RESUMEN
BACKGROUND: There is a lack of predictive preclinical animal models combining atherosclerosis and type 2 diabetes. APOE∗3-Leiden (E3L) mice are a well-established model for diet-induced hyperlipidemia and atherosclerosis, and glucokinase+/- (GK+/-) mice are a translatable disease model for glucose control in type 2 diabetes. The respective mice respond similarly to lipid-lowering and antidiabetic drugs as humans. The objective of this study was to evaluate/characterize the APOE∗3-Leiden.glucokinase+/- (E3L.GK+/-) mouse as a novel disease model to study the metabolic syndrome and diabetic complications. METHODS: Female E3L.GK+/-, E3L, and GK+/- mice were fed fat- and cholesterol-containing diets for 37 weeks, and plasma parameters were measured throughout. Development of diabetic macro- and microvascular complications was evaluated. RESULTS: Cholesterol and triglyceride levels were significantly elevated in E3L and E3L.GK+/- mice compared to GK+/- mice, whereas fasting glucose was significantly increased in E3L.GK+/- and GK+/- mice compared to E3L. Atherosclerotic lesion size was increased 2.2-fold in E3L.GK+/- mice as compared to E3L (p = 0.037), which was predicted by glucose exposure (R 2 = 0.636, p = 0.001). E3L and E3L.GK+/- mice developed NASH with severe inflammation and fibrosis which, however, was not altered by introduction of the defective GK phenotype, whereas mild kidney pathology with tubular vacuolization was present in all three phenotypes. CONCLUSIONS: We conclude that the E3L.GK+/- mouse is a promising novel diet-inducible disease model for investigation of the etiology and evaluation of drug treatment on diabetic atherosclerosis.
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Apolipoproteína E3/genética , Aterosclerosis/genética , Complicaciones de la Diabetes/genética , Diabetes Mellitus Tipo 2/genética , Modelos Animales de Enfermedad , Dislipidemias/genética , Animales , Aterosclerosis/sangre , Glucemia/metabolismo , Colesterol/sangre , Complicaciones de la Diabetes/sangre , Diabetes Mellitus Tipo 2/sangre , Dislipidemias/sangre , Femenino , Heterocigoto , Inflamación , Lípidos/sangre , Ratones , Ratones Noqueados , Fenotipo , Riesgo , Investigación Biomédica Traslacional , Triglicéridos/metabolismoRESUMEN
The mechanisms responsible for macrovascular complications in diabetes remain to be fully understood. Recent studies have identified impaired vascular repair as a possible cause of plaque vulnerability in diabetes. This notion is supported by observations of a reduced content of fibrous proteins and smooth muscle cell mitogens in carotid endarterectomy from diabetic patients along with findings of decreased circulating levels of endothelial progenitor cells. In the present study we used a diabetic mouse model to characterize how hyperglycemia affects arterial repair responses. We induced atherosclerotic plaque formation in ApoE-deficient (ApoE-/-) and heterozygous glucokinase knockout ApoE-deficient mice (ApoE-/- GK+/-) mice with a shear stress-modifying cast. There were no differences in cholesterol or triglyceride levels between the ApoE-/- and ApoE-/- GK+/- mice. Hyperglycemia did not affect the size of the formed atherosclerotic plaques, and no effects were seen on activation of cell proliferation, smooth muscle cell content or on the expression and localization of collagen, elastin and several other extracellular matrix proteins. The present study demonstrates that hyperglycemia per se has no significant effects on tissue repair processes in injured mouse carotid arteries, suggesting that other mechanisms are involved in diabetic plaque vulnerability.
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Glucoquinasa/genética , Hiperglucemia/genética , Placa Aterosclerótica/genética , Animales , Traumatismos de las Arterias Carótidas , Proliferación Celular , Colesterol/análisis , Modelos Animales de Enfermedad , Hiperglucemia/complicaciones , Hiperglucemia/metabolismo , Masculino , Ratones , Ratones Noqueados para ApoE , Placa Aterosclerótica/etiología , Placa Aterosclerótica/metabolismo , Resistencia al Corte , Estrés Mecánico , Triglicéridos/análisisRESUMEN
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 ß 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.
