General Surgery Resident; Post-doctoral fellow University of Michigan Ann Arbor, Michigan, United States
Objective: In humans with deep venous thrombosis (DVT), leukocyte expression of Toll-like receptor 4 (TLR4) is persistently elevated during the transformation to chronic DVT and in non-resolving thrombi. Though the role of TLR4 signaling in acute thrombosis is established, the function of leukocyte TLR4 during thrombus clearance remains to be explored. We hypothesized that TLR4 signaling regulated the expression of fibrinolytic factors in monocytes and macrophages (MO/Mφs), the predominant leukocyte involved in thrombus resolution.
Methods: Chronic human thrombus samples were obtained under an approved institutional protocol. Tlr4-/- mice and wild-type (WT) controls underwent IVC stasis/ligation to yield VT at 2h, 2d, and 8d. Human and murine thrombi underwent immunofluorescent staining for indicated targets. Weight to length ratios of murine thrombi were calculated. For in vitro studies, small interfering RNA (siRNA) mediated silencing of TLR4 (using two independent siRNAs), or selective abrogation of the Myd88-dependent pathway using siRNAs specific to Myd88, or the Myd88-independent pathway, using silencing of TRIF or TRAM was performed using immortalized Mφs (RAW264.7 cells). Quantitative real-time PCR (qRT-PCR) for a limited panel of validated fibrinolytic factors, or immunoblotting for urokinase was performed on transfected cells.
Results: In human (not shown) and murine chronic thrombi, TLR4 staining was observed to co-localize with the Mφ marker CCR2 (Fig. 1A). Though global knockout of TLR4 in mice did not influence early thrombogenesis (Fig. 1B, left data sets), impaired thrombus resolution was observed as early as 2d (Fig. 1B, right data sets). Silencing of TLR4 in RAW264.7 cells yielded suppression of urokinase (Plau, a potent fibrinolytic factor) mRNA levels and protein levels (Fig. 1C and data not shown). Selective knockdown of TRIF, but not TRAM or Myd88 similarly yielded suppressed urokinase mRNA (Fig. 1D) and protein levels (not shown), implicating Myd88-independent signaling in promoting urokinase expression in Mφs.
Conclusions: TLR4 signaling promotes VT resolution in vivo. TLR4-Myd88-independent signaling in MO/Mφs promotes the expression of urokinase, a potent fibrinolytic factor, which has been described as critical in experimental VT resolution. Agonism of this pathway represents a novel therapeutic target for addressing VT resolution.