Hematoxylin/eosin staining of virus induced scars generated by different PDGF isoforms.

<p>Hematoxylin/eosin staining of cryo sectioned hearts: (A, B) empty vector control; (C, D) PDGF-A_short virus; (E, F) PDGF-A_long virus; (G, H) PDGF-B virus; (I, J) PDGF-C virus; (K, L) PDGF-D virus. Scale bar is 250 μm.</p

Comparison of scar size and heart functionality.

<p>(A) One-way ANOVA analysis of scar volume corrected with Tukey´s multiple comparisons test. (B) Circle diagram illustrating the average volume of scar tissue (yellow) relative to the volume of cardiac tissue (red) for each different type of virus. (C) Ejection fraction of the left ventricle (indicating the ventricular functionality) before, and 15 days after, virus injections. (D) Left ventricle wall thickness before, and 15 days after virus injection. Error bars in D, E show std.dev and asterisk indicate a statistical significant difference (p<0.05). E = empty vector; As = PDGF-A_short; Al = PDGF-A_long; B = PDGF-B; C = PDGF-C; D = PDGF-D.</p

Distribution of PDGF receptors.

<p>(A-F) Expression of PDGFRα (green nuclei) and PDGFRβ (red) in cells within the fibrotic scars. Lipid membranes are highlighted in grey. Scale bars are 30μm. (G-J) One-way ANOVA analyses of GFP expression in different locations of the scar, corrected with Tukey´s multiple comparisons test. * = p<0.05; ** = p<0.01; *** = p<0.001, **** = p<0.0001. (G) PDGFRα positive cells in the center (non-peripheral area) of the scar. (H) PDGFRα positive cells in close association to vessels. (I) PDGFRα positive cells in peripheral regions. (J) Relation of PDGFRα positive cells in the peripheral- and central region of the scar. E = empty vector; As = PDGF-A_short; Al = PDGF-A_long; B = PDGF-B; C = PDGF-C; D = PDGF-D. (K) Schematic representation of PDGF receptor expression, scar size, myofibroblasts and vessel morphology in scars from different experimental groups.</p

Angiogenic response in virus-induced scars.

<p>Endothelial CD31 expression (red) marks blood vessels, PDGFRα in green and lipid membranes in grey. (Left column, A, C, E, G, I, K) Overview, showing changes in vessel morphology and density in PDGF induced scars. (Right column, B, D, F, H, J, L) Asterisks mark big vessels with altered morphology. (M) Quantification of small vessels in the different experimental groups, normalized to the empty vector control. (N) Quantification of vessel size (area of cross-sectioned vessels) for the different experimental groups, normalized to the empty vector control. Analyses were performed on 3–4 mice per experimental group. E = empty vector; As = PDGF-A_short; Al = PDGF-A_long; B = PDGF-B; C = PDGF-C; D = PDGF-D. Scale bars are 100 μm.</p

Myofibroblasts in the inflammatory response to viruses.

<p>Alpha-SMA (red) marks myofibroblasts and mural cells in the scars, PDGFRα is in green and lipid membranes in grey. Arrows in (G) and (J) point at α-SMA positive cardiomyocytes outside of scars induced by PDGF-B and PDGF-C viruses. Scale bars are 100 μm.</p

Inflammatory response in virus-induced scars.

<p>Expression of the pan-inflammatory marker CD45 (red), PDGFRα (green) and lipid membranes (grey) in center and periphery of scars. (Left column, A, C, E, G, I, K) Abundant expression of CD45 in scars of all viruses. (B, D, F, H, J, L) Arrows indicate inflammatory cells outside of the scar area. (M, N) Arrows mark inflammatory cells that accumulate around blood vessels in PDGF-B induced scars. (O, P) The myofibrillar organization is lost where inflammatory cells invade the myocardium in PDGF-B induced scars (arrows). The red channel has been removed in (P) to visualize absence of grey staining in the lost myofibrils. (Q-T) CD45 and PDGFRα are inversely expressed in PDGF-B and PDGF-C induced scars. The green channel has been removed in (R and T) to visualize the inverse correlation. (U) Quantification of cells expressing CD45 in the different experimental groups by fluorescence intensity measurements. All values are normalized to the empty vector control group, error bars show standard deviation and statistical significance is represented by asterisk (p<0.05). E = empty vector; As = PDGF-A_short; Al = PDGF-A_long; B = PDGF-B; C = PDGF-C; D = PDGF-D. Scale bars are 100μm.</p

Image_1_AAV8-mediated sVEGFR2 and sVEGFR3 gene therapy combined with chemotherapy reduces the growth and microvasculature of human ovarian cancer and prolongs the survival in mice.pdf