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Apolipoproteínas B/deficiencia , Aterosclerosis/prevención & control , Tronco Braquiocefálico/efectos de los fármacos , Factor II del Crecimiento Similar a la Insulina/deficiencia , Factores de Transcripción NFATC/antagonistas & inhibidores , Placa Aterosclerótica , Pirazoles/farmacología , Receptores de LDL/deficiencia , Animales , Apolipoproteína B-100 , Apolipoproteínas B/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Tronco Braquiocefálico/metabolismo , Tronco Braquiocefálico/patología , Catalasa/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Factor II del Crecimiento Similar a la Insulina/genética , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 4/metabolismo , Factores de Transcripción NFATC/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fenotipo , Receptores de LDL/genética , Transducción de SeñalRESUMEN
Aim. Models combining diabetes and atherosclerosis are important in evaluating the cardiovascular (CV) effects and safety of antidiabetes drugs in the development of treatments targeting CV complications. Our aim was to evaluate if crossing the heterozygous glucokinase knockout mouse (GK+/-) and hyperlipidemic mouse deficient in apolipoprotein E (ApoE-/-) will generate a disease model exhibiting a diabetic and macrovascular phenotype. Methods. The effects of defective glucokinase on the glucose metabolism and on the progression and regression of atherosclerosis on high-fat diets were studied in both genders of GK+/-ApoE-/- and ApoE-/- mice. Coronary vascular function of the female GK+/-ApoE-/- and ApoE-/- mice was also investigated. Results. GK+/-ApoE-/- mice show a stable hyperglycemia which was increased on Western diet. In oral glucose tolerance test, GK+/-ApoE-/- mice showed significant glucose intolerance and impaired glucose-stimulated insulin secretion. Plasma lipids were comparable with ApoE-/- mice; nevertheless the GK+/-ApoE-/- mice showed slightly increased atherosclerosis development. Conclusions. The GK+/-ApoE-/- mice showed a stable and reproducible hyperglycemia, accelerated atherosclerotic lesion progression, and no lesion regression after lipid lowering. This novel model provides a promising tool for drug discovery, enabling the evaluation of compound effects against both diabetic and cardiovascular endpoints simultaneously in one animal model.
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Apolipoproteínas E/metabolismo , Aterosclerosis/metabolismo , Glucoquinasa/metabolismo , Hiperglucemia/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/patología , Dieta Alta en Grasa , Progresión de la Enfermedad , Femenino , Glucoquinasa/genética , Hiperglucemia/genética , Hiperglucemia/patología , Lípidos/sangre , Masculino , Ratones , Ratones Noqueados , Miocardio/metabolismo , Miocardio/patologíaRESUMEN
BACKGROUND: Type 2 diabetes is associated with macro- and microvascular complications in man. Microvascular dysfunction affects both cardiac and renal function and is now recognized as a main driver of cardiovascular mortality and morbidity. However, progression of microvascular dysfunction in experimental models is often obscured by macrovascular pathology and consequently demanding to study. The obese type 2 diabetic leptin-deficient (ob/ob) mouse lacks macrovascular complications, i.e. occlusive atherosclerotic disease, and may therefore be a potential model for microvascular dysfunction. The present study aimed to test the hypothesis that these mice with an insulin resistant phenotype might display microvascular dysfunction in both coronary and renal vascular beds. METHODS AND RESULTS: In this study we used non-invasive Doppler ultrasound imaging to characterize microvascular dysfunction during the progression of diabetes in ob/ob mice. Impaired coronary flow velocity reserve was observed in the ob/ob mice at 16 and 21 weeks of age compared to lean controls. In addition, renal resistivity index as well as pulsatility index was higher in the ob/ob mice at 21 weeks compared to lean controls. Moreover, plasma L-arginine was lower in ob/ob mice, while asymmetric dimethylarginine was unaltered. Furthermore, a decrease in renal vascular density was observed in the ob/ob mice. CONCLUSION: In parallel to previously described metabolic disturbances, the leptin-deficient ob/ob mice also display cardiac and renal microvascular dysfunction. This model may therefore be suitable for translational, mechanistic and interventional studies to improve the understanding of microvascular complications in type 2 diabetes.
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Circulación Coronaria , Diabetes Mellitus Tipo 2/diagnóstico por imagen , Leptina/deficiencia , Circulación Renal , Animales , Diabetes Mellitus Tipo 2/genética , Flujometría por Láser-Doppler , Leptina/genética , Masculino , Ratones , UltrasonografíaRESUMEN
Diabetes mellitus is a lifelong, incapacitating metabolic disease associated with chronic macrovascular complications (coronary heart disease, stroke, and peripheral vascular disease) and microvascular disorders leading to damage of the kidneys (nephropathy) and eyes (retinopathy). Based on the current trends, the rising prevalence of diabetes worldwide will lead to increased cardiovascular morbidity and mortality. Therefore, novel means to prevent and treat these complications are needed. Under the auspices of the IMI (Innovative Medicines Initiative), the SUMMIT (SUrrogate markers for Micro- and Macrovascular hard end points for Innovative diabetes Tools) consortium is working on the development of novel animal models that better replicate vascular complications of diabetes and on the characterization of the available models. In the past years, with the high level of genomic information available and more advanced molecular tools, a very large number of models has been created. Selecting the right model for a specific study is not a trivial task and will have an impact on the study results and their interpretation. This review gathers information on the available experimental animal models of diabetic macrovascular complications and evaluates their pros and cons for research purposes as well as for drug development.
