PS204. Toll-like Receptors 2 and 4, but Not RAGE Deficiency Results in Greater Muscle Necrosis 2 Weeks After Hind-limb Ischemia: Implications for Muscle Recovery After Ischemic Injury

Abstract

Innate immunity is the first line of defense against invading pathogens through receptors such as TLR2 and TLR4. These receptors also mediate responses to tissue injury through endogenous danger signals. We have shown that HMGB1, a danger signal, is released by hypoxic cells and promotes angiogenesis in vitro. Anti-HMGB1 antibody increases muscle necrosis in murine hind-limb ischemia but did not change endothelial density, supporting a role for HMGB1 and innate immunity in skeletal muscle recovery following ischemia. In this study, we examined the roles of the HMGB1 receptors TLR2, TLR4 and RAGE in mediating this protection. 10-12 wk old mice genetically deficient for TLR4 (C3H/HeJ, control HeOuJ; N = 4), TLR2 (N = 3), or RAGE (N = 4; control C57B/6, N = 4) underwent right femoral artery ligation. At 2wks, calf muscles were fixed, sectioned, and stained with H&E to quantify muscle necrosis (% area) and vWF for endothelial density. Statistical significance was determined by ANOVA. TLR4 mutant mice developed greater muscle necrosis following hind-limb ischemia than control HeOuJ mice (16.4% ± 4.6 vs 4.6 ± 2.1; p = 0.04). Muscle necrosis was also markedly increased in TLR2 KO (23.5 ± 17.3; P-0.04) while no necrosis was detected in the C57B/6 controls. RAGE KO mice also showed little necrosis. In contrast, the C57B/6 and RAGE KO mice exhibited significant muscle regeneration in the ischemic hind-limb. Endothelial density was similar between the knockout and control mice. The absence of TLR2 and TLR4 signaling is associated with increased skeletal muscle necrosis following ischemia and suggests that these receptors are necessary for muscle protection. In contrast, RAGE signaling is not involved. The protection offered by TLR2 and TLR4 did not involve endothelial proliferation but may involve regenerative processes. These data suggest that TLRs may be critical in tissue recovery from ischemia independent of angiogenesis.