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      Liposome-Mediated High-Efficiency Transfection of Human Endothelial Cells

      a,b , b

      Journal of Vascular Research

      S. Karger AG

      Gene transfer, Cell culture, Vasculature

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          Liposome-mediated transfection of endothelial cells provides a valuable experimental technique to study cellular gene expression and may also be adapted for gene therapy studies. However, the widely recognized disadvantage of liposome-mediated transfection is low efficiency. Therefore, studies were performed to optimize transfection techniques in human endothelial cells. The majority of the experiments were performed with primary cultures of human umbilical vein endothelial cells (HUVEC). In addition, selected experiments were performed using human brain microvascular endothelial cells and human dermal microvascular endothelial cells. To study transfection rates, HUVEC were transfected with the pGL3 vector, containing the luciferase reporter gene, complexed with several currently available liposomes, such as different Perfect Lipid (pFx) mixtures, DMRIE-C, or lipofectin. The optimal transfection rate was achieved in HUVEC transfected for 1.5 h with 5 µg/ml of DNA plasmid in the presence of 36 µg/ml of pFx-7. In addition, transfection with the VR-3301 vector encoding for human placental alkaline phosphatase revealed that, under the described conditions, transfection efficiency in HUVEC was approximately 32%. Transfections mediated by other liposomes were less efficient. The usefulness of the optimized transfection technique was confirmed in HUVEC transfected with NF-ĸB or AP-1-responsive constructs and stimulated with TNF or LPS. We conclude that among several currently available liposomes, pFx-7 appears to be the most suitable for transfections of cultured human endothelial cells.

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          Most cited references 6

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          Transfection of human endothelial cells.

           F C Tanner,  D Carr,  G Nabel (1997)
          The introduction of recombinant genes into endothelial cells provides a method to study specific gene products and their effect on cell function. In addition, endothelial cells can be used for implantation into vessels or prosthetic vascular grafts. Because transfection efficiencies in human endothelial cells have been low, it is important to develop improved gene transfer techniques. Therefore, several transfection methods were optimized and transfection efficiencies were determined.
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            Effect of linoleic acid on endothelial cell inflammatory mediators.

             F. Yang,  C. Young,  C McClain (1998)
            Selected lipids may influence the inflammatory cascade within the vascular endothelium. To test this hypothesis, endothelial cells were treated with linoleic acid (18:2, n - 6) for 12 hours and/or tumor necrosis factor-alpha (TNF) for 4 hours. For a combined exposure to 18:2 and TNF (18:2 + TNF), cells were first preenriched with 18:2 for 8 hours before exposure to TNF for an additional 4 hours. Exposure to 18:2 increased cellular oxidative stress, activated nuclear factor-kappaB (NF-kappaB), increased interleukin-8 (IL-8) production, and elevated intercellular adhesion molecule-1 (ICAM-1) levels. A combined exposure to 18:2+ TNF resulted in decreased NF-kappaB activation compared with TNF treatment alone. In addition, preexposure to 18:2 altered TNF-mediated IkappaB-alpha signaling. Within the first 15 minutes of a 90-minute period, cytoplasmic levels of IkappaB-alpha decreased more rapidly in cells treated with 18:2 + TNF compared with TNF, suggesting translocation and activation of NF-kappaB in cultures that were pretreated with 18:2 before TNF exposure. A combined exposure to 18:2+TNF had various effects on IL-8 production and ICAM-1 levels depending on the time of exposure. For example, 18:2 + TNF treatment increased ICAM-1 levels at 12 hours but decreased ICAM-1 levels at 24 hours compared with treatment with TNF alone. These data suggest that selected fatty acids such as 18:2 can exert proinflammatory effects and, in addition, may markedly alter TNF-mediated inflammatory events.
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              Toxicity, Uptake Kinetics and Efficacy of New Transfection Reagents: Increase of Oligonucleotide Uptake

              Human arterial smooth muscle cell (haSMC) proliferation is stimulated by platelet-derived growth factor (PDGF) release of human arterial endothelial cells (haEC) whereas transforming growth factor-β 1 (TGF-β 1 ) secretion by haSMC promotes extracellular matrix formation. Inhibitory concepts with antisense oligonucleotides (ASO) against those growth factors might be promising, requiring, however, sufficient transfection efficacy. Thus, toxicity and efficacy of new transfection reagents were examined. MTT tests showed that high doses >1.6 μg/ml of the liposome Cytofectin GSV ® (CF) and the dendrimer SuperFect ® (SF) reduced mitochondrial activity of haEC after ≥4 h transfection whereas viability of haSMC was not influenced. DAC-30 ® showed significant toxic effects on haEC and haSMC at each dose after ≥4 h and Lipofectin ® (LF) caused complete detachment of haEC and haSMC in medium containing 10% serum. Uptake studies demonstrated that ‘naked’ ASO were not incorporated intracellularly whereas transfection within CF or SF resulted in a strong cytoplasmic and nuclear labeling after 2–5 h. With DAC-30 ® , only a slight cytoplasmic fluorescence was found. SF caused an unexpected stimulation of endothelial PDGF-AB synthesis. Thus, CF was favored for inhibition studies. ELISA, Western and Northern blotting showed a significant inhibition of endothelial PDGF-B and smooth muscle TGF-β 1 mRNA expression and synthesis after transfection for 3–5 h using 0.1–1.0 μ M ASO versus control oligonucleotides. We conclude that Cytofectin GSV ® is superior to the other transfection reagents, predominantly at haEC, showing an improved efficacy and less toxicity than the classical liposome Lipofectin ® . Cytofectin GSV ® might offer a promising tool for antisense strategies in the treatment of vascular disorders.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                April 2001
                13 April 2001
                : 38
                : 2
                : 133-143
                aUniversity of Potsdam, Potsdam, Germany and bDepartment of Surgery University of Kentucky, Lexington, Ky., USA
                51040 J Vasc Res 2001;38:133–143
                © 2001 S. Karger AG, Basel

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                Page count
                Figures: 7, Tables: 1, References: 31, Pages: 11
                Research Paper


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