scholarly journals SAT-632 Involvement of NF-κB-p65 in BAG3 Regulation After Stress Stimuli

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Karthik Dhanabalan ◽  
Madhu V Singh ◽  
Thomas Wong ◽  
Ayotunde O Dokun
Keyword(s):  
Cell Survival ◽  
Total Protein ◽  
Hind Limb ◽  
Cell Damage ◽  
Limb Ischemia ◽  
Arterial Disease ◽  
Hind Limb Ischemia ◽  
Human Skeletal Muscle Cells ◽  
Control Plasmid ◽  
Peripheral Arterial

Abstract We previously identified the limb salvage QTL1 (LSQ-1) on mouse chromosome 7 as a locus that offered protection against ischemic injury following induction of hind limb ischemia (HLI) a model of experimental peripheral arterial disease (PAD) in mice. To better understand the role of the LSQ-1 locus in post ischemic adaptation we characterized several genes within this locus and identified a number of genes that were important in tissue adaptation to ischemia, including BCL2-associated athanogene 3 (BAG3). BAG3 is an anti-apoptotic protein that plays an important role in cell survival through the regulation of autophagy. BAG3 expression is induced in the gastrocnemius muscles of mice after hind limb ischemia but how ischemia regulates BAG3 expression is poorly understood. Additionally, the activation of NF-κB transcription factor is essential for cell survival after stress stimuli. We hypothesized that BAG3 upregulation following stress stimuli is regulated by NF-κB. We determined whether NF-κB is involved in BAG3-mediated survival of primary human skeletal muscle cells (HSMC) during ischemia and diabetic conditions. Within 6 hours of treatment, ischemia induced BAG3 mRNA (no ischemia vs ischemia: 1.0 ±0.09 vs 1.41±0.02; p<0.01) and protein expression (BAG3/total protein, no ischemia vs ischemia 1.0±0.01 vs 1.38±0.06; p<0.01). Knockdown of BAG3 expression by shRNA induced early cell damage in HMSC under ischemic conditions as measured by HMGB1 expression (HMGB1/total protein; control plasmid vs shRNA, 1.0±0.09 vs 1.71±0.04; p<0.01). Knockdown of p65 subunit of the NF-κB by shRNA significantly diminished BAG3 mRNA expression after ischemia (control plasmid vs shRNA: 2.11±0.18 vs 1.48±0.05; p<0.05). Moreover, treatment of HSMC with 750 uM palmitic acid (PA) and 100nM insulin for 3 days to mimic diabetic conditions significantly increased the expression of BAG3 mRNA (control vs PAL+Ins, 1.0±0.14 vs 2.27±0.08; p<0.01) and protein (BAG3/total protein, control vs PAL+Ins, 1.0±0.03 vs 1.39±0.11, p<0.01). Knocking down p65 attenuated these increase in BAG3 mRNA (PAL+Ins vs shRNA+PAL+Ins, 2.27±0.08 vs 1.56±0.02 p<0.01) and protein (BAG3/total protein; PAL+Ins vs shRNA+PAL+Ins, 1.39±0.11 vs 0.99±0.1; p<0.05) Thus, 1) BAG3 expression is important in cell survival under ischemic conditions, and 2) NF-kB plays a key role in upregulating the expression of BAG3 under diabetic and ischemic conditions.

scholarly journals Variations in Surgical Procedures for Inducing Hind Limb Ischemia in Mice and the Impact of These Variations on Neovascularization Assessment

2019 ◽  
Vol 20 (15) ◽  
pp. 3704 ◽  
Author(s):  
Zeen Aref ◽  
Margreet R. de Vries ◽  
Paul H.A. Quax
Keyword(s):  
Vascular Remodeling ◽  
Hind Limb ◽  
Limb Ischemia ◽  
Arterial Disease ◽  
Preclinical Model ◽  
Comprehensive Overview ◽  
Hind Limb Ischemia ◽  
Surgical Methods ◽  
Peripheral Arterial ◽  
The Impact

Mouse hind limb ischemia is the most common used preclinical model for peripheral arterial disease and critical limb ischemia. This model is used to investigate the mechanisms of neovascularization and to develop new therapeutic agents. The literature shows many variations in the model, including the method of occlusion, the number of occlusions, and the position at which the occlusions are made to induce hind limb ischemia. Furthermore, predefined end points and the histopathological and radiological analysis vary. These differences hamper the correlation of results between different studies. In this review, variations in surgical methods of inducing hind limb ischemia in mice are described, and the consequences of these variations on perfusion restoration and vascular remodeling are discussed. This study aims at providing the reader with a comprehensive overview of the methods so far described, and proposing uniformity in research of hind limb ischemia in a mouse model.

