Abstract 15312: Genetic Deletion of Toll-like Receptor 9 Accelerates Blood Flow Recovery after Hindlimb Ischemia

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Sachiko Nishimoto ◽  
Daiju Fukuda ◽  
Yasutomi Higashikuni ◽  
Kimie Tanaka ◽  
Yoichiro Hirata ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Femoral Artery ◽  
Cpg Odn ◽  
Toll Like Receptor ◽  
Artery Ligation ◽  
Ischemic Limb ◽  
Tnf Α ◽  
Genetic Deletion ◽  
Inflammatory Molecules

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.

scholarly journals TNF-α Activates High-Mobility Group Box 1 - Toll-Like Receptor 4 Signaling Pathway in Human Aortic Endothelial Cells

2016 ◽  
Vol 38 (6) ◽  
pp. 2139-2151 ◽  
Author(s):  
Won Seok Yang ◽  
Nam Jeong Han ◽  
Jin Ju Kim ◽  
Mee Jeong Lee ◽  
Su-Kil Park
Keyword(s):  
Signal Transduction ◽  
Endothelial Cells ◽  
High Mobility ◽  
Neutralizing Antibody ◽  
High Mobility Group ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Aortic Endothelial Cells ◽  
Tnf Α ◽  
Human Aortic Endothelial Cells

Background/Aims: Toll-like receptor 4 (TLR4) interacts with endogenous substances as well as lipopolysaccharide. We explored whether TLR4 is implicated in tumor necrosis factor-α (TNF-α) signal transduction in human aortic endothelial cells. Methods: The pathway was evaluated by transfection of siRNAs, immunoprecipitation and Western blot analysis. Results: TNF-α activated spleen tyrosine kinase (Syk) within 10 min, which led to endothelin-1 (ET-1) production. TLR4 was also rapidly activated by TNF-α stimulation, as shown by recruitment of interleukin-1 receptor-associated kinase 1 to TLR4 and its adaptor molecule, myeloid differentiation factor 88 (MyD88). siRNA depletion of TLR4 markedly attenuated TNF-α-induced Syk activation and ET-1 production. TLR4 inhibitor (CLI-095), TLR4-neutralizing antibody and siRNA depletion of MyD88 also attenuated TNF-α-induced Syk activation. Syk was co-immunoprecipitated with TLR4, and TNF-α activated Syk bound to TLR4. High-mobility group box 1 (HMGB1) was rapidly released and associated with TLR4 after TNF-α stimulation with a peak at 5 min, which was prevented by N-acetylcysteine, an antioxidant. Glycyrrhizin (HMGB1 inhibitor), HMGB1-neutralizing antibody and siRNA depletion of HMGB1 all suppressed TNF-α-induced Syk activation and ET-1 production. Conclusion: Upon TNF-α stimulation, TLR4 is activated by HMGB1 that is immediately released after the generation of reactive oxygen species, and plays a crucial role in the signal transduction.

scholarly journals Human venous valve disease caused by mutations in FOXC2 and GJC2

2017 ◽  
Vol 214 (8) ◽  
pp. 2437-2452 ◽  
Author(s):  
Oliver Lyons ◽  
Prakash Saha ◽  
Christopher Seet ◽  
Adam Kuchta ◽  
Andrew Arnold ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Transcription Factors ◽  
Gap Junction ◽  
Venous Hypertension ◽  
Valve Disease ◽  
Venous Valve ◽  
Venous Valves ◽  
Genetic Deletion ◽  
Junction Proteins

Venous valves (VVs) prevent venous hypertension and ulceration. We report that FOXC2 and GJC2 mutations are associated with reduced VV number and length. In mice, early VV formation is marked by elongation and reorientation (“organization”) of Prox1hi endothelial cells by postnatal day 0. The expression of the transcription factors Foxc2 and Nfatc1 and the gap junction proteins Gjc2, Gja1, and Gja4 were temporospatially regulated during this process. Foxc2 and Nfatc1 were coexpressed at P0, and combined Foxc2 deletion with calcineurin-Nfat inhibition disrupted early Prox1hi endothelial organization, suggesting cooperative Foxc2–Nfatc1 patterning of these events. Genetic deletion of Gjc2, Gja4, or Gja1 also disrupted early VV Prox1hi endothelial organization at postnatal day 0, and this likely underlies the VV defects seen in patients with GJC2 mutations. Knockout of Gja4 or Gjc2 resulted in reduced proliferation of Prox1hi valve-forming cells. At later stages of blood flow, Foxc2 and calcineurin-Nfat signaling are each required for growth of the valve leaflets, whereas Foxc2 is not required for VV maintenance.

