Abstract 554: Monocyte Toll-like Receptor 4 (tlr4) Signaling Is Essential For Post Thrombotic Upa Expression

Abstract

Background: Leukocyte expression of Toll-like receptor 4 (TLR4) increases with transformation to chronic deep venous thrombosis (DVT) & in non-resolving thrombi, but TLR4 function during thrombus clearance is unclear. We hypothesized TLR4 signaling to regulate expression of fibrinolytic factors in monocytes & macrophages (MO/MFs) for VT resolution. Methods: Small interfering RNA (siRNA) knockdown (KD) of TLR4 & disruption of Myd88-dependent/independent pathways occurred in immortalized MFs (RAW264.7), with quantitative real-time PCR (qRT-PCR) for a panel of fibrinolytic factors & immunoblotting for urokinase (uPA). Tlr4-/- mice & wild-type controls underwent IVC stasis/ligation, yielding VT at 2h, 2d, & 8d. Murine/chronic human thrombi underwent immunofluorescent staining for indicated targets & murine thrombi weight-length ratios were found. Splenic & Bone marrow-derived MFs from Tlr4 Cre mice were immunoblotted for uPA. Results: TLR4 silencing in RAW264.7 cells yielded suppression of uPA mRNA (Fig 1B) & protein levels (not shown). TRIF KD yielded decreased uPA mRNA (Fig 1C) & protein levels (not shown), implicating Myd88-independent signaling in MO/MF uPA production. Human (not shown) & murine chronic thrombi staining saw TLR4 co-localization with MF marker CCR2 (Fig 1D). Global knockout (KO) of murine TLR4 did not affect early thrombogenesis (Fig 1E left), with delayed VT resolution seen from 2d (Fig 1E right). Decreased uPA protein levels were observed in Tlr4 KO mice (Fig 1F). Conclusions: TLR4 signaling stimulates in vivo VT resolution. Expression of fibrinolytic factor uPA is enforced in MO/MFs by TLR4-Myd88-independent signaling, a potential target for VT resolution.