Activation of Toll-Like Receptor 9 Impairs Blood Flow Recovery After Hind-Limb Ischemia
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, whi...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2018-10-01
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| Series: | Frontiers in Cardiovascular Medicine |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fcvm.2018.00144/full |
| _version_ | 1828356977889116160 |
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| author | Sachiko Nishimoto Kunduziayi Aini Daiju Fukuda Daiju Fukuda Yasutomi Higashikuni Kimie Tanaka Yoichiro Hirata Shusuke Yagi Kenya Kusunose Hirotsugu Yamada Takeshi Soeki Michio Shimabukuro Michio Shimabukuro Masataka Sata |
| author_facet | Sachiko Nishimoto Kunduziayi Aini Daiju Fukuda Daiju Fukuda Yasutomi Higashikuni Kimie Tanaka Yoichiro Hirata Shusuke Yagi Kenya Kusunose Hirotsugu Yamada Takeshi Soeki Michio Shimabukuro Michio Shimabukuro Masataka Sata |
| author_sort | Sachiko Nishimoto |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-14T03:07:55Z |
| format | Article |
| id | doaj.art-28d36596f04e4b49b48269fc85654a9a |
| institution | Directory Open Access Journal |
| issn | 2297-055X |
| language | English |
| last_indexed | 2024-04-14T03:07:55Z |
| publishDate | 2018-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cardiovascular Medicine |
| spelling | doaj.art-28d36596f04e4b49b48269fc85654a9a2022-12-22T02:15:40ZengFrontiers Media S.A.Frontiers in Cardiovascular Medicine2297-055X2018-10-01510.3389/fcvm.2018.00144343682Activation of Toll-Like Receptor 9 Impairs Blood Flow Recovery After Hind-Limb IschemiaSachiko Nishimoto0Kunduziayi Aini1Daiju Fukuda2Daiju Fukuda3Yasutomi Higashikuni4Kimie Tanaka5Yoichiro Hirata6Shusuke Yagi7Kenya Kusunose8Hirotsugu Yamada9Takeshi Soeki10Michio Shimabukuro11Michio Shimabukuro12Masataka Sata13Department of Cardiovascular Medicine, Tokushima University Graduate School of Biomedical Sciences, Tokushima, JapanDepartment of Cardiovascular Medicine, Tokushima University Graduate School of Biomedical Sciences, Tokushima, JapanDepartment of Cardiovascular Medicine, Tokushima University Graduate School of Biomedical Sciences, Tokushima, JapanDepartment of Cardio-Diabetes Medicine, Tokushima University Graduate School of Biomedical Sciences, Tokushima, JapanDepartment of Cardiovascular Medicine, The University of Tokyo, Tokyo, JapanDivision for Health Service Promotion, The University of Tokyo, Tokyo, JapanDepartment of Pediatrics, The University of Tokyo Hospital, Tokyo, JapanDepartment of Cardiovascular Medicine, Tokushima University Graduate School of Biomedical Sciences, Tokushima, JapanDepartment of Cardiovascular Medicine, Tokushima University Graduate School of Biomedical Sciences, Tokushima, JapanDepartment of Cardiovascular Medicine, Tokushima University Graduate School of Biomedical Sciences, Tokushima, JapanDepartment of Cardiovascular Medicine, Tokushima University Graduate School of Biomedical Sciences, Tokushima, JapanDepartment of Cardio-Diabetes Medicine, Tokushima University Graduate School of Biomedical Sciences, Tokushima, JapanDepartment of Diabetes, Endocrinology and Metabolism, School of Medicine, Fukushima Medical University, Fukushima, JapanDepartment of Cardiovascular Medicine, Tokushima University Graduate School of Biomedical Sciences, Tokushima, JapanBackground: 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.https://www.frontiersin.org/article/10.3389/fcvm.2018.00144/fullhind-limb ischemiablood flow recoveryToll-like receptor 9inflammationmacrophage |
| spellingShingle | Sachiko Nishimoto Kunduziayi Aini Daiju Fukuda Daiju Fukuda Yasutomi Higashikuni Kimie Tanaka Yoichiro Hirata Shusuke Yagi Kenya Kusunose Hirotsugu Yamada Takeshi Soeki Michio Shimabukuro Michio Shimabukuro Masataka Sata Activation of Toll-Like Receptor 9 Impairs Blood Flow Recovery After Hind-Limb Ischemia Frontiers in Cardiovascular Medicine hind-limb ischemia blood flow recovery Toll-like receptor 9 inflammation macrophage |
| title | Activation of Toll-Like Receptor 9 Impairs Blood Flow Recovery After Hind-Limb Ischemia |
| title_full | Activation of Toll-Like Receptor 9 Impairs Blood Flow Recovery After Hind-Limb Ischemia |
| title_fullStr | Activation of Toll-Like Receptor 9 Impairs Blood Flow Recovery After Hind-Limb Ischemia |
| title_full_unstemmed | Activation of Toll-Like Receptor 9 Impairs Blood Flow Recovery After Hind-Limb Ischemia |
| title_short | Activation of Toll-Like Receptor 9 Impairs Blood Flow Recovery After Hind-Limb Ischemia |
| title_sort | activation of toll like receptor 9 impairs blood flow recovery after hind limb ischemia |
| topic | hind-limb ischemia blood flow recovery Toll-like receptor 9 inflammation macrophage |
| url | https://www.frontiersin.org/article/10.3389/fcvm.2018.00144/full |
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