scholarly journals Studying the fate of tumor extracellular vesicles at high spatio-temporal resolution using the zebrafish embryo

2018 ◽  
Author(s):  
Vincent Hyenne ◽  
Shima Ghoroghi ◽  
Mayeul Collot ◽  
Sébastien Harlepp ◽  
Jack Bauer ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Animal Model ◽  
Endothelial Cells ◽  
Tumor Progression ◽  
Extracellular Vesicles ◽  
Zebrafish Embryo ◽  
Living Organism ◽  
List Type ◽  
Membrane Probes

SummaryTumor extracellular vesicles (tumor EVs) mediate the communication between tumor and stromal cells mostly to the benefit of tumor progression. Notably, tumor EVs have been reported to travel in the blood circulation, reach specific distant organs and locally modify the microenvironment. However, visualizing these eventsin vivostill faces major hurdles. Here, we show a new method for tracking individual circulating tumor EVs in a living organism: we combine novel, bright and specific fluorescent membrane probes, MemBright, with the transparent zebrafish embryo as an animal model. We provide the first description of tumor EVs’ hemodynamic behavior and document their arrest before internalization. Using transgenic lines, we show that circulating tumor EVs are uptaken by endothelial cells and blood patrolling macrophages, but not by leukocytes, and subsequently stored in acidic degradative compartments. Finally, we prove that the MemBright can be used to follow naturally released tumor EVsin vivo. Overall, our study demonstrates the usefulness and prospects of zebrafish embryo to track tumor EVsin vivo.HighlightsMemBright, a new family of membrane probes, allows for bright and specific staining of EVsZebrafish melanoma EVs are very similar to human and mouse melanoma EVs in morphology and protein contentThe zebrafish embryo is an adapted model to precisely track tumor EVs dynamics and fate in a living organism from light to electron microscopyCirculating tumor EVs are rapidly uptaken by endothelial cells and patrolling macrophagesCorrelated light and electron microscopy can be used in zebrafish to identify cells and compartments uptaking tumor EVsBlurbDispersion of tumor extracellular vesicles (EVs) throughout the body promotes tumor progression. However the behavior of tumor EVs in body fluids remains mysterious due to their small size and the absence of adapted animal model. Here we show that the zebrafish embryo can be used to track circulating tumor EVsin vivoand provide the first high-resolution description of their dissemination and uptake.

scholarly journals Live tracking of inter-organ communication by endogenous exosomes in vivo

2018 ◽  
Author(s):  
Frederik J Verweij ◽  
Celine Revenu ◽  
Guillaume Arras ◽  
Florent Dingli ◽  
Damarys Loew ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Molecular Mechanisms ◽  
Scavenger Receptor ◽  
Model Organisms ◽  
Zebrafish Embryos ◽  
List Type ◽  
Dependent Manner ◽  
Fluorescent Reporter

SUMMARYExtracellular vesicles (EVs) are released by most cell types but the definitive demonstration of their functional relevance remains challenging due to the lack of appropriate model organisms. Here we developed anin vivomodel to study EV physiology by expressing CD63-pHluorin in zebrafish embryos. A combination of microscopy techniques and proteomic analysis allowed us to study the biogenesis, composition, transfer, uptake and fate of individual endogenous EVsin vivo. We identified an exosome population released in a syntenin-dependent manner from the Yolk Syncytial Layer into the blood circulation. These exosomes were specifically captured, endocytosed and degraded by patrolling macrophages and endothelial cells in the Caudal Vein Plexus (CVP) in a scavenger receptor and dynamin-dependent manner. Interference with exosome secretion affected CVP growth, supporting their trophic role. Altogether, our work provides a unique model to track in vivo inter-organ communication by endogenous exosomes at individual vesicle level and high spatio-temporal accuracy.Highlights- Single endogenous EVs can be live-visualized in the whole embryo with CD63-pHluorin- In the YSL, syntenin regulates exosome release into the blood for their propagation- YSL exosomes reach the tail to be taken up by macrophages and endothelial cells- Uptake is scavenger receptor and dynamin-dependent and provides trophic supportBlurbWe propose zebrafish embryos expressing a fluorescent reporter for exosomes as a relevant model organism to live-track production, journey and fate of individual extracellular vesicles in vivo. Our model allows investigation of the composition of EVs and the molecular mechanisms controlling their biogenesis and fate and functions in receiving cells.

Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 909-914 ◽  
Author(s):  
Enid Yi Ni Lam ◽  
Christopher J. Hall ◽  
Philip S. Crosier ◽  
Kathryn E. Crosier ◽  
Maria Vega Flores
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Fluorescent Protein ◽  
Living Organism ◽  
Time Lapse ◽  
Dorsal Aorta ◽  
Transgenic Zebrafish ◽  
Hematopoietic Stem ◽  
Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

Metaphase-Based Cytogenetic Approach Identifies Radiation-Induced Chromosome and Chromatid Aberrations in Zebrafish Embryos

2021 ◽  
Author(s):  
Halida Thanveer Asana Marican ◽  
Hongyuan Shen
Keyword(s):  
Animal Model ◽  
Radiation Exposure ◽  
Progenitor Cells ◽  
Chromosome Aberrations ◽  
Structural Changes ◽  
Current Knowledge ◽  
Living Organism ◽  
Zebrafish Embryos ◽  
Radiation Induced

Metaphase-based cytogenetic methods based on scoring of chromosome aberrations for the estimation of the radiation dose received provide a powerful approach for evaluating the associated risk upon radiation exposure and form the bulk of our current knowledge of radiation-induced chromosome damages. They mainly rely on inducing quiescent peripheral lymphocytes into proliferation and blocking them at metaphases to quantify the damages at the chromosome level. However, human organs and tissues demonstrate various sensitivity towards radiation and within them, self-proliferating progenitor/stem cells are believed to be the most sensitive populations. The radiation-induced chromosome aberrations in these cells remain largely unknown, especially in the context of an intact living organism. Zebrafish is an ideal animal model for research into this aspect due to their small size and the large quantities of progenitor cells present during the embryonic stages. In this study, we employ a novel metaphase-based cytogenetic approach on zebrafish embryos and demonstrate that chromosome-type and chromatid-type aberrations could be identified in progenitor cells at different cell-cycle stages at the point of radiation exposure. Our work positions zebrafish at the forefront as a useful animal model for studying radiation-induced chromosome structural changes in vivo.

scholarly journals Electro-Metabolic Sensing Through Capillary ATP-Sensitive K+ Channels and Adenosine to Control Cerebral Blood Flow

2021 ◽  
Author(s):  
Maria Sancho ◽  
Nicholas R. Klug ◽  
Amreen Mughal ◽  
Thomas J. Heppner ◽  
David Hill-Eubanks ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Cerebral Blood Flow ◽  
Vascular Function ◽  
Brain Activity ◽  
List Type ◽  
Capillary Endothelial Cells ◽  
Metabolic Sensing ◽  
Cellular Components

SUMMARYThe dense network of capillaries composed of capillary endothelial cells (cECs) and pericytes lies in close proximity to all neurons, ideally positioning it to sense neuro/glial-derived compounds that regulate regional and global cerebral perfusion. The membrane potential (VM) of vascular cells serves as the essential output in this scenario, linking brain activity to vascular function. The ATP-sensitive K+ channel (KATP) is a key regulator of vascular VM in other beds, but whether brain capillaries possess functional KATP channels remains unknown. Here, we demonstrate that brain capillary ECs and pericytes express KATP channels that robustly control VM. We further show that the endogenous mediator adenosine acts through A2A receptors and the Gs/cAMP/PKA pathway to activate capillary KATP channels. Moreover, KATP channel stimulation in vivo causes vasodilation and increases cerebral blood flow (CBF). These findings establish the presence of KATP channels in cECs and pericytes and suggest their significant influence on CBF.HIGHLIGHTSCapillary network cellular components—endothelial cells and pericytes—possess functional KATP channels.Activation of KATP channels causes profound hyperpolarization of capillary cell membranes.Capillary KATP channels are activated by exogenous adenosine via A2A receptors and cAMP-dependent protein kinase.KATP channel activation by adenosine or synthetic openers increases cerebral blood flow.

