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.
Tissue regeneration requires coordinated "teamwork" of growth factors, proteases, progenitor and immune cells producing inflammatory cytokines. Mesenchymal stem cells (MSC) might play a pivotal role by substituting cells or by secretion of growth factors or cytokines, and attraction of progenitor and inflammatory cells, which participate in initial stages of tissue repair. Due to obvious impact of inflammation on regeneration it seems promising to explore whether inflammatory factors could influence proangiogenic abilities of MSC. In this study we investigated effects of TNF-α on activity of adipose-derived stem cells (ADSC). We found that treatment with TNF-α enhances ADSC proliferation, F-actin microfilament assembly, increases cell motility and migration through extracellular matrix. Exposure of ADSC to TNF-α led to increased mRNA expression of proangiogenic factors (FGF-2, VEGF, IL-8, and MCP-1), inflammatory cytokines (IL-1β, IL-6), proteases (MMPs, uPA) and adhesion molecule ICAM-1. At the protein level, VEGF, IL-8, MCP-1, and ICAM-1 production was also up-regulated. Pre-incubation of ADSC with TNF-α-enhanced adhesion of monocytes to ADSC but suppressed adherence of ADSC to endothelial cells (HUVEC). Stimulation with TNF-α triggers ROS generation and activates a number of key intracellular signaling mediators known to positively regulate angiogenesis (Akt, small GTPase Rac1, ERK1/2, and p38 MAP-kinases). Pre-treatment with TNF-α-enhanced ADSC ability to promote growth of microvessels in a fibrin gel assay and accelerate blood flow recovery, which was accompanied by increased arteriole density and reduction of necrosis in mouse hind limb ischemia model. These findings indicate that TNF-α plays a role in activation of ADSC angiogenic and regenerative potential.
The adult heart contains small populations of multipotent cardiac progenitor cells (CPC) that present a convenient and efficient resource for treatment of myocardial infarction. Several clinical studies of direct CPC delivery by injection have already been performed but showed low engraftment rate that limited beneficial effects of procedure. «Cell sheet» technology has been developed to facilitate longer retention of grafted cells and show new directions for cell-based therapy using this strategy. In this study we hypothesized that СPC-based cell sheet transplantation could improve regeneration after myocardial infarction. We demonstrated that c-kit+ CPC were able to form cell sheets on temperature-responsive surfaces. Cell sheet represented a well-organized structure, in which CPC survived, retained ability to proliferate, expressed progenitor cell marker Gata-4 formed connexin-43+ gap junctions, and were surrounded by significant amount of extracellular matrix proteins. Transplantation of cell sheets after myocardial infarction resulted in CPC engraftment as well as their proliferation, migration, and differentiation; cell sheets also stimulated neovascularization and cardiomyocyte proliferation in underlining myocardium and ameliorated left ventricular remodeling. Obtained data strongly supported potential use of CPC sheet transplantation for repair of damaged heart.
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