ligase, and report that Pdzrn3 deficiency impairs embryonic angiogenic remodelling and postnatal retinal vascular patterning, with a loss of two-dimensional polarized orientation of the intermediate retinal plexus. Using in vitro and ex vivo Pdzrn3 loss-of-function and gain-of-function experiments, we demonstrate a key role of PDZRN3 in endothelial cell directional and coordinated extension. PDZRN3 ubiquitinates Dishevelled 3 (Dvl3), to promote endocytosis of the Frizzled/Dvl3 complex, for PCP signal transduction. These results highlight the role of PDZRN3 to direct Wnt PCP signalling, and broadly implicate this pathway in the planar orientation and highly branched organization of vascular plexuses.

Aims Vascular permeability is essential for the health of normal tissues and is an important characteristic of many disease states. The role of the Wnt/frizzled pathway in vascular biology has recently been reported. The objectives of this study are to analyse the role of Frizzled7 (Fzd7) receptor in the control of vascular integrity. Methods and results Fzd7 is expressed in endothelial cells and accumulates at the points of cell–cell contact in association with VE-cadherin and β-catenin, two major adherens junction molecules. To selectively delete fzd7 in the vasculature, we developed gene targeting approaches using CreLox strategy in mice. Genetic fzd7 inhibition in the endothelium increases vascular permeability in basal and factor-induced conditions. On the cellular level, fzd7 knockdown or depletion leads to an increase in paracellular permeability with a loss of adherens junction organization. These impairments are associated with a decrease in both VE-Cadherin and β-catenin expression, a decrease in their association and an increase of tyrosine phosphorylation of VE-cadherin/β-catenin. Fzd7 transduces a Wnt/β-catenin signalling cascade that is required to regulate β-catenin and canonical target gene expression. Finally, LiCl, a GSK3 inhibitor, and β-catenin overexpression rescued endothelial integrity and adherens junction organization, induced by fzd7 deletion. Conclusion These findings establish that Fzd7 is a new partner of adherens junctional complex and represents a novel molecular switch for the control of vascular permeability via activation of the Wnt-canonical pathway.

International audience; Left-right (L/R) asymmetries in the brain are thought to underlie lateralised cognitive functions. Understanding how neuroanatomicalasymmetries are established has been achieved through the study of the zebrafish epithalamus. Morphological symmetry in theepithalamus is broken by leftward migration of the parapineal, which is required for the subsequent elaboration of left habenular identity;the habenular nuclei flank the midline and show L/R asymmetries in marker expression and connectivity. The Nodal target pitx2c isexpressed in the left epithalamus, but nothing is known about its role during the establishment of asymmetry in the brain. We show thatabrogating Pitx2c function leads to the right habenula adopting aspects of left character, and to an increase in parapineal cell numbers. Parapineal ablation in Pitx2c loss of function results in right habenular isomerism, indicating that the parapineal is required for the left character detected in the right habenula in this context. Partial parapineal ablation in the absence of Pitx2c, however, reduces the number of parapineal cells to wild-type levels and restores habenular asymmetry. We provide evidence suggesting that antagonism between Nodal and Pitx2c activities sets an upper limit on parapinealcell numbers. We conclude that restricting parapineal cell number is crucial for the correct elaboration of epithalamic asymmetry.

Discrepancies between success in experimental animals with a variety of pharmacologic strategies and failure with such agents in clinical trials have raised questions concerning the mechanism of restenosis. Recent observations suggest a potential implication for the adventitial (Adv) layer in neointimal formation.The purpose of this study was to examine the Adv changes in the rat carotid artery subjected to balloon injury. These changes were characterized by morphometric, immunohistochemical, and electron microscopy analyses, with special attention devoted to early time-points post-injury.We report that the most important adventitial changes occurred in the first 48 h post-injury. Within 2 h there was extensive cell-loss by apoptosis and oncosis in the Adv and in the media; this was followed by the rapid onset of proliferation and a parallel slow increase in Adv thickening, reaching a maximum at 7 days. We further demonstrate an early migration of these Adv cells to the media and neointima. Moreover, we characterize the Adv cell phenotype with a panel of antibodies. Within 48 h after injury, a population of Adv cells expressed alpha-actin and vinculin with a maximum expression 7 days post-injury. At that time, these Adv cells started to express smooth muscle myosin heavy chain, a specific marker of smooth muscle cells. In parallel, we report an impaired production of elastic fibres in the Adv and medial layer.We reported a detailed time-course of adventitial changes after rat carotid injury (cell death, proliferation, migration and differentiation) that supports an important role of adventitia in neointima formation.

