scholarly journals Endothelial cells act as gatekeepers for LTβR-dependent thymocyte emigration

2018 ◽  
Vol 215 (12) ◽  
pp. 2984-2993 ◽  
Author(s):  
Kieran D. James ◽  
Emilie J. Cosway ◽  
Beth Lucas ◽  
Andrea J. White ◽  
Sonia M. Parnell ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Gene Targeting ◽  
Developmental Stages ◽  
Perivascular Space ◽  
Specific Gene ◽  
Thymic Epithelium ◽  
Cell Compartments ◽  
Rate Limit ◽  
Test Current ◽  
Limit Access

The emigration of mature thymocytes from the thymus is critical for establishing peripheral T cell compartments. However, the pathways controlling this process and the timing of egress in relation to postselection developmental stages are poorly defined. Here, we reexamine thymocyte egress and test current and opposing models in relation to the requirement for LTβR, a regulator of thymic microenvironments and thymocyte emigration. Using cell-specific gene targeting, we show that the requirement for LTβR in thymocyte egress is distinct from its control of thymic epithelium and instead maps to expression by endothelial cells. By separating emigration into sequential phases of perivascular space (PVS) entry and transendothelial migration, we reveal a developmentally ordered program of egress where LTβR operates to rate limit access to the PVS. Collectively, we show the process of thymic emigration ensures only the most mature thymocytes leave the thymus and demonstrate a role for LTβR in the initiation of thymus emigration that segregates from its control of medulla organization.

scholarly journals Antiviral effect of Bosentan and Valsartan during coxsackievirus B3 infection of human endothelial cells

2010 ◽  
Vol 91 (8) ◽  
pp. 1959-1970 ◽  
Author(s):  
Carsten Funke ◽  
Martin Farr ◽  
Bianca Werner ◽  
Sven Dittmann ◽  
Klaus Überla ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Viral Entry ◽  
Umbilical Vein ◽  
Viral Myocarditis ◽  
Antiviral Effect ◽  
Specific Gene ◽  
Human Endothelial Cells ◽  
Drug Induced ◽  
Adenoviral Infection ◽  
Cvb3 Infection

In viral myocarditis, adeno- and enteroviruses have most commonly been implicated as causes of infection. Both viruses require the human coxsackie-adenovirus receptor (CAR) to infect the myocardium. Due to its crucial role for viral entry, CAR-downregulation may lead to novel approaches for treatment for viral myocarditis. In this study, we report on pharmaceutical drug influences on CAR levels in human umbilical vein endothelial cells (HUVEC) and cervical carcinoma cells (HeLa) detected by immunoblotting, quantitative real time-PCR and cellular susceptibility to the cardiotropic coxsackie-B3 virus strain Nancy (CVB3). Our results indicate, for the first time, a dose-dependent CAR mRNA and protein downregulation upon Valsartan and Bosentan treatment. Most interestingly, drug-induced CAR diminution significantly reduced the viral load in CVB3-infected HUVEC. In order to assess the regulatory effects of both drugs in detail, we knocked down their protein targets, the G-protein coupled receptors angiotensin-II type-1 receptor (AT1R) and endothelin-1 type-A and -B receptors (ETAR/ETBR) in HUVEC. Receptor-specific gene silencing indicates that CAR gene expression is regulated by agonistic and antagonistic binding to ETBR, but not ETAR. In addition, neither stimulation nor inhibition of AT1R seemed to be involved in CAR gene regulatory processes. Our study indicates that Valsartan and Bosentan protected human endothelial cells from CVB3-infection. Therefore, besides their well-known anti-hypertensive effects these drugs may also protect the myocardium and other tissues from coxsackie- and adenoviral infection.

scholarly journals Deletion of the DNA Ligase IV Gene in Candida glabrata Significantly Increases Gene-Targeting Efficiency

2015 ◽  
Vol 14 (8) ◽  
pp. 783-791 ◽  
Author(s):  
Yuke Cen ◽  
Alessandro Fiori ◽  
Patrick Van Dijck
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Candida Glabrata ◽  
The United States ◽  
Genetic Alterations ◽  
Nonhomologous End Joining ◽  
Specific Gene ◽  
Phylogenetic Distance ◽  
End Joining ◽  
Content Type

