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1.
STAR Protoc ; 2(2): 100428, 2021 06 18.
Article in English | MEDLINE | ID: mdl-33870229

ABSTRACT

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).


Subject(s)
Cell Adhesion/physiology , Cytological Techniques/methods , Embryo, Nonmammalian/cytology , Integrins/metabolism , Mesoderm/cytology , Animals , Cells, Cultured , Zebrafish
2.
Dev Cell ; 49(5): 681-696.e6, 2019 06 03.
Article in English | MEDLINE | ID: mdl-31006651

ABSTRACT

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 ß1 to enhance PLPM cell adhesion to fibronectin localized at the somite boundary. Rap1b-induced integrin-ß1-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.


Subject(s)
Cell Adhesion , Fibronectins/metabolism , Hematopoietic Stem Cells/cytology , Integrin beta1/metabolism , Receptors, Notch/metabolism , Zebrafish Proteins/metabolism , rap GTP-Binding Proteins/metabolism , Animals , Fibronectins/genetics , Hematopoietic Stem Cells/metabolism , Integrin beta1/genetics , Receptors, Notch/genetics , Zebrafish , Zebrafish Proteins/genetics , rap GTP-Binding Proteins/genetics
3.
BMB Rep ; 51(1): 21-26, 2018 Jan.
Article in English | MEDLINE | ID: mdl-28946938

ABSTRACT

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].


Subject(s)
Endothelial Cells/metabolism , Intercellular Signaling Peptides and Proteins/biosynthesis , Intracellular Signaling Peptides and Proteins/biosynthesis , LIM Domain Proteins/metabolism , Transcription Factors/metabolism , Zebrafish Proteins/biosynthesis , Zebrafish Proteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Animals, Genetically Modified , Calcium-Binding Proteins , Gene Knockdown Techniques , Human Umbilical Vein Endothelial Cells , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/genetics , LIM Domain Proteins/deficiency , LIM Domain Proteins/genetics , Neovascularization, Physiologic , Promoter Regions, Genetic , RNA, Small Interfering/genetics , Signal Transduction , Transcription Factors/deficiency , Transcription Factors/genetics , Transcription, Genetic , Vascular Endothelial Growth Factor A/metabolism , Zebrafish , Zebrafish Proteins/deficiency , Zebrafish Proteins/genetics
4.
J Nippon Med Sch ; 84(4): 148-159, 2017.
Article in English | MEDLINE | ID: mdl-28978894

ABSTRACT

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 ß-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.


Subject(s)
Antigens, CD/metabolism , Antigens, CD/physiology , Cadherins/metabolism , Cadherins/physiology , Endothelial Cells/metabolism , Endothelial Cells/physiology , Intercellular Junctions/metabolism , Intercellular Junctions/physiology , Actin Cytoskeleton/metabolism , Cell Adhesion/genetics , Cell Adhesion/physiology , Humans , Phosphorylation , rap1 GTP-Binding Proteins/physiology
5.
J Clin Invest ; 127(2): 457-471, 2017 Feb 01.
Article in English | MEDLINE | ID: mdl-27991863

ABSTRACT

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.


Subject(s)
Gene Expression Regulation, Neoplastic , Lectins, C-Type/metabolism , Lymphangiogenesis , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Neoplasms, Experimental/blood supply , Neovascularization, Pathologic/metabolism , Signal Transduction , Vascular Endothelial Growth Factor Receptor-2/metabolism , Vascular Endothelial Growth Factor Receptor-3/metabolism , Animals , Human Umbilical Vein Endothelial Cells , Humans , Lectins, C-Type/genetics , Membrane Proteins/genetics , Mice , Mice, Knockout , Neoplasm Proteins/genetics , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/pathology , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-3/genetics
6.
Oncotarget ; 5(9): 2761-77, 2014 May 15.
Article in English | MEDLINE | ID: mdl-24811731

ABSTRACT

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.