Heparin Increases Exercise-Induced Collateral Blood Flow in Rats With Femoral Artery Ligation

1995 ◽  
Vol 76 (3) ◽  
pp. 448-456 ◽  
Author(s):  
H. T. Yang ◽  
Robert W. Ogilvie ◽  
Ronald L. Terjung
Keyword(s):  
Blood Flow ◽  
Femoral Artery ◽  
Collateral Blood Flow ◽  
Artery Ligation ◽  
Exercise Induced

Interaction of Femoral Artery Ligation and TVaining on Microvascular PO2 and Blood Flow in Rat Soleus Muscle

2007 ◽  
Vol 39 (Supplement) ◽  
pp. S429
Author(s):  
William L. Sexton ◽  
Kyra Carpenter-Timm ◽  
Marietta Squire ◽  
Neja Valeja ◽  
Matthew J. Wessner ◽  
...  
Keyword(s):  
Blood Flow ◽  
Soleus Muscle ◽  
Femoral Artery ◽  
Artery Ligation ◽  
Rat Soleus Muscle

bFGF increases collateral blood flow in aged rats with femoral artery ligation

2000 ◽  
Vol 278 (1) ◽  
pp. H85-H93 ◽  
Author(s):  
H. T. Yang ◽  
Y. Feng
Keyword(s):  
Blood Flow ◽  
Young Adult ◽  
Femoral Artery ◽  
Age Groups ◽  
Calf Muscle ◽  
Treadmill Running ◽  
Dependent Manner ◽  
Aged Rats ◽  
Artery Ligation ◽  
Similar Treatment

We tested the hypothesis that aged animals are as responsive as the young adult animals in expanding collateral vasculature under a similar treatment of basic fibroblast growth factor (bFGF). Two age groups of male Fischer 344 rats (11 mo old; n = 32, 23 mo old; n = 43) weighing ∼385 g were subdivided into normal, acute ligation [femoral artery (FA) ligated 3 days before blood flow (BF) measurement] or ligated groups for 16 days and received recombinant human bFGF intra-arterial infusion at doses of 0, 0.5, 5, and 50 μg ⋅ kg− 1 ⋅ day− 1. BF was determined with 85Sr- and 141Ce-labeled microspheres during treadmill running at 15 and 20 m/min at 15% grade. Blood presure (BP) values were ∼149 and ∼163 mmHg ( p < 0.05); heart rates were ∼496 and ∼512 beats/min in the aged and young adult groups during running, respectively. Maximal collateral BF values were confirmed by no additional BF increase in the calf muscle at the higher speed. Ligation of the FA for 3 days reduced the BF reserve to the calf muscle by ∼90%. Calf muscle BF was modestly greater (10 ml ⋅ min− 1 ⋅ 100 g− 1) by 16 days in the carrier group. bFGF infusion expanded collateral BF in a dose-dependent manner with an increase of 33 and 42 ml ⋅ min− 1 ⋅ 100 g− 1 ( P < 0.001) in the 5 and 50 μg ⋅ kg− 1 ⋅ day− 1bFGF groups, respectively. Aged animals showed similar BF improvements as observed with the adult groups in response to ligation surgery and bFGF treatment. Our data indicate that the aged rats (∼23 mo old) remain responsive to exogenous bFGF induced in developing collateral-dependent BF as the young adult (∼11 mo old) controls. This suggests that the influence of bFGF in expanding collateral BF should not be preempted in the aged group, the population most affected by peripheral arterial insufficiency.

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