RESUMO
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.
Assuntos
Aterosclerose/etiologia , Hipercolesterolemia/complicações , Lipoproteínas/metabolismo , Vasos Linfáticos/fisiopatologia , Animais , Apolipoproteínas B/fisiologia , Colesterol/metabolismo , Humanos , Lipídeos/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de LDL/fisiologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologiaRESUMO
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.
Assuntos
Aortite/metabolismo , Doença da Artéria Coronariana/metabolismo , Fluordesoxiglucose F18/farmacocinética , Reserva Fracionada de Fluxo Miocárdico , Tomografia por Emissão de Pósitrons/métodos , Animais , Aortite/complicações , Aortite/diagnóstico por imagem , Simulação por Computador , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/diagnóstico por imagem , Feminino , Interpretação de Imagem Assistida por Computador/métodos , Masculino , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Cardiovasculares , Compostos Radiofarmacêuticos/farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Apolipoproteína B-100/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Receptores de LDL/deficiência , Doenças Retinianas/patologia , Animais , Apoptose , Atrofia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Contagem de Células , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Dieta , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras de Vertebrados/metabolismo , Receptores de LDL/metabolismo , Degeneração Retiniana/complicações , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Doenças Retinianas/complicações , Doenças Retinianas/metabolismoRESUMO
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.
Assuntos
Endotélio/metabolismo , Membro Posterior/irrigação sanguínea , Isquemia/metabolismo , Neoplasias Experimentais/patologia , Transgenes/genética , Fator D de Crescimento do Endotélio Vascular/genética , Fator D de Crescimento do Endotélio Vascular/metabolismo , Análise de Variância , Animais , Vetores Genéticos/genética , Membro Posterior/patologia , Humanos , Imuno-Histoquímica , Lentivirus , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/genética , Regiões Promotoras Genéticas/genética , Receptor de TIE-1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de SobrevidaRESUMO
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.
Assuntos
Aorta/diagnóstico por imagem , Aterosclerose/diagnóstico por imagem , Etanidazol/análogos & derivados , Radioisótopos de Flúor , Hidrocarbonetos Fluorados , Hipóxia/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Análise de Variância , Animais , Aorta/metabolismo , Aorta/patologia , Apolipoproteína B-100/deficiência , Apolipoproteína B-100/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Autorradiografia , Modelos Animais de Doenças , Etanidazol/farmacocinética , Feminino , Radioisótopos de Flúor/farmacocinética , Genótipo , Hidrocarbonetos Fluorados/farmacocinética , Hipóxia/genética , Hipóxia/metabolismo , Hipóxia/patologia , Imuno-Histoquímica , Fator de Crescimento Insulin-Like II/deficiência , Fator de Crescimento Insulin-Like II/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitroimidazóis , Fenótipo , Compostos Radiofarmacêuticos/farmacocinética , Receptores de LDL/deficiência , Receptores de LDL/genética , Distribuição TecidualRESUMO
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.
Assuntos
Células Endoteliais/metabolismo , Glicoproteínas/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Neuropilina-2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/metabolismo , Animais , Apolipoproteína B-100/deficiência , Apolipoproteína B-100/genética , Western Blotting , Proliferação de Células , Células Cultivadas , Perfilação da Expressão Gênica , Membro Posterior , Humanos , Camundongos , Camundongos Knockout , Neuropilina-1/metabolismo , Neuropilina-2/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptores de LDL/deficiência , Receptores de LDL/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo , Transdução Genética , Regulação para Cima , Fator D de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Apolipoproteína B-100/metabolismo , Diabetes Mellitus Tipo 2/complicações , Angiopatias Diabéticas/complicações , Coração/fisiopatologia , Receptores de LDL/deficiência , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/fisiopatologia , Envelhecimento/fisiologia , Animais , Apolipoproteína B-100/genética , Cardiotônicos/farmacologia , Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/fisiopatologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Dobutamina/farmacologia , Feminino , Coração/efeitos dos fármacos , Hipercolesterolemia/complicações , Hipercolesterolemia/fisiopatologia , Fator de Crescimento Insulin-Like II/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/patologia , Miocárdio/ultraestrutura , Receptores de LDL/genéticaRESUMO
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.
