Erika Gurzeler

AAV9-mediated VEGF-B Gene Transfer Improves Systolic Function in Progressive Left Ventricular Hypertrophy

Mechanisms of the transition from compensatory hypertrophy to heart failure are poorly understood and the roles of vascular endothelial growth factors (VEGFs) in this process have not been fully clarified. We determined the expression profile of VEGFs and relevant receptors during the progression of left ventricular hypertrophy (LVH). C57BL mice were exposed to transversal aortic constriction (TAC) and the outcome was studied at different time points (1 day, 2, 4, and 10 weeks). A clear compensatory phase (2 weeks after TAC) was seen with following heart failure (4 weeks after TAC). Interestingly, VEGF-C and VEGF-D as well as VEGF receptor-3 (VEGFR-3) were upregulated in the compensatory hypertrophy and VEGF-B was downregulated in the heart failure. After treatment with adeno-associated virus serotype 9 (AAV9)-VEGF-B(186) gene therapy in the compensatory phase for 4 weeks the function of the heart was preserved due to angiogenesis, inhibition of apoptosis, and promotion of cardiomyocyte proliferation. Also, the genetic programming towards fetal gene expression, a known phenomenon in heart failure, was partly reversed in AAV9-VEGF-B(186)-treated mice. We conclude that VEGF-C and VEGF-D are associated with the compensatory LVH and that AAV9-VEGF-B(186) gene transfer can rescue the function of the failing heart and postpone the transition towards heart failure.

The effects of VEGF-A on atherosclerosis, lipoprotein profile, and lipoprotein lipase in hyperlipidaemic mouse models

AIMS The role of vascular endothelial growth factor (VEGF-A) in atherogenesis has remained controversial. We addressed this by comparing the effects of adenoviral VEGF-A gene transfer on atherosclerosis and lipoproteins in ApoE(-/-), LDLR(-/-), LDLR(-/-)ApoE(-/-), and LDLR(-/-)ApoB(100/100) mice. METHODS AND RESULTS After 4 weeks on western diet, systemic adenoviral gene transfer was performed with hVEGF-A or control vectors. Effects on atherosclerotic lesion area and composition, lipoprotein profiles, and plasma lipoprotein lipase (LPL) activity were examined. On day 4, VEGF-A induced alterations in lipoprotein profiles and a significant negative correlation was observed between plasma LPL activity and VEGF-A levels. One month after gene transfer, no changes in atherosclerosis were observed in LDLR(-/-) and LDLR(-/-)ApoB(100/100) models, whereas both ApoE(-/-) models displayed increased en face lesion areas in thoracic and abdominal aortas. VEGF-A also reduced LPL mRNA in heart and white adipose tissue, whereas Angptl4 was increased, potentially providing further mechanistic explanation for the findings. CONCLUSION VEGF-A gene transfer induced pro-atherogenic changes in lipoprotein profiles in all models. As a novel finding, VEGF-A also reduced LPL activity, which might underlie the observed changes in lipid profiles. However, VEGF-A was observed to increase atherosclerosis only in the ApoE(-/-) background, clearly indicating some mouse model-specific effects.

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 B100/100) 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.

Regulation of endothelial lipase and systemic HDL cholesterol levels by SREBPs and VEGF-A

OBJECTIVE Endothelial lipase (EL) regulates HDL cholesterol levels and in inflammatory states, like atherosclerosis, EL expression is increased contributing to low HDL cholesterol. The regulation of EL expression is poorly understood and has mainly been attributed to inflammatory stimuli. As sterol regulatory element binding proteins (SREBPs) are regulators of genes involved in lipid metabolism, we hypothesized that EL is regulated by SREBPs and that EL expression is modified by the SREBP activator vascular endothelial growth factor A (VEGF-A). METHODS and results: Quantitative PCR and Western blot results demonstrated that starvation increased EL expression in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). Also, 25-hydroxycholesterol (25HC), an inhibitor of SREBP activation inhibited EL expression. With siRNA-mediated inhibition of SREBPs the effect of starvation was shown to be SREBP-2 dependent. VEGF-A decreased EL expression in both endothelial cell lines used, most likely via inhibition of SREBP-2 binding determined by chromatin immunoprecipitation (ChIP). Furthermore, in atherosclerosis prone LDLR(-/-)ApoB(100/100) mice, systemic adenoviral gene transfer with human VEGF-A decreased EL mRNA in peripheral tissues and increased plasma HDL cholesterol. CONCLUSIONS These results identify SREBPs as novel regulators of EL expression. VEGF-A as an endogenous EL inhibitor could be therapeutically relevant in atherosclerosis by increasing systemic HDL cholesterol levels.

