The Vascular Endothelial-Cadherin Promoter Directs Endothelial-Specific Expression in Transgenic Mice

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 184-192 ◽  
Author(s):  
S. Gory ◽  
M. Vernet ◽  
M. Laurent ◽  
E. Dejana ◽  
J. Dalmon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transgenic Mice ◽  
Dna Sequences ◽  
Vascular System ◽  
Proximal Region ◽  
Vascular Endothelial ◽  
Specific Expression ◽  
Functional Regions ◽  
Vascular Endothelial Cadherin

Vascular endothelial-cadherin (VE-cadherin) is a calcium-dependent adhesive molecule, exclusively and constitutively expressed in endothelial cells. Analysis of the VE-cadherin promoter fused to a reporter gene in bovine aortic endothelial cells showed three major functional regions. The proximal region alone (−139, +24) promoted nonspecific transcription; the addition of the (−289, −140) and (−2226, −1190) domains abolished transcription in fibroblasts while expression in endothelial cells remained unchanged, suggesting that fragments (−2226, +24) and longer contain the full endogenous promoter activity. To study the transcriptional specificity of the promoter region in vivo, we generated transgenic mice carrying the chimeric construct containing the (−2486, +24) region. The promoter directed reporter expression in all examined organs of adult transgenic mice. During embryonic development, transgene expression was detected at the early steps of vasculogenesis. Later, the expression persisted during development of the vascular system and was restricted to the endothelial layer of the vessels. Together, these data provide evidence for specific regulatory regions within the VE-cadherinpromoter. Furthermore, the identification of DNA sequences restricting gene expression to the endothelium has many potential applications for the development of animal models of cardiovascular or angiogenic diseases or for the delivery of therapeutic molecules.

The Vascular Endothelial-Cadherin Promoter Directs Endothelial-Specific Expression in Transgenic Mice

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 184-192 ◽  
Author(s):  
S. Gory ◽  
M. Vernet ◽  
M. Laurent ◽  
E. Dejana ◽  
J. Dalmon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transgenic Mice ◽  
Dna Sequences ◽  
Vascular System ◽  
Proximal Region ◽  
Vascular Endothelial ◽  
Specific Expression ◽  
Functional Regions ◽  
Vascular Endothelial Cadherin

Abstract Vascular endothelial-cadherin (VE-cadherin) is a calcium-dependent adhesive molecule, exclusively and constitutively expressed in endothelial cells. Analysis of the VE-cadherin promoter fused to a reporter gene in bovine aortic endothelial cells showed three major functional regions. The proximal region alone (−139, +24) promoted nonspecific transcription; the addition of the (−289, −140) and (−2226, −1190) domains abolished transcription in fibroblasts while expression in endothelial cells remained unchanged, suggesting that fragments (−2226, +24) and longer contain the full endogenous promoter activity. To study the transcriptional specificity of the promoter region in vivo, we generated transgenic mice carrying the chimeric construct containing the (−2486, +24) region. The promoter directed reporter expression in all examined organs of adult transgenic mice. During embryonic development, transgene expression was detected at the early steps of vasculogenesis. Later, the expression persisted during development of the vascular system and was restricted to the endothelial layer of the vessels. Together, these data provide evidence for specific regulatory regions within the VE-cadherinpromoter. Furthermore, the identification of DNA sequences restricting gene expression to the endothelium has many potential applications for the development of animal models of cardiovascular or angiogenic diseases or for the delivery of therapeutic molecules.

Vascular Endothelial Cadherin-Expressing Cells Involved in Both Developmental and Adult Neovascularization.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2610-2610
Author(s):  
Naoko Kogata ◽  
Yuji Arai ◽  
Kazuaki Hashimoto ◽  
Minetaro Ogawa ◽  
Naoki Mochizuki
Keyword(s):  
Endothelial Cells ◽  
Myocardial Ischemia ◽  
Transgenic Mice ◽  
Vascular Endothelial ◽  
Lacz Reporter ◽  
Reporter Expression ◽  
Vessel Formation ◽  
Vascular Endothelial Cadherin ◽  
Reporter Mice ◽  
Vasculogenesis And Angiogenesis

