Treatment effect with anti-RAGE F(ab′)2 antibody improves hind limb angiogenesis and blood flow in Type 1 diabetic mice with left femoral artery ligation

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.

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IL10 Alters Peri-Collateral Macrophage Polarization and Hind-Limb Reperfusion in Mice after Femoral Artery Ligation

International Journal of Molecular Sciences ◽

10.3390/ijms21082821 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2821 ◽

Cited By ~ 1

Author(s):

Alexander M. Götze ◽

Christian Schubert ◽

Georg Jung ◽

Oliver Dörr ◽

Christoph Liebetrau ◽

...

Keyword(s):

Femoral Artery ◽

Hind Limb ◽

Macrophage Activation ◽

Macrophage Polarization ◽

Doppler Imaging ◽

Activated Macrophages ◽

Artery Ligation ◽

Alternatively Activated Macrophages ◽

Collateral Growth ◽

Alternatively Activated

Arteriogenesis is a process by which a pre-existing arterioarterial anastomosis develops into a functional collateral network following an arterial occlusion. Alternatively activated macrophages polarized by IL10 have been described to promote collateral growth. This study investigates the effect of different levels of IL10 on hind-limb reperfusion and the distribution of perivascular macrophage activation types in mice after femoral artery ligation (FAL). IL10 and anti-IL10 were administered before FAL and the arteriogenic response was measured by Laser-Doppler-Imaging perioperatively, after 3, 7, and 14 d. Reperfusion recovery was accelerated when treated with IL10 and impaired with anti-IL10. Furthermore, symptoms of ischemia on ligated hind-limbs had the highest incidence after application of anti-IL10. Perivascular macrophages were immunohistologically phenotyped using CD163 and CD68 in adductor muscle segments. The proportion of alternatively activated macrophages (CD163+/CD68+) in relation to classically activated macrophages (CD163−/CD68+) observed was the highest when treated with IL10 and suppressed with anti-IL10. This study underlines the proarteriogenic response with increased levels of IL10 and demonstrates an in-vivo alteration of macrophage activation types in the perivascular bed of growing collaterals.

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Recovery from hind limb ischemia is less effective in type 2 than in type 1 diabetic mice: Roles of endothelial nitric oxide synthase and endothelial progenitor cells

Journal of Vascular Surgery ◽

10.1016/j.jvs.2009.08.007 ◽

2009 ◽

Vol 50 (6) ◽

pp. 1412-1422 ◽

Cited By ~ 73

Author(s):

Jinglian Yan ◽

Guodong Tie ◽

Brian Park ◽

Yagai Yan ◽

Philip T. Nowicki ◽

...

Keyword(s):

Nitric Oxide ◽

Progenitor Cells ◽

Endothelial Nitric Oxide Synthase ◽

Hind Limb ◽

Limb Ischemia ◽

Diabetic Mice ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

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bFGF INFUSION INCREASES COLLATERAL BLOOD FLOW IN AGED RATS WITH FEMORAL ARTERY LIGATION 800

Medicine & Science in Sports & Exercise ◽

10.1097/00005768-199705001-00799 ◽

1997 ◽

Vol 29 (Supplement) ◽

pp. 139

Author(s):

Y. Feng ◽

H. T. Yang

Keyword(s):

Blood Flow ◽

Femoral Artery ◽

Aged Rats ◽

Collateral Blood Flow ◽

Artery Ligation

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Secretoneurin Gene Therapy Improves Blood Flow in an Ischemia Model in Type 1 Diabetic Mice by Enhancing Therapeutic Neovascularization

PLoS ONE ◽

10.1371/journal.pone.0074029 ◽

2013 ◽

Vol 8 (9) ◽

pp. e74029 ◽

Cited By ~ 13

Author(s):

Wilfried Schgoer ◽

Markus Theurl ◽

Karin Albrecht-Schgoer ◽

Verena Jonach ◽

Bernhard Koller ◽

...

Keyword(s):

Gene Therapy ◽

Blood Flow ◽

Diabetic Mice

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PRIOR TRAINING INCREASES COLLATERAL BLOOD FLOW IN RATS FOLLOWING ACUTE FEMORAL ARTERY LIGATION 1026

Medicine & Science in Sports & Exercise ◽

10.1097/00005768-199605001-01023 ◽

1996 ◽

Vol 28 (Supplement) ◽

pp. 172

Author(s):

H. T. Yang ◽

R. L. Terjung

Keyword(s):

Blood Flow ◽

Femoral Artery ◽

Collateral Blood Flow ◽

Prior Training ◽

Artery Ligation

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Modified multipotent stromal cells with epidermal growth factor restore vasculogenesis and blood flow in ischemic hind-limb of type II diabetic mice

Laboratory Investigation ◽

10.1038/labinvest.2010.86 ◽

2010 ◽

Vol 90 (7) ◽

pp. 985-996 ◽

Cited By ~ 64

Author(s):

Ali H Amin ◽

Zakaria Y Abd Elmageed ◽

Devika Nair ◽

Megan I Partyka ◽

Philip J Kadowitz ◽

...