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Complicaciones de la Diabetes/fisiopatología , Complicaciones de la Diabetes/terapia , Diabetes Mellitus Experimental/terapia , Modelos Animales de Enfermedad , Animales , Aterosclerosis/complicaciones , Enfermedades Cardiovasculares/complicaciones , Enfermedades Cardiovasculares/terapia , Ensayos Clínicos como Asunto , Enfermedad de la Arteria Coronaria/complicaciones , Angiopatías Diabéticas/terapia , Humanos , Hipoglucemiantes/uso terapéutico , Ratones , Microcirculación , Modelos Animales , Ratas , Especificidad de la EspecieRESUMEN
OBJECTIVES: Coronary microvascular dysfunction, observed as impaired coronary vasodilator capacity, is an early manifestation of coronary artery disease. Inflammation plays an important role in different stages of atherogenesis. To study the role of vessel wall inflammation in the development of coronary dysfunction, we compared [(18)F]fluorodeoxyglucose (FDG) uptake in the aorta and coronary flow reserve (CFR) in atherosclerotic mice. METHODS: We studied healthy young C57BL/6 mice fed a normal diet (n = 7) as well as hypercholesterolemic low-density lipoprotein receptor-disrupted/apolipoprotein B100-expressing (LDLR(-/-)ApoB(100/100)) mice (n = 15) and hypercholesterolemic and diabetic LDLR(-/-)ApoB(100/100)insulinlike growth factor II-overexpressing mice (n = 14) fed a western-type diet, aged 4 to 6 months. Doppler sonography was used to measure CFR as the ratio of coronary flow velocity during isoflurane-induced hyperemia and at rest. Uptake of [(18)F]FDG into the aorta was measured by autoradiography of tissue sections. RESULTS: Histologic sections showed extensive atherosclerosis in the aorta, but coronary arteries were not obstructed. Both hyperemic coronary flow velocity and CFR were reduced (P < .05) in hypercholesterolemic mice with and without diabetes in comparison to healthy young C57BL/6 controls. Among hypercholesterolemic mice, both hyperemic flow velocity and CFR inversely correlated with atherosclerotic plaque [(18)F]FDG uptake in the aorta (r = -0.73; P < .001; r = -0.63; P = .001, respectively). In a multivariate analysis, including animal weight, aortic plaque burden, plasma glucose, plasma cholesterol, and [(18)F]FDG uptake in atherosclerotic plaques, only [(18)F]FDG uptake remained an independent predictor of reduced CFR (ß = 0.736; P = .001). CONCLUSIONS: The inflammatory activity in atherosclerotic plaques of the aorta independently predicts reduced CFR in atherosclerotic mice without obstructive coronary artery disease. This finding suggests that atherosclerotic inflammation contributes to coronary dysfunction.
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Aortitis/metabolismo , Enfermedad de la Arteria Coronaria/metabolismo , Fluorodesoxiglucosa F18/farmacocinética , Reserva del Flujo Fraccional Miocárdico , Tomografía de Emisión de Positrones/métodos , Animales , Aortitis/complicaciones , Aortitis/diagnóstico por imagen , Simulación por Computador , Enfermedad de la Arteria Coronaria/complicaciones , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Femenino , Interpretación de Imagen Asistida por Computador/métodos , Masculino , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Cardiovasculares , Radiofármacos/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: Lymphatic vessels collect extravasated fluid and proteins from tissues to blood circulation as well as play an essential role in lipid metabolism by taking up intestinal chylomicrons. Previous studies have shown that impairment of lymphatic vessel function causes lymphedema and fat accumulation, but clear connections between arterial pathologies and lymphatic vessels have not been described. APPROACH AND RESULTS: Two transgenic mouse strains with lymphatic insufficiency (soluble vascular endothelial growth factor 3 [sVEGFR3] and Chy) were crossed with atherosclerotic mice deficient of low-density lipoprotein receptor and apolipoprotein B48 (LDLR(-/-)/ApoB(100/100)) to study the effects of insufficient lymphatic vessel transport on lipoprotein metabolism and atherosclerosis. Both sVEGFR3×LDLR(-/-)/ApoB(100/100) mice and Chy×LDLR(-/-)/ApoB(100/100) mice had higher plasma cholesterol levels compared with LDLR(-/-)/ApoB(100/100) control mice during both normal chow diet (16.3 and 13.7 versus 8.2 mmol/L, respectively) and Western-type high-fat diet (eg, after 2 weeks of fat diet, 45.9 and 42.6 versus 30.2 mmol/L, respectively). Cholesterol and triglyceride levels in very-low-density lipoprotein and low-density lipoprotein fractions were increased. Atherosclerotic lesions in young and intermediate cohorts of sVEGFR3×LDLR(-/-)/ApoB(100/100) mice progressed faster than in control mice (eg, intermediate cohort mice at 6 weeks, 18.3% versus 7.7% of the whole aorta, respectively). In addition, lesions in sVEGFR3×LDLR(-/-)/ApoB(100/100) mice and Chy×LDLR(-/-)/ApoB(100/100) mice had much less lymphatic vessels than lesions in control mice (0.33% and 1.07% versus 7.45% of podoplanin-positive vessels, respectively). CONCLUSIONS: We show a novel finding linking impaired lymphatic vessels to lipoprotein metabolism, increased plasma cholesterol levels, and enhanced atherogenesis.
