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During wound healing, the distribution, availability, and signaling of growth factors (GFs) are orchestrated by their binding to extracellular matrix components in the wound microenvironment. Extracellular matrix proteins have been shown to modulate angiogenesis and promote wound healing through GF binding. The hemostatic protein von Willebrand factor (VWF) released by endothelial cells (ECs) in plasma and in the subendothelial matrix has been shown to regulate angiogenesis; this function is relevant to patients in whom VWF deficiency or dysfunction is associated with vascular malformations. Here, we show that VWF deficiency in mice causes delayed wound healing accompanied by decreased angiogenesis and decreased amounts of angiogenic GFs in the wound. We show that in vitro VWF binds to several GFs, including vascular endothelial growth factor-A (VEGF-A) isoforms and platelet-derived growth factor-BB (PDGF-BB), mainly through the heparin-binding domain (HBD) within the VWF A1 domain. VWF also binds to VEGF-A and fibroblast growth factor-2 (FGF-2) in human plasma and colocalizes with VEGF-A in ECs. Incorporation of the VWF A1 HBD into fibrin matrices enables sequestration and slow release of incorporated GFs. In vivo, VWF A1 HBD-functionalized fibrin matrices increased angiogenesis and GF retention in VWF-deficient mice. Treatment of chronic skin wounds in diabetic mice with VEGF-A165 and PDGF-BB incorporated within VWF A1 HBD-functionalized fibrin matrices accelerated wound healing, with increased angiogenesis and smooth muscle cell proliferation. Therefore, the VWF A1 HBD can function as a GF reservoir, leading to effective angiogenesis and tissue regeneration.

A striking feature of severe COVID-19 is thrombosis in large as well as small vessels of multiple organs. This has led to the assumption that SARS-CoV-2 virus directly infects and damages the vascular endothelium. However, endothelial expression of ACE2, the cellular receptor for SARS-CoV-2, has not been convincingly demonstrated. Interrogating human bulk and single-cell transcriptomic data, we found ACE2 expression in endothelial cells to be extremely low or absent in vivo and not upregulated by exposure to inflammatory agents in vitro. Also, the endothelial chromatin landscape at the ACE2 locus showed presence of repressive and absence of activation marks, suggesting that the gene is inactive in endothelial cells. Finally, we failed to achieve infection and replication of SARS-CoV-2 in cultured human endothelial cells, which were permissive to productive infection by coronavirus 229E that uses CD13 as the receptor. Our data suggest that SARS-Cov-2 is unlikely to infect endothelial cells directly; these findings are consistent with a scenario where endothelial injury is indirectly caused by the infection of neighbouring epithelial cells and/or due to systemic effects mediated by immune cells, platelets, complement activation, and/or proinflammatory cytokines.

The role of the endothelium in protecting from chronic liver disease and TGFβ-mediated fibrosis remains unclear. Here we describe how the endothelial transcription factor ETS-related gene (ERG) promotes liver homoeostasis by controlling canonical TGFβ-SMAD signalling, driving the SMAD1 pathway while repressing SMAD3 activity. Molecular analysis shows that ERG binds to SMAD3, restricting its access to DNA. Ablation of ERG expression results in endothelial-to-mesenchymal transition (EndMT) and spontaneous liver fibrogenesis in EC-specific constitutive hemi-deficient (Erg

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.

