| Summary: | 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 (<i>VEGF165</i>) + hepatocyte growth factor (<i>HGF</i>). Our recent works have demonstrated that a bicistronic pDNA that carries both human <i>HGF</i> and <i>VEGF165</i> coding sequences has a potential for clinical application in peripheral artery disease (PAD). The present study aimed to test <i>HGF/VEGF</i> 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 <i>m.tibialis anterior</i>, 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. <i>HGF/VEGF</i> plasmid injection aggravated glucose intolerance in HFD conditions. Blood flow recovery was not changed by <i>HGF/VEGF</i> 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, <i>HGF/VEGF</i> plasmid enhanced neuritogenesis and enlarged NF200+ area on muscle sections. In HFD conditions, <i>HGF/VEGF</i> 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. <i>HGF/VEGF</i> 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 <i>HGF/VEGF</i> combined plasmid a very promising tool for PAD therapy in impaired glucose tolerance conditions.
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