ABSTRACTCandida glabratais reported as the second most prevalent human opportunistic fungal pathogen in the United States. Over the last decades, its incidence increased, whereas that ofCandida albicansdecreased slightly. One of the main reasons for this shift is attributed to the inherent tolerance ofC. glabratatoward the commonly used azole antifungal drugs. Despite a close phylogenetic distance toSaccharomyces cerevisiae, homologous recombination works with poor efficiency inC. glabratacompared to baker's yeast, in fact limiting targeted genetic alterations of the pathogen's genome. It has been shown that nonhomologous DNA end joining is dominant over specific gene targeting inC. glabrata. To improve the homologous recombination efficiency, we have generated a strain in which theLIG4gene has been deleted, which resulted in a significant increase in correct gene targeting. The very specific function of Lig4 in mediating nonhomologous end joining is the reason for the absence of clear side effects, some of which affect theku80mutant, another mutant with reduced nonhomologous end joining. We also generated aLIG4reintegration cassette. Our results show that thelig4mutant strain may be a valuable tool for theC. glabrataresearch community.

scholarly journals Gata2 is required for HSC generation and survival

2013 ◽  
Vol 210 (13) ◽  
pp. 2843-2850 ◽  
Author(s):  
Emma de Pater ◽  
Polynikis Kaimakis ◽  
Chris S. Vink ◽  
Tomomasa Yokomizo ◽  
Tomoko Yamada-Inagawa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Developmental Stages ◽  
Vascular Endothelial ◽  
Vascular Integrity ◽  
Hematopoietic Stem ◽  
Vascular Endothelial Cadherin ◽  
Umbilical Arteries ◽  
Specific Deletion

Knowledge of the key transcription factors that drive hematopoietic stem cell (HSC) generation is of particular importance for current hematopoietic regenerative approaches and reprogramming strategies. Whereas GATA2 has long been implicated as a hematopoietic transcription factor and its dysregulated expression is associated with human immunodeficiency syndromes and vascular integrity, it is as yet unknown how GATA2 functions in the generation of HSCs. HSCs are generated from endothelial cells of the major embryonic vasculature (aorta, vitelline, and umbilical arteries) and are found in intra-aortic hematopoietic clusters. In this study, we find that GATA2 function is essential for the generation of HSCs during the stage of endothelial-to-hematopoietic cell transition. Specific deletion of Gata2 in Vec (Vascular Endothelial Cadherin)-expressing endothelial cells results in a deficiency of long-term repopulating HSCs and intra-aortic cluster cells. By specific deletion of Gata2 in Vav-expressing hematopoietic cells (after HSC generation), we further show that GATA2 is essential for HSC survival. This is in contrast to the known activity of the RUNX1 transcription factor, which functions only in the generation of HSCs, and highlights the unique requirement for GATA2 function in HSCs throughout all developmental stages.

Abstract 225: Chronic Alcohol Consumption Alters the Epigenetic Fingerprint of Cardiac Cell Types

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Alexander R Mackie ◽  
Erin E Vaughan ◽  
Mohsin Khan ◽  
Suresh K Verma ◽  
Srikanth Garikipati ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Ejection Fraction ◽  
Cell Types ◽  
Chronic Ethanol ◽  
Ethanol Ingestion ◽  
Specific Gene ◽  
Cardiac Cell ◽  
Functional Changes ◽  
Chronic Ethanol Consumption ◽  
High Ethanol