Assuntos
Epigênese Genética , Terapia Genética/métodos , Membro Posterior/irrigação sanguínea , Isquemia/terapia , Lentivirus , Regiões Promotoras Genéticas , RNA/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Linhagem Celular , Células Endoteliais/metabolismo , Isquemia/genética , Camundongos , RNA/genética , Transcrição Gênica , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Besides their well-characterized proinflammatory and proatherogenic effects, oxidized phospholipids, such as oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine) have been shown to have beneficial responses in vascular cells via induction of antioxidant enzymes such as heme oxygenase-1. We therefore hypothesized that oxPAPC could evoke a general cytoprotective response via activation of antioxidative transcription factor Nrf2. Here, we show that oxPAPC increases nuclear accumulation of Nrf2. Using the small interfering RNA approach, we demonstrate that Nrf2 is critical in mediating the induction of glutamate-cysteine ligase modifier subunit (GCLM) and NAD(P)H quinone oxidoreductase-1 (NQO1) by oxPAPC in human endothelial cells, whereas the contribution to the induction of heme oxygenase-1 was less significant. The induction of GCLM and NQO1 was attenuated by reduction of electrophilic groups with sodium borohydrate, as well as treatment with thiol antioxidant N-acetylcysteine, suggesting that the thiol reactivity of oxPAPC is largely mediating its effect on Nrf2-responsive genes. Moreover, we show that oxidized phospholipid having a highly electrophilic isoprostane ring in its sn-2 position is a potent inducer of Nrf2 target genes. Finally, we demonstrate that the oxPAPC-inducible expression of heme oxygenase-1, GCLM, and NQO1 is lower in Nrf2-null than wild-type mouse carotid arteries in vivo. We suggest that the activation of Nrf2 by oxidized phospholipids provides a mechanism by which their deleterious effects are limited in the vasculature.
Assuntos
Antioxidantes/metabolismo , Artérias Carótidas/enzimologia , Núcleo Celular/metabolismo , Células Endoteliais/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Fosfatidilcolinas/farmacocinética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Artérias Carótidas/citologia , Núcleo Celular/genética , Células Endoteliais/citologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Glutamato-Cisteína Ligase/biossíntese , Glutamato-Cisteína Ligase/genética , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/genética , Camundongos , Camundongos Mutantes , NAD(P)H Desidrogenase (Quinona) , NADPH Desidrogenase/biossíntese , NADPH Desidrogenase/genética , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Oxirredução/efeitos dos fármacos , Fosfatidilcolinas/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
No mouse model is currently available where the induction of type 2 diabetes on an atherosclerotic background could be achieved without significant concomitant changes in plasma lipid levels. We crossbred 2 genetically modified mouse strains to achieve a model expressing both atherosclerosis and characteristics of type 2 diabetes. For atherosclerotic background we used low-density lipoprotein receptor-deficient mice synthetizing only apolipoprotein B100 (LDLR(-/-) ApoB(100/100)). Diabetic background was obtained from transgenic mice overexpressing insulin-like growth factor-II (IGF-II) in pancreatic beta cells. Thorough phenotypic characterization was performed in 6- and 15-month-old mice on both normal and high-fat Western diet. Results indicated that IGF-II transgenic LDLR(-/-)ApoB(100/100) mice demonstrated insulin resistance, hyperglycemia, and mild hyperinsulinemia compared with hypercholesterolemic LDLR(-/-)ApoB(100/100) controls. In addition, old IGF-II/LDLR(-/-)ApoB(100/100) mice displayed significantly increased lesion calcification, which was more related to insulin resistance than glucose levels, and significantly higher baseline expression in aorta of several genes related to calcification and inflammation. Lipid levels of IGF-II/LDLR(-/-)ApoB(100/100) mice did not differ from LDLR(-/-)ApoB(100/100) controls at any time. In conclusion, type 2 diabetic factors induce increased calcification and lesion progression without any lipid changes in a new mouse model of diabetic macroangiopathy.
Assuntos
Calcinose/complicações , Angiopatias Diabéticas/complicações , Modelos Animais de Doenças , Hipercolesterolemia/complicações , Hiperglicemia/complicações , Camundongos Transgênicos , Animais , Apolipoproteínas B/genética , Aterosclerose/complicações , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Glicemia , Calcinose/patologia , Calcinose/fisiopatologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/patologia , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/patologia , Angiopatias Diabéticas/fisiopatologia , Hipercolesterolemia/patologia , Hipercolesterolemia/fisiopatologia , Hiperglicemia/patologia , Hiperglicemia/fisiopatologia , Resistência à Insulina , Fator de Crescimento Insulin-Like II/genética , Lipídeos/sangue , Camundongos , Camundongos Endogâmicos C57BL , Osteoprotegerina/sangue , Receptores de LDL/genéticaRESUMO
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.