Pre-Clinical Studies of Therapeutic Angiogenesis Induced by Human Hepatocyte Growth Factor (HGF) Gene in Rat and Rabbit Hind Limb Ischemia Models

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 706-706
Author(s):  
Yoshiaki Taniyama
Keyword(s):  
Gene Therapy ◽  
Peripheral Arterial Disease ◽  
Intramuscular Injection ◽  
Hind Limb ◽  
Limb Ischemia ◽  
Arterial Disease ◽  
Therapeutic Angiogenesis ◽  
Hindlimb Ischemia ◽  
Angiogenic Growth Factors ◽  
Peripheral Arterial

P72 A novel therapeutic strategy for ischemic diseases using angiogenic growth factors to expedite and/or augment collateral artery development has been proposed. In this study, we examined the feasibility of gene therapy using HGF to treat peripheral arterial disease rather than recombinant therapy. Initially, we examined the transfection of “naked” human HGF plasmid into a rat hindlimb ischemia model. Intramuscular injection of human HGF plasmid resulted in a significant increase in blood flow as assessed by laser Doppler imager. Importantly, at 5 weeks after transfection, the degree of angiogenesis induced by transfection of HGF plasmid was significantly greater than that caused by a single injection of recombinant HGF. As an approach to human gene therapy, we also employed a rabbit hindlimb ischemia model as a preclinical study. Naked HGF plasmid was intramuscularly injected in the ischemic hindlimb of rabbits, to evaluate its angiogenic activity. Intramuscular injection of HGF plasmid produced significant augmentation of collateral vessel development on day 30 in the ischemia model, as assessed by angiography (C100% vs H 245%, P<0.01). Overall, intramuscular injection of naked human HGF plasmid induced therapeutic angiogenesis in rat and rabbit ischemic hind limb models, as potential therapy for peripheral arterial disease.

scholarly journals FACS-purified myoblasts producing controlled VEGF levels induce safe and stable angiogenesis in chronic hind limb ischemia

2011 ◽  
Vol 16 (1) ◽  
pp. 107-117 ◽  
Author(s):  
Thomas Wolff ◽  
Edin Mujagic ◽  
Roberto Gianni-Barrera ◽  
Philipp Fueglistaler ◽  
Uta Helmrich ◽  
...  
Keyword(s):  
Hind Limb ◽  
Limb Ischemia ◽  
Hind Limb Ischemia

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

10.3791/62402 ◽  
2021 ◽  
Author(s):  
Kaixuan Yan ◽  
Jiaxing Zheng ◽  
Frank G. Zöllner ◽  
Kay Schwenke ◽  
Prama Pallavi ◽  
...  
Keyword(s):  
Hind Limb ◽  
Limb Ischemia ◽  
Hind Limb Ischemia ◽  
Surgical Model

Abstract 17597: Endogenous Del-1 is a Negative Regulator of Ischemia-induced Angiogenesis by Interacting With the Leukocytic LFA-1 Integrin

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Sebastian Cremer ◽  
Anne Klotzsche-von Ameln ◽  
Alessia Orlandi ◽  
Irina Korovina ◽  
Bettina Gercken ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Hind Limb ◽  
Hematopoietic Cells ◽  
Limb Ischemia ◽  
Inflammatory Cells ◽  
Capillary Density ◽  
Negative Regulator ◽  
Hind Limb Ischemia ◽  
Autonomous Pathway

Developmental endothelial locus-1 (Del-1) is an endothelial cell-derived secreted protein circulating in blood and associated with the cell surface and the extracellular matrix. As we previously demonstrated, Del-1 restricts leukocyte recruitment by inhibiting the β2-integrin, LFA-1. Leukocytes and progenitor cells (PC) may contribute to angiogenesis. The role of endogenous Del-1 in angiogenesis is elusive. We found, that physiological angiogenesis of the developing retina was not affected in the Del-1-/- mice compared to the wildtype (WT) mice. Surprisingly, Del-1-/- mice displayed a significantly increased angiogenic response compared to WT mice after induction of hind limb ischemia (144 ± 6 % increase of capillary density) and retinal ischemia (retinopathy of prematurity model) suggesting that endogenous Del-1 is an inhibitor of ischemia-induced neovascularization. Silencing of Del-1 with siRNA did not affect the angiogenic sprouting of endothelial cell (EC) spheroids, indicating that Del-1 blocks angiogenesis in a non-endothelial cell autonomous pathway. Soluble Del-1 blocked the adhesion of inflammatory cells on EC monolayers. In line with these results, ischemic muscles and ischemic retinae from Del-1-/- mice displayed an enhanced infiltration with inflammatory cells compared to the WT mice. Since Del-1 blocks inflammatory cell homing by inhibiting the leukocytic LFA-1-integrin, we addressed the role of the Del-1/LFA-1-integrin interaction on the inhibitory function of endogenous Del-1 on angiogenesis. Indeed, Del-1/LFA-1-double deficiency reversed the pro-angiogenic phenotype of the Del-1-/- mice to the level of WT mice in the model of hind limb ischemia. Thus, the inhibitory role of Del-1 on neovascularization is mediated by the interaction of Del-1 with the LFA-1-integrin. Moreover, Del-1-deficiency led to an increased homing of intravenously injected murine fluorescence-labeled WT Lin- BM PC in ischemic muscles in comparison to WT mice after the induction of hind limb ischemia. Taken together, Del-1 acts as a negative regulator of ischemia-induced angiogenesis by interacting with the LFA-1-integrin expressed in hematopoietic cells, thereby inhibiting the homing of hematopoietic cells to ischemic tissues.

scholarly journals Apolipoprotein E–/– mice have delayed skeletal muscle healing after hind limb ischemia–reperfusion

2008 ◽  
Vol 48 (3) ◽  
pp. 701-708 ◽  
Author(s):  
Jeanwan Kang ◽  
Hassan Albadawi ◽  
Virendra I. Patel ◽  
Thomas A. Abbruzzese ◽  
Jin-Hyung Yoo ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Apolipoprotein E ◽  
Hind Limb ◽  
Ischemia Reperfusion ◽  
Limb Ischemia ◽  
Hind Limb Ischemia
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