The cardiac influence of chronic ethanol (EtOH) consumption in humans is dictated by the frequency of ethanol ingestion. Whereas moderate consumption (i.e. 1-2 alcoholic drinks/day) imparts a cardiac benefit to patients by reducing adverse cardiovascular event (ACE) occurrences, consumption below or beyond these moderate levels significantly increases the incidence of ACEs. Despite these observations, little is known regarding the functional impact of chronic EtOH consumption on post-myocardial infarct repair or the cellular mechanisms involved in this process. Thus, we investigated the post-AMI functional consequences of chronic ethanol consumption in mice. Mice received chronic ethanol via the Lieber-DeCarli paradigm (i.e. 0%, 1% (moderate) or 5% (high) ethanol v/v) in an isocaloric fashion for 8 weeks. After 8 weeks, mice underwent a 60-minute ischemic/reperfusion injury and the subsequent assessment of their cardiac function for 4 weeks post-AMI. As early as two weeks post-AMI, mice fed the 1% EtOH displayed modest yet significant improvements in ejection fraction and systolic ventricular volumes as compared to control mice. Conversely, the 5% EtOH diet group displayed diminished ejection fraction and increases in both systolic chamber volume and infarct size. To explore the cellular basis of these observed functional changes, primary cardiac cell types (fibroblasts and endothelial cells) treated chronically (5 days) with ethanol in vitro (i.e. 0%, 0.1% (moderate) or 0.5% (high) ethanol v/v) displayed robust changes in their epigenetic histone-modification profiles indicating a high likelihood for changes in cell specific gene expression. In addition, conditioned media from ethanol treated primary cardiac fibroblasts (PCFBs) altered the tube forming capacity of endothelial cells in a matrigel tube-formation assay in a dose pattern akin to what was observed in vivo. This data suggests that chronic ethanol directly invokes epigenetic changes in PCFBs that can modify their contribution to cardiac repair processes following AMI. Lastly, we are actively investigating whether ethanol-induced changes to endothelial cell epigenetic patterns alter the cells responses in the face of an ischemic insult.

Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice

2001 ◽  
Vol 114 (4) ◽  
pp. 671-676 ◽  
Author(s):  
E. Gustafsson ◽  
C. Brakebusch ◽  
K. Hietanen ◽  
R. Fassler
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Cre Recombinase ◽  
Embryoid Bodies ◽  
Lymphoid Cells ◽  
Adult Brain ◽  
Specific Gene

Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth, LacZ activity was detected in most endothelial cells within the embryo and of extra-embryonic tissues such as yolk sac and chorioallantoic placenta. Ectopic expression of Cre was observed in approximately 12–20% of the adult erythroid, myeloid and lymphoid cells and in subregions of the adult brain. These results show that the tie-1-Cre transgenic strain can efficiently direct deletion of floxed genes in endothelial cells in vivo.

Xanthan gum-functionalised span nanoparticles for gene targeting to endothelial cells

2018 ◽  
Vol 170 ◽  
pp. 411-420 ◽  
Author(s):  
I. Fernandez-Piñeiro ◽  
J. Alvarez-Trabado ◽  
J. Márquez ◽  
I. Badiola ◽  
A. Sanchez
Keyword(s):  
Endothelial Cells ◽  
Gene Targeting ◽  
Xanthan Gum

scholarly journals Effect of mechanical stretch on HIF-1α and MMP-2 expression in capillaries isolated from overloaded skeletal muscles: laser capture microdissection study

2005 ◽  
Vol 289 (3) ◽  
pp. H1315-H1320 ◽  
Author(s):  
Malgorzata Milkiewicz ◽  
Tara L. Haas
Keyword(s):  
Endothelial Cells ◽  
Laser Capture Microdissection ◽  
Skeletal Muscles ◽  
Hypoxia Inducible Factor ◽  
Fold Increase ◽  
Mechanical Stretch ◽  
Specific Gene ◽  
Capillary Endothelial Cells ◽  
Laser Capture

Under physiological nonhypoxic conditions, angiogenesis can be driven by mechanical forces. However, because of the limitations of the specific gene expression analysis of microvessels from in vivo experiments, the mechanisms regulating the coordinated expression of angiogenic factors implicated in the process remain intangible. In this study, the technique of laser capture microdissection (LCM) was adapted for the study of angiogenesis in skeletal muscles. With a combination of LCM and real-time quantitative PCR it was demonstrated that capillary endothelial cells produce matrix metalloproteinase (MMP)-2 and that mechanical stretch of capillaries within muscle tissue markedly increases MMP-2 mRNA (2.5-fold increase vs. control; P < 0.05). In addition, we showed that transcription factor hypoxia-inducible factor (HIF)-1α expression was 13.5-fold higher in capillaries subjected to stretch compared with controls ( P < 0.05). These findings demonstrate the feasibility of this approach to study angiogenic gene regulation and provide novel evidence of HIF-1α induction in stretched capillary endothelial cells.

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