Essentials To reliably study the respective roles of blood and endothelial cells in hemostasis, mouse models with a strong and specific endothelial expression of the Cre recombinase are needed. Using mT/mG reporter mice and conditional JAK2V617F/WT mice, we compared Pdgfb-iCreERT2 and Cdh5(PAC)-CreERT2 with well-characterized Tie2-Cre mice. Comparison of recombination efficiency and specificity towards blood lineage reveals major differences between endothelial transgenic mice. Cre-mediated recombination occurs in a small number of adult hematopoietic stem cells in Pdgfb-iCreERT2;JAK2V617F/WT transgenic mice. SUMMARY: Background The vessel wall, and particularly blood endothelial cells (BECs), are intensively studied to better understand hemostasis and target thrombosis. To understand the specific role of BECs, it is important to have mouse models that allow specific and homogeneous expression of genes of interest in all BEC beds without concomitant expression in blood cells. Inducible Pdgfb-iCreERT2 and Cdh5(PAC)-CreERT2 transgenic mice are widely used for BEC targeting. However, issues remain in terms of recombination efficiency and specificity regarding hematopoietic cells. Objectives To determine which mouse model to choose when strong expression of a transgene is required in adult BECs from various organs, without concomitant expression in hematopoietic cells. Methods Using mT/mG reporter mice to measure recombination efficiency and conditional JAK2V617F/ WT mice to assess specificity regarding hematopoietic cells, we compared Pdgfb-iCreERT2 and Cdh5(PAC)-CreERT2 with well-characterized Tie2-Cre mice. Results Adult Cdh5(PAC)-CreERT2 mice are endothelial specific but require a dose of 10 mg of tamoxifen to allow constant Cre expression. Pdgfb-iCreERT2 mice injected with 5 mg of tamoxifen are appropriate for most endothelial research fields except liver studies, as hepatic sinusoid ECs are not recombined. Surprisingly, 2 months after induction of Cre-mediated recombination, all Pdgfb-iCreERT2;JAK2V617F/ WT mice developed a myeloproliferative neoplasm that is related to the presence of JAK2V617F in hematopoietic cells, showing for the first time that Cre-mediated recombination occurs in a small number of adult hematopoietic stem cells in Pdgfb-iCreERT2 transgenic mice. Conclusion This study provides useful guidelines for choosing the best mouse line to study the role of BECs in hemostasis and thrombosis.

Endothelial cells actively maintain an anti-thrombotic environment; loss of this protective function may lead to thrombosis and systemic coagulopathy. The transcription factor ERG is essential to maintain endothelial homeostasis. Here, we show that inducible endothelial ERG deletion (ErgiEC-KO) in mice is associated with spontaneous thrombosis, hemorrhages and systemic coagulopathy. We find that ERG drives transcription of the anticoagulant thrombomodulin (TM), as shown by reporter assays and chromatin immunoprecipitation. TM expression is regulated by shear stress (SS) via Krüppel-like factor 2 (KLF2). In vitro, ERG regulates TM expression under low SS conditions, by facilitating KLF2 binding to the TM promoter. However, ERG is dispensable for TM expression in high SS conditions. In ErgiEC-KO mice, TM expression is decreased in liver and lung microvasculature exposed to low SS but not in blood vessels exposed to high SS. Our study identifies an endogenous, vascular bed-specific anticoagulant pathway in microvasculature exposed to low SS., The endothelium actively maintains an anticoagulant surface through expression of thrombomodulin. Here, Peghaire et al. identify an anti-thrombotic pathway that controls thrombomodulin expression selectively in regions of low shear stress, via cooperation of the transcription factors ERG and KLF2.

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.

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.

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.

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

Background Pathological angiogenesis in the retina is the cause of many human diseases, including retinopathy of prematurity and proliferative diabetic retinopathy. A better understanding of the mechanisms involved in the abnormal proliferation of blood vessel observed in these blinding pathologies appears to be essential to develop potential therapeutic drugs against new molecular targets. Growing evidence has shown that Wnt/Frizzled (Fzd) are directly involved in vascular development. Recently, we have evidenced that Fzd7 receptor is required for postnatal angiogenesis of retina by controlling endothelial cells (EC) proliferation and migration through a β-catenin canonical pathway. This study aimed to investigate the role of Fzd7 during aberrant angiogenesis in a model of ischemic retinopathy. Methods/Findings We used mice model of oxygen-induced retinopathy (OIR) to explore the involvement of Fzd7 during initial vaso-obliteration (VO) and subsequent neovascularization (NV) phases. First we observed Fzd7 was expressed in pathologic neovessels. Second, by transgenic approaches, we observed that specific deletion of fzd7 in the endothelium (fzd7ECKO) resulted in increased retinal tissue sensitivity to hypoxia during the vaso-obliterative phase of the OIR model. Moreover, fzd7 deletion in EC reduced the ectopic growth of pathologic neovessels into the vitreous during the second phase of the OIR model demonstrating that Fzd7 was involved in pathological NV formation in mice retina after ischemia. To determine the molecular mechanisms by which Fzd7 may regulate ischemic retinopathy, we explore canonical β-catenin and Notch signaling during the two different phases of OIR. Preliminary results showed that transcript expressions of β-catenin (lef1, axin2) and partners of Notch signaling were strongly decreased in fzd7ECKO retina mice as compared to control. Conclusion By controlling pathogenesis of ischemic retinopathy, Fzd7 could be an efficient and specific target to develop anti-angiogenic drugs in the treatment of ischemic retinopathies.