Factor VIIa induces extracellular vesicles from the endothelium: A potential mechanism for its hemostatic effect

Blood ◽  
2021 ◽  
Author(s):  
Kaushik Das ◽  
Shiva Keshava ◽  
Shabbir A Ansari ◽  
Vijay Kumar Reddy Kondreddy ◽  
Charles Esmon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Factor Viia ◽  
Mutant Mice ◽  
In Vivo Studies ◽  
Cell Protein ◽  
Wild Type ◽  
Hemostatic Effect

Recombinant FVIIa (rFVIIa) is used as a hemostatic agent to treat bleeding disorders in hemophilia patients with inhibitors and other groups of patients. Our recent studies showed that FVIIa binds endothelial cell protein C receptor (EPCR) and induces protease-activated receptor 1 (PAR1)-mediated biased signaling. The importance of FVIIa-EPCR-PAR1-mediated signaling in hemostasis is unknown. In the present study, we show that FVIIa induces the release of extracellular vesicles (EVs) from endothelial cells both in vitro and in vivo. Silencing of EPCR or PAR1 in endothelial cells blocked the FVIIa-induced generation of EVs. Consistent with these data, FVIIa treatment enhanced the release of EVs from murine brain endothelial cells isolated from wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice. In vivo studies revealed that administration of FVIIa to wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice, increase the number of circulating EVs. EVs released in response to FVIIa treatment exhibit enhanced procoagulant activity. Infusion of FVIIa-generated EVs and not control EVs to platelet-depleted mice increased thrombin generation at the site of injury and reduced blood loss. Administration of FVIIa-generated EVs or generation of EVs endogenously by administering FVIIa augmented the hemostatic effect of FVIIa. Overall, our data reveal that FVIIa treatment, through FVIIa-EPCR-PAR1 signaling, releases EVs from the endothelium into the circulation, and these EVs contribute to the hemostatic effect of FVIIa.

A New Absorbable Pressure-Equalizing Tube

2002 ◽  
Vol 127 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Riccardo D'Eredità ◽  
Roger R. Marsh ◽  
Silvano Lora ◽  
Ken Kazahaya
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Animal Model ◽  
Clinical Use ◽  
Survival Times ◽  
Disintegration Rate ◽  
Tube Insertion ◽  
Absorbable Material ◽  
Scanning Electron

OBJECTIVE: We investigated pressure-equalizing (PE) tubes made of biodegradable, absorbable material in an animal model. METHODS: PE tubes, made of poly-bis(ethylanate)phosphazene (PBE) were inserted in 55 ears of 28 Hartley guinea pigs, with survival times of 10, 30, and 60 days after tube insertion. In vivo reactions between the PBE-PE tube and the tympanic membrane (TM) were studied. Tubes, TMs, and middle ears were examined by scanning electron microscopy and light microscopy. RESULTS: There was neither infection nor an inflammatory reaction to the tube within the middle ear in any animal. At 30 days, 53% of the tubes had disintegrated. At 60 days, tubes were still functioning in the 25% of ears. CONCLUSION: More research must be performed before these new PBE PE tubes can be considered for clinical use. Nonetheless, these tubes are promising. The disintegration rate can be controlled by varying the formulation of the polymer, so treatment can be adjusted to the needs of each patient.

