scholarly journals Integration of Endothelial Cells in Multicellular Spheroids Prevents Apoptosis and Induces Differentiation

1998 ◽  
Vol 143 (5) ◽  
pp. 1341-1352 ◽  
Author(s):  
Thomas Korff ◽  
Hellmut G. Augustin
Keyword(s):  
Endothelial Cells ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Multicellular Spheroids ◽  
Survival Factors ◽  
Cell Contacts ◽  
Survival Factor ◽  
Cell Culture Systems ◽  
Polarized Surface ◽  
Cell Cell

Single endothelial cells (EC) seeded in suspension culture rapidly undergo apoptosis. Addition of survival factors, such as VEGF and FGF-2, does not prevent apoptosis of suspended EC. However, when cells are allowed to establish cell–cell contacts, they become responsive to the activities of survival factors. These observations have led to the development of a three-dimensional spheroid model of EC differentiation. EC spheroids remodel over time to establish a differentiated surface layer of EC and a center of unorganized EC that subsequently undergo apoptosis. Surface EC become quiescent, establish firm cell–cell contacts, and can be induced to express differentiation antigens (e.g., induction of CD34 expression by VEGF). In contrast, the unorganized center spheroid cells undergo apoptosis if they are not rescued by survival factors. The responsiveness to the survival factor activities of VEGF and FGF-2 was not dependent on cell shape changes since it was retained after cytochalasin D treatment. Taken together, these findings characterize survival factor requirements of unorganized EC and indicate that polarized surface EC differentiate to become independent of exogenous survival factors. Furthermore, they demonstrate that spheroid cell culture systems are useful not just for the study of tumor cells and embryonic stem cells but also for the analysis of differentiated functions of nontransformed cells.

Cell confluence regulates tyrosine phosphorylation of adherens junction components in endothelial cells

1997 ◽  
Vol 110 (17) ◽  
pp. 2065-2077 ◽  
Author(s):  
M.G. Lampugnani ◽  
M. Corada ◽  
P. Andriopoulou ◽  
S. Esser ◽  
W. Risau ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Phosphorylation ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Molecular Organization ◽  
Beta Catenin ◽  
Cell Contacts ◽  
Cell Type Specific ◽  
Dynamic Structures ◽  
Cell Cell

In src- and ras-transformed cells, tyrosine phosphorylation of adherens junction (AJ) components is related to impairment of cell-cell adhesion. In this paper we report that in human endothelial cells (EC), tyrosine phosphorylation of AJ can be a physiological process regulated by cell density. Immunofluorescence analysis revealed that a phosphotyrosine (P-tyr) antibody could stain cell-cell junctions only in sparse or loosely confluent EC, while the staining was markedly reduced in tightly confluent cultures. This process was reversible, since on artificial wounding of EC monolayers, the cells at the migrating front reacquired P-tyr labelling at cell contacts. In EC, the major cadherin at intercellular AJ is the cell-type-specific VE-cadherin. We therefore analyzed whether this molecule was at least in part responsible for the changes in P-tyr content at cell junctions. Tyrosine phosphorylation of VE-cadherin, beta-catenin and p120, occurred in looser AJ, i.e. in recently confluent cells, and was notably reduced in tightly confluent cultures. Changes in P-tyr content paralleled changes in the molecular organization of AJ. VE-cadherin was mostly associated with beta-catenin and p120 in loose EC monolayers, while in long-confluent cells, these two catenins were largely replaced by plakoglobin. Inhibition of P-tyr phosphatases (PTPases) by PV markedly augmented the P-tyr content of VE-cadherin, which bound p120 and beta-catenin more efficiently, but not plakoglobin. Transfection experiments in CHO cells showed that p120 could bind to a VE-cadherin cytoplasmic region different from that responsible for beta-catenin binding, and PV stabilized this association. Overall these data indicate that endothelial AJ are dynamic structures that can be affected by the state of confluence of the cells. Tyrosine phosphorylation of VE-cadherin and its association to p120 and beta-catenin characterizes early cell contacts, while the formation of mature and cytoskeleton-connected junctions is accompanied by dephosphorylation and plakoglobin association.

Enhancement of connexin30.3 expression in mouse embryonic stem cell line EB3 in response to cell–cell contacts

Human Cell ◽  
2019 ◽  
Vol 32 (2) ◽  
pp. 95-102
Author(s):  
Naruwa Tokunaga ◽  
Ryota Kishi ◽  
Tomoko Sasai ◽  
Mikako Saito
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Line ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Stem Cell Line ◽  
Cell Contacts ◽  
Cell Cell

scholarly journals Identification of Fer Tyrosine Kinase Localized on Microtubules as a Platelet Endothelial Cell Adhesion Molecule-1 Phosphorylating Kinase in Vascular Endothelial Cells

2003 ◽  
Vol 14 (9) ◽  
pp. 3553-3564 ◽  
Author(s):  
Naoko Kogata ◽  
Michitaka Masuda ◽  
Yuji Kamioka ◽  
Akiko Yamagishi ◽  
Akira Endo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Adhesion Molecule ◽  
Intracellular Signaling ◽  
Vascular Endothelial Cells ◽  
Expression Cloning ◽  
Vascular Endothelial ◽  
Cell Contacts ◽  
Cell Cell

Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.

