scholarly journals Neutrophil Extracellular Traps as an Adhesion Substrate for Different Tumor Cells Expressing RGD-Binding Integrins

2018 ◽  
Vol 19 (8) ◽  
pp. 2350 ◽  
Author(s):  
Marcello Monti ◽  
Viviana De Rosa ◽  
Francesca Iommelli ◽  
Maria Vincenza Carriero ◽  
Cristina Terlizzi ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cancer Cells ◽  
Cell Lines ◽  
Defense Mechanism ◽  
Cell Attachment ◽  
Thrombus Formation ◽  
Neutrophil Extracellular Traps ◽  
Adhesion Properties ◽  
Extracellular Traps ◽  
Relevant Role

Neutrophil extracellular traps (NETs), in addition to their function as a host defense mechanism, play a relevant role in thrombus formation and metastatic dissemination of cancer cells. Here we screened different cancer cell lines endogenously expressing a variety of integrins for their ability to bind to NETs. To this end, we used NETs isolated from neutrophil-like cells as a substrate for adhesion assays of HT1080, U-87 MG, H1975, DU 145, PC-3 and A-431 cells. Levels of α5, αIIb, αv, β1, β3 and β5 chains were determined by western blot analysis in all cell lines and levels of whole integrins on the plasma membrane were assessed by fluorescence-activated cell sorting (FACS) analysis. We found that high levels of α5β1, αvβ3 and αvβ5 enhance cell adhesion to NETs, whereas low expression of α5β1 prevents cell attachment to NETs. Excess of cyclic RGD peptide inhibited cell adhesion to NETs by competing with fibronectin within NETs. The maximal reduction of such adhesion was similar to that obtained by DNase 1 treatment causing DNA degradation. Our findings indicate that NETs from neutrophil-like cells may be used as a substrate for large screening of the adhesion properties of cancer cells expressing a variety of RGD-binding integrins.

scholarly journals Testing the in vitro performance of hydroxyapatite coated magnesium (AZ91D) and titanium concerning cell adhesion and osteogenic differentiation

2015 ◽  
Vol 16 (1) ◽  
Author(s):  
Claudia Kleinhans ◽  
Gabriele Vacun ◽  
Roman Surmenev ◽  
Maria Surmeneva ◽  
Petra Juliane Kluger
Keyword(s):  
Cell Adhesion ◽  
Osteogenic Differentiation ◽  
Cell Attachment ◽  
Human Mesenchymal Stem Cell ◽  
Adhesion Properties ◽  
Sputtering Deposition ◽  
Initial Cell ◽  
Tissue Culture Polystyrene ◽  
Diffraction Patterns

AbstractIn the current study the in vitro outcome of a degradable magnesium alloy (AZ91D) and standard titanium modified by nanostructured-hydroxyapatite (n-HA) coatings concerning cell adhesion and osteogenic differentiation was investigated by direct cell culture. The n-HA modification was prepared via radio-frequency magnetron sputtering deposition and proven by field emission scanning electron microscopy and X-ray powder diffraction patterns revealing a homogenous surface coating. Human mesenchymal stem cell (hMSCs) adhesion was examined after one and 14 days displaying an enhanced initial cell adhesion on the n-HA modified samples. The osteogenic lineage commitment of the cells was determined by alkaline phosphatase (ALP) quantification. On day one n-HA coated AZ91D exhibited a comparable ALP expression to standard tissue culture polystyrene samples. However, after 14 days solely little DNA and ALP amounts were measurable on n-HA coated AZ91D due to the lack of adherent cells. Titanium displayed excellent cell adhesion properties and ALP was detectable after 14 days. An increased pH of the culture was measured for AZ91D as well as for n-HA coated AZ91D. We conclude that n-HA modification improves initial cell attachment on AZ91D within the first 24 h. However, the effect does not persist for 14 days in in vitro conditions.