SRY gene transferred by extracellular vesicles accelerates atherosclerosis by promotion of leucocyte adherence to endothelial cells

2015 ◽  
Vol 129 (3) ◽  
pp. 259-269 ◽  
Author(s):  
Jin Cai ◽  
Weiwei Guan ◽  
Xiaorong Tan ◽  
Caiyu Chen ◽  
Liangpeng Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Gene Copy Number ◽  
Intercellular Adhesion Molecule ◽  
Gene Copy ◽  
Sry Gene ◽  
Intercellular Adhesion Molecule 1

We set out to investigate whether and how SRY (sex-determining region, Y) DNAs in plasma EVs (extracellular vesicles) is involved in the pathogenesis of atherosclerosis. PCR and gene sequencing found the SRY gene fragment in plasma EVs from male, but not female, patients; EVs from male patients with CAD (coronary artery disease) had a higher SRY GCN (gene copy number) than healthy subjects. Additional studies found that leucocytes, the major source of plasma EVs, had higher SRY GCN and mRNA and protein expression in male CAD patients than controls. After incubation with EVs from SRY-transfected HEK (human embryonic kidney)-293 cells, monocytes (THP-1) and HUVECs (human umbilical vein endothelial cells), which do not endogenously express SRY protein, were found to express newly synthesized SRY protein. This resulted in an increase in the adherence factors CD11-a in THP-1 cells and ICAM-1 (intercellular adhesion molecule 1) in HUVECs. EMSA showed that SRY protein increased the promoter activity of CD11-a in THP-1 cells and ICAM-1 in HUVECs. There was an increase in THP-1 cells adherent to HUVECs after incubation with SRY-EVs. SRY DNAs transferred from EVs have pathophysiological significance in vivo; injection of SRY EVs into ApoE−/− (apolipoprotein-knockout) mice accelerated atherosclerosis. The SRY gene in plasma EVs transferred to vascular endothelial cells may play an important role in the pathogenesis of atherosclerosis; this mechanism provides a new approach to the understanding of inheritable CAD in men.

scholarly journals Activation of iCaspase-9 in Neovessels Inhibits Oral Tumor Progression

2006 ◽  
Vol 85 (5) ◽  
pp. 436-441 ◽  
Author(s):  
M.S. Pinsky ◽  
W. Song ◽  
Z. Dong ◽  
K. Warner ◽  
B. Zeitlin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Oral Cancer ◽  
Tumor Progression ◽  
Cancer Cells ◽  
Immunodeficient Mice ◽  
Oral Tumor ◽  
Biodegradable Scaffolds ◽  
Oral Tumors ◽  
Oral Cancer Cells

Tumors of the oral cavity are highly vascularized malignancies. Disruption of neovascular networks was shown to limit the access of nutrients and oxygen to tumor cells and inhibit tumor progression. Here, we evaluated the effect of the activation of an artificial death switch (iCaspase-9) expressed in neovascular endothelial cells on the progression of oral tumors. We used biodegradable scaffolds to co-implant human dermal microvascular endothelial cells stably expressing iCaspase-9 (HDMEC-iCasp9) with oral cancer cells expressing luciferase (OSCC3-luc or UM-SCC-17B-luc) in immunodeficient mice. Alternatively, untransduced HDMEC were co-implanted with oral cancer cells, and a transcriptionaly targeted adenovirus (Ad-VEGFR2-iCasp-9) was injected locally to deliver iCaspase-9 to neovascular endothelial cells. In vivo bioluminescence demonstrated that tumor progression was inhibited, and immunohistochemistry showed that microvessel density was decreased, when iCaspase-9 was activated in tumor-associated microvessels. We conclude that activation of iCaspase-9 in neovascular endothelial cells is sufficient to inhibit the progression of xenografted oral tumors.

scholarly journals Hypoxia Inducible Factor-1α Overexpression Enhances the Efficacy of Mesenchymal Stem Cells Derived Extracellular Vesicles for Cardiac Repair After Myocardial Infarction

2021 ◽  
Author(s):  
Qingjie Wang ◽  
Le Zhang ◽  
Zhiqin Sun ◽  
Boyu Chi ◽  
Ailin Zou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Mesenchymal Stem Cells ◽  
Cardiac Function ◽  
Extracellular Vesicles ◽  
Biological Effects ◽  
Hypoxia Inducible Factor ◽  
Sprague Dawley