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Aterosclerosis/etiología , Hipercolesterolemia/complicaciones , Lipoproteínas/metabolismo , Vasos Linfáticos/fisiopatología , Animales , Apolipoproteínas B/fisiología , Colesterol/metabolismo , Humanos , Lípidos/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de LDL/fisiología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
PURPOSE: The aim of this study was to characterize the ocular morphology of low-density lipoprotein receptor-deficient apolipoprotein B-100-only mice, where overexpression of insulin-like growth factor II (IGF-II) has been shown to induce glucose intolerance and increase atherosclerotic lesion progression and calcification. METHODS: Fifteen-month-old mice were examined on a normal chow diet and after 3 months of a high-fat Western diet. IGF-II-negative LDLR(-/-)ApoB(100/100) littermates and C57Bl/6J mice served as controls. In vivo color images of the fundi were obtained, and eyes were processed either for retinal flat mounts for assessment of neovascularization or for paraffin-embedded samples for immunohistochemical analyses. RESULTS: IGF-II overexpression and the resulting prediabetic phenotype did not induce microvascular damage when assessed in fundus photographs and retinal whole mounts, and the number of capillaries in IGF-II/LDLR(-/-)ApoB(100/100) mice was not significantly different from LDLR(-/-)ApoB(100/100) mice. However, morphology of the inner nuclear, outer plexiform, and outer nuclear layers was altered in the IGF-II/LDLR(-/-)ApoB(100/100) mice. Moreover, photoreceptor atrophy and thinning of the outer nuclear layer were present. Caspase-3 staining was positive in the photoreceptor inner segment. In addition, retinas of the IGF-II/LDLR(-/-)ApoB(100/100) mice displayed reduced rhodopsin positivity, consistent with the decreased number of photoreceptor cells. CONCLUSIONS: This study reports a novel form of retinopathy with photoreceptor atrophy and abundant changes in retinal morphology in a mouse model of prediabetes and atherosclerosis.
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Apolipoproteína B-100/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Receptores de LDL/deficiencia , Enfermedades de la Retina/patología , Animales , Apoptosis , Atrofia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Recuento de Células , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Dieta , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Células Fotorreceptoras de Vertebrados/metabolismo , Receptores de LDL/metabolismo , Degeneración Retiniana/complicaciones , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Enfermedades de la Retina/complicaciones , Enfermedades de la Retina/metabolismoRESUMEN
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.
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Aterosclerosis/etiología , Terapia Genética , Hiperlipidemias/terapia , Lipoproteína Lipasa/metabolismo , Lipoproteínas/sangre , Factor A de Crecimiento Endotelial Vascular/genética , Adenoviridae/genética , Animales , Apolipoproteínas B/deficiencia , Apolipoproteínas B/fisiología , Modelos Animales de Enfermedad , Femenino , Hiperlipidemias/sangre , Hiperlipidemias/patología , Lípidos/sangre , Lipoproteína Lipasa/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de LDL/fisiologíaRESUMEN
AIMS: The loss of nuclear factor E2-related factor 2 (Nrf2) has been shown to protect against atherogenesis in apoE-deficient mice. The mechanism by which Nrf2 deficiency affords atheroprotection in this model is currently unknown, but combined systemic and local vascular effects on lesion macrophages have been proposed. We investigated the effect of bone marrow-specific loss of Nrf2 on early atherogenesis in low-density lipoprotein (LDL) receptor-deficient (LDLR(-/-)) mice, and assessed the effect of Nrf2 on cellular accumulation of modified LDLs and the expression of inflammatory markers in macrophages. METHODS AND RESULTS: The effect of bone marrow-specific loss of Nrf2 on atherogenesis was studied using bone marrow transplantation of wild-type (WT) or Nrf2(-/-) bone marrow to LDLR(-/-) mice. Mice transplanted with Nrf2(-/-) bone marrow and fed a high-fat diet for 6 weeks exhibited significantly larger atherosclerotic lesions than WT bone marrow transplanted mice. Moreover, in thioglycollate-elicited Nrf2(-/-) macrophages, the uptake of acetylated and malondialdehyde-modified LDLs was increased in comparison with WT controls, with the concomitant increase in the expression of scavenger receptor A and toll-like receptor 4. In addition, the expression of pro-inflammatory monocyte chemoattractant protein-1 and interleukin-6 were increased in Nrf2(-/-) vs. WT macrophages. CONCLUSION: Nrf2 deficiency specific to bone marrow-derived cells aggravates atherosclerosis in LDLR(-/-) mice. Furthermore, the loss of Nrf2 in macrophages enhances foam cell formation and promotes the pro-inflammatory phenotype.