Thrombopoietin Stimulates the Formation of Endothelial Cell-Cell Contacts and Activates the Small GTPase Rap1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3713-3713
Author(s):  
Marloes R. Tijssen ◽  
Franca di Summa ◽  
Anne-Marieke van Stalborch ◽  
C. Ellen Van der Schoot ◽  
Jaap D. van Buul ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Electrical Resistance ◽  
Umbilical Vein ◽  
Small Gtpase ◽  
Endothelial Monolayer ◽  
Impedance Sensing ◽  
Cell Contacts ◽  
Umbilical Vein Endothelial Cells ◽  
Cell Cell

Abstract In addition to hematopoietic cells, the receptor for thrombopoietin (Tpo), c-Mpl, is expressed on human umbilical vein endothelial cells (HUVEC). In this study, we demonstrated that Mpl mRNA and protein can also be detected in immortalized human bone marrow endothelial cells (HBMEC). Furthermore, it was investigated whether Tpo affects endothelial monolayer integrity by either cell spreading or cell-cell contacts. By continuously monitoring the electrical resistance of freshly plated HUVEC or HBMEC with ECIS (electric cell-substrate impedance sensing), we observed a significant increase in the electrical resistance when endothelial cells were treated with 100 ng/ml of Tpo just prior to seeding, as shown in the figure below. This increase in electrical resistance was not due to increased cell adhesion or proliferation of the endothelial cells. However, a significant increase in initial spreading could be detected. Besides its effect on spreading, Tpo increased the electrical resistance of a confluent endothelial monolayer when compared to untreated monolayers. This indicates that Tpo promotes the formation of endothelial cell-cell contacts. Indeed, a decrease in immunostaining levels of phosphotyrosine could be observed in monolayers treated with Tpo. Also the junctional protein VE-cadherin showed diminished co-localization with phosphotyrosine staining when cells were treated with Tpo. The small GTPase Rap1 plays an important role in the formation of endothelial cell-cell contacts. In a pull-down experiment, using RalGDS as bait for active Rap1, we could demonstrate that Tpo transiently activates Rap1 in HUVEC. Thus, these results may put Tpo forward as a regulator of endothelial cell-cell contacts, possibly after vascular damage and subsequent release of Tpo by activated platelets. To our knowledge, Tpo is the first cytokine exerting this effect on endothelial cells. Figure Figure

The Expression of Microfilament-Associated Cell-Cell Contacts in Brain Endothelial Cells Is Modified by IFN-β1a (Rebif®)

2004 ◽  
Vol 24 (12) ◽  
pp. 711-716 ◽  
Author(s):  
Michael Harzheim ◽  
Manuela Stepien-Mering ◽  
Rolf Schröder ◽  
Stephan Schmidt
Keyword(s):  
Endothelial Cells ◽  
Brain Endothelial Cells ◽  
Cell Contacts ◽  
Cell Cell

scholarly journals Physical confinement signals regulate the organization of stem cells in three dimensions

2016 ◽  
Vol 13 (123) ◽  
pp. 20160613 ◽  
Author(s):  
Sebastian V. Hadjiantoniou ◽  
David Sean ◽  
Maxime Ignacio ◽  
Michel Godin ◽  
Gary W. Slater ◽  
...  
Keyword(s):  
Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Cell Interactions ◽  
Three Dimensions ◽  
Cell Substrate ◽  
Cell Cell

During embryogenesis, the spherical inner cell mass (ICM) proliferates in the confined environment of a blastocyst. Embryonic stem cells (ESCs) are derived from the ICM, and mimicking embryogenesis in vitro , mouse ESCs (mESCs) are often cultured in hanging droplets. This promotes the formation of a spheroid as the cells sediment and aggregate owing to increased physical confinement and cell–cell interactions. In contrast, mESCs form two-dimensional monolayers on flat substrates and it remains unclear if the difference in organization is owing to a lack of physical confinement or increased cell–substrate versus cell–cell interactions. Employing microfabricated substrates, we demonstrate that a single geometric degree of physical confinement on a surface can also initiate spherogenesis. Experiment and computation reveal that a balance between cell–cell and cell–substrate interactions finely controls the morphology and organization of mESC aggregates. Physical confinement is thus an important regulatory cue in the three-dimensional organization and morphogenesis of developing cells.

scholarly journals Key Proteins Involved in Spheroid Formation and Angiogenesis in Endothelial Cells After Long-Term Exposure to Simulated Microgravity

2018 ◽  
Vol 45 (2) ◽  
pp. 429-445 ◽  
Author(s):  
Anita Dittrich ◽  
Daniela Grimm ◽  
Jayashree Sahana ◽  
Johann Bauer ◽  
Marcus Krüger ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Adhesion Molecule ◽  
Cell Structure ◽  
Three Dimensional ◽  
Cardiovascular Complications ◽  
Intercellular Adhesion Molecule ◽  
Intercellular Adhesion Molecule 1 ◽  
Multicellular Spheroids ◽  
Monocyte Chemoattractant Protein 1 ◽  
Molecular Approaches