Keyword(s):

Blood Flow ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Stromal Cells ◽

Hind Limb ◽

Multipotent Stromal Cells ◽

Type Ii ◽

Diabetic Mice ◽

Epidermal Growth

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Abstract 17955: Thereapeutic Angiogenesis Induced by Human Trx-1 Gene in Mice Hind Limb Ischemia Model: Preclinical Study for Treatment of Peripheral Artery Disease

Circulation ◽

10.1161/circ.130.suppl_2.17955 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Mahesh Thirunavukkarasu ◽

Inam A Shaikh ◽

Vaithinathan Selvaraju ◽

J.Alexandar Palesty ◽

Nilanjana Maulik

Keyword(s):

Gene Therapy ◽

Peripheral Artery Disease ◽

Femoral Artery ◽

Hind Limb ◽

Blood Perfusion ◽

Limb Ischemia ◽

Heme Oxygenase 1 ◽

Peripheral Artery ◽

Artery Ligation ◽

Artery Disease

Introduction: Peripheral artery disease affects 12-20% Americans over the age of 60. Thioredoxin-1 (Trx-1) is a class of small redox proteins. We have demonstrated earlier that Trx-1 reduces oxidative stress resulting in less inflammation and increased angiogenesis in cardiac muscle via heme oxygenase-1 (HO-1) and VEGF after myocardial infarction. In the current study, we evaluate the effect of Trx-1 on post-ischemic hindlimb recovery. Methods: Peripheral artery disease was mimicked using a hindlimb ischemia (HLI) model. Wild type (WT) and Trx-1 transgenic (Trx-1Tg/+) mice (8-12 weeks old) were subjected to femoral artery ligation. Following surgery, mice were observed for 5 weeks. Serial laser doppler images were obtained, and perfusion ratios between the ischemic and non-ischemic limbs were calculated at set time intervals. The perfusion ratios were compared between WT and Trx-1Tg/+ groups. Immunohistochemical analysis of the skeletal muscle was performed to quantify the extent of fibrosis, capillary and arteriolar density 35 days after surgery. In addition, another set of experiments was designed with Ad.Trx-1 gene therapy after femoral artery ligation to study the molecular mechanism of neovascularization with Trx-1. Results: The recovery of hind limb perfusion was significantly increased in Trx-1Tg/+ mice at day 7 (0.19 ± 0.03 vs. 0.36 ± 0.07 (n=12-9), day-21 (0.37 ± 0.05 vs. 0.62 ± 0.03 (n=12-9), and day 28 (0.40 ± 0.04 vs. 0.79 ± 0.04 (n=10-9); p<0.05). Capillary density [1265 ± 87.8 vs. 762.4 ± 86.6 counts/mm2 ; (n=5); p<0.05] and arteriolar density [36.2 ± 2.96 vs. 22± 1.33 counts/mm2 ; (n=5); p<0.05] staining showed significant increase in Trx-1Tg/+ mice as compared to WT mice. Picrosirrus Red and immunofluorescence staining showed decreased fibrosis [8.3 ± 0.46 vs. 22.2 ± 1.04 (n=5); p<0.0001] and increased HO-1 expression respectively in Trx-1Tg/+ mice group as compared to WT mice, respectively. Trx-1 gene therapy study also revealed by Western blot analysis, increased Trx-1 (4.2 fold) and HO-1 (8.2 fold) expression in Ad.Trx-1-HLI as compared to Ad.LacZ-HLI. Conclusions: Our results suggest that Trx-1 is a potential therapeutic agent to increase blood perfusion and angiogenesis for the treatment of critical limb ischemia patients.

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Abstract 321: Prasugrel, a Platelet P2y 12 Receptor Antagonist, Improves Abnormal Gait in a Novel Murine Model of Thrombotic Hind Limb Ischaemia

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.321 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Kousaku Ohno ◽

Atsuyuki Tomizawa ◽

Makoto Mizuno ◽

Joseph A Jakubowski ◽

Atsuhiro Sugidachi

Keyword(s):

Blood Flow ◽

Femoral Artery ◽

Murine Model ◽

Hind Limb ◽

Limb Ischaemia ◽

Phase Duration ◽

Flow Reduction ◽

Blood Flow Reduction ◽

Gait Abnormalities ◽

Deficient Mice

Background: The efficacy of P2Y 12 inhibition for the prevention of cardiovascular events in patients with peripheral arterial disease (PAD) has been established. However the therapeutic effects on ischaemic limb manifestations are less clear. Accordingly, we developed a novel murine model of thrombotic hind limb ischaemia to reflect that found in patients with PAD exhibiting ischaemic limb symptoms. We further investigated the effects of P2Y 12 inhibition by prasugrel in this model. Methods and Results: Thrombus formation induced by application of ferric chloride to the femoral artery resulted in a significant reduction in blood flow in the injured limb. In gait analysis of the ischaemic limb using the CatWalk system, maximum contact area and stance phase duration were reduced and swing phase duration increased in this model. Blood flow reduction and gait abnormalities gradually recovered over 21 days to levels present before arterial injury. Compared to wild-type (WT) mice, significant increases in blood flow and improvement in gait were observed in P2Y 12 -deficient mice. In addition, daily oral administration of prasugrel (3 mg/kg/day) to WT mice resulted in significant inhibition of blood flow reduction and gait abnormalities to levels found in P2Y 12 deficient mice. Conclusions: In conclusion, acute femoral artery thrombosis resulted in hind limb ischaemia and moderate gait abnormalities in mice. In addition, the present study suggests a therapeutic role for P2Y 12 antagonism in reducing the manifestations of limb ischaemia in PAD.

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