Vascular Endothelial Growth Factor-B Induces a Distinct Electrophysiological Phenotype in Mouse Heart

Vascular endothelial growth factor B (VEGF-B) is a potentmediator of vascular, metabolic, growth, and stress responses in the heart, but the effects on cardiac muscle and cardiomyocyte function are not known. The purpose of this study was to assess the effects of VEGF-B on the energy metabolism, contractile, and electrophysiological properties of mouse cardiac muscle and cardiac muscle cells. In vivo and ex vivo analysis of cardiac-specific VEGF-B TG mice indicated that the contractile function of the TG hearts was normal. Neither the oxidative metabolism of isolated TG cardiomyocytes nor their energy substrate preference showed any difference to WT cardiomyocytes. Similarly, myocyte Ca2+ signaling showed only minor changes compared to WT myocytes. However, VEGF-B overexpression induced a distinct electrophysiological phenotype characterized by ECG changes such as an increase in QRSp time and decreases in S and R amplitudes. At the level of isolated TG cardiomyocytes, these changes were accompanied with decreased action potential upstroke velocity and increased duration (APD60-70). These changes were partly caused by downregulation of sodium current (INa) due to reduced expression of Nav1.5. Furthermore, TG myocytes had alterations in voltage-gated K + currents, namely decreased density of transient outward current (Ito) and total K + current (Ipeak). At the level of transcription, these were accompanied by downregulation of Kv channel-interacting protein 2 (Kcnip2), a knownmodulatory subunit for Kv4.2/3 channel. Cardiac VEGF-B overexpression induces a distinct electrophysiological phenotype including remodeling of cardiomyocyte ion currents, which in turn induce changes in action potential waveform and ECG.Peer reviewe

Angiopoietin-2 blocking antibodies reduce early atherosclerotic plaque development in mice

Objective: Angiopoietin-2 (Ang-2) blocking agents are currently undergoing clinical trials for use in cancer treatment. Ang-2 has also been associated with rupture-prone atherosclerotic plaques in humans, suggesting a role for Ang-2 in plaque stability. Despite the availability of Ang-2 blocking agents, their clinical use is still lacking. Our aim was to establish if Ang-2 has a role in atheroma development and in the transition of subclinical to clinically relevant atherosclerosis. We investigated the effect of antibody-mediated Ang-2 blockage on atherogenesis after in a mouse model of atherosclerosis. Methods: Hypercholesterolemic (low-density lipoprotein receptor(-/-) apolipoprotein B-100/100) mice were subjected to high-cholesterol diet for eight weeks, one group with and one group without Ang-2 blocking antibody treatment during weeks 4-8. To enhance plaque development, a peri-adventitial collar was placed around the carotid arteries at the start of antibody treatment. Aortic root, carotid arteries and brachiocephalic arteries were analyzed to evaluate the effect of Ang-2 blockage on atherosclerotic plaque size and stable plaque characteristics. Results: Anti-Ang-2 treatment reduced the size of fatty streaks in the brachiocephalic artery (-72%, p <0.05). In addition, antibody-mediated Ang-2 blockage reduced plasma triglycerides (-27%, p <0.05). In contrast, Ang-2 blockage did not have any effect on the size or composition (collagen content, macrophage percentage, adventitial microvessel density) of pre-existing plaques in the aortic root or collar-induced plaques in the carotid artery. Conclusions: Ang-2 blockage was beneficial as it decreased fatty streak formation and plasma triglyceride levels, but had no adverse effect on pre-existing atherosclerosis in hypercholesterolemic mice. (C) 2015 The Authors. Published by Elsevier Ireland Ltd.Peer reviewe

In vivo inhibition of nuclear factor of activated T-cells leads to atherosclerotic plaque regression in IGF-II/LDLR -/-ApoB100/100 mice

AIMS: Despite vast clinical experience linking diabetes and atherosclerosis, the molecular mechanisms leading to accelerated vascular damage are still unclear. Here, we investigated the effects of nuclear factor of activated T-cells inhibition on plaque burden in a novel mouse model of type 2 diabetes that better replicates human disease.METHODS & RESULTS: IGF-II/LDLR-/-ApoB100/100mice were generated by crossbreeding low-density lipoprotein receptor-deficient mice that synthesize only apolipoprotein B100 (LDLR-/-ApoB100/100) with transgenic mice overexpressing insulin-like growth factor-II in pancreatic β cells. Mice have mild hyperglycaemia and hyperinsulinaemia and develop complex atherosclerotic lesions. In vivo treatment with the nuclear factor of activated T-cells blocker A-285222 for 4 weeks reduced atherosclerotic plaque area and degree of stenosis in the brachiocephalic artery of IGF-II/LDLR-/-ApoB100/100mice, as assessed non-invasively using ultrasound biomicroscopy prior and after treatment, and histologically after termination. Treatment had no impact on plaque composition (i.e. muscle, collagen, macrophages). The reduced plaque area could not be explained by effects of A-285222 on plasma glucose, insulin or lipids. Inhibition of nuclear factor of activated T-cells was associated with increased expression of atheroprotective NOX4 and of the anti-oxidant enzyme catalase in aortic vascular smooth muscle cells.CONCLUSION: Targeting the nuclear factor of activated T-cells signalling pathway may be an attractive approach for the treatment of diabetic macrovascular complications

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

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

dvdres-sep-2017-00131-File003 – Supplemental material for <i>In vivo</i> inhibition of nuclear factor of activated T-cells leads to atherosclerotic plaque regression in IGF-II/LDLR^–/–ApoB^100/100 mice

<p>Supplemental material, dvdres-sep-2017-00131-File003 for <i>In vivo</i> inhibition of nuclear factor of activated T-cells leads to atherosclerotic plaque regression in IGF-II/LDLR^–/–ApoB^100/100 mice by Fabiana Blanco, Suvi E Heinonen, Erika Gurzeler, Lisa M Berglund, Anna-Maria Dutius Andersson, Olga Kotova, Ann-Cathrine Jönsson-Rylander, Seppo Ylä-Herttuala and Maria F Gomez in Diabetes & Vascular Disease Research</p

dvdres-sep-2017-00131-File004 – Supplemental material for <i>In vivo</i> inhibition of nuclear factor of activated T-cells leads to atherosclerotic plaque regression in IGF-II/LDLR^–/–ApoB^100/100 mice

<p>Supplemental material, dvdres-sep-2017-00131-File004 for <i>In vivo</i> inhibition of nuclear factor of activated T-cells leads to atherosclerotic plaque regression in IGF-II/LDLR^–/–ApoB^100/100 mice by Fabiana Blanco, Suvi E Heinonen, Erika Gurzeler, Lisa M Berglund, Anna-Maria Dutius Andersson, Olga Kotova, Ann-Cathrine Jönsson-Rylander, Seppo Ylä-Herttuala and Maria F Gomez in Diabetes & Vascular Disease Research</p