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Aterosclerosis/etiología , Macrófagos/fisiología , Factor 2 Relacionado con NF-E2/fisiología , Animales , Quimiocina CCL2/genética , Colesterol/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Receptores de LDL/fisiología , Receptores Depuradores/análisisRESUMEN
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.
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HDL-Colesterol/sangre , Células Endoteliales/enzimología , Lipasa/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adenoviridae/genética , Animales , Apolipoproteína B-100/deficiencia , Apolipoproteína B-100/genética , Aterosclerosis/sangre , Aterosclerosis/enzimología , Aterosclerosis/genética , Sitios de Unión , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Hidroxicolesteroles/farmacología , Lipasa/genética , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Mensajero/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/antagonistas & inhibidores , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Factores de Tiempo , Transfección , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
BACKGROUND: Vascular endothelial growth factors (VEGFs) are central mediators in vascular development and lymphangiogenesis. VEGF-D contributes to the growth and formation of blood and lymphatic vessels, although its biological role is still somewhat unclear. METHODS: Transgenic mice, which express the mature form of human VEGF-D under endothelium-specific Tie1 promoter, were produced by the lentiviral perivitelline-injection method. The mice were followed up to generation F(5) and the effect of the transgene was analyzed. RESULTS: Transgenic mice had a high expression of human (h)VEGF-D in the endothelium in several tissues, such as kidney, liver, lung and spleen. However, transgenic mice developed tumors in lungs, kidneys, liver, mammary glands and lymph nodes upon aging and their mortality was also increased as a result of other pathological conditions. Hind limb ischemia was surgically induced in these mice and they were analyzed 1, 2 and 3 weeks after the ischemia operation. No significant differences were found in hVEGF-D mRNA expression, the number of capillaries or tissue repair between ischemic transgenic mice and transgene negative littermates. CONCLUSIONS: It is concluded that targeted unregulated long-term expression of hVEGF-D in endothelium may not be useful and reduces the life span of transgenic mice.
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Endotelio/metabolismo , Miembro Posterior/irrigación sanguínea , Isquemia/metabolismo , Neoplasias Experimentales/patología , Transgenes/genética , Factor D de Crecimiento Endotelial Vascular/genética , Factor D de Crecimiento Endotelial Vascular/metabolismo , Análisis de Varianza , Animales , Vectores Genéticos/genética , Miembro Posterior/patología , Humanos , Inmunohistoquímica , Lentivirus , Ratones , Ratones Transgénicos , Neoplasias Experimentales/genética , Regiones Promotoras Genéticas/genética , Receptor TIE-1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de SupervivenciaRESUMEN
OBJECTIVES: This study investigated the effects of age, duration of a high-fat diet, and type 2 diabetes on atherosclerotic plaque development and uptake of (18)F-fluorodeoxyglucose ((18)F-FDG) in 2 mouse models. BACKGROUND: The animal's age and start time and duration of a high-fat diet have effects on plaque composition in atherosclerotic mice. METHODS: The aortas of atherosclerotic low-density lipoprotein receptor deficient mice expressing only apolipoprotein B100 (LDLR(-/-)ApoB(100/100)) and atherosclerotic and diabetic mice overexpressing insulin-like growth factor II (IGF-II/LDLR(-/-)ApoB(100/100)) were investigated at 4, 6, and 12 months of age and older after varying durations of high-fat diet. C57BL/6N mice on normal chow served as controls. Plaque size (intima-to-media ratio), macrophage density (Mac-3 staining), and plaque uptake of (18)F-FDG were studied by means of in vivo positron emission tomography/computed tomography by ex vivo autoradiography and by histological and immunohistochemical methods. RESULTS: From the ages of 4 to 6 months and 12 months and older, the plaque size increased and the macrophage density decreased. Compared with the controls, the in vivo imaging showed increased aortic (18)F-FDG uptake at 4 and 6 months, but not at 12 months and older. Autoradiography showed focal (18)F-FDG uptake in plaques at all time points (average plaque-to-normal vessel wall ratio: 2.4 ± 0.4, p < 0.001) with the highest uptake in plaques with high macrophage density. There were no differences in the plaque size, macrophage density, or uptake of (18)F-FDG between LDLR(-/-)ApoB(100/100) and IGF-II/LDLR(-/-)ApoB(100/100) mice at any time point. CONCLUSIONS: The 6-month-old LDLR(-/-)ApoB(100/100) and IGF-II/LDLR(-/-)ApoB(100/100) mice demonstrated highly inflamed, large, and extensive atherosclerotic plaques after 4 months of a high-fat diet, presenting a suitable model for studying the imaging of atherosclerotic plaque inflammation with (18)F-FDG. The presence of type 2 diabetes did not confound evaluation of plaque inflammation with (18)F-FDG.