Abstract Vessel formation, including vasculogenesis and angiogenesis, is thought to be directly mediated by adhesive molecules between vascular endothelial cells and adjacent cells. Vascular endothelial cadherin (VE-cadherin, CD144) is a adhesive molecule exclusively expressed in adherens junctions of cultured endothelial cells. VE-cadherin transcripts have been detected at first in mesodermal cells. However, no work has focused on which cell type expresses VE-cadherin during vessel formation and after vessel stabilization. To monitor VE-cadherin in vivo, we generated transgenic mice expressing a VE-cadherin promoter-driven Cre (VE-cad-Cre). By crossing VE-cad-Cre with loxP-based EGFP reporter mice (CAG-CAT-EGFP) and LacZ reporter mice (cAct-XstopX-LacZ), we obtained two double transgenic mice; one expressing EGFP (VE/EG) another expressing LacZ (VE/Z) dependently upon Cre driven by VE-cadherin promoter. Both reporter expression was increased in developing vasculature during embryogenesis, while it was decreased in postnatal vessel maturation. Intriguingly, LacZ reporter was found in the budding cells and lining cells of the dorsal aorta in the aorta-gonad-mesonephros region, suggesting that VE-cadherin-expressing cells belongs to hemangioblasts. Consistently, although VE-cadherin and its mRNA was detected in vasculature by immunohistochemistry and in situ hybridization, respectively, during embryogenesis, it was not detected in maturated vessels. These results indicated that VE-cadherin promoter activity reflects the endogeneous VE-cadherin expression and that VE-cadherin promoter is activated during developmental vasculogenesis and angiogenesis. To test whether the VE-cadherin promoter is re-activated in postnatal neovascularization, we used two models; a VEGF-induced cornea angiogenesis model and a myocardial ischemia model. VEGF implanted in the VE/EG eyes promoted the neovascularization visualized by EGFP expression. Myocardial ischemia in which the coronary artery was banded triggered the VE-cadherin promoter, thereby inducing reporter expression in vascular cells (endothelial cells and mural cells) of undamaged region and in infiltrated cells of damaged region. Furthermore, by flow cytometric analyses, we confirmed that myocardial ischemia re-activated the VE-cadherin promoter of both bone marrow cells and peripheral blood cells, as indicated by the remarkable increase of EGFP-positive cells. These data show that the VE-cadherin promoter is re-activated in pre-existing vascular cells and infiltrated cells during adult neovascularization. Collectively, VE-cadherin-expressing cells participates in both developmental and postnatal vasulogenesis and angiogenesis.

A monoclonal antibody to vascular endothelial–cadherin inhibits tumor angiogenesis without side effects on endothelial permeability

Blood ◽  
2002 ◽  
Vol 100 (3) ◽  
pp. 905-911 ◽  
Author(s):  
Monica Corada ◽  
Lucia Zanetta ◽  
Fabrizio Orsenigo ◽  
Ferruccio Breviario ◽  
Maria Grazia Lampugnani ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cells ◽  
Cell Adhesion ◽  
Vascular Permeability ◽  
Antibody Binding ◽  
Vascular Endothelial ◽  
Collagen Gels ◽  
Vascular Endothelial Cadherin

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

scholarly journals Complete lack of NF-κB activity in IKK1 and IKK2 double-deficient mice: additional defect in neurulation

2000 ◽  
Vol 14 (14) ◽  
pp. 1729-1733 ◽  
Author(s):  
Qiutang Li ◽  
Gabriela Estepa ◽  
Sylvie Memet ◽  
Alain Israel ◽  
Inder M. Verma
Keyword(s):  
Endothelial Cells ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Additional Defect ◽  
Deficient Mice

NF-κB activity is induced by cytokines, stress, and pathogens. IKK1 and IKK2 are critical IκB kinases in NF-κB activation. In this study mice lacking IKK1 and IKK2 died at E12. Additional defect in neurulation associated with enhanced apoptosis in the neuroepithelium was also observed. MEF cells fromIKK1−/−/IKK2−/−embryos did not respond to NF-κB inducers. Upon crossing withκB–lacZ transgenic mice, double-deficient embryos also lost lacZ transgene expression in vascular endothelial cells during development. Our data suggest that IKK1 and IKK2 are essential for NF-κB activation in vivo and have an important role in protecting neurons against excessive apoptosis during development.

