Covalent attachment of maleimide-activated human transferrin to cysteine-modified Ad vectors and their transduction efficiency in hTfR-positive human brain microvascular endothelial cells.

<p>A) Schematic illustration of Ad vector particles containing a solvent-exposed cysteine either on fiber (LIGGG<u>C</u>GGGID) or hexon (HRV5, alanine to cysteine substitution), to which maleimide-activated transferrin is covalently attached (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045977#s4" target="_blank">materials and methods</a>). B–C) Relative transduction efficiency of fiber (AdFiberCys) and hexon-modified (AdHexonCys) vectors with or without covalently attached transferrin in K562 (B) and hCMEC/D3 (C) cells at 24 hrs p.t. by flow cytometry (multiplicities of infection based on particles (pMOIs) 200 and 5000). Relative mean fluorescence and standard deviations are shown (n = 3, 10.000 cells/sample). D) Cellular uptake of the fluid-phase endocytosis marker 70 kDa FITC-Dextran in untransduced K562 and hCMEC/D3 and transduced hCMEC/D3 cells (pMOI 5000) after 1 hr uptake at 37°C determined by flow cytometry. Relative mean fluorescence normalized to untransduced hCMEC/D3 cells, as well as standard deviations are shown (n = 3, 10.000 cells/sample).</p

Delivery of transferrin-coupled hexon-modified Ad vectors across the endothelium barrier.

<p>A) qPCR detection of the transcytosed hTf-coupled vectors after DNAse treatment and viral DNA isolation. Heat and DNAse treated vectors, as well as untreated vectors were used as controls. The percentages were calculated by comparing the detected copy numbers of the untreated vector to the copy numbers of the transcytosed vector. B–C) Gene transfer efficiency of transcytosed hTf-coupled vectors in 293 cells at 16 hrs p.t. as determined by fluorescence microscopy. For quantification, six to eight random areas were imaged and the cells were counted with the help of ImageJ Cell Counter. Transduction units (T.U.) presented were counted from the total sample volume obtained on the basolateral side of the hCMEC/D3 cells (n = 500 cells, mean ± S.E.). Scale bars in all images, 20 µm.</p

Delivery of transferrin-coupled fiber-modified Ad vectors across the endothelium barrier.

<p>A, B) After transcytosis experiments in Transwell plates, qPCR was performed from the basolateral media using Ad fiber and E4 primers. The corresponding Ad copy number was determined by the standard curve of linearized pGS66 plasmid. The detected vector copy numbers of fiber- (A) and hexon-modified vectors (B) are shown (2×10⁸, n = 2–3 monolayers; and 1×10⁹ VPs, <i>P</i><0.05, n = 6 monolayers; mean ± S.E.). C) Transcytosis percentages of the vectors after transcellular delivery detected by qPCR across hCMEC/D3 and PBCEC cells (1×10⁹ and 5×10⁹ VPs). The percentages were calculated by comparing the detected copy numbers of the input vector to the copy numbers on the basolateral side.</p

Integrity of hcmec/d3 endothelium in the presence of Ad vectors and their transduction efficiencies in polarized cells.

<p>A) Schematic illustration of the <i>in vitro</i> hCMEC/D3 endothelium model on collagen-coated 0.4 µm-Transwell filters. Cells are grown to confluency for 6–7 days in EBM-2 media, after which the barrier properties of the endothelium are measured by voltohmeter and permeability assays with fluorescent markers. Extracellular matrix (ECM), filter, and apical and basolateral sides of the Transwell chamber are shown. B) Transendothelial electrical resistance (TEER) values of hCMEC/D3 monolayers after 6 days in culture (59.4±1.5 Ω/cm²; mean±S.E., n = 30 monolayers). Boxplot data is shown, containing median (bar), quartile range (box) and minimum and maximum values (whiskers). C) Representative TEER values of hCMEC/D3 monolayers before and after 4 hrs incubation with hexon (AdHexonCys, 5×10⁹ VPs/monolayer) or fiber-modified (AdFiberCys, 1×10⁹ VPs/monolayer) vectors with or without human transferrin (n = 3–4 monolayers/vector). In all experiments, TEER measurements were performed as triplicates with Milli-Cell ERS equipment (mean Ω/cm²±SD, <i>P</i>>0.1). D–E) Transduction efficiencies of fiber or hexon-modified vectors with or without transferrin in unpolarized or transwell-cultured, polarized hCMEC/D3 cells at 24 hrs p.t. detected by fluorimetry (D; n = 3 monolayers, TEER >60 Ω/cm²) or fluorescence microscopy (E; polarized cells). Relative mean fluorescence and standard deviation is calculated from the obtained mean fluorescence values. Scale bars in the images, 50 µm.</p

Epigenetic Upregulation of Endogenous VEGF-A Reduces Myocardial Infarct Size in Mice