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Envejecimiento , Aterosclerosis/diagnóstico por imagen , Aterosclerosis/etiología , Diabetes Mellitus Tipo 2/complicaciones , Dieta Alta en Grasa/efectos adversos , Fluorodesoxiglucosa F18 , Imagen Multimodal , Placa Aterosclerótica/diagnóstico por imagen , Placa Aterosclerótica/etiología , Tomografía de Emisión de Positrones , Radiofármacos , Tomografía Computarizada por Rayos X , Factores de Edad , Animales , Aorta/diagnóstico por imagen , Aorta/patología , Aortografía , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Autorradiografía , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Fluorodesoxiglucosa F18/farmacocinética , Inmunohistoquímica , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Radiofármacos/farmacocinética , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Factores de RiesgoRESUMEN
BACKGROUND: Lack of suitable mouse models has hindered the studying of diabetic macrovascular complications. We examined the effects of type 2 diabetes on coronary artery disease and cardiac function in hypercholesterolemic low-density lipoprotein receptor-deficient apolipoprotein B100-only mice (LDLR-/-ApoB100/100). METHODS AND RESULTS: 18-month-old LDLR-/-ApoB100/100 (n = 12), diabetic LDLR-/-ApoB100/100 mice overexpressing insulin-like growth factor-II (IGF-II) in pancreatic beta cells (IGF-II/LDLR-/-ApoB100/100, n = 14) and age-matched C57Bl/6 mice (n = 15) were studied after three months of high-fat Western diet. Compared to LDLR-/-ApoB100/100 mice, diabetic IGF-II/LDLR-/-ApoB100/100 mice demonstrated more calcified atherosclerotic lesions in aorta. However, compensatory vascular enlargement was similar in both diabetic and non-diabetic mice with equal atherosclerosis (cross-sectional lesion area ~60%) and consequently the lumen area was preserved. In coronary arteries, both hypercholesterolemic models showed significant stenosis (~80%) despite positive remodeling. Echocardiography revealed severe left ventricular systolic dysfunction and anteroapical akinesia in both LDLR-/-ApoB100/100 and IGF-II/LDLR-/-ApoB100/100 mice. Myocardial scarring was not detected, cardiac reserve after dobutamine challenge was preserved and ultrasructural changes revealed ischemic yet viable myocardium, which together with coronary artery stenosis and slightly impaired myocardial perfusion suggest myocardial hibernation resulting from chronic hypoperfusion. CONCLUSIONS: LDLR-/-ApoB100/100 mice develop significant coronary atherosclerosis, severe left ventricular dysfunction with preserved but diminished cardiac reserve and signs of chronic myocardial hibernation. However, the cardiac outcome is not worsened by type 2 diabetes, despite more advanced aortic atherosclerosis in diabetic animals.
Asunto(s)
Apolipoproteína B-100/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Angiopatías Diabéticas/complicaciones , Corazón/fisiopatología , Receptores de LDL/deficiencia , Disfunción Ventricular Izquierda/etiología , Disfunción Ventricular Izquierda/fisiopatología , Envejecimiento/fisiología , Animales , Apolipoproteína B-100/genética , Cardiotónicos/farmacología , Enfermedad de la Arteria Coronaria/etiología , Enfermedad de la Arteria Coronaria/fisiopatología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Angiopatías Diabéticas/genética , Angiopatías Diabéticas/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Dobutamina/farmacología , Femenino , Corazón/efectos de los fármacos , Hipercolesterolemia/complicaciones , Hipercolesterolemia/fisiopatología , Factor II del Crecimiento Similar a la Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/patología , Miocardio/ultraestructura , Receptores de LDL/genéticaRESUMEN
OBJECTIVE: The mature form of human vascular endothelial growth factor-D (hVEGF-D(ΔNΔC)) is an efficient angiogenic factor, but its full mechanism of action has remained unclear. We studied the effects of hVEGF-D(ΔNΔC) in endothelial cells using gene array, signaling, cell culture, and in vivo gene transfer techniques. METHODS AND RESULTS: Concomitant with the angiogenic and proliferative responses, hVEGF-D(ΔNΔC) enhanced the phosphorylation of VEGF receptor-2, Akt, and endothelial nitric oxide synthase. Gene arrays, quantitative reverse transcription-polymerase chain reaction, and Western blot revealed increases in VEGF-A, stanniocalcin-1 (STC1), and neuropilin (NRP) 2 expression by hVEGF-D(ΔNΔC) stimulation, whereas induction with hVEGF-A(165) altered the expression of STC1 and NRP1, another coreceptor for VEGFs. The effects of hVEGF-D(ΔNΔC) were seen only under high-serum conditions, whereas for hVEGF-A(165), the strongest response was observed under low-serum conditions. The hVEGF-D(ΔNΔC)-induced upregulation of STC1 and NRP2 was also evident in vivo in mouse skeletal muscle treated with hVEGF-D(ΔNΔC) by adenoviral gene delivery. The importance of NRP2 in hVEGF-D(ΔNΔC) signaling was further studied with NRP2 small interfering RNA and NRP antagonist, which were able to block hVEGF-D(ΔNΔC)-induced survival of endothelial cells. CONCLUSIONS: In this study, the importance of serum and upregulation of NRP2 and STC1 for VEGF-D(ΔNΔC) effects were demonstrated. Better knowledge of VEGF-D(ΔNΔC) signaling and regulation is valuable for the development of efficient and safe VEGF-D(ΔNΔC)-based therapeutic applications for cardiovascular diseases.