Subject(s)
Antigens, CD/metabolism , Cadherins/metabolism , Carcinoma, Lewis Lung/prevention & control , Endothelium, Vascular/drug effects , Lung Neoplasms/prevention & control , Melanoma, Experimental/prevention & control , Neovascularization, Pathologic/prevention & control , Saponins/pharmacology , Animals , Antigens, CD/genetics , Apoptosis/drug effects , Blotting, Western , Cadherins/genetics , Carcinoma, Lewis Lung/blood supply , Carcinoma, Lewis Lung/metabolism , Carcinoma, Lewis Lung/pathology , Cell Movement/drug effects , Cell Proliferation/drug effects , Diabetic Retinopathy/metabolism , Diabetic Retinopathy/pathology , Diabetic Retinopathy/prevention & control , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Epithelial-Mesenchymal Transition/drug effects , HeLa Cells , Humans , Immunoenzyme Techniques , Lung Neoplasms/blood supply , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Lymphatic Metastasis , MCF-7 Cells , Male , Melanoma, Experimental/blood supply , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
7.
Biochem Biophys Res Commun ; 421(2): 305-11, 2012 May 04.
Article in English | MEDLINE | ID: mdl-22708120

ABSTRACT

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-a-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.


Subject(s)
Cell Nucleus/metabolism , Endothelial Cells/metabolism , Interleukins/metabolism , Transcription Factor RelA/genetics , Transcriptional Activation , Cell Adhesion/genetics , Cells, Cultured , Gene Knockdown Techniques , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Inflammation/genetics , Intercellular Adhesion Molecule-1/genetics , Interleukin-33 , Monocytes/physiology , Promoter Regions, Genetic , Tumor Necrosis Factor-alpha/pharmacology , Vascular Cell Adhesion Molecule-1/genetics
8.
J Clin Invest ; 121(5): 1882-93, 2011 May.
Article in English | MEDLINE | ID: mdl-21540552

ABSTRACT

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.


Subject(s)
Endothelial Cells/cytology , Gene Expression Regulation , Intercellular Signaling Peptides and Proteins/metabolism , Neovascularization, Pathologic , Proteins/metabolism , Wnt Proteins/metabolism , Animals , Aorta/metabolism , Humans , Ischemia/pathology , Male , Mice , Mice, Inbred C57BL , Myocardial Infarction/pathology , Rats , Rats, Sprague-Dawley , Umbilical Cord/cytology
9.
Biochem Biophys Res Commun ; 408(1): 186-92, 2011 Apr 29.
Article in English | MEDLINE | ID: mdl-21501586

ABSTRACT

Homeobox (HOX) family genes, major transcription factors for embryonic development, have been also implicated in vascular development and angiogenesis, particularly with regulation of genes involved in cell-cell or cell-extracellular matrix (ECM) interactions. However, the cellular and molecular functions of HOXD1 in endothelial cells (ECs) are yet to be explored. We here report that HOXD1 is prominently expressed in human ECs and regulates angiogenic activities. Knockdown of HOXD1 in ECs resulted in significant inhibition of migration and adhesion as well as tube like structure formation. These effects were correlated with the reduced expression of integrin ß1 (ITGB1), an important signaling component of angiogenesis. Consistently, ITGB1 promoter activity was decreased by HOXD1 knockdown in ECs. Furthermore, we identified the putative HOXD1-binding sites in the promoter region of ITGB1. Together, these findings suggest that HOXD1 plays a significant role in EC functions by regulating the expression of ITGB1.


Subject(s)
Endothelium, Vascular/physiology , Gene Expression Regulation , Homeodomain Proteins/metabolism , Integrin beta1/genetics , Neovascularization, Physiologic/genetics , Transcription Factors/metabolism , Binding Sites , Cell Adhesion/genetics , Cell Movement/genetics , Cells, Cultured , Endothelium, Vascular/metabolism , Gene Knockdown Techniques , Homeodomain Proteins/genetics , Humans , Promoter Regions, Genetic , Transcription Factors/genetics , Transcription, Genetic
10.
Biochem Biophys Res Commun ; 404(1): 103-8, 2011 Jan 07.
Article in English | MEDLINE | ID: mdl-21095181

ABSTRACT

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.


Subject(s)
Cell Adhesion Molecules/physiology , Endothelial Cells/physiology , Endothelium, Vascular/physiology , Lectins, C-Type/physiology , Membrane Proteins/physiology , Neovascularization, Physiologic , Animals , Cell Adhesion , Cell Adhesion Molecules/genetics , Cells, Cultured , Endothelium, Vascular/cytology , Female , Gene Deletion , Gene Silencing , Humans , Lectins, C-Type/genetics , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL
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