The Efficacy of HGF/VEGF Gene Therapy for Limb Ischemia in Mice with Impaired Glucose Tolerance: Shift from Angiogenesis to Axonal Growth and Oxidative Potential in Skeletal Muscle

Abstract

Background: Combined non-viral gene therapy (GT) of ischemia and cardiovascular disease is a promising tool for potential clinical translation. In previous studies our group has developed combined gene therapy by vascular endothelial growth factor 165 (VEGF165) + hepatocyte growth factor (HGF). Our recent works have demonstrated that a bicistronic pDNA that carries both human HGF and VEGF165 coding sequences has a potential for clinical application in peripheral artery disease (PAD). The present study aimed to test HGF/VEGF combined plasmid efficacy in ischemic skeletal muscle comorbid with predominant complications of PAD-impaired glucose tolerance and type 2 diabetes mellitus (T2DM).

Methods: Male C57BL mice were housed on low-fat (LFD) or high-fat diet (HFD) for 10 weeks and metabolic parameters including FBG level, ITT, and GTT were evaluated. Hindlimb ischemia induction and plasmid administration were performed at 10 weeks with 3 weeks for post-surgical follow-up. Limb blood flow was assessed by laser Doppler scanning at 7, 14, and 21 days after ischemia induction. The necrotic area of m.tibialis anterior, macrophage infiltration, angio- and neuritogenesis were evaluated in tissue sections. The mitochondrial status of skeletal muscle (total mitochondria content, ETC proteins content) was assessed by Western blotting of muscle lysates.

Results: At 10 weeks, the HFD group demonstrated impaired glucose tolerance in comparison with the LFD group. HGF/VEGF plasmid injection aggravated glucose intolerance in HFD conditions. Blood flow recovery was not changed by HGF/VEGF plasmid injection either in LFD or HFD conditions. GT in LFD, but not in HFD conditions, enlarged the necrotic area and CD68+ cells infiltration. However, HGF/VEGF plasmid enhanced neuritogenesis and enlarged NF200+ area on muscle sections. In HFD conditions, HGF/VEGF plasmid injection significantly increased mitochondria content and ETC proteins content.

Conclusions: The current study demonstrated a significant role of dietary conditions in pre-clinical testing of non-viral GT drugs. HGF/VEGF combined plasmid demonstrated a novel aspect of potential participation in ischemic skeletal muscle regeneration, through regulation of innervation and bioenergetics of muscle. The obtained results made HGF/VEGF combined plasmid a very promising tool for PAD therapy in impaired glucose tolerance conditions.

Keywords: HGF; VEGF; gene therapy; high-fat diet; limb ischemia; obesity; plasmid.

Figure 1

HGF/VEGF plasmid injection to ischemic muscle impairs glucose tolerance, but not other metabolic parameters, at 3 weeks after surgery (10 + 3 weeks of experiment). (A) body weight dynamics during post-surgical period; (B) FBG level dynamics during post-surgical period; (C) GTT curves on 10 + 3 weeks of experiment; (D) ITT curves on 10 + 3 weeks of experiment. Abbreviations: LFD, low fat diet; HFD, high fat diet; FBG, fasting blood glucose; GTT, glucose tolerance test; ITT, insulin tolerance test. Data is presented as mean ± SEM; Kruskal–Wallis test with post-hoc Dunn’s test; * p < 0.05.

Figure 2

HGF/VEGF plasmid injection impairs glucose tolerance compared to pre-surgery state. (A) GTT curves for LFD group on 10 and 10 + 3 weeks of experiment; (B) GTT curves for LFD HGF/VEGF group on 10 and 10 + 3 weeks of experiment; (C) GTT curves for HFD group on 10 and 10 + 3 weeks of experiment; (D) GTT curves for HFD HGF/VEGF group on 10 and 10 + 3 weeks of experiment. Abbreviations: LFD, low fat diet; HFD, high fat diet; FBG, fasting blood glucose; GTT, glucose tolerance test. Data is presented as mean ± SEM, Mann–Whitney U-test; * p < 0.05.

Figure 3

HGF/VEGF plasmid injection induces a transient delay of blood flow recovery in HFD group. (10 + 3 weeks of experiment). (A) representative Laser Doppler scanning images subcutaneous blood flow in experimental groups; (B) dynamics of blood flow recovery after HGF/VEGF therapy under LFD or HFD dietary intervention. Data is presented as mean ± SEM; Kruskal–Wallis test with post hoc Dunn’s test; * p < 0.05.

Figure 4

HGF/VEGF plasmid injection enhances ischemic-induced necrosis and macrophage infiltration under LFD conditions, but does not affect post-ischemic recovery process under HFD conditions. (10 + 3 weeks of experiment). (A) representative images of hematoxylin/eosin stained m.tibialis anterior sections, dotted line has marked necrotic area; (B) statistical analysis of necrotic area morphometry data; (C) representative images panel of CD68+ cells infiltration in m.tibialis anterior, scale bar 200 μm, dotted line has marked muscle area; (D) statistical analysis of CD68+ cells infiltration data. Data is presented as mean ± SEM; Kruskal–Wallis test with post hoc Dunn’s test; * p < 0.05.

Figure 5

HGF/VEGF plasmid injection demonstrated limited impact on angiogenesis and activated both arteriogenesis and neuritogenesis under LFD conditions while under HFD was limited to neuritogenesis. (10 + 3 weeks of experiment). (A) representative images panel of NF200 and CD31 staining in m.tibialis anterior, scale bar 50 μm; (B–D) graphical presentation of blood vessel density analysis with average group values per field of view: CD31+ vessels without lumen (B), CD31+ vessels with lumen < 30 μm (C), CD31+ vessels with lumen >30 μm (D); (E) graphical presentation of neurites density analysis with average group values per field of view. Data is presented as mean ± SEM; Kruskal–Wallis test with post hoc Dunn’s test; * p < 0.05.

Figure 6

HGF/VEGF combined plasmid suppresses mitochondrial biogenesis and electron transport chain (ETC) input components expression in LFD conditions, but enhances these parameters in HFD conditions. (10 + 3 weeks of experiment). (A) representative Western blots panel; (B) mitochondria content in ischemic muscle; (C) NADPH-dehydrogenase (Complex I) expression level in ischemic muscle, (D) succinate dehydrogenase (Complex II) expression level in ischemic muscle; (E) cytochrome C oxidase (Complex IV) expression level in ischemic muscle; (F) ATP-synthase (Complex V) expression level in ischemic muscle. Data is presented as mean ± SEM, the Kruskal–Wallis test with post hoc Dunn’s test; * p < 0.05.