Background/Aims: Cardiovascular complications are common in astronauts returning from a prolonged spaceflight. These health problems might be driven by complex modulations of gene expression and protein synthesis in endothelial cells (ECs). Studies on the influence of microgravity on phenotype, growth pattern and biological processes of ECs can help to understand these complications. Methods: We exposed ECs (EA.hy926) to a Random Positioning Machine (RPM). Proteins associated with cell structure, angiogenesis and endothelial dysfunction were investigated in distinct pools of multicellular spheroids (MCS), adherent cells (AD) and tubular structures (TS) formed after a 35-day RPM-exposure. Results: Combining morphological and molecular approaches, we found AD, MCS and TS with changes in the synthesis and release of proteins involved in three-dimensional growth. Fibronectin and monocyte chemoattractant protein-1 (MCP-1) mRNAs and protein contents were elevated along with an increased secretion of vascular endothelial growth factor (VEGF), interleukin (IL)-6, IL-8, MCP-1, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), neutrophil gelatinase-associated lipocalin (NGAL) and regulated on activation, normal T cell expressed and secreted (RANTES) proteins in the culture supernatant as determined by multianalyte profiling technology. Together they form a network of interaction. Conclusions: These results show that a prolonged RPM-exposure of ECs induced TS and MCS formation. The factors VEGF, NGAL, IL-6, IL-8, MCP-1, VCAM-1, ICAM-1, fibronectin and RANTES seem to be affected when gravity is omitted.

scholarly journals Three-Dimensional Cell Culture Systems in Radiopharmaceutical Cancer Research

Cancers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2765
Author(s):  
Alina Doctor ◽  
Verena Seifert ◽  
Martin Ullrich ◽  
Sandra Hauser ◽  
Jens Pietzsch
Keyword(s):  
Cell Culture ◽  
Cancer Research ◽  
Animal Experiments ◽  
Three Dimensional ◽  
3D Models ◽  
Ductal Adenocarcinoma ◽  
Multicellular Spheroids ◽  
Culture Systems ◽  
Advantages And Disadvantages ◽  
Cell Culture Systems

In preclinical cancer research, three-dimensional (3D) cell culture systems such as multicellular spheroids and organoids are becoming increasingly important. They provide valuable information before studies on animal models begin and, in some cases, are even suitable for reducing or replacing animal experiments. Furthermore, they recapitulate microtumors, metastases, and the tumor microenvironment much better than monolayer culture systems could. Three-dimensional models show higher structural complexity and diverse cell interactions while reflecting (patho)physiological phenomena such as oxygen and nutrient gradients in the course of their growth or development. These interactions and properties are of great importance for understanding the pathophysiological importance of stromal cells and the extracellular matrix for tumor progression, treatment response, or resistance mechanisms of solid tumors. Special emphasis is placed on co-cultivation with tumor-associated cells, which further increases the predictive value of 3D models, e.g., for drug development. The aim of this overview is to shed light on selected 3D models and their advantages and disadvantages, especially from the radiopharmacist’s point of view with focus on the suitability of 3D models for the radiopharmacological characterization of novel radiotracers and radiotherapeutics. Special attention is paid to pancreatic ductal adenocarcinoma (PDAC) as a predestined target for the development of new radionuclide-based theranostics.

scholarly journals Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device

Lab on a Chip ◽  
2013 ◽  
Vol 13 (18) ◽  
pp. 3562 ◽  
Author(s):  
Andries D. van der Meer ◽  
Valeria V. Orlova ◽  
Peter ten Dijke ◽  
Albert van den Berg ◽  
Christine L. Mummery
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Microfluidic Device ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Human Endothelial Cells

scholarly journals Molecular cloning of CD31, a putative intercellular adhesion molecule closely related to carcinoembryonic antigen.

1990 ◽  
Vol 171 (6) ◽  
pp. 2147-2152 ◽  
Author(s):  
D L Simmons ◽  
C Walker ◽  
C Power ◽  
R Pigott
Keyword(s):  
Endothelial Cells ◽  
Carcinoembryonic Antigen ◽  
Adhesion Molecule ◽  
Umbilical Vein ◽  
Sequence Similarity ◽  
Intercellular Adhesion ◽  
Intercellular Adhesion Molecule ◽  
Cdna Clones ◽  
Cell Contacts ◽  
Cell Cell

cDNA clones encoding CD31 have been isolated by transient expression. The sequence of CD31 expressed on human umbilical vein endothelial cells (HUVEC) is identical to that expressed on the monocyte-like cell line HL60. In HUVEC. CD31 is concentrated in regions of cell-cell contacts. CD31 is a member of the Ig superfamily and is most closely related to the carcinoembryonic antigen CEA, consisting of four contiguous C2 domains. The localization of CD31 to regions of cell-cell contacts, and the sequence similarity to CEA, a known intercellular adhesion molecule (ICAM), strongly suggest that CD31 may function as an ICAM, possibly mediating endothelial cell-cell contacts and also promoting interactions between leukocytes and endothelial cells.

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