The APOE3-Leiden Heterozygous Glucokinase Knockout Mouse as Novel Translational Disease Model for Type 2 Diabetes, Dyslipidemia, and Diabetic Atherosclerosis.

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.

Hyperglycemia does not affect tissue repair responses in shear stress-induced atherosclerotic plaques in ApoE-/- mice.

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 vivo inhibition of nuclear factor of activated T-cells leads to atherosclerotic plaque regression in IGF-II/LDLRApoB mice.

Aims: Despite vast clinical experience linking diabetes and atherosclerosis, the molecular mechanisms leading to accelerated vascular damage are still unclear. Here, we investigated the effects of nuclear factor of activated T-cells inhibition on plaque burden in a novel mouse model of type 2 diabetes that better replicates human disease.

Methods & Results: IGF-II/LDLRApoB mice were generated by crossbreeding low-density lipoprotein receptor-deficient mice that synthesize only apolipoprotein B100 (LDLRApoB) with transgenic mice overexpressing insulin-like growth factor-II in pancreatic β cells.

Hyperglycemia Induced by Glucokinase Deficiency Accelerates Atherosclerosis Development and Impairs Lesion Regression in Combined Heterozygous Glucokinase and the Apolipoprotein E-Knockout Mice.

. 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.

Impaired Coronary and Renal Vascular Function in Spontaneously Type 2 Diabetic Leptin-Deficient Mice.

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.

Animal models of diabetic macrovascular complications: key players in the development of new therapeutic approaches.

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.

[18F]fluorodeoxyglucose uptake in atherosclerotic plaques is associated with reduced coronary flow reserve in mice.

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.

Lymphatic vessel insufficiency in hypercholesterolemic mice alters lipoprotein levels and promotes atherogenesis.

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.

LDLR-/-ApoB100/100 mice with insulin-like growth factor II overexpression reveal a novel form of retinopathy with photoreceptor atrophy and altered morphology of the retina.

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.

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

Aims: The role of vascular endothelial growth factor (VEGF-A) in atherogenesis has remained controversial. We addressed this by comparing the effects of adenoviral VEGF-A gene transfer on atherosclerosis and lipoproteins in ApoE(-/-), LDLR(-/-), LDLR(-/-)ApoE(-/-), and LDLR(-/-)ApoB(100/100) mice.

Methods And Results: After 4 weeks on western diet, systemic adenoviral gene transfer was performed with hVEGF-A or control vectors.

The absence of macrophage Nrf2 promotes early atherogenesis.

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.

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

Objective: Endothelial lipase (EL) regulates HDL cholesterol levels and in inflammatory states, like atherosclerosis, EL expression is increased contributing to low HDL cholesterol. The regulation of EL expression is poorly understood and has mainly been attributed to inflammatory stimuli. As sterol regulatory element binding proteins (SREBPs) are regulators of genes involved in lipid metabolism, we hypothesized that EL is regulated by SREBPs and that EL expression is modified by the SREBP activator vascular endothelial growth factor A (VEGF-A).

Endothelium-specific overexpression of human vascular endothelial growth factor-D in mice leads to increased tumor frequency and a reduced lifespan.

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.

Effects of age, diet, and type 2 diabetes on the development and FDG uptake of atherosclerotic plaques.

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.

Left ventricular dysfunction with reduced functional cardiac reserve in diabetic and non-diabetic LDL-receptor deficient apolipoprotein B100-only mice.

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.

Vascular endothelial growth factor (VEGF)-D stimulates VEGF-A, stanniocalcin-1, and neuropilin-2 and has potent angiogenic effects.

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.

Detection of hypoxia by [18F]EF5 in atherosclerotic plaques in mice.

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.

Silencing of either SR-A or CD36 reduces atherosclerosis in hyperlipidaemic mice and reveals reciprocal upregulation of these receptors.

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.

The effects of VEGF-R1 and VEGF-R2 ligands on angiogenic responses and left ventricular function in mice.

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.

Efficient regulation of VEGF expression by promoter-targeted lentiviral shRNAs based on epigenetic mechanism: a novel example of epigenetherapy.

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.

Vascular endothelial growth factor-D transgenic mice show enhanced blood capillary density, improved postischemic muscle regeneration, and increased susceptibility to tumor formation.

Vascular endothelial growth factor-D (VEGF-D) has angiogenic and lymphangiogenic activity, but its biologic role has remained unclear because knockout mice showed no clear phenotype. Transgenic (TG) mice expressing the mature form of human VEGF-D (hVEGF-D) were produced by lentiviral (LV) transgenesis using the perivitelline injection method. Several viable founders showed a macroscopically normal phenotype and the transgene transmitted through germ line.

Nrf2 regulates antioxidant gene expression evoked by oxidized phospholipids in endothelial cells and murine arteries in vivo.

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.

Umbilical cord blood-derived progenitor cells enhance muscle regeneration in mouse hindlimb ischemia model.

Progenitor cell therapy is a potential new treatment option for ischemic conditions in the myocardium and skeletal muscles. However, it remains unclear whether umbilical cord blood (UCB)-derived progenitor cells can provide therapeutic effects in ischemic muscles and whether ex vivo gene transfer can be used for improving the effect. In this study, the use of a lentiviral vector led to efficient transduction of both UCB-derived hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs).

Increased atherosclerotic lesion calcification in a novel mouse model combining insulin resistance, hyperglycemia, and hypercholesterolemia.

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)).

Transduction patterns and efficiencies in rabbit uterine tissues after intraluminal uterine adenovirus administration vary with the reproductive cycle.

Background: The options for destroying the full thickness of endometrium are very limited, relying mainly on surgical endometrial ablation. A nonsurgical, safe method would benefit many women in clinical settings. This study was undertaken to investigate the potential of gene therapy to transfect and destroy endometriun at different stages of the reproductive cycle in rabbits.