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Células Endoteliales/metabolismo , Glicoproteínas/metabolismo , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Neuropilina-2/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor D de Crecimiento Endotelial Vascular/metabolismo , Animales , Apolipoproteína B-100/deficiencia , Apolipoproteína B-100/genética , Western Blotting , Proliferación Celular , Células Cultivadas , Perfilación de la Expresión Génica , Miembro Posterior , Humanos , Ratones , Ratones Noqueados , Neuropilina-1/metabolismo , Neuropilina-2/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Receptores de LDL/deficiencia , Receptores de LDL/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Transducción Genética , Regulación hacia Arriba , Factor D de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: Atherosclerotic plaques with large lipid cores and inflammation contain regions of hypoxia. We examined the uptake of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide ([18F]EF5), a specific marker of hypoxia labeled for positron emission tomography, in mouse atherosclerotic plaques. METHODS AND RESULTS: Atherosclerotic mice of 2 different genetic backgrounds (low-density lipoprotein receptor-/- apolipoprotein B100/100 and insulin-like growth factor II/low-density lipoprotein receptor-/- apolipoprotein B100/100) were first fed a Western diet to induce development of plaques with variable phenotypes and then injected with [18F]EF5. C57BL/6N mice served as controls. Aortas were dissected for biodistribution studies, autoradiography, histology, and immunohistochemistry. Uptake of [18F]EF5 was significantly higher in the aortas of mice with large atherosclerotic plaques than in the C57BL/6N controls. Furthermore, autoradiography demonstrated, on average, 2.0-fold higher [18F]EF5 uptake in atherosclerotic plaques than in the adjacent normal vessel wall. Hypoxia in plaques was verified by using an EF5 adduct-specific antibody and pimonidazole. The blood clearance of [18F]EF5 was slow, with blood radioactivity remaining relatively high up to 180 minutes after injection. CONCLUSIONS: Large atherosclerotic plaques in mice contained hypoxic areas and showed uptake of [18F]EF5. Despite its slow blood clearance, the high uptake of [18F]EF5 in plaques suggested that plaque hypoxia is a potential target for identifying high-risk plaques noninvasively.
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Aorta/diagnóstico por imagen , Aterosclerosis/diagnóstico por imagen , Etanidazol/análogos & derivados , Radioisótopos de Flúor , Hidrocarburos Fluorados , Hipoxia/diagnóstico por imagen , Tomografía de Emisión de Positrones , Radiofármacos , Análisis de Varianza , Animales , Aorta/metabolismo , Aorta/patología , Apolipoproteína B-100/deficiencia , Apolipoproteína B-100/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Autorradiografía , Modelos Animales de Enfermedad , Etanidazol/farmacocinética , Femenino , Radioisótopos de Flúor/farmacocinética , Genotipo , Hidrocarburos Fluorados/farmacocinética , Hipoxia/genética , Hipoxia/metabolismo , Hipoxia/patología , Inmunohistoquímica , Factor II del Crecimiento Similar a la Insulina/deficiencia , Factor II del Crecimiento Similar a la Insulina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nitroimidazoles , Fenotipo , Radiofármacos/farmacocinética , Receptores de LDL/deficiencia , Receptores de LDL/genética , Distribución TisularRESUMEN
AIMS: Macrophage scavenger receptor A (SR-A) and class B scavenger receptor CD36 (CD36) contribute to foam cell formation and atherogenesis via uptake of modified lipoproteins. So far, the role of these scavenger receptors has been studied mainly using knockout models totally lacking these receptors. We studied the role of SR-A and CD36 in foam cell formation and atherogenesis by RNA interference (RNAi)-mediated silencing, which is a clinically feasible method to down-regulate the expression of these receptors. METHODS AND RESULTS: We constructed lentivirus vectors encoding short hairpin RNAs (shRNAs) against mouse SR-A and CD36. Decreased SR-A but not CD36 expression led to reduced foam cell formation caused by acetylated low-density lipoprotein (LDL) in mouse macrophages, whereas the uptake of oxidized LDL was not altered. More importantly, silencing of SR-A upregulates CD36 and vice versa. In LDL receptor-deficient apolipoprotein B100 (LDLR(-/-)ApoB(100/100)) mice kept on a western diet, silencing of either SR-A or CD36 in bone marrow cells led to a marked decrease (37.4 and 34.2%, respectively) in cross-sectional lesion area, whereas simultaneous silencing of both receptors was not effective. CONCLUSION: Our results suggest that silencing of either SR-A or CD36 alone reduces atherogenesis in mice. However, due to reciprocal upregulation, silencing of both SR-A and CD36 is not effective.