The Wnt genes encode a large family of secreted proteins that play a key role in embryonic development and tissue differentiation in many species ( Rijsewijk et al., 1987 , Nusse and Varmus, 1992 ). Genetic and biochemical studies have suggested that the frizzled proteins are cell surface receptors for Wnts ( Vinson et al., 1989 , Chan et al., 1992 , Bhanot et al., 1996 , Wang et al., 1996 ). In parallel, a number of secreted frizzled-like proteins with a conserved N-terminal frizzled motif have been identified ( Finch et al., 1997 , Melkonyan et al., 1997 , Rattner et al., 1997 ). One of these proteins, FrzA, the bovine counterpart of the murine sFRP-1 (93% identity) is involved in vascular cell growth control, binds Wg in vitro and antagonizes Xwnt-8 and hWnt-2 signaling in Xenopus embryos ( Xu et al., 1998 , Duplaa et al., 1999 ). In this study, we report that sFRP-1 is expressed in the heart and in the visceral yolk sac during mouse development, and that sFRP-1 and mWnt-8 display overlapping expression patterns during heart morphogenesis. From 8.5 to 12.5 d.p.c., sFRP-1 is expressed in cardiomyocytes together with mWnt-8 but neither in the pericardium nor in the endocardium; at 17.5 d.p.c., they are no longer present in the heart. In mouse adult tissues, while sFRP-1 is highly detected in the aortic endothelium and media and in cardiomyocytes, mWnt-8 is not detected in these areas. Immunoprecipitation experiments demonstrates that FrzA binds to mWnt-8 in cell culture experiments.

In nematodes, flies, trypanosomes, and planarians, introduction of double-stranded RNA results in sequence-specific inactivation of gene function, a process termed RNA interference (RNAi). We demonstrate that RNAi against the Caenorhabditis elegans gene lir-1, which is part of the lir-1/lin-26 operon, induced phenotypes very different from a newly isolated lir-1 null mutation. Specifically, lir-1(RNAi) induced embryonic lethality reminiscent of moderately strong lin-26 alleles, whereas the lir-1 null mutant was viable. We show that the lir-1(RNAi) phenotypes resulted from a severe loss of lin-26 gene expression. In addition, we found that RNAi directed against lir-1 or lin-26 introns induced similar phenotypes, so we conclude that lir-1(RNAi) targets the lir-1/lin-26 pre-mRNA. This provides direct evidence that RNA interference can prevent gene expression by targeting nuclear transcripts. Our results highlight that caution may be necessary when interpreting RNA interference without the benefit of mutant alleles.

The Caenorhabditis elegans LIN-26 protein is required to specify and/or maintain the fates of all non-neuronal ectodermal cells. Here we show that lin-26 is expressed until the somatic gonad primordium stage in all cells of the somatic gonad, except in distal tip cells, and later in all uterine cells. To determine if lin-26 functions in the somatic gonad, we have generated gonad-specific lin-26 alleles obtained by integration of lin-26 promoter deletion derivatives into a lin-26 null mutant background. In this way, we rescued the lethal phenotype imparted by lin-26 null mutations and uncovered a highly penetrant sterile phenotype. Specifically, the strongest of these new alleles was characterized by the absence of lin-26 expression in the somatic gonad, the presence of endomitotic oocytes, decreased germline proliferation, a protruding vulva and a less penetrant absence of gonad arms. Lineage analysis of mutant somatic gonads and examination of several markers expressed in the spermatheca, sheath cells, distal tip cells and the uterus, suggest that LIN-26 is required in sheath, spermatheca and uterine precursors, and in uterine cells. We conclude that lin-26 performs a similar function in the non-neuronal ectoderm and the somatic gonad, a mesoderm derivative, and we speculate that lin-26 is required to express epithelial characteristics.

