Background/Purpose: Neutrophil-mediated activation of the endothelium plays a role in the pathogenesis of diverse disease states ranging from autoimmunity to cancer to COVID-19. Neutralization of cationic proteins (such as neutrophil extracellular trap/NET-derived histones) with polyanionic compounds has been suggested as a strategy for protecting the endothelium from such insults. Here, we investigated the role of the FDA-approved agent defibrotide (a pleotropic mixture of oligonucleotides) in engaging histones and blocking their pathological effects on endothelium.

Methods: Human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMVECs) were cultured with purified NETs or histone H4. Cell activation was determined by specific gene expression (e.g., E-selectin), neutrophil adhesion, enzymatic activity of tissue factor, and RNA sequencing. Cell viability was tested by staining with crystal violet and annexin V. Surface plasmon resonance was used to verify molecular interactions. Thrombosis was modeled in mice via stenosis of the inferior vena cava.

Results: HUVECs and HMVECs were treated with NETs (1 μg DNA content/ml) in the presence or absence of defibrotide (10 μg/ml) for 4 hours. Defibrotide significantly (p< 0.0001) mitigated NET-mediated upregulation of E-selectin (30% reduction), ICAM-1 (70%), and VCAM-1 (60%). Calcein-AM-labeled neutrophils adhered 10-times more strongly to NET-activated endothelial cells, an effect that was reduced by 50% in the presence of defibrotide (p< 0.01). NET-mediated tissue factor upregulation was also significantly reduced by defibrotide, whether measured by gene expression or enzymatic activity. Network analysis of upregulated genes in NET-stimulated endothelial cells revealed an inflammatory signature highlighted by meta groups such as the TNF and NF-ĸB signaling pathways, which were downregulated by defibrotide. Narrowing the focus from polymolecular NETs to specific NET components, a strong interaction between histone H4 and defibrotide was confirmed by surface plasmon resonance (K_D 53.5 nM). Purified histone H4 (25 μg/ml) increased expression of E-selectin and ICAM-1 by endothelial cells, effects that were completely abolished by defibrotide. Furthermore, defibrotide markedly protected the viability of endothelial cells over a 24-hour period of exposure to histone H4 (100 μg/ml). Notably, histone H4-mediated cell death was on the spectrum of pyroptosis with increased IL-1β and cleavage of gasdermin D (both inhibited by defibrotide). Finally, in a mouse model of thrombosis, injection of histones (10 mg/kg) markedly increased thrombus accretion at 24 hours (mean thrombus weight 8 mg vs. 2 mg), with thrombus weight returned to baseline by infusion of 150 mg/kg defibrotide at the time of histone injection. Notably, soluble E-selectin tracked closely with thrombus accretion, as did infiltration of thrombus leukocytes.

Conclusion: These data provide insights into the potential role of polyanionic compounds in protecting the endothelium from thromboinflammation with potential implications for myriad NET- and histone-accelerated disease states.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/endothelium-protective-histone-neutralizing-properties-of-the-polyanionic-agent-defibrotide/