Endothelial cells lining blood vessels regulate vascular barrier function, which controls the passage of plasma proteins and circulating cells across the endothelium. In most normal adult tissues, ...endothelial cells preserve basal vascular permeability at a low level, while they increase permeability in response to inflammation. Therefore, vascular permeability is tightly controlled by a number of extracellular stimuli and mediators to maintain tissue homeostasis. Accordingly, impaired regulation of endothelial permeability causes various diseases, including chronic inflammation, asthma, edema, sepsis, acute respiratory distress syndrome, anaphylaxis, tumor angiogenesis, and diabetic retinopathy. Vascular endothelial (VE)-cadherin, a member of the classical cadherin superfamily, is a component of cell-to-cell adherens junctions in endothelial cells and plays an important role in regulating vascular permeability. VE-cadherin mediates intercellular adhesion through trans-interactions formed by its extracellular domain, while its cytoplasmic domain is anchored to the actin cytoskeleton via α- and β-catenins, leading to stabilization of VE-cadherin at cell-cell junctions. VE-cadherin-mediated cell adhesions are dynamically, but tightly, controlled by mechanisms that involve protein phosphorylation and reorganization of the actomyosin cytoskeleton. Phosphorylation of VE-cadherin, and its associated-catenins, results in dissociation of the VE-cadherin/catenin complex and internalization of VE-cadherin, leading to increased vascular permeability. Furthermore, reorganization of the actomyosin cytoskeleton by Rap1, a small GTPase that belongs to the Ras subfamily, and Rho family small GTPases, regulates VE-cadherin-mediated cell adhesions to control vascular permeability. In this review, we describe recent progress in understanding the signaling mechanisms that enable dynamic regulation of VE-cadherin adhesions and vascular permeability. In addition, we discuss the possibility of novel therapeutic approaches targeting the signaling pathways controlling VE-cadherin-mediated cell adhesion in diseases associated with vascular hyper-permeability.
Hematopoietic stem cells (HSCs) emerge from hemogenic endothelium (HE) within the ventral portion of the dorsal aorta during vertebrate development. In zebrafish, Notch signaling induces HE ...specification from posterior lateral plate mesoderm (PLPM) cells as they migrate over the ventral surface of the somite. During migration, PLPM cells make close contact with Notch-ligand-expressing somitic cells to acquire HE identity. Herein, we show in zebrafish that the small GTPase Rap1b regulates HSC development by potentiating Notch-mediated HE specification. PLPM cells migrate toward the midline along the somite boundary where fibronectin accumulates. Rap1b stimulates integrin β1 to enhance PLPM cell adhesion to fibronectin localized at the somite boundary. Rap1b-induced integrin-β1-mediated adhesion to fibronectin leads to the spreading of PLPM cells to facilitate their physical contact with the Notch-ligand-expressing somitic cells, thereby promoting Notch-mediated HE specification. Thus, we have revealed an unexpected role of Rap1-induced integrin-mediated cell adhesion in HSC development.
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•Rap1b is involved in hematopoietic stem cell development in zebrafish•Rap1b promotes Notch-mediated hemogenic endothelium specification of PLPM cells•Fibronectin guides PLPM cells to migrate along somite boundary through integrin β1•Rap1b activates Notch by inducing integrin-β1-mediated PLPM adhesion to somite
Rho et al. show a role for the small GTPase Rap1b in hematopoietic stem cell development in zebrafish. Rap1b promotes Notch-signal-mediated specification of posterior lateral plate mesoderm cells by inducing their adhesion to Notch-ligand-expressing somitic cells through integrin-β1-mediated adhesion to fibronectin localized at the somite boundary.
► IL-33 as nuclear factor regulated expression of ICAM-1 and VCAM-1. ► Nuclear IL-33 increased the transcription of NF-κB p65 by binding to the p65 promoter. ► Nuclear IL-33 controls NF-κB-dependent ...inflammatory responses.
