Gene therapy with Pellino-1 improves perfusion and decreases tissue loss in Flk-1 heterozygous mice but fails in MAPKAP Kinase-2 knockout murine hind limb ischemia model

Introduction: Peripheral vascular disease (PVD) is a major cause of morbidity in the US. Vascular endothelial growth factor (VEGF) induces angiogenesis through Fetal-Liver Kinase-1 (Flk-1 or VEGFR-2), an endothelial tyrosine kinase receptor which activates a downstream signaling cascade involving MAPKAPKinase2 (MK2). We have demonstrated poorer recovery in hind limb ischemia models using Flk-1 +/- and MK2 -/- knockout mice compared to wild type mice. We also demonstrated overespression of Pellino1 (Peli1), an E3 Ligase, can attenuate ischemic damage and increase neovascularization and survivability of ischemic myocardium and skin flaps. Therefore, in this study we aim to show if Peli1 gene therapy can help rescue function in Flk-1 +/- and MK2 -/- after femoral artery ligation. Methods: 8-12 week-old Flk-1 +/- and MK2 -/- mice were subjected to femoral artery ligation of the right hind limb and treated with either adenovirus carrying Peli1 (Ad.Peli1) or LacZ (Ad.LacZ), for a total of 4 groups: Flk-1 +/- Ad.LacZ, Flk-1 +/- Ad.Peli1, MK2 -/- Ad.LacZ and MK2 -/- Ad.Peli1. Perfusion was assessed with laser Doppler imaging preop and on days 0, 3, 7, 14, 21 and 28. Extent of limb ischemia was also quantified using a standardized scoring scale (0 = no ischemia, to 8 = full limb amputation). Results: Perfusion was significantly higher in the Flk-1 +/- Ad.Peli1 group compared to Flk-1 +/- Ad.LacZ on days 7 (0.19±0.03 vs 0.08±0.02), 14 (0.41±0.07 vs 0.09±0.03), 21 (0.33±0.05 vs 0.14±0.05) and 28 (0.31±0.05 vs 0.15±0.02) (n=9-15, p<0.05). The level of ischemia (ischemic score) was significantly lower in the Flk-1 +/- Ad.Peli1 group when compared to Flk-1 +/- Ad.LacZ on days 3 (1.89±0.56 vs 4.88±0.85), 7 (2.11±0.54 vs 5.69±0.92), 14 (2.18±0.52 vs 6.31±1.02), 21 (1.83±0.58 vs 6.50±1.05) and 28 (1.67±0.62 vs 6.50±1.05) (n=8-9, p<0.05). There was no difference in perfusion or ischemic score between the MK2 -/- Ad.Peli1 and MK2 -/- Ad.LacZ groups. Values are mean +/- SEM. Conclusions: Treatment with Ad.Peli1 resulted in improved perfusion and decreased tissue ischemia in Flk-1 +/- mice. These results were not paralleled in MK2 -/- mice suggesting that Peli1 acts upstream to MK2 but downstream of VEGF/Flk-1. Overall, Peli1 gene therapy is a promising candidate in the management of PVD.

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Heat shock protein A12B gene therapy improves perfusion, promotes neovascularization, and decreases fibrosis in a murine model of hind limb ischemia

Surgery ◽

10.1016/j.surg.2021.05.006 ◽

2021 ◽

Author(s):

Mahesh Thirunavukkarasu ◽

Muhammad Tipu Rishi ◽

Seetur R. Pradeep ◽

Santosh Swaminathan ◽

Diego Accorsi ◽

...

Keyword(s):

Gene Therapy ◽

Heat Shock ◽

Heat Shock Protein ◽

Murine Model ◽

Hind Limb ◽

Limb Ischemia ◽

Hind Limb Ischemia

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Faculty Opinions recommendation of Soluble CD146 displays angiogenic properties and promotes neovascularization in experimental hind-limb ischemia.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.3963958.3722058 ◽

2010 ◽

Author(s):

Lindsey A Miles ◽

Shahrzad Lighvani

Keyword(s):

Hind Limb ◽

Limb Ischemia ◽

Hind Limb Ischemia ◽

Soluble Cd146

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

Journal of Cellular and Molecular Medicine ◽

10.1111/j.1582-4934.2011.01308.x ◽

2011 ◽

Vol 16 (1) ◽

pp. 107-117 ◽

Cited By ~ 16

Author(s):

Thomas Wolff ◽

Edin Mujagic ◽

Roberto Gianni-Barrera ◽

Philipp Fueglistaler ◽

Uta Helmrich ◽

...

Keyword(s):

Hind Limb ◽

Limb Ischemia ◽

Hind Limb Ischemia

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WITHDRAWN: Melatonin attenuates lung injury in a hind limb ischemia–reperfusion rat model

Revista Portuguesa de Pneumologia ◽

10.1016/j.rppneu.2014.01.007 ◽

2014 ◽

Author(s):

Hamed Takhtfooladi ◽

Mohammad Takhtfooladi ◽

Fariborz Moayer ◽

Sayed Mobarakeh

Keyword(s):

Lung Injury ◽

Rat Model ◽

Hind Limb ◽

Ischemia Reperfusion ◽

Limb Ischemia ◽

Hind Limb Ischemia

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

Journal of Visualized Experiments ◽

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

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

Circulation ◽

10.1161/circ.130.suppl_2.17597 ◽

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.

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