Abstract
Background: Peripheral artery disease causes significant functional disability and results in impaired quality of life. Ischemic tissue injury releases various endogenous ligands for Toll-like receptors (TLRs), suggesting the involvement of TLRs in blood flow recovery. However, the role of TLR9, which was originally known as a sensor for bacterial DNA, remains unknown. This study investigated the role of TLR9 in blood flow recovery in the ischemic limb using a mouse hind-limb ischemia model.Methods and Results: Unilateral femoral artery ligation was performed in TLR9-deficient (Tlr9−/−) mice and wild-type mice. In wild-type mice, femoral artery ligation significantly increased mRNA expression of TLR9 in the ischemic limb (P < 0.001) and plasma levels of cell-free DNA (cfDNA) as determined by single-stranded DNA (ssDNA) (P < 0.05) and double-stranded DNA (dsDNA) (P < 0.01), which are endogenous ligands for TLR9, compared with the sham-operated group. Laser Doppler perfusion imaging demonstrated significantly improved ratio of blood flow in the ischemic to non-ischemic limb in Tlr9−/− mice compared with wild-type mice at 2 weeks after ligation (P < 0.05). Tlr9−/− mice showed increased capillary density and reduced macrophage infiltration in ischemic limb. Genetic deletion of TLR9 reduced the expression of TNF-α, and attenuated NF-κB activation in ischemic muscle compared with wild-type mice (P < 0.05, respectively) at 3 days after the surgery. ODN1826, a synthetic agonistic oligonucleotide for TLR9, or plasma obtained from mice with ischemic muscle promoted the expression of TNF-α in wild-type macrophages (P < 0.05), but not in Tlr9−/− macrophages. ODN1826 also activated NF-κB signaling as determined by the degradation of IκBα in wild-type macrophages (P < 0.05), but not in Tlr9−/− macrophages. In vitro experiments using human umbilical vein endothelial cells demonstrated that TNF-α, or conditioned medium obtained from wild-type macrophages treated with ODN1826 accelerated cell death as determined by MTS assay (P < 0.05 and P < 0.01, respectively).Conclusion: Our results suggest that ischemic muscle releases cfDNA, which activates TLR9 and enhances inflammation, leading to impairment of blood flow recovery in the ischemic limb. cfDNA-TLR9 signaling may serve as a potential therapeutic target in ischemic limb disease.
Highlights
Peripheral artery disease (PAD), due to partial or complete obstruction of the arteries, is one of the most common manifestations of atherosclerosis, which is associated with increased risk of cardiovascular events [1]
To determine the participation of TLR9 in blood flow recovery of the ischemic limb, we examined the expression of TLR9 and circulating level of TLR9 ligand at 3 days after femoral artery ligation in wild-type mice
Results of Quantitative real-time RT-PCR (qPCR) demonstrated that ligation of the femoral artery markedly increased Tlr9 expression in the ischemic limb compared to the sham operated group (Figure 1A)
Summary
Peripheral artery disease (PAD), due to partial or complete obstruction of the arteries, is one of the most common manifestations of atherosclerosis, which is associated with increased risk of cardiovascular events [1]. Previous studies indicated that an inflammatory process regulates neovascularization and subsequent blood flow recovery [2] In this process, the immune system plays a pivotal role [3]. The immune system plays a pivotal role [3] Exogenous ligands such as microbial-associated molecules activate macrophages through Toll-like receptors (TLRs), whereas endogenous molecules derived from damaged and dead host cells activate macrophages though TLRs and promote sterile inflammation by inducing cytokines [4]. Ischemia induces cellular and tissue damage [5], causing the release of cellular debris which contains various endogenous ligands for TLRs. several studies reported that TLR-2, 3 and 4 participate in blood flow recovery in ischemic tissues by modulating inflammation and angiogenesis [6,7,8,9,10,11]. This study investigated the role of TLR9 in blood flow recovery in the ischemic limb using a mouse hind-limb ischemia model
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
