Synthesis of Human Neutrophil Extracellular Traps Contributes to Angiopoietin-Mediated In Vitro Proinflammatory and Proangiogenic Activities

Abstract

Neutrophil extracellular traps (NETs) are composed of nuclear DNA in a web-like structure extruded from neutrophils in response to either bacterial infection or inflammation. We previously reported the expression of angiopoietin Tie2 receptor on human neutrophils and the capacity of both angiopoietins (Ang1 and Ang2) to induce proinflammatory activities, such as synthesis and release of platelet-activating factor, upregulation of β integrin complex (CD11/CD18), and neutrophil chemotaxis. In contrast, only Ang1 but not Ang2 is capable of promoting translational and transcriptional activities in neutrophils. In this article, we addressed whether Ang1 and/or Ang2 could modulate the release of NETs and if they contribute to angiopoietin-mediated proinflammatory activities. We observed that Ang1 and Ang2, alone or combined (10 nM, 3 h), increase NET synthesis and release by ≈2.5-fold as compared with PBS-treated neutrophils. The release of NETs is Tie2 dependent and requires downstream intracellular participation of PI3K, p38, and p42/44 MAPK pathways; reactive oxygen species production; intracellular calcium store depletion; and protein arginine deiminase 4 activation. These isolated NETs induced neutrophil and endothelial cell activation, leading to neutrophil adhesion onto human extracellular matrix and HUVEC and in vitro formation of capillary-like tubes by endothelial cells. Our study reports the capacity of Ang1 and Ang2 to promote the release of NETs and that these NETs contribute to angiopoietin-mediated in vitro proinflammatory and proangiogenic activities.

Circulating Neutrophil Extracellular Traps and Neutrophil Activation Are Increased in Proportion to Disease Severity in Human Malaria

“…Glycocalyx loss is considered a key event in SM pathogenesis [48]. Angiopoietin-induced NET release may also contribute to angiopoietin-mediated endothelial activation [21] and accentuate the microvascular dysfunction due to parasite sequestration [49]. Furthermore, the increase in NETs may exacerbate intravascular thrombosis [22] and promote ischemia and reduced tissue perfusion [24].…”

“…A key mechanism underlying both the pathogen-killing and pathogenic effects of neutrophils in other human infections is the release of neutrophil extracellular traps (NETs). Activated neutrophils form NETs by releasing extracellular deoxyribonucleic acid (DNA), chromatin, and granule proteins capable of capturing and destroying pathogens [20]; however, they can also exacerbate angiopoietin-mediated endothelial activation [21] and tissue damage [22, 23]. Plasmodium falciparum -infected red blood cells (RBCs) stimulate human neutrophils to release NETs in vitro [19].…”

Increased Circulating Levels of Neutrophil Extracellular Traps During Cardiopulmonary Bypass

“…Consequently, it is possible that NETs formation in the perioperative period is triggered by direct contact with the extracorporeal circuit or with other pathways, such as complement activation, platelet activation, and endothelial cell activation. On the basis of our in vitro study, 40 in which the peak of NETs synthesis was observed within 3 hours poststimulation with proinflammatory mediators, we elected to measure circulating NETs 3 hours postprocedure. Additionally, it is possible that measuring NETs at this time point after CPB separation does not allow enough time for the triggering of NETs formation by the general inflammatory response associated with CPB.…”

CRP Induces NETosis in Heart Failure Patients with or without Diabetes

“…Another 25 ml sample of venous blood was mixed with Anticoagulant Citrate Dextrose Solution USP Formula A (MP Biomedicals, Solon, OH). Neutrophils were isolated and resuspended in RPMI medium supplemented with 25 mM HEPES and 1% penicillin/streptomycin, as described previously (27). Contamination with PBMCs was ,0.1% as determined by morphological analysis and flow cytometry (data not shown), and viability was .98% (trypan blue dye exclusion) (27).…”

“…High-sensitivity CRP (hsCRP) in serum samples was quantified by nephelometry at the biochemistry laboratory at the MHI. (27).…”