epidermal-growth-factor and 1-2-dioleoyloxy-3-(trimethylammonium)propane

We aim to investigate the feasibility and efficacy of cholesterol (Chol)+DOTAP liposome (CD liposome) based human vascular endothelial growth factor-165 (hVEGF(165)) gene transfer into human skeletal myoblasts (hSkM) for cardiac repair. The feasibility and efficacy of CD liposome for gene transfer with hSkM was characterized using plasmid carrying enhanced green fluorescent protein (pEGFP). Based on the optimized transfection procedure, hSkM were transfected with CD lipoplexes carrying plasmid-hVEGF(165) (CD-phVEGF(165)). The genetically modified hSkM were transplanted into rat heart model of acute myocardial infarction. Flow cytometry revealed that about 7.99% hSkM could be transfected with pEGFP. Based on the optimized transfection condition, transfected hSkM expressed hVEGF(165) up to day-18 (1.7+/-0.1ng/ml) with peak at day-2 (13.1+/-0.52ng/ml) with >85% cell viability. Animal studies revealed that reduced apoptosis, improved angiogenesis with blood flow in group-3 animal's heart were achieved as compared to group-1 and 2. Ejection fraction was best recovered in group-3 animals. The study demonstrates that though gene transfection efficiency using CD liposome mediated hVEGF(165) gene transfer with hSkM was low; hVEGF(165) gene expression efficiency was sufficient to induce neovascularization, improve blood flow and injured heart function.

Topics: Animals; Apoptosis; Cells, Cultured; Cholesterol; Epidermal Growth Factor; Fatty Acids, Monounsaturated; Heart Transplantation; Liposomes; Microscopy, Electron, Scanning; Myoblasts, Skeletal; Neovascularization, Physiologic; Particle Size; Quaternary Ammonium Compounds; Rats; Regional Blood Flow; Transfection; Vascular Endothelial Growth Factor A

Cationic liposomes have provided many advantages over viral vector formulations; however, the problem of inefficient gene expression remains. This is due in part to the nuclear membrane, which limits DNA entry into the nucleus. Cytoplasmic expression systems using T7 RNA polymerase have been developed to express genes in the cytoplasm and avoid the need for nuclear import of DNA. Although these systems show improved transgene expression, little is known about how they function in transfected cells. Direct comparisons between a cytoplasmic and nuclear expression system were carried out with a 293 cell line stably expressing T7 RNA polymerase. A formulation for optimal reporter gene expression was developed and used in conjunction with a variety of subcellular trafficking inhibitors to study the process of DNA endocytosis. Transfected cells were also studied at different stages of the cell cycle to determine the dependence of each system on mitosis. These results showed that cytoplasmic and nuclear expression systems utilize similar endocytosis pathways to the point of endosomal release. Once DNA is released into the cytoplasm, the cytoplasmic expression system shows immediate expression that is proportional to the amount of DNA released. In contrast, DNA targeted for nuclear expression requires additional time for nuclear entry. The level of nuclear expression is also restricted by the limited amount of DNA that is imported into the nucleus. Finally, mitosis is required for effective nuclear expression but not for cytoplasmic expression. Therefore, the cytoplasmic expression system has considerable advantages over traditional nuclear expression systems and may be an effective method for transfecting nondividing cells. Efficient expression of genes delivered by nonviral vectors is hindered owing to poor nuclear transport of plasmid DNA. A potential solution to this problem would be to use a cytoplasmic expression system. Previous studies have shown that this method produces enhanced gene expression when compared with traditional nuclear expression systems; however, the actual mechanisms by which the cytoplasmic expression system works remains unknown. This article focuses on a direct comparison between cytoplasmic and nuclear expression in terms of optimal DNA delivery formulations, intracellular trafficking of DNA, and cell cycle dependence. These results indicate that the cytoplasmic expression system has two primary advantages over nuclear expression in

Topics: Anti-Bacterial Agents; Cation Exchange Resins; Cell Line; Cell Nucleus; Chloroquine; Cytochalasin B; Cytoplasm; DNA; Endocytosis; Enzyme Inhibitors; Epidermal Growth Factor; Fatty Acids, Monounsaturated; Gene Expression Regulation; Humans; Kidney; Lipids; Liposomes; Luciferases; Macrolides; Mitosis; Nocodazole; Promoter Regions, Genetic; Protamines; Quaternary Ammonium Compounds; Subcellular Fractions