Background:
Peripheral artery disease causes significant functional disability and results in impaired quality of life. Toll-like receptor (TLR)-2, 3 and 4 are suggested to participate in blood flow recovery in ischemic limb by modulating inflammation and angiogenesis, however, the role of TLR9 remains unknown. TLR9 recognizes bacterial unmethylated DNA and plays a role in innate defense, although it can also provoke inflammation in response to fragmented DNA released from regenerated mammalian cells. This study tested the hypothesis that genetic deletion of TLR9 accelerates blood flow recovery after femoral artery ligation by inhibiting inflammation and improving endothelial cell function.
Methods and Results:
Unilateral femoral artery ligation was performed in TLR9-deficient (TLR9KO) mice and wild type (WT) mice. Femoral artery ligation significantly increased RNA expression of TLR9 (20-times) in WT mice and plasma levels of single-stranded DNA and double-stranded DNA, endogenous ligands for TLR9, in both strains of mice compared with each sham-operated group (P<0.05). Laser Doppler perfusion imaging demonstrated that TLR9KO mice significantly improved the ratio of the blood flow in the ischemic to non-ischemic limb compared with WT mice at 2 weeks after ligation (P<0.05). TLR9KO mice showed less accumulation of macrophages and less expression of inflammatory molecules (e.g., TNF-α, MCP-1 and IL-1β in ischemic muscle compared with WT mice (P<0.05, respectively). In vitro experiments using thioglycolate-stimulated peritoneal macrophages demonstrated that CpG ODN, agonistic oligonucleotide for TLR9, promoted the expression of pro-inflammatory molecules (e.g., MCP-1 and TNF-α) in WT macrophages (P<0.05, respectively) but not in TLR9 KO macrophages. Furthermore, activation of TLR9 by CpG ODN inhibited migration and proliferation of endothelial cells as determined by scratch-wound assay and MTS assay, respectively (P<0.05).
Conclusion:
Our results suggested that TLR9 enhances inflammation and affects migration and proliferation of endothelial cells, leading to impaired blood flow recovery in ischemic limb. TLR9 may serve as a potential therapeutic target for ischemic limb disease.
A bi-adjuvant nanovaccine that potentiates immunogenicity of neoantigen for combination immunotherapy of colorectal cancer