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      Magnetofection--a highly efficient tool for antisense oligonucleotide delivery in vitro and in vivo.

      Molecular Therapy

      metabolism, Animals, Cell Survival, radiation effects, Cytochromes c, Drug Delivery Systems, methods, Electromagnetic Fields, Endothelium, Vascular, drug effects, Fluorescent Antibody Technique, Humans, Membrane Transport Proteins, Mice, Mice, Inbred C57BL, Multienzyme Complexes, Muscles, cytology, NADH, NADPH Oxidoreductases, NADPH Dehydrogenase, genetics, NADPH Oxidase, Oligonucleotides, Antisense, administration & dosage, Phosphoproteins, Superoxides, Transfection, Umbilical Veins

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          Abstract

          Delivery of antisense oligodesoxynucleotides (ODN) into primary cells is a specific strategy for research with therapeutic perspectives but transfection-associated difficulties. We established the technique of magnetofection to enhance ODN delivery at low toxicity and procedure time in vitro and in vivo. In vitro, target knockout was assessed at protein and mRNA levels and by measuring superoxide generation after antisense magnetofection against the p22(phox) subunit of endothelial NAD(P)H-oxidase. Under magnetic field guidance, low-dose magnetic particle-bound ODN were transfected to 84% human umbilical vein endothelial cells within 15 min followed by nuclear accumulation within 2 h, which required 24 h using standard methods. Antisense magnetofection against p22(phox) significantly decreased basal and prevented stimulated superoxide release due to loss of NAD(P)H-oxidase activity by mRNA knockout as assessed after 24 h. Knockout of endothelial phosphatase SHP-1 and connexin 37 proteins confirmed the method's efficiency. Transfection-associated toxicity was minimal. Twenty-four hours after injection of fluorescence-labeled ODN into femoral arteries of male mice, there was specific ODN uptake only into cremaster vessels exposed to magnetic fields during injection. Magnetofection is an ideal tool for delivery of functionally active ODN to difficult-to-transfect cells to study gene/protein function and a promising strategy for targeted ODN delivery in vivo.

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          Most cited references 32

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          Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase.

          Increased superoxide production contributes to reduced vascular nitric oxide (NO) bioactivity and endothelial dysfunction in experimental models of diabetes. We characterized the sources and mechanisms underlying vascular superoxide production in human blood vessels from diabetic patients with coronary artery disease compared with nondiabetic patients. Vascular superoxide production was quantified in both saphenous veins and internal mammary arteries from 45 diabetic and 45 matched nondiabetic patients undergoing coronary artery bypass surgery. NAD(P)H-dependent oxidases were important sources of vascular superoxide in both diabetic and nondiabetic patients, but both the activity of this enzyme system and the levels of NAD(P)H oxidase protein subunits (p22phox, p67phox, and p47phox) were significantly increased in diabetic veins and arteries. In nondiabetic vessels, endothelial NO synthase produced NO that scavenged superoxide. However, in diabetic vessels, the endothelium was an additional net source of superoxide production because of dysfunctional endothelial NO synthase that was corrected by intracellular tetrahydrobiopterin supplementation. Furthermore, increased superoxide production in diabetes was abrogated by the protein kinase C inhibitor chelerythrine. These observations suggest important roles for NAD(P)H oxidases, endothelial NO synthase uncoupling, and protein kinase C signaling in mediating increased vascular superoxide production and endothelial dysfunction in human diabetes mellitus.
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            Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo.

            Low efficiencies of nonviral gene vectors, the receptor-dependent host tropism of adenoviral or low titers of retroviral vectors limit their utility in gene therapy. To overcome these deficiencies, we associated gene vectors with superparamagnetic nanoparticles and targeted gene delivery by application of a magnetic field. This potentiated the efficacy of any vector up to several hundred-fold, allowed reduction of the duration of gene delivery to minutes, extended the host tropism of adenoviral vectors to nonpermissive cells and compensated for low retroviral titer. More importantly, the high transduction efficiency observed in vitro was reproduced in vivo with magnetic field-guided local transfection in the gastrointestinal tract and in blood vessels. Magnetofection provides a novel tool for high throughput gene screening in vitro and can help to overcome fundamental limitations to gene therapy in vivo.
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              Novel competitive inhibitor of NAD(P)H oxidase assembly attenuates vascular O(2)(-) and systolic blood pressure in mice.

              We previously reported enhanced expression of the p67(phox) and gp91(phox) components of NAD(P)H oxidase in angiotensin (Ang) II-induced hypertension, suggesting de novo assembly in response to Ang II. To examine the direct involvement of NAD(P)H oxidases in Ang II-induced O(2)(-) production, we designed a chimeric peptide that inhibits p47(phox) association with gp91(phox) in NAD(P)H oxidase (gp91ds-tat). This was achieved by linking a 9-amino acid peptide (aa) derived from HIV-coat protein (tat) to a 9-aa sequence of gp91(phox) (known to interact with p47(phox)). As a control, we constructed a chimera containing tat and a scrambled gp91 sequence (scramb-tat). We found that gp91ds-tat decreased O(2)(-) levels in aortic rings treated with Ang II (10 pmol/L) but had no effect on either the O(2)(-)-generating enzyme xanthine oxidase or potassium superoxide-generated O(2)(-). We infused vehicle, Ang II (0.75 mg. kg(-1). d(-1)), Ang II+gp91ds-tat (10 mg. kg(-1). d(-1)), or Ang II+scramb-tat intraperitoneally in C57Bl/6 mice and measured systolic blood pressure (SBP) on days 0, 3, 5, and 7 of infusion. SBP increased by day 3 in mice given Ang II and Ang II+scramb-tat but was significantly lower with Ang II+gp91-tat. On day 7, SBP was still significantly inhibited in mice given Ang II+gp91ds-tat, whereas Ang II-induced O(2)(-) production was inhibited throughout the aorta as detected by dihydroethidium staining, consistent with the ability of this inhibitor to block the various vascular NAD(P)H oxidase isoforms. These data support the hypothesis that inhibition of the interaction of p47(phox) and gp91(phox) (or its homologues) can block O(2)(-) production and attenuate blood pressure elevation in mice.
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                Author and article information

                Journal
                12718913
                10.1016/S1525-0016(03)00065-0

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