scholarly journals How Neutrophil Extracellular Traps Become Visible

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Nicole de Buhr ◽  
Maren von Köckritz-Blickwede
Keyword(s):  
Electron Microscopy ◽  
Nuclear Dna ◽  
Defense Mechanism ◽  
Neutrophil Extracellular Traps ◽  
Immune Defense ◽  
Extracellular Traps ◽  
Activated Neutrophils ◽  
Microscopy Techniques

Neutrophil extracellular traps (NETs) have been identified as a fundamental innate immune defense mechanism against different pathogens. NETs are characterized as released nuclear DNA associated with histones and granule proteins, which form an extracellular web-like structure that is able to entrap and occasionally kill certain microbes. Furthermore, NETs have been shown to contribute to several noninfectious disease conditions when released by activated neutrophils during inflammation. The identification of NETs has mainly been succeeded by various microscopy techniques, for example, immunofluorescence microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Since the last years the development and improvement of new immunofluorescence-based techniques enabled optimized visualization and quantification of NETs. On the one handin vitrolive-cell imaging led to profound new ideas about the mechanisms involved in the formation and functionality of NETs. On the other hand different intravital,in vivo, andin situmicroscopy techniques led to deeper insights into the role of NET formation during health and disease. This paper presents an overview of the main used microscopy techniques to visualize NETs and describes their advantages as well as disadvantages.

Neutrophil Extracellular Traps Induce Platelet Adhesion and Thrombus Formation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1345-1345 ◽  
Author(s):  
Tobias Fuchs ◽  
Alexander Brill ◽  
Daniel Dürschmied ◽  
Daphne Schatzberg ◽  
John H. Hartwig ◽  
...  
Keyword(s):  
Whole Blood ◽  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Neutrophil Extracellular Traps ◽  
Thrombin Inhibitor ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Extracellular Traps ◽  
Deep Vein

Abstract Abstract 1345 Introduction Thrombus stability is provided by very large polymers adhering to platelets and anchoring the thrombus to the vessel wall. The best described polymers are fibrin and von Willebrand Factor (VWF). Activated neutrophils and other leukocytes can form an extracellular fibrous network which is composed of DNA, histones, and granular proteins. These neutrophil extracellular traps (NETs) are present in various inflammatory diseases. In deep vein thrombosis (DVT) inflammation closely cooperates with thrombosis. Here we examine whether NETs provide a new means to support the adhesion and recruitment of platelets and whether NETs are present in DVT. Methods and Results: To study the interaction of platelets with NETs, we isolated human neutrophils, induced NET formation and perfused over the NETs human platelets in plasma or whole blood anticoagulated with the thrombin inhibitor PPACK. Microscopic analysis revealed that under flow platelets adhere avidly to NETs. Perfusion of whole blood at physiological shear resulted in formation of thrombi on NETs in a time dependent manner. Addition of DNase1 degraded NETs and removed all platelets and thrombi demonstrating their adhesion to NETs. Thrombus formation on NETs was absent if blood was supplemented with EDTA indicating the requirement for divalent cations. Perfusion of NETs with heparinized blood dismantled NETs and prevented thrombus formation. Incubation of NETs with heparin alone released histones from NETs, indicating that heparin destroys the chromatin backbone of NETs. Furthermore, immunocytochemistry revealed that NETs were able to bind platelet adhesion molecules VWF and fibronectin from human plasma. Immunohistochemical analysis of a baboon deep vein thrombus showed abundant extracellular chromatin which co-localized with fibronectin and VWF. Conclusions: We show that extracellular traps are able to promote thrombosis in vitro and are abundant in vivo in DVT. We propose that extracellular chromatin provides a new type of scaffold that promotes platelet adhesion, activation, and aggregation and may be important for thrombus initiation or stability. Disclosures No relevant conflicts of interest to declare.

