Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease (Aug 2024)
Extracellular RNA Induces Neutrophil Recruitment Via Toll‐Like Receptor 3 During Venous Thrombosis After Vascular Injury
Abstract
Background Venous thromboembolism is associated with endothelial cell activation that contributes to the inflammation‐dependent activation of the coagulation system. Cellular damage is associated with the release of different species of extracellular RNA (eRNA) involved in inflammation and coagulation. TLR3 (toll‐like receptor 3), which recognizes (viral) single‐stranded or double‐stranded RNAs and self‐RNA fragments, might be the receptor of these species of eRNA during venous thromboembolism. Here, we investigate how the TLR3/eRNA axis contributes to venous thromboembolism. Methods and Results Thrombus formation and size in wild‐type and TLR3 deficient (−/−) mice were monitored by ultrasonography after venous thrombosis induction using the ferric chloride and stasis models. Mice were treated with RNase I, with polyinosinic‐polycytidylic acid, a TLR3 agonist, or with RNA extracted from murine endothelial cells. Gene expression and signaling pathway activation were analyzed in HEK293T cells overexpressing TLR3 in response to eRNA or in human umbilical vein endothelial cells transfected with a small interference RNA against TLR3. Plasma clot formation on treated human umbilical vein endothelial cells was analyzed. Thrombosis exacerbated eRNA release in vivo and increased eRNA content within the thrombus. RNase I treatment reduced thrombus size compared with vehicle‐treated mice (P<0.05). Polyinosinic‐polycytidylic acid and eRNA treatments increased thrombus size in wild‐type mice (P<0.01 and P<0.05), but not in TLR3−/− mice, by reinforcing neutrophil recruitment (P<0.05). Mechanistically, TLR3 activation in endothelial cells promotes CXCL5 (C‐X‐C motif chemokine 5) secretion (P<0.001) and NFκB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells) activation (P<0.05). Finally, eRNA triggered plasma clot formation in vitro (P<0.01). Conclusions We show that eRNA and TLR3 activation enhance venous thromboembolism through neutrophil recruitment possibly through secretion of CXCL5, a potent neutrophil chemoattractant.
Keywords