scholarly journals Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations

2016 ◽  
Vol 124 (6) ◽  
pp. 1780-1787 ◽  
Author(s):  
Zhenjun Zhao ◽  
Michael S. Johnson ◽  
Biyi Chen ◽  
Michael Grace ◽  
Jaysree Ukath ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Endothelial Cells ◽  
Ionizing Radiation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Vascular Targeting ◽  
Radiation Doses ◽  
Radiation Induced ◽  
Polarity Sensitive

OBJECT Stereotactic radiosurgery (SRS) is an established intervention for brain arteriovenous malformations (AVMs). The processes of AVM vessel occlusion after SRS are poorly understood. To improve SRS efficacy, it is important to understand the cellular response of blood vessels to radiation. The molecular changes on the surface of AVM endothelial cells after irradiation may also be used for vascular targeting. This study investigates radiation-induced externalization of phosphatidylserine (PS) on endothelial cells using live-cell imaging. METHODS An immortalized cell line generated from mouse brain endothelium, bEnd.3 cells, was cultured and irradiated at different radiation doses using a linear accelerator. PS externalization in the cells was subsequently visualized using polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, a polarity-sensitive probe. Live-cell imaging was used to monitor PS externalization in real time. The effects of radiation on the cell cycle of bEnd.3 cells were also examined by flow cytometry. RESULTS Ionizing radiation effects are dose dependent. Reduction in the cell proliferation rate was observed after exposure to 5 Gy radiation, whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with cells treated with sham radiation, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA-positive cells were significantly higher (p = 0.04) 24 hours after treatment in the cultures that received 25- and 15-Gy doses of radiation. This effect was sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation controls at any time points (p > 0.15). Flow cytometric analysis data indicate that irradiation induced growth arrest of bEnd.3 cells, with cells accumulating in the G2 phase of the cell cycle. CONCLUSIONS Ionizing radiation causes remarkable cellular changes in endothelial cells. Significant PS externalization is induced by radiation at doses of 15 Gy or higher, concomitant with a block in the cell cycle. Radiation-induced markers/targets may have high discriminating power to be harnessed in vascular targeting for AVM treatment.

scholarly journals ANGI-03OBSERVATION OF GLIOMA STEM CELLS TRANSFORM INTO ENDOTHELIAL CELLS VIA LIVE CELL IMAGING

2015 ◽  
Vol 17 (suppl 5) ◽  
pp. v41.3-v41
Author(s):  
Xin Mei ◽  
Yinsheng Chen ◽  
Zhongping Chen
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Glioma Stem Cells

scholarly journals Live Cell Imaging of Telomerase RNA Dynamics Reveals Cell Cycle-Dependent Clustering of Telomerase at Elongating Telomeres

2011 ◽  
Vol 44 (5) ◽  
pp. 819-827 ◽  
Author(s):  
Franck Gallardo ◽  
Nancy Laterreur ◽  
Emilio Cusanelli ◽  
Faissal Ouenzar ◽  
Emmanuelle Querido ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Telomerase Rna ◽  
Rna Dynamics ◽  
Cycle Dependent

Human and equine endothelial cells in a live cell imaging scratch assay in vitro

2019 ◽  
Vol 70 (4) ◽  
pp. 495-509 ◽  
Author(s):  
Juliane Rieger ◽  
Carsten Hopperdietzel ◽  
Sabine Kaessmeyer ◽  
Ilka Slosarek ◽  
Sebastian Diecke ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Scratch Assay