Interleukin (IL)-33, an IL-1 family member, acts as an extracellular cytokine by binding its cognate receptor, ST2. IL-33 is also a chromatin-binding transcriptional regulator highly expressed in the nuclei of endothelial cells. However, the function of IL-33 as a nuclear factor is poorly defined. Here, we show that IL-33 is a novel transcriptional regulator of the p65 subunit of the NF-κB complex and is involved in endothelial cell activation. Quantitative reverse transcriptase PCR and Western blot analyses indicated that IL-33 mediates the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in endothelial cells basally and in response to tumor necrosis factor-α-treatment. IL-33-induced ICAM-1/VCAM-1 expression was dependent on the regulatory effect of IL-33 on the nuclear factor (NF)-κB pathway; NF-κB p65 expression was enhanced by IL-33 overexpression and, conversely, reduced by IL-33 knockdown. Moreover, NF-κB p65 promoter activity and chromatin immunoprecipitation analysis revealed that IL-33 binds to the p65 promoter region in the nucleus. Our data provide the first evidence that IL-33 in the nucleus of endothelial cells participates in inflammatory reactions as a transcriptional regulator of NF-κB p65.
Delta-like ligand 4 (DLL4) expression in endothelial cells is intimately associated with angiogenic sprouting and vascular remodeling, but the precise mechanism of transcriptional regulation of DLL4 ...remains incompletely understood. Here, we showed that LIM-domain binding protein 2 (LDB2) plays an important role in regulating basal DLL4 and VEGF-induced DLL4 expression. Knockdown of LDB2 using siRNA enhanced endothelial sprouting and tubular network formation in vitro. Injection of ldb2-morpholino resulted in defective development of intersegmental vessels in zebrafish. Reduction or overexpression of LDB2 in endothelial cells decreased or increased DLL4 expression. LDB2 regulated DLL4 promoter activity by binding to its promoter region and the same promoter region was occupied and regulated by the LMO2/TAL1/GATA2 complex. Interestingly, LDB2 also mediated VEGF-induced DLL4 expression in endothelial cells. The regulation of DLL4 by the LDB2 complex provides a novel mechanism of DLL4 transcriptional control that may be exploited to develop therapeutics for aberrant vascular remodeling. BMB Reports 2018; 51(1): 21-26.
Neovessel formation is a complex process governed by the orchestrated action of multiple factors that regulate EC specification and dynamics within a growing vascular tree. These factors have been ...widely exploited to develop therapies for angiogenesis-related diseases such as diabetic retinopathy and tumor growth and metastasis. WNT signaling has been implicated in the regulation and development of the vascular system, but the detailed mechanism of this process remains unclear. Here, we report that Dickkopf1 (DKK1) and Dickkopf2 (DKK2), originally known as WNT antagonists, play opposite functional roles in regulating angiogenesis. DKK2 induced during EC morphogenesis promoted angiogenesis in cultured human endothelial cells and in in vivo assays using mice. Its structural homolog, DKK1, suppressed angiogenesis and was repressed upon induction of morphogenesis. Importantly, local injection of DKK2 protein significantly improved tissue repair, with enhanced neovascularization in animal models of both hind limb ischemia and myocardial infarction. We further showed that DKK2 stimulated filopodial dynamics and angiogenic sprouting of ECs via a signaling cascade involving LRP6-mediated APC/Asef2/Cdc42 activation. Thus, our findings demonstrate the distinct functions of DKK1 and DKK2 in controlling angiogenesis and suggest that DKK2 may be a viable therapeutic target in the treatment of ischemic vascular diseases.
Controlled angiogenesis and lymphangiogenesis are essential for tissue development, function, and repair. However, aberrant neovascularization is an essential pathogenic mechanism in many human ...diseases, including diseases involving tumor growth and survival. Here, we have demonstrated that mice deficient in C-type lectin family 14 member A (CLEC14A) display enhanced angiogenic sprouting and hemorrhage as well as enlarged jugular lymph sacs and lymphatic vessels. CLEC14A formed a complex with VEGFR-3 in endothelial cells (ECs), and CLEC14A KO resulted in a marked reduction in VEGFR-3 that was concomitant with increases in VEGFR-2 expression and downstream signaling. Implanted tumor growth was profoundly reduced in CLEC14A-KO mice compared with that seen in WT littermates, but tumor-bearing CLEC14A-KO mice died sooner. Tumors in CLEC14A-KO mice had increased numbers of nonfunctional blood vessels and severe hemorrhaging. Blockade of VEGFR-2 signaling suppressed these vascular abnormalities and enhanced the survival of tumor-bearing CLEC14A-KO mice. We conclude that CLEC14A acts in vascular homeostasis by fine-tuning VEGFR-2 and VEGFR-3 signaling in ECs, suggesting its relevance in the pathogenesis of angiogenesis-related human disorders.