Sequences in Intron 51 of the VWF Gene Target Promoter Activation to a Subset of Lung Endothelial Cells in Transgenic Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1798-1798
Author(s):  
Nadia Jahroudi ◽  
Marjan Nassiri ◽  
Molly S. Stitt ◽  
Karla Wasserloos ◽  
Simon C. Watkins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Transgenic Mice ◽  
Promoter Activity ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Sv40 Promoter ◽  
Lung Endothelial Cells

Abstract The VWF gene, located on chromosome 12, is 178 kb long and contains 52 exons. VWF is synthesized exclusively by endothelial cells and megakaryocytes. We have previously characterized a region of the VWF gene spanning sequences −487 to +247 that functions as an endothelial specific promoter in vitro. Subsequently a number of transacting factors including, NF1, Oct 1, Ets, GATA6, NFY and Ebp4 that positively and negatively regulate the activity of this promoter were identified by us and others. However, in vivo analysis of the promoter demonstrated that this promoter fragment (sequences −487 to +247) targets activation of fused heterologous transgenes (LacZ and amyloid precursor proteins) specifically and exclusively to brain vascular endothelial cells of transgenic mice. A longer VWF promoter fragment, including 2182 bp of the 5′ flanking sequences, the first exon and the first intron was reported by Aird et al to activate LacZ transgene expression in endothelial cells of the heart and muscle as well as brain of transgenic mice. Considering that endogenous VWF expression is observed in almost all endothelial cells, these results suggested that additional VWF gene sequences were required for transcriptional activation of the VWF promoter in vascular endothelial cells of multiple other organs in vivo; and that distinct regions of the VWF gene are required to achieve promoter activity in endothelial cells of distinct organs. To identify additional cis acting elements within the VWF gene that may participate in its transcriptional regulation generally and/or in distinct organs, we explored the possibility that such sequences may be located in VWF chromatin regions that show hypersensitivity to DNase I. We have now identified a region within intron 51 of the VWF gene that is DNase I hypersensitive (HSS) specifically in non-endothelial cells. This region was shown to interact with YY1 transcription factor in a manner that forms endothelial and non-endothelial specific complexes. In vitro transfection analyses demonstrated that HSS sequences containing this YY1 binding site significantly increased a heterologous SV40 promoter activity specifically in endothelial cell and that this increase was dependent on the presence of an intact YY1 binding site. In contrast, the HSS sequences significantly decreased the SV40 promoter activity in non-endothelial cells. These results suggested that the HSS sequences may participate in activation of gene expression in an YY1 dependent manner in endothelial cells, while repress gene expression in non-endothelial cells. Nevertheless the HSS sequences did not significantly affect the homologous VWF promoter activity that was analyzed by in vitro transfection analyses. However, in vivo analyses demonstrated that addition of these sequences to the VWF promoter (−487 to +247) results in promoter activation in lung and brain vascular endothelial cells. These results demonstrate that the HSS sequences in intron 51 of the VWF gene participate in organ specific regulation of VWF gene expression, an observation that could not be determined by in vitro analysis. These analyses suggest that the HSS sequences contain cis-acting elements that are specifically necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo.

Stealthy Nanoparticles Protecting Endothelium Barrier from Leakiness by Resisting the Absorption of VE-cadherin

Nanoscale ◽  
2021 ◽  
Author(s):  
Yuan Huang ◽  
Suxiao Wang ◽  
Jin-Zhi Zhang ◽  
Hang-Xing Wang ◽  
Qichao Zou ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Interstitial Space ◽  
Cell Interactions ◽  
Vascular Endothelial ◽  
Vascular Endothelial Cadherin ◽  
Cell Cell

Nanomaterial induced endothelial cells leakiness (NanoEL) is caused because nanomaterials enter the interstitial space of endothelial cells and disrupt the endothelial cell-cell interactions by interacting with vascular endothelial cadherin (VE-cad)....

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

scholarly journals Intestinal Apolipoprotein A-IV Gene Transcription Is Controlled by Two Hormone-Responsive Elements: A Role for Hepatic Nuclear Factor-4 Isoforms

2005 ◽  
Vol 19 (9) ◽  
pp. 2320-2334 ◽  
Author(s):  
Amena Archer ◽  
Dominique Sauvaget ◽  
Valérie Chauffeton ◽  
Pierre-Etienne Bouchet ◽  
Jean Chambaz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Distal Region ◽  
Proximal Region ◽  
Dominant Negative ◽  
Specific Expression ◽  
Nuclear Factors ◽  
Dominant Negative Form ◽  
Responsive Element ◽  
Hormone Responsive Element