Left ventricular remodeling leads to heart failure in mice with cardiac‐specific overexpression of VEGF‐B167: echocardiography and magnetic resonance imaging study

Cardiac‐specific overexpression of vascular endothelial growth factor (VEGF)‐B167 is known to induce left ventricular hypertrophy due to altered lipid metabolism, in which ceramides accumulate to the heart and cause mitochondrial damage. The aim of this study was to evaluate and compare different imaging methods to find the most sensitive way to diagnose at early stage the progressive left ventricular remodeling leading to heart failure. Echocardiography and cardiovascular magnetic resonance imaging were compared for imaging the hearts of transgenic mice with cardiac‐specific overexpression of VEGF‐B167 and wild‐type mice from 5 to 14 months of age at several time points. Disease progression was verified by molecular biology methods and histology. We showed that left ventricular remodeling is already ongoing at the age of 5 months in transgenic mice leading to heart failure by the age of 14 months. Measurements from echocardiography and cardiovascular magnetic resonance imaging revealed similar changes in cardiac structure and function in the transgenic mice. Changes in histology, gene expressions, and electrocardiography supported the progression of left ventricular hypertrophy. Longitudinal relaxation time in rotating frame (T1ρ) in cardiovascular magnetic resonance imaging could be suitable for detecting severe fibrosis in the heart. We conclude that cardiac‐specific overexpression of VEGF‐B167 leads to left ventricular remodeling at early age and is a suitable model to study heart failure development with different imaging methods.

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

Vascular endothelial growth factor-A (VEGF-A) is the master regulator of angiogenesis, vascular permeability and growth. However, its role in mature blood vessels is still not well understood. To better understand the role of VEGF-A in the adult vasculature, we generated a VEGF-A knockdown mouse model carrying a doxycycline (dox)-regulatable short hairpin RNA (shRNA) transgene, which silences VEGF-A. The aim was to find the critical level of VEGF-A reduction for vascular well-being in vivo. In vitro, the dox-inducible lentiviral shRNA vector decreased VEGF-A expression efficiently and dose-dependently in mouse endothelial cells and cardiomyocytes. In the generated transgenic mice plasma VEGF-A levels decreased shortly after the dox treatment but returned back to normal after two weeks. VEGF-A expression decreased shortly after the dox treatment only in some tissues. Surprisingly, increasing the dox exposure time and dose led to elevated VEGF-A expression in some tissues of both wildtype and knockdown mice, suggesting that dox itself has an effect on VEGF-A expression. When the effect of dox on VEGF-A levels was further tested in naïve/non-transduced cells, the dox administration led to a decreased VEGF-A expression in endothelial cells but to an increased expression in cardiomyocytes. In conclusion, the VEGF-A knockdown was achieved in a dox-regulatable fashion with a VEGF-A shRNA vector in vitro, but not in the knockdown mouse model in vivo. Dox itself was found to regulate VEGF-A expression explaining the unexpected results in mice. The effect of dox on VEGF-A levels might at least partly explain its previously reported beneficial effects on myocardial and brain ischemia. Also, this effect on VEGF-A should be taken into account in all studies using dox-regulated vectors.

High Plasma Lipid Levels Reduce Efficacy of Adenovirus-Mediated Gene Therapy

Adenoviruses are very efficient vectors for delivering therapeutic genes in preclinical and clinical trials. However, randomized controlled human trials have often been lacking clear clinically relevant results. We hypothesized that high lipid levels and specific lipoproteins could significantly decrease adenoviral transduction efficiency in vivo. Here we demonstrate that mice on a high fat diet have lower transgene expression compared to mice on a regular chow. In addition, on a high fat diet, ApoE−/− mice have much higher plasma transgene levels compared to LDLR-deficient mice. We also found that specific lipoprotein receptors play an important role in adenoviral transduction. These findings suggest that high plasma lipid levels, especially apoE-containing lipoproteins, reduce efficacy of adenoviral transduction in mice, which implies that high cholesterol levels in humans could be protective against viral infections and also lead to insufficient transgene expression in clinical trials using adenoviral vectors.

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

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

Vascular endothelial growth factor B (VEGF-B) is a potent mediator 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 known modulatory 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.