B. Xie1, M. Xie1, X. Cui1, J. Xu1, E. Tzeng1, T. Billiar1, U. Sachdev1 1University Of Pittsburgh,Surgery,Pittsburgh, PA, USA
Introduction: We have previously shown that lack of Toll-like Receptor 4 (TLR4), a pattern recognition receptor in the innate immune system, promotes perfusion recovery, angiogenesis and muscle fiber regeneration in a murine hindlimb ischemia model. However, the cell-type responsible for this protective effect has not been elucidated. In other experimental models, platelet-derived TLR4 has been shown to a mediator of damage, possibly through expression of the platelet activation marker P-selectin. We therefore hypothesized that platelet-specific TLR4 similarly mediates inflammation from muscle hypoxia, and that targeted deletion of TLR4 in platelets is protective in limb ischemia.
Methods: Targeted deletion of TLR4 was performed using Cre-Lox site-specific recombinase technology. To generate endothelial cell, platelet and myeloid specific TLR4 knockout mice, TLR4-floxed mice were bred with VE-cadherein-Cre (VEC), platelet-factor 4-Cre (PF4) and LysM-Cre(LysM) mice, respectively. Homozygous mutation was confirmed using PCR. In cell-specific TLR4KO as well as TLR4-floxed (control) mice, femoral artery ligation (FAL) was performed on the right hindlimb, while vessels were exposed without ligation on the left. Perfusion was assessed at baseline, 1, 7 and 14 days after FAL using Laser Doppler perfusion imaging (LDPI). IL-6 ELISA levels 6 hours after FAL were measured from control and PF4-TLR4KO mice. Platelets from control and global TLR4KO mice were isolated from whole blood and activated with buffer, LPS (TLR4 agonist), Pam3CSK4 (TLR2 agonist), HMGB1 (TLR2 and TLR4 agonist) or thrombin (0.25U), and subjected to flow cytometry to assess P-selectin expression.
Results: FAL uniformly resulted in significant, unilateral ischemia. Two weeks after FAL, platelet-specific TLR4KO mice (PF4) demonstrated more perfusion recovery compared to endothelial cell specific (VEC) or myeloid specific (LysM) TLR4KO mice (Figure 1, p<0.02; N=5-8/group; ANOVA). PF4-TLR4KO mice had less serum IL-6 levels compared with controls six hours after FAL (p<0.03, N=3-5/group;t-test). Platelets from control and global TLR4KO mice responded to treatment with thrombin and Pam3CSK4 with increased P-selectin expression. However, when exposed to either LPS or HMGB1, platelet P-Selectin expression did not increase significantly over baseline.
Conclusion: As opposed to myeloid and endothelial cells, platelet sources of TLR4 negatively mediated perfusion recovery following FAL, promoting release of IL-6 after injury. However, platelets with functional TLR4 did not respond to traditional TLR4 agonists with P-selectin expression. Thus, the mechanism for a protective effect of platelet –specific TLR4 deletion in muscle ischemia may be P-selectin independent.