Abstract In the small intestine, the expression of the apolipoprotein (apo) C-III and A-IV genes is restricted to the enterocytes of the villi. We have previously shown that, in transgenic mice, specific expression of the human apo C-III requires a hormone-responsive element (HRE) located in the distal region of the human apoA-IV promoter. This HRE binds the hepatic nuclear factors (HNF)-4α and γ. Here, intraduodenal injections in mice and infections of human enterocytic Caco-2/TC7 cells with an adenovirus expressing a dominant-negative form of HNF-4α repress the expression of the apoA-IV gene, demonstrating that HNF-4 controls the apoA-IV gene expression in enterocytes. We show that HNF-4α and γ functionally interact with a second HRE present in the proximal region of the human apoA-IV promoter. New sets of transgenic mice expressing mutated forms of the promoter, combined with the human apo C-III enhancer, demonstrate that, whereas a single HRE is sufficient to reproduce the physiological cephalo-caudal gradient of apoA-IV gene expression, both HREs are required for expression that is restricted to villi. The combination of multiple HREs may specifically recruit regulatory complexes associating HNF-4 and either coactivators in villi or corepressors in crypts.

scholarly journals Synthesis and physiological activity of heterodimers comprising different splice forms of vascular endothelial growth factor and placenta growth factor

1996 ◽  
Vol 316 (3) ◽  
pp. 703-707 ◽  
Author(s):  
Ralf BIRKENHÄGER ◽  
Bernard SCHNEPPE ◽  
Wolfgang RÖCKL ◽  
Jörg WILTING ◽  
Herbert A. WEICH ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Vascular Endothelial Cells ◽  
Physiological Activity ◽  
Expression System ◽  
Vascular Endothelial ◽  
Placenta Growth Factor ◽  
Splice Forms

Vascular endothilial growth factor (VEGF) and placenta growth factor (PIGF) are members of a dimeric-growth-factor family with angiogenic properties. VEGF is a highly potent and specific mitogen for endothelial cells, playing a vital role in angiogenesis in vivo. The role of PIGF is less clear. We expressed the monomeric splice forms VEGF-165, VEGF-121, PIGF-1 and PlGF-2 as unfused genes in Escherichia coli using the pCYTEXP expression system. In vitro dimerization experiments revealed that both homo- and hetero-dimers can be formed from these monomeric proteins. The dimers were tested for their ability to promote capillary growth in vivo and stimulate DNA synthesis in cultured human vascular endothelial cells. Heterodimers comprising different VEGF splice forms, or combinations of VEGF/PlGF splice forms, showed mitogenic activity. The results demonstrate that four different heterodimeric growth factors are likely to have as yet uncharacterized functions in vivo.

A Single Pitx1 Binding Site Is Essential for Activity of the LHβ Promoter in Transgenic Mice

2001 ◽  
Vol 15 (5) ◽  
pp. 734-746 ◽  
Author(s):  
Christine C. Quirk ◽  
Kristen L. Lozada ◽  
Ruth A. Keri ◽  
John H. Nilson
Keyword(s):  
Transgenic Mice ◽  
Binding Site ◽  
Cell Lines ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Vital Role ◽  
Proximal Region ◽  
Expression Vectors ◽  
Homeodomain Protein

Abstract Reproduction depends on regulated expression of the LHβ gene. Tandem copies of regulatory elements that bind early growth response protein 1 (Egr-1) and steroidogenic factor 1 (SF-1) are located in the proximal region of the LHβ promoter and make essential contributions to its activity as well as mediate responsiveness to GnRH. Located between these tandem elements is a single site capable of binding the homeodomain protein Pitx1. From studies that employ overexpression paradigms performed in heterologous cell lines, it appears that Egr-1, SF-1, and Pitx1 interact cooperatively through a mechanism that does not require the binding of Pitx1 to its site. Since the physiological ramifications of these overexpression studies remain unclear, we reassessed the requirement for a Pitx1 element in the promoter of the LHβ gene using homologous cell lines and transgenic mice, both of which obviate the need for overexpression of transcription factors. Our analysis indicated a striking requirement for the Pitx1 regulatory element. When assayed by transient transfection using a gonadotrope-derived cell line (LβT2), an LHβ promoter construct harboring a mutant Pitx1 element displayed attenuated transcriptional activity but retained responsiveness to GnRH. In contrast, analysis of wild-type and mutant expression vectors in transgenic mice indicated that LHβ promoter activity is completely dependent on the presence of a functional Pitx1 binding site. Indeed, the dependence on an intact Pitx1 binding site in transgenic mice is so strict that responsiveness to GnRH is also lost, suggesting that the mutant promoter is inactive. Collectively, our data reinforce the concept that activity of the LHβ promoter is determined, in part, through highly cooperative interactions between SF-1, Egr-1, and Pitx1. While Egr-1 can be regarded as a key downstream effector of GnRH, and Pitx1 as a critical partner that activates SF-1, our data firmly establish that the Pitx1 element plays a vital role in permitting these functions to occur in vivo.

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