Abstract —Vitronectin (VN) is a plasma glycoprotein that promotes cell attachment and induces migration of human smooth muscle cells (SMCs) in culture. VN has been observed to accumulate in human atherosclerotic plaques, although its origin and role in atherosclerosis are not yet established. In the present experiments, synthesis of VN by intimal cells and its colocalization with receptors, αvβ3 and αvβ5, were studied by in situ hybridization and immunohistochemistry on 15 human atherosclerotic plaques from carotid arteries obtained after surgery. Strong VN protein and mRNA expression was observed in the intima and in the media. In the intima, VN mRNA expression was colocalized with SMCs, indicating that these cells produce VN, which may account for its accumulation in atherosclerotic plaques. In SMCs in culture, immunoprecipitation after metabolic labeling demonstrated that human SMCs do synthesize vitronectin. Confocal microscopic examination showed that VN colocalized with its receptors, αvβ3 and αvβ5, in the atherosclerotic intima. However, the distribution of the VN receptors on SMCs in culture in contact with VN was different. These observations suggest that VN plays various parts in atherogenesis via different SMC membrane receptors. ( Arterioscler Thromb Vasc Biol. 1998;18:168-176.)

See related article, pages 1329–1337 Understanding how smooth muscle cells transition from a steady state to a proliferative state will have an important impact on our understanding of vascular pathology. In the present issue of Circulation Research , Quasnichka et al take an indepth look at the role of the β-catenin/T-cell factor (TCF) signaling pathway in vascular smooth muscle cell (VSMC) proliferation.1 Although β-catenin is the central component in the Wnt canonical pathway, and is regarded as a hallmark of Wnt pathway activation, the present article and other recent reports elucidate new insights into activation of the β-catenin pathway and indicates, that β-catenin activation appears to be intricately orchestrated by both Wnt and growth factor networks. At the plasma membrane, it controls cell-cell adhesion through its binding with cadherin in adherence junctions and also mediates the link between cadherin and the actin cytoskeleton through the molecular switch α-catenin.2,3 In addition, β-catenin acts as a transcriptional activator and regulates transcription of target genes responsible for cell proliferation and differentiation.4 Literature diverges on the interpretation of the interplay between these pathways. It is still unclear if these 2 processes act in concert or independently.5,6 The Wnt system is 1 of the well-known potent pathways, which activates nuclear β-catenin. In the absence of Wnt signal, free cytoplasmic β-catenin is phosphorylated by serine/threonine kinases, casein Kinase Iα (CKIα) and GSK3β in a large APC/axin scaffolding complex that targets β-catenin for degradation. In the presence of Wnt signaling, this destruction complex is disrupted, and dissociation of GSK3β prevents phosphorylation of β-catenin. The increase stability of β-catenin following Wnt activation leads to its translocation in the nucleus and induces transcriptional activation of target genes by β-catenin interaction with TCF/LEF (lymphoid enhancer factor) DNA-binding proteins (Figure). The multiple routes of β-catenin activation. …

Hypoxia induces intercellular adhesion molecule-1 on cultured human tubular cells. The adverse effects of acute renal ischemia are partly mediated through an infiltration of inflammatory cells into the tubulointerstitium. The expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) by resident renal cells (endothelial cells and tubular cells) may facilitate this process. We investigated whether hypoxia stimulates the expression of ICAM-1 by cultured human proximal tubular cells (HPTC). Hypoxic culture conditions (PO2 < 4 kPa) stimulated the expression of ICAM-1 by HPTC in a time-dependent manner (P < 0.0001) as demonstrated by quantitative flow cytometry analysis. Quantitative PCR demonstrated an increase in ICAM-1 transcription. Re-oxygenation of tubular cells did not increase ICAM-1 expression further. TNFα concentration in culture supernatants increased with hypoxia, but blocking experiments demonstrated that TNFα was not implicated in hypoxia-induced expression of ICAM-1. Furthermore, the cytokines IL-6 and IL-1β were not involved, but the effect of hypoxia was blocked by PDTC, an antioxidant that may inhibit the activation of the transcription factor NF-κN. These data demonstrate that hypoxia is a stimulus that induces the synthesis and expression of the adhesion molecule ICAM-1, presumably via the activation of NF-κB.