Comparative cell adhesion properties of cysteine extended peptide architectures

RSC Advances ◽  
2016 ◽  
Vol 6 (4) ◽  
pp. 2695-2702 ◽  
Author(s):  
Saniye Soylemez ◽  
Bilal Demir ◽  
Gizem Oyman Eyrilmez ◽  
Seçkin Kesici ◽  
Aytül Saylam ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Attachment ◽  
Rgd Peptide ◽  
Adhesion Properties ◽  
Modified Surfaces

This study presents the comparative cell attachment investigation of TAT and well-known RGD peptide modified surfaces.

scholarly journals DNA, histones and neutrophil extracellular traps exert anti-fibrinolytic effects in a plasma environment

2015 ◽  
Vol 113 (06) ◽  
pp. 1289-1298 ◽  
Author(s):  
Imre Varjú ◽  
Colin Longstaff ◽  
László Szabó ◽  
Ádám Zoltán Farkas ◽  
Veronika Judit Varga-Szabó ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Fibre Diameter ◽  
Neutrophil Extracellular Traps ◽  
Enzymatic Digestion ◽  
Enzyme Inactivation ◽  
Tissue Type ◽  
Confocal Laser ◽  
Sem Images ◽  
Permeability Constant ◽  
Extracellular Traps

SummaryIn response to various inflammatory stimuli, neutrophils secrete neutrophil extracellular traps (NETs), web-like meshworks of DNA, histones and granular components forming supplementary scaffolds in venous and arterial thrombi. Isolated DNA and histones are known to promote thrombus formation and render fibrin clots more resistant to mechanical forces and tissue-type plasminogen activator (tPA)-induced enzymatic digestion. The present study extends our earlier observations to a physiologically more relevant environment including plasma clots and NET-forming neutrophils. A range of techniques was employed including imaging (scanning electron microscopy (SEM), confocal laser microscopy, and photoscanning of macroscopic lysis fronts), clot permeability measurements, turbidimetric lysis and enzyme inactivation assays. Addition of DNA and histones increased the median fibre diameter of plasma clots formed with 16 nM thrombin from 108 to 121 and 119 nm, respectively, and decreased their permeability constant from 6.4 to 3.1 and 3.7×10−9 cm2. Histones effectively protected thrombin from antithrombin-induced inactivation, while DNA inhibited plasminogen activation on the surface of plasma clots and their plasmin-induced resolution by 20 and 40 %, respectively. DNA and histones, as well as NETs secreted by phorbol-myristate-acetate-activated neutrophils, slowed down the tPA-driven lysis of plasma clots and the latter effect could be reversed by the addition of DNase (streptodornase). SEM images taken after complete digestion of fibrin in NET-containing plasma clots evidenced retained NET scaffold that was absent in DNase-treated clots. Our results show that DNA and histones alter the fibrin architecture in plasma clots, while NETs contribute to a decreased lytic susceptibility that can be overcome by DNase.

scholarly journals The amount range of membrane cholesterol required for robust cell adhesion and proliferation in serum-free condition.

2021 ◽  
Author(s):  
Shino Takii ◽  
Jun Wu ◽  
Daiji Okamura
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cell Attachment ◽  
Cancer Cell Lines ◽  
Membrane Cholesterol ◽  
Cholesterol Depletion ◽  
Serum Free ◽  
Beneficial Effects ◽  
Cells In Culture

Serum-containing medium is widely used to support cell attachment, stable growth and serial passaging of various cancer cell lines.   However, the presence of cholesterols and lipids in serum greatly hinders the analysis of the effects of cholesterol depletion on cells in culture.   In this study, we develop a defined serum-free culture condition accessible to a variety of different types of adherent cancer cells. We tested different factors that are considered essential for cell culture and various extracellular matrix for plate coating, and found cells cultured in Dulbecco's Modified Eagle's Medium (DMEM) basal media supplemented with Albumin (BSA) and insulin-transferrin-selenium-ethanolamine (ITS-X) on fibronectin-precoated well (called as “DA-X condition”) showed comparable proliferation and survival to those in a serum-containing medium. Interestingly, we observed that DA-X condition could be adapted to a wide variety of adherent cancer cell lines, which enabled the analysis of how cholesterol depletion affected cancer cells in culture. Mechanistically, we found the beneficial effects of the DA-X condition in part can be attributed to the appropriate level of membrane cholesterol, and fibronectin-mediated signaling plays an important role in the suppression of cholesterol production.

