AUTHOR=Nishimoto Sachiko , Aini Kunduziayi , Fukuda Daiju , Higashikuni Yasutomi , Tanaka Kimie , Hirata Yoichiro , Yagi Shusuke , Kusunose Kenya , Yamada Hirotsugu , Soeki Takeshi , Shimabukuro Michio , Sata Masataka 

TITLE=Activation of Toll-Like Receptor 9 Impairs Blood Flow Recovery After Hind-Limb Ischemia

JOURNAL=Frontiers in Cardiovascular Medicine

VOLUME=5

YEAR=2018

URL=https://www.frontiersin.org/journals/cardiovascular-medicine/articles/10.3389/fcvm.2018.00144

DOI=10.3389/fcvm.2018.00144

ISSN=2297-055X

ABSTRACT=<p><bold>Background:</bold> 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.</p><p><bold>Methods and Results:</bold> Unilateral femoral artery ligation was performed in TLR9-deficient (<italic>Tlr9</italic><sup>−/−</sup>) mice and wild-type mice. In wild-type mice, femoral artery ligation significantly increased mRNA expression of TLR9 in the ischemic limb (<italic>P</italic> &lt; 0.001) and plasma levels of cell-free DNA (cfDNA) as determined by single-stranded DNA (ssDNA) (<italic>P</italic> &lt; 0.05) and double-stranded DNA (dsDNA) (<italic>P</italic> &lt; 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 <italic>Tlr9</italic><sup>−/−</sup> mice compared with wild-type mice at 2 weeks after ligation (<italic>P</italic> &lt; 0.05). <italic>Tlr9</italic><sup>−/−</sup> 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 (<italic>P</italic> &lt; 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 (<italic>P</italic> &lt; 0.05), but not in <italic>Tlr9</italic><sup>−/−</sup> macrophages. ODN1826 also activated NF-κB signaling as determined by the degradation of IκBα in wild-type macrophages (<italic>P</italic> &lt; 0.05), but not in <italic>Tlr9</italic><sup>−/−</sup> 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 (<italic>P</italic> &lt; 0.05 and <italic>P</italic> &lt; 0.01, respectively).</p><p><bold>Conclusion:</bold> 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.</p>