<div><p>“Epigenetherapy” alters epigenetic status of the targeted chromatin and modifies expression of the endogenous therapeutic gene. In this study we used lentiviral <i>in vivo</i> delivery of small hairpin RNA (shRNA) into hearts in a murine infarction model. shRNA complementary to the promoter of vascular endothelial growth factor (VEGF-A) was able to upregulate endogenous VEGF-A expression. Histological and multiphoton microscope analysis confirmed the therapeutic effect in the transduced hearts. Magnetic resonance imaging (MRI) showed <i>in vivo</i> that the infarct size was significantly reduced in the treatment group 14 days after the epigenetherapy. Importantly, we show that promoter-targeted shRNA upregulates all isoforms of endogenous VEGF-A and that an intact hairpin structure is required for the shRNA activity. In conclusion, regulation of gene expression at the promoter level is a promising new treatment strategy for myocardial infarction and also potentially useful for the upregulation of other endogenous genes.</p></div

Multiphoton microscopy and histology analysis of myocardial infarction animals.

<p>(a) Multiphoton laser scanning microscopy (MPLSM) analysis of GFP expression in transduced mouse heart, (b) Immunohistological analysis of GFP expression in mouse heart, (c) antibody omitted control, (d and k) Massons Trichrome staining from mouse heart transduced with VEGF-A upregulating LV-451 and shRNA control, respectively, (e and l) insert from infarcted area of d and k, respectively, (h and o) insert from infarct borderzone (f, i, m, p) alpha-SMA staining of smooth muscle cells, arrows point to arteriols formed, (g, j, n, q) CD-31 staining of endothelial cells. Scale bars (a) 100 µm, (d and k) 2000 µm, (e, f, g, h, i, j, l, m, n, o, p, q) 200 µm.</p

ELISA assay of myocardial infarction samples and analysis for single-stranded vectors for a mechanistical view.

<p>(a) ELISA analysis of VEGF-A protein from transduced hearts, (b) ELISA assay from growth medium of C166 cells transduced with LV-451 and corresponding single stranded vectors using MOI 10, 7 days time point. (c) RT-PCR analysis of VEGF-A mRNA levels. C166 cells were transduced with LV-451 and corresponding single stranded vectors using MOI 10, 11 days time point. (d) qChIP assay of C166 cells using antibodies against H3K4me2. Cells were transduced with LV-451 and corresponding single stranded vectors using MOI 10, 11 days timepoint. All results are shown as mean ± SD.</p

Intracellular distribution of LV-451 expressed RNA and VEGF-A mRNA in transduced cells.

<p>C166 cells were subjected to RNA-FISH analysis with LV-451 or VEGF mRNA probes. (a) Confocal microscopy images of LV-451 transduced (MOI 10) cells 72 h post transduction. Distribution of LV-451 RNA (green) and VEGF-A mRNA (red) probe binding induced signals is shown. Nuclei were visualized with DAPI (grey). Scale bars, 5 µm. (b) Quantification of LV-451 RNA or VEGF-A mRNA RNA-FISH signal spots detected in LV-451 transduced (MOI 4, 40, 200) cells at 72 h post transduction and in nontransduced control cells. The amount of signal was calculated in the nucleus (white), the cytosol (grey) and whole cell (black). Error bars = SD. (c) Nucleus size in response to LV transduction. CTRL sample is nontransduced C166 cells and LV-451 is C166 cells transduced with LV-451 vector.</p

MRI analysis of murine myocardial infarction.

<p>Infarct size in VEGF-A upregulated (shRNA) and in control (shRNA Control) groups measured using MRI (a), and representative examples of short axis cine images with outlined (red lines) infarcts in late diastole at days 4 and 14 in both shRNA and shRNA control animals (b).</p

Analysis for mechanism of action for promoter targeted shRNAs.

<p>(a) RT-PCR analysis for different VEGF-A isoforms. The expression levels for different isoforms were studied using primers specific to each isoform. Total VEGF-A protein level was measured with ELISA. (b) Reversing DNA methylation with 5-Azacytidine treatment induces responses in MS1 cells but erases responses in C166 cells. Cells were treated with 1 µM 5-Azacytidine, transduced with different vectors on day 3 and samples were collected on day 8. qRT-PCR analysis of VEGF-A and B-actin mRNA levels in MS1 cells and C166 cells. (c) qChIP assay in MS1 cells using antibody against H3K27me3. (d) The VEGF-A gene promoter in C166 cells was also analyzed for basal DNA methylation levels without 5-Azacytidine treatment using MeDIP. Cells were transduced with different vectors using MOI 10, 10 days timepoint. (e) RT-PCR analysis of VEGF-A mRNA levels after C166 cells were transfected with siRNA oligos. Results are calculated in reference to housekeeping gene ACTB and control oligo. (f) CBP-CREB interaction inhibitor (7.5 µM) abolishes the upregulation of VEGF-A by LV-451 in C166 cells. For all results, mean ± SD shown.</p