Assuntos
Apolipoproteína E3/genética , Aterosclerose/genética , Complicações do Diabetes/genética , Diabetes Mellitus Tipo 2/genética , Modelos Animais de Doenças , Dislipidemias/genética , Animais , Aterosclerose/sangue , Glicemia/metabolismo , Colesterol/sangue , Complicações do Diabetes/sangue , Diabetes Mellitus Tipo 2/sangue , Dislipidemias/sangue , Feminino , Heterozigoto , Inflamação , Lipídeos/sangue , Camundongos , Camundongos Knockout , Fenótipo , Risco , Pesquisa Translacional Biomédica , Triglicerídeos/metabolismoRESUMO
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.
Assuntos
Glucoquinase/genética , Hiperglicemia/genética , Placa Aterosclerótica/genética , Animais , Lesões das Artérias Carótidas , Proliferação de Células , Colesterol/análise , Modelos Animais de Doenças , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Masculino , Camundongos , Camundongos Knockout para ApoE , Placa Aterosclerótica/etiologia , Placa Aterosclerótica/metabolismo , Resistência ao Cisalhamento , Estresse Mecânico , Triglicerídeos/análiseRESUMO
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.
Assuntos
Apolipoproteínas B/deficiência , Aterosclerose/prevenção & controle , Tronco Braquiocefálico/efeitos dos fármacos , Fator de Crescimento Insulin-Like II/deficiência , Fatores de Transcrição NFATC/antagonistas & inibidores , Placa Aterosclerótica , Pirazóis/farmacologia , Receptores de LDL/deficiência , Animais , Apolipoproteína B-100 , Apolipoproteínas B/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Tronco Braquiocefálico/metabolismo , Tronco Braquiocefálico/patologia , Catalase/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Fator de Crescimento Insulin-Like II/genética , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 4/metabolismo , Fatores de Transcrição NFATC/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Receptores de LDL/genética , Transdução de SinaisRESUMO
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.
Assuntos
Apolipoproteínas E/metabolismo , Aterosclerose/metabolismo , Glucoquinase/metabolismo , Hiperglicemia/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Dieta Hiperlipídica , Progressão da Doença , Feminino , Glucoquinase/genética , Hiperglicemia/genética , Hiperglicemia/patologia , Lipídeos/sangue , Masculino , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologiaRESUMO
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.
Assuntos
Circulação Coronária , Diabetes Mellitus Tipo 2/diagnóstico por imagem , Leptina/deficiência , Circulação Renal , Animais , Diabetes Mellitus Tipo 2/genética , Fluxometria por Laser-Doppler , Leptina/genética , Masculino , Camundongos , UltrassonografiaRESUMO
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.
Assuntos
Complicações do Diabetes/fisiopatologia , Complicações do Diabetes/terapia , Diabetes Mellitus Experimental/terapia , Modelos Animais de Doenças , Animais , Aterosclerose/complicações , Doenças Cardiovasculares/complicações , Doenças Cardiovasculares/terapia , Ensaios Clínicos como Assunto , Doença da Artéria Coronariana/complicações , Angiopatias Diabéticas/terapia , Humanos , Hipoglicemiantes/uso terapêutico , Camundongos , Microcirculação , Modelos Animais , Ratos , Especificidade da EspécieRESUMO
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.
Assuntos
Aterosclerose/etiologia , Terapia Genética , Hiperlipidemias/terapia , Lipase Lipoproteica/metabolismo , Lipoproteínas/sangue , Fator A de Crescimento do Endotélio Vascular/genética , Adenoviridae/genética , Animais , Apolipoproteínas B/deficiência , Apolipoproteínas B/fisiologia , Modelos Animais de Doenças , Feminino , Hiperlipidemias/sangue , Hiperlipidemias/patologia , Lipídeos/sangue , Lipase Lipoproteica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de LDL/fisiologiaRESUMO
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.
Assuntos
Aterosclerose/etiologia , Macrófagos/fisiologia , Fator 2 Relacionado a NF-E2/fisiologia , Animais , Quimiocina CCL2/genética , Colesterol/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de LDL/fisiologia , Receptores Depuradores/análiseRESUMO
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.
Assuntos
HDL-Colesterol/sangue , Células Endoteliais/enzimologia , Lipase/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adenoviridae/genética , Animais , Apolipoproteína B-100/deficiência , Apolipoproteína B-100/genética , Aterosclerose/sangue , Aterosclerose/enzimologia , Aterosclerose/genética , Sítios de Ligação , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Hidroxicolesteróis/farmacologia , Lipase/genética , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Interferência de RNA , RNA Mensageiro/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/antagonistas & inibidores , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Fatores de Tempo , Transfecção , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
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.