Vascular cell adhesion molecule-1 (VCAM-1) and its counterreceptor, the integrin very late antigen-4 (VLA-4), have recently been identified in smooth muscle cells during intimal thickening in humans and in newly forming vessels during ontogeny in mice, respectively. We examined the coexpression of VCAM-1 and the α 4 integrin subunit in human smooth muscle cells. The expression of VCAM-1 and α 4 subunit were studied during development of the aorta. In the 10-week-old human fetal aorta, VCAM-1 and α 4 were strongly expressed in smooth muscle cells. Their expression was dramatically reduced within the 24th week of gestation and disappeared in the adult aortic media. However, smooth muscle cells from intimal atherosclerotic thickening of adult aorta reexpressed both VCAM-1 and α 4 . In a culture model mimicking smooth muscle differentiation, VCAM-1 mRNA and protein and α 4 integrin protein were coexpressed with smooth muscle–specific variants of cytoskeletal and contractile proteins, smooth muscle myosin heavy chain, caldesmon heavy chain, and desmin. Treatment with antibodies against VCAM-1 or α 4 integrin subunit interfered with the mRNA induction of smooth muscle–specific markers of differentiation. These results in vitro, associated with the transitory expression of VCAM-1 and VLA-4 during vascular ontogeny and the atherosclerosis process, point to a possible role of VCAM-1 and VLA-4 in the induction of smooth muscle differentiation.

Blinding diseases including retinopathy of prematurity and proliferative diabetic retinopathy are the consequence of pathological angiogenesis in the retina. A better understanding of the mechanisms involved in this abnormal proliferation of neovessels appears to be essential to develop drugs against new molecular targets. Recently, we have evidenced that Fzd7 receptor, a member of the Wnt/Fzd family, is required for retinal postnatal angiogenesis. This study aimed to investigate the role of Fzd7 during aberrant angiogenesis in a model of ischemic retinopathy. We used mice model of OIR to explore the involvement of Fzd7 during initial vaso-obliteration (VO) and subsequent neovascularization (NV) phases. By transgenic approaches, we observed that deletion of fzd7 in the endothelium ( fzd7 ECKO ) resulted in increased retinal tissue sensitivity to hypoxia during the VO phase of OIR. Moreover, fzd7 deletion in EC reduced the ectopic growth of pathologic neovessels into the vitreous during the NV phase (P17) demonstrating that Fzd7 was involved in pathological NV formation in mice retina during OIR. To determine how Fzd7 may regulate ischemic retinopathy, we explored canonical β-catenin and Notch signaling during OIR and showed that transcript expressions of lef1, axin2 and partners of Notch signaling were strongly decreased in fzd7 ECKO retina mice as compared to control. To evaluate therapeutic potential of Fzd7 blocking, we experimented 2 strategies: a monoclonal antibody (MabFzd7) and a soluble Fzd7 receptor (CRD domain). In vitro both MabFzd7 and Fzd7CRD decreased the activity of β-catenin reporter gene induced by Wnt3A in EC. In vivo intravitreal microinjection of MabFzd7 or intraperitoneal injection of Fzd7CRD in mice at P12 decreased neovessels in the treated eye compared to the control eye at P17. By controlling development of abnormal vascularization, Fzd7 could be a new target to develop promising anti-angiogenic drugs in the treatment of ischemic retinopathies. The author hereby declares no conflict of interest

Thrombomodulin is a membrane glycoprotein expressed by endothelium and is a receptor for thrombin. The thrombin-thrombomodulin complex inactivates the procoagulant activity of thrombin and catalyzes activation of protein C(1). The decrease in thrombomodulin expression at the surface of the vascular endothelium may contribute to the development of thrombosis. In vitro studies have shown that cytokines (TNF, IL-1) or endotoxins down regulate thrombomodulin expression (2, 3, 4) whereas histamine (5), retinoic acid (6) and cAMP analogs (7, 8) increase its expression. Hypoxia is frequently related to disorders of the vascular system such as atheromatous arterial disease and venous insufficiency (9). Clinically, hypoxia induces an increase in vascular permeability (10) and a prothrombotic tendency (12). In vitro, it has been demonstrated that hypoxia is able to modify endothelial properties. In effect, hypoxia has been shown to down regulate thrombornodulin expression on bovine arterial endothelial cells (BAEC) and microvascular endothelial cells (11, 13, 14). Moreover, it has been demonstrated that hypoxia decreases endothelial cell barrier function by lowering cAMP levels (15). cAMP is the major up regulator of thrombomodulin expression, so we investigated the effect of agents able to increase cAMP levels on membrane thrombomodulin expression in hypoxic conditions on HUVEC