Abstract Aims Naturally secreted extracellular vesicles (EVs) play important roles in stem-mediated cardioprotection. This study aimed to investigate the cardioprotective function and underlying mechanisms of EVs derived from HIF-1a engineered mesenchymal stem cells (MSCs) in a rat model of AMI.Methods and Results EVs isolated from HIF-1a engineered MSCs (HIF-1a-EVs) and control MSCs (MSCs-EVs) were prepared. In in vitro experiments, the EVs were incubated with cardiomyocytes and endothelial cells exposed to hypoxia and serum deprivation (H/SD); in in vivo experiments, the EVs were injected in the acutely infarcted hearts of Sprague-Dawley rats. Compared with MSCs-EVs, HIF-1a-EVs significantly inhibited the apoptosis of cardiomyocytes and enhanced angiogenesis of endothelial cells; meanwhile, HIF-1a-EVs also significantly shrunk fibrotic area and strengthened cardiac function in infarcted rats. After treatment with EVs/RGD-biotin hydrogels, we observed longer retention, higher stability in HIF-1a-EVs, and stronger cardiac function in the rats. Quantitative real-time PCR (qRT-PCR) displayed that miRNA-221-3p was highly expressed in HIF-1a-EVs. After miR-221-3p was inhibited in HIF-1a-EVs, the biological effects of HIF-1a EVs on apoptosis and angiogenesis were attenuated.Conclusion EVs released by MSCs with HIF-1a overexpression can promote the angiogenesis of endothelial cells and the apoptosis of cardiomyocytes via upregulating the expression of miR-221-3p. RGD hydrogels can enhance the therapeutic efficacy of HIF-1a engineered MSC-derived EVs.

scholarly journals Human endothelial cells express integrin receptors on the luminal aspect of their membrane

Blood ◽  
1992 ◽  
Vol 80 (2) ◽  
pp. 437-446 ◽  
Author(s):  
G Conforti ◽  
C Dominguez-Jimenez ◽  
A Zanetti ◽  
MA Jr Gimbrone ◽  
O Cremona ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endothelial Cells ◽  
Plasma Proteins ◽  
The Body ◽  
Matrix Proteins ◽  
Integrin Receptors ◽  
Immunogold Staining ◽  
Circulating Cells ◽  
Alpha V Beta 3

Endothelial cells (EC) form a dynamic interface between blood and the rest of the body. EC surface properties promoting adhesion of reactive plasma proteins and/or circulating cells might be of pivotal importance for the homeostasis of blood and tissues. EC express multiple integrin receptors that promote their attachment to the subendothelial matrix proteins. Among these receptors, alpha v beta 3 is of particular relevance on EC, since it is abundantly expressed and can bind many different matrix and plasma proteins. It is still unknown whether integrin receptors are selectively located to the basal side of EC membrane or may also be exposed on the cell surface in contact with blood. This issue was addressed using different experimental approaches. First, selective surface radioiodination using lactoperoxidase (LPO)-latex beads and immunoprecipitation analysis were performed. We found that cultured EC, similarly to human skin fibroblasts (HSF), expose alpha v beta 3 on both their apical (free) and basal (substratum-attached) surfaces. This held also for other integrins such as alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1. Immunoprecipitation data were verified by morphological techniques. Immunofluorescence and immunogold-staining of EC with alpha v beta 3, as well as with beta 1 subfamily antibodies, showed a diffuse and granular distribution of these integrins on EC surface. alpha v beta 3 and beta 1 integrins were also detected on the apical membrane of EC at higher magnification by scanning electron microscopy (SEM). Finally, data obtained on cultured EC were confirmed in vivo on immunogold-labeled ultrathin cryosections of human vessels by transmission electron microscopy (TEM). Data indicate, that in addition to their role in promoting EC attachment to extracellular matrix proteins, integrin receptors of EC can be exposed to blood-stream and eventually be available for binding of plasma proteins and circulating cells.

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