► Clec14a is specifically expressed in various kinds of endothelial cells (ECs) and vasculature of mice. ► Clec14a mediates endothelial cell–cell adhesion via C-type lectin domain. ► Knockdown of ...Clec14a in ECs resulted in suppression of endothelial cell migration and tube formation on Matrigel.
Clec14a is a member of the thrombomodulin (TM) family, but its function has not yet been determined. Here, we report that Clec14a is a plasma membrane protein of endothelial cells (ECs) expressed specifically in the vasculature of mice. Deletion mutant analysis revealed that Clec14a mediates cell–cell adhesion through its C-type lectin-like domain. Knockdown of Clec14a in ECs suppressed cell migratory activity and filopodial protrusion, and delayed formation of tube-like structures. These findings demonstrate that Clec14a is a novel EC-specific protein that appears to play a role in cell–cell adhesion and angiogenesis.
Lateral plate mesoderm (LPM) cells differentiate into various cell types including endothelial and hematopoietic cells. In zebrafish embryos, LPM cells migrate toward the midline along the ventral ...surfaces of somites during which their cell fate specification depends upon efficient integrin-mediated cell adhesion and migration. Herein, we present a protocol for analysis of integrin-mediated cell adhesion of LPM cells isolated from zebrafish embryos. This allows the study of the molecular mechanisms underlying integrin activation required for LPM cell fate specification.
For complete details on the use and execution of this protocol, please refer to Rho et al. (2019).
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•Zebrafish breeding and preparation of the LPM cells dissociated from the embryos•Protocol for LPM cell adhesion to the fibronectin-coated dish•Imaging the LPM cells adhering to the fibronectin-coated dish•Analysis of cell spreading and focal adhesion formations in the LPM cells
Lateral plate mesoderm (LPM) cells differentiate into various cell types including endothelial and hematopoietic cells. In zebrafish embryos, LPM cells migrate toward the midline along the ventral surfaces of somites during which their cell fate specification depends upon efficient integrin-mediated cell adhesion and migration. Herein, we present a protocol for analysis of integrin-mediated cell adhesion of LPM cells isolated from zebrafish embryos. This allows the study of the molecular mechanisms underlying integrin activation required for LPM cell fate specification.
Tumor blood vessels are leaky and immature, which causes inadequate blood supply to tumor tissues resulting in hypoxic microenvironment and promotes metastasis. Here we have explored tumor vessel ...modulating activity of Sac-1004, a recently developed molecule in our lab, which directly potentiates VE-cadherin-mediated endothelial cell junction. Sac-1004 could enhance vascular junction integrity in tumor vessels and thereby inhibit vascular leakage and enhance vascular perfusion. Improved perfusion enabled Sac-1004 to have synergistic anti-tumor effect on cisplatin-mediated apoptosis of tumor cells. Interestingly, characteristics of normalized blood vessels namely reduced hypoxia, improved pericyte coverage and decreased basement membrane thickness were readily observed in tumors treated with Sac-1004. Remarkably, Sac-1004 was also able to inhibit lung and lymph node metastasis in MMTV and B16BL6 tumor models. This was in correlation with a reduction in epithelial-to-mesenchymal transition of tumor cells with considerable diminution in expression of related transcription factors. Moreover, cancer stem cell population dropped substantially in Sac-1004 treated tumor tissues. Taken together, our results showed that direct restoration of vascular junction could be a significant strategy to induce normalization of tumor blood vessels and reduce metastasis.
Delta-like ligand 4 (DLL4) expression in endothelial cells is intimately associated with angiogenic sprouting and vascular remodeling, but the precise mechanism of transcriptional regulation of DLL4 ...remains incompletely understood. Here, we showed that LIM-domain binding protein 2 (LDB2) plays an important role in regulating basal DLL4 and VEGF-induced DLL4 expression. Knockdown of LDB2 using siRNA enhanced endothelial sprouting and tubular network formation in vitro. Injection of ldb2-morpholino resulted in defective development of intersegmental vessels in zebrafish. Reduction or overexpression of LDB2 in endothelial cells decreased or increased DLL4 expression. LDB2 regulated DLL4 promoter activity by binding to its promoter region and the same promoter region was occupied and regulated by the LMO2/TAL1/GATA2 complex. Interestingly, LDB2 also mediated VEGF-induced DLL4 expression in endothelial cells. The regulation of DLL4 by the LDB2 complex provides a novel mechanism of DLL4 transcriptional control that may be exploited to develop therapeutics for aberrant vascular remodeling. BMB Reports 2018; 51(1): 21-26