scholarly journals Coronavirus disease 2019 (COVID-19): NETosis-associated mechanisms of progression and prospects for therapy regulating the formation of neutrophil extracellular traps (NETs)

2021 ◽  
Vol 6 (4) ◽  
pp. 64-73
Author(s):  
K. A. Aitbaev ◽  
I. T. Murkamilov ◽  
V. V. Fomin ◽  
I. O. Kudaibergenova ◽  
F. A. Yusupov
Keyword(s):  
Molecular Mechanisms ◽  
Adverse Reactions ◽  
Thrombus Formation ◽  
The Elderly ◽  
Neutrophil Extracellular Traps ◽  
The Body ◽  
Surrounding Tissue ◽  
Chronic Respiratory Diseases ◽  
Small Vessels ◽  
Extracellular Traps

Infectious disease COVID-19 caused by the SARS-CoV-2 coronavirus is characterized by high contagiousness, complexity of pathogenesis and unpredictability of the clinical course. In severe cases, which are especially susceptible to men, the elderly and people with underlying medical conditions such as obesity, diabetes, hypertension, cardiovascular and chronic respiratory diseases, the infection leads to respiratory failure and death due to the development of an extensive inflammatory reaction. As a result of many studies, it has been established that one of the leading causes of the severe course and death of patients with COVID-19 is the development of coagulopathy, that is, increased thrombus formation in small vessels due to excessive activity of neutrophils, which form the so-called neutrophil extracellular traps (NETs). Although NETs play a useful role in protecting their host from pathogens, their overgrowth can trigger a cascade of adverse reactions including: the production of antibodies against the host’s DNA (autoimmunization); damage to surrounding tissue; or the occurrence of thromboembolic complications. Therefore, extracellular neutrophil traps and their markers have been identified as targets for new therapeutic strategies aimed at reducing the severity of COVID-19 disease and/or mortality. This article describes the structure of NETs, as well as analyzes the molecular mechanisms that contribute to their overgeneration. In addition, the prospects for COVID-19 therapy aimed at regulating the formation of extracellular traps by creating drugs both limiting the production of NET structures and dissolving their excess amounts in the body of patients are discussed.

scholarly journals Neutrophil Extracellular Traps in Arterial and Venous Thrombosis

2019 ◽  
Vol 45 (01) ◽  
pp. 086-093 ◽  
Author(s):  
Elodie Laridan ◽  
Kimberly Martinod ◽  
Simon De Meyer
Keyword(s):  
Myocardial Infarction ◽  
Thrombus Formation ◽  
Neutrophil Extracellular Traps ◽  
Major Health ◽  
Vein Thrombosis ◽  
Extracellular Traps ◽  
Thrombotic Complications ◽  
Decondensed Chromatin ◽  
Deep Vein ◽  
Novel Therapeutic

AbstractThrombotic complications are still a major health risk worldwide. Our view on the pathophysiology of thrombosis has significantly changed since the discovery of neutrophil extracellular traps (NETs) and their prothrombotic characteristics. Generated by neutrophils that release their decondensed chromatin as a network of extracellular fibers, NETs promote thrombus formation by serving as a scaffold that activates platelets and coagulation. The thrombogenic involvement of NETs has been described in various settings of thrombosis, including stroke, myocardial infarction, and deep vein thrombosis. The aim of this review is to summarize existing evidence showing the presence of NETs in human thrombus material. Following an introduction on NETs and their role in thrombus formation, the authors address studies showing the presence of NETs in arterial or venous thrombi. In addition, they focus on potential novel therapeutic opportunities to resolve or prevent thrombosis by targeting NETs.

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