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Poster Networking

Vascular Biology, Blood Cells, and Immunothrombosis

Session: MiR-146a regulates NET formation through different molecular mechanisms increasing cardiovascular and thrombotic risk"

PB0828 - "MiR-146a regulates NET formation through different molecular mechanisms increasing cardiovascular and thrombotic risk"

Monday, June 24, 2024
13:45 – 14:45 ICT
Room: Exhibition Hall

    Presenting Author(s)

  • Laura Zapata Martínez, PhD

    PhD Student
    Regional Center of Hemodonation, Department of Hematology and Medical Oncology, University General Hospital Morales Meseguer, University of Murcia, IMIB-Pascual Parrilla, 30003, Murcia, Spain
    Blanca (Murcia), Murcia, Spain

    • Co-Author(s)

  • S

    Sonia Águila

    Doctor
    Regional Center of Hemodonation, Department of Hematology and Medical Oncology, University General Hospital Morales Meseguer, University of Murcia, IMIB-Pascual Parrilla, 30003, Murcia, Spain
    Murcia, Murcia, Spain

  • NG

    Nuria García-Barberá

    Laboratory Technician
    Regional Center of Hemodonation, Department of Hematology and Medical Oncology, University General Hospital Morales Meseguer, University of Murcia, IMIB-Pascual Parrilla, 30003, Murcia, Spain
    Murcia, Murcia, Spain

  • MF

    María Piedad Fernández-Pérez

    Doctor
    Regional Center of Hemodonation, Department of Hematology and Medical Oncology, University General Hospital Morales Meseguer, University of Murcia, IMIB-Pascual Parrilla, 30003, Murcia, Spain
    Murcia, Murcia, Spain

  • AA

    Alejandra Aroca-Crevillén

    PhD Student
    CNIC - Spanish National Center for Cardiovascular Research
    Murcia, Murcia, Spain

  • PG

    Pedro J. Guijarro

    Laboratory Technician
    Regional Center of Hemodonation, Department of Hematology and Medical Oncology, University General Hospital Morales Meseguer, University of Murcia, IMIB-Pascual Parrilla, 30003, Murcia, Spain
    Murcia, Murcia, Spain

  • AJ

    Ana Jiménez Espín

    n
    n
    Murcia, Murcia, Spain

  • SC

    Salvador Carrillo-Tornel

    PhD Student
    Regional Center of Hemodonation, Department of Hematology and Medical Oncology, University General Hospital Morales Meseguer, University of Murcia, IMIB-Pascual Parrilla, 30003, Murcia, Spain
    Murcia, Murcia, Spain

  • AH

    Andrés Hidalgo

    Doctor
    CNIC - Spanish National Center for Cardiovascular Research
    Murcia, Murcia, Spain

  • RG

    Rocío González-Conejero

    Doctor
    Regional Center of Hemodonation, Department of Hematology and Medical Oncology, University General Hospital Morales Meseguer, University of Murcia, IMIB-Pascual Parrilla, 30003, Murcia, Spain
    Murcia, Murcia, Spain

  • CM

    Constantino Martínez

    Doctor
    Regional Center of Hemodonation, Department of Hematology and Medical Oncology, University General Hospital Morales Meseguer, University of Murcia, IMIB-Pascual Parrilla, 30003, Murcia, Spain
    Murcia, Murcia, Spain

Background: Neutrophil extracellular traps (NETs) have a crucial role in thrombosis. We
described for the first time that miR-146a regulates NET formation although the mechanisms are not known. Indeed, we showed that decreased miR-146a levels due to rs2431697-T are associated with increased cardiovascular events and higher NET markers in patients with thromboinflammatory diseases such as atrial fibrillation, IAM or sepsis.

Aims: To determine the mechanisms of miR-146a to trigger enhanced NET formation.

Methods: miR-146a -/- and WT mice 8-14 week-old were used. Bone marrow derived
neutrophils were purified. Reactive oxygen species (ROS) production was determined by H2DCFDA or MitoSOX, untargeted metabolomics was performed using mass spectrometry, signaling pathways and autophagy were evaluated by western blot, and inflammasome activation by ELISA. Finally, intravital microscopy was performed in cremaster in mice injected with anti-Ly6G antibody and TNF-α.

Results: Immunofluorescence studies showed that miR-146a deficiency enhanced NET
formation in vitro. Moreover, miR-146a-/- neutrophils presented higher respiratory burst and ROS generation than WT following PMA activation. However, mitochondrial ROS production and Nox2 expression were similar to WT neutrophils indicating that NADPH oxidase activation was enhanced in miR-146a-/- neutrophils. The metabolic pathways (pentose phosphate pathway, glycolysis, and TCA cycle) were accelerated in deficient neutrophils causing elevated NADP(H) and glutathione pools fueling ROS generation. Molecular pathways implicated in NETosis were more activated in miR-146 -/- neutrophils: higher p-ERK1/2 expression and increased IL-1β secretion in miR-146a -/- neutrophils incubated with LPS+ATP. On the other hand, macroautophagy (LC3II/LC3I) and chaperone mediated autophagy (LAMP2A) were altered in miR-146a -/- neutrophils compared with WT. Finally, miR-146a -/- neutrophils presented reduced rolling and increased adhesion to the endothelium following TNF-α activation.

Conclusion(s): miR-146a controls NETosis through different pathways which might contribute to
the increased cardiovascular and thrombotic events observed in rs2431697-T patients with thromboinflammatory diseases and pointing to miRNA-based therapy as a promising new therapeutic approach.