Search

In the United States, over 8.5 million people suffer from peripheral arterial disease (PAD). Previously we reported that Pellino-1(Peli1) gene therapy reduces ischemic damage in the myocardium and skin flaps in Flk-1 [Fetal Liver kinase receptor-1 (Flk-1)/ Vascular endothelial growth factor receptor-2/VEGFR2] heterozygous (Flk-1+/‐−) mice. The present study compares the angiogenic response and perfusion efficiency following hind limb ischemia (HLI) in, Flk-1+/− and, MAPKAPKINASE2 (MK2−/−) knockout (KO) mice to their control wild type (WT). We also demonstrated the use of Peli1 gene therapy to improve loss of function following HLI. Femoral artery ligation (HLI) was performed in both Flk-1+/− and MK2−/− mice along with their corresponding WT. Another set of Flk-1+/− and MK2−/− were injected with either Adeno-LacZ (Ad.LacZ) or Adeno-Peli1 (Ad.Peli1) after HLI. Hind limb perfusion was assessed by laser doppler imaging at specific time points. A standardized scoring scale is used to quantify the extent of ischemia. Histology analysis performed includes capillary density, fibrosis, pro-angiogenic and anti-apoptotic proteins. Flk-1+/− and MK2−/− had a slower recovery of perfusion efficiency in the ischemic limbs than controls. Both Flk-1+/− and MK2−/− KO mice showed decreased capillary density and capillary myocyte ratios with increased fibrosis than their corresponding wild types. Ad.Peli1 injected ischemic Flk-1+/− limb showed improved perfusion, increased capillary density, and pro-angiogenic molecules with reduced fibrosis compared to Ad.LacZ group. No significant improvement in perfusion was observed in MK2−/− ischemic limb after Ad. Peli1 injection. Deletion of Flk-1 and MK2 impairs neovascularization and perfusion following HLI. Treatment with Ad. Peli1 results in increased angiogenesis and improved perfusion in Flk-1+/− mice but fails to rectify perfusion in MK2 KO mice. Overall, Peli1 gene therapy is a promising candidate for the treatment of PAD. • Therapeutic vascularization in the hind-limb ischemia model with Pellino-1 gene therapy, an E3 ligase is promising. • Pellino-1 repairs and rectifies downstream abnormal VEGF signaling in ischemic tissues. • Pellino-1 is more effective and appropriate in inducing angiogenic response than VEGF-A. • VEGF-A increases vascular permeability and thrombosis, whereas Pellino-1 bypasses these effects. • The role of E3 Ligases in therapeutic angiogenesis is new. [ABSTRACT FROM AUTHOR]

Abstract: We have used time-resolved phosphorescence anisotropy (TPA) of actin to evaluate domains of dystrophin and utrophin, with implications for gene therapy in muscular dystrophy. Dystrophin and its homolog utrophin bind to cytoskeletal actin to form mechanical linkages that prevent muscular damage. Because these proteins are too large for most gene therapy vectors, much effort is currently devoted to smaller constructs. We previously used TPA to show that both dystrophin and utrophin have a paradoxical effect on actin rotational dynamics—restricting amplitude while increasing rate, thus increasing resilience, with utrophin more effective than dystrophin. Here, we have evaluated individual domains of these proteins. We found that a “mini-dystrophin,” lacking one of the two actin-binding domains, is less effective than dystrophin in regulating actin dynamics, correlating with its moderate effectiveness in rescuing the dystrophic phenotype in mice. In contrast, we found that a “micro-utrophin,” with more extensive internal deletions, is as effective as full-length dystrophin in the regulation of actin dynamics. Each of utrophin''s actin-binding domains promotes resilience in actin, while dystrophin constructs require the presence of both actin-binding domains and the C-terminal domain for full function. This work supports the use of a utrophin template for gene or protein therapy designs. Resilience of the actin–protein complex, measured by TPA, correlates remarkably well with previous reports of functional rescue by dystrophin and utrophin constructs in mdx mice. We propose the use of TPA as an in vitro method to aid in the design and testing of emerging gene therapy constructs. [Copyright &y& Elsevier]