Combined Transfer of Human VEGF165 and HGF Genes Renders Potent Angiogenic Effect in Ischemic Skeletal Muscle

Abstract

Increased interest in development of combined gene therapy emerges from results of recent clinical trials that indicate good safety yet unexpected low efficacy of “single-gene” administration. Multiple studies showed that vascular endothelial growth factor 165 aminoacid form (VEGF165) and hepatocyte growth factor (HGF) can be used for induction of angiogenesis in ischemic myocardium and skeletal muscle. Gene transfer system composed of a novel cytomegalovirus-based (CMV) plasmid vector and codon-optimized human VEGF165 and HGF genes combined with intramuscular low-voltage electroporation was developed and tested in vitro and in vivo . Studies in HEK293T cell culture, murine skeletal muscle explants and ELISA of tissue homogenates showed efficacy of constructed plasmids. Functional activity of angiogenic proteins secreted by HEK293T after transfection by induction of tube formation in human umbilical vein endothelial cell (HUVEC) culture. HUVEC cells were used for in vitro experiments to assay the putative signaling pathways to be responsible for combined administration effect one of which could be the ERK1/2 pathway. In vivo tests of VEGF165 and HGF genes co-transfer were conceived in mouse model of hind limb ischemia. Intramuscular administration of plasmid encoding either VEGF165 or HGF gene resulted in increased perfusion compared to empty vector administration. Mice injected with a mixture of two plasmids (VEGF165+HGF) showed significant increase in perfusion compared to single plasmid injection. These findings were supported by increased CD31+ capillary and SMA+ vessel density in animals that received combined VEGF165 and HGF gene therapy compared to single gene therapy. Results of the study suggest that co-transfer of VEGF and HGF genes renders a robust angiogenic effect in ischemic skeletal muscle and may present interest as a potential therapeutic combination for treatment of ischemic disorders.

Non-viral transfer of BDNF and uPA stimulates peripheral nerve regeneration

“…4 days after treatment mice were sacrificed by anesthetic overdose, tibial anterior muscles were extracted and homogenized in 450 ml of buffer (0.5 M NaCl, 20 mM Tris and 1 mM EDTA) [10]; the homogenates were studied for the presence of muPA using muPA ELISA kit (''Cell Sciences'', cat #CSI19903A) according to the manufacturer instructions. Injection of pVax1 at the same dose was used as a control.…”

Therapeutic Angiogenesis: Foundations and Practical Application

“…This puts the basis for combined gene therapy to treat ischemia with higher eicacy and it has been supported by experimental indings using VEGF165 combined with another pro-angiogenic growth factor: bFGF [71], PDGF [72], angiopoietin-1 [73], or Stromal cell-derived factor-1α (SDF-1α) [74]. Our previous experience in mouse hind limb ischemia model showed that combination of VEGF165 and uPA [75] or HGF [76] induced angiogenic response more efectively than each factor alone or allowed to reduce pDNA dose for combined delivery [75]. A crucial transcription factor in angiogenesis, HIF-1α, was also used for combined gene therapy with VEGF165 showing good results in animal model [77] as well as bFGF + heme oxygenase-1 (HO-1) [78].…”

Formation and development of therapeutic angiogenesis in cardiovascular surgery in Russia

“…М.В. Ломоносова, на моделях ишемии задних конечно-стей и инфаркта миокарда у мышей выявлены аддитив-ный ангио-и артериогенный эффекты VEGF165 и HGF, способствовавшие максимальному восстановлению пер-фузии пораженных тканей (до 70%) [44]. Помимо иссле-дований вышеуказанных конструкций авторы работают над созданием генномодицированных клеток, которые, возможно, будут способствовать индукции ангиогенеза и реперфузии ишемизированных тканей [61].…”