Asunto(s)
Aterosclerosis/prevención & control , Antígenos CD36/genética , Hiperlipidemias/complicaciones , Interferencia de ARN , Receptores Depuradores de Clase A/genética , Regulación hacia Arriba/genética , Animales , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Aterosclerosis/genética , Aterosclerosis/metabolismo , Antígenos CD36/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Femenino , Células Espumosas/metabolismo , Células Espumosas/patología , Humanos , Hiperlipidemias/metabolismo , Hiperlipidemias/patología , Lentivirus/genética , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Noqueados , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores Depuradores de Clase A/metabolismoRESUMEN
AIMS: Vascular endothelial growth factors (VEGFs) and their receptors (VEGF-Rs) are among the most powerful factors regulating vascular growth. However, it has remained unknown whether stimulation of VEGF-R1, VEGF-R2 or both of the receptors produces the best angiogenic responses in myocardium. The aim of this study was to compare the VEGF-R1-specific ligand VEGF-B(186), VEGF-R2-specific ligand VEGF-E and VEGF-A(165,) which stimulates both receptors, regarding their effects on angiogenesis and left ventricular function in mice. METHODS AND RESULTS: High-resolution echocardiography was used to guide the closed-chest injections of adenoviral (Ad) vectors expressing VEGF-B(186,) VEGF-E, and VEGF-A(165) into the anterior wall of the left ventricle in C57Bl/6J mice. Angiogenic and functional effects were analysed using histology, ultrasound and perfusion analyses 6 (D6) and 14 (D14) days after the Ad injection. AdVEGF-A(165) induced a strong angiogenic response seen as an enlargement of myocardial capillaries whereas angiogenesis induced by AdVEGF-B(186) and AdVEGF-E seemed more physiological. The increase in the capillary area was accompanied with an increase in myocardial perfusion at D6 after the gene injection. AdVEGF-A(165) and AdVEGF-E induced endothelial-specific proliferation whereas AdVEGF-B(186) mostly induced proliferation of cardiomyocytes. AdVEGF-A(165) induced more pronounced tissue damage than AdVEGF-B(186) and AdVEGF-E. Left ventricular function measured as ejection fraction did not change during the follow-up. AdVEGF-A(165) increased both VEGF-R1 and VEGF-R2 protein expression whereas AdVEGF-B(186) and AdVEGF-E did not affect endogenous receptor expression levels. CONCLUSION: AdVEGF-B(186) and AdVEGF-E are equally potent in inducing therapeutic angiogenesis in mouse myocardium and produce less side effects than AdVEGF-A(165).
Asunto(s)
Neovascularización Fisiológica/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Función Ventricular Izquierda/fisiología , Adenoviridae/genética , Animales , Western Blotting , División Celular/efectos de los fármacos , División Celular/fisiología , Circulación Coronaria/efectos de los fármacos , Circulación Coronaria/fisiología , Ecocardiografía , Ensayo de Inmunoadsorción Enzimática , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Cardiovasculares , Miocarditis/inducido químicamente , Miocarditis/metabolismo , Miocarditis/fisiopatología , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Volumen Sistólico/efectos de los fármacos , Volumen Sistólico/fisiología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/farmacología , Factor B de Crecimiento Endotelial Vascular/genética , Factor B de Crecimiento Endotelial Vascular/metabolismo , Factor B de Crecimiento Endotelial Vascular/farmacología , Función Ventricular Izquierda/efectos de los fármacos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Virales/farmacologíaRESUMEN
RATIONALE: We studied a possibility that shRNAs can lead to transcriptional gene activation at the promoter level via epigenetic mechanism. OBJECTIVE: The purpose of this study was to test the effects on vascular endothelial growth factor (VEGF-A) expression by promoter targeted small hairpin RNAs (shRNAs) in vitro and in experimental animals in vivo using stable local lentiviral gene transfer. METHODS AND RESULTS: One shRNA was identified which strongly increased VEGF-A expression in C166 endothelial cells at mRNA and protein level whereas another shRNA decreased VEGF-A expression. Quantitative chromatin immunoprecipitation analysis revealed that the repressing shRNA caused epigenetic changes, which increased nucleosome density within the promoter and transcription start site and led to repression of VEGF-A expression. Epigenetic changes caused by the activating shRNA were opposite to those caused by the repressing shRNA. These results were confirmed in vivo in an ischemic mouse hindlimb model after local gene transfer where VEGF-A upregulation achieved by promoter-targeted shRNA increased vascularity and blood flow. CONCLUSIONS: We show that lentivirus-mediated delivery of shRNA molecules targeted to specific regions in the mVEGF-A promoter either induce or repress VEGF-A expression via epigenetic modulation. Thus, we describe a new approach of gene therapy, epigenetherapy, based on an epigenetic mechanism at the promoter level. Controlling transcription through manipulation of specific epigenetic marks provides a novel approach for the treatment of several diseases.