BackgroundVascular endothelial growth factors (VEGFs) are major regulators of intratumoral angiogenesis in ovarian cancer (OVCA). Overexpression of VEGFs is associated with increased tumor growth and metastatic tendency and VEGF-targeting therapies are thus considered as potential treatments for OVCA. Here, we examined the antiangiogenic and antitumoral effects on OVCA of adeno-associated virus 8 (AAV8)-mediated expression of soluble VEGF receptors (sVEGFRs) sVEGFR2 and sVEGFR3 together with paclitaxel and carboplatin chemotherapy.Materials and methodsImmunodeficient mice were inoculated with human OVCA cell line SKOV-3m. Development of tumors was confirmed with magnetic resonance imaging (MRI) and mice were treated with gene therapy and paclitaxel and carboplatin chemotherapy. The study groups included (I) non-treated control group, (II) blank control vector AAV8-CMV, (III) AAV8-CMV with chemotherapy, (IV) AAV8-sVEGFR2, (V) AAV8-sVEGFR3, (VI) AAV8-sVEGFR2 and AAV8-sVEGFR3, and (VII) AAV8-sVEGFR2 and AAV8-sVEGFR3 with chemotherapy. Antiangiogenic and antitumoral effects were evaluated with immunohistochemical stainings and serial MRI.ResultsReduced intratumoral angiogenesis was observed in all antiangiogenic gene therapy groups. The combined use of AAV8-sVEGFR2 and AAV8-sVEGFR3 with chemotherapy suppressed ascites fluid formation and tumor growth, thus improving the overall survival of mice. Antitumoral effect was mainly caused by AAV8-sVEGFR2 while the benefits of AAV8-sVEGFR3 and chemotherapy were less prominent.ConclusionCombined use of the AAV8-sVEGFR2 and AAV8-sVEGFR3 with chemotherapy reduces intratumoral angiogenesis and tumor growth in OVCA mouse model. Results provide preclinical proof-of-concept for the use of soluble decoy VEGFRs and especially the AAV8-sVEGFR2 in the treatment of OVCA.</p

Doxycycline modulates VEGF-A expression: Failure of doxycycline-inducible lentivirus shRNA vector to knockdown VEGF-A expression in transgenic mice - Fig 3

<p><b>Plasma VEGF-A concentration in response to dox treatment (a). Tissue VEGF-A mRNA and protein levels after two weeks of 1 mg/ml dox treatment and five weeks of 2 mg/ml dox dose treatment (b-k).</b> Dox treatment decreased plasma VEGF-A levels in TG mice after 1 week, after which the plasma VEGF-A increased. In WT mice a decreasing effect on plasma VEGF-A was seen (a). *p<0.05, **p<0.01 and ***p<0.001 compared to baseline within each group, 1-way ANOVA with Dunnett´s post hoc test, n = 9-10/group. In the selected tissues, aorta, heart and kidney, the dox treatment with the 1 mg/ml dox dose for 2 weeks showed decreasing trend in VEGF-A mRNA expression in TG mice in comparison to WT mice (b, d, h), which was associated with increased cardiac VEGF-A protein (e) and decreased kidney VEGF-A protein levels (i). When the dox dose was doubled and the treatment time increased to 5 weeks, a trend towards increasing VEGF-A expression was seen in all three tissues in both WT and TG mice (c, f, j). However, no changes were detected in protein levels (g, k). **p<0.01 and ***p<0.001 compared to WT+dox group in 2 weeks experiment (b, d, e, h, i) or to no dox group (-dox) in 5 weeks experiment (c, f, g, j, k), <i>t-test</i>, n = 6-24/group.</p

Schematic drawing of the dox-regulatable VEGF-A shRNA lentivirus vector.

<p>Lentivirus vector for inducible knockdown permits constitutive expression of a Tet-transactivating component (rtTA3) with a Venus selection marker (green fluorescent protein -like protein) and tetracycline-regulated expression of VEGF-A-shRNAs. The shRNA transcripts were designed as primary microRNA mimics i.e. they were embedded in the primary transcript of human miRNA30. The lentiviruses were self-inactivating (SIN) third generation vectors, in which part of the viral 3´LTR has been deleted preventing the viral replication. The vectors contain a central polypurine tract (indicated as FLAP) for enhancement of viral titers and a woodchuck hepatitis virus posttranscriptional regulatory element (WRE) for better transgene expression [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190981#pone.0190981.ref024" target="_blank">24</a>].</p

VEGF-A knockdown via RNAi in mouse endothelial cells and cardiomyocytes in a doxycycline-regulatable fashion.

<p>Secreted VEGF-A protein amount was most efficiently decreased with sh1 -knockdown vector (T-1 and TT-1) in mouse endothelial cells in comparison to sh2 (T-2 and TT-2) and sh3 (T-3 and TT-3) vectors. With the sh1 construct the normal TRE promoter (T-1) seemed to be slightly more efficient and less leaky than the tight TRE promoter (TT-1) (a). The magnitude of VEGF-A knockdown with the selected T-1 vector increased with increasing dox doses in endothelial cells (b). With the increasing dox doses also the amount of Venus expressing cells (%) was increased as follows: 40 ± 7% (–dox), 46 ± 8% (dox 10 ng/ml), 85 ± 3% (dox 100 ng/ml) and 78 ± 2% (dox 1000 ng/ml) (c). In cardiomyocytes the knockdown of VEGF-A with the T-1 vector was shown to be dependent of the amount of virus (MOI) and dox exposure time and the decrease was larger at the cellular level in comparison to the secreted protein (d-e). Results are shown as mean ± S.D., n = 3/group. The percentage of VEGF-A protein concentration compared to control +dox group (a) or non-transduced (NT) group (b, d, e) are shown above bars. *p<0.05, **p<0.01 and ***p<0.001 compared to control +dox group (a) or non-transduced (NT) group (b, d, e), 1-way ANOVA with Dunnett´s post hoc test. NT = non-transduced cells, MOI = multiplicity of infection, Contr = Control lentivirus vector targeting Luciferase. Scale bar 200 μm (c).</p