Imaging of Von Willebrand Factor Remodeling Upon Secretion From Vascular Endothelial Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 263-263
Author(s):  
Marjon J Mourik ◽  
Karine M Valentijn ◽  
Jack A Valentijn ◽  
Jan Voorberg ◽  
Abraham J Koster ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Light And Electron Microscopy ◽  
Live Cell ◽  
String Formation ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 263 In response to vascular injury, endothelial cells rapidly secrete high molecular weight multimers of the coagulation protein Von Willebrand factor (VWF). Once expelled from the cells, VWF unfurls in long strings that bind platelets from the bloodstream to induce primary hemostasis. VWF secreted upon stimulation is released from specialized storage compartments called Weibel Palade bodies (WPB) which have a typical rod or cigar shape. They emerge from the Trans Golgi network in a process driven by the formation of helical tubules consisting of VWF multimers and the VWF propeptide. When WPBs undergo exocytosis and release VWF, rapid structural changes occur which eventually result in platelet capturing VWF strings. It has been postulated that the tubular storage of VWF in WPBs is required for sufficient unfolding of the protein during string formation as agents disrupting the VWF tubules were shown to result in less strings. Recently we described a novel structure involved in VWF exocytosis which is formed only upon stimulation. We refer to this structure as a “secretory pod” as it seemed to derive from multiple WPBs and was identified as a VWF release site where strings seemed to be formed. By transmission electron microscopy (TEM) we identified this structure to be a membrane-delimited organelle containing filamentous material resembling unfurled VWF. The VWF tubules as seen in WPBs are absent in secretory pods suggesting that tubular packaging of VWF is not essential for sufficient release and string formation. To study the formation of secretory pods and the subsequent release and remodeling of VWF, several imaging techniques were used such as live-cell imaging and correlative light and electron microscopy. We expressed propeptide-EGFP in endothelial cells to label the WPBs and stimulated them with PMA. By live-cell imaging we visualized the exocytotic events. We observed, apart from single WPB exocytosis, the formation of secretory pods which occurred by the coalescence of several WPBs. In some cases the individual WPBs rounded up first, before they joined into one round structure while in other cases the coalescence event seemed to happen at once. After coalescence, fusion with the plasma membrane occurred to release the pooled VWF which resulted in the disappearance of the fluorescent signal as the propeptide rapidly diffused into the extracellular medium. How the secreted VWF is remodeled after secretion into VWF strings was studied by correlative light and electron microscopy. We correlated confocal pictures of stimulated endothelial cells, which were stained with VWF specific fluorescent antibodies, to consecutive TEM sections. We found that fluorescently labeled VWF dots that were connected to strings, correlated to secretory pods but also to globular mass of secreted VWF. Interestingly, when we analyzed consecutive EM sections, the globular masses were found to originate from the secretory pods. From the globular masses we also observed deriving strings indicating that once VWF is expelled, remodeling occurs independently from secretion. We hypothesize that fluid flow remodels the secreted globular VWF mass into strings. To study this we stimulated endothelial cells under flow. The intracellular VWF pool in the WPBs was labeled green by transient expression of propeptide-EGFP and the secreted VWF was labeled red with strongly diluted red fluorescent VWF specific antibodies in the perfusate. Using live-cell imaging we observed that upon fusion of EGFP labeled WPBs, the green signal transformed into a red signal revealing dots of labeled secreted VWF. These dots rolled, in the direction of the flow, to the edge of the cell where they aggregated and only then formed strings. In non-transfected cells we performed similar experiments and there we observed the same pattern, confirming even more the VWF aggregation and string formation at the edges of the cell. In conclusion, we demonstrated that several WPBs can fuse with each other to form secretory pods and that VWF is secreted as a globular mass of protein. From these globular masses strings originated indicating that string formation occurs independently from the mechanism of secretion in which the tubular packaging of VWF in WPBs does not seem to be of importance. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Glioblastoma stem cell differentiation into endothelial cells evidenced through live-cell imaging

2017 ◽  
Vol 19 (8) ◽  
pp. 1109-1118 ◽  
Author(s):  
Xin Mei ◽  
Yin-Sheng Chen ◽  
Fu-Rong Chen ◽  
Shao-Yan Xi ◽  
Zhong-Ping Chen
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Stem Cell Differentiation ◽  
Live Cell ◽  
Glioblastoma Stem Cell

scholarly journals Long-term fate of etoposide-induced micronuclei and micronucleated cells in Hela-H2B-GFP cells

2020 ◽  
Vol 94 (10) ◽  
pp. 3553-3561
Author(s):  
Hauke Reimann ◽  
Helga Stopper ◽  
Henning Hintzsche
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Cellular Structures ◽  
Death Rates ◽  
Daughter Cells

Abstract Micronuclei are small nuclear cellular structures containing whole chromosomes or chromosomal fragments. While there is a lot of information available about the origin and formation of micronuclei, less is known about the fate of micronuclei and micronucleated cells. Possible fates include extrusion, degradation, reincorporation and persistence. Live cell imaging was performed to quantitatively analyse the fates of micronuclei and micronucleated cells occurring in vitro. Imaging was conducted for up to 96 h in HeLa-H2B-GFP cells treated with 0.5, 1 and 2 µg/ml etoposide. While a minority of micronuclei was reincorporated into the main nucleus during mitosis, the majority of micronuclei persisted without any alterations. Degradation and extrusion were observed rarely or never. The presence of micronuclei affected the proliferation of the daughter cells and also had an influence on cell death rates. Mitotic errors were found to be clearly increased in micronucleus-containing cells. The results show that micronuclei and micronucleated cells can, although delayed in cell cycle, sustain for multiple divisions.

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