Venous thromboembolism (VTE), which includes deep vein thrombosis and pulmonary embolism remains the third most common cause of mortality among all cardiovascular diseases. Studies support intertwined roles of innate inflammatory systems and thrombosis in the pathogenesis of VTE. Toll-like receptors (TLRs) are transmembrane proteins that play pivotal roles in the innate immune system by detecting « pathologic ligands » and transducing signals into a prothrombotic state. Endogenous RNA, released from cells under pathological conditions, has been identified as a potent pro-coagulant factor and a ligand for TLR3. We hypothesize that eRNA and TLR3 promote venous thrombosis development after vessel injury.
The ferric chloride (FeCl3) model was used to induce venous thrombosis in mice. WT or TLR3 deficient (-/-) mice received an i.v. injection of either vehicle, a specific fluorescent probe for RNA (Syto RNA Select), RNase1, poly (I:C) or RNA extracted from murine endothelial cells (eRNA). Human umbilical vein endothelial cells (HUVECs) were treated with siRNA directed against TLR3 or an IL-8 receptor (CXCR2) antagonist.
Using a cell-permeant nucleic acid stain, we demonstrated that FeCl3 promoted RNA release in vivo and thus increased the RNA content in the thrombus. Therefore, RNase 1 treatment reduced thrombus size compared to mice injected with vehicle. In contrast, eRNA and poly (I:C) increased thrombus size in WT mice whereas no modifications were observed in TLR3-/- mice. Poly (I:C) and eRNA treatments bolstered neutrophil infiltration in WT but not in TLR3-/- mice. Similarly, the expression of citrullinated histone 3 (CitH3), a neutrophil extracellular traps (NETs) biomarker, was heightened by poly (I:C) and eRNA in WT but not in TLR3/- mice. In vitro, eRNA stimulated CXCL5 mRNA expression, a murine isoform of human IL-8, in WT but not in TLR3-/- endothelial cells. ELISA analysis corroborated that CXCL5 expression is induced by eRNA via TLR3 in endothelial cells. To confirm these data, human endothelial cells treated with poly (I:C) were transfected with siRNA targeting TLR3. This contributes to the abolition of neutrophil recruitment as compared with control siRNA. The administration of an IL-8 receptor (CXCR2) antagonist to the human endothelial cells abrogated neutrophil infiltration.
These results suggest that eRNA through TLR3 enhances neutrophil recruitment and NET formation leading to venous thrombosis.Le texte complet de cet article est